Emma West

Cell Carriage, Delivery, and Selective Replication of an Oncolytic Virus in Tumor in Patients

Oncolytic reovirus is carried by cells to tumors and protected from neutralizing antibodies in the circulation. Therapeutic Virus Hide-and-Seek Oncolytic viruses (OVs) selectively kill cancer cells by direct lysis as well as by stimulating an antitumor immune response. However, the lack of a method for widespread delivery of OVs to tumor cells hangs like an enthusiasm-squelching dark cloud over the field. Direct intratumoral injection is an option but limits this therapy to easily accessible tumors. Mouse studies suggest that the intravenous route would be blocked by preexisting neutralizing antibodies to the virus—the fast immune response that prevents recurrent infection would block the virus from getting to the tumor. Adair et al. now show in human patients with colorectal cancer that, after intravenous injection, reovirus can be escorted to the tumor by immune cells, which protect it from neutralizing antibodies in the plasma. The authors performed a window-of-opportunity clinical trial in 10 colorectal cancer patients scheduled to have surgery to remove liver metastases. Before the planned surgery, the patients were injected with oncolytic reovirus. Replication-competent cytotoxic reovirus was recovered from blood cells, but not from plasma taken from these patients, and reovirus protein was identified preferentially in malignant cells compared with nonmalignant liver tissue from surgical specimens. These data suggest that in contrast to observations in mice, human immune cells may shield reovirus from neutralizing antibodies and deliver the oncolytic reovirus to tumors in patients. Although the mechanism behind the delivery process and the efficacy of the OVs remain to be determined, these potentially cloud-lifting studies support intravenous administration of reovirus for cancer therapy. Oncolytic viruses, which preferentially lyse cancer cells and stimulate an antitumor immune response, represent a promising approach to the treatment of cancer. However, how they evade the antiviral immune response and their selective delivery to, and replication in, tumor over normal tissue has not been investigated in humans. Here, we treated patients with a single cycle of intravenous reovirus before planned surgery to resect colorectal cancer metastases in the liver. Tracking the viral genome in the circulation showed that reovirus could be detected in plasma and blood mononuclear, granulocyte, and platelet cell compartments after infusion. Despite the presence of neutralizing antibodies before viral infusion in all patients, replication-competent reovirus that retained cytotoxicity was recovered from blood cells but not plasma, suggesting that transport by cells could protect virus for potential delivery to tumors. Analysis of surgical specimens demonstrated greater, preferential expression of reovirus protein in malignant cells compared to either tumor stroma or surrounding normal liver tissue. There was evidence of viral factories within tumor, and recovery of replicating virus from tumor (but not normal liver) was achieved in all four patients from whom fresh tissue was available. Hence, reovirus could be protected from neutralizing antibodies after systemic administration by immune cell carriage, which delivered reovirus to tumor. These findings suggest new preclinical and clinical scheduling and treatment combination strategies to enhance in vivo immune evasion and effective intravenous delivery of oncolytic viruses to patients in vivo.

Optimization of Dendritic Cell Loading With Tumor Cell Lysates for Cancer Immunotherapy

The immune response to cancer is critically determined by the way in which tumor cells die. As necrotic, stress-associated death can be associated with activation of antitumor immunity, whole tumor cell antigen loading strategies for dendritic cell (DC)-based vaccination have commonly used freeze-thaw “necrotic” lysates as an immunogenic source of tumor-associated antigens. In this study, the effect of such lysates on the ability of DCs to mature in response to well-established maturation stimuli was examined, and methods to enhance lysate-induced DC activation explored. Freeze-thaw lysates were prepared from murine tumor cell lines and their effects on bone marrow-derived DC maturation and function examined. Unmodified freeze-thaw tumor cell lysates inhibited the toll-like receptor-induced maturation and function of bone marrow-derived DCs, preventing up-regulation of CD40, CD86, and major histocompatibility complex class II, and reducing secretion of inflammatory cytokines [interleukin (IL)-12 p70, tumor necrosis factor-α, and IL-6]. Although IL-10 secretion was increased by lysate-pulsed DCs, this was not responsible for the observed suppression of IL-12. Although activation of the nuclear factor-κB pathway remained intact, the kinase activity of phosphorylated p38 mitogen-activated protein kinase was inhibited in lysate-pulsed DCs. Lysate-induced DC suppression was partially reversed in vitro by induction of tumor cell stress before lysis, and only DCs loaded with stressed lysates afforded protection against tumor challenge in vivo. These data suggest that ex vivo freeze-thaw of tumor cells does not effectively mimic in vivo immunogenic necrosis, and advocates careful characterization and optimization of tumor cell-derived vaccine sources for cancer immunotherapy.

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.

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 (IFNɣ, 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.

Evidence for Oncolytic Virotherapy: Where Have We Got to and Where Are We Going?

The last few years have seen an increased interest in immunotherapy in the treatment of malignant disease. In particular, there has been significant enthusiasm for oncolytic virotherapy, with a large amount of pre-clinical data showing promise in animal models in a wide range of tumour types. How do we move forward into the clinical setting and translate something which has such potential into meaningful clinical outcomes? Here, we review how the field of oncolytic virotherapy has developed thus far and what the future may hold.

Immune activation by combination human lymphokine-activated killer and dendritic cell therapy

Background:Optimal cellular immunotherapy for cancer should ideally harness both the innate and adaptive arms of the immune response. Lymphokine-activated killer cells (LAKs) can trigger early innate killing of tumour targets, whereas long-term adaptive-specific tumour control requires priming of CD8+ cytotoxic lymphocytes (CTLs) following acquisition of tumour-associated antigens (TAAs) by antigen-presenting cells such as dendritic cells (DCs). As DCs stimulate both innate and adaptive effectors, combination cell therapy using LAKs and DCs has the potential to maximise anti-tumour immune priming.Methods:Reciprocal activation between human clinical grade LAKs and DCs on co-culture, and its immune consequences, was monitored by cell phenotype, cytokine release and priming of both innate and adaptive cytotoxicity against melanoma targets.Results:Co-culture of DCs and LAKs led to phenotypic activation of natural killer (NK) cells within the LAK population, which was associated with increased production of inflammatory cytokines and enhanced innate cytotoxicity against tumour cell targets. The LAKs reciprocally matured DCs, and the combination of LAKs and DCs, on addition of melanoma cells, supported priming of specific anti-tumour CTLs better than DCs alone.Conclusion:Clinical-grade LAKs/DCs represents a practical, effective combination cell immunotherapy for stimulation of both innate and adaptive anti-tumour immunity in cancer patients.

Clinical grade OK432-activated dendritic cells: In vitro characterization and tracking during intralymphatic delivery

Dendritic cells (DC) are under intense preclinical and early clinical evaluation for the immunotherapy of cancer. However, the optimal culture conditions and route of delivery for DC vaccination have not been established. Here we describe the first human application of DC matured with the bacterial agent OK432 (OK-DC), using a short-term serum-free culture protocol, which generates mature DC from CD14+ precursors after 5 days. These cells were prepared within the framework of a National Blood Service facility, demonstrating that DC represent a product which is potentially deliverable alongside current standardized cell therapies within the UK National Health Service. In vitro analysis confirmed that OK-DC were mature, secreted tumor necrosis factor-α, interleukin-6, and interleukin-12, and stimulated both T cell and natural killer cell function. To explore effective delivery of OK-DC to lymph nodes, we performed an initial clinical tracking study of radioactively labeled, unpulsed OK-DC after intralymphatic injection into the dorsum of the foot. We showed that injected DC rapidly localized to ipsilateral pelvic lymph nodes, but did not disseminate to more distant nodes over a 48-hour period. There was no significant toxicity associated with OK-DC delivery. These results show that OK-DC are suitable for clinical use, and that intralymphatic delivery is feasible for localizing cells to sites where optimal priming of innate and adaptive antitumor immunity is likely to occur.

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.

Talimogene laherparepvec in the treatment of melanoma

Introduction: Metastatic melanoma continues to present a significant therapeutic challenge, with an incidence rate rising faster than that of any other cancer. The last 5 years have seen a revolution in the development of new treatments for advanced melanoma, with oncogene targeted agents and checkpoint inhibitor immunotherapies providing the first convincing evidence of a positive shift in overall survival. The role of oncolytic virotherapy in this rapidly evolving field has long been the subject of debate. However, it is with the development of Talimogene Laheparepvec (T-Vec), an intratumourally administered, genetically modified clinical herpes simplex virus-1 strain that has shown positive results in Phase III testing, that the potential for the use of OV may be realised. Areas covered: This review will outline some of the recent advances in the treatment of advanced melanoma, with a detailed overview of evidence surrounding the development of T-Vec. A literature search was conducted using the databases ‘Medline’ and ‘Pubmed’, including a subsequent manual search of references to identify papers of further relevance. Expert opinion: As the pivotal OPTiM trial concludes, we outline some of the potential new directions for T-Vec and OV therapy and evaluate the ever-increasing role these novel agents are likely to play in the future landscape of cancer immunotherapy.

Abstract A49: Systemic oncolytic reovirus for the treatment of primary and secondary brain tumors

Reovirus is a systemically delivered oncolytic agent with evidence of activity in both pre-clinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against many human/murine tumor cells, as well as activating anti-tumor innate and adaptive immunity. Having previously shown that intravenously delivered reovirus selectively accesses colorectal cancer metastatic to the liver in patients, in this study we explored the potential of systemic reovirus for the treatment of both primary and secondary brain tumors. We first showed that intravenous reovirus can be detected in tumors implanted into the brains of immunocompetent mice. We then tested our current most potent reovirus-based therapy (systemic reovirus plus granulocyte-macrophage colony stimulating factor [GMCSF] in reovirus pre-immune mice), and showed effective therapy in immunocompetent murine models of primary glioma, and of both directly reovirus sensitive, and insensitive, models of melanoma brain metastases. We also found that addition of reovirus/GMCSF to clinical ‘standard of care’ (radiotherapy and temozolamide chemotherapy) significantly enhances survival. In parallel to these pre-clinical experiments, we have initiated an open-label, non-randomized study of intravenous reovirus administered to patients prior to planned surgery for recurrent high grade glioma or metastatic brain tumors, to test whether the data showing access of the agent to tumors in the brains in mice, also applies to humans. In total, 10 patients will be treated with a single infusion of 1x1010 TCID50 of reovirus, of which 9 have completed the study to date. The primary objective is the presence of reovirus in the resected tumors as assessed by immunohistochemistry, RNA in-situ hybridization and retrieval of infectious virions. Early analysis of the first 3 patients, comprising one glioblastoma multiforme, one grade 3 oligodendroglioma and one colorectal brain metastasis, has shown that all 3 resected tumors contained reovirus RNA and protein. There was also evidence for productive reovirus infection in 2 of the tumors. Within all patients to date, the only grade 3-4 adverse reaction has been neutropaenia in 1 patient. Further tissue analysis is ongoing, as is testing of blood samples from these patients to further charactize how reovirus is carried in the blood and protected from neutralizing antibodies. This clinical study shows, for the first time that an oncolytic virus, reovirus, infects and replicates in brain tumors following intravenous administration. Together with pre-clinical data showing the efficacy of systemic reovirus in combination with GMCSF/radiation/temozolamide, these findings support the future development of trials and combination studies using reovirus in patients with high grade gliomas and brain metastases. Citation Format: Adel Jebar, Liz Ilett, Tim Kottke, Emma West, Karen Scott, Simon Thomson, Matt Coffey, Gerard Nuovo, Susan Short, Richard Vile, Alan Melcher. Systemic oncolytic reovirus for the treatment of primary and secondary brain tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A49.