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.

Complement inhibition prevents oncolytic vaccinia virus neutralization in immune humans and cynomolgus macaques.

Oncolytic viruses (OVs) have shown promising clinical activity when administered by direct intratumoral injection. However, natural barriers in the blood, including antibodies and complement, are likely to limit the ability to repeatedly administer OVs by the intravenous route. We demonstrate here that for a prototype of the clinical vaccinia virus based product Pexa-Vec, the neutralizing activity of antibodies elicited by smallpox vaccination, as well as the anamnestic response in hyperimmune virus treated cancer patients, is strictly dependent on the activation of complement. In immunized rats, complement depletion stabilized vaccinia virus in the blood and led to improved delivery to tumors. Complement depletion also enhanced tumor infection when virus was directly injected into tumors in immunized animals. The feasibility and safety of using a complement inhibitor, CP40, in combination with vaccinia virus was tested in cynomolgus macaques. CP40 pretreatment elicited an average 10-fold increase in infectious titer in the blood early after the infusion and prolonged the time during which infectious virus was detectable in the blood of animals with preexisting immunity. Capitalizing on the complement dependence of antivaccinia antibody with adjunct complement inhibitors may increase the infectious dose of oncolytic vaccinia virus delivered to tumors in virus in immune hosts.

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?

Development, optimization, and scale-up of suspension Vero cell culture process for high titer production of oncolytic herpes simplex virus-1.

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, and four OVs have been approved for cancer immunotherapy. However, high-yield and cost-effective production processes remain to be developed for most OVs. Here suspension-adapted Vero cell culture processes were developed for high titer production of an OV model, herpes simplex virus type 1 (HSV-1). Our study showed the HSV-1 productivity was significantly affected by multiplicity of infection, cell density, and nutritional supplies. Cell culture conditions were first optimized in shake flask experiments and then scaled up to 3 L bioreactors for virus production under batch and perfusion modes. A titer of 2.7 × 108 TCID50 mL-1 was obtained in 3 L batch culture infected at a cell density of 1.4 × 106 cells mL-1 , and was further improved to 1.1 × 109 TCID50 mL-1 in perfusion culture infected at 4.6 × 106 cells mL-1 . These titers are similar to or better than the previously reported best titer of 8.6 × 107 TCID50 mL-1 and 8.1 × 108 TCID50 mL-1 respectively obtained in labor-intensive adherent Vero batch and perfusion cultures. HSV-1 production in batch culture was successfully scaled up to 60 L pilot-scale bioreactor to demonstrate the scalability. The work reported here is the first study demonstrating high titer production of HSV-1 in suspension Vero cell culture under different bioreactor operating modes.

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.

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 VSV's 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.

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.

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 ß-galactosidase (ß-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.

Synergistic interaction between oncolytic viruses augments tumor killing.

A major barrier to all oncolytic viruses (OVs) in clinical development is cellular innate immunity, which is variably active in a spectrum of human malignancies. To overcome the heterogeneity of tumor response, we combined complementary OVs that attack cancers in distinct ways to improve therapeutic outcome. Two genetically distinct viruses, vesicular stomatitis virus (VSV) and vaccinia virus (VV), were used to eliminate the risk of recombination. The combination was tested in a variety of tumor types in vitro, in immunodeficient and immunocompetent mouse tumor models, and ex vivo, in a panel of primary human cancer samples. We found that VV synergistically enhanced VSV antitumor activity, dependent in large part on the activity of the VV B18R gene product. A recombinant version of VSV expressing the fusion-associated small-transmembrane (p14FAST) protein also further enhanced the ability of VV to spread through an infected monolayer, resulting in a "ping pong" oncolytic effect wherein each virus enhanced the ability of the other to replicate and/or spread in tumor cells. Our strategy is the first example where OVs are rationally combined to utilize attributes of different OVs to overcome the heterogeneity of malignancies and demonstrates the feasibility of combining complementary OVs to improve therapeutic outcome.

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 VSV(ΔM51). 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).

Carrier cell-based delivery of an oncolytic virus circumvents antiviral immunity.

Oncolytic viruses capable of tumor-selective replication and cytolysis have shown early promise as cancer therapeutics. However, the host immune system remains a significant obstacle to effective systemic administration of virus in a clinical setting. Here, we demonstrate the severe negative impact of the adaptive immune response on the systemic delivery of oncolytic vesicular stomatitis virus (VSV) in an immune-competent murine tumor model, an effect mediated primarily by the neutralization of injected virions by circulating antibodies. We show that this obstacle can be overcome by administering virus within carrier cells that conceal viral antigen during delivery. Infected cells were delivered to tumor beds and released virus to infect malignant cells while sparing normal tissues. Repeated administration of VSV in carrier cells to animals bearing metastatic tumors greatly improved therapeutic efficacy when compared with naked virion injection. Whole-body molecular imaging revealed that carrier cells derived from solid tumors accumulate primarily in the lungs following intravenous injection, whereas leukemic carriers disseminate extensively throughout the body. Furthermore, xenogeneic cells were equally effective at delivering virus as syngeneic cells. These findings emphasize the importance of establishing cell-based delivery platforms in order to maximize the efficacy of oncolytic therapeutics.

Correction: Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence.

[This corrects the article DOI: 10.1371/journal.pone.0155947.].

Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence.

Anti-tumor CD8+ T cells are a key determinant for overall survival in patients following surgical resection for solid malignancies. Using a mouse model of cancer vaccination (adenovirus expressing melanoma tumor-associated antigen (TAA)-dopachrome tautomerase (AdDCT) and resection resulting in major surgical stress (abdominal nephrectomy), we demonstrate that surgical stress results in a reduction in the number of CD8+ T cell that produce cytokines (IFNγ, TNFα, Granzyme B) in response to TAA. This effect is secondary to both reduced proliferation and impaired T cell function following antigen binding. In a prophylactic model, surgical stress completely abrogates tumor protection conferred by vaccination in the immediate postoperative period. In a clinically relevant surgical resection model, vaccinated mice undergoing a positive margin resection with surgical stress had decreased survival compared to mice with positive margin resection alone. Preoperative immunotherapy with IFNα significantly extends survival in surgically stressed mice. Importantly, myeloid derived suppressor cell (MDSC) population numbers and functional impairment of TAA-specific CD8+ T cell were altered in surgically stressed mice. Our observations suggest that cancer progression may result from surgery-induced suppression of tumor-specific CD8+ T cells. Preoperative immunotherapies aimed at targeting the prometastatic effects of cancer surgery will reduce recurrence and improve survival in cancer surgery patients.

Pexa-Vec double agent engineered vaccinia: oncolytic and active immunotherapeutic.

Oncolytic immunotherapies (OI) selectively infect, amplify within and destroy cancer cells, thereby representing a novel class of anti-cancer therapy. In addition to this primary mechanism-of-action (MOA), OI based on vaccinia have been shown to selectively target tumor-associated vasculature, triggering an acute reduction in tumor perfusion. This review focuses on a third complementary MOA for this product class: the induction of active immunotherapy. While the active immunotherapy approach has been validated by recent product approvals, the field is still faced with significant challenges. Tumors have evolved diverse mechanisms to hide from immune-mediated destruction. Here we hypothesize that oncolytic immunotherapy replication within tumors may tip the immune balance to allow for the effective induction and execution of adaptive anti-tumor immunity, resulting in long-term tumor control following OI clearance. This immune activation against the cancer can be augmented through OI 'arming' for the expression of immunostimulatory transgene products from the virus genome. With the first vaccinia OI (Pexa-Vec, thymidine kinase-inactivated vaccinia expressing Granulocyte-colony stimulating factor [GM-CSF]) now in advanced-stage clinical trials, it has become more important than ever to understand the complimentary MOA that contributes to tumor destruction and control in patients.

Leukemia cell-rhabdovirus vaccine: personalized immunotherapy for acute lymphoblastic leukemia.

PURPOSE: Acute lymphoblastic leukemia (ALL) remains incurable in most adults. It has been difficult to provide effective immunotherapy to improve outcomes for the majority of patients. Rhabdoviruses induce strong antiviral immune responses. We hypothesized that mice administered ex vivo rhabdovirus-infected ALL cells [immunotherapy by leukemia-oncotropic virus (iLOV)] would develop robust antileukemic immune responses capable of controlling ALL. EXPERIMENTAL DESIGN: Viral protein production, replication, and cytopathy were measured in human and murine ALL cells exposed to attenuated rhabdovirus. Survival following injection of graded amounts of ALL cells was compared between cohorts of mice administered γ-irradiated rhabdovirus-infected ALL cells (iLOV) or multiple control vaccines to determine key immunotherapeutic components and characteristics. Host immune requirements were assessed in immunodeficient and bone marrow-transplanted mice or by adoptive splenocyte transfer from immunized donors. Antileukemic immune memory was ascertained by second leukemic challenge in long-term survivors. RESULTS: Human and murine ALL cells were infected and killed by rhabdovirus; this produced a potent antileukemia vaccine. iLOV protected mice from otherwise lethal ALL by developing durable leukemia-specific immune-mediated responses (P < 0.0001), which required an intact CTL compartment. Preexisting antiviral immunity augmented iLOV potency. Splenocytes from iLOV-vaccinated donors protected 60% of naïve recipients from ALL challenge (P = 0.0001). Injecting leukemia cells activated by, or concurrent with, multiple Toll-like receptor agonists could not reproduce the protective effect of iLOV. Similarly, injecting uninfected irradiated viable, apoptotic, or necrotic leukemia cells with/without concurrent rhabdovirus administration was ineffective. CONCLUSION: Rhabdovirus-infected leukemia cells can be used to produce a vaccine that induces robust specific immunity against aggressive leukemia.

Disparate regulation of LPS-induced MAPK signaling and IL-12p40 expression between different myeloid cell types with and without HIV infection.

Studies from our laboratory and those of others have implicated lipopolysaccharide (LPS)-induced MAPK signaling as an important pathway in the regulation of cytokine expression. In this article, the regulation of IL-12 expression in two different human myeloid cell populations was evaluated. In primary monocytes, the inhibition of p38 enhanced IL-12 production, whereas it downregulated IL-12 production in THP-1 cells. The role of MAPK signaling in transcription factor binding to the IL-12p40 promoter was subsequently determined. In primary monocytes, ERK and p38 inhibition increased binding of AP-1 and Sp1, respectively, to the IL-12p40 promoter, while JNK inhibition increased NF-kappaB, AP-1, and Sp1 binding. In THP-1 cells, p38, ERK, and JNK inhibition increased NF-kappaB and Sp1 binding to the IL-12p40 promoter, while inhibiting AP-1 binding. In monocytes, mutations in the NF-kappaB, AP-1, Sp1, or Ets-2 binding sites resulted in complete inhibition of LPS-stimulated IL-12p40 promoter activity using a luciferase-based assay. In contrast, promoter activity was abrogated in THP-1 cells only when the Sp1 or Ets-2 binding sites were mutated. Transcription factor binding to the IL-12p40 promoter following in-vitro HIV infection demonstrated several differences between monocytes and THP-1 cells. Infection with HIV produced an increase in NF-kappaB, AP-1, and Sp1 binding in primary monocytes. In contrast, binding of Ets-2 was dramatically impaired following HIV infection of monocytes, but was unaffected in THP-1 cells. These data clearly show that although LPS induces IL-12p40 expression in primary monocytes and THP-1 cells, the signaling pathways involved and the effect of HIV infection differ and can have disparate effects in these two cell types.

Diplomatic immunity: turning a foe into an ally.

Striking a balance between subversion of antiviral immune responses and enhancement of potentially therapeutic, antitumor cytotoxic responses is a challenge at the forefront of oncolytic virus (OV) research. Some of the immune hurdles that must be overcome to maximize OV delivery to spread throughout tumor beds, and an outline of some of the strategies developed to deal with these obstacles are reviewed. In addition, current research that may lead to antitumor immunity during, or subsequent to, OV therapy is discussed. Finally speculations are made upon emerging areas of viral and immune research that could be merged to create new therapeutic paradigms.

Targeted inflammation during oncolytic virus therapy severely compromises tumor blood flow.

Oncolytic viruses (OVs) are selected or designed to eliminate malignancies by direct infection and lysis of cancer cells. In contrast to this concept of direct tumor lysis by viral infection, we observed that a significant portion of the in vivo tumor killing activity of two OVs, vesicular stomatitis virus (VSV) and vaccinia virus is caused by indirect killing of uninfected tumor cells. Shortly after administering the oncolytic virus we observed limited virus infection, coincident with a loss of blood flow to the interior of the tumor that correlated with induction of apoptosis in tumor cells. Transcript profiling of tumors showed that virus infection resulted in a dramatic transcriptional activation of pro-inflammatory genes including the neutrophil chemoattractants CXCL1 and CXCL5. Immunohistochemical examination of infected tumors revealed infiltration by neutrophils correlating with chemokine induction. Depletion of neutrophils in animals prior to VSV administration eliminated uninfected tumor cell apoptosis and permitted more extensive replication and spreading of the virus throughout the tumor. Taken all together, these results indicate that targeted recruitment of neutrophils to infected tumor beds enhances the killing of malignant cells. We propose that activation of inflammatory cells can be used for enhancing the effectiveness of oncolytic virus therapeutics, and that this approach should influence the planning of therapeutic doses.