Sam Illingworth

Tropism ablation and stealthing of oncolytic adenovirus enhances systemic delivery to tumors and improves virotherapy of cancer

Intravenous delivery of therapeutic virus particles remains a major goal for virotherapy of metastatic cancer. Avoiding phagocytic capture and unwanted infection of nontarget cells is essential for extended plasma particle kinetics, and simply ablating one or the other does not give extended plasma circulation. Here we show that polymer coating of adenovirus type 5 (Ad5) can combine with predosing strategies or Kupffer cell ablation to achieve systemic kinetics with a half-life >60 min, allowing ready access to peripheral tumors. Accumulation of virus particles within tumor nodules is proportional to the area under the plasma concentration/time curve. Polymer coating wild-type Ad5 in this way is known to decrease hepatic toxicity, increasing the dose of virus particles that can be safely administered. Using polymer-coating technology to deliver a replicating Ad5 systemically, virus replication and transgene expression was almost totally confined to tumor tissues, giving a much improved therapeutic index compared with uncoated virus, and complete control of human HepG2 tumor xenografts.

Oncolytic Group B Adenovirus Enadenotucirev Mediates Non-apoptotic Cell Death with Membrane Disruption and Release of Inflammatory Mediators

Enadenotucirev (EnAd) is a chimeric group B adenovirus isolated by bioselection from a library of adenovirus serotypes. It replicates selectively in and kills a diverse range of carcinoma cells, shows effective anticancer activity in preclinical systems, and is currently undergoing phase I/II clinical trials. EnAd kills cells more quickly than type 5 adenovirus, and speed of cytotoxicity is dose dependent. The EnAd death pathway does not involve p53, is predominantly caspase independent, and appears to involve a rapid fall in cellular ATP. Infected cells show early loss of membrane integrity; increased exposure of calreticulin; extracellular release of ATP, HSP70, and HMGB1; and influx of calcium. The virus also causes an obvious single membrane blister reminiscent of ischemic cell death by oncosis. In human tumor biopsies maintained in ex vivo culture, EnAd mediated release of pro-inflammatory mediators such as TNF-α, IL-6, and HMGB1. In accordance with this, EnAd-infected tumor cells showed potent stimulation of dendritic cells and CD4+ T cells in a mixed tumor-leukocyte reaction in vitro. Whereas many viruses have evolved for efficient propagation with minimal inflammation, bioselection of EnAd for rapid killing has yielded a virus with a short life cycle that combines potent cytotoxicity with a proinflammatory mechanism of cell death.

Preclinical Safety Studies of Enadenotucirev, a Chimeric Group B Human-Specific Oncolytic Adenovirus

Enadenotucirev is an oncolytic group B adenovirus identified by a process of bio-selection for the ability to selectively propagate in and rapidly kill carcinoma cells. It is resistant to inactivation by human blood components, potentially enabling intravenous dosing in patients with metastatic cancer. However, there are no known permissive animal models described for group B adenoviruses that could facilitate a conventional approach to preclinical safety studies. In this manuscript, we describe our tailored preclinical strategy designed to evaluate the key biological properties of enadenotucirev. As enadenotucirev does not replicate in animal cells, a panel of primary human cells was used to evaluate enadenotucirev replication selectivity in vitro, demonstrating that virus genome levels were >100-fold lower in normal cells relative to tumor cells. Acute intravenous tolerability in mice was used to assess virus particle-mediated toxicology and effects on innate immunity. These studies showed that particle toxicity could be ameliorated by dose fractionation, using an initial dose of virus to condition the host such that cytokine responses to subsequent doses were significantly attenuated. This, in turn, supported the initiation of a phase I intravenous clinical trial with a starting dose of 1 × 1010 virus particles given on days 1, 3, and 5.

Development of a versatile oncolytic virus platform for local intra-tumoural expression of therapeutic transgenes

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.

Abstract 4875: Developing tumor-localized, combination immunotherapies

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 IFNa, MIP1a 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.

Arming the oncolytic virus enadenotucirev (EnAd) to generate enhanced locally-acting immunotherapies for cancer

Background EnAd is a potent, chimeric Ad11p/Ad3 adenovirus with selective oncolytic activity against a range of epithelial cancer cells. Ongoing clinical studies have shown that i. v. dosed EnAd infects and selectively replicates in tumor cells, producing detectable levels of viral protein, often with an associated high frequency of intra-tumoral CD8 cells. These data indicate that transgene-encoded proteins will also be effectively produced within tumors using an “armed” EnAd virus.

Abstract 295: Delivery of checkpoint inhibitor antibodies and other therapeutics directly to tumors by encoding them within the oncolytic adenovirus enadenotucirev

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