Novel oncolytic chimeric orthopoxvirus causes regression of pancreatic cancer xenografts and exhibits abscopal effect at a single low dose.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been increasing by 0.5% per year in the United States. PDAC portends a dismal prognosis and novel therapies are needed. This study describes the generation and characterization of a novel oncolytic chimeric orthopoxvirus for the treatment of pancreatic cancer. METHODS: After chimerization and high-throughput screening, CF33 was chosen from 100 new chimeric orthopoxvirus isolates for its ability to kill pancreatic cancer cells. In vitro cytotoxicity was assayed in six pancreatic cancer cell lines. In vivo efficacy and toxicity were evaluated in PANC-1 and MIA PaCa-2 xenograft models. RESULTS: CF33 caused rapid killing of six pancreatic cancer cells lines in vitro, releasing damage-associated molecular patterns, and regression of PANC-1 injected and non-injected distant xenografts in vivo after a single low intratumoral dose of 103 plaque-forming units. Using luciferase imaging, CF33 was noted to preferentially replicate in tumors which corresponds to the low viral titers found in solid organs. CONCLUSION: The low dose of CF33 required to treat pancreatic cancer in this preclinical study may ease the manufacturing and dosing challenges currently facing oncolytic viral therapy.

Isolated limb perfusion with melphalan, tumour necrosis factor-alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma.

Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in-transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer-selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour-targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor-alpha (TNF-α) and Lister strain vaccinia virus (GLV-1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF-α, melphalan and GLV-1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV-1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF-α/GLV-1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF-α/melphalan-based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted.

Microglia and astrocytes attenuate the replication of the oncolytic vaccinia virus LIVP 1.1.1 in murine GL261 gliomas by acting as vaccinia virus traps.

BACKGROUND: Oncolytic virotherapy is a novel approach for the treatment of glioblastoma multiforme (GBM) which is still a fatal disease. Pathologic features of GBM are characterized by the infiltration with microglia/macrophages and a strong interaction between immune- and glioma cells. The aim of this study was to determine the role of microglia and astrocytes for oncolytic vaccinia virus (VACV) therapy of GBM. METHODS: VACV LIVP 1.1.1 replication in C57BL/6 and Foxn1(nu/nu) mice with and without GL261 gliomas was analyzed. Furthermore, immunohistochemical analysis of microglia and astrocytes was investigated in non-, mock-, and LIVP 1.1.1-infected orthotopic GL261 gliomas in C57BL/6 mice. In cell culture studies virus replication and virus-mediated cell death of GL261 glioma cells was examined, as well as in BV-2 microglia and IMA2.1 astrocytes with M1 or M2 phenotypes. Co-culture experiments between BV-2 and GL261 cells and apoptosis/necrosis studies were performed. Organotypic slice cultures with implanted GL261 tumor spheres were used as additional cell culture system. RESULTS: We discovered that orthotopic GL261 gliomas upon intracranial virus delivery did not support replication of LIVP 1.1.1, similar to VACV-infected brains without gliomas. In addition, recruitment of Iba1(+) microglia and GFAP(+) astrocytes to orthotopically implanted GL261 glioma sites occurred already without virus injection. GL261 cells in culture showed high virus replication, while replication in BV-2 and IMA2.1 cells was barely detectable. The reduced viral replication in BV-2 cells might be due to rapid VACV-induced apoptotic cell death. In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected. Application of BV-2 microglial cells with M1 phenotype onto organotypic slice cultures with implanted GL261 gliomas resulted in reduced infection of BV-2 cells, whereas GL261 cells were well infected. CONCLUSION: Our results indicate that microglia and astrocytes, dependent on their activation state, may preferentially clear viral particles by immediate uptake after delivery. By acting as VACV traps they further reduce efficient virus infection of the tumor cells. These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.

Vaccinia virus GLV-1h153 in combination with 131I shows increased efficiency in treating triple-negative breast cancer.

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.

A novel vaccinia virus with dual oncolytic and anti-angiogenic therapeutic effects against triple-negative breast cancer.

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. 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. 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 422 mm(3) (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). This is the first study to demonstrate efficient combination of oncolytic and anti-angiogenic activity of a novel VACV on TNBC xenografts. Our results suggest that GLV-1h164 is a promising therapeutic agent that warrants testing for patients with TNBC.

Expression of a non-coding RNA in ectromelia virus is required for normal plaque formation.

Poxviruses are dsDNA viruses with large genomes. Many genes in the genome remain uncharacterized, and recent studies have demonstrated that the poxvirus transcriptome includes numerous so-called anomalous transcripts not associated with open reading frames. Here, we characterize the expression and role of an apparently non-coding RNA in orthopoxviruses, which we call viral hairpin RNA (vhRNA). Using a bioinformatics approach, we predicted expression of a transcript not associated with an open reading frame that is likely to form a stem-loop structure due to the presence of a 21 nt palindromic sequence. Expression of the transcript as early as 2 h post-infection was confirmed by northern blot and analysis of publicly available vaccinia virus infected cell transcriptomes. The transcription start site was determined by RACE PCE and transcriptome analysis, and early and late promoter sequences were identified. Finally, to test the function of the transcript we generated an ectromelia virus knockout, which failed to form plaques in cell culture. The important role of the transcript in viral replication was further demonstrated using siRNA. Although the function of the transcript remains unknown, our work contributes to evidence of an increasingly complex poxvirus transcriptome, suggesting that transcripts such as vhRNA not associated with an annotated open reading frame can play an important role in viral replication.

Vaccinia virus-mediated expression of human erythropoietin in tumors enhances virotherapy and alleviates cancer-related anemia in mice.

Recombinant human erythropoietin (rhEPO), a glycoprotein hormone regulating red blood cell (RBC) formation, is used for the treatment of cancer-related anemia. The effect of rhEPO on tumor growth, however, remains controversial. Here, we report the construction and characterization of the recombinant vaccinia virus (VACV) GLV-1h210, expressing hEPO. GLV-1h210 was shown to replicate in and kill A549 lung cancer cells in culture efficiently. In mice bearing A549 lung cancer xenografts, treatment with a single intravenous dose of GLV-1h210 resulted in tumor-specific production and secretion of functional hEPO, which exerted an effect on RBC progenitors and precursors in the mouse bone marrow, leading to a significant increase in the number of RBCs and in the level of hemoglobin. Furthermore, virally expressed hEPO, but not exogenously added rhEPO, enhanced virus-mediated green fluorescent protein (GFP) expression in tumors and subsequently accelerated tumor regression when compared with the treatment with the parental virus GLV-1h68 or GLV-1h209 that expressed a nonfunctional hEPO protein. Moreover, intratumorally expressed hEPO caused enlarged tumoral microvessels, likely facilitating virus spreading. Taken together, VACV-mediated intratumorally expressed hEPO not only enhanced oncolytic virotherapy but also simultaneously alleviated cancer-related anemia.

Vaccinia virus GLV-1h153 is a novel agent for detection and effective local control of positive surgical margins for breast cancer.

INTRODUCTION: Surgery 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. METHODS: GLV-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. RESULTS: Viral 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). CONCLUSIONS: This 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.

Combination of fractionated irradiation with anti-VEGF expressing vaccinia virus therapy enhances tumor control by simultaneous radiosensitization of tumor associated endothelium.

Oncolytic viruses are currently in clinical trials for a variety of tumors, including high grade gliomas. A characteristic feature of high grade gliomas is their high vascularity and treatment approaches targeting tumor endothelium are under investigation, including bevacizumab. The aim of this study was to improve oncolytic viral therapy by combining it with ionizing radiation and to radiosensitize tumor vasculature through a viral encoded anti-angiogenic payload. Here, we show how vaccinia virus-mediated expression of a single-chain antibody targeting VEGF resulted in radiosensitization of the tumor-associated vasculature. Cell culture experiments demonstrated that purified vaccinia virus encoded antibody targeting VEGF reversed VEGF-induced radioresistance specifically in endothelial cells but not tumor cells. In a subcutaneous model of U-87 glioma, systemically administered oncolytic vaccinia virus expressing anti-VEGF antibody (GLV-1h164) in combination with fractionated irradiation resulted in enhanced tumor growth inhibition when compared to nonanti-VEGF expressing oncolytic virus (GLV-1h68) and irradiation. Irradiation of tumor xenografts resulted in an increase in VACV replication of both GLV-1h68 and GLV-1h164. However, GLV-1h164 in combination with irradiation resulted in a drastic decrease in intratumoral VEGF levels and tumor vessel numbers in comparison to GLV-1h68 and irradiation. These findings demonstrate the incorporation of an oncolytic virus expressing an anti-VEGF antibody (GLV-1h164) into a fractionated radiation scheme to target tumor cells by enhanced VACV replication in irradiated tumors as well as to radiosensitize tumor endothelium which results in enhanced efficacy of combination therapy of human glioma xenografts.

Growth inhibition of different human colorectal cancer xenografts after a single intravenous injection of oncolytic vaccinia virus GLV-1h68.

BACKGROUND: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers. METHODS: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models. RESULTS: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke's type A-stage HCT-116 and Duke's type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls. CONCLUSION: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.

Vaccinia virus expressing bone morphogenetic protein-4 in novel glioblastoma orthotopic models facilitates enhanced tumor regression and long-term survival.

BACKGROUND: Glioblastoma multiforme (GBM) is one of the most aggressive forms of cancer with a high rate of recurrence. We propose a novel oncolytic vaccinia virus (VACV)-based therapy using expression of the bone morphogenetic protein (BMP)-4 for treating GBM and preventing recurrence. METHODS: We have utilized clinically relevant, orthotopic xenograft models of GBM based on tumor-biopsy derived, primary cancer stem cell (CSC) lines. One of the cell lines, after being transduced with a cDNA encoding firefly luciferase, could be used for real time tumor imaging. A VACV that expresses BMP-4 was constructed and utilized for infecting several primary glioma cultures besides conventional serum-grown glioma cell lines. This virus was also delivered intracranially upon implantation of the GBM CSCs in mice to determine effects on tumor growth. RESULTS: We found that the VACV that overexpresses BMP-4 demonstrated heightened replication and cytotoxic activity in GBM CSC cultures with a broad spectrum of activity across several different patient-biopsy cultures. Intracranial inoculation of mice with this virus resulted in a tumor size equal to or below that at the time of injection. This resulted in survival of 100% of the treated mice up to 84 days post inoculation, significantly superior to that of a VACV lacking BMP-4 expression. When mice with a higher tumor burden were injected with the VACV lacking BMP-4, 80% of the mice showed tumor recurrence. In contrast, no recurrence was seen when mice were injected with the VACV expressing BMP-4, possibly due to induction of differentiation in the CSC population and subsequently serving as a better host for VACV infection and oncolysis. This lack of recurrence resulted in superior survival in the BMP-4 VACV treated group. CONCLUSIONS: Based on these findings we propose a novel VACV therapy for treating GBM, which would allow tumor specific production of drugs in the future in combination with BMPs which would simultaneously control tumor maintenance and facilitate CSC differentiation, respectively, thereby causing sustained tumor regression without recurrence.

Vaccinia virus GLV-1h153 is effective in treating and preventing metastatic triple-negative breast cancer.

OBJECTIVE: This study aimed to investigate the therapeutic impact of a new oncolytic vaccinia virus in a triple-negative breast cancer (TNBC) murine model and its potential for treating distant metastatic disease. BACKGROUND: TNBCs are aggressive tumors associated with a high metastatic rate. Their lack of targets for hormonal/biological therapy presents significant clinical challenges and a dire need for novel therapies. METHODS: GLV-1h153, a replication-competent vaccinia virus, was tested against multiple cell lines. Cytotoxicity and viral replication were determined. Intratumoral (IT) or intravenous (IV) injection of GLV-1h153 (1 × 10(7) plaque-forming units) or phosphate buffered saline was tested in an orthotopic murine model, which reliably produces systemic metastasis. Tumors, lymph nodes, and metastatic organs (lung, liver, and brain) were harvested 5 and 8 weeks after treatment and prepared for histopathological review. Demonstration of metastasis was performed using immunofluorescence and hematoxylin and eosin (H&E) staining. RESULTS: GLV-1h153 infected, replicated in, and killed all TNBC cell lines in vitro. In vivo, mean tumor volume 2 weeks after treatment was 22 (IT), 29 (IV) versus 245 mm(3) (control; P < 0.002). Five weeks after treatment, all harvested lymph nodes and organs showed no evidence of metastatic cells. All harvested tumors showed complete response to treatment, with only necrosis and fibrosis on H&E staining 8 weeks after treatment. CONCLUSIONS: This is the first study to demonstrate that TNBCs are killed by a novel vaccinia virus both in vitro and in vivo. Our results suggest that GLV-1h153 is a promising therapeutic agent for preventing and treating metastatic TNBC and warrants further clinical testing in patients.

Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells.

BACKGROUND: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. METHODS: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44(+)CD24(+)ESA(+) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. RESULTS: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44(+)CD24(+)ESA(+) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44(+)CD24(-)ESA(+) cells. CONCLUSIONS: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.

Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy.

BACKGROUND: Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic. METHODS: Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects. RESULTS: We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia. CONCLUSION: Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects.

A novel genetically modified oncolytic vaccinia virus in experimental models is effective against a wide range of human cancers.

BACKGROUND: Replication-competent oncolytic viruses have shown great promise as a potential cancer treatment. This study aimed to determine whether a novel vaccinia virus, GLV-1h151, with genetic modifications enhancing cancer specificity and enabling virus detection, is effective against a range of human cancers and is safe when administered in preclinical models. METHODS: GLV-1h151 was modified with deletion of thymidine kinase enhancing specificity and insertion of the green fluorescent protein (GFP) gene. The virus was tested in several human cancer cell lines for cytotoxicity including breast, lung, pancreatic, and colorectal. Virus replication was assessed via visualization of GFP expression and bioluminescence, and viral plaque assays. Finally, GLV-1h151 was administered systemically or intratumorally in mice with pancreatic cancer xenografts (PANC-1) to assess virus biodistribution, toxicity, and effect on tumor growth. RESULTS: GLV-1h151 effectively infected, replicated in, and killed several cancer cell types. Detection and visualization of virus replication was successful via fluorescence imaging of GFP expression, which was dose dependent. When administered intravenously or intratumorally in vivo, GLV-1h151 regressed tumor growth (P < 0.001) and displayed a good biosafety profile. GLV-1h151 infection and replication in tumors was successfully visualized via GFP and bioluminescence, with virus presence in tumors confirmed histologically. CONCLUSIONS: GLV-1h151 is effective as an oncolytic agent against a wide range of cancers in cell culture and is effective against pancreatic human xenografts displaying a good biosafety profile and ability to be detected via optical imaging. GLV-1h151 thus adds another potential medium for the killing of cancer and detection of virus in infected tissue.

Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors.

BACKGROUND: Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. METHODS: For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. RESULTS: GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. CONCLUSIONS: Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.

Effective oncolytic vaccinia therapy for human sarcomas.

BACKGROUND: Approximately one fourth of bone and soft-tissue sarcomas recur after prior treatment. GLV-1h68 is a recombinant, replication-competent vaccinia virus that has been shown to have oncolytic effects against many human cancer types. We sought to determine whether GLV-1h68 could selectively target and lyse a panel of human bone and soft-tissue sarcoma cell lines in vitro and in vivo. METHODS: GLV-1h68 was tested in a panel of four cell lines including: fibrosarcoma HT-1080, osteosarcoma U-2OS, fibrohistiocytoma M-805, and rhabdomyosarcoma HTB-82. Gene expression, infectivity, viral proliferation, and cytotoxicity were characterized in vitro. HT-1080 xenograft flank tumors grown in vivo were injected intratumorally with a single dose of GLV-1h68. RESULTS: All four cell lines supported robust viral transgene expression in vitro. At a multiplicity of infection (MOI) of five, GLV-1h68 was cytotoxic to three cell lines, resulting in >80% cytotoxicity over 7 d. In vivo, a single injection of GLV-1h68 into HT-1080 xenografts exhibited localized intratumoral luciferase activity peaking at d 2-4, with gradual resolution over 8 d and no evidence of spread to normal tissues. Treated animals exhibited near-complete tumor regression over a 28-d period without observed toxicity. CONCLUSION: GLV-1h68 has potent direct oncolytic effects against human sarcoma in vitro and in vivo. Recombinant vaccinia oncolytic virotherapy could provide a new platform for the treatment of patients with bone and soft tissue sarcomas. Future clinical trials investigating oncolytic vaccinia as a therapy for sarcomas are warranted.

Anti-VEGF single-chain antibody GLAF-1 encoded by oncolytic vaccinia virus significantly enhances antitumor therapy.

We previously reported that the replication-competent vaccinia virus (VACV) GLV-1h68 shows remarkable oncolytic activity and efficacy in different animal models as a single treatment modality and also in combination with chemotherapy [Yu YA, et al. (2009) Mol Cancer Ther 8:141-151]. Here, we report the construction of 3 VACV strains encoding GLAF-1, a previously undescribed engineered single-chain antibody (scAb). This unique scAb is transcribed from 3 vaccinia promoters (synthetic early, early/late, and late) and directed against both human and murine VEGFs. The expression of GLAF-1 was demonstrated in cell cultures. Also, the replication efficiency of all GLAF-1-expressing VACV strains in cell culture was similar to that of the parental GLV-1h68 virus. Successful tumor-specific delivery and continued production of functional scAb derived from individual VACV strains were obtained in tumor xenografts following a single intravenous injection of the virus. The VACV strains expressing the scAb exhibited significantly enhanced therapeutic efficacy in comparison to treatment of human tumor xenografts with the parental virus GLV-1h68. This enhanced efficacy was comparable to the concomitant treatment of tumors with a one-time i.v. injection of GLV-1h68 and multiple i.p. injections of Avastin. Taken together, the VACV-mediated delivery and production of immunotherapeutic anti-VEGF scAb in colonized tumors may open the way for a unique therapy concept: tumor-specific, locally amplified drug therapy in humans.

Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.