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.

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.

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.

The optimal route for clinical delivery of oncolytic viruses (OVs) is thought to be intravenous (IV) injection; however, the immune system is armed with several highly efficient mechanisms to remove pathogens from the circulatory system. To overcome the challenges in trying to deliver OVs IV, cell carriers have been investigated to determine their suitability as delivery vehicles for systemic administration of OVs. Here we demonstrate the utility of a Drosophila melanogaster cell platform for the production and in vivo delivery of multi-gene biotherapeutic systems. We show that cultured Drosophila S2 cell carriers can stably propagate OV therapeutics that are highly cytotoxic for mammalian cancer cells without adverse effects on insect cell viability or cellular gene expression. Drosophila cell carriers administered systemically to immunocompetent animals trafficked to tumours to deliver multiple biotherapeutics with little apparent off-target tissue homing or toxicity, resulting in a therapeutic effect. S2 cells provide a genetically tractable platform supporting the integration of complex, multi-gene biotherapies while avoiding many of the barriers to systemic administration of mammalian cell carriers. Once OVs are delivered to tumour beds, they initiate replication in tumour cells, which often possess defects in antiviral pathways and are thus susceptible to infection. However, not all tumours have defects in their antiviral defenses and thus virus replication in these tumours is rather limited. Identifying and modulating host factors that regulate virus replication in OV-resistant cancer cells, but not normal cells, could lead to increased replication in these tumours and potentially improve therapeutic outcomes. We therefore conducted an RNA interference screen using Sindbis virus (SINV) in order to identify host factors that modulate OV replication in tumour cells. Specifically, serial passage of a SINV- artificial microRNA (amiRNA) library in a tumour cell line followed by deep sequencing of ii the selected virus populations led to the identification of several amiRNA sequences that were enriched. Furthermore, the identified amiRNA sequences increased the replication of various OVs both in vitro and in vivo, ultimately resulting in an enhanced therapeutic effect. Overall, the work presented here highlights strategies in which both the systemic delivery and tumour-specific replication of OVs can be improved.

Pancreatic cancer (PDAC) affects 4400 Canadians each year and with 5year survival rates <8%, there is clearly an unmet need for new therapeutic approaches for treating this deadly disease. Herein I report the development of a surgical model of PDAC that recapitulates many of the hallmarks of human disease and has an immune infiltrate consisting of T cells and suppressive regulatory T cells and myeloid derived suppressor cells. This model allows the exploration of new therapeutics that can be used in combination with surgical resection of primary tumours. Furthermore, I propose that the use of neoadjuvant administration of a prime-boost oncolytic vaccine targeting a pancreatic tumour associated antigen (TAA) - mesothelin - could potentiate pancreatic tumour specific immune responses to improve patient prognosis. We demonstrate that immune tolerance to this self antigen can be broken by the complete depletion of circulating Tregs at the time of vaccination, which leads to the activation of a population of CD8+ T cells responsive to mesothelin. We demonstrate that these T cells respond to mesothelin expressing tumour cells ex vivo, and that CD8+ T cells are recruited to the site of tumour challenge. However, despite the generated CD8+ T cell response, oncolytic vaccine strategies targeting mesothelin provide no protection against Pan02 tumours, or against other mesothelin expressing murine tumour lines. I demonstrate that this is not through common tumour escape mechanisms, nor through the upregulation of suppressive immune populations. Any efficacy observed was found to be provided solely by depletion of Tregs, as the depletion of CD8+ T cells did not reduce protection from tumour outgrowth in vaccinated mice. While mesothelin represents a promising target, it is not an ideal target for oncolytic vaccine platforms, potentially due to its nature as a self antigen.

Cancer is a multifaceted and intricate disease that poses a significant global health burden and impacts millions of individuals worldwide. Among the diverse subtypes of cancer, sarcoma stands out as a rare yet highly aggressive malignancy originating from connective tissues such as bone, cartilage, and muscle, and presents challenges in diagnosis and treatment. Despite remarkable progress in cancer research and therapy, the prognosis for sarcoma patients remains low, requiring development of novel therapeutic avenues. Cancer immunotherapies focused on generating tumor-specific responses are emerging as promising alternatives to traditional cancer treatments. T cell-based immunotherapies, such as cancer vaccines and CAR-T cells, are designed to target tumor antigens and generate long term immune memory capable of constant surveillance against recurrence. Therefore, the objective of this study is to establish the groundwork for a novel T cell-based immunotherapeutic approach tailored specifically to sarcoma. Throughout the study, we explored various critical aspects associated with the development of immunotherapy. First, we conducted a proof-of-concept study, evaluating a novel prime-boost vaccine combination employing anti-DEC205 and oncolytic rhabdoviruses targeting a model antigen in a pre- clinical model of melanoma. This study showed that using the DEC205 dendritic cell-targeting antibody as a vector for antigen delivery is a promising alternative to other prime-boost strategies being evaluated in the clinic (NCT02285816). To facilitate the translation of this therapeutic approach to clinical applications, a comprehensive understanding of the human sarcoma tumor immune microenvironment and the identification of a suitable target antigen are essential. Therefore, we conducted an in-depth immune profiling of a high grade and aggressive dedifferentiated liposarcoma (DDLS) using gene expression profiling and immunohistochemistry. We gained valuable insights into the tumor biology and the complex immunological mechanisms within the tumor immune microenvironment. Notably, we identified a novel antigen that is highly expressed in human DDLS and absent in normal tissues, that could be used as a potential antigenic target for immunotherapy. Finally, we evaluated a range of prime and boost vaccine vectors targeting the newly discovered target antigen in pre-clinical murine sarcoma models. Ultimately, we found that an oncolytic rhabdovirus prime and a modified vaccinia Ankara virus boost targeting the sarcoma antigen generates strong antigen specific cellular and humoral responses and protects against tumor growth in a prophylactic model of sarcoma. Altogether, this study lays the foundations for the development of a T cell-based immunotherapy employing an oncolytic rhabdovirus and targeting a novel antigen for the treatment of sarcoma.

Glioblastoma multiforme is the most common and lethal primary brain tumour in adults. Its aggressive and invasive phenotype makes it resistant to current standards of care, with a patient median survival following treatment of only 14 months. Potent and safe therapeutics are necessary to improve patient prognosis. Globally, efforts are being made in immunotherapies to combat such deleterious tumours. Preliminary work in the Stojdl lab has developed a novel oncolytic virus platform for brain cancer therapy that is non-toxic and exhibits potent anti-tumour efficacy. This platform is based on the rhabdovirus Farmington, identified for its potent oncolytic properties and engineering malleability. Herein, we begin to show our capability to discover and vaccinate against immunogenic neoepitopes derived from a mouse cancer mutanome. Engineering Farmington virus to express neoepitopes, allows for robust tumour specific immune proliferation following a prime vaccination. Overcoming problems of targeting self-antigen and antigen loss variants, a multi-neoepitope vaccine, presented here, is one of many alternative approaches to help combat cancer resistance. Despite achieving robust anti-tumour immunity by vaccination, selectivity of the tumour microenvironment remains an enormous challenge. Cumulative efforts in immunotherapy research will help drive novel therapeutics, like Farmington, into clinic and, ultimately, improve patient’s prognosis and quality of life.