Hereditary breast and ovarian cancers (HBOC) represent 5-10% of breast and 10-15% of ovarian cancer cases. These cancers tend to be aggressive and curative treatment strategies are scarce. Poly(ADP-ribose) polymerase inhibitors (PARPi), a family of drugs that inhibit DNA repair, are a promising therapy for cancers harbouring mutations in their DNA repair machinery, such as HBOC. Unfortunately, nearly all patients ultimately become resistant to PARPi, leaving limited options for definitive treatment. Oncolytic or “cancer-killing” viruses are an innovative immunotherapeutic platform capable of selectively targeting cancer cells, leaving healthy tissues unharmed. Our group has demonstrated that oncolytic rhabdoviruses may be used to deliver therapeutic payloads by encoding targeting sequences to act on genes via RNA interference. In the present work, I have engineered the oncolytic virus, vesicular stomatitis virus (VSV), to express a variety of microRNA (miRNA) sequences that target genes essential for DNA repair, sensitizing resistant cancer cells to PARPi therapy. After initial experiments revealed hurdles concerning the functionality of artificial miRNAs which specifically target BRCA1 and BRCA2 I encoded the naturally occurring hsa-miR-182 into VSV to knockdown BRCA1 and additional genes essential for DNA repair. Using a 3D spheroid model, I have demonstrated sensitization of initially resistant MDA-MB-231 breast cancer cells to the PARPi, rucaparib. Complementary work exploring the shuttling of miRNAs into small extracellular vesicles, or EVs, has also shown that we can take advantage of the EV packaging facilities in infected cells, inducing the packaging of miRNAs over-expressed by VSV (EV-miRNAs) into EVs. Future work will address the functionality of these EV-miRNAs, testing their ability to knockdown targets in uninfected cancer cells.

Oncolytic viruses (OVs) have begun to show their promise in the clinical setting, however these results have been predominantly associated with loco-regional administration of virus. The treatment of metastatic disease necessitates a systemic approach to virus delivery. The circulatory system, though, is a hostile environment for viruses and the advantages associated with intravenous (IV) delivery come at a heavy cost that must be understood and brokered. Pre-existing immunity, specifically through the function of antibody and complement, poses a significant hurdle to the IV delivery of infectious virus to dispersed tumor beds. This is of particular importance for therapeutic vaccinia viruses as a majority of today’s cancer patients were vaccinated during the smallpox eradication campaign. In vitro neutralization assays of oncolytic vaccinia virus demonstrated that the antibodies elicited from smallpox vaccination, and also the anamnestic response in patients undergoing Pexa-Vec treatment, was minimally neutralizing in the absence of functional complement. Accordingly, in a Fischer rat model, complement depletion stabilized virus in the blood of pre-immunized hosts and correlated with improved delivery to mammary adenocarcinoma tumors. Complement depletion additionally enhanced infection of tumors following direct intratumoral injection of virus. The feasibility and safety of using a complement inhibitor, CP40, was tested in a cynomolgus macaque model. Immune animals saw an average 10-fold increase in infectious virus titer at an early point after the infusion, and a prolongation of the time during which infectious virus was still detectable in the blood. We have also demonstrated that vaccinia virus engages in promiscuous interactions with cells in the blood and that these interactions may be partially complement-dependent. Additionally, we have translated this complement inhibition approach to other OV candidates and found that reovirus, measles virus and a virus pseudo typed with the LCMV glycoprotein all elicit antibodies, that to some degree, are dependent on complement activation to neutralize their target viruses. We show here that capitalizing on the complement dependence of anti-viral antibody with adjunct complement inhibitors may increase the effective dose to enable successful delivery of multiple rounds of OV in immune hosts.

Oncolytic viruses (OVs) are an emerging cancer therapy that use an oncotropic virus to selectively infect and kill cancer cells, as well as stimulate long-lasting anti-tumor immune responses. In order to achieve high therapeutic efficacy, OVs need sufficient replication within the tumor tissue to mediate these effects. However, OV’s infectivity varies between different tumors and the host’s immune system can rapidly clear the virus, hampering treatment efficiency. Oncolytic virus sensitizers are chemical compounds that specifically enhance OV’s infectivity and efficacy. In our lab, I found that treatment of various cancer cell lines with BI-D1870, a pan-RSK (ribosomal S6 kinase) inhibitor, resulted in augmented Herpes Simplex Virus-1 (HSV1) and Vesicular Stomatitis Virus (VSVΔ51) infectivity. I also demonstrated that the effects of BI-D1870 on viral infection are virus-specific, and that RSK inhibition is not the primary target causing the enhancement of HSV1 and VSVΔ51 infection. Finally, BI-D1870 structural analogs were generated in an attempt to enhance the efficacy and selectivity of BI-D1870-based OV sensitizers. One of the analogs synthesized, KA-019, showed an improvement in the augmentation of OV infection over BI-D1870. As a genetically engineered strain of HSV1 has been approved by FDA for treatment of melanoma, the results of my project propose a novel viral sensitizer to improve viral replication within tumour cells with the hope of improving therapeutic efficacy.

Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are blood cancers that are often treated with stem cell transplantation (SCT). Since SCT treatments have variable success, especially in adults with AML whose disease frequently relapses, novel and more effective solutions must be considered. In this thesis, I will explore one type of immunotherapy in murine models for ALL (L1210) and AML (C1498) using in vitro and in vivo techniques such as flow cytometry and transcriptomics. In my approach, I am attempting to enhance the immunogenicity of whole cell vaccines by pre-infecting the leukemia cells with oncolytic virus (OV) and thus producing leukemia infected cell vaccines (ICVs). While it has been previously shown that L1210-ICV pre-treatment works well in protecting mice from ALL challenge, I have found that pre-immunization with C1498-ICV has a limited efficacy in protecting animals from AML progression. By investigating the downstream effects of ICV, I was able to show that unlike C1498 cells, L1210 cells produce previously unknown immunogenic factors following OV infection.

Vaccinia virus has a large and still incompletely understood genome although several strains of this virus are already in clinical development. For the most part, clinical candidates have been attenuated from their wild type vaccine strains through deletion of metabolic genes like the viral thymidine kinase gene.In the present work, we thoroughly examined the genetic elements of vaccinia which could be modulated to tailor the virus as a cancer therapeutic. Using a variety of cancer cell lines and primary tumor explants, we performed a fitness assay that directly compares multiple wild-type Vaccinia strains to identify the genetic elements that together create an optimal “oncolytic engine”. Using a transposon insertion strategy and deep sequencing of viral populations we systematically examined Vaccinia genes that do or do not play a role in the therapeutic activity of the virus. Our studies allowed us to identify a variety of genes in the vaccinia genome that when deleted, augment the oncolytic activity of a newly engineered Vaccinia virus. In the context of this thesis, I define enhanced oncolytic activity as superior therapeutic activity, increased immunogenicity and an improved safety profile, all aspects which we used to compare this novel virus to Vaccinia viruses currently in the clinic.

Oncolytic viruses (OV) are a promising alternative cancer therapy due to their specificity and lack of debilitating side effects, such as those which typically accompany conventional therapeutics such as chemotherapy and radiation. Bovine herpesvirus type 1 (BHV-1) is an alphaherpesvirus with the ability to infect and kill multiple human tumor cell types. In comparison to other species-specific viruses, for which deficiencies in type I interferon signalling pathways dictates cellular sensitivity to infection, mutations in KRAS were found to correlate with high levels of BHV-1 replication. Interestingly, BHV-1 is able to induce cellular cytotoxicity in the absence of a productive infection. In contrast to current breast cancer (BC) treatments, which are largely based on receptor expression status, BHV-1 is able to infect and kill BC cells and breast cancer initiating cells (BCICs) from luminal and basal subtypes. Furthermore, BHV-1-infected BC cells are significantly diminished in their capacity to form tumors in vivo, suggesting that BHV-1 reduces the tumor forming capacity of BCICs. Combination therapy involving OVs has been used to exploit differences in the mechanism of tumor cell death elicited by individual treatments. Treatment with epigenetic modifiers such as 5-Azacytidine (5-Aza), a DNA methyltransferase inhibitor, has been shown to increase the antitumor activity of OVs. Our data indicates that 5-Aza strongly synergises with BHV-1, increasing virus replication and cytotoxicity in vitro. In vivo, BHV-1 monotherapy did not significantly impact tumor growth or survival of CR bearing subcutaneous breast tumors; however, combination therapy with 5-Aza significantly decreased the number of secondary lesions compared to BHV-1 monotherapy. Overall, the data presented in this dissertation indicate that BHV-1 is a promising broad spectrum OV with a unique mechanism of tumor cell targeting, and the ability to infect and kill tumor cells independent of a productive infection. / Thesis / Doctor of Philosophy (Medical Science)

Treatment of permissive tumours with the oncolytic virus (OV) VSV-Δ51 leads to a robust anti-tumour T cell response, which contributes to efficacy; however, many tumours are not permissive to in vivo treatment with VSV-Δ51. In an attempt to channel the immune stimulatory properties of VSV-Δ51 and broaden the scope of tumours that can be treated by an OV, a potent oncolytic vaccine platform was developed, consisting of tumour cells infected with VSV-Δ51. I demonstrate that prophylactic immunization with this infected cell vaccine (ICV) protected mice from subsequent tumour challenge, and expression of GM-CSF by the virus (VSVgm-ICV) increased efficacy. Immunization with VSVgm-ICV in the VSV-resistant B16-F10 model induced maturation of dendritic cells, natural killer (NK) cells, and T cells. I demonstrate that this approach is robust enough to control the growth of established and spontaneous tumours. This strategy is broadly applicable because of VSV’s extremely broad tropism, allowing nearly all cell types to be infected at high MOIs in vitro, where the virus replication kinetics outpace the cellular IFN response. It is also personalized to the unique tumour antigen(s) displayed by the cancer cell. Histone deacetylase inhibitors (HDIs) can augment viral replication, making them particularly interesting complements to OV therapy. However, the impact of HDIs on the generation and re-stimulation of immune responses remains to be clearly elucidated. Along with my collaborators at McMaster University, I demonstrate that MS-275, but not SAHA, selectively depletes naïve and regulatory lymphocytes. Memory lymphocytes that are being boosted remain unscathed and even have enhanced cytokine production, potentially as a consequence of the depleted lymphocyte compartment. This leads to a delay in anti-VSV neutralizing antibodies and T cell responses. Interestingly, HDI treatment of B16-F10 cells appears to inhibit VSV replication but allows for a longer persistence within the tumour. When used in an oncolytic prime/boost vaccination model, MS-275 potently enhanced survival. Though the anti-tumour immune response is enhanced, a near complete reduction in autoimmune vitiligo is observed with MS-275 administration. Therefore, this HDI uniquely modulates the immune response to enhance anti-tumour immunity and decrease the anti-viral response, while also decreasing autoimmune sequelae.

Treatment of permissive tumours with the oncolytic virus (OV) VSV-Δ51 leads to a robust anti-tumour T cell response, which contributes to efficacy; however, many tumours are not permissive to in vivo treatment with VSV-Δ51. In an attempt to channel the immune stimulatory properties of VSV-Δ51 and broaden the scope of tumours that can be treated by an OV, a potent oncolytic vaccine platform was developed, consisting of tumour cells infected with VSV-Δ51. I demonstrate that prophylactic immunization with this infected cell vaccine (ICV) protected mice from subsequent tumour challenge, and expression of GM-CSF by the virus (VSVgm-ICV) increased efficacy. Immunization with VSVgm-ICV in the VSV-resistant B16-F10 model induced maturation of dendritic cells, natural killer (NK) cells, and T cells. I demonstrate that this approach is robust enough to control the growth of established and spontaneous tumours. This strategy is broadly applicable because of VSV’s extremely broad tropism, allowing nearly all cell types to be infected at high MOIs in vitro, where the virus replication kinetics outpace the cellular IFN response. It is also personalized to the unique tumour antigen(s) displayed by the cancer cell. Histone deacetylase inhibitors (HDIs) can augment viral replication, making them particularly interesting complements to OV therapy. However, the impact of HDIs on the generation and re-stimulation of immune responses remains to be clearly elucidated. Along with my collaborators at McMaster University, I demonstrate that MS-275, but not SAHA, selectively depletes naïve and regulatory lymphocytes. Memory lymphocytes that are being boosted remain unscathed and even have enhanced cytokine production, potentially as a consequence of the depleted lymphocyte compartment. This leads to a delay in anti-VSV neutralizing antibodies and T cell responses. Interestingly, HDI treatment of B16-F10 cells appears to inhibit VSV replication but allows for a longer persistence within the tumour. When used in an oncolytic prime/boost vaccination model, MS-275 potently enhanced survival. Though the anti-tumour immune response is enhanced, a near complete reduction in autoimmune vitiligo is observed with MS-275 administration. Therefore, this HDI uniquely modulates the immune response to enhance anti-tumour immunity and decrease the anti-viral response, while also decreasing autoimmune sequelae.

Oncolytic viruses are currently under investigation as anti - cancer therapies due to their innate ability to selectively infect and destroy cancer cells. Major barriers to this anti - tumour effect include inefficient viral spread and immune - mediated neutralization. This study aims to overcome these limitations by taking advantage of the life cycle of the oncolytic clinical candidate known as vaccinia virus (VACV). Naturally, a small proportion (<1%) of VACV progeny are released from infected cells with a cell - derived membrane and become known as extra - cellular enveloped virus (EEV). Due to this additional membrane, EEV can be shielded from many anti -viral immune factors , allowing it to travel further and largely avoid host - mediated neutralization. This form of VACV is important for long range virus dissemination as well as sustained infection. Though the exact mechanism remains to be elucidated, it has been demonstrated that EEV release can be influenced by Abl tyrosine kinase (Abl TK) function. Specific point mutations in viral envelope proteins are known to bring about enhanced viral release, resulting in an elevated proportion of produced EEV. In this study, we investigate the effect of EEV enhancing modifications within various oncolytic VACV strains. Our data reveals that this augmentation of EEV production through the A34R L151E mutation within the Copenhagen (Cop) backbone can enhance the oncolytic potential of VACV in vivo through enhanced spread and immunoevasion.

Oncolytic viruses are characterised by their ability to selectively infect and kill tumour cells. Recently it has emerged that they can exert an additional anticancer mechanism stimulating adaptive immune-mediated cancer cell killing. Enadenotucirev (EnAd, formerly known as ColoAd1), is a chimeric Ad11p/Ad3 virus group B oncolytic adenovirus that binds CD46 and is under development for the systemic treatment of metastatic carcinomas. The central aim of this thesis was to to assess whether EnAd provides an adjuvant effect on tumour-associated antigen presenting cells (APCs) that could drive T_H1 polarisation for an effective anti-tumour immune response. This thesis describes the potent oncolytic properties, fast replication and high numbers of virus progeny production by EnAd in cancer cells. Recombinant EnAd variants were engineered to investigate the roles of the mutant regions in the genome of EnAd, and how these influence the modified phenotype. A chemical drug panel was used to identify pathways and cellular factors involved in cellular production of EnAd, finding that several mTOR inhibitors and microtubule inhibitors could improve virus replication. An in vitro system using partially matured human monocyte-derived dendritic cells (DCs), which displayed a similar phenotype to tumour-infiltrating DCs, was used to explore the effect of EnAd on APC responses. EnAd induced a strong adjuvant effect on these cells by up-regulating surface markers and secretion of pro-inflammatory factors. Further mechanistic experiments, alongside a CAR-binding group C adenovirus 5, indicated these adjuvant effects were virus particle-mediated and dependent on CD46 binding. To understand the functional implications downstream of these interactions, T cell activation and phenotype was assessed using a mixed lymphocyte reaction approach. The data indicated EnAd was a good candidate compared to other adenoviruses, that may steer the response of activated T-cells towards a T_H1 phenotype, for an effective immune response. In conclusion, the potent oncolytic properties of EnAd virus may provide an adjuvant effect on tumour-associated APCs, helping to harness an adaptive immune response.