Julia Cockle

Intravenous delivery of oncolytic reovirus to brain tumor patients immunologically primes for subsequent checkpoint blockade

Intravenous infusion of oncolytic reovirus in patients leads to infection of brain tumors, infiltration by cytotoxic T cells, and up-regulation of PD-L1. Viruses team up with cancer immunotherapy Immune checkpoint inhibitors have shown great promise for cancer therapy, but they do not treat all cancers, and neither breast nor brain tumors are usually treatable with these drugs. However, Bourgeois-Daigneault et al. discovered a way to address this for breast cancer, and Samson et al. discovered a way to address this for brain tumors. In both cases, the authors found that oncolytic virus treatment given early, before surgical resection, alters the antitumor immune response and potentiates the effects of subsequent treatment with immune checkpoint inhibitors. Although these studies differ in the details of their methods and the immune effects induced by the oncolytic viruses, they indicate the potential of such viruses for enhancing the potential of checkpoint therapy and expanding it to new types of cancer. Immune checkpoint inhibitors, including those targeting programmed cell death protein 1 (PD-1), are reshaping cancer therapeutic strategies. Evidence suggests, however, that tumor response and patient survival are determined by tumor programmed death ligand 1 (PD-L1) expression. We hypothesized that preconditioning of the tumor immune microenvironment using targeted, virus-mediated interferon (IFN) stimulation would up-regulate tumor PD-L1 protein expression and increase cytotoxic T cell infiltration, improving the efficacy of subsequent checkpoint blockade. Oncolytic viruses (OVs) represent a promising form of cancer immunotherapy. For brain tumors, almost all studies to date have used direct intralesional injection of OV, because of the largely untested belief that intravenous administration will not deliver virus to this site. We show, in a window-of-opportunity clinical study, that intravenous infusion of oncolytic human Orthoreovirus (referred to herein as reovirus) leads to infection of tumor cells subsequently resected as part of standard clinical care, both in high-grade glioma and in brain metastases, and increases cytotoxic T cell tumor infiltration relative to patients not treated with virus. We further show that reovirus up-regulates IFN-regulated gene expression, as well as the PD-1/PD-L1 axis in tumors, via an IFN-mediated mechanism. Finally, we show that addition of PD-1 blockade to reovirus enhances systemic therapy in a preclinical glioma model. These results support the development of combined systemic immunovirotherapy strategies for the treatment of both primary and secondary tumors in the brain.

Combination viroimmunotherapy with checkpoint inhibition to treat glioma, based on location-specific tumor profiling.

BACKGROUND Systemic delivery of a complementary cDNA library expressed from the vesicular stomatitis virus (VSV) treats tumors by vaccinating against a wide range of tumor associated antigens (TAAs). For subcutaneous B16 melanomas, therapy was achieved using a specific combination of self-TAAs (neuroblastoma-Ras, cytochrome c, and tyrosinase-related protein 1) expressed from VSV. However, for intracranial B16 tumors, a different combination was therapeutic (consisting of VSV-expressed hypoxia-inducible factor [HIF]-2α, Sox-10, c-Myc, and tyrosinase-related protein 1). Therefore, we tested the hypothesis that tumors of different histological types growing in the brain share a common immunogenic signature which can be exploited for immunotherapy. METHODS Syngeneic tumors, including GL261 gliomas, in the brains of immune competent mice were analyzed for their antigenic profiles or were treated with systemic viroimmunotherapy. RESULTS Several different histological types of tumors growing intracranially, as well as freshly resected human brain tumor explants, expressed a HIF-2α(Hi) phenotype imposed by brain-derived CD11b+ cells. This location-specific antigen expression was exploited therapeutically against intracranial GL261 gliomas using systemically delivered VSV expressing HIF-2α, Sox-10, and c-Myc. Viroimmunotherapy was enhanced by immune checkpoint inhibitors, associated with the de-repression of antitumor T-helper cell type 1 (Th1) interferon-γ and Th17 T cell responses. CONCLUSIONS Since different tumor types growing in the same location in the brain share a location-specific phenotype, we suggest that antigen-specific immunotherapies should be based upon expression of both histological type-specific tumor antigens and location-specific antigens. Our findings support clinical application of VSV-TAA therapy with checkpoint inhibition for aggressive brain tumors and highlight the importance of the intracranial microenvironment in sculpting a location-specific profile of tumor antigen expression.

Cell migration in paediatric glioma; characterisation and potential therapeutic targeting.

Background:Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response, and novel treatments that block brain tumour invasion are needed.Methods:Here, we examine the migratory characteristics and treatment effect of small molecule glycogen synthase kinase-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), previously shown to inhibit the migration of adult glioma cells, on two pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG line (HSJD-DIPG-007) using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion in collagen) assays.Results:All lines were migratory, but there were differences in morphology and migration rates. Both LiCl and BIO reduced migration and instigated cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. In the presence of drugs, loss of polarity and differences in cellular movement were observed by live cell imaging.Conclusions:Ours is the first study to demonstrate that it is possible to pharmacologically target migration of paediatric glioma in vitro using LiCl and BIO, and we conclude that these agents and their derivatives warrant further preclinical investigation as potential anti-migratory therapeutics for these devastating tumours.

Oncolytic Herpes Simplex Virus Inhibits Pediatric Brain Tumor Migration and Invasion

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are invasive tumors with poor survival. Oncolytic virotherapy, initially devised as a direct cytotoxic treatment, is now also known to act via immune-mediated mechanisms. Here we investigate a previously unreported mechanism of action: the inhibition of migration and invasion in pediatric brain tumors. We evaluated the effect of oncolytic herpes simplex virus 1716 (HSV1716) on the migration and invasion of pHGG and DIPG both in vitro using 2D (scratch assay, live cell imaging) and 3D (spheroid invasion in collagen) assays and in vivo using an orthotopic xenograft model of DIPG invasion. HSV1716 inhibited migration and invasion in pHGG and DIPG cell lines. pHGG cells demonstrated reduced velocity and changed morphology in the presence of virus. HSV1716 altered pHGG cytoskeletal dynamics by stabilizing microtubules, inhibiting glycogen synthase kinase-3, and preventing localized clustering of adenomatous polyposis coli (APC) to the leading edge of cells. HSV1716 treatment also reduced tumor infiltration in a mouse orthotopic xenograft DIPG model. Our results demonstrate that HSV1716 targets the migration and invasion of pHGG and DIPG and indicates the potential of an oncolytic virus (OV) to be used as a novel anti-invasive treatment strategy for pediatric brain tumors.

Future clinical potential of oncolytic virotherapy for pediatric CNS tumors

307 ISSN 2045-0907 10.2217/CNS.13.25

197. Balancing Anti-Tumor Efficacy with Local Inflammatory Toxicity for the Treatment of Diffuse Intrinsic Pontine Glioma and Other Brain Tumors

We have shown previously that multiple rounds of a systemic treatment of GM-CSF, followed by intravenous reovirus, leads to effective treatment of subcutaneous melanomas (Ilett et al., Mol. Ther. 2015). We show here that a similar regimen is also effective at treating both melanoma (B16) and glioma (GL261) tumors growing intra-cranially. As a result of these pre-clinical studies, we initiated a Phase I clinical trial in paediatric patients with gliomas of GM-CSF and reovirus therapy. To date, three patients have been treated. In two of three of these patients there were possible indications of pseudoprogression and intra-cranial inflammation, both of which were clinically resolved upon treatment with dexamethasone. Whilst this trial continues to recruit, we have investigated additional treatments for paediatric brain tumors, especially those in which direct virus injection may allow greater local access to tumor. In this respect, there is a constant tension between the pro-inflammatory, anti-tumor nature of oncolytic viroimmunotherapy and the need to reduce potentially toxic local inflammatory reactions within the brain. Our overall hypothesis is that it will be possible to balance the anti tumor effects of viral oncolysis, in part caused by inflammatory reactions to the virus, with the associated toxicity. Nowhere is this more relevant than for the treatment of Diffuse Pontine Gliomas (DIPG), which grow in the brain stem. Our experiments show that it is indeed possible to treat tumors growing in the pons/and or medulla, with inflammatory, immune based therapies (both T cell mediated and locally cytotoxic) without increasing toxicity over that caused by tumor growth alone. Therefore, we have screened multiple oncolytic virus types for their ability to kill DIPG cell lines both in vitro and in vivo. In particular, we have compared viruses with different speeds of oncolysis and different inflammatory properties to investigate how the efficacy can be balanced with local toxicity, using standard of care anti inflammatory treatments (with dexamethasone) as well as with novel agents and viruses designed to suppress local inflammatory reactions.