Jennelle Francis

E3 gene manipulations affect oncolytic adenovirus activity in immunocompetent tumor models

Oncolytic replication-selective adenoviruses constitute a rapidly growing therapeutic platform for cancer. However, the role of the host immune response and the E3 immunoregulatory genes of the human adenovirus were unknown until now. We identified four mouse carcinoma lines of variable permissivity for adenoviral gene expression, cytopathic effects and/or burst size. To determine E3 gene effects in immunocompetent tumor-bearing hosts, we injected tumors with one of three adenoviruses: Ad5 (E3 wild type), dl309 (del. E3 10.4/14.5, 14.7 kDa) or dl704 (del. E3 gp19 kDa). Compared with Ad5 and dl704, dl309 was cleared much more rapidly and/or its activity was lower in all four models. Intratumoral injection with dl309 resulted in markedly greater macrophage infiltration and expression of both tumor necrosis factor and interferon-γ. Adenovirus replication, CD8+ lymphocyte infiltration and efficacy were similar upon intratumoral injection with either dl704 or Ad5. E3-dependent differences were not evident in athymic mice. These findings have important implications for the design of oncolytic adenoviruses and may explain the rapid clearance of E3-10.4/14.5,14.7-deleted adenoviruses in patients.

Novel Immunocompetent Murine Tumor Models for the Assessment of Replication-Competent Oncolytic Adenovirus Efficacy

Oncolytic replication-selective adenoviruses constitute a rapidly expanding experimental approach to the treatment of cancer. However, due to the lack of an immunocompetent and replication-competent efficacy model, the role of the host immune response and viral E3 immunoregulatory genes remained unknown. We screened nine murine carcinoma lines for adenovirus (Ad5) uptake, gene expression, replication, and cytopathic effects. In seven of these murine cell lines the infectability and cytopathic effects were similar to those seen with human carcinoma lines. Surprisingly, productive viral replication was demonstrated in several lines; replication varied from levels similar to those for some human carcinoma lines (e.g., CMT-64) to very low levels. Seven of these lines were grown as subcutaneous xenografts in immunocompetent mice and were subsequently injected directly with Ad5, saline, or a replication-deficient control adenovirus particle to assess intratumoral viral gene expression, replication, and antitumoral effects. E1A, coat protein expression, and cytopathic effects were documented in five xenografts; Ad5 replication was demonstrated in CMT-64 and JC xenografts. Ad5 demonstrated significant efficacy compared to saline and nonreplicating control Ad particles in both replication-permissive xenografts (CMT-64, JC) and poorly permissive tumors (CMT-93); efficacy against CMT-93 tumors was significantly greater in immunocompetent mice compared to athymic mice. These murine tumor xenograft models have potential for elucidating viral and host immune mechanisms involved in oncolytic adenovirus antitumoral effects.

Lister strain of vaccinia virus armed with endostatin–angiostatin fusion gene as a novel therapeutic agent for human pancreatic cancer

Survival after pancreatic cancer remains poor despite incremental advances in surgical and adjuvant therapy, and new strategies for treatment are needed. Oncolytic virotherapy is an attractive approach for cancer treatment. In this study, we have evaluated the effectiveness of the Lister vaccine strain of vaccinia virus armed with the endostatin–angiostatin fusion gene (VVhEA) as a novel therapeutic approach for pancreatic cancer. The Lister vaccine strain of vaccinia virus was effective against all human pancreatic carcinoma cells tested in vitro, especially those insensitive to oncolytic adenovirus. The virus displayed inherently high selectivity for cancer cells, sparing normal cells both in vitro and in vivo, with effective infection of tumors after both intravenous (i.v.) and intratumoral (i.t.) administrations. The expression of the endostatin–angiostatin fusion protein was confirmed in a pancreatic cancer model both in vitro and in vivo, with evidence of inhibition of angiogenesis. This novel vaccinia virus showed significant antitumor potency in vivo against the Suit-2 model by i.t. administration. This study suggests that the novel Lister strain of vaccinia virus armed with the endostatin–angiostatin fusion gene is a potential therapeutic agent for pancreatic cancer.

Virus-Associated RNA I-Deleted Adenovirus, a Potential Oncolytic Agent Targeting EBV-Associated Tumors

Given the growing number of tumor types recognizably associated with EBV infection, it is critically important that therapeutic strategies are developed to treat such tumors. Replication-selective oncolytic adenoviruses represent a promising new platform for anticancer therapy. Virus-associated I (VAI) RNAs of adenoviruses are required for efficient translation of viral mRNAs. When the VAI gene is deleted, adenovirus replication is impeded in most cells (including HEK 293 cells). EBV-encoded small RNA1 is uniformly expressed in most EBV-associated human tumors and can functionally substitute for the VAI RNAs of adenovirus. It enables replication to proceed through complementation of VAI-deletion mutants. We hypothesized that VAI-deleted adenovirus would selectively replicate in EBV-positive tumor cells due to the presence of EBV-encoded small RNA1 with no (or poor) replication in normal or EBV-negative tumor cells. In this report, we show that high levels of replication occurred in the VAI-deleted mutant in the EBV-positive tumor cells compared with low (or negligible) levels in EBV-negative and normal human primary cells. Correspondingly, high toxicity levels were observed in EBV-positive tumor cells but not in EBV-negative tumor or normal human primary cells. In vivo, VAI-deleted adenovirus showed superior antitumoral efficacy to wild-type adenovirus in EBV-positive tumor xenografts, with lower hepatotoxicity than wild-type adenovirus. Our data suggest that VAI-deleted adenovirus is a promising replication-selective oncolytic virus with targeting specificity for EBV-associated tumors.

CEACAM6 attenuates adenovirus infection by antagonizing viral trafficking in cancer cells

The changes in cancer cell surface molecules and intracellular signaling pathways during tumorigenesis make delivery of adenovirus-based cancer therapies inefficient. Here we have identified carcinoembryonic antigen- related cell adhesion molecule 6 (CEACAM6) as a cellular protein that restricts the ability of adenoviral vectors to infect cancer cells. We have demonstrated that CEACAM6 can antagonize the Src signaling pathway, downregulate cancer cell cytoskeleton proteins, and block adenovirus trafficking to the nucleus of human pancreatic cancer cells. Similar to CEACAM6 overexpression, treatment with a Src-selective inhibitor significantly reduced adenovirus replication in these cancer cells and normal human epithelial cells. In a mouse xenograft tumor model, siRNA-mediated knockdown of CEACAM6 also significantly enhanced the antitumor effect of an oncolytic adenovirus. We propose that CEACAM6-associated signaling pathways could be potential targets for the development of biomarkers to predict the response of patients to adenovirus-based therapies, as well as for the development of more potent adenovirus-based therapeutics.

An oncolytic adenovirus defective in pRb-binding (dl922-947) can efficiently eliminate pancreatic cancer cells and tumors in vivo in combination with 5-FU or gemcitabine.

Pancreatic adenocarcinoma has a poor prognosis and frequently develops resistance to standard chemotherapeutics. Oncolytic adenoviruses represent a promising approach to overcome treatment resistance. The replication-selective dl922–947 adenovirus, defective in pRb binding, targets cancers with deregulated cell cycle control, such as the majority of pancreatic tumors. Cell killing efficacy was higher for dl922–947 than for adenovirus type 5 (Ad5) and the clinically approved dl1520 in pancreatic cancer cells with K-ras, p16 and p53 mutations. Combinations of dl922–947 and 5-fluorouracil or gemcitabine (2′2′-difluoro-2-deoxytidine) resulted in strong synergistic cell killing in Suit-2 and the highly drug- and virus-resistant Hs766T cells. Viral uptake increased in response to drugs, but was independent of the expression levels of the viral attachment receptor coxsackie and adenovirus receptor (CAR), whereas expression levels of the internalization receptors αvβ3- and αvβ5-integrins were increased. Early viral E1A expression was potently induced with drugs contributing to the synergistic effects. The dl922–947 mutant was more efficacious than Ad5 in vivo in Hs766T and Suit-2 xenograft models. In combination with gemcitabine, median survival was further prolonged. We demonstrate that dl922–947 is highly efficacious in pancreatic cancers and conclude that oncolytic adenoviruses harboring the E1ACR2 deletion have great potential for development into future clinical candidates for pancreatic cancer.

608. VAI deleted adenovirus as an oncolytic agent targeting human EBV-associated tumours

The EBV genome is frequently present in Burkitts lymphomas, B-Cell lymphomas of immunocompromised patients, nasopharyngeal carcinomas, Hodgkins disease, T-cell lymphomas, natural killer cell leukemia/lymphoma, smooth-muscle tumors and gastric cancer. More recently, there have been scattered reports linking EBV with conventional epithelial cancers of other primary sites including breast cancers, lung cancers, prostate cancers, liver cancers, colon cancers and lymphoepithelioma-like carcinoma of the esophagus. Given the ever-growing number of tumor types associated with EBV infection, it is therefore critically important to develop therapeutic strategies to treat EBV-associated tumors. Replication-selective oncolytic adenovirus is a promising new platform for anti-cancer therapy. Data from numerous clinical trials have demonstrated several attenuated oncolytic viral mutants to be safe and to replicate selectively in tumor cells with significant anti-tumor potency. Virus associated (VA) 1 and 2 RNA are two low molecular weight adenoviral RNAs that are synthesized late during the viral replication cycle and are required for efficient translation of late viral mRNAs. When the VA1 gene is deleted, replication is impeded in most cells (including HEK 293 cells). The Epstein Barr Virus (EBV) genome encodes two small, non-polyadenylated nuclear RNAs (EBER 1 and 2) that are uniformly expressed in most EBV-associated human tumors. EBER 1 and 2 can functionally substitute for the VA1 RNAs of adenovirus, enabling replication to proceed normally through complementation of VA1 deletion mutants. We hypothesized that VA1-deleted adenovirus would selectively replicate in EBV-positive tumor cells due to complementation by EBER 1, with poor or no replication in normal cells and EBV-negative tumor cells. Our data demonstrate that cytotoxicity of VA1 RNA deletion mutants was restricted to EBV-positive tumor cells (e.g. c666, AGS-EBV, Raji, Jiyoye). Cytopathic effects were much lower in EBV-negative tumor cells (Hep-2, AGS-ATCC, Panc-1). The replication of the VA1 deletion mutant virus was 14–25 times higher in EBV-positive tumor cells than in EBV-negative tumor cells and comparable with wild type adenovirus. The VA1 mutant did not replicate in normal cells in culture such as normal human bronchial epithelial cells (NHBE), and replicated poorly in prostate epithelial cells (PrEC). In vivo, VAI deleted adenovirus showed the same efficacy as wild type adenovirus in xenograft models of human EBV-positive tumor cells, but much lower cytotoxicity than wildtype adenovirus. Our data suggest that a VA1 RNA-deleted viral mutant is a promising replication-selective oncolytic virus specifically targeting EBV-associated cancers.

284. Oncolytic Adenovirus E1B-19kD Deletion and E3B Retention Results in Both Enhanced Potency and Tumor Necrosis Factor - Mediated Selectivity

Replication-selective oncolytic adenoviruses hold promise as a new cancer treatment platform, but novel mechanisms must be identified to maximize virus safety, potency and exogenous therapeutic gene-carrying capacity. The E1B-19kD and E3B proteins independently antagonize tumor necrosis factor (TNF-α)-mediated cell death, but their functional interactions have not been previously described. Since tumors have numerous blocks in apoptotic pathways, we hypothesized that deletion of one or both gene regions would result in cancer-selectivity in the presence of TNF-α. We therefore compared the potency and selectivity of E1B-19kD(-), E3B(-) and E1B-19kD(-)/E3B(-) mutants to wildtype adenovirus in vitro and in vivo. In cancer cells in vitro, the E1B-19kD(-) mutant had superior replication, spread and cytolysis (+) or (-) TNF-α; deletion of both E1B-19kD and E3B was relatively antagonistic. In normal cells without TNF-α, similar results were obtained. In contrast, all three mutants were dramatically inhibited in the presence of TNF-α. In immunocompetent mice, all three mutants were significantly inhibited in normal tissue compared to wildtype adenovirus. In tumors, only the E1B-19kD(-) mutant demonstrated enhanced replication, spread and antitumoral efficacy versus wildytpe. In summary, E1B-19kD-deletion plus E3B retention results in both enhanced potency and tumor necrosis factor-mediated selectivity.