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.

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.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

Research Doctorate - Doctor of Philosophy (PhD) / A number of oncolytic picornaviruses are currently under evaluation as potential therapeutic agents for a range of human malignancies. In particular, a subset of naturally occurring human C-cluster enteroviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15), Coxsackievirus A18 (CVA18) and Coxsackievirus A21 (CVA21) and the human B-cluster enterovirus, Echovirus 1 (EV1), display promising pre-clinical oncolytic activity against a wide variety of neoplastic cells. CVA21 is currently under clinical evaluation for the control of melanoma, breast, prostate and head/neck cancer. The preferential targeting of cancer cells by this subset of viruses is based on extracellular capsid interactions with specific viral receptors (intercellular adhesion molecule-1 [ICAM-1], decay-accelerating factor [DAF] or integrin α2β1), on the surface of malignant cells. In the present study, the therapeutic potential of this subset of enteroviruses was evaluated as a novel treatment strategy for the control of human malignancies of the gastrointestinal system. In Chapter 3, the capacity of the aforementioned enteroviruses for oncolytic activity was assessed in a panel of in vitro human gastric cancer cell cultures. Flow cytometric analysis revealed low-to-medium levels of ICAM-1, in addition to abundant α2β1 and DAF expression on the surface of gastric cancer cell lines. Cell monolayer lytic infectivity assays demonstrated that, of the viruses under evaluation, EV1 displayed the most potent and widespread in vitro lytic activity against the gastric cancer cell lines. Monoclonal antibody blockade confirmed the specific integrin α2β1-mediated route of EV1 cell infection in the gastric cancer MKN-45 cell line. Subsequently, an in vivo dose ranging study assessing the efficacy of oncolytic EV1 was undertaken in an immune-compromised MKN-45-Luc mouse model of human gastric cancer peritoneal carcinomatosis (PC). In this model, an intra-peritoneal dose of as little as 1x103 TCID50 EV1 resulted in a significant reduction in peritoneal tumour burden. In Chapter 4, the oncolytic capacity of this enterovirus subset was further evaluated, as a potential therapeutic option for the control of colorectal cancer (CRC). Flow cytometric analysis of a panel of CRC cell lines demonstrated abundant levels of DAF and integrin α2β1, and low-to-moderate levels of ICAM-1 expression on the surface of CRC cells. Of the subset of viruses examined, a DAF-using variant of CVA21 (CVA21-DAFv) displayed the most potent and widespread oncolytic activity against in vitro CRC cell cultures. Consequently, the potential in vivo oncolytic capacity of CVA21-DAFv and the wild-type CVA21 was evaluated in three individual immune-compromised mouse sub-cutaneous xenograft models of human CRC. However, despite the immunohistochemical detection of ICAM-1/DAF on cells of the CRC xenografts, and the detection of infectious virus in the blood of treated tumour-bearing mice, a detectable reduction in tumour burden was not observed. On account of the varying degrees of oncolytic efficacy observed in colorectal and gastric cancers, global gene expression profiling was employed in Chapters 5 and 6, to further elucidate the molecular mechanisms of enterovirus-mediated tumour cell tropism and cell death. As the most extensively characterised virus in pre-clinical studies, and the only virus of this subset under current clinical evaluation, CVA21 was selected as the challenge virus for analysis of the transcriptional response to enterovirus infection. Malignant cells that displayed reproducible susceptibility to in vitro and in vivo lytic CVA21 challenge were necessary for extensive characterisation, therefore, melanoma SK-Mel-28 and breast cancer MDA-MB-231-Luc cell lines, rather than CRC cell lines, were utilised. In Chapter 5, the response of SK-Mel-28 and MDA-MB-231-Luc cell monolayers, and a supporting panel of malignant and normal cell lines, to in vitro CVA21 challenge was assessed. In Chapter 6, the transcriptional response of immune-compromised mouse SK-Mel-28 and MDA-MB-231-Luc xenograft cells to systemic CVA21 administration was characterised. The transcriptional response of cells propagated as in vitro monolayers differed markedly when compared to that of in vivo xenografts generated from the same cell lines. In Chapter 5, a delayed rate of CVA21 replication and cell lysis was observed in normal cell cultures, as compared to malignant cell lines. Gene expression profiling suggested that the normal human lung fibroblast cell line, MRC-5, mounted an interferon (IFN)-mediated innate immune response against CVA21 challenge, a phenomenon not observed following challenge of the malignant cell line panel. Such findings suggest a potential role for the functional status of the IFN-mediated innate immune system in the tumour cell tropism of oncolytic CVA21. Somewhat surprisingly, in Chapter 6, an IFN-mediated transcriptional response was observed in the SK-Mel-28/MDA-MB-231-Luc xenograft cells, potentially attributed to the ‘priming’ effects of in vivo endogenous murine IFN activity. Furthermore, in Chapters 5 and 6, the potential contributions of transcriptionally regulated genes, in respect to their biological roles in cell cycle regulation, apoptosis, oxidative stress, stimulation of anti-tumoural immunity, and inhibition of angiogenesis in CVA21-mediated oncolysis were considered. Moreover, in Chapter 6, a distinct genetic signature of infection was identified, comprising a total of 9 individual genes, significantly upregulated in response to infection in each xenograft model at 24 and 72 h following the systemic administration of CVA21. The identified genes involved in this core transcriptional response to infection may serve as effective molecular biomarkers for the evaluation of oncolytic CVA21 efficacy.

Research Doctorate - Doctor of Philosophy (PhD) / A number of oncolytic picornaviruses are currently under evaluation as potential therapeutic agents for a range of human malignancies. In particular, a subset of naturally occurring human C-cluster enteroviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15), Coxsackievirus A18 (CVA18) and Coxsackievirus A21 (CVA21) and the human B-cluster enterovirus, Echovirus 1 (EV1), display promising pre-clinical oncolytic activity against a wide variety of neoplastic cells. CVA21 is currently under clinical evaluation for the control of melanoma, breast, prostate and head/neck cancer. The preferential targeting of cancer cells by this subset of viruses is based on extracellular capsid interactions with specific viral receptors (intercellular adhesion molecule-1 [ICAM-1], decay-accelerating factor [DAF] or integrin α2β1), on the surface of malignant cells. In the present study, the therapeutic potential of this subset of enteroviruses was evaluated as a novel treatment strategy for the control of human malignancies of the gastrointestinal system. In Chapter 3, the capacity of the aforementioned enteroviruses for oncolytic activity was assessed in a panel of in vitro human gastric cancer cell cultures. Flow cytometric analysis revealed low-to-medium levels of ICAM-1, in addition to abundant α2β1 and DAF expression on the surface of gastric cancer cell lines. Cell monolayer lytic infectivity assays demonstrated that, of the viruses under evaluation, EV1 displayed the most potent and widespread in vitro lytic activity against the gastric cancer cell lines. Monoclonal antibody blockade confirmed the specific integrin α2β1-mediated route of EV1 cell infection in the gastric cancer MKN-45 cell line. Subsequently, an in vivo dose ranging study assessing the efficacy of oncolytic EV1 was undertaken in an immune-compromised MKN-45-Luc mouse model of human gastric cancer peritoneal carcinomatosis (PC). In this model, an intra-peritoneal dose of as little as 1x103 TCID50 EV1 resulted in a significant reduction in peritoneal tumour burden. In Chapter 4, the oncolytic capacity of this enterovirus subset was further evaluated, as a potential therapeutic option for the control of colorectal cancer (CRC). Flow cytometric analysis of a panel of CRC cell lines demonstrated abundant levels of DAF and integrin α2β1, and low-to-moderate levels of ICAM-1 expression on the surface of CRC cells. Of the subset of viruses examined, a DAF-using variant of CVA21 (CVA21-DAFv) displayed the most potent and widespread oncolytic activity against in vitro CRC cell cultures. Consequently, the potential in vivo oncolytic capacity of CVA21-DAFv and the wild-type CVA21 was evaluated in three individual immune-compromised mouse sub-cutaneous xenograft models of human CRC. However, despite the immunohistochemical detection of ICAM-1/DAF on cells of the CRC xenografts, and the detection of infectious virus in the blood of treated tumour-bearing mice, a detectable reduction in tumour burden was not observed. On account of the varying degrees of oncolytic efficacy observed in colorectal and gastric cancers, global gene expression profiling was employed in Chapters 5 and 6, to further elucidate the molecular mechanisms of enterovirus-mediated tumour cell tropism and cell death. As the most extensively characterised virus in pre-clinical studies, and the only virus of this subset under current clinical evaluation, CVA21 was selected as the challenge virus for analysis of the transcriptional response to enterovirus infection. Malignant cells that displayed reproducible susceptibility to in vitro and in vivo lytic CVA21 challenge were necessary for extensive characterisation, therefore, melanoma SK-Mel-28 and breast cancer MDA-MB-231-Luc cell lines, rather than CRC cell lines, were utilised. In Chapter 5, the response of SK-Mel-28 and MDA-MB-231-Luc cell monolayers, and a supporting panel of malignant and normal cell lines, to in vitro CVA21 challenge was assessed. In Chapter 6, the transcriptional response of immune-compromised mouse SK-Mel-28 and MDA-MB-231-Luc xenograft cells to systemic CVA21 administration was characterised. The transcriptional response of cells propagated as in vitro monolayers differed markedly when compared to that of in vivo xenografts generated from the same cell lines. In Chapter 5, a delayed rate of CVA21 replication and cell lysis was observed in normal cell cultures, as compared to malignant cell lines. Gene expression profiling suggested that the normal human lung fibroblast cell line, MRC-5, mounted an interferon (IFN)-mediated innate immune response against CVA21 challenge, a phenomenon not observed following challenge of the malignant cell line panel. Such findings suggest a potential role for the functional status of the IFN-mediated innate immune system in the tumour cell tropism of oncolytic CVA21. Somewhat surprisingly, in Chapter 6, an IFN-mediated transcriptional response was observed in the SK-Mel-28/MDA-MB-231-Luc xenograft cells, potentially attributed to the ‘priming’ effects of in vivo endogenous murine IFN activity. Furthermore, in Chapters 5 and 6, the potential contributions of transcriptionally regulated genes, in respect to their biological roles in cell cycle regulation, apoptosis, oxidative stress, stimulation of anti-tumoural immunity, and inhibition of angiogenesis in CVA21-mediated oncolysis were considered. Moreover, in Chapter 6, a distinct genetic signature of infection was identified, comprising a total of 9 individual genes, significantly upregulated in response to infection in each xenograft model at 24 and 72 h following the systemic administration of CVA21. The identified genes involved in this core transcriptional response to infection may serve as effective molecular biomarkers for the evaluation of oncolytic CVA21 efficacy.

Despite improvements in chemotherapy and radical surgical debulking, peritoneal carcinomatosis (PC) remains among the most common causes of death for abdominal cancers. Immunotherapies have demonstrated efficacy in selected solid malignancies but their potential in PC is poorly explored. Here I report that intraperitoneal injection of an infected cell vaccine (ICV), consisting of autologous tumor cells infected ex-vivo with an oncolytic Maraba MG1 virus expressing interleukin-12 (IL-12), promotes the migration of activated natural killer (NK) cells to the peritoneal cavity in response to the secretion of interferon gamma-induced protein-10 (IP-10) from dendritic cells. This recruitment of cytotoxic, IFNγ-secreting NK cells is associated with a dramatic reduction in tumor burden and improved survival in a colon cancer model of PC. Even in mice with bulky PC (tumors >8 mm), a complete radiological response was demonstrated within 8-14 weeks, associated with 100% long-term survival. Importantly, these results were recapitulated in human lymphocytes exposed to human tumor cell lines infected with MG1-IL12. Finally, I demonstrate that MG1-IL12-ICV generates an effective CD4 and CD8 T cell response in mice following prophylactic immunization associated with the maturation of peritoneal dendritic cells and enrichment of tumor-specific peritoneal T cells. The research presented in this thesis suggests that an MG1-IL12-ICV is a promising therapy that could provide benefit to the thousands of patients diagnosed with PC each year.