Purpose Vascular endothelial growth factor (VEGF) expression is higher in triple-negative breast cancers (TNBC) compared to other subtypes and is reported to predict incidence of distant metastases and shorter overall survival. We investigated the therapeutic impact of a vaccinia virus (VACV) GLV-1h164 (derived from its parent virus GLV-1h100), encoding a single-chain antibody (scAb) against VEGF (GLAF-2) in an orthotopic TNBC murine model. Methods GLV-1h164 was tested against multiple TNBC cell lines. Viral infectivity, cytotoxicity, and replication were determined. Mammary fat pad tumors were generated in athymic nude mice using MDA-MB-231 cells. Xenografts were treated with GLV-1h164, GLV-1h100, or PBS and followed for tumor growth. Results Viral infectivity was time- and concentration-dependent. GLV-1h164 killed TNBC cell lines in a dose-dependent fashion with greater than 90% cytotoxicity within 4 days at a multiplicity of infection of 5.0. In vitro, cytotoxicity of GLV-1h164 was identical to GLV-1h100. GLV-1h164 replicated efficiently in all cell lines with an over 400-fold increase in copy numbers from the initial viral dose within 4 days. In vivo, mean tumor volumes after 2 weeks of treatment were 73, 191, and 422mm3 (GLV-1h164, GLV-1h100, and PBS, respectively) (p<0.05). Both in vivo Doppler ultrasonography and immuno-staining showed decreased neo-angiogenesis in GLV-1h164-treated tumors compared to both GLV-1h100 and PBS controls (p<0.05). Conclusion This is the first study to demonstrate efficient combination of oncolytic and anti-angiogenic activity of a novel vaccinia virus on TNBC xenografts. Our results suggest that GLV-1h164 is a promising therapeutic agent that warrants testing for patients with TNBC.

IntroductionSurgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model.MethodsGLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 μCi of 124I-iodide.ResultsViral infectivity, measured by green fluorescent protein (GFP) expression, was time- and concentration-dependent. All cell lines showed less than 10% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( <10,000-fold increase from the initial viral dose ) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm3 versus 168 mm3 in untreated controls (P < 0.05).ConclusionsThis is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors.

Background The purpose of this original work is to examine the ability of an oncolytic vaccinia virus expressing the human sodium iodine transporter (hNIS) to provide real time monitoring of viral therapy and effective treatment of malignant pleural mesothelioma (MPM). Methods Infectivity and cytotoxic effect of GLV-1h153 on mesothelioma cell lines of all histologic subtypes was assayed in vitro. Viral replication was examined by standard viral plaque assay. Orthotopic MPM xenografts were generated in athymic nude mice and treated with intrapleural GLV-1h153 and assessed for effect on tumor burden and survival. Orthotopic tumors were also imaged on SPECT/CT after 131I administration. Results GLV-1h153 infected and killed all cell lines in a time and concentration dependent manner. Viral replication demonstrated over a 2.5 log increase in titer over 4 days. Intrapleural treatment of orthotopic MPM xenografts resulted in a significant reduction in tumor burden one week after treatment and an improvement in survival. Infection of orthotopic xenografts was both therapeutic and facilitated monitoring by 131I-SPECT/CT via expression of hNIS in infected tissue. Conclusions Our results suggest GLV-1h153 is a promising therapeutic agent for MPM and warrants further investigation.

We investigated the therapeutic efficacy of a replication-competent oncolytic vaccinia virus, GLV-1h153, carrying human sodium iodide symporter (hNIS), in combination with radioiodine in an orthotopic triple-negative breast cancer (TNBC) murine model. In vitro viral infection was confirmed by immunoblotting and radioiodine uptake assays. Orthotopic xenografts (MDA-MB-231 cells) received intratumoral injection of GLV-1h153 or PBS. One week after viral injection, xenografts were randomized into 4 treatment groups: GLV-1h153 alone, GLV-1h153 and (131)I (∼ 5 mCi), (131)I alone, or PBS, and followed for tumor growth. Kruskal-Wallis and Wilcoxon tests were performed for statistical analysis. Radiouptake assay showed a 178-fold increase of radioiodine uptake in hNIS-expressing infected cells compared with PBS control. Systemic (131)I-iodide in combination with GLV-1h153 resulted in a 6-fold increase in tumor regression (24 compared to 146 mm(3) for the virus-only treatment group; P<0.05; d 40). We demonstrated that a novel vaccinia virus, GLV-1h153, expresses hNIS, increases the expression of the symporter in TNBC cells, and serves both as a gene marker for noninvasive imaging of virus and as a vehicle for targeted radionuclide therapy with (131)I.

Background Sorafenib is the standard systemic therapy for un-resectable or recurrent hepatocellular carcinoma (HCC) with minimal increase in survival. Therefore, there is a great need to develop novel therapies for advanced or recurrent HCC. One emerging field of cancer treatment involves oncolytic viruses that specifically infect, replicate within, and kill cancer cells. In this study we look at the ability of GLV-1h68, a recombinant vaccinia virus derived from the vaccine strain that was used to eradicate smallpox, to kill sorafenib-resistant HCC. Methods Four sorafenib-resistant HCC cell lines were generated by repeated passage in the presence of sorafenib. Median inhibitory concentration was determined for all cell lines. The infectivity, viral replication and cytotoxicity of GLV-1h68 were assayed for both parental and sorafenib-resistant HCC cells. Results Infectivity increased in a time and concentration dependent manner in all cell lines. All cell lines supported efficient replication of virus. No significant difference between the rates of cell death between the parental and sorafenib-resistant cell lines was observed. Conclusions Our results demonstrate that oncolytic vaccinia virus GLV-1h68 efficiently kills both parental and sorafenib-resistant HCC cell lines. This study indicates that patients who have failed treatment with sorafenib remain viable candidates for oncolytic therapy.