Characterization of Morreton virus as an oncolytic virotherapy platform for liver cancers.

BACKGROUND: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). AIM: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. APPROACH AND RESULTS: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells. CONCLUSIONS: Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

Systems neuroimmunology: a review of multiomics methodologies to characterize neuroimmunological interactions in spinal and cranial diseases.

Neuroimmunology plays a critical role in our understanding of the pathophysiological processes that underlie a variety of diseases treated by neurosurgeons, including degenerative disc disease (DDD), glioblastoma (GBM), aneurysmal subarachnoid hemorrhage (aSAH), and others. Compared with traditional methods in neuroimmunology, which study one pathway or gene at a time, emerging multiomics methodologies allow for holistic interrogation of multiple immune-signaling pathways to test hypotheses and the effects of therapeutics at a systems level. In this review, the authors summarize key concepts for gathering and analyzing multiomics data so that neurosurgeons can contribute to the emerging field of systems neuroimmunology. Additionally, they describe 3 use cases, based on original research published by their group and others, that utilize transcriptomic, metabolomic, and proteomic analyses to study immune-signaling pathways in DDD, aSAH, and GBM. Through these use cases, techniques for performing machine learning and network-based analyses to generate new clinical insights from multiomics data are shared. The authors hope that neurosurgeons might use this review as a summary of common tools and principles in systems immunology to better engage in creating the immunotherapies of tomorrow.

Brain cancer induces systemic immunosuppression through release of non-steroid soluble mediators.

Immunosuppression of unknown aetiology is a hallmark feature of glioblastoma and is characterized by decreased CD4 T-cell counts and downregulation of major histocompatibility complex class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for glioblastoma. We recapitulated the immunosuppression observed in glioblastoma patients in the C57BL/6 mouse and investigated the aetiology of low CD4 T-cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of brain cancer, including mice harbouring GL261 glioma, B16 melanoma, and in a spontaneous model of diffuse intrinsic pontine glioma. In addition to thymic involution, we determined that tumour growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC II expression on blood leucocytes, and a modest increase in bone marrow resident CD4 T cells. Using parabiosis we report that thymic involution, declines in peripheral T-cell counts, and reduced major histocompatibility complex class II expression levels were mediated through circulating blood-derived factors. Conversely, T-cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is non-steroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the immunosuppression was not unique to cancer itself, but rather occurs in response to brain injury. Non-cancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that brain cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity.

Vesicular Stomatitis Virus Encoding a Destabilized Tumor Antigen Improves Activation of Anti-tumor T Cell Responses.

Enhancing the immunogenicity of tumor-associated antigens would represent a major advance for anti-tumor vaccination strategies. Here, we investigated structure-directed antigen destabilization as a strategy to improve the degradation, immunogenic epitope presentation, and T cell activation against a vesicular stomatitis virus (VSV)-encoded tumor antigen. We used the crystal structure of the model antigen ovalbumin to identify charge-disrupting amino acid mutations that were predicted to decrease the stability of the protein. One mutation, OVA-C12R, significantly reduced the half-life of the protein and was preferentially degraded in a 26-S proteasomal-dependent manner. The destabilized ovalbumin protein exhibited enhanced presentation of the major histocompatibility complex (MHC) class I immunogenic epitope, SIINFEKL, on the surface of B16F10 cells or murine bone marrow-derived dendritic cells (BMDCs) in vitro. Enhanced presentation correlated with better recognition by cognate CD8 OT-I T cells as measured by activation, proliferation, and effector cytokine production. Finally, VSV encoding the degradation-prone antigen was better able to prime an antigen ovalbumin-specific CD8 T cell response in vivo without altering the anti-viral CD8 T cell response. Our studies highlight that not only is the choice of antigen in cancer vaccines of importance, but that emphasis should be placed on modifying antigen quality to ensure optimal priming of anti-tumor responses.

Synthetic and systems biology principles in the design of programmable oncolytic virus immunotherapies for glioblastoma.

Oncolytic viruses (OVs) are a class of immunotherapeutic agents with promising preclinical results for the treatment of glioblastoma (GBM) but have shown limited success in recent clinical trials. Advanced bioengineering principles from disciplines such as synthetic and systems biology are needed to overcome the current challenges faced in developing effective OV-based immunotherapies for GBMs, including off-target effects and poor clinical responses. Synthetic biology is an emerging field that focuses on the development of synthetic DNA constructs that encode networks of genes and proteins (synthetic genetic circuits) to perform novel functions, whereas systems biology is an analytical framework that enables the study of complex interactions between host pathways and these synthetic genetic circuits. In this review, the authors summarize synthetic and systems biology concepts for developing programmable, logic-based OVs to treat GBMs. Programmable OVs can increase selectivity for tumor cells and enhance the local immunological response using synthetic genetic circuits. The authors discuss key principles for developing programmable OV-based immunotherapies, including how to 1) select an appropriate chassis, a vector that carries a synthetic genetic circuit, and 2) design a synthetic genetic circuit that can be programmed to sense key signals in the GBM microenvironment and trigger release of a therapeutic payload. To illustrate these principles, some original laboratory data are included, highlighting the need for systems biology studies, as well as some preliminary network analyses in preparation for synthetic biology applications. Examples from the literature of state-of-the-art synthetic genetic circuits that can be packaged into leading candidate OV chassis are also surveyed and discussed.

Potentiating Oncolytic Virus-Induced Immune-Mediated Tumor Cell Killing Using Histone Deacetylase Inhibition.

A clinical oncolytic herpes simplex virus (HSV) encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), talimogene laherparepvec, causes regression of injected and non-injected melanoma lesions in patients and is now licensed for clinical use in advanced melanoma. To date, limited data are available regarding the mechanisms of human anti-tumor immune priming, an improved understanding of which could inform the development of future combination strategies with improved efficacy. This study addressed direct oncolysis and innate and adaptive human immune-mediated effects of a closely related HSV encoding GM-CSF (HSVGM-CSF) alone and in combination with histone deacetylase inhibition. We found that HSVGM-CSF supported activation of anti-melanoma immunity via monocyte-mediated type I interferon production, which activates NK cells, and viral maturation of immature dendritic cells (iDCs) into potent antigen-presenting cells for cytotoxic T lymphocyte (CTL) priming. Addition of the histone deacetylase inhibitor valproic acid (VPA) to HSVGM-CSF treatment of tumor cells increased viral replication, viral GM-CSF production, and oncolysis and augmented the development of anti-tumor immunity. Mechanistically, VPA increased expression of activating ligands for NK cell recognition and induced expression of tumor-associated antigens, supporting innate NK cell killing and CTL priming. These data support the clinical combination of talimogene laherparepvec with histone deacetylase inhibition to enhance oncolysis and anti-tumor immunity.

Oncolytic reovirus as a combined antiviral and anti-tumour agent for the treatment of liver cancer.

OBJECTIVE: Oncolytic viruses (OVs) represent promising, proinflammatory cancer treatments. Here, we explored whether OV-induced innate immune responses could simultaneously inhibit HCV while suppressing hepatocellular carcinoma (HCC). Furthermore, we extended this exemplar to other models of virus-associated cancer. DESIGN AND RESULTS: Clinical grade oncolytic orthoreovirus (Reo) elicited innate immune activation within primary human liver tissue in the absence of cytotoxicity and independently of viral genome replication. As well as achieving therapy in preclinical models of HCC through the activation of innate degranulating immune cells, Reo-induced cytokine responses efficiently suppressed HCV replication both in vitro and in vivo. Furthermore, Reo-induced innate responses were also effective against models of HBV-associated HCC, as well as an alternative endogenous model of Epstein-Barr virus-associated lymphoma. Interestingly, Reo appeared superior to the majority of OVs in its ability to elicit innate inflammatory responses from primary liver tissue. CONCLUSIONS: We propose that Reo and other select proinflammatory OV may be used in the treatment of multiple cancers associated with oncogenic virus infections, simultaneously reducing both virus-associated oncogenic drive and tumour burden. In the case of HCV-associated HCC (HCV-HCC), Reo should be considered as an alternative agent to supplement and support current HCV-HCC therapies, particularly in those countries where access to new HCV antiviral treatments may be limited.

Inhibitory Receptors Induced by VSV Viroimmunotherapy Are Not Necessarily Targets for Improving Treatment Efficacy.

Systemic viroimmunotherapy activates endogenous innate and adaptive immune responses against both viral and tumor antigens. We have shown that therapy with vesicular stomatitis virus (VSV) engineered to express a tumor-associated antigen activates antigen-specific adoptively transferred T cells (adoptive cell therapy, ACT) in vivo to generate effective therapy. The overall goal of this study was to phenotypically characterize the immune response to VSV+ACT therapy and use the information gained to rationally improve combination therapy. We observed rapid expansion of blood CD8+ effector cells acutely following VSV therapy with markedly high expression of the immune checkpoint molecules PD-1 and TIM-3. Using these data, we tested a treatment schedule incorporating mAb immune checkpoint inhibitors with VSV+ACT treatment. Unlike clinical scenarios, we delivered therapy at early time points following tumor establishment and treatment. Our goal was to potentiate the immune response generated by VSV therapy to achieve durable control of metastatic disease. Despite the high frequency of endogenous PD-1+ TIM-3+ CD8+ T cells following virus administration, antibody blockade did not improve survival. These findings provide highly significant information about response kinetics to viroimmunotherapy and juxtapose the clinical use of checkpoint inhibitors against chronically dysfunctional T cells and the acute T cell response to oncolytic viruses.

Combination Therapy With Reovirus and Anti-PD-1 Blockade Controls Tumor Growth Through Innate and Adaptive Immune Responses.

Oncolytic reovirus can be delivered both systemically and intratumorally, in both preclinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against a variety of tumor types and antitumor activity is directly associated with immune activation by virus replication in tumors. Immune mechanisms of therapy include both innate immune activation against virally infected tumor cells, and the generation of adaptive antitumor immune responses as a result of in vivo priming against tumor-associated antigens. We tested the combination of local oncolytic reovirus therapy with systemic immune checkpoint inhibition. We show that treatment of subcutaneous B16 melanomas with a combination of intravenous (i.v.) anti-PD-1 antibody and intratumoral (i.t.) reovirus significantly enhanced survival of mice compared to i.t. reovirus (P < 0.01) or anti-PD-1 therapy alone. In vitro immune analysis demonstrated that checkpoint inhibition improved the ability of NK cells to kill reovirus-infected tumor cells, reduced T(reg) activity, and increased the adaptive CD8(+) T-cell-dependent antitumor T-cell response. PD-1 blockade also enhanced the antiviral immune response but through effector mechanisms which overlapped with but also differed from those affecting the antitumor response. Therefore, combination with checkpoint inhibition represents a readily translatable next step in the clinical development of reovirus viroimmunotherapy.

Mutated BRAF Emerges as a Major Effector of Recurrence in a Murine Melanoma Model After Treatment With Immunomodulatory Agents.

We used a VSV-cDNA library to treat recurrent melanoma, identifying immunogenic antigens, allowing us to target recurrences with immunotherapy or chemotherapy. Primary B16 melanoma tumors were induced to regress by frontline therapy. Mice with recurrent tumors were treated with VSV-cDNA immunotherapy. A Th17 recall response was used to screen the VSV-cDNA library for individual viruses encoding rejection antigens, subsequently targeted using immunotherapy or chemotherapy. Recurrent tumors were effectively treated with a VSV-cDNA library using cDNA from recurrent B16 tumors. Recurrence-associated rejection antigens identified included Topoisomerase-IIα, YB-1, cdc7 kinase, and BRAF. Fourteen out of 16 recurrent tumors carried BRAF mutations (595-605 region) following frontline therapy, even though the parental B16 tumors were BRAF wild type. The emergence of mutated BRAF-containing recurrences served as an excellent target for BRAF-specific immune-(VSV-BRAF), or chemo-(PLX-4720) therapies. Successful PLX-4720 therapy of recurrent tumors was associated with the development of a broad spectrum of T-cell responses. VSV-cDNA technology can be used to identify recurrence specific antigens. Emergence of mutated BRAF may be a major effector of melanoma recurrence which could serve as a target for chemo or immune therapy. This study suggests a rationale for offering patients with initially wild-type BRAF melanomas an additional biopsy to screen for mutant BRAF upon recurrence.

BRAF- and MEK-Targeted Small Molecule Inhibitors Exert Enhanced Antimelanoma Effects in Combination With Oncolytic Reovirus Through ER Stress.

Reovirus type 3 (Dearing) (RT3D) infection is selective for cells harboring a mutated/activated RAS pathway. Therefore, in a panel of melanoma cell lines (including RAS mutant, BRAF mutant and RAS/BRAF wild-type), we assessed therapeutic combinations that enhance/suppress ERK1/2 signaling through use of BRAF/MEK inhibitors. In RAS mutant cells, the combination of RT3D with the BRAF inhibitor PLX4720 (paradoxically increasing ERK1/2 signaling in this context) did not enhance reoviral cytotoxicity. Instead, and somewhat surprisingly, RT3D and BRAF inhibition led to enhanced cell kill in BRAF mutated cell lines. Likewise, ERK1/2 inhibition, using the MEK inhibitor PD184352, in combination with RT3D resulted in enhanced cell kill in the entire panel. Interestingly, TCID50 assays showed that BRAF and MEK inhibitors did not affect viral replication. Instead, enhanced efficacy was mediated through ER stress-induced apoptosis, induced by the combination of ERK1/2 inhibition and reovirus infection. In vivo, combined treatments of RT3D and PLX4720 showed significantly increased activity in BRAF mutant tumors in both immune-deficient and immune-competent models. These data provide a strong rationale for clinical translation of strategies in which RT3D is combined with BRAF inhibitors (in BRAF mutant melanoma) and/or MEK inhibitors (in BRAF and RAS mutant melanoma).

Progress in clinical oncolytic virus-based therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) carries a dismal prognosis, with advanced disease being resistant to both radiotherapy and conventional cytotoxic drugs, whilst anti-angiogenic drugs are marginally efficacious. Oncolytic viruses (OVs) offer the promise of selective cancer therapy through direct and immune-mediated mechanisms. The premise of OVs lies in their preferential genomic replication, protein expression and productive infection of malignant cells. Numerous OVs are being tested in preclinical models of HCC, with good evidence of direct and immune-mediated anti-tumour efficacy. Efforts to enhance the performance of these agents have concentrated on engineering OV cellular specificity, immune evasion, enhancing anti-tumour potency and improving delivery. The lead agent in HCC clinical trials, JX-594, a recombinant Wyeth strain vaccinia virus, has demonstrated evidence for significant benefit and earned orphan drug status. Thus, JX-594 appears to be transcending the barrier between novel laboratory science and credible clinical therapy. Relatively few other OVs have entered clinical testing, a hurdle that must be overcome if significant progress is to be made in this field. This review summarizes the preclinical and clinical experience of OV therapy in the difficult-to-treat area of HCC.

Cytokine conditioning enhances systemic delivery and therapy of an oncolytic virus.

Optimum clinical protocols require systemic delivery of oncolytic viruses in the presence of an intact immune system. We show that preconditioning with immune modulators, or loading virus onto carrier cells ex vivo, enhances virus-mediated antitumor activity. Our early trials of systemic reovirus delivery showed that after infusion reovirus could be recovered from blood cells--but not from plasma--suggesting that rapid association with blood cells may protect virus from neutralizing antibody. We therefore postulated that stimulation of potential carrier cells directly in vivo before intravenous viral delivery would enhance delivery of cell-associated virus to tumor. We show that mobilization of the CD11b(+) cell compartment by granulocyte macrophage-colony stimulating factor immediately before intravenous reovirus, eliminated detectable tumor in mice with small B16 melanomas, and achieved highly significant therapy in mice bearing well-established tumors. Unexpectedly, cytokine conditioning therapy was most effective in the presence of preexisting neutralizing antibody. Consistent with this, reovirus bound by neutralizing antibody effectively accessed monocytes/macrophages and was handed off to tumor cells. Thus, preconditioning with cytokine stimulated recipient cells in vivo for enhanced viral delivery to tumors. Moreover, preexisting neutralizing antibody to an oncolytic virus may, therefore, even be exploited for systemic delivery to tumors in the clinic.

The profile of tumor antigens which can be targeted by immunotherapy depends upon the tumor's anatomical site.

Previously, we showed that vesicular stomatitis virus (VSV) engineered to express a cDNA library from human melanoma cells (ASMEL, Altered Self Melanoma Epitope Library) was an effective systemic therapy to treat subcutaneous (s.c.) murine B16 melanomas. Here, we show that intravenous treatment with the same ASMEL VSV-cDNA library was an effective treatment for established intra-cranial (i.c.) melanoma brain tumors. The optimal combination of antigens identified from the ASMEL which treated s.c. B16 tumors (VSV-N-RAS+VSV-CYTC-C+VSV-TYRP-1) was ineffective against i.c. B16 brain tumors. In contrast, combination of VSV-expressed antigens-VSV-HIF-2α+VSV-SOX-10+VSV-C-MYC+VSV-TYRP1-from ASMEL which was highly effective against i.c. B16 brain tumors, had no efficacy against the same tumors growing subcutaneously. Correspondingly, i.c. B16 tumors expressed a HIF-2α(Hi), SOX-10(Hi), c-myc(Hi), TYRP1, N-RAS(lo)Cytc(lo) antigen profile, which differed significantly from the HIF-2α(lo), SOX-10(lo), c-myc(lo), TYRP1, N-RAS(Hi)Cytc(Hi) phenotype of s.c. B16 tumors, and was imposed upon the tumor cells by CD11b(+) cells within the local brain tumor microenvironment. Combining T-cell costimulation with systemic VSV-cDNA treatment, long-term cures of mice with established i.c. tumors were achieved in about 75% of mice. Our data show that the anatomical location of a tumor profoundly affects the profile of antigens that it expresses.

The efficacy versus toxicity profile of combination virotherapy and TLR immunotherapy highlights the danger of administering TLR agonists to oncolytic virus-treated mice.

Injection of oncolytic vesicular stomatitis virus (VSV) into established B16ova melanomas results in tumor regression, in large part by inducing innate immune reactivity against the viral infection, mediated by MyD88- and type III interferon (IFN)-, but not TLR-4-, signaling. We show here that intratumoral (IT) treatment with lipopolysaccharide (LPS), a TLR-4 agonist, significantly enhanced the local therapy induced by VSV by combining activation of different innate immune pathways. Therapy was further enhanced by co-recruiting a potent antitumor, adaptive T-cell response by using a VSV engineered to express the ovalbumin tumor-associated antigen ova, in combination with LPS. However, the combination of IT LPS with systemically delivered VSV resulted in rapid morbidity and mortality in the majority of mice. Decreasing the intravenous (IV) dose of VSV to levels at which toxicity was ameliorated did not enhance therapy compared with IT LPS alone. Toxicity of the systemic VSV + IT LPS regimen was associated with rapidly elevated levels of serum tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, which neither systemic VSV, nor IT LPS, alone induced. These data show that therapy associated with direct IT injections of oncolytic viruses can be significantly enhanced by combination with agonists of innate immune activation pathways, which are not themselves activated by the virus alone. Importantly, they also highlight possible, unforeseen dangers of combination therapies in which an immunotherapy, even delivered locally at the tumor site, may systemically sensitize the patient to a cytokine shock-like response triggered by IV delivery of oncolytic virus.

Functional cloning of recurrence-specific antigens identifies molecular targets to treat tumor relapse.

Aggressive regrowth of recurrent tumors following treatment-induced dormancy represents a major clinical challenge for treatment of malignant disease. We reported previously that recurrent prostate tumors, which underwent complete macroscopic regression followed by aggressive regrowth, could be cured with a vesicular stomatitis virus (VSV)-expressed cDNA library derived from recurrent tumor cells. By screening the protective, recurrence-derived VSV-cDNA library, here we identify topoisomerase-IIα (TOPO-IIα) as a recurrence-specific tumor antigen against which tolerance can be broken. Tumor recurrences, in two different types of tumor (prostate and melanoma), which had evaded two different frontline treatments (immunotherapy or chemotherapy), significantly overexpressed TOPO-IIα compared with their primary tumor counterparts, which conferred a novel sensitivity to doxorubicin (DOX) chemotherapy upon the recurrent tumors. This was exploited in vivo using combination therapies to cure mice, which would otherwise have relapsed, after suboptimal primary therapy in both models. Our data show that recurrent tumors-across histologies and primary treatments-express distinct antigens compared with the primary tumor which can be identified using the VSV-cDNA library technology. These results suggest that it may be possible to design a few common second-line therapies against a variety of tumor recurrences, in some cases using agents with no obvious activity against the primary tumor.

Expression of tumor antigens within an oncolytic virus enhances the anti-tumor T cell response.

Although patients benefit from immune checkpoint inhibition (ICI) therapy in a broad variety of tumors, resistance may arise from immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, OVs could potentially restore ICI responsiveness via recruitment, priming, and activation of anti-tumor T cells. Here we find that on the contrary, an oncolytic vesicular stomatitis virus, expressing interferon-ß (VSV-IFNß), antagonizes the effect of anti-PD-L1 therapy in a partially anti-PD-L1-responsive model of HCC. Cytometry by Time of Flight shows that VSV-IFNß expands dominant anti-viral effector CD8 T cells with concomitant relative disappearance of anti-tumor T cell populations, which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, combination OV and anti-PD-L1 therapeutic benefit could be restored. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, through encoding tumor antigens within the virus, oncolytic virotherapy can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.

Cytotoxic and immune-mediated killing of human colorectal cancer by reovirus-loaded blood and liver mononuclear cells.

Reovirus is a promising oncolytic virus, acting by both direct and immune-mediated mechanisms, although its potential may be limited by inactivation after systemic delivery. Our study addressed whether systemically delivered reovirus might be shielded from neutralising antibodies by cell carriage and whether virus-loaded blood or hepatic innate immune effector cells become activated to kill colorectal cancer cells metastatic to the liver in human systems. We found that reovirus was directly cytotoxic against tumour cells but not against fresh hepatocytes. Although direct tumour cell killing by neat virus was significantly reduced in the presence of neutralising serum, reovirus was protected when loaded onto peripheral blood mononuclear cells, which may carry virus after intravenous administration in patients. As well as handing off virus for direct oncolytic killing, natural killer (NK) cells within reovirus-treated blood mononuclear cells were stimulated to kill tumour targets, but not normal hepatocytes, in a Type I interferon-dependent manner. Similarly, NK cells within liver mononuclear cells became selectively cytotoxic towards tumour cells when activated by reovirus. Hence, intravenous reovirus may evade neutralisation by serum via binding to circulating mononuclear cells, and this blood cell carriage has the potential to investigate both direct and innate immune-mediated therapy against human colorectal or other cancers metastatic to the liver.

Phase II trial of intravenous administration of Reolysin(®) (Reovirus Serotype-3-dearing Strain) in patients with metastatic melanoma.

Reovirus, a replication competent RNA virus, has preclinical activity against melanoma lines and xenografts. We conducted a phase II trial of reovirus in metastatic melanoma patients. Patients received 3 × 10(10) TCID50 on days 1-5 of each 28 day cycle, administered intravenously. Twenty-one eligible patients were enrolled. Treatment was well tolerated without any dose reductions having to be implemented. Post-treatment biopsy samples were obtained in 15 patients, 13/15 contained adequate tumor for correlative analysis. In two patients, productive reoviral replication (viral antigen coexpression with tubulin) was demonstrated, despite increase in neutralizing antibody titers. There were no objective responses although 75-90% tumor necrosis, consistent with treatment effect, was observed in one patient who had metastatic lesions surgically removed. Median time to progression and survival were 45 days (range 13-96 days) and 165 days (range 15 days-15.8 months) respectively. In conclusion, reovirus treatment was well tolerated in metastatic melanoma patients; viral replication was demonstrated in biopsy samples. Based on preclinical data showing synergy with taxane and platinum compounds, a phase II combination trial in metastatic melanoma patients is ongoing.