Oncolytic viruses which infect and kill tumour cells can also be genetically modified to express therapeutic genes that augment their anti-cancer activities. Modifying oncolytic viruses to produce effective cancer therapies is challenging as encoding transgenes often attenuates virus activity or prevents systemic delivery in patients due to the risk of off-target expression of transgenes in healthy tissues. To overcome these issues we aimed to generate a readily modifiable virus platform using the oncolytic adenovirus, enadenotucirev. Enadenotucirev replicates in human tumour cells but not cells from healthy tissues and can be delivered intravenously because it is stable in human blood. Here, the enadenotucirev genome was used to generate plasmids into which synthesised transgene cassettes could be directly cloned in a single step reaction. The platform enabled generation of panels of reporter viruses to identify cloning sites and transgene cassette designs where transgene expression could be linked to the virus life cycle. It was demonstrated using these viruses that encoded transgene proteins could be successfully expressed in tumour cells in vitro and tumours in vivo. The expression of transgenes did not impact either the oncolytic activity or selective properties of the virus. The effectiveness of this approach as a drug delivery platform for complex therapeutics was demonstrated by inserting multiple genes in the virus genome to encode full length anti-VEGF antibodies. Functional antibody could be synthesised and secreted from infected tumour cells without impacting the activity of the virus particle in terms of oncolytic potency, manufacturing yields or selectivity for tumour cells. In vivo, viral particles could be efficaciously delivered intravenously to disseminated orthotopic tumours.
Building on the recent clinical successes of checkpoint inhibitor antibodies, the field of cancer immunotherapy is now focussing on combination treatment regimens to further improve efficacy benefits to patients. However, combining such systemically dosed agents is associated with a number of challenges including enhanced side effect profiles and high costs. One strategy being explored to overcome such issues is to dose the therapeutics directly into the tumor rather than systemically but many tumors will not be accessible for this type of treatment. We have developed a broadly applicable vector platform system, based on the potent chimeric oncolytic group B adenovirus enadenotucirev (EnAd), for directing the efficient local production of a combination of immunotherapeutic agents selectively within the tumor. The versatility and fidelity of the platform has been exemplified by encoding up to three separate biomolecules in the same virus, including antibodies, cytokines, chemokines and tumor-associated antigens, without altering other virus properties. A systemic clinical dosing regimen has been established for EnAd, with data directly demonstrating selective virus delivery to and protein production from colorectal and other tumor types. The advantage of this approach is that immunotherapeutics encoded in the virus can be produced locally, both in tumors that are not directly injectable and in metastases, while minimising systemic off-target effects. A candidate virus NG-345 has been designed to produce a combination of three secreted immunomodulatory agents (human IFNá, MIP1á and Flt3L) aimed at enhancing the recruitment and activation of immune cells into tumor cell nests. We have shown that NG-345 retains the full oncolytic properties (potency and selectivity) of the parental EnAd virus, with infected human tumor cells producing high levels of all three cytokine/chemokines in the culture supernatants. Functional activity of individual encoded agents has also been demonstrated using relevant cell-based assays. EnAd is highly human-tumor selective and does not replicate, produce infectious progeny or express endogenously regulated transgenes in non-human cells. In vivo evaluation of immuno-modulatory activities of armed viruses is therefore challenging and requires the application of multiple approaches that can collectively provide informative data. In particular, studies are focusing on using surrogate candidate viruses expressing murine gene homologs in human tumor xenografts in immunodeficient mice with or without a reconstituted immune system. Citation Format: Brian R. Champion, Nalini Rasiah, Sam Illingworth, Matthieu Besneux, Rochelle Lear, Darren Plumb, Prithvi Kodialbail, Alice C.N. Brown. Developing tumor-localized, combination immunotherapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4875.
NG-348 is a transgene-modified variant of enadenotucirev, a chimeric oncolytic group B adenovirus with potent and selective anti-tumor activity against a range of epithelial cancer cells. Enadenotucirev has a blood stability profile that enables systemic dosing and has been administered intravenously to over 90 cancer patients. These studies have demonstrated that IV dosed enadenotucirev is delivered to tumors and subsequent virus activity is associated with CD8+ T-cell infiltration in tumor cell nests, consistent with immune stimulation within the tumor. NG-348 encodes two immunomodulatory proteins in its genome: full-length human CD80, and a membrane anchored single chain variable fragment of the mouse anti-human CD3ϵ monoclonal antibody OKT3. Together these membrane proteins provide both T-cell receptor (signal 1) and costimulatory (signal 2) activation signals required to polyclonally activate tumor-infiltrating T-cells. When expressed on the surface of NG-348 infected tumor cells the transgenes therefore enhance the potency of the virus by driving local T-cell immune responses selectively in the tumor microenvironment. The expression of both transgenes encoded in the NG-348 virus is controlled by the endogenous virus major late promoter. This restricts expression of the proteins to the surface of cells permissive to virus infection (i.e. tumor cells) and prevents off-target expression in the cells from healthy tissues. Using co-cultures of human T-cells with human tumor cell lines, we have shown that NG-348 infected tumor cells potently activate both CD4 and CD8 T-cells. This was demonstrated by analysis of activation marker expression (CD25, CD69), intracellular and secreted cytokines (IL-2, TNF, IFNγ) and induction of T-cell mediated tumor cell death by apoptosis (prior to oncolytic death by the virus). Treatment of different human non-tumor cells (e.g. fibroblasts, T-cells, PBMCs) with NG-348 did not lead to transgene protein expression or activation of T-cells in co-cultures. NG-348 has also been shown to activate human T-cells in vivo, using a human tumor xenograft model system in immunodeficient mice reconstituted with human PBMCs. Collectively these data indicate that following delivery to tumor tissues of patients, NG-348 oncolytic virus can selectively replicate and express it’s payload of T-cell activating ligands. NG-348 should therefore stimulate potent antigen-independent, polyclonal activation of T-cells already present in the tumor, as well as those recruited into the tumor in response to the virus infection, to drive effective anti-tumor immunity. NG-348 is currently in preclinical development with a first phase I study planned to initiate in Q4 2017. Citation Format: Brian R. Champion, Matthieu Besneux, Nalini Marino, Darren Plumb, Prithvi Kodialbail, Sam Illingworth, Rochelle Lear, Alice C. Brown. NG-348: a novel oncolytic virus designed to mediate anti-tumour activity via the potent and selective polyclonal activation of tumor-infiltrating T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5098. doi:10.1158/1538-7445.AM2017-5098
We are developing “armed” versions of the oncolytic adenovirus, enadenotucirev (EnAd), that will selectively infect and deliver immunotherapeutics to tumours following systemic dosing. EnAd is a potent, chimeric Ad11p/Ad3 adenovirus active against a range of epithelial cancer cells. In normal cells, EnAd is attenuated and shows little or no activity by either cytotoxicity assay or qPCR. In vivo, EnAd shows efficacy in a range of xenograft human tumor models following intra-tumoural, intravenous and intra-peritoneal injection and is currently being evaluated clinically for treatment of several different epithelial cancers. Data from ongoing clinical studies have shown that i.v. dosed EnAd infects and selectively replicates in tumor cells, producing significant amounts of viral protein (hexon). This is associated with CD8 cell accumulation and also indicates that transgene encoded proteins will be made in significant amounts by tumors following i.v. delivery of an armed EnAd virus. To develop armed EnAd variants we have developed a novel, efficient cloning system for rapid generation of viruses that can produce antibodies and other payloads under the control of the virus replication cycle or exogenous promoters. As an initial exemplification of the platform we have successfully produced EnAd variants encoding full-length (NG-135) and ScFv (NG-76) forms of anti-human VEGF antibodies. These have similar virus activity profiles to EnAd in cancer cell lines in vitro (virus replication, gene expression and oncolytic action), but also express and release the respective anti-VEGF antibody forms into the culture supernatant of tumor cells but not non-transformed cells. Using HCT-116 or DLD human colon carcinoma xenograft models we have shown that the virus infection profile following intra-tumoral injection is also similar to the parental EnAd virus (virus replication and hexon gene expression). Anti-VEGF antibody expression could be detected in the tumor tissue as both mRNA and functional antibody. Antibodies were detectable early (within 3 days of infection) and sustained over several weeks. Using an orthotopic A549 lung tumor model, NG-135 virus dosed i.v. following the development of tumors was able to decrease tumor burden by >90%. Viruses similarly expressing IgG1 or ScFv versions of anti-PDL1 and anti-CTLA4 checkpoint inhibitor antibodies have also been made and shown to produce functional antibodies that inhibit the respective receptor-ligand interactions in a range of in vitro assays, including upregulation of T-cell responses. In conclusion, our data show that EnAd can be modified to produce different antibody “payloads” following infection of human tumor cells in vitro and in vivo. Evaluation of the in vivo impact of these armed oncolytic viruses on the growth and microenvironment of tumors is now in progress. Citation Format: Brian R. Champion, Prithvi Kodialbail, Sam Illingworth, Nalini Rasiah, Daniel Cochrane, John Beadle, Kerry Fisher, Alice Brown. Delivery of checkpoint inhibitor antibodies and other therapeutics directly to tumors by encoding them within the oncolytic adenovirus enadenotucirev. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2015-295
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