Potent Oncolytic Virus Research Articles

PEX5-mediated modulation of apoptotic pathways in response to Newcastle disease virus infection1

Newcastle disease virus (NDV) is a highly lethal and contagious viral pathogen; it is also a potent oncolytic virus that selectively replicates in tumor cells. NDV demonstrates high replication efficiency in avian and tumor cells, causing various types of cell death, including ferroptosis, necrosis, apoptosis and autophagic cell death, with apoptosis being the most thoroughly studied. Organelles play critical and distinctive roles in the regulation and execution of apoptosis. However, the involvement of peroxisomes, an important organelle that regulates redox balance and lipid biosynthesis, in virus-induced apoptosis remains unclear. Our findings reveal that NDV infection promotes the downregulation of several peroxisome biogenesis factors (PEXs) at the mRNA level. Peroxisomal biogenesis factor 5 (PEX5), a critical peroxisomal shuttle protein, was identified to be significantly downregulated at both the mRNA and protein levels. Further, gain- and loss-of-function experiments demonstrated the negative regulation of NDV-induced apoptosis by PEX5. In addition, PEX5 inhibits NDV-induced apoptosis by regulating the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) expression. These findings reveal a novel mechanism by which NDV-induced apoptosis is modulated through the downregulation of PEXs, particularly PEX5, shedding light on the potential role of peroxisome in apoptosis regulation in response to virus infection.

Therapeutic Efficacy of a VSV-GP-based Human Papilloma Virus Vaccine in a Murine Cancer Model

Human papilloma virus (HPV) infections are associated with almost all cervical cancers and to a lower extend also with anogenital or oropharyngeal cancers. HPV proteins expressed in HPV-associated tumors are attractive antigens for cancer vaccination strategies as self-tolerance, which is associated with most endogenous tumor-associated antigens, does not need to be overcome. In this study, we generated a live attenuated cancer vaccine based on the chimeric vesicular stomatitis virus VSV-GP, which has previously proven to be a potent vaccine vector and oncolytic virus. Genes at an earlier position in the genome more to the 3′ end are expressed stronger compared to genes located further downstream. By inserting an HPV16-derived antigen cassette consisting of E2, E6 and E7 into VSV-GP either at first (HPVp1) or fifth (HPVp5) position in VSV-GP’s genome we aimed to analyze the effect of vaccine antigen position and consequently expression level on viral fitness, immunogenicity, and anti-tumoral efficacy in a syngeneic mouse tumor model. HPVp1 expressed higher amounts of HPV antigens compared to HPVp5 in vitro but had a slightly delayed replication kinetic which overall translated into increased HPV-specific T cell responses upon vaccination of mice. Immunization with both vectors protected mice in prophylactic and in therapeutic TC-1 tumor models with HPVp1 being more effective in the prophylactic setting. Taken together, VSV-GP is a promising candidate as therapeutic HPV vaccine and first position of the vaccine antigen in a VSV-derived vector seems to be superior to fifth position.

Abstract 6218: Oncolytic Seneca Valley virus (SVV-001) overcomes checkpoint inhibitor resistance and demonstrates a systemic anti-tumor immune response in a syngeneic pancreatic cancer murine model

Abstract Here we evaluated the efficacy of Seneca Valley Virus (SVV), an oncolytic virus in the Picornaviridae family, in combination with checkpoint inhibitors (CPI) using the CPI-resistant murine syngeneic pancreatic tumor model Pan02. SVV, serendipitously identified in 2001, is an extremely tumor selective and potent oncolytic virus for a variety of solid human cancers, primarily due to the expression of the receptor of SVV, TEM8, being selectively expressed on the surface of malignant cancer cells and cancer stem cells within a variety of solid tumors. SVV has been tested in three clinical trials of patients having neuroendocrine neoplasms as a single intravenous dose monotherapy. Despite this, the results were encouraging with patients having evidence of clinical benefit and only one DLT was reported in the 76 patients treated. Recent data have demonstrated that some oncolytic viruses can enhance efficacy in preclinical models and in clinical trials when the oncolytic virus was injected intratumorally in combination with systemic administration of a CPI(s). The mechanism is primarily believed to be due to the oncolytic virus enhancing T cell infiltration and fostering an anti-tumor immune response. SVV was injected intra-tumorally in Pan02 tumors along with aPD1and/or aCTLA4. SVV reversed any resistance to CPIs and enhanced efficacy over CPI(s) alone resulting in complete cures in >83% of mice with primary and abscopal tumors. SVV plus aPD1+aCTLA4 resulted in 5 of 6 mice cured of their primary tumor. The animals were challenged on their contralateral flank with Pan02 to evaluate systemic and abscopal immune effects. All mice (5/5) that cleared the primary Pan02 tumor also eradicated the abscopal secondary tumors. Control treated Pan02 tumor-bearing mice were all sacrificed due to tumor burden by day 70 (median survival <50 days). Control animals treated with aPD1+aCTLA4 showed transient tumor regressions but these animals all grew large tumors, requiring sacrifice. In contrast, the SVV+aPD1+aCTLA4 primary tumors were eliminated within 44 days and these animals remain tumor-free out to >110+ days. Contralateral tumors were eradicated in all 5 animals that rejected the primary tumor, demonstrating potent systemic immunity. In the control aPD1+aCTLA4 group, only one animal with contralateral tumor showed prolonged tumor regression. SVV treatment caused increases in T-cell infiltration; the combination of SVV+aPD1+aCTLA4 showing the highest infiltration. Phenotypic and gene expression analyses of infiltrating cells will be presented. These studies serve as a foundation for translating SVV oncolytic virotherapy combined with anti-PD-1 and anti-CTLA4 antibodies in patients with neuroendocrine neoplasms with a clinical trial anticipated to begin in H1, 2021. Citation Format: Paul L. Hallenbeck, Sunil Chada. Oncolytic Seneca Valley virus (SVV-001) overcomes checkpoint inhibitor resistance and demonstrates a systemic anti-tumor immune response in a syngeneic pancreatic cancer murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6218.

Modulation of the tumor microenvironment by armed vesicular stomatitis virus in a syngeneic pancreatic cancer model

BackgroundThe immunosuppressive microenvironment in pancreatic ductal adenocarcinoma is a major factor that limits the benefits of immunotherapy, especially immune checkpoint blockade. One viable strategy for reverting the immunosuppressive conditions is the use of an oncolytic virus (OV) in combination with other immunotherapy approaches. Infection of PDAC cells with a robust OV can change the tumor microenvironment and increase tumor antigen release by its lytic activities. These changes in the tumor may improve responses to immunotherapy, including immune checkpoint blockade. However, a more potent OV may be required for efficiently infecting pancreatic tumors that may be resistant to OV.MethodsVesicular stomatitis virus, a rapid replicating OV, was armed to express the Smac protein during virus infection (VSV-S). Adaptation by limited dilution largely increased the selective infection of pancreatic cancer cells by VSV-S. The engineered OV was propagated to a large quantity and evaluated for their antitumor activities in an animal model.ResultsIn a syngeneic KPC model, intratumoral injection of VSV-S inhibited tumor growth, and induced increasing tumor infiltration of neutrophils and elimination of myeloid derived suppressor cells and macrophages in the tumor. More importantly, M2-like macrophages were eliminated preferentially over those with an M1 phenotype. Reduced levels of arginase 1, TGF-β and IL-10 in the tumor also provided evidence for reversion of the immunosuppressive conditions by VSV-S infection. In several cases, tumors were completely cleared by VSV-S treatment, especially when combined with anti-PD-1 therapy. A long-term survival of 44% was achieved.ConclusionsThe improved OV, VSV-S, was shown to drastically alter the immune suppressive tumor microenvironment when intratumorally injected. Our results suggest that the combination of potent OV treatment with immune checkpoint blockade may be a promising strategy to treat pancreatic cancer more effectively.

Abstract PO-016: Directed evolution generates novel oncolytic H-1 parvoviruses with improved therapeutic efficacy in virus-resistant pancreatic cancer cells

Abstract Despite considerable promise and emerging clinical success, several challenges impede the broader implementation of novel immunotherapies such as oncolytic virus(OV)-based gene therapy, including for patients with pancreatic cancer (PDAC). One of such challenge is inter-patient variability that may impact on OV selectivity and killing efficacy for tumor cells. For this study, we selected the rat parvovirus H-1 (H-1PV) that is nonpathogenic in humans and has a natural oncolytic activity in several cancer models. The safety and tolerability of H-1PV was recently demonstrated in early clinical trials for glioma and PDAC. However, we report here that H-1PV infection, oncolytic and pro-apoptotic activity are limited in PDAC cells, including patient-derived primary cells. To address this concern, we applied a directed evolution strategy to generate H-1PV variants with specific activity towards PDAC cells. Following selection using patient primary cells, we managed to isolate clonal, PDAC-adapted H-1PVs that induce PDAC cells lysis as compared to parental H-1PV while infection of normal pancreatic cells remained negligeable. Genome sequencing of the tumor adapted virus reveals mutations in promoting and viral capsid sequences. In vivo, the tumor-adapted H1PV demonstrates greater anti-tumor effect than parental H-1PV, following intravenous administration in an experimental model of orthotopic pancreatic tumors engrafted in immunodeficient mice. To our knowledge, we report here for the first time the production of highly selective and potent OV using directed evolution to override PDAC resistance to virotherapy. While the molecular mechanisms involved are still under investigation, this project is a first step towards precision medicine strategies based on OV. Citation Format: Pierre Garcin, Monireh Kazemimanesh, Hubert Lulka, Nelson Dusetti, Guillaume Labrousse, Emilie Benuzzi, Louis Buscail, Pierre Cordelier. Directed evolution generates novel oncolytic H-1 parvoviruses with improved therapeutic efficacy in virus-resistant pancreatic cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-016.

In Vivo Priming of Peritoneal Tumor-Reactive Lymphocytes With a Potent Oncolytic Virus for Adoptive Cell Therapy.

Adoptive cell therapy (ACT) using autologous tumor infiltrating lymphocytes (TIL) achieves durable clinical benefit for patients from whom these cells can be derived in advanced metastatic melanoma but is limited in most solid tumors as a result of immune escape and exclusion. A tumor microenvironment (TME) priming strategy to improve the quantity and quality of TIL represents an important tactic to explore. Oncolytic viruses expressing immune stimulatory cytokines induce a potent inflammatory response that may enhance infiltration and activation of T cells. In this study, we examined the ability of an attenuated oncolytic vaccinia virus expressing IL15/IL15Rα (vvDD-IL15/Rα) to enhance recovery of lavage T cells in peritoneal carcinomatosis (PC). We found that intraperitoneal (IP) vvDD-IL15/Rα treatment of animals bearing PC resulted in a significant increase in cytotoxic function and memory formation in CD8+ T cells in peritoneal fluid. Using tetramers for vaccinia virus B8R antigen and tumor rejection antigen p15E, we found that the expanded population of peritoneal CD8+ T cells are specific for vaccinia or tumor with increased tumor-specificity over time, reinforced with viral clearance. Application of these vvDD-IL15/Rα induced CD8+ T cells in ACT of a lethal model of PC significantly increased survival. In addition, we found in patients with peritoneal metastases from various primary solid tumors that peritoneal T cells could be recovered but were exhausted with infrequent tumor-reactivity. If clinically translatable, vvDD-IL15/Rα in vivo priming would greatly expand the number of patients with advanced metastatic cancers responsive to T cell therapy.

IL-21 arming potentiates the anti-tumor activity of an oncolytic vaccinia virus in monotherapy and combination therapy

BackgroundOncolytic viruses (OVs) have shown promise in containing cancer progression in both animal models and clinical trials. How to further improve the efficacy of OVs are intensively explored. Arming OVs...

Abstract 4576: Oncolytic vaccinia virus TG6010 expressing cytidine deaminase induces DNA damage and shows potent anti-tumor effects

Abstract The recombinant Vaccinia virus Copenhagen strain deleted in Thymidine Kinase and Ribonucleotide Reductase (VVCOPTK−RR−) is a potent and versatile oncolytic platform that has demonstrated strong activity in various preclinical models. The deletion of such VV genes inhibits viral replication in normal cells, while retaining therapeutic replication in tumor cells. TG6002, a VVCOPTK−RR− expressing the suicide gene FCU1, is under investigation in Phase I trials in patients with advanced gastrointestinal tumors. We have developed a new product based on the VVCOPTK−RR− vector, named TG6010, expressing the human cytidine deaminase (CDA) that efficiently catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. The tumor specific expression of CDA by the VV leads to a depletion of cytidine and deoxycytidine. This cytidine/deoxycytidine depletion resulting from CDA overexpression, activated a DNA damage response highlighted by an induction of H2AX phosphorylation. Next, to validate the potential therapeutic use of TG6010, we analyzed the effects of the virus on human xenograft tumors implanted in mice. We observed, after systemic injection of TG6010, high expression of CDA in the tumors with a significant increase in DNA damage in vivo as revealed by the γH2AX foci assay. In addition, intraperitoneal administration of TG6010 in a xenograft model of human ovarian carcinomatosis resulted in significantly prolonged animal survival. In conclusion, the oncolytic vaccinia virus expressing the cytidine deaminase (TG6010) has shown potent anti-tumor effects. Mechanistically, due to the cytidine deaminase overexpression, we observed induction of a DNA damage response. These are data encouraging of clinical development for this highly potent oncolytic Vaccinia virus TG6010 against ovarian cancer. Citation Format: Johann Foloppe, Juliette Kempf, Christelle Pichon, Annie Findeli, Isabelle Farine, Julie Hortelano, Baptiste Moreau, Michèle Klein, Sandrine Cochin, Renée Brandely, Véronique Koerper, Nathalie Silvestre, Christelle Machon, Jérôme Guitton, Eric Quéméneur, Philippe Erbs. Oncolytic vaccinia virus TG6010 expressing cytidine deaminase induces DNA damage and shows potent anti-tumor effects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4576.

Abstract 4578: Development of MEM-288, a dual-transgene armed and conditionally replication-enhanced oncolytic adenovirus with potent systemic antitumor immunity

Abstract MEM-288 is a conditionally replication-enhanced and dual-transgene armed oncolytic adenovirus designed for the treatment of both solid and liquid tumor indications as either a standalone product or in combination with immune checkpoint inhibitors (ICI). Multiple modifications to the adenovirus backbone have been incorporated to confer tumor-selective replication that includes delta-24 (Δ24) E1, E1b 55kb and E3 viral genome deletions. Furthermore, MEM-288 is armed with two potent immune agonist transgenes: MEM40, a novel chimeric CD40 ligand (CD40L) designed for stable cell surface expression of CD40L that functions as a potent activator of CD40 expressing dendritic cells (DCs), and the DC and T cell maturation and activating cytokine IFNβ. MEM-288 is rationally designed to overcome limitations of other OV by conferring enhanced tumor selectivity, oncolytic activity, and ability to directly enhance dendritic cell (DC) and T cell functionality. MEM-288 exhibited ~100-fold greater replication and oncolytic activity in a broad range of human solid tumors and blood tumor types, including a variety of human lung tumor cell lines, in comparison with control Δ24, ΔE1b 55kb and ΔE3 backbone adenoviruses. MEM-288 also exhibited ~1,000-fold greater viral replication in tumor cells compared to normal cell types, including immature dendritic cells and cancer associated human lung fibroblasts cells. These positive in vitro results were expanded upon in multiple in vivo immunocompetent mouse tumor models. Intratumoral administration of MEM-288 in the syngeneic mouse lung metastatic model 344SQ demonstrated potent anti-metastatic activity of MEM-288 compared to control OVs. MEM-288 also generated a highly efficacious response against both intratumoral injected and distant non-injected contralateral tumors in the B16-F10 melanoma model, including significantly greater inhibition of tumor growth with MEM-288 compared to the combination of anti-CTLA4 + PD-1. In addition, the combination of MEM-288 with anti-CTLA4 + PD-1 significantly enhanced anti-tumor activity with tumor regressions and 100% mouse survival at day 30 compared to 56% and 0% survival with individual treatments of either MEM-288 or anti-CTLA4 + PD-1, respectively. MEM-288 generated significantly better T cell tumor antigen clonal expansion following intratumoral administration in the B16-OVA melanoma model compared to a matched control OV that only expresses MEM40, suggesting IFNβ is a critical mediator of the T cell clonal expansion in combination with MEM40. These positive preclinical data suggest MEM-288 is a potent tumor-selective oncolytic virus with desirable mechanistic features that can be ideally combined with ICI, even in the ICI refractory setting. These studies warrant clinical evaluation for lung cancer and other tumor types that are currently under development. Citation Format: Hong Zheng, Wenjie Dai, Scott Antonia, Mark J. Cantwell, Amer A. Beg. Development of MEM-288, a dual-transgene armed and conditionally replication-enhanced oncolytic adenovirus with potent systemic antitumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4578.

Novel recombinant coxsackievirus B3 with genetically inserted basic peptide elicits robust antitumor activity against lung cancer.

Cancer therapy that utilizes oncolytic virus may offer an exciting alternative, and coxsackievirus B3 (CVB3) is a potent oncolytic virus. This study was to assess the oncolytic activities of novel recombinant CVB3 with genetically inserted basic peptides in lung cancer. Recombinant CVB3 was produced in Vero cells, with or without genetically inserted basic peptides. In vitro and in vivo experiments with nude mouse models bearing human lung carcinoma xenografts were performed to examine the antitumor activities. Cytokines and immune responses to the recombinant CVB3 were determined in cynomolgus monkeys. Recombinant CVB3 with genetically inserted basic peptides was associated with significantly higher pH values within tumors. Mice treated with recombinant CVB3 showed significantly less tumor progression, and recombinant CVB3 with genetically inserted basic peptides appeared to enhance tumor suppression. Recombinant CVB3 was associated with significantly less proliferation of various lung cancer cells without affecting proliferation of normal lung fibroblasts. The cytokine profiles of the cynomolgus monkeys were comparable among control group (normal saline solution) and those given recombinant CVB3 with or without fused basic peptides, with no induction of excessive cytokine or immune responses. In conclusions, recombinant CVB3, especially those with fused basic peptides, possess strong antitumor activities without eliciting excessive immune responses.

MicroRNA Modification of Coxsackievirus B3 Decreases Its Toxicity, while Retaining Oncolytic Potency against Lung Cancer

We recently discovered that coxsackievirus B3 (CVB3) is a potent oncolytic virus against KRAS mutant lung adenocarcinoma. Nevertheless, the evident toxicity restricts the use of wild-type (WT)-CVB3 for cancer therapy. The current study aims to engineer the CVB3 to decrease its toxicity and to extend our previous research to determine its safety and efficacy in treating TP53/RB1 mutant small-cell lung cancer (SCLC). A microRNA-modified CVB3 (miR-CVB3) was generated via inserting multiple copies of tumor-suppressive miR-145/miR-143 target sequences into the viral genome. In vitro experiments revealed that miR-CVB3 retained the ability to infect and lyse KRAS mutant lung adenocarcinoma and TP53/RB1-mutant SCLC cells, but with a markedly reduced cytotoxicity toward cardiomyocytes. In vivo study using a TP53/RB1-mutant SCLC xenograft model demonstrated that a single dose of miR-CVB3 via systemic administration resulted in a significant tumor regression. Most strikingly, mice treated with miR-CVB3 exhibited greatly attenuated cardiotoxicities and decreased viral titers compared to WT-CVB3-treated mice. Collectively, we generated a recombinant CVB3 that is powerful in destroying both KRAS mutant lung adenocarcinoma and TP53/RB1-mutant SCLC, with a negligible toxicity toward normal tissues. Future investigation is needed to address the issue of genome instability of miR-CVB3, which was observed in ~40% of mice after a prolonged treatment.

MicroRNA Modification of Coxsackievirus B3 Decreases Its Cardiotoxicity, While Retaining Oncolytic Potency Against Lung Cancer

Background: We recently discovered that coxsackievirus B3 (CVB3) is a potent oncolytic virus against KRAS-mutant (KRASmut) lung adenocarcinoma. Nevertheless, the evident cardiotoxicity restricts the use of wild-type (WT)-CVB3 for lung cancer therapy. The current study aims to genetically engineer the CVB3 genome to decrease its toxicity towards normal tissues and to extend our previous research to determine the safety and oncolytic efficacy of the modified CVB3 in treating TP53-and/or RB1-mutant small cell lung cancer (TP53mut/RB1mut SCLC). Methods: A microRNA-modified CVB3 (miR-CVB3) was generated via inserting multiple copies of tumor-suppressive miR-145 and miR-143 target sequences into the 5' untranslated region of the viral genome. The safety and effectiveness of the miR-CVB3 were then examined in cultured cells and using a TP53mut/RB1mut SCLC cell-derived xenograft model. Findings: In vitro experiments revealed that miR-CVB3 retained the ability to infect and lyse KRASmut lung adenocarcinoma and TP53mut/RB1mut SCLC cells, but with a markedly reduced cytotoxicity towards cardiomyocytes. In vivo study demonstrated that a single dose of miR-CVB3 via systemic administration resulted in a significant tumor regression. Most strikingly, mice treated with miR-CVB3 all survived the time-course of the experiment with greatly attenuated cardiotoxicities and decreased viral titers as compared to WT-CVB3-treated mice. Interpretation: We generated a recombinant CVB3 that was powerful in destroying both KRASmut lung adenocarcinoma and TP53mut/RB1mut SCLC, with a negligible toxicity towards normal tissues. Fundings: This work was supported by the BC Lung Association, Providence Health Care Research Institute and Vancouver Coastal Health Research Institute Innovation and Translational Research Award. Declaration of Interest: WWGJ is the Chief Scientific Officer at Virogin Biotech Ltd. HL (H Liu) and CSN are partially sponsored by Virogin Biotech Ltd. through MITACS Accelerate Program. No conflicts of interest were disclosed by others. Ethical Approval: The protocol was approved by the University Animal Care Committee (A18-0275).

Abstract 4773: Development of ONCR-NEP, a lipid nanoparticle delivered oncolytic virus capable of robust in situ amplification resulting in tumor lysis and regression

Abstract Oncolytic viruses (OV) have shown clinical efficacy for treatment of malignancies when administered intratumorally. Attempts to use OV for systemic treatment of cancer have shown very limited success due to the presence of neutralizing antibodies. Development of an intravenously delivered oncolytic viral therapy that maintains its potency and effectiveness in the presence of neutralizing antibodies is greatly needed for patients with visceral lesions that are metastatic and difficult to inject. Therefore, the OV field is moving toward the design of vector platforms that can be used for systemic therapy to take full advantage of the oncolytic activity and the induction of anti-tumor immunity. Oncorus is developing a new strategy for systemic delivery of OV genomes by using tumor targeted nanoparticles, designed to be immunologically inert systemically, but can initiate a local robust intratumoral oncolytic virus infection. The Nanoparticle Encapsulated Polynucleotide (NEP) platform is designed for systemic delivery of replication competent OV genomes encoded by a plasmid DNA loaded into tumor-homing nanoparticles. To initiate viral replication intratumorally from a plasmid DNA we engineered a mammalian mRNA cassette that codes for an infectious +sense picornaviral RNA. This RNA is liberated from the larger capped and polyadenylated mammalian transcript by combination of ribozymes and siRNA cleavage, enabling rapid expression of viral proteins and viral replication. We selected Seneca Valley Virus (SVV) as our first candidate virus for the NEP platform. SVV is a potent oncolytic virus in neuroendocrine tumors previously evaluated in clinical trial after IV administration and well tolerated in the patients. The robust initiation of SVV replication from the pDNA expression construct results in cellular lysis in permissive cells when transfected in vitro. In vivo, intratumoral injection of a lipid based formulated SVV plasmid resulted in robust viral replication and dramatic tumor growth inhibition in multiple tumor models. This viral construct has also been modified to express immunomodulatory payloads to enhance the adaptive immune response by recruitment of immune effector cells. Finally, we have enabled miRNA attenuation of his construct to add a genetic tissue selectivity switch for safety, and we have shown that both miR-1 and miR-122 effectively and specifically attenuate viral replication. To enable ONCR-NEP therapy for intravenously administration, we have begun developing a lipid nanoparticle formulation with an active tumor targeting moiety. We have obtained lipid nanoparticle formulations with favorable physical and functional characteristics that are effective both in vitro and in vivo. We show here for the first time the development of a synthetic virus, a breakthrough therapeutic technology that is highly effective in vivo. Citation Format: Edward M. Kennedy, Jennifer S. Lee, Judy Jacques, Terry Farkaly, Prajna Behara, Peter Grzesik, Brian B. Haines, Agnieszka Denslow, Jacqueline Gursha, Lingxin Kong, Mitchell Finer, Christophe Quéva, Lorena Lerner. Development of ONCR-NEP, a lipid nanoparticle delivered oncolytic virus capable of robust in situ amplification resulting in tumor lysis and regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4773.

Abstract 2267: Therapeutic synergy of the chimeric, oncolytic virus VSV-GP with immune modulators

Abstract Introduction: Oncolytic viruses (OVs), which selectively replicate in and destroy tumor cells, represent a highly promising class of cancer therapeutics. In recent years, the focus in the OV field has significantly shifted from a pure oncolytic mode of action to their strong immune stimulatory potential. Tumor cells lysed by the viruses release pro-inflammatory signals, tumor associated antigens as well as display hallmarks of immunogenic cell death. VSV-GP, a chimeric VSV pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) is a potent tumor cell-lysing agent and capable of jump-starting α-tumor immunity. The current study explores the immune promoting properties of VSV-GP and investigates clinically relevant combination therapies with checkpoint inhibition (α-PD-1) as well as a “second mitochondria-derived activator of caspases“ mimetic (SMACm). Experimental Procedures: Immune competent mice bearing established LLC1-IFNARKO, B16-F1-OVA or CT26.CL25-IFNARKO tumors were subject to treatments comprising VSV-GP, α-PD-1, a SMACm or combinations of VSV-GP with either of the latter therapies. α-tumor effects were determined by tumor growth inhibition and/or changes in overall survival. VSV-GP's tumor cell selective replication, resulting in immune activation, was assessed using FFPE based IHC staining for the viral N-protein, PD-L1 and different immune cell subsets as well as the NanoString platform (nCounter Pan Cancer Immune Profiling Panel). Data Summary: Here we present data on VSV-GP, which displays potent α-tumor efficacy and upon systemic delivery increases the local T-cell infiltration, expression of chemokines, T-cell and myeloid cell specific activation markers as well as expression of immune checkpoints, such as PD-1 and PD-L1 within the tumor microenvironment. Building on the observed therapeutic potential of VSV-GP as monotherapy, its immune promoting properties and the delineated compensatory mechanisms, such as PD-1/-L1 upregulation, we explored therapeutic combinations with a PD-1 blocking antibody, which resulted in a strong improvement of VSV-GP-mediated α-tumor efficacy as well as overall survival. Cured, tumor free animals were protected from tumor rechallange indicating immunological memory formation. We further explored the therapeutic combination of VSV-GP with a SMACm, which also resulted in a strongly improved therapeutic benefit and increased overall survival. Conclusion: In summary VSV-GP induces a pro-inflammatory microenvironment within infected tumors, increases immune cell infiltration and synergizes with α-PD-1 as well as a SMACm. VSV-GP is a potent OV with a strong α-tumor immunity promoting potential. Preclinical data justify further testing of VSV-GP and the combinations with PD-1 blockade or a SMACm in clinical trials. Citation Format: Philipp Mueller, Patrik Erlmann, Monika Petersson, Tobias Nolden, Carles Urbiola, Liesa-Marie Schreiber, Bart Spiesschaert, Klaus Erb, Birgit Stierstorfer, Maria Antonietta Impagnatiello, Eric Borges, Knut Elbers, Guido Wollmann, John Park. Therapeutic synergy of the chimeric, oncolytic virus VSV-GP with immune modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2267.

Characterizing Cellular Responses During Oncolytic Maraba Virus Infection.

The rising demand for powerful oncolytic virotherapy agents has led to the identification of Maraba virus, one of the most potent oncolytic viruses from Rhabdoviridae family which displays high selectivity for killing malignant cells and low cytotoxicity in normal cells. Although the virus is readied to be used for clinical trials, the interactions between the virus and the host cells is still unclear. Using a newly developed interferon-sensitive mutant Maraba virus (MG1), we have identified two key regulators of global translation (4E-BP1 and eIF2α) as being involved in the regulation of protein synthesis in the infected cells. Despite the translational arrest upon viral stress, we showed an up-regulation of anti-apoptotic Bcl-xL protein that provides a survival benefit for the host cell, yet facilitates effective viral propagation. Given the fact that eIF5B canonically regulates 60S ribosome subunit end joining and is able to replace the role of eIF2 in delivering initiator tRNA to the 40S ribosome subunit upon the phosphorylation of eIF2α we have tested whether eIF5B mediates the translation of target mRNAs during MG1 infection. Our results show that the inhibition of eIF5B significantly down-regulates the level of Bcl-xL steady-state mRNA, thus indirectly attenuates viral propagation.

Abstract 729: CD40 and 4-1BB pathway activation using oncolytic viruses kills tumor cells by oncolysis and CD40-mediated apoptosis while activating immune cells

Abstract LOAd703 is a tumor-selective oncolytic adenovirus (serotype 5/35) encoding two immunostimulatory transgenes: trimerized membrane-bound CD40L and 4-1BBL. We have previously shown the immunostimulatory effect of LOAd703 in pancreatic cancer models and a clinical trial enrolling patients with pancreatic cancer is ongoing. LOAd703 can potentially be used in a variety of cancers and its applicability across various cancer types was investigated herein. The immune function of the transgenes was further elucidated, including the tumor-specific apoptotic effect of TMZ-CD40L. A panel of human tumor cell lines including B-cell lymphoma (Karpas-422, BC-3, Daudi, DG-75, U-698), multiple myeloma (RPMI-8226), colorectal (HT29), ovarian (SKOV3), lung (A549), neuroendocrine (H727), bladder (T24) and pancreas (Panc01) was selected for evaluation of the LOAd703 oncolytic capacity. The killing capability of LOAd viruses was analyzed using CellTiter96AQueous One Solution Cell Proliferation Assay and Incucyte. Further, LOAd703 was investigated in xenograft models (one per indication). Replication was confirmed by virus-specific quantitative PCR in selected lines. Immunomodulatory capacity on myeloid cells including dendritic cells and macrophages was evaluated in in vitro cultures by flow cytometry and cytokine assays. Apoptosis was determined using flow cytometry. LOAd703 infection resulted in robust virus expansion and killing of tumor cell lines across indications while primary cultures obtained from the pancreas, lung, heart and kidney were not affected. Cell lines such as T24 expressing CD40 showed enhanced sensitivity to LOAd703 that contains TMZ-CD40L compared to LOAd(-) lacking transgenes. The enhanced cell death was due to increased apoptosis frequency. In contrast, the CD40 negative cell lines such as SKOV-3 was equally sensitive to LOAd703 and LOAd(-). Likewise, LOAd703 could effectively control established tumors in xenograft models and the effect was more pronounced in CD40+ cell lines. Interestingly, infected B cell lines showed an enhanced immunogenic phenotype (MHC molecules, Fas, ICAM-I, CD80 and CD86) post infection likely due to their origin as antigen-presenting cells. Stimulation of immature DCs with LOAd703 forced differentiation into a mature phenotype with high capacity of antigen-presentation and expansion of both T cells and NK cells while stimulation of macrophages favored a high M1:M2 ratio. In conclusion, the LOAd703 oncolytic virus can kill a broad range of tumor cells both by CD40-mediated apoptosis and replication-induced oncolysis and the effect is most potent in CD40+ tumor cells. CD40+ myeloid cells such as DCs and macrophages are instead activated by LOAd703. Hence, LOAd703 is a potent multi-functional oncolytic virus. Citation Format: Emma Eriksson, Jessica Wenthe, Sedigheh Naseri, Ann-Charlotte Hellström, Angelica Loskog. CD40 and 4-1BB pathway activation using oncolytic viruses kills tumor cells by oncolysis and CD40-mediated apoptosis while activating immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 729.

Transforming the prostatic tumor microenvironment with oncolytic virotherapy

ABSTRACTProstate cancer (PCa) was estimated to have the second highest global incidence rate for male non-skin tumors and is the fifth most deadly in men thus mandating the need for novel treatment options. MG1-Maraba is a potent and versatile oncolytic virus capable of lethally infecting a variety of prostatic tumor cell lines alongside primary PCa biopsies and exerts direct oncolytic effects against large TRAMP-C2 tumors in vivo. An oncolytic immunotherapeutic strategy utilizing a priming vaccine and intravenously administered MG1-Maraba both expressing the human six-transmembrane antigen of the prostate (STEAP) protein generated specific CD8+ T-cell responses against multiple STEAP epitopes and resulted in functional breach of tolerance. Treatment of mice with bulky TRAMP-C2 tumors using oncolytic STEAP immunotherapy induced an overt delay in tumor progression, marked intratumoral lymphocytic infiltration with an active transcriptional profile and up-regulation of MHC class I. The preclinical data generated here offers clear rationale for clinically evaluating this approach for men with advanced PCa.

Production and Purification of High-Titer Newcastle Disease Virus for Use in Preclinical Mouse Models of Cancer

Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus in the Paramyxoviridae family. Although primarily an avian pathogen, NDV is a potent oncolytic virus that has been shown to be safe and effective in a variety of preclinical cancer models and human clinical trials. To produce virus for oncolytic trials, NDV is commonly amplified in embryonated chicken eggs and purified from the allantoic fluid. Conventional methods for purifying virus from allantoic fluid often result in relatively low-titer preparations containing high levels of impurities, including immunogenic chicken host cell proteins from allantoic fluid. However, large quantities of virus need to be delivered intravenously to administer oncolytic NDV systemically to mice. This route of administration requires virus preparations that are both highly concentrated (to enable delivery of small volumes) and highly pure (to limit toxic effects from contaminants). Given the accumulation of promising preclinical and clinical data demonstrating the efficacy of NDV as an oncolytic agent, strategies for increasing the titer and purity of NDV preparations are sorely needed to allow for effective intravenous administration in mice. Here, we describe an optimized protocol for the rescue, production, and purification of high-titer in vivo-grade NDV for preclinical studies in mouse models.

135P - Coxsackievirus type B3 is a potent oncolytic virus against KRAS-mutant non-small cell lung cancer

135P - Coxsackievirus type B3 is a potent oncolytic virus against KRAS-mutant non-small cell lung cancer

Upregulation of Coxsackie Adenovirus Receptor Sensitizes Cisplatin-Resistant Lung Cancer Cells to CRAd-Induced Inhibition.

Objective. Conditionally replicating adenoviruses (CRAds) have been proven potent oncolytic viruses in previous studies. They selectively replicate in the tumor cells because of incorporated survivin promoter and ultimately lead to their killing with minimal side effects on normal tissue. Chemotherapy with cisplatin is commonly employed for treating tumors, but its cytotoxic effects and development of resistance remained major concerns to be dealt with. The aim of this study was to explore the anticancer potential of survivin regulated CRAd alone or in combination with cisplatin in the A549 lung cancer cell line and cisplatin-resistant lung cancer cell line, A549-DDPR.Methods. CRAd was genetically engineered in our laboratory by removing its E1B region and adding survivin promoter to control its replication. A549, H292, and H661 lung cancer cell lines were procured from the CAS-China. The anti-tumor effectiveness of combined treatment (cisplatin plus CRAd) was evaluated in vitro through MTS assays and in vivo through mouse model experimentation. RT- PCR was used to assess MDR gene and mRNA expression of coxsackie adenoviral receptor (CAR).Results. Results of in vitro studies established that A549 lung cancer cells were highly sensitive to cisplatin showing dose-dependent inhibition. The resistant cells of A549-DDPR exhibited very less sensitivity to cisplatin but were infected with CRAd more efficiently as compared to A549. A549-DDPR cells exhibited higher expression of MDR gene and CAR in the RT-PCR analysis. The nearly similar rise in the CAR expression was seen when lung cancer cell lines received cisplatin in combined treatment (cisplatin plus CRAd). Combined anti-cancer therapy (cisplatin plus oncolytic virus) proved more efficient than monotherapy in the killing of cancer cells. Results of in vivo experiments recapitulated nearly similar tumor inhibition activities.Conclusion. This study highlighted the significant role of survivin in gene therapy as it has the potential to render CRAd more tumor specific. It also establishes that higher CAR expression plays a vital role in the success of adenovirus-based therapies. Furthermore, a careful combination of chemotherapy drugs and oncolytic viruses can culminate in significant therapeutic achievements against cancer.