Novel Chimeric Poxvirus CF17 Improves Survival in a Murine Model of Intraperitoneal Ovarian Cancer Metastasis.

Despite improvements in surgical techniques and chemotherapy, ovarian cancer remains the most lethal gynecologic cancer. Thus, there is an urgent need for more effective therapeutics, particularly for chemo-resistant peritoneal ovarian cancer metastases. Oncolytic virotherapy represents an innovative treatment paradigm; however, for oncolytic viruses tested from the last generation of genetically engineered viruses, the therapeutic benefits have been modest. To overcome these limitations, we generated a chimeric poxvirus, CF17, through the chimerization of nine species of orthopoxviruses. Compared with its parental viruses, CF17 has demonstrated superior oncolytic characteristics. Here, we report the oncolytic potential of CF17 in ovarian cancer. Replication of CF17 and its resulting cytotoxicity were observed at multiplicities of infection (MOIs) as low as 0.001 in human and mouse cancer cell lines in vitro. Furthermore, CF17 exerted potent antitumor effects in a syngeneic mouse model of ovarian cancer at doses as low as 6 × 106 plaque-forming units. Together, these data merit further investigation of the potential use of this novel chimeric poxvirus as an effective treatment for aggressive intraperitoneal ovarian cancer.

Effective combination immunotherapy using oncolytic viruses to deliver CAR targets to solid tumors.

Chimeric antigen receptor (CAR)-engineered T cell therapy for solid tumors is limited by the lack of both tumor-restricted and homogeneously expressed tumor antigens. Therefore, we engineered an oncolytic virus to express a nonsignaling, truncated CD19 (CD19t) protein for tumor-selective delivery, enabling targeting by CD19-CAR T cells. Infecting tumor cells with an oncolytic vaccinia virus coding for CD19t (OV19t) produced de novo CD19 at the cell surface before virus-mediated tumor lysis. Cocultured CD19-CAR T cells secreted cytokines and exhibited potent cytolytic activity against infected tumors. Using several mouse tumor models, delivery of OV19t promoted tumor control after CD19-CAR T cell administration. OV19t induced local immunity characterized by tumor infiltration of endogenous and adoptively transferred T cells. CAR T cell-mediated tumor killing also induced release of virus from dying tumor cells, which propagated tumor expression of CD19t. Our study features a combination immunotherapy approach using oncolytic viruses to promote de novo CAR T cell targeting of solid tumors.

Neural Stem Cells Improve the Delivery of Oncolytic Chimeric Orthopoxvirus in a Metastatic Ovarian Cancer Model.

Oncolytic virotherapy represents a promising approach for treating recurrent and/or drug-resistant ovarian cancer. However, its successful application in the clinic has been hampered by rapid immune-mediated clearance, which reduces viral delivery to the tumor. Patient-derived mesenchymal stem cells that home to tumors have been used as viral delivery tools, but variability associated with autologous cell isolations limits the clinical applicability of this approach. We previously developed an allogeneic, clonal neural stem cell (NSC) line (HB1.F3.CD21) that can be used to deliver viral cargo. Here, we demonstrate that this NSC line can improve the delivery of a thymidine kinase gene-deficient conditionally replication-competent orthopoxvirus, CF33, in a preclinical cisplatin-resistant peritoneal ovarian metastases model. Overall, our findings provide the basis for using off-the-shelf allogeneic cell-based delivery platforms for oncolytic viruses, thus providing a more efficient delivery alternative compared with the free virus administration approach.

A chimeric poxvirus with J2R (thymidine kinase) deletion shows safety and anti-tumor activity in lung cancer models.

Oncolytic viruses have shown excellent safety profiles in preclinical and clinical studies; however, in most cases therapeutic benefits have been modest. We have previously reported the generation of a chimeric poxvirus (CF33), with significantly improved oncolytic characteristics, through chimerization among different poxviruses. Here we report the sequence analysis of CF33 and oncolytic potential of a GFP-encoding CF33 virus (CF33-GFP) with a J2R deletion in lung cancer models. Replication of CF33-GFP and the resulting cytotoxicity were higher in cancer cell lines compared to a normal cell line, in vitro. After infection with virus, cancer cells expressed markers for immunogenic cell death in vitro. Furthermore, CF33-GFP was safe and exerted potent anti-tumor effects at a dose as low as 1000 plaque forming units in both virus-injected and un-injected distant tumors in A549 tumor xenograft model in mice. Likewise, in a syngeneic model of lung cancer in mice, the virus showed significant anti-tumor effect and was found to increase tumor infiltration by CD8+ T cells. Collectively, these data warrant further investigation of this novel chimeric poxvirus for its potential use as a cancer bio-therapeutic.

Oncolytic Viruses and Immune Checkpoint Inhibition: The Best of Both Worlds.

Cancer immunotherapy and the emergence of immune checkpoint inhibitors have markedly changed the treatment paradigm for many cancers. They function to disrupt cancer cell evasion of the immune response and activate sustained anti-tumor immunity. Oncolytic viruses have also emerged as an additional therapeutic agent for cancer treatment. These viruses are designed to target and kill tumor cells while leaving the normal cells unharmed. As part of this process, oncolytic virus infection stimulates anti-cancer immune responses that augment the efficacy of checkpoint inhibition. These viruses have the capability of transforming a "cold" tumor microenvironment with few immune effector cells into a "hot" environment with increased immune cell and cytokine infiltration. For this reason, there are multiple ongoing clinical trials that combine oncolytic virotherapy and immune checkpoint inhibitors. This review will detail the key oncolytic viruses in preclinical and clinical studies and highlight the results of their testing with checkpoint inhibitors.

A Novel Chimeric Poxvirus Encoding hNIS Is Tumor-Tropic, Imageable, and Synergistic with Radioiodine to Sustain Colon Cancer Regression.

Colon cancer has a high rate of recurrence even with good response to modern therapies. Novel curative adjuncts are needed. Oncolytic viral therapy has shown preclinical promise against colon cancer but lacks robust efficacy in clinical trials and raises regulatory concerns without real-time tracking of viral replication. Novel potent vectors are needed with adjunctive features to enhance clinical efficacy. We have thus used homologous recombination and high-throughput screening to create a novel chimeric poxvirus encoding a human sodium iodide symporter (hNIS) at a redundant tk locus. The resulting virus (CF33-hNIS) consistently expresses hNIS and demonstrates replication efficiency and immunogenic cell death in colon cancer cells in vitro. Tumor-specific CF33-hNIS efficacy against colon cancer results in tumor regression in vivo in colon cancer xenograft models. Early expression of hNIS by infected cells makes viral replication reliably imageable via positron emission tomography (PET) of I-124 uptake. The intensity of I-124 uptake mirrors viral replication and tumor regression. Finally, systemic delivery of radiotherapeutic I-131 isotope following CF33-hNIS infection of colon cancer xenografts enhances and sustains tumor regression compared with virus treatment alone in HCT116 xenografts, demonstrating synergy of oncolytic viral therapy with radioablation in vivo.

Endogenous Akt Activity Promotes Virus Entry and Predicts Efficacy of Novel Chimeric Orthopoxvirus in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high recurrence rate and poor prognosis. Here, we describe a novel, chimeric orthopoxvirus (CF33) that efficiently kills TNBC. Cytotoxicity was assayed in vitro in four TNBC cell lines. Viral replication was examined through standard plaque assay. Two orthotopic TNBC xenograft models were generated in athymic nude mice and were injected with CF33 intratumorally. CF33 was effective in vitro with potent cytotoxicity and efficient intracellular replication observed in TNBC lines with phosphatidylinositol 3-kinase (PI3K)/Akt pathway mutations that resulted in endogenous phospho-Akt (p-Akt) activity (BT549, Hs578T, and MDA-MB-468). Relative resistance to CF33 by wild-type PI3K/Akt pathway cell line MDA-MB-231 was overcome using higher MOI. The virus was effective in vivo with significant tumor size reduction in both xenograft models. Mechanistically, CF33 appears to share similar properties to vaccinia virus with respect to Akt-mediated and low-pH-mediated viral entry. In summary, CF33 demonstrated potent antitumoral effect in vitro and in vivo, with the most potent effect predicted by the presence of endogenous Akt activity in the TNBC cell line. Further investigation of its mechanism of action as well as genetic modifications to enhance its natural viral tropism are warranted for preclinical development.

A Novel Oncolytic Chimeric Orthopoxvirus Encoding Luciferase Enables Real-Time View of Colorectal Cancer Cell Infection.

This study hypothesizes that a novel oncolytic chimeric orthopoxvirus CF33-Fluc is imageable and targets colorectal cancer cells (CRCs). A novel chimeric orthopoxvirus (CF33) was constructed. The thymidine kinase locus was replaced with firefly luciferase (Fluc) to yield a recombinant virus-CF33-Fluc. In vitro cytotoxicity and viral replication assays were performed. In vivo CRC flank xenografts received single doses of intratumoral or intravenous CF33-Fluc. Viral biodistribution was analyzed via luciferase imaging and organ titers. CF33-Fluc infects, replicates in, and kills CRCs in vitro in a dose-dependent manner. CF33 has superior secretion of extracellular-enveloped virus versus all but one parental strain. Rapid tumor regression or stabilization occurred in vivo at a low dose over a short time period, regardless of the viral delivery method in the HCT-116 colorectal tumor xenograft model. Rapid luciferase expression in virus-infected tumor cells was associated with treatment response. CRC death occurs via necroptotic pathways. CF33-Fluc replicates in and kills colorectal cancer cells in vitro and in vivo regardless of delivery method. Expression of luciferase enables real-time tracking of viral replication. Despite the chimerism, CRC death occurs via standard poxvirus-induced mechanisms. Further studies are warranted in immunocompetent models.

Oncolytic Virotherapy versus Cancer Stem Cells: A Review of Approaches and Mechanisms.

A growing body of evidence suggests that a subset of cells within tumors are resistant to conventional treatment modalities and may be responsible for disease recurrence. These cells are called cancer stem cells (CSC), which share properties with normal stem cells including self-renewal, pluripotency, drug resistance, and the ability to maintain quiescence. While most conventional therapies can efficiently destroy rapidly dividing cancer cells comprising the bulk of a tumor, they often fail to kill the less abundant and quiescent CSCs. Furthermore, killing of only differentiated cells in the tumor may actually allow for enrichment of CSCs and thereby portend a bad prognosis. Therefore, targeting of CSCs is important to achieve long-term success in cancer therapy. Oncolytic viruses represent a completely different class of therapeutics that can kill cancer cells in a variety of ways, which differ from those of conventional therapies. Hence, CSCs that are inherently resistant to conventional therapies may be susceptible to oncolytic virus-mediated killing. Recent studies have shown that oncolytic viruses can efficiently kill CSCs in many types of cancer. Here, we discuss the mechanism through which CSCs can escape conventional therapies and how they may still be susceptible to different classes of oncolytic viruses. Furthermore, we provide a summary of recent studies that have tested oncolytic viruses on CSCs of different origins and discuss possible future directions for this fascinating subset of oncolytic virus research.

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.

Oncolytic viruses and immunity.

Initially, direct oncolysis was thought to be the sole mechanism through which oncolytic viruses (OVs) exert their anti-tumor effect, and the immune system was perceived as the major obstacle in oncolytic virotherapy. Over the last decade, there has been a lot of debate on whether the immune system is a friend or foe of OVs. However, we are now at a stage where the initial thinking has been reversed as a result of compelling evidence that the immune system plays a critical role in the success of oncolytic virotherapy. In this review we discuss the importance of the involvement of innate and adaptive immunity for therapeutic efficacy of OVs, and the rational combination of OVs with other immunotherapies for further enhancement of overall therapeutic outcome.

Novel chimeric parapoxvirus CF189 as an oncolytic immunotherapy in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer is an aggressive subtype of breast cancer with high recurrence rate and poor prognosis. Here we describe a novel, genetically engineered parapoxvirus that efficiently kills triple-negative breast cancer. METHODS: A novel chimeric parapoxvirus (CF189) was generated via homologous recombination and identified through high-throughput screening. Cytotoxicity was assayed in vitro in 4 triple-negative breast cancer cell lines. Viral replication was examined through standard plaque assay. Orthotopic triple-negative breast cancer xenografts were generated by MDA-MB-468 implantation into the 2nd and 4th mammary fat pads of athymic nude mice and treated with the virus. RESULTS: Chimeric parapoxvirus (CF189) demonstrated dose-dependent cytotoxicity at low multiplicity of infection, with > 80% cell death 6 days after treatment. Significant reductions in tumor size were observed 2 weeks after intratumoral injection at doses as low as 103 plaque-forming units (PFU) compared with control (P < 0.01). In addition, abscopal effect (shrinkage of noninjected remote tumors) was clearly demonstrated. CONCLUSION: Chimeric parapoxvirus (CF189) demonstrated efficient cytotoxicity in vitro and potent antitumor effect in vivo at doses as low as 103 PFU. These are data encouraging of clinical development for this highly potent agent against triple-negative breast cancer.

Molecular network, pathway, and functional analysis of time-dependent gene changes associated with pancreatic cancer susceptibility to oncolytic vaccinia virotherapy.

BACKGROUND: Pancreatic cancer is a fatal disease associated with resistance to conventional therapies. This study aimed to determine changes in gene expression patterns associated with infection and susceptibility of pancreatic cancer cells to an oncolyticvaccinia virus, GLV-1h153, carrying the human sodium iodide symporter for deep tissue imaging of virotherapy. METHODS: Replication and susceptibility of pancreatic adenocarcinoma PANC-1 cells to GLV-1h153 was confirmed with replication and cytotoxicity assays. PANC-1 cells were then infected with GLV-1h153 and near-synchronous infection confirmed via flow cytometry of viral-induced green fluorescent protein (GFP) expression. Six and 24 hours after infection, three samples of each time point were harvested, and gene expression patterns assessed using HG-U133A cDNA microarray chips as compared to uninfected control. Differentially expressed genes were identified using Bioconductor LIMMA statistical analysis package. A fold change of 2.0 or above was used as a cutoff, with a P value of 0.01. The gene list was then analyzed using Ingenuity Pathways Analysis software. RESULTS: Differential gene analysis revealed a total of 12,412 up- and 11,065 downregulated genes at 6 and 24 hours postinfection with GLV-1h153 as compared to control. At 6 hours postinfection. A total of 139 genes were either up or downregulated >twofold (false discovery rate < 0.05), of which 124 were mapped by Ingenuity Pathway Analysis (IPA). By 24 hours postinfection, a total of 5,698 genes were identified and 5,563 mapped by IPA. Microarray revealed gene expression changes, with gene networks demonstrating downregulation of processes such as cell death, cell cycle, and DNA repair, and upregulation of infection mechanisms (P < 0.01). Six hours after infection, gene changes involved pathways such as HMGB-1, interleukin (IL)-2, IL-6, IL-8, janus kinase/signal tranducer and activator of transcription (JAK/STAT), interferon, and ERK 5 signaling (P < 0.01). By 24 hours, prominent pathways included P53- and Myc-induced apoptotic processes, pancreatic adenocarcinoma signaling, and phosphoinositide 3-kinase/v-akt murine thymoma vial oncogene homolog 1 (PI3/AKT) pathways. CONCLUSIONS: Our study reveals the ability to assess time-dependent changes in gene expression patterns in pancreatic cancer cells associated with infection and susceptibility to vaccinia viruses. This suggests that molecular assays may be useful to develop safer and more efficacious oncolyticvirotherapies and support the idea that these treatments may target pathways implicated in pancreatic cancer resistance to conventional therapies.

From Benchtop to Bedside: A Review of Oncolytic Virotherapy.

Oncolytic viruses (OVs) demonstrate the ability to replicate selectively in cancer cells, resulting in antitumor effects by a variety of mechanisms, including direct cell lysis and indirect cell death through immune-mediate host responses. Although the mechanisms of action of OVs are still not fully understood, major advances have been made in our understanding of how OVs function and interact with the host immune system, resulting in the recent FDA approval of the first OV for cancer therapy in the USA. This review provides an overview of the history of OVs, their selectivity for cancer cells, and their multifaceted mechanism of antitumor action, as well as strategies employed to augment selectivity and efficacy of OVs. OVs in combination with standard cancer therapies are also discussed, as well as a review of ongoing human clinical trials.

Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus.

BACKGROUND: Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. METHODS: Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. RESULTS: GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral ß-galactosidase expression as compared to PBS. CONCLUSION: The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes.

Recombinant vaccinia virus GLV-1h68 is a promising oncolytic vector in the treatment of cholangiocarcinoma.

Although early stage cholangiocarcinoma (CC) can be cured by surgical extirpation, the options for treatment of advanced stage CC are very few and suboptimal. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) is a promising new strategy to treat human cancers. The ability of oncolytic VACV GLV-1h68 to infect, replicate in, and lyse three human CC cell lines was assayed in vitro and in subcutaneous flank xenografts in athymic nude mice. In this study, we have demonstrated that GLV-1h68 effectively infects and lyses three CC cell lines (KMC-1, KMBC, and KMCH-1) in vitro. Expression of the viral marker gene ruc-gfp facilitated real-time monitoring of infection and replication. Furthermore in athymic nude mice, a single dose of GLV-1h68 significantly suppressed tumor growth. The treatment was well tolerated in all animals. Recombinant VACV GLV-1h68 has significant oncolytic ability against CC both in vitro and in vivo. GLV-1h68 has the potential to be used clinically as a therapeutic agent against CC.

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.

Oncolytic gene therapy with recombinant vaccinia strain GLV-2b372 efficiently kills hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) commonly presents at a late stage when surgery is no longer a curative option. As such, novel therapies for advanced HCC are needed. Oncolytic viruses are a viable option for cancer therapy owing to their ability to specifically infect, replicate within, and kill cancer cells. In this study, we have investigated the ability of GLV-2b372, a novel light-emitting recombinant vaccinia virus derived from a wild-type Lister strain, to kill HCC. METHODS: Four human HCC cell lines were assayed in vitro for infectivity and cytotoxicity. Viral replication was quantified via standard viral plaque assays. Flank HCC xenografts generated in athymic nude mice were treated with intratumoral GLV-2b372 to assess for tumor growth inhibition and viral biodistribution. RESULTS: Infectivity occurred in a time- and concentration-dependent manner with 70% cell death in all cell lines by day 5. All cell lines supported efficient viral replication. At 25 days after infection, flank tumor volumes decreased by 50% whereas controls increased by 400%. Tumor tissue demonstrated substantial GLV-2b372 infection at 24 hours, 48 hours, and 2 weeks. CONCLUSION: We demonstrate that GLV-2b372 efficiently kills human HCC in vitro and in vivo and is a viable treatment option for patients with HCC.