Kelley Parato

Recent progress in the battle between oncolytic viruses and tumours

In the past 5 years, the field of oncolytic virus research has matured significantly and is moving past the stage of being a laboratory novelty into a new era of preclinical and clinical trials. What have recent anticancer trials of oncolytic viruses taught us about this exciting new line of therapeutics?

Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans

The efficacy and safety of biological molecules in cancer therapy, such as peptides and small interfering RNAs (siRNAs), could be markedly increased if high concentrations could be achieved and amplified selectively in tumour tissues versus normal tissues after intravenous administration. This has not been achievable so far in humans. We hypothesized that a poxvirus, which evolved for blood-borne systemic spread in mammals, could be engineered for cancer-selective replication and used as a vehicle for the intravenous delivery and expression of transgenes in tumours. JX-594 is an oncolytic poxvirus engineered for replication, transgene expression and amplification in cancer cells harbouring activation of the epidermal growth factor receptor (EGFR)/Ras pathway, followed by cell lysis and anticancer immunity. Here we show in a clinical trial that JX-594 selectively infects, replicates and expresses transgene products in cancer tissue after intravenous infusion, in a dose-related fashion. Normal tissues were not affected clinically. This platform technology opens up the possibility of multifunctional products that selectively express high concentrations of several complementary therapeutic and imaging molecules in metastatic solid tumours in humans.

Thunder and Lightning: Immunotherapy and Oncolytic Viruses Collide

For the last several decades, the development of antitumor immune-based strategies and the engineering and testing of oncolytic viruses (OVs) has occurred largely in parallel tracks. Indeed, the immune system is often thought of as an impediment to successful oncolytic virus delivery and efficacy. More recently, however, both preclinical and clinical results have revealed potential synergy between these two promising therapeutic strategies. Here, we summarize some of the evidence that supports combining OVs with immuno-therapeutics and suggest new ways to mount a multipronged biological attack against cancers.

The Oncolytic Poxvirus JX-594 Selectively Replicates in and Destroys Cancer Cells Driven by Genetic Pathways Commonly Activated in Cancers

Oncolytic viruses are generally designed to be cancer selective on the basis of a single genetic mutation. JX-594 is a thymidine kinase (TK) gene-inactivated oncolytic vaccinia virus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and lac-Z transgenes that is designed to destroy cancer cells through replication-dependent cell lysis and stimulation of antitumoral immunity. JX-594 has demonstrated a favorable safety profile and reproducible tumor necrosis in a variety of solid cancer types in clinical trials. However, the mechanism(s) responsible for its cancer-selectivity have not yet been well described. We analyzed the replication of JX-594 in three model systems: primary normal and cancer cells, surgical explants, and murine tumor models. JX-594 replication, transgene expression, and cytopathic effects were highly cancer-selective, and broad spectrum activity was demonstrated. JX-594 cancer-selectivity was multi-mechanistic; replication was activated by epidermal growth factor receptor (EGFR)/Ras pathway signaling, cellular TK levels, and cancer cell resistance to type-I interferons (IFNs). These findings confirm a large therapeutic index for JX-594 that is driven by common genetic abnormalities in human solid tumors. This appears to be the first description of multiple selectivity mechanisms, both inherent and engineered, for an oncolytic virus. These findings have implications for oncolytic viruses in general, and suggest that their cancer targeting is a complex and multifactorial process.

A mechanistic proof-of-concept clinical trial with JX-594, a targeted multi-mechanistic oncolytic poxvirus, in patients with metastatic melanoma.

JX-594 is a targeted and granulocyte macrophage-colony stimulating factor (GM-CSF)-expressing oncolytic poxvirus designed to selectively replicate in and destroy cancer cells through viral oncolysis and tumor-specific immunity. In order to study the mechanisms-of-action (MOA) of JX-594 in humans, a mechanistic proof-of-concept clinical trial was performed at a low dose equivalent to ≤10% of the maximum-tolerated dose (MTD) in other clinical trials. Ten patients with previously treated stage IV melanoma were enrolled. Tumors were injected weekly for up to nine total treatments. Blood samples and tumor biopsies were analyzed for evidence of transgene activity, virus replication, and immune stimulation. The β-galactosidase (β-gal) transgene was expressed in all patients as evidenced by antibody induction. Six patients had significant induction of GM-CSF-responsive white blood cell (WBC) subsets such as neutrophils (25-300% increase). JX-594 replication and subsequent shedding into blood was detectable in five patients after cycles 1-9. Tumor biopsies demonstrated JX-594 replication, perivascular lymphocytic infiltration, and diffuse tumor necrosis. Mild flu-like symptoms were the most common adverse events. In sum, JX-594 replication, oncolysis, and expression of both transgenes were demonstrated; replication was still evident after multiple cycles. These findings have implications for further clinical development of JX-594 and other transgene-armed oncolytic viruses.

Targeting Tumor Vasculature With an Oncolytic Virus

Oncolytic viruses (OVs) have been engineered or selected for cancer cell-specific infection however, we have found that following intravenous administration of vesicular stomatitis virus (VSV), tumor cell killing rapidly extends far beyond the initial sites of infection. We show here for the first time that VSV directly infects and destroys tumor vasculature in vivo but leaves normal vasculature intact. Three-dimensional (3D) reconstruction of infected tumors revealed that the majority of the tumor mass lacks significant blood flow in contrast to uninfected tumors, which exhibit relatively uniform perfusion. VSV replication in tumor neovasculature and spread within the tumor mass, initiates an inflammatory reaction including a neutrophil-dependent initiation of microclots within tumor blood vessels. Within 6 hours of intravenous administration of VSV and continuing for at least 24 hours, we observed the initiation of blood clots within the tumor vasculature whereas normal vasculature remained clot free. Blocking blood clot formation with thrombin inhibitors prevented tumor vascular collapse. Our results demonstrate that the therapeutic activity of an OV can go far beyond simple infection and lysis of malignant cells.

Sequential Therapy With JX-594, A Targeted Oncolytic Poxvirus, Followed by Sorafenib in Hepatocellular Carcinoma: Preclinical and Clinical Demonstration of Combination Efficacy

JX-594 is a targeted and granulocyte-macrophage colony stimulating factor (GM-CSF) expressing oncolytic poxvirus designed to selectively replicate in and destroy cancer cells through viral oncolysis and tumor-specific immunity. In a phase 1 trial, JX-594 injection into hepatocellular carcinoma (HCC) was well-tolerated and associated with viral replication, decreased tumor perfusion, and tumor necrosis. We hypothesized that JX-594 and sorafenib, a small molecule inhibitor of B-raf and vascular endothelial growth factor receptor (VEGFR) approved for HCC, would have clinical benefit in combination given their demonstrated efficacy in HCC patients and their complementary mechanisms-of-action. HCC cell lines were uniformly sensitive to JX-594. Anti-raf kinase effects of concurrent sorafenib inhibited JX-594 replication in vitro, whereas sequential therapy was superior to either agent alone in murine tumor models. We therefore explored pilot safety and efficacy of JX-594 followed by sorafenib in three HCC patients. In all three patients, sequential treatment was (i) well-tolerated, (ii) associated with significantly decreased tumor perfusion, and (iii) associated with objective tumor responses (Choi criteria; up to 100% necrosis). HCC historical control patients on sorafenib alone at the same institutions had no objective tumor responses (0 of 15). Treatment of HCC with JX-594 followed by sorafenib has antitumoral activity, and JX-594 may sensitize tumors to subsequent therapy with VEGF/VEGFR inhibitors.

Loading of oncolytic vesicular stomatitis virus onto antigen-specific T cells enhances the efficacy of adoptive T-cell therapy of tumors

Although adoptive T-cell therapy has shown clinical success, efficacy is limited by low levels of T-cell trafficking to, and survival in, the immunosuppressive environment of an established tumor. Oncolytic virotherapy has recently emerged as a promising approach to induce both direct tumor cell killing and local proinflammatory environments within tumors. However, inefficient systemic delivery of oncolytic viruses remains a barrier to use of these agents against metastatic disease that is not directly accessible to the end of a needle. Here we show that the ability of antigen-specific T cells to circulate freely, and to localize to tumors, can be exploited to achieve the systemic delivery of replication-competent, oncolytic vesicular stomatitis virus (VSV). Thus, VSV loaded onto OT-I T cells, specific for the SIINFEKL epitope of the ovalbumin antigen, was efficiently delivered to established B16ova tumors in the lungs of fully immune-competent C57Bl/6 mice leading to significant increases in therapy compared to the use of virus, or T cells, alone. Although OT-I T-cell-mediated delivery of VSV led to viral replication within tumors and direct viral oncolysis, therapy was also dependent upon an intact host immune system. Moreover, VSV loading onto the T cells increased both T-cell activation in vitro and T-cell trafficking in vivo. The combination of adoptive T-cell transfer of antigen-specific T cells, along with oncolytic virotherapy, can, therefore, increase the therapeutic utility of both approaches through multiple mechanisms and should be of direct translational value.

Efficacy and Safety/Toxicity Study of Recombinant Vaccinia Virus JX-594 in Two Immunocompetent Animal Models of Glioma

The purpose of this study was to investigate the oncolytic potential of the recombinant, granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus (VV) JX-594 in experimental malignant glioma (MGs) in vitro and in immunocompetent rodent models. We have found that JX-594 killed all MG cell lines tested in vitro. Intratumoral (i.t.) administration of JX-594 significantly inhibited tumor growth and prolonged survival in rats-bearing RG2 intracranial (i.c.) tumors and mice-bearing GL261 brain tumors. Combination therapy with JX-594 and rapamycin significantly increased viral replication and further prolonged survival in both immunocompetent i.c. MG models with several animals considered “cured” (three out of seven rats >120 days, terminated experiment). JX-594 infected and killed brain tumor-initiating cells (BTICs) from patient samples grown ex vivo, and did so more efficiently than other oncolytic viruses MYXV, Reovirus type-3, and VSVΔM51. Additional safety/toxicity studies in nontumor-bearing rodents treated with a supratherapeutic dose of JX-594 demonstrated GM-CSF-dependent inflammation and necrosis. These results suggest that i.c. administered JX-594 triggers a predictable GM-CSF-mediated inflammation in murine models. Before proceeding to clinical trials, JX-594 should be evaluated in the brains of nonhuman primates and optimized for the viral doses, delivery routes as well as the combination agents (e.g., mTOR inhibitors).

Harnessing Oncolytic Virus-mediated Antitumor Immunity in an Infected Cell Vaccine

Treatment of permissive tumors with the oncolytic virus (OV) VSV-Δ51 leads to a robust antitumor T-cell response, which contributes to efficacy; however, many tumors are not permissive to in vivo treatment with VSV-Δ51. In an attempt to channel the immune stimulatory properties of VSV-Δ51 and broaden the scope of tumors that can be treated by an OV, we have developed a potent oncolytic vaccine platform, consisting of tumor cells infected with VSV-Δ51. We demonstrate that prophylactic immunization with this infected cell vaccine (ICV) protected mice from subsequent tumor challenge, and expression of granulocyte-monocyte colony stimulating factor (GM-CSF) by the virus (VSVgm-ICV) increased efficacy. Immunization with VSVgm-ICV in the VSV-resistant B16-F10 model induced maturation of dendritic and natural killer (NK) cell populations. The challenge tumor is rapidly infiltrated by a large number of interferon γ (IFNγ)-producing T and NK cells. Finally, we demonstrate that this approach is robust enough to control the growth of established tumors. This strategy is broadly applicable because of VSVs extremely broad tropism, allowing nearly all cell types to be infected at high multiplicities of infection in vitro, where the virus replication kinetics outpace the cellular IFN response. It is also personalized to the unique tumor antigen(s) displayed by the cancer cell.