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Oncolytic vaccinia viruses have promising efficacy and safety profiles in cancer therapy. Although antitumor activity can be increased by manipulating viral genes, the relative efficacy of individual modifications has been difficult to assess without side-by-side comparisons. This study sought to compare the initial antitumor activity after intravenous administration of five vaccinia virus variants of the same Western Reserve backbone and thymidine kinase gene deletion in RIP-Tag2 transgenic mice with spontaneous pancreatic neuroendocrine tumors. Tumors had focal regions of infection at 5 days after all viruses. Natural killer (NK) cells were restricted to these sites of infection, but CD8+ T cells and tumor cell apoptosis were widespread and varied among the viruses. Antitumor activity of virus VV-A34, bearing amino acid substitution A34K151E to increase viral spreading, and virus VV-IL2v, expressing a mouse IL2 variant (mIL2v) with attenuated IL2 receptor alpha subunit binding, was similar to control virus VV-GFP. However, antitumor activity was significantly greater after virus VV-A34/IL2v, which expressed mIL2v together with A34K151E mutation and viral B18R gene deletion, and virus VV-GMCSF that expressed mouse GM-CSF. Both viruses greatly increased expression of CD8 antigens Cd8a/Cd8b1 and cytotoxicity genes granzyme A, granzyme B, Fas ligand, and perforin-1 in tumors. VV-A34/IL2v led to higher serum IL2 and greater tumor expression of death receptor ligand TRAIL, but VV-GMCSF led to higher serum GM-CSF, greater expression of leukocyte chemokines and adhesion molecules, and more neutrophil recruitment. Together, the results show that antitumor activity is similarly increased by viral expression of GM-CSF or IL2v combined with additional genetic modifications.

Introduction Adeno-associated virus-mediated (AAV-mediated) gene therapy has emerged as a promising treatment approach for a spectrum of monogenic genetic disorders (1-4). With the approval of the first recombinant AAV vector-based [...], BACKGROUND. Systemic administration of adeno-associated virus (AAV) can trigger life-threatening inflammatory responses, including thrombotic microangiopathy (TMA), acute kidney injury due to atypical hemolytic uremic syndrome- like complement activation, immune-mediated myocardial inflammation, and hepatic toxicity. METHODS. We describe the kinetics of immune activation following systemic AAV serotype 9 (AAV9) administration in 38 individuals following 2 distinct prophylactic immunomodulation regimens. Group 1 received corticosteroids and Group 2 received rituximab plus sirolimus in addition to steroids to prevent anti-AAV antibody formation. RESULTS. Group 1 participants had a rapid increase in immunoglobulin M (IgM) and IgG. Increase in D-dimer, decline in platelet count, and complement activation are indicative of TMA. All Group 1 participants demonstrated activation of both classical and alternative complement pathways, as indicated by depleted C4 and elevated soluble C5b-9, Ba, and Bb antigens. Group 2 patients did not have a significant change in IgM or IgG and had minimal complement activation. CONCLUSIONS. This study demonstrates that TMA in the setting of AAV gene therapy is antibody dependent (classical pathway) and amplified by the alternative complement pathway. Critical time points and interventions are identified to allow for management of immune-mediated events that impact the safety and efficacy of systemic gene therapy.

Oncolytic viruses pose many questions in their use in cancer therapy. In this study, we assessed the potential of mpJX-594 (mouse-prototype JX-594), a replication-competent vaccinia virus administered by intravenous injection, to target the tumor vasculature, produce immune activation and tumor cell killing more widespread than the infection, and suppress invasion and metastasis. These actions were examined in RIP-Tag2 transgenic mice with pancreatic neuroendocrine tumors that developed spontaneously and progressed as in humans. mpJX-594 initially infected tumor vascular endothelial cells, leading to vascular pruning and prolonged leakage in tumors but not in normal organs; parallel effects were observed in U87 gliomas. Viral infection spread to tumor cells, where tumor cell killing was much more widespread than the infection. Widespread tumor cell killing at 5 days was prevented by depletion of CD8+ T lymphocytes and did not require GM-CSF, as mpJX-594 variants that expressed human, mouse, or no GM-CSF produced equivalent amounts of killing. The antivascular, antitumor, and antimetastatic effects of mpJX-594 were amplified by concurrent or sequential administration of sunitinib, a multitargeted receptor tyrosine kinase inhibitor. These effects were not mimicked by selective inhibition of VEGFR2 despite equivalent vascular pruning, but were accompanied by suppression of regulatory T cells and greater influx of activated CD8+ T cells. Together, our results showed that mpJX-594 targets tumor blood vessels, spreads secondarily to tumor cells, and produces widespread CD8+ T-cell-dependent tumor cell killing in primary tumors and metastases, and that these effects can be amplified by coadministration of sunitinib.Significance: These findings reveal multiple unrecognized features of the antitumor properties of oncolytic vaccinia viruses, all of which can be amplified by the multitargeted kinase inhibitor sunitinib. Cancer Res; 78(4); 922-37. ©2017 AACR.

Despite advances in cancer treatment over the past 30 years with chemotherapy and biologics, the majority of solid tumors remain incurable once they are metastatic. Truly new agents with multiple [...], Oncolytic viruses and active immunotherapeutics have complementary mechanisms of action (MOA) that are both self amplifying in tumors, yet the impact of dose on subject outcome is unclear. JX-594 (Pexa-Vec) is an oncolytic and immunotherapeutic vaccinia virus. To determine the optimal JX-594 dose in subjects with advanced hepatocellular carcinoma (HCC), we conducted a randomized phase 2 dose-finding trial (n = 30). Radiologists infused low- or high-dose JX-594 into liver tumors (days 1, 15 and 29); infusions resulted in acute detectable intravascular JX-594 genomes. Objective intrahepatic Modified Response Evaluation Criteria in Solid Tumors (mRECIST) (15%) and Choi (62%) response rates and intrahepatic disease control (50%) were equivalent in injected and distant noninjected tumors at both doses. JX-594 replication and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression preceded the induction of anticancer immunity. In contrast to tumor response rate and immune endpoints, subject survival duration was significantly related to dose (median survival of 14.1 months compared to 6.7 months on the high and low dose, respectively; hazard ratio 0.39; P = 0.020). JX-594 demonstrated oncolytic and immunotherapy MOA, tumor responses and dose-related survival in individuals with HCC.

Introduction Oncolytic viruses have promise as cancer therapeutics due to their targeted nature and ability to destroy cancer cells through novel mechanisms-of-action (oncolysis and/or necrosis) [1-3]. Selective intratumoral replication of [...], Background Oncolytic viruses hold much promise for clinical treatment of many cancers, but a lack of systemic delivery and insufficient tumor cell killing have limited their usefulness. We have previously demonstrated that vaccinia virus strains are capable of systemic delivery to tumors in mouse models, but infection of normal tissues remains an issue. We hypothesized that interferon-beta (IFN-β) expression from an oncolytic vaccinia strain incapable of responding to this cytokine would have dual benefits as a cancer therapeutic: increased anticancer effects and enhanced virus inactivation in normal tissues. We report the construction and preclinical testing of this virus. Methods and Findings In vitro screening of viral strains by cytotoxicity and replication assay was coupled to cellular characterization by phospho-flow cytometry in order to select a novel oncolytic vaccinia virus. This virus was then examined in vivo in mouse models by non-invasive imaging techniques. A vaccinia B18R deletion mutant was selected as the backbone for IFN-β expression, because the B18R gene product neutralizes secreted type-I IFNs. The oncolytic B18R deletion mutant demonstrated IFN-dependent cancer selectivity and efficacy in vitro, and tumor targeting and efficacy in mouse models in vivo. Both tumor cells and tumor-associated vascular endothelial cells were targeted. Complete tumor responses in preclinical models were accompanied by immune-mediated protection against tumor rechallenge. Cancer selectivity was also demonstrated in primary human tumor explant tissues and adjacent normal tissues. The IFN-β gene was then cloned into the thymidine kinase (TK) region of this virus to create JX-795 ([TK.sup.-]/ [B18R.sup.-]/IFN-[β.sup.+]). JX-795 had superior tumor selectivity and systemic intravenous efficacy when compared with the [TK.sup.-]/[B18R.sup.-] control or wild-type vaccinia in preclinical models. Conclusions By combining IFN-dependent cancer selectivity with IFN-β expression to optimize both anticancer effects and normal tissue antiviral effects, we were able to achieve, to our knowledge for the first time, tumor-specific replication, IFN-β gene expression, and efficacy following systemic delivery in preclinical models.

Replication-selective oncolytic viruses (virotherapeutics) are being developed as novel cancer therapies with unique mechanisms of action, but limitations in i.v. delivery to tumors and systemic efficacy have highlighted the need [...]