BACKGROUND: Patients with relapsed or refractory large B-cell lymphoma (LBCL) who have relapsed after at least 2 lines of therapy had a poor prognosis before the introduction of chimeric antigen receptor (CAR) T-cell therapy. The FDA approved three CD19 CAR T-cell products, axicabtagene ciloleucel (axi-cel), tisagenlecleucel, and lisocabtagene maraleucel (liso-cel), based on the results of pivotal phase 2 clinical trials. High response rates and long-term remissions in these multiply relapsed patients led to randomized trials as a second-line therapy against the current standard of care for primary refractory and early relapsing LBCL. Axi-cel and liso-cel are now approved as second-line treatments for patients with relapsed or refractory large B-cell lymphoma based on these trials, while tisagenlecleucel failed to improve upon second-line standard of care. This has led to greater axi-cel and liso-cel usage as compared with tisagenlecleucel. Clinical trials and real-world trials show a higher toxicity profile for axi-cel as compared to liso-cel with similar efficacy outcomes, leading to selection of liso-cel for older patients with more medical comorbidities. However, axi-cel manufacturing is faster and more reliable making it a preferred choice for rapidly progressive lymphomas. No direct comparison has been made between the two in order to optimally inform product selection. OBJECTIVE: We aimed to compare the toxicity profile and efficacy outcomes between two cohorts, one treated with axi-cel and the other with liso-cel, ideally well matched, during the same period of time. METHODS: We retroactively gathered patient data for patients treated between June 2021 to September 2022 with both products. We compared the cohorts for patient characteristics that are proven to affect the toxicity and efficacy in order to identify significant differences that could influence our results and to increase the likelihood that the two cohorts were well matched. We then assessed associated toxicities and long-term efficacy outcomes. RESULTS: The two cohorts were comparable for all patient and disease variables other than age (median age of 62 years old in axi-cel compared to 71 years old liso-cel [p < 0.001]). There was no significant difference between high-grade cytokine release syndrome (CRS) (3% vs 5% for axi-cel vs. liso-cel cohorts, respectively; p = 0.58), high-grade immune effector cell-associated neurotoxicity syndrome (ICANS) (18% [ASTCT] or 19% [CTCAE], 14% [ASTCT] or 12% [CTCAE] for axi-cel vs. liso-cel cohorts, respectively, p = 0.055). There were higher rates of any grade CRS with axi-cel, and duration of hospitalization was longer for axi-cel vs. liso-cel (10 vs. 14 days, respectively). Best overall response rates (ORR) (93% vs. 84% axi-cel vs. liso-cel, respectively) and complete response (CR) rates (71% vs. 56% axi-cel vs liso-cel, respectively) did not statistically differ between the two groups. 12-month overall survival (OS) (76% vs. 81% axi-cel vs. liso-cel, respectively) and progression free survival (PFS) (61% vs. 45% of patients axi-cel vs. liso-cel, respectively) did not statistically differ between the two groups (p =0.94, p =0.51 for OS and PFS, respectively). CONCLUSIONS: Our study showed both products are similar in their high-grade toxicity profile as well as their efficacy. While axi-cel has more any grade CRS and ICANS, the lack of significantly higher high-grade toxicities likely reflects better and more aggressive toxicity mitigation strategies when patients present with low grade side effects. As a result, axi-cel in our study was found to be less toxic than previously seen in past clinical trials as well as real-world studies. Many factors go into selection of a CAR T-cell product, ranging from product performance attributes like safety and efficacy, to product manufacturing qualities like turnaround time and fidelity of manufacturing. With equivalency with regards to product performance, manufacturing qualities may then be most important in guiding product selection for LBCL patients.

Cancer is an uncontrolled growth of the body's own cells. While cancer rates increase with age, this disease afflicts both young and old. Traditional cancer therapy has had three major facets: 1) chemotherapy, which can non-specifically damage healthy tissue, 2) radiation, which can make some types of cancer more likely in the future, and 3) surgery, which can be physically traumatic and is not effective in removing unseen microtumors or circulating metastases. Immunotherapy, by its very nature, is drastically different. Immunotherapy seeks to employ cells or molecules from the immune system, in their original or a modified form, to augment, assist or replace missing elements of the native functioning immune system. Our immunotherapeutic approach has been to develop novel chimeric antigen receptors (CAR) and deliver the engineered transgene into hematopoietic stem cells (HSC). We have developed a novel single chain TCR (scTCR) in which the TCR V-alpha and V-beta segments are joined by a flexible linker. In addition to our scTCR we developed a single chain antibody molecule (scFv) to increase avidity to the tumor antigen and avoid the potential limitation of MHC restriction. Our lab has previously developed a signaling cassette based on the CD3 zeta chain, CD28 and p56Lck proteins which are prominent in the T-cell signaling pathway. The single chain specificities are linked to the signaling cassette that we have shown to function in T-cells. With specificity and signaling coupled, the chimeric antigen receptor can be transduced into hematopoietic stem cells (HSC) via a lentivirus vector. This adoptive immunotherapy can potentially eliminate malignant cells or supplement traditional therapies by providing engineered specificity and a useful method to transfer and expand tumor specific T-cells. We show in this study that the CAR can be delivered effectively to HSC and that the introduced transgene is expressed in multiple cell lineages. We also have developed a novel method of increasing lentiviral transduction efficiency. Both transduced fraction of cells and overall expression can be increased by proper timing and coordination of cell growth, cell cycle phase, vector addition and treatment with heat shock.

Few cancers arising in fluid organ systems can be cured with localized therapeutic modalities, such as radiation or surgical organ removal. Chemotherapy and hematopoietic stem cell transplants have long been employed as the standard of care for patients diagnosed with leukemias and lymphomas. Though research continues to propose new, more potent chemotherapeutic agents, a new paradigm of treating cancerous malignancies with tumor-specific monoclonal antibodies, adoptively transferred tumor-fighting cells, and other exogenously administered immunomodulatory agents, has emerged over the past decade. These immunotherapies have dramatically improved the outcomes of patients diagnosed with cancers of B lymphocytes, referred to as B-cell malignancies. Though curative FDA-approved therapies for patients diagnosed with B-cell malignancies have yet to be established, recent research in the field of adoptive T-cell therapy has produced promising results. Tumor infiltrating lymphocyte therapy (TIL therapy), T-cell Receptor Therapy (TCR therapy) and Chimeric Antigen Receptor T-cell Therapy (CAR T-cell therapy) are the three most extensively studied adoptive T-cell immunotherapies in the context of B-cell malignancies. TIL and TCR therapies, in which patients are provided with either the patient’s own tumor-specific T-cells or T-cells expressing engineered, tumor-specific TCRs, respectively, enhance the patient’s immune system to mount a more potent, anti-tumor response. However, these adoptive T-cell therapies do not change the mechanisms of the immune response. Cancerous cells can evade immune attack and dampen immune responses to survive and thrive in the body. By down-regulating their expression of human major histocompatibility complex I (MHC I), for example, cancer cells escape T-cell recognition, which is dependent on MHC expression. A chimeric antigen receptor (CAR), is composed of an antibody-derived (B-cell derived) extracellular, antigen-recognition domain, and T-cell derived intracellular domains. CAR T-cells, therefore, exploit the cytotoxic nature of CD8+ T-cells, and the MHC independent recognition of B-cell receptors, to identify and destroy all cells expressing a specific target. Consequently, many of the cancer cell’s mechanisms of immune evasion are less effective in the presence of CAR T-cells. Progressive generations of CAR T-cell designs couple these receptors with costimulatory molecules to amplify the activation, efficacy, and potency of these cells in-vivo. Over the past five years, phase I and IIa clinical trials have produced remarkable results in the treatment of advanced stage, high-risk B-cell malignancies, namely Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), and Non-Hodgkin’s Lymphoma (NHL). However, the significant oncogenic risks and fatal adverse events associated with this therapy necessitate further research to improve safety and reliable clinical efficacy of CAR T-cell therapy. In spite of these risks, the adoptive transfer of CD19-targeting, CAR expressing, cytotoxic T-cells (anti-CD19 CAR-T-cells) has produced sustained, complete remissions in patients diagnosed with progressive, advanced-stage, B-cell malignancies, for whom alternative treatments were not available. The unprecedented results of early clinical trials, as well as ongoing preclinical studies aimed at improving the design and production of CAR T-cells suggest a promising future for CAR T-cell therapy as a cure for B-cell malignancies.

Advances in our understanding of the molecular events leading to cancer have facilitated the development of next-generation targeted therapies. Among the most promising new approaches is immuno-oncology, where therapeutic agents engage the immune system to fight cancer. One exciting strategy therein is the adoptive transfer of ex vivo cultivated tumor-specific T lymphocytes into a cancer patient. Tumor-specific T cells can be produced by engineering a patient’s own T cells with synthetic receptors (e.g. chimeric antigen receptors (CARs)) designed to redirect T cell cytotoxicity against a tumor target. CAR-engineered T cells (CAR-T cells) were expected to be a non-toxic cellular therapy which would seek out and specifically eliminate disseminated tumors. The clinical experience supports the promise of CAR-T cell therapy (striking efficacy has been observed in the treatment of hematological malignancies), while highlighting areas for improvement; CAR-T cell use has been associated with a host of toxicities and robust clinical efficacy has yet to be replicated in solid tumors. This thesis uses pre-clinical models to describe previously unappreciated aspects of CAR-T cell-associated toxicity and novel synthetic receptor strategies, including: i. The capacity of NKG2D-based CAR-T cells to mediate toxicity. ii. The utility of designed ankyrin repeat proteins as CAR antigen-binding domains. iii. The discovery that variables intrinsic to human CAR-T cell products contribute to toxicity. iv. A novel synthetic receptor capable of redirecting T cell specificity against a tumor target – the T cell antigen coupler (TAC). Unlike equivalent CAR-T cells, TAC-T cells are capable of mediating efficacy against a solid tumor in the absence of toxicity. We anticipate that these results will contribute towards the development of next-generation synthetic receptor-engineered T cell products that can deliver upon the promise of safe, systemic cancer therapeutics. / Thesis / Doctor of Philosophy (PhD) / The human immune system has the unique capacity to “seek and destroy” tumor cells throughout the body. A novel class of drugs, immuno-oncology agents, harness this ability to fight cancer. Within this class is a new cellular drug where genetic engineering is used to create killer immune cells (called T cells) capable of recognizing and eliminating tumors. Two of these cellular drugs have recently received FDA approval, supporting the feasibility of this approach. However, further research is needed to improve the safety of engineered-T cells and increase the number of patients whom can benefit from their use. This thesis uses laboratory investigations to better understand the side-effects associated with anti-cancer engineered-T cells and evaluate new engineering strategies. We anticipate that these results will contribute towards the development of next-generation engineered-T cell drugs which retain the ability to function systemically against cancer but offer an enhanced safety profile.

In the last decade cancer immunotherapy has taken huge strides forward from bench to bedside and being approved as drugs. Cancer immunotherapy harnesses the power of patient’s own immune system to fight cancer. Approaches are diverse and include antibodies, therapeutic vaccines, adoptively transferred T-cells, immune checkpoint inhibitors, oncolytic viruses and immune cell activators such as toll-like receptor (TLR) agonists. Excellent clinical responses have been observed for certain cancers with checkpoint antibodies and chimeric antigen receptor (CAR)-engineered T-cells. It is however becoming evident that strategies need to be combined for broader effective treatment responses because cancers evolve to escape immune recognition. A conditionally replication-competent oncolytic adenovirus (Ad5PTDf35-[Δ24]) was engineered to secrete Helicobacter pylori Neutrophil Activating Protein (HP-NAP, a TLR-2 agonist) to combine viral oncolysis and immune stimulation. Treatment with Ad5PTDf35-[Δ24-sNAP] improved survival of mice bearing human neuroendocrine tumors (BON). Expression of HP-NAP in the tumor microenvironment promoted neutrophil infiltration, proinflammatory cytokine secretion and increased necrosis. We further studied the ability of HP-NAP to activate dendritic cells (DCs) a key player in priming T-cell responses. HP-NAP phenotypically matured and activated DCs to secrete the T-helper type-1 (Th-1) polarizing cytokine IL-12. HP-NAP-matured DCs were functional; able to migrate to draining lymph nodes and prime antigen-specific T-cell proliferation. CAR T-cells were engineered to secrete HP-NAP upon T-cell activation. Secreted HP-NAP was able to mature DCs, leading to a reciprocal effect on the CAR T-cells with improved cytotoxicity in vitro. Semliki Forest virus (SFV), an oncolytic virus with natural neuro-tropism was tagged with central nervous system (CNS)-specific microRNA target sequences for miR124, miR125 and miR134 to selectively attenuate virus replication in healthy CNS cells. Systemic infection of mice with the SFV4miRT did not cause encephalitis, while it retained its ability to replicate in tumor cells and cure a big proportion of mice bearing syngeneic neuroblastoma and gliomas. Therapeutic efficacy of SFV4miRT inversely correlated with type-I antiviral interferon response (IFN-β) mounted by tumor cells. In summary, combining immunotherapeutic strategies with HP-NAP is a promising approach to combat cancers and SFV4miRT is an excellent candidate for treatment of neuroblastomas and gliomas.

<p>Dysregulated inflammation underlies the pathogenesis of a myriad of human diseases ranging from cancer to autoimmunity. As coordinators, executers and sentinels of host immunity, T cells represent a compelling target population for immune-modulation. In fact, the antigen-specificity, cytotoxicity and promise of long-lived of immune-protection make T cells ideal vehicles for cancer immunotherapy. Interventions for autoimmune disorders, on the other hand, aim to dampen T cell-mediated inflammation and promote their regulatory functions. Although significant strides have been made in targeting T cells for immune-modulation, current approaches remain less than ideal and leave room for improvement. In this dissertation, I seek to improve on current T cell-targeted immunotherapies, by identifying and preclinically characterizing their mechanisms of action and in vivo therapeutic efficacy.</p><p>CD8+ cytotoxic T cells have potent antitumor activity and therefore are leading candidates for use in cancer immunotherapy. The application of CD8+ T cells for clinical use has been limited by the susceptibility of ex vivo-expanded CD8+ T cells to become dysfunctional in response to immunosuppressive microenvironments. To enhance the efficacy of adoptive cell transfer therapy (ACT), we established a novel microRNA-targeting approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor Blimp-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR-23a was upregulated in tumor-infiltrating CD8+ T cells, and its expression correlated with impaired antitumor potential of patient CD8+ T cells. We determined that tumor-derived TGF-&#946; directly suppresses CD8+ T cell immune function by elevating miR-23a and downregulating Blimp-1. Functional blockade of miR-23a in human CD8+ T cells enhanced granzyme B expression; and in mice with established tumors, immunotherapy with just a small number of tumor-specific CD8+ T cells in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CD8+ T cells that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGF&#946;-mediated tumor immune-evasion pathway.</p><p>Having established that miR-23a-inhibition can enhance the quality and functional-resilience of anti-tumor CD8+ T cells, especially within the immune-suppressive tumor microenvironment, we went on to interrogate the translational applicability of this strategy in the context of chimeric antigen receptor (CAR)-modified CD8+ T cells. Although CAR T cells hold immense promise for ACT, CAR T cells are not completely curative due to their in vivo functional suppression by immune barriers &#8210; such as TGF&#946; &#8210; within the tumor microenvironment. Since TGF&#946; poses a substantial immune barrier in the tumor microenvironment, we sought to investigate whether inhibiting miR-23a in CAR T cells can confer immune-competence to afford enhanced tumor clearance. To this end, we retrovirally transduced wildtype and miR-23a-deficient CD8+ T cells with the EGFRvIII-CAR, which targets the PepvIII tumor-specific epitope expressed by glioblastomas (GBM). Our in vitro studies demonstrated that while wildtype EGFRvIII-CAR T cells were vulnerable to functional suppression by TGF&#946;, miR-23a abrogation rendered EGFRvIII-CAR T cells immune-resistant to TGF&#946;. Rigorous preclinical studies are currently underway to evaluate the efficacy of miR-23a-deficient EGFRvIII-CAR T cells for GBM immunotherapy. </p><p>Lastly, we explored novel immune-suppressive therapies by the biological characterization of pharmacological agents that could target T cells. Although immune-suppressive drugs are classical therapies for a wide range of autoimmune diseases, they are accompanied by severe adverse effects. This motivated our search for novel immune-suppressive agents that are efficacious and lack undesirable side effects. To this end, we explored the potential utility of subglutinol A, a natural product isolated from the endophytic fungus Fusarium subglutinans. We showed that subglutinol A exerts multimodal immune-suppressive effects on activated T cells in vitro: subglutinol A effectively blocked T cell proliferation and survival, while profoundly inhibiting pro-inflammatory IFN&#947; and IL-17 production by fully-differentiated effector Th1 and Th17 cells. Our data further revealed that subglutinol A might exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells, but not in innate immune cells or fibroblasts. Additionally, we demonstrated that subglutinol A significantly reduced lymphocytic infiltration into the footpad and ameliorated footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of subglutinol A as a novel therapeutic for inflammatory diseases.</p> / Dissertation

The field of synthetic biology seeks to revolutionize the scope and scale of what is currently feasible by genetic engineering. By focusing on engineering general signal processing platforms using modular genetic parts and devices rather than `one-off' systems, synthetic biologists aim to enable plug-and-play genetic circuits readily adaptable to different contexts. For mammalian systems, the goal of synthetic biology is to create sophisticated research tools and gene therapies. While several isolated parts and devices exist for mammalian systems there are few signal processing platforms available. We addressed this need by creating a transcriptional regulatory framework using programmable zinc finger (ZF) and TALE transcription factors and a conceptual framework for logical T-cell receptor signaling. We first engineered a large set of ZF activator and repressor transcription factors and response promoters. ZFs are scalable elements as they can be engineered to bind to given DNA sequences. We demonstrated that we could ‘tune’ the activity of the ZF transcription factors by fusing them to protein homo-dimerization domains and by modifying their response promoters. We also created OR and NOR logic gates using hybrid promoters and AND and NAND logic gates by reconstituting split zinc finger activators and repressors with split inteins. Next, using a computational algorithm we designed a series of TALE transcriptional activators and repressors predicted to be orthogonal to all 2kb human promoter regions and thus minimally interfere with endogenous gene expression. TALEs can be designed to bind to even longer DNA sequences than ZFs, however off-target binding is predicted to occur. We tested our computationally designed TALEs in human cells demonstrating that they activated their intended target genes, but not their likely endogenous off-target genes, nor synthetic promoters with binding site mismatches. Finally, we created a conceptual framework for logical T-cell-mediated killing of target cells expressing combinations of surface antigens. The systems consist of conventional and novel chimeric antigen receptors (CARs) containing inhibitory or co-stimulatory cytoplasmic signaling domains. In co-incubation assays of engineered T-cells with target cells we demonstrated a functioning OR-Gate system and progress toward development of a functional NOT-Gate system using the CD300a and CD45 inhibitory receptor domains.

Liver metastases are the most common cause of death in colorectal cancer patients. The standard of care and potential for cure for colorectal liver metastases is resection, but often times disease it too extensive for this treatment. Over the years, cancer research has made way for advances in treating progressive disease through immunotherapy. By genetically modifying an individual’s immune system using virally transduced chimeric antigen receptor T cells (CAR-T), patients are better able to receive exquisitely specific T cells to target specific tumors. Furthermore, selective delivery strategies may enhance efficacy while limiting detrimental, systemic adverse effects. Not only this, CAR-Ts have also lead to complete remission in some liquid tumors while maintaining the potential for remission in solid tumors as well. This literature review takes readers through the emergence of the different generations of CAR-T and the various studies including clinical trials that have demonstrated the safety and efficacy of CAR-T. The second portion of this paper will outline the design for a phase II clinical trial using intrahepatic CAR-T therapy in addition to selective internal radiation therapy (SIRT) for refractory CEA+ colorectal liver metastases. Benefits and limitations of using these therapies are further discussed.

No description available.

Through the years, cancer therapies have progressed rapidly, pouring out novel treatments such as gene therapy, small molecule therapies and immunotherapy. One such immunotherapy, adoptive cell transfer (ACT), augmented through the addition of a chimeric antigen receptor (CAR), has proven success in treatment of hematological malignancies. Additionally, oncolytic viruses (OV) and OV-based (OVV) therapies, have shown promising results in both clinical and pre-clinical studies. In most instances, when applied as a monotherapy, the aforementioned treatment methods are incapable of inducing complete tumour remission. The Wan lab has developed an approach combining ACT with OVV therapies that dramatically increase therapeutic benefit resulting in complete regression of well-established solid tumours. Despite promising results, certain tumours can still escape this combination therapy through antigen loss resulting in antigen negative relapse (ANR). To further augment the therapy, the addition of a secondary receptor (CAR) provides the ACT multiple avenues of attack to prevent ANR. In this dissertation, we define culture conditions that promote strong expression of the CAR alongside confirmation of function in an in vitro setting. Following, it is demonstrated that OVV boosted dual-targeting T cells carry strong T cell activity by measure of cytokine release in vivo. Despite promising T cell activity data, dual-specific T cells are unable to improve tumour control and survival once relapse occurs. The failure to control relapse remains unclear however evidence points towards lack of T cell persistence, poor CAR function in vivo and a lack of endogenous T cell response leading to compounding effects that prevent dual-targeting T cells from preventing ANR. Although dual specific therapies have shown poor efficacy in preventing ANR, further study must be completed to identify areas of improvement – such as persistence, as the potential for success in using dual-targeting T cells coupled with OVVs still lies untapped. / Thesis / Master of Science (MSc)