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.

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.

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.

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.

Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice.

BACKGROUND: We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, ß-galactosidase, and ß-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV-1h68. This strain shows tumor-specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in mice METHODS: A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV-1h68 with short non-coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts. RESULTS: we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice. CONCLUSIONS: These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the purpose of insertional mutagenesis did modulate the efficiency of virus replication together with antitumor efficacy as well as virulence. Replication efficiency of oncolytic VACV in cell cultures can predict the virulence and therapeutic efficacy in nude mice. These findings may be essential for rational design of safe and potent VACV strains for vaccination and virotherapy of cancer in humans and animals.

Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus.

INTRODUCTION: Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153. METHODS: GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free 124I radiotracer. RESULTS: GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via 124I-PET. CONCLUSION: Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy.

Viral-mediated oncolysis is the most critical factor in the late-phase of the tumor regression process upon vaccinia virus infection.

BACKGROUND: In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms--e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV). METHODS: Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII+-cell depletion) in nude mice. RESULTS: Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII+/CD31+ vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII+-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis. CONCLUSIONS: Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.

Regression of human prostate tumors and metastases in nude mice following treatment with the recombinant oncolytic vaccinia virus GLV-1h68.

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In the current study, we analyzed the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 against two human prostate cancer cell lines DU-145 and PC-3 in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 was able to infect, replicate in, and lyse these prostate cancer cells in culture. In DU-145 and PC-3 tumor xenograft models, a single intravenous injection with GLV-1h68 resulted in a significant reduction of primary tumor size. In addition, the GLV-1h68-infection led to strong inflammatory and oncolytic effects resulting in drastic reduction of regional lymph nodes with PC-3 metastases. Our data documented that the GLV-1h68 virus has a great potential for treatment of human prostate carcinoma.

Imaging a Genetically Engineered Oncolytic Vaccinia Virus (GLV-1h99) Using a Human Norepinephrine Transporter Reporter Gene.

PURPOSE: Oncolytic viral therapy continues to be investigated for the treatment of cancer, and future studies in patients would benefit greatly from a noninvasive modality for assessing virus dissemination, targeting, and persistence. The purpose of this study was to determine if a genetically modified vaccinia virus, GLV-1h99, containing a human norepinephrine transporter (hNET) reporter gene, could be sequentially monitored by [(123)I]metaiodobenzylguanidine (MIBG) gamma-camera and [(124)I]MIBG positron emission tomography (PET) imaging. EXPERIMENTAL DESIGN: GLV-1h99 was tested in human malignant mesothelioma and pancreatic cancer cell lines for cytotoxicity, expression of the hNET protein using immunoblot analysis, and [(123)I]MIBG uptake in cell culture assays. In vivo [(123)I]MIBG gamma-camera and serial [(124)I]MIBG PET imaging was done in MSTO-211H orthotopic pleural mesothelioma tumors. RESULTS: GLV-1h99 successfully infected and provided dose-dependent levels of transgene hNET expression in human malignant mesothelioma and pancreatic cancer cells. The time course of [(123)I]MIBG accumulation showed a peak of radiotracer uptake at 48 hours after virus infection in vitro. In vivo hNET expression in MSTO-211H pleural tumors could be imaged by [(123)I]MIBG scintigraphy and [(124)I]MIBG PET 48 and 72 hours after GLV-1h99 virus administration. Histologic analysis confirmed the presence of GLV-1h99 in tumors. CONCLUSION: GLV-1h99 shows high mesothelioma tumor cell infectivity and cytotoxic efficacy. The feasibility of imaging virus-targeted tumor using the hNET reporter system with [(123)I]MIBG gamma-camera and [(124)I]MIBG PET was shown in an orthotopic pleural mesothelioma tumor model. The inclusion of human reporter genes into recombinant oncolytic viruses enhances the potential for translation to clinical monitoring of oncolytic viral therapy.

Regression of human pancreatic tumor xenografts in mice after a single systemic injection of recombinant vaccinia virus GLV-1h68.

Oncolytic virotherapy of tumors has shown promising results in both preclinical and clinical studies. Here, we investigated the therapeutic efficacy of a replication-competent vaccinia virus, GLV-1h68, against human pancreatic carcinomas in cell cultures and in nude mice. We found that GLV-1h68 was able to infect, replicate in, and lyse tumor cells in vitro. Virus-mediated marker gene expressions were readily detected. Moreover, s.c. PANC-1 pancreatic tumor xenografts were effectively treated by a single i.v. dose of GLV-1h68. Cancer killing was achieved with minimal toxicity. Viral titer analyses in homogenized organs and PANC-1 tumors showed that the mutant virus resides almost exclusively in the tumors and not in healthy organs. Except mild spleen enlargements, no histopathology changes were observed in any other organs 2 months after virus injection. Surprisingly, s.c. MIA PaCa-2 pancreatic tumors were treated with similar efficiency as PANC-1 tumors, although they differ significantly in sensitivity to viral lysis in cell cultures. When GLV-1h68 oncolytic viral therapy was used together with cisplatin or gemcitabine to treat PANC-1 tumors, the combination therapy resulted in enhanced and accelerated therapeutic results compared with the virus treatment alone. Profiling of proteins related to immune response revealed a significant proinflammatory immune response and marked activation of innate immunity in virus-colonized tumors. In conclusion, the GLV-1h68 strain showed outstanding therapeutic effects and a documented safety profile in mice, with great promise for future clinical development.

Systemic treatment of xenografts with vaccinia virus GLV-1h68 reveals the immunologic facet of oncolytic therapy.

BACKGROUND: GLV-1h68 is an attenuated recombinant vaccinia virus (VACV) that selectively colonizes established human xenografts inducing their complete regression. RESULTS: Here, we explored xenograft/VACV/host interactions in vivo adopting organism-specific expression arrays and tumor cell/VACV in vitro comparing VACV replication patterns. There were no clear-cut differences in vitro among responding and non-responding tumors, however, tumor rejection was associated in vivo with activation of interferon-stimulated genes (ISGs) and innate immune host's effector functions (IEFs) correlating with VACV colonization of the xenografts. These signatures precisely reproduce those observed in humans during immune-mediated tissue-specific destruction (TSD) that causes tumor or allograft rejection, autoimmunity or clearance of pathogens. We recently defined these common pathways in the "immunologic constant of rejection" hypothesis (ICR). CONCLUSION: This study provides the first prospective validation of a universal mechanism associated with TSD. Thus, xenograft infection by oncolytic VACV, beyond offering a promising therapy of established cancers, may represent a reliable pre-clinical model to test therapeutic strategies aimed at modulating the central pathways leading to TSD; this information may lead to the identification of principles that could refine the treatment of cancer and chronic infection by immune stimulation or autoimmunity and allograft rejection through immune tolerance.

Oncolytic vaccinia therapy of squamous cell carcinoma.

BACKGROUND: Novel therapies are necessary to improve outcomes for patients with squamous cell carcinomas (SCC) of the head and neck. Historically, vaccinia virus was administered widely to humans as a vaccine and led to the eradication of smallpox. We examined the therapeutic effects of an attenuated, replication-competent vaccinia virus (GLV-1h68) as an oncolytic agent against a panel of six human head and neck SCC cell lines. RESULTS: All six cell lines supported viral transgene expression (beta-galactosidase, green fluorescent protein, and luciferase) as early as 6 hours after viral exposure. Efficient transgene expression and viral replication (>150-fold titer increase over 72 hrs) were observed in four of the cell lines. At a multiplicity of infection (MOI) of 1, GLV-1h68 was highly cytotoxic to the four cell lines, resulting in > or = 90% cytotoxicity over 6 days, and the remaining two cell lines exhibited >45% cytotoxicity. Even at a very low MOI of 0.01, three cell lines still demonstrated >60% cell death over 6 days. A single injection of GLV-1h68 (5 x 10(6) pfu) intratumorally into MSKQLL2 xenografts in mice exhibited localized intratumoral luciferase activity peaking at days 2-4, with gradual resolution over 10 days and no evidence of spread to normal organs. Treated animals exhibited near-complete tumor regression over a 24-day period without any observed toxicity, while control animals demonstrated rapid tumor progression. CONCLUSION: These results demonstrate significant oncolytic efficacy by an attenuated vaccinia virus for infecting and lysing head and neck SCC both in vitro and in vivo, and support its continued investigation in future clinical trials.

Real-time intraoperative detection of melanoma lymph node metastases using recombinant vaccinia virus GLV-1h68 in an immunocompetent animal model.

There is a clinical need for improved intraoperative detection of lymph node metastases from malignant melanoma (MM). We aimed to investigate the use of recombinant vaccinia virus GLV-1h68, expressing green fluorescent protein (GFP), for real-time intraoperative detection of melanoma lymph node metastases in an immunocompetent animal model. Mice bearing foot pad tumors received intratumoral injections of GLV-1h68, and 48 hr later were evaluated for popliteal lymph node metastasis using noninvasive bioluminescence imaging and fluorescence imaging. Histologic analysis of lymph nodes was performed to determine sensitivity and specificity of virus-mediated detection. Intratumoral injection of GLV-1h68 into primary foot pad melanoma tumors resulted in viral transmission to popliteal lymph nodes, infection of lymphatic metastases, and transgene expression that was reliably and easily detected. Histologic confirmation demonstrated favorable operating characteristics of this assay (sensitivity 80%, specificity 100%, positive predictive value [PPV] 100%, negative predictive value [NPV] 91%). Detection of marker gene expression by GLV-1h68 allowed the detection of lymphatic metastases in an immunocompetent animal model of MM. This assay is rapid, sensitive, specific and easy to perform and interpret. As a candidate gene therapy virus for killing cancer, GLV-1h68 may also have significant concomitant diagnostic utility in the staging of cancer patients.

A novel recombinant vaccinia virus expressing the human norepinephrine transporter retains oncolytic potential and facilitates deep-tissue imaging.

Noninvasive and repetitive monitoring of a virus in target tissues and/or specific organs of the body is highly desirable for the development of safe and efficient cancer virotherapeutics. We have previously shown that the oncolytic vaccinia virus GLV-1h68 can target and eradicate human tumors in mice and that its therapeutic effects can be monitored by using optical imaging. Here, we report on the development of a derivative of GLV-1h68, a novel recombinant vaccinia virus (VACV) GLV-1h99, which was constructed to carry the human norepinephrine transporter gene (hNET) under the VACV synthetic early promoter placed at the F14.5L locus for deep-tissue imaging. The hNET protein was expressed at high levels on the membranes of cells infected with this virus. Expression of the hNET protein did not negatively affect virus replication, cytolytic activity in cell culture, or in vivo virotherpeutic efficacy. GLV-1h99-mediated expression of the hNET protein in infected cells resulted in specific uptake of the radiotracer [131I]-meta-iodobenzylguanidine (MIBG). In mice, GLV-1h99-infected tumors were readily imaged by [124I]-MIBG positron emission tomography. To our knowledge, GLV-1h99 is the first oncolytic virus expressing the hNET protein that can efficiently eliminate tumors and simultaneously allow deep-tissue imaging of infected tumors.

The highly attenuated oncolytic recombinant vaccinia virus GLV-1h68: comparative genomic features and the contribution of F14.5L inactivation.

As a new anticancer treatment option, vaccinia virus (VACV) has shown remarkable antitumor activities (oncolysis) in preclinical studies, but potential infection of other organs remains a safety concern. We present here genome comparisons between the de novo sequence of GLV-1h68, a recombinant VACV, and other VACVs. The identified differences in open reading frames (ORFs) include genes encoding host-range selection, virulence and immune modulation proteins, e.g., ankyrin-like proteins, serine proteinase inhibitor SPI-2/CrmA, tumor necrosis factor (TNF) receptor homolog CrmC, semaphorin-like and interleukin-1 receptor homolog proteins. Phylogenetic analyses indicate that GLV-1h68 is closest to Lister strains but has lost several ORFs present in its parental LIVP strain, including genes encoding CrmE and a viral Golgi anti-apoptotic protein, v-GAAP. The reduced pathogenicity of GLV-1h68 is confirmed in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma. The contribution of foreign gene expression cassettes in the F14.5L, J2R and A56R loci is analyzed, in particular the contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence of GLV-1h68 with its F14.5L-null and revertant viruses. GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism.

Eradication of solid human breast tumors in nude mice with an intravenously injected light-emitting oncolytic vaccinia virus.

Previously, we reported that a recombinant vaccinia virus (VACV) carrying a light-emitting fusion gene enters, replicates in, and reveals the locations of tumors in mice. A new recombinant VACV, GLV-1h68, as a simultaneous diagnostic and therapeutic agent, was constructed by inserting three expression cassettes (encoding Renilla luciferase-Aequorea green fluorescent protein fusion, beta-galactosidase, and beta-glucuronidase) into the F14.5L, J2R (encoding thymidine kinase) and A56R (encoding hemagglutinin) loci of the viral genome, respectively. I.v. injections of GLV-1h68 (1x10(7) plaque-forming unit per mouse) into nude mice with established (approximately 300-500 mm3) s.c. GI-101A human breast tumors were used to evaluate its toxicity, tumor targeting specificity, and oncolytic efficacy. GLV-1h68 showed an enhanced tumor targeting specificity and much reduced toxicity compared with its parental LIVP strains. The tumors colonized by GLV-1h68 exhibited growth, inhibition, and regression phases followed by tumor eradication within 130 days in 95% of the mice tested. Tumor regression in live animals was monitored in real time based on decreasing light emission, hence demonstrating the concept of a combined oncolytic virus-mediated tumor diagnosis and therapy system. Transcriptional profiling of regressing tumors based on a mouse-specific platform revealed gene expression signatures consistent with immune defense activation, inclusive of IFN-stimulated genes (STAT-1 and IRF-7), cytokines, chemokines, and innate immune effector function. These findings suggest that immune activation may combine with viral oncolysis to induce tumor eradication in this model, providing a novel perspective for the design of oncolytic viral therapies for human cancers.

Oncolytic vaccinia virotherapy of anaplastic thyroid cancer in vivo.

CONTEXT: Anaplastic thyroid carcinoma (ATC) is a fatal disease with a median survival of only 6 months. Novel therapies are needed to improve dismal outcomes. OBJECTIVE: A mutated, replication-competent, vaccinia virus (GLV-1h68) has oncolytic effects on human ATC cell lines in vitro. We assessed the utility of GLV-1h68 in treating anaplastic thyroid cancer in vivo. DESIGN: Athymic nude mice with xenograft flank tumors of human ATCs (8505C and DRO90-1) were treated with a single intratumoral injection of GLV-1h68 at low dose (5x10(5) plaque-forming unit), high dose (5x10(6) plaque-forming unit), or PBS. Virus-mediated marker gene expression (luciferase, green fluorescent protein, and beta-galactosidase), viral biodistribution, and flank tumor volumes were measured. RESULTS: Luciferase expression was detected 2 d after injection. Continuous viral replication within tumors was reflected by increasing luciferase activity to d 9. At d 10, tumor viral recovery was increased more than 50-fold as compared with the injected dose, and minimal virus was recovered from the lung, liver, brain, heart, spleen, and kidneys. High-dose virus directly injected into normal tissues was undetectable at d 10. The mean volume of control 8505C tumors increased 50.8-fold by d 45, in contrast to 10.5-fold (low dose) and 2.1-fold (high dose; P=0.028) increases for treated tumors. DRO90-1 tumors also showed significant growth inhibition by high-dose virus. No virus-related toxicity was observed throughout the study. CONCLUSIONS: GLV-1h68 efficiently infects, expresses transgenes within, and inhibits the growth of ATC in vivo. These promising findings support future clinical trials for patients with ATC.

Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria.

Intravenously injected bacteria have the ability to enter and replicate within tumors in mice. Here, we further characterized this dynamic process by investigating the conditions required for tumor colonization by bacteria. We discovered that a broad range of bacteria, including both Gram-negative and Gram-positive strains, colonize a panel of syngeneic and xenogeneic tumors, as well as spontaneous tumors in mice. The colonization process is dependent on the stage of tumor development, but independent of tumor type. The entry and replication of bacteria in tumors was consistently achieved in nude mice when approximately 1 x 10(5) bacteria were injected. The majority of the bacteria were found in the central portion of the tumors, coinciding with the necrotic regions of sectioned tumors. We also found that, although cancer can be characterized as a chronic inflammation, inflammation alone, as seen in cutaneous wounds and in sites artificially induced by Sephadex, was not sufficient to sustain bacterial colonization. Furthermore, we found that "sensitizing" the tumors by oncolytic vaccinia virus colonization prior to bacterial delivery may help to enhance tumor colonization at least twofold by bacteria that were delivered subsequently. Taken together, the data from this and previous studies, lead us to propose the concept that blood-borne bacteria enter tumors to amplify in the central necrotic region; and that, due to impaired immunosurveillance in the tumors as known previously, the clearance of bacteria is inhibited. Bacterial colonization of tumors could be achieved without any specific gene modifications either in bacteria or in the host.