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Multiple myeloma is a plasma cell cancer that progressively evolves to an aggressive, multi-drug resistant disease, which presents an unmet clinical need. In clinical trials, myeloma shows susceptibility to novel immunotherapeutic agents, particularly those targeting B-cell maturation antigen (BCMA). Among different classes of immunotherapies, T cell-based approaches have progressed the most due to their ability to induce durable responses in patients with advanced drug-resistant blood cancers. Most T cell engineering strategies rely on the use of chimeric antigen receptors (CARs), which although effective, can cause serious life-threatening toxicities. We created a new synthetic receptor, T cell antigen coupler (TAC), which recruits the endogenous T cell receptor and allows T cells to autoregulate their activity. Our experience in solid tumor models has shown that TAC-T cells are similarly efficacious and significantly less toxic than CAR-T cells. This thesis describes our optimization of BCMA-specific TAC-T cells and analysis of different anti-BCMA antigen-binding domains. TAC receptor functions by engaging endogenous TCR-CD3 complex and redirecting it to the target of interest. In Chapter 3, we characterize optimization and humanization of the CD3-recruitment domain in the TAC scaffold and provide evidence that TAC-T cells are effective against multiple myeloma, irrespective of receptor surface levels. In Chapter 4, we describe selection of the human BCMA-binding domain and the creation of a fully humanized TAC receptor against BCMA. Chapters 5 and 6 describe how a BCMA-targeting antigen-binding domain that cross-reacts with an unknown antigen in mice augments in vivo efficacy of TAC- and CAR-T cells, respectively. The work described in Chapters 3 and 4 presents an optimized, fully human BCMA-TAC that is being moved into clinical testing. The work in Chapters 5 and 6 improves our understanding of how antigen-targeting domains in synthetic receptors influence the functionality of engineered T cells. / Thesis / Doctor of Science (PhD) / Multiple myeloma is an incurable blood cancer that has a remarkable ability to develop resistance to different types of chemotherapy. In recent years, treatments redirecting immune cells against tumors have shown impressive clinical responses against different types of chemotherapy-resistant blood cancers, including multiple myeloma. Our lab has developed a new technology for redirecting T cells against tumors, called T cell antigen coupler (TAC) receptor. This thesis describes optimization of a fully human TAC receptor specific for a target on the surface of myeloma cells, known as BCMA. Durable remissions induced by TAC-engineered T cells in a preclinical mouse model of myeloma in the absence of toxicity warrant further testing of this therapeutic in a clinical trial.

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Glioblastoma (GBM) remains the most aggressive primary brain tumor in adults. Since 2005, Standard of Care (SoC) consists of surgical resection followed by radiation and adjuvant chemotherapy with temozolomide. Treatment failure is attributed to intratumoral heterogeneity with populations capable of mechanisms to repair damaged DNA. Given the lack of progress to improve patient outcomes, the current work encompasses how multi-omic approaches can be utilized to uncover novel biology in GBM and develop precision medicines to exploit these cancer specific phenomena. Using patient derived GBM samples I first used the surface marker CD133 to interrogate glioblastoma stem cells, a subpopulation of cells identified to withstand conventional therapies and lead to tumor relapse. I used a genome-wide CRISPR-Cas9 library to conduct an unbiased loss-of-function phenotypic screen to identify regulators of CD133. I then validated SOX2 as a direct transcription factor to PROM1 encoding CD133. These findings further show the untapped potential of CRISPR to uncover novel biology to directly apply to broader fields of stem cells and cancer biology. Next, I combed GBM data sets at transcriptomic and proteomic levels to identify understudied proteins as potential targets for immunotherapies. Glycoprotein nonmetastatic melanoma protein B (GPNMB) has previously been identified as a clinically relevant target in GBM and shown to be active in the tumor immune microenvironment. I found GPNMB to be upregulated in recurrent GBM and macrophage populations which can be exploited in a more comprehensive manner to treat GBM. Through a series of models, I elucidated how GPNMB influences GBM biology, its effectiveness as a target for Chimeric Antigen Receptor T-cells, and how it can be paired with CD133 therapies to provide better coverage of tumor cells. Together, these studies highlight how advances in pre-clinical models and technologies can be leveraged to develop new therapies in a rational manner. / Thesis / Doctor of Science (PhD) / Glioblastoma (GBM) remains an aggressive and incurable brain cancer despite decades of intense research. Treatment failure is due to the untargeted approaches currently undertaken in the clinic. The current work uses multiples methods to interrogate how GBM grows and develops over time. Using GBM samples from consenting patients, I investigated an important population of the tumor using a surface marker CD133 and CRISPR to study which genes influenced it. I then successfully validated SOX2 as a direct regulator of CD133 expression. Next, I combed multiple data sets for a target to kill GBM cells without harming healthy tissue in patients. I found Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB) to be exploitable and used several experimental methods to investigate its role in GBM progression. Finally, we used a novel immunotherapy to eliminate cells which express GPNMB. Together, these findings could apply to the broader field of stem cell biology and be used for a more targeted method to eliminate the cancer entirely.

Suppression of anti-tumour immunity by the tumour microenvironment remains a major barrier to the development of broadly effective immunotherapies to treat solid tumours. Cytotoxic natural killer (NK) cells are vital to anti-cancer immunity and have shown clinical efficacy for treating hematologic malignancies. However, NK cell therapies have failed to be effective against solid tumours as cytotoxic NK cells become dysfunctional in the tumour microenvironment. While tumours hinder cytotoxic NK cells, they stimulate the tumour-promoting functions of regulatory NK cells. The mechanisms that dictate NK cell polarization and their fate in the tumour microenvironment remain poorly defined but harbour key therapeutic potential. Glucose-driven cellular metabolism has emerged as a central regulator of NK cell anti-tumour activity. Notably, tumour cells have deregulated metabolism, causing a metabolically hostile environment that is low in glucose and oxygen and high in metabolic waste. In the work presented, we demonstrate that NK cells expanded from cancer patients or healthy donors exert strong anti-tumour activity and dismantle the immunosuppressive tumour microenvironments of advanced ovarian and lung cancer. As a result, expanded NK cells were capable of sensitising initially non-responsive patient tumours to PD1 checkpoint-blockade therapy. Further, we uncover that the activity of cellular metabolic pathways plays a key role in NK cell functional fate in tumour microenvironment. We show that the tumour microenvironment induces paralysis of cytotoxic NK cell glucose metabolism to cause their dysfunction. However, reprogramming of NK cell metabolism through expansion arms expanded NK cells with enhanced metabolic flexibility which enabled their anti- tumour activity to be paradoxically strengthened by the tumour microenvironment. We further identify that regulatory NK cells have a distinct metabolic program compared to cytotoxic NK cells, including lower glucose-driven metabolism, that is amenable with the tumour microenvironment. Our work provides new mechanistic insight into how NK cell fate is regulated and how the pathological environment of a tumour capitalizes on this. This knowledge provides new therapeutic targets to intervene with the suppression of cytotoxic immunity in tumours. Further, this work identifies that expanded NK cells are a promising therapeutic candidate that exploit the metabolic hostility of the tumour microenvironment and synergize with other immunotherapies. / Thesis / Candidate in Philosophy / Harnessing the body’s natural immune defenses against cancer in the form of immunotherapy has emerged as a powerful treatment modality. Over the past decade, immune cell therapies have revolutionized the treatment of blood cancers like leukemia and lymphoma. Yet despite the potential, immune cell therapies have failed to be broadly effective against solid tumours because the anti-cancer activity of immune cells, such as Natural Killer (NK) cells, becomes severely impaired by the tumour environment. In this work, we identify that NK cells expanded from cancer patients and healthy donors overcome suppression by tumours and eliminate detectable tumour in pre-clinical models of advanced ovarian and lung cancer. These expanded NK cells also enhanced the functions of other immunotherapies. Further, we shed new light on how NK cells become dysfunctional in tumours. We uncover that NK cells undergo a metabolic energy crisis in tumours that causes their dysfunction, but that expanded NK cells have increased metabolic fitness which allows them to overcome this energy crisis and remain highly functional. Finally, we also characterize the metabolism of a subset of NK cells that are tumour-promoting and find that they harbour metabolic advantages to thrive in tumours. Overall, our work provides new insight as to how to overcome immunosuppression by tumours. This work identifies that expanded NK cells are a promising therapeutic candidate that exploit the hostility of tumours and synergize with other immunotherapies.

Yes / Background: The European Academy of Allergy and Clinical Immunology (EAACI) is in the process of developing Guidelines on Allergen Immunotherapy (AIT) for Allergic Rhinoconjunctivitis. To inform the development of clinical recommendations, we undertook a systematic review to assess the effectiveness, cost-effectiveness, and safety of AIT in the management of allergic rhinoconjunctivitis. Methods: We searched nine international biomedical databases for published, in-progress, and unpublished evidence. Studies were independently screened by two reviewers against predefined eligibility criteria and critically appraised using established instruments. Our primary outcomes of interest were symptom, medication,and combined symptom and medication scores. Secondary outcomes of interest included cost-effectiveness and safety. Data were descriptively summarized and then quantitatively synthesized using random-effects meta-analyses. Results: We identified 5960 studies of which 160 studies satisfied our eligibility criteria. There was a substantial body of evidence demonstrating significant reductions in standardized mean differences (SMD) of symptom (SMD -0.53, 95% CI -0.63,-0.42), medication (SMD -0.37, 95% CI -0.49, -0.26), and combined symptom and medication (SMD -0.49, 95% CI -0.69, -0.30) scores while on treatment that were robust to prespecified sensitivity analyses. There was in comparison a more modest body of evidence on effectiveness post-discontinuation of AIT, suggesting a benefit in relation to symptom scores.Conclusions: AIT is effective in improving symptom, medication, and combined symptom and medication scores in patients with allergic rhinoconjunctivitis while on treatment, and there is some evidence suggesting that these benefits are maintained in relation to symptom scores after discontinuation of therapy. / EAACI and BM4SIT project (grant number601763) in the European Union’s Seventh Framework Programme FP7.

The therapy suppressive tumour microenvironment (TME) continues to hinder anti-cancer therapies. Local delivery of therapeutic proteins, including potentially toxic factors, is increasingly needed to enhance immunotherapeutic bioactivities and minimize systemic toxicity. To this end, we are developing vehicles that immobilize to extracellular matrix (ECM) components upregulated in TME for localization of polymer-grafted bioactive cytokines with tunable degradation rates to control cytokine clearance. The grafted cytokine would be bioactive, and the length of the therapy would be governed by the degradation kinetics of the hydrolytic linker between the cytokine and polymer. The cytokines were expressed and purified, and their biological activity was confirmed. Click chemistry was used to graft the therapeutic proteins and collagen-binding peptides to the copolymer. Production of the therapeutic carriers was confirmed by SEC and fluorescent measurements. Biolayer interferometry and tracking immobilization inside collagen gel confirmed the binding affinity between carriers and collagen type 1. In vitro studies confirmed the bioactivity of the carriers in the presence of T-cells and macrophages. In summary, ECM binding vehicles for local sustained protein release will aid in the local delivery of therapeutic proteins to alter TME and promote immunotherapies. Screens will be conducted in multicellular spheroid models to identify bioactive formulations. / Thesis / Master of Science (MSc)

Cancer is the leading cause of death in Canada and one of the leading causes of death worldwide. Conventional cancer therapies such as chemotherapy often include severe side effects that can decrease the quality of life of patients undergoing treatment. Immunotherapy is designed to harness the host immune response and enhance its ability to seek out and kill cancer cells. Immunotherapy has gained traction in the past decade due to its improved safety and efficacy over conventional cancer therapies. However, there is room for improvement as most patients fail to respond to immunotherapy. The work described in this dissertation involves the development of therapeutic combination platforms that are designed to improve upon immunotherapy outcomes. Murine tumor models were used to develop a better understanding of biological processes associated with therapeutic efficacy. These findings can be used for the development of therapeutic strategies that can further improve the efficacy of cancer immunotherapy. / Cancer immunotherapy has demonstrated immense promise in the past decade. Immune checkpoint therapy has shown unprecedented responses in many cancers; however most patients fail to respond to checkpoint therapy. This highlights the need to develop a better understanding of factors in the tumor microenvironment that can influence therapeutic outcomes. In this body of work, we have utilized oncolytic viruses (OVs) to enhance immunogenicity in the tumor and study the cellular mechanisms that enable a therapeutic response. We utilize a combination of OVs and low dose chemotherapy to further sensitize murine models of mismatch repair-deficient colorectal cancer to checkpoint therapy. Using a Clariom S transcriptome assay we found that the combination induced gene signatures associated with the recruitment and activation of myeloid subsets. When we assessed tumor infiltrates, we found that the combination induced the chemoattraction of several myeloid subsets, including type I conventional DCs (cDC1s) which are known for their role in antigen presentation. Using Batf3-/- mice, we demonstrated that the therapeutic efficacy of our combination platform was dependent on the presence of cDC1s. In this dissertation, we also studied the role of OV-induced type I IFN (IFNI) in enabling or suppressing antitumor immunity. We found that OVs induced the upregulation of PD-L1 in an IFN-I-dependent manner in cancer cells and circulating immune cells. Inhibition of IFN-I signaling using an anti-IFNAR monoclonal antibody partially prevented OV-induced upregulation of PD-L1. Furthermore, the combination of OV and v | P a g e IFNAR blockade enhanced the effector functions of tumor-specific T cells and led to better tumor control compared to OV monotherapy. Altogether, these findings demonstrate that OVs can be an effective agent for enhancing immunogenicity in the tumor and promoting the infiltration of inflammatory myeloid subsets. By combining OVs with checkpoint or IFNAR inhibitors, we prevent the onset of immunosuppression and enable a favorable therapeutic response. / Thesis / Doctor of Philosophy (Medical Science)

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