Marianne Stanford

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.

Oncolytic Virotherapy Synergism with Signaling Inhibitors: Rapamycin Increases Myxoma Virus Tropism for Human Tumor Cells

ABSTRACT Myxoma virus is a rabbit-specific poxvirus pathogen that also exhibits a unique tropism for human tumor cells and is dramatically oncolytic for human cancer xenografts. Most tumor cell lines tested are permissive for myxoma infection in a fashion intimately tied to the activation state of Akt kinase. A host range factor of myxoma virus, M-T5, directly interacts with Akt and mediates myxoma virus tumor cell tropism. mTOR is a regulator of cell growth and metabolism downstream of Akt and is specifically inhibited by rapamycin. We report that treatment of nonpermissive human tumor cell lines, which normally restrict myxoma virus replication, with rapamycin dramatically increased virus tropism and spread in vitro. This increased myxoma replication is concomitant with global effects on mTOR signaling, specifically, an increase in Akt kinase. In contrast to the effects on human cancer cells, rapamycin does not increase myxoma virus replication in rabbit cell lines or permissive human tumor cell lines with constitutively active Akt. This indicates that rapamycin increases the oncolytic capacity of myxoma virus for human cancer cells by reconfiguring the internal cell signaling environment to one that is optimal for productive virus replication and suggests the possibility of a potentially therapeutic synergism between kinase signaling inhibitors and oncolytic poxviruses for cancer treatment.

Myxoma virus and oncolytic virotherapy: a new biologic weapon in the war against cancer.

Oncolytic virotherapy is an innovative alternative to more conventional cancer therapies. The ability of some viruses to specifically target and kill malignant cancerous cells while leaving normal tissue unscathed has opened a large repertoire of new and selective cancer killing therapeutic candidates. Poxviruses, such as vaccinia virus, have a long history of use in humans as live vaccines and have more recently been studied as potential platforms for delivery of immunotherapeutics and attenuated variants of vaccinia have been explored as oncolytic candidates. In contrast, the poxvirus myxoma virus is a novel oncolytic candidate that has no history of use in humans directly, as it has a distinct and absolute host species tropism to lagomorphs (rabbits). Myxoma virus has been recently shown to be able to also selectively infect and kill human tumor cells, a unique tropism that is linked to dysregulated intracellular signalling pathways found in the majority of human cancers. This review outlines the existing knowledge on the tropism of myxoma virus for human cancer cells, as well as preclinical data exhibiting its ability to infect and clear tumors in animal models of cancer. This is an exciting new therapeutic option for treating cancer, and myxoma virus joins a growing group of oncolytic virus candidates that are being developed as a new class of cancer therapies in man.

Immunopathogenesis of poxvirus infections: forecasting the impending storm.

Variola virus, the causative agent of smallpox, is a member of the poxvirus family and one of the most virulent human pathogens known. Although smallpox was eradicated almost 30 years ago, it is not understood why the mortality rates associated with the disease were high, why some patients recovered, and what constitutes an effective host response against infection. As variola virus infects only humans, our current understanding of poxvirus infections comes largely from historical clinical data from smallpox patients and from animal studies using closely related viruses such as ectromelia, myxoma and monkeypox. The outcome of an infection is determined by a complex interaction between the type of immune response mounted by the host and by evasion mechanisms that the virus has evolved to subvert it. Disease pathogenesis is also a function of both host and viral factors. Poxviruses are not only cytopathic, causing host tissue damage, but also encode an array of immunomodulatory molecules that affect the severity of disease. The ability of the host to control virus replication is therefore critical in limiting tissue damage. However, in addition to targeting virus, the immune response can inadvertently damage the host to such a degree that it causes illness and even death. There is growing evidence that many of the symptoms associated with serious poxvirus infections are a result of a ‘cytokine storm’ or sepsis and that this may be the underlying cause of pathology.

The relative activity of CXCR3 and CCR5 ligands in T lymphocyte migration: concordant and disparate activities in vitro and in vivo

In chronic inflammatory reactions such as rheumatoid arthritis and multiple sclerosis, T cells in the inflamed tissue express the chemokine receptors CXCR3 and CCR5, and the chemokine ligands (CCL) of these receptors are present in the inflammatory lesions. However, the contribution of these chemokines to T cell recruitment to sites of inflammation is unclear. In addition, the relative roles of the chemokines that bind CXCR3 (CXCL9, CXCL10, CXCL11) and CCR5 (CCL3, CCL4, CCL5) in this process are unknown. The in vitro chemotaxis and in vivo migration of antigen‐activated T lymphoblasts and unactivated spleen T cells to chemokines were examined. T lymphoblasts migrated in vitro to CXCR3 ligands with a relative potency of CXCL10 > CXCL11 > CXCL9, but these cells demonstrated much less chemotaxis to the CCR5 ligands. In vivo, T lymphocytes were recruited in large numbers with rapid kinetics to skin sites injected with CXCL10 and CCL5 and less to CCL3, CCL4, CXCL9, and CXCL11. The combination of CCL5 with CXCL10 but not the other chemokines markedly increased recruitment. Coinjection of interferon‐γ, tumor necrosis factor α, and interleukin‐1α to up‐regulate endothelial cell adhesion molecule expression with CXCL10 or CCL5 induced an additive increase in lymphoblast migration. Thus, CXCR3 ligands are more chemotactic than CCR5 ligands in vitro; however, in vivo, CXCL10 and CCL5 have comparable T cell‐recruiting activities to cutaneous sites and are more potent than the other CXCR3 and CCR5 chemokines. Therefore, in vitro chemotaxis induced by these chemokines is not necessarily predictive of their in vivo lymphocyte‐recruiting activity.

Intravenously Administered Alphavirus Vector VA7 Eradicates Orthotopic Human Glioma Xenografts in Nude Mice

Background VA7 is a neurotropic alphavirus vector based on an attenuated strain of Semliki Forest virus. We have previously shown that VA7 exhibits oncolytic activity against human melanoma xenografts in immunodeficient mice. The purpose of this study was to determine if intravenously administered VA7 would be effective against human glioma. Methodology/Principal Findings In vitro, U87, U251, and A172 human glioma cells were infected and killed by VA7-EGFP. In vivo, antiglioma activity of VA7 was tested in Balb/c nude mice using U87 cells stably expressing firefly luciferase in subcutaneous and orthotopic tumor models. Intravenously administered VA7-EGFP completely eradicated 100% of small and 50% of large subcutaneous U87Fluc tumors. A single intravenous injection of either VA7-EGFP or VA7 expressing Renilla luciferase (VA7-Rluc) into mice bearing orthotopic U87Fluc tumors caused a complete quenching of intracranial firefly bioluminescence and long-term survival in total 16 of 17 animals. In tumor-bearing mice injected with VA7-Rluc, transient intracranial and peripheral Renilla bioluminescence was observed. Virus was well tolerated and no damage to heart, liver, spleen, or brain was observed upon pathological assessment at three and ninety days post injection, despite detectable virus titers in these organs during the earlier time point. Conclusion VA7 vector is apathogenic and can enter and destroy brain tumors in nude mice when administered systemically. This study warrants further elucidation of the mechanism of tumor destruction and attenuation of the VA7 virus.

First-in-man application of a novel therapeutic cancer vaccine formulation with the capacity to induce multi-functional T cell responses in ovarian, breast and prostate cancer patients

BackgroundDepoVaxTM is a novel non-emulsion depot-forming vaccine platform with the capacity to significantly enhance the immunogenicity of peptide cancer antigens. Naturally processed HLA-A2 restricted peptides presented by breast, ovarian and prostate cancer cells were used as antigens to create a therapeutic cancer vaccine, DPX-0907.MethodsA phase I clinical study was designed to examine the safety and immune activating potential of DPX-0907 in advanced stage breast, ovarian and prostate cancer patients. A total of 23 late stage cancer patients were recruited and were divided into two dose/volume cohorts in a three immunization protocol.ResultsDPX-0907 was shown to be safe with injection site reactions being the most commonly reported adverse event. All breast cancer patients (3/3), most of ovarian (5/6) and one third of prostate (3/9) cancer patients exhibited detectable immune responses, resulting in a 61% immunological response rate. Immune responses were generally observed in patients with better disease control after their last prior treatment. Antigen-specific responses were detected in 73% of immune responders (44% of evaluable patients) after the first vaccination. In 83% of immune responders (50% of evaluable patients), peptide-specific T cell responses were detected at ≥2 time points post vaccination with 64% of the responders (39% of evaluable patients) showing evidence of immune persistence. Immune monitoring also demonstrated the generation of antigen-specific T cell memory with the ability to secrete multiple Type 1 cytokines.ConclusionsThe novel DepoVax formulation promotes multifunctional effector memory responses to peptide-based tumor associated antigens. The data supports the capacity of DPX-0907 to elicit Type-1 biased immune responses, warranting further clinical development of the vaccine. This study underscores the importance of applying vaccines in clinical settings in which patients are more likely to be immune competent.Trial RegistrationClinicalTrials.gov NCT01095848

M-T5, the Ankyrin Repeat, Host Range Protein of Myxoma Virus, Activates Akt and Can Be Functionally Replaced by Cellular PIKE-A

ABSTRACT The myxoma virus (MV) ankyrin repeat, host range factor M-T5 has the ability to bind and activate cellular Akt, leading to permissive MV replication in a variety of diverse human cancer cell lines (G. Wang, J. W. Barrett, M. Stanford, S. J. Werden, J. B. Johnston, X. Gao, M. Sun, J. Q. Cheng, and G. McFadden, Proc. Natl. Acad. Sci. USA 103:4640-4645, 2006). The susceptibility of permissive human cancer cells to MV infection is directly correlated with the basal or induced levels of phosphorylated Akt. When M-T5 is deleted from MV, the knockout virus, vMyxT5KO, can no longer productively infect a subset of human cancer cells (designated type II) that exhibit little or no endogenous phosphorylated Akt. In searching for a host counterpart of M-T5, we noted sequence similarity of M-T5 to a recently identified ankyrin repeat cellular binding protein of Akt called PIKE-A. PIKE-A binds and activates the kinase activity of Akt in a GTP-dependent manner and promotes the invasiveness of human cancer cell lines. Here, we demonstrate that transfected PIKE-A is able to rescue the ability of vMyxT5KO to productively infect type II human cancer cells that were previously resistant to infection. Also, cancer cells that were completely nonpermissive for both wild-type and vMyxT5KO infection (called type III) were rendered fully permissive following ectopic expression of PIKE-A. We conclude that the MV M-T5 host range protein is functionally interchangeable with the host PIKE-A protein and that the activation of host Akt by either M-T5 or PIKE-A is critical for the permissiveness of human cancer cells for MV.

Survivin-targeted immunotherapy drives robust polyfunctional T cell generation and differentiation in advanced ovarian cancer patients

DepoVax™ is an innovative and strongly immunogenic vaccine platform. Survivin is highly expressed in many tumor types and has reported prognostic value. To generate tumor-specific immune response, a novel cancer vaccine was formulated in DepoVax platform (DPX-Survivac) using survivin HLA class I peptides. Safety and immune potency of DPX-Survivac was tested in combination with immune-modulator metronomic cyclophosphamide in ovarian cancer patients. All the patients receiving the therapy produced antigen-specific immune responses; higher dose vaccine and cyclophosphamide treatment generating significantly higher magnitude responses. Strong T cell responses were associated with differentiation of naïve T cells into central/effector memory (CM/EM) and late differentiated (LD) polyfunctional antigen-specific CD4+ and CD8+ T cells. This approach enabled rapid de novo activation/expansion of vaccine antigen-specific CD8+ T cells and provided a strong rationale for further testing to determine clinical benefits associated with this immune activation. These data represent vaccine-induced T cell activation in a clinical setting to a self-tumor antigen previously described only in animal models.

Identification of host range mutants of myxoma virus with altered oncolytic potential in human glioma cells

The authors have recently demonstrated that wild-type myxoma virus (MV) tagged with gfp (vMyxgfp) can generate a tumor-specific infection that productively infects and clears human tumor-derived xenografts when injected intratumorly into human gliomas transplanted into immunodeficient mice (Lun et al, 2005). To expand the understanding of MV tropism in cancer cells from a specific tissue lineage, the authors have screened a series of human glioma cells (U87, U118, U251, U343, U373) for myxoma virus replication and oncolysis. To assess the viral tropism determinants for these infections, the authors have screened myxoma virus knockout constructs that have been deleted for specific host range genes (M-T2, M-T4, M-T5, M11L, and M063), as well as a control MV gene knockout construct with no known host range function (vMyx135KO) but is highly attenuated in rabbits. The authors report wide variation in the ability of various vMyx-hrKOs to replicate and spread in the human glioma cells as measured by early and late viral gene expression. This differential ability to support vMyx-hrKO productive viral replication is consistent with levels of endogenous activated Akt in the various gliomas. The authors have identified one vMyx-hrKO virus (vMyx63KO) and one nonhost range knock-out construct (vMyx135KO) that appear to replicate in the gliomas even more efficiently than the wild-type virus and that reduce the viability of the infected gliomas. These knockout viruses also inhibit the proliferation of gliomas in a manner similar to the wild-type virus. Together these data, as well as the fact that these knockout viruses are attenuated in their natural hosts, may represent environmentally safer candidate oncolytic viruses for usage in human trials.