Preventing postoperative metastatic disease by inhibiting surgery-induced dysfunction in natural killer cells.

Natural killer (NK) cell clearance of tumor cell emboli following surgery is thought to be vital in preventing postoperative metastases. Using a mouse model of surgical stress, we transferred surgically stressed NK cells into NK-deficient mice and observed enhanced lung metastases in tumor-bearing mice as compared with mice that received untreated NK cells. These results establish that NK cells play a crucial role in mediating tumor clearance following surgery. Surgery markedly reduced NK cell total numbers in the spleen and affected NK cell migration. Ex vivo and in vivo tumor cell killing by NK cells were significantly reduced in surgically stressed mice. Furthermore, secreted tissue signals and myeloid-derived suppressor cell populations were altered in surgically stressed mice. Significantly, perioperative administration of oncolytic parapoxvirus ovis (ORFV) and vaccinia virus can reverse NK cell suppression, which correlates with a reduction in the postoperative formation of metastases. In human studies, postoperative cancer surgery patients had reduced NK cell cytotoxicity, and we show for the first time that oncolytic vaccinia virus markedly increases NK cell activity in patients with cancer. These data provide direct in vivo evidence that surgical stress impairs global NK cell function. Perioperative therapies aimed at enhancing NK cell function will reduce metastatic recurrence and improve survival in surgical cancer patients.

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.

ORFV: a novel oncolytic and immune stimulating parapoxvirus therapeutic.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. Parapoxvirus ovis or Orf virus (ORFV) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent T(h)-1-dominated immune response. Here, we show that live replicating ORFV induces an antitumor immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumoricidal natural killer (NK) cells. We have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer.

A selectable and excisable marker system for the rapid creation of recombinant poxviruses.

BACKGROUND: Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene. CONCLUSION/SIGNIFICANCE: The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.

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.

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.

Potent oncolytic activity of raccoonpox virus in the absence of natural pathogenicity.

A number of oncolytic virus (OV) candidates currently in clinical trials are human viruses that have been engineered to be safer for patient administration by limiting normal cell targeting and replication. The newest OVs include viruses that cause no disease in humans, yet still have natural tumor tropism. Raccoonpox virus (RCNV) is a member of the Orthopoxvirus genus of Poxviridae and closely related to vaccinia virus, yet has no known pathogenicity in any mammalian species. A screen of cells from the NCI-60 cancer cell panel using growth curves demonstrated greater than a log increase in replication of RCNV in nearly 74% of the cell lines tested, similar to other tested OV poxviruses. In normal cell lines, pretreatment with interferon (IFN)-alpha/beta resulted in significant inhibition of RCNV replication. In both xenograft and syngeneic models of solid tumors, injection of RCNV resulted in significantly slower tumor progression and increased survival of mice. RCNV treatment also prolonged survival in treatment-resistant models of brain tumors and decreased tumor burden by systemic administration in models of lung metastasis.

Tetrahydro-1,8-naphthyridinol analogues of alpha-tocopherol as antioxidants in lipid membranes and low-density lipoproteins.

Recently we demonstrated that the C(7)-unsubstituted tetrahydro-1,8-naphthyridin-3-ol has more than an order of magnitude better peroxyl radical trapping activity than alpha-tocopherol (alpha-TOH) in inhibited autoxidations in benzene. In order to prepare analogues more structurally related to alpha-TOH for further studies in vitro and in vivo, we developed synthetic approaches to C(7)-monoalkyl and C(7)-dialkyl analogues using a sequence involving (1) AgNO3-mediated hydroxymethyl radical addition to 1,8-naphthyridine, (2) regioselective alkyllithium addition by cyclic chelation in a nonpolar solvent, (3) iodination of the naphthyridine at C(3), and (4) CuI-medidated benzyloxylation of the aryl iodide followed by catalytic hydrogenolysis. An alpha-TOH isostere was prepared by a Wittig coupling of a C16 side chain identical to that of alpha-TOH to the naphthyridinols. The C(7)-mono- and dialkyl analogues exhibited more than an order of magnitude higher antioxidant activity (k(inh) = (5.3-6.1) x 10(7) M(-1) s(-1)) than alpha-TOH (k(inh) = 0.35 x 10(7) M(-) s(-1)) in benzene, as determined by a newly developed peroxyl radical clock. In addition to the strong antioxidant activity in benzene, the closest alpha-TOH analogue (naphthyridinol-based tocopherol, N-TOH) showed excellent inhibition of the oxidation of cholesteryl esters in human low-density lipoprotein and spared endogenous alpha-TOH in these experiments. Lateral diffusion of N-TOH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes was comparable to that of alpha-TOH, suggesting that it will have good antioxidant characteristics in both membranes and lipoproteins. Furthermore, a binding assay using a fluorescent tocopherol analogue showed that N-TOH binds to recombinant human tocopherol transfer protein better than alpha-TOH itself, suggesting that distribution of unnatural antioxidants such as N-TOH in vivo is possible.