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-gamma. 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.

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.

A novel assay to assess primary human cancer infectibility by replication-selective oncolytic adenoviruses.

PURPOSE: Replication-selective oncolytic adenoviruses hold promise for cancer treatment, but the predictive use of cell lines, dissociated tumor tissue, and animal models for efficacy against primary cancers are unclear. To further evaluate cytotoxicity and the potential for efficacy of replication-competent adenoviruses we therefore developed a novel methodology using primary human cancer specimens ex vivo; ovarian, colon, rectal, and breast carcinomas were included. EXPERIMENTAL DESIGN: Tissue culture conditions were developed to maintain viability of adenocarcinomas ex vivo for 48 hours postsurgery. Explants were infected by replication-competent (wild type 5 and E1A mutant dl922-947) and replication-defective (dl312) adenoviruses; early (E1A) and late (hexon) viral gene expression, alphav integrins, coxsackievirus and adenovirus receptor (CAR) and tissue viability were assessed by immunohistochemistry and histopathology. Viral replication was verified by replication assays on selected samples. RESULTS: Viral gene expression varied dramatically among cancer specimens (n = 41). With Ad5, hexon expression was high in 8 of 11 tested specimens, whereas E1A levels were detectable in 16 of 27 tumor explants. Viral gene expression, distribution, and cytopathic effects were greater postinfection with dl922-947. Specimens that supported early gene expression (E1A) also supported viral replication in 13 of 14 tested cases, determined by recovery of infectious units. As predicted, the replication-defective adenovirus dl312 was not associated with viral gene expression. CONCLUSIONS: Primary human tumor tissue remained viable when cultured ex vivo enabling evaluation of viral mutants in tissue with intact morphology. This assay may have great use in determining treatment-sensitive cancers and assess specific oncolytic mutants in individual cases.

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 E1B-19 kDa gene deletion mutant adenovirus demonstrates tumor necrosis factor-enhanced cancer selectivity and enhanced oncolytic potency.

Oncolytic adenoviruses hold promise as a new treatment platform for cancer, but limitations have been identified, including limited spread and potency. The adenoviral protein E1B-19 kDa is a Bcl-2 homologue that blocks apoptosis induction via the intrinsic and extrinsic pathways, specifically including tumor necrosis factor-mediated cell death. We demonstrate that an E1B-19 kDa gene deletion mutant had tumor necrosis factor-enhanced cancer selectivity, in vitro and in vivo, due to genetic blocks in apoptosis pathways in cancer cells. In addition, this mutant demonstrated significantly enhanced viral spread and antitumoral potency relative to dl1520 (aka Onyx-015) and wild-type adenovirus in vitro. Significant antitumoral efficacy was demonstrated in vivo by intratumoral and intravenous routes of administration. E1B-19 kDa deletion should be considered as a feature of oncolytic adenoviruses to enhance their safety, spread, and efficacy.

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.