Prime-boost using separate oncolytic viruses in combination with checkpoint blockade improves anti-tumour therapy.

The anti-tumour effects associated with oncolytic virus therapy are mediated significantly through immune-mediated mechanisms, which depend both on the type of virus and the route of delivery. Here, we show that intra-tumoral oncolysis by Reovirus induced the priming of a CD8+, Th1-type anti-tumour response. By contrast, systemically delivered Vesicular Stomatitis Virus expressing a cDNA library of melanoma antigens (VSV-ASMEL) promoted a potent anti-tumour CD4+ Th17 response. Therefore, we hypothesised that combining the Reovirus-induced CD8+ T cell response, with the VSV-ASMEL CD4+ Th17 helper response, would produce enhanced anti-tumour activity. Consistent with this, priming with intra-tumoral Reovirus, followed by an intra-venous VSV-ASMEL Th17 boost, significantly improved survival of mice bearing established subcutaneous B16 melanoma tumours. We also show that combination of either therapy alone with anti-PD-1 immune checkpoint blockade augmented both the Th1 response induced by systemically delivered Reovirus in combination with GM-CSF, and also the Th17 response induced by VSV-ASMEL. Significantly, anti-PD-1 also uncovered an anti-tumour Th1 response following VSV-ASMEL treatment that was not seen in the absence of checkpoint blockade. Finally, the combination of all three treatments (priming with systemically delivered Reovirus, followed by double boosting with systemic VSV-ASMEL and anti-PD-1) significantly enhanced survival, with long-term cures, compared to any individual, or double, combination therapies, associated with strong Th1 and Th17 responses to tumour antigens. Our data show that it is possible to generate fully systemic, highly effective anti-tumour immunovirotherapy by combining oncolytic viruses, along with immune checkpoint blockade, to induce complementary mechanisms of anti-tumour immune responses.

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.

Lymphokine-activated killer and dendritic cell carriage enhances oncolytic reovirus therapy for ovarian cancer by overcoming antibody neutralization in ascites.

Reovirus is an oncolytic virus (OV), which acts by both direct tumor cell killing and priming of antitumor immunity. A major obstacle for effective oncolytic virotherapy is effective delivery of OV to tumor cells. Ovarian cancer is often confined to the peritoneal cavity and therefore i.p. delivery of reovirus may provide the ideal locoregional delivery, avoiding systemic dissemination. However, ovarian cancer is associated with an accumulation of ascitic fluid, which may interfere with oncolytic viral therapy. Here, we investigated the effect of ascites on reovirus-induced oncolysis against primary ovarian cancer cells and ovarian cancer cell lines. In the absence of ascites, reovirus was cytotoxic against ovarian cancer cells; however, cytotoxicity was abrogated in the presence of ascitic fluid. Neutralizing antibodies (NAb) were identified as the cause of this inhibition. Loading OV onto cell carriers may facilitate virus delivery in the presence of NAb and immune cells which have their own antitumor effector activity are particularly appealing. Immature dendritic cells (iDC), Lymphokine-activated killer (LAK) cells and LAKDC cocultures were tested as potential carriers for reovirus for tumor cell killing and immune cell priming. Reovirus-loaded LAKDC, and to a lesser degree iDC, were able to: (i) protect from NAb and hand-off reovirus for tumor cell killing; (ii) induce a proinflammatory cytokine milieu (IFNÉ£, IL-12, IFNα and TNFα) and (iii) generate an innate and specific antitumor adaptive immune response. Hence, LAKDC pulsed with reovirus represent a novel, clinically practical treatment for ovarian cancer to maximise both direct and innate/adaptive immune-mediated tumor cell killing.

Dendritic cells and T cells deliver oncolytic reovirus for tumour killing despite pre-existing anti-viral immunity.

Reovirus is a naturally occurring oncolytic virus currently in early clinical trials. However, the rapid induction of neutralizing antibodies represents a major obstacle to successful systemic delivery. This study addresses, for the first time, the ability of cellular carriers in the form of T cells and dendritic cells (DC) to protect reovirus from systemic neutralization. In addition, the ability of these cellular carriers to manipulate the subsequent balance of anti-viral versus anti-tumour immune response is explored. Reovirus, either neat or loaded onto DC or T cells, was delivered intravenously into reovirus-naive or reovirus-immune C57Bl/6 mice bearing lymph node B16tk melanoma metastases. Three and 10 days after treatment, reovirus delivery, carrier cell trafficking, metastatic clearance and priming of anti-tumour/anti-viral immunity were assessed. In naive mice, reovirus delivered either neat or through cell carriage was detectable in the tumour-draining lymph nodes 3 days after treatment, though complete clearance of metastases was only obtained when the virus was delivered on T cells or mature DC (mDC); neat reovirus or loaded immature DC (iDC) gave only partial early tumour clearance. Furthermore, only T cells carrying reovirus generated anti-tumour immune responses and long-term tumour clearance; reovirus-loaded DC, in contrast, generated only an anti-viral immune response. In reovirus-immune mice, however, the results were different. Neat reovirus was completely ineffective as a therapy, whereas mDC--though not iDC--as well as T cells, effectively delivered reovirus to melanoma in vivo for therapy and anti-tumour immune priming. Moreover, mDC were more effective than T cells over a range of viral loads. These data show that systemically administered neat reovirus is not optimal for therapy, and that DC may be an appropriate vehicle for carriage of significant levels of reovirus to tumours. The pre-existing immune status against the virus is critical in determining the balance between anti-viral and anti-tumour immunity elicited when reovirus is delivered by cell carriage, and the viral dose and mode of delivery, as well as the immune status of patients, may profoundly affect the success of any clinical anti-tumour viral therapy. These findings are therefore of direct translational relevance for the future design of clinical trials.

The immune system--is it relevant to cancer development, progression and treatment?

The ability of the immune system to effectively respond to human tumours is a matter of long-term controversy. There is an increasing body of recent evidence to support a role for the immune system in eliminating pre-clinical cancers, an old concept termed 'immunosurveillance'. 'Immunoediting' is an updated hypothesis, in which selection pressures applied by the immune response to tumours modulate tumour immunogenicity and growth. Tumour infiltration by immune cells has been shown to have powerful prognostic significance in a host of cancer types. Paradoxically, in some circumstances the immune system can promote tumour development. Cytotoxic therapies, including radiotherapy and chemotherapy, induce potentially immunogenic cell death, releasing tumour-associated antigens in the context of a 'danger' signal to the immune system. An understanding of the interaction between immune cells, tumour cells and treatment modalities will therefore guide the future combination of immunotherapy with conventional therapy to achieve optimal anti-tumour effects.

Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia.

The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.

The evolving role of dendritic cells in cancer therapy.

IMPORTANCE OF THE FIELD: Dendritic cells (DC) are a clear choice for use in cancer immunotherapy, and much research has focused on generating DC for clinical use. Although DC therapy has been successful in inducing specific anti-tumour immune responses, these have rarely translated into clinical efficacy. AREAS COVERED IN THIS REVIEW: We examine some of the components of generating DC for therapy, including their culture, antigen loading and delivery, and discuss why DC therapy has not yet delivered substantial clinical benefit. We also examine more novel approaches, such as the potential for combination DC-based immunomodulatory strategies. WHAT THE READER WILL GAIN: Given the highly immunosuppressive tumour environment, many of the approaches to DC vaccination are unlikely to result in effective therapy, as even successfully primed T cells may fail to infiltrate tumours or be anergized after entry. Broader approaches against multiple tumour-associated antigens in the context of overcoming tumour immune suppression are likely to prove more successful. The combination of oncolytic viral therapy with DC vaccines may promote an inflammatory tumour environment, inducing optimal DC activation, T cell priming and effective therapy. TAKE HOME MESSAGE: Evolving DC-based therapeutic strategies addressing multiple components of tumour-immune system interactions may yield substantial benefits for patients.