Multiple myeloma is an aggressive plasma cell cancer that consistently acquires multi-drug resistance and relapses despite initial treatment successes. Patients may go through greater than 10-lines of therapy, highlighting the need for more effective treatment options. Immunotherapies are the latest evolution in targeted cancer treatments, and thus far have displayed impressive results in several hematological cancers, including multiple myeloma. T cells possess robust anti-tumor functions which can be harnessed and refined for the treatment of cancers. Genetic engineering of T cells to express a chimeric antigen receptor (CAR) confers antigen-specific tumor-targeting, and adoptive transfer of patient-derived CAR-engineered T (CAR T) cells has been efficacious in relapsed/refractory multiple myeloma. Despite the high efficacy, CAR T cell therapy for myeloma is associated with serious adverse events, which limits dose levels and patient eligibility. We have developed a novel synthetic antigen receptor platform, called the T cell antigen coupler (TAC) receptor, which has shown comparatively higher efficacy with a reduced pro-inflammatory profile compared with CAR T cells in pre-clinical models. The TAC receptor was purpose-built to co-opt the natural T cell activation machinery and lacks the costimulatory signaling typically incorporated in CAR designs. This thesis investigates strategies to augment TAC T cell function against for multiple myeloma through the evaluation of ancillary pharmacological and protein stimuli that would complement the anti-tumor functions of TAC T cells without modifying the TAC receptor design. In chapter 2, I investigated a strategy combining TAC T cells with the SMAC mimetic LCL161 to provide transient costimulatory effects. While LCL161 boosted TAC T cells survival and proliferation, the drug also enhanced susceptibility of TAC T cells to apoptosis and offered no advantage to the TAC T cells when challenged with myeloma. In chapter 3, I engineered TAC T cells to secrete IL-27 in an attempt to modulate the myeloma microenvironment and support T cell cytolytic function. IL-27 did not enhance the anti-tumor activity of TAC T cells but forced expression of IL-27 led to a reduction in the production of pro-inflammatory cytokines without altering cytotoxicity. In appendix I, I describe the process of optimizing CRISPR/Cas9 editing of primary TAC T cells. This methodology was required for much of the work in chapter 2. Ph.D. Thesis – Arya Afsahi McMaster University – Biochemistry and Biomedical Sciences v In appendix II, I describe an assessment of mRNA-engineering as a method to produce TAC T cells. This approach proved to be therapeutically futile and was not pursued beyond the work described herein. The work presented here highlights methods of combining TAC T cells with a clinically relevant SMAC mimetic, or the cytokine IL-27, and provides insights into the biological mechanisms that are affected by these approaches. / Thesis / Doctor of Philosophy (PhD)

The regulated cell death apoptosis enables redundant or compromised cells in ontogeny and homeostasis to remove themselves receptor-dependent after extrinsic signaling or after internal stress by BCL-2 proteins on the outer mitochondrial membrane (OMM). Mitochondrial BCL-2 proteins are also often needed for receptor-mediated signaling in apoptosis. Then, the truncated BH3-only protein BID (tBID) blocks retrotranslocation of the pro-apoptotic BCL-2 proteins BAX and BAK from the mitochondria into the cytosol. BAX and BAK in turn permeabilize the OMM. Although the BCL-2 proteins are controlled by a complex regulatory network, a specific mechanism for the inhibition of tBID remained unknown. Curiously, it was suggested that hexokinases, which channel glucose into the metabolism, have an intriguing function in the regulation of apoptosis. Recent analysis of transient hexokinase interactions with BAX revealed its participation in the inhibition of BAX and also BAK by retrotranslocation from mitochondria to the cytosol. In contrast to general apoptosis inhibition by anti-apoptotic BCL-2 proteins, hexokinase I and hexokinase 2 specifically inhibit tBID and thus the mitochondrial apoptosis pathway in response to death receptor signaling. Mitochondrial hexokinase localization and BH3 binding of cytosolic hexokinase domains are prerequisites for protection against receptor-mediated cell death, whereas glucose metabolism is not. This mechanism protects cells from apoptosis induced by cytotoxic T cells.

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Hematological cancers account for nearly ten percent of cancer cases diagnosed annually in the United States. Patients who fail to respond to chemotherapy or radiotherapy must often undergo a bone marrow transplant to treat their malignancy. A significant complication following this procedure is Graft versus Host Disease (GvHD), which occurs when donor T cells mount an immune response against recipient tissues. Immunological research has highlighted the role of aberrant T cell metabolism, specifically a shift toward aerobic glycolysis, as a key driver behind the occurrence of this condition. The transcription factor FoxK1 has been revealed to be a key regulator of the cell's ability to induce aerobic glycolysis. Utilizing established GvHD murine models and novel CRISPR-Cas9 techniques, this study investigates how controlling this important pathway by FoxK1 may limit the damage inflicted by GvHD. Our studies reveal that depleting FoxK1 in donor T cells has a protective effect following transplants by promoting an immunosuppressive phenotype in donor T cells. These results suggest that FoxK1 may hold promise as a future cellular target for cellular therapies administered to transplant patients to prevent the occurrence of GvHD. Continued research is needed to ascertain the precise mechanisms that afford FoxK1 this protective role.

Hematopoietic stem cell transplantation (HSCT) is an important form of therapy for hematological genetic disorders and malignancies, particularly hematological cancers. However, common usage of this procedure is obstructed by graft-versus-host disease (GvHD), in which transplanted donor T cells wage an attack on recipient antigens, causing severe tissue damage and mortality. GvHD prognosis remains poor, and current treatment methods continue to be insufficient, especially for patients with more advanced and severe GvHD. T cells have been identified as the fundamental force behind GvHD, and their cellular metabolism is deemed vital to their fate and function, especially in pathogenic environments. A hallmark of T cell metabolism in GvHD microenvironments is aerobic glycolysis, which maximizes biomass accumulation and supports growth and proliferation. Lactate dehydrogenase A (LDHA) is an essential enzyme that sustains this pathway and may be a potential therapeutic target. Using murine and in-vitro GvHD models, this study investigates the ameliorative impacts of LDHA inhibition on the fate and function of T cells following HSCT. The results reveal that LDHA depletion leads to an immunosuppressive donor T cell characterization that minimizes recipient harm induced by GvHD. Future studies should focus on investigating LDHA inhibition in in-vivo models to introduce a paradigm shift in the development of clinically relevant therapeutics.

<p><strong>INTRODUCTION: </strong>Pre-clinical and clinical data strongly support the feasibility of employing immunotherapy as a strategy to treat cancer.</p> <p><strong>METHODS: </strong>Using the B16F10 murine melanoma model, we have been investigating mechanisms of T cell-mediated antitumor immunity following immunization with dopachrome tautomerase (DCT), a melanoma-associated antigen.</p> <p><strong>RESULTS: </strong>In <strong>Chapter 2</strong>, we uncovered an interesting dichotomy whereby DCT-specific CD4⁺ T cell-mediated tumor protection and autoimmunity are dependent on IL-4/STAT-6 and IFN-g/STAT-4, respectively. Our data also revealed that this phenomenon is extrinsic of CD4⁺ T cell polarization.</p> <p>To gain further insight into the targets recognized by CD4⁺ T cells, we conducted in <strong>Chapter 3</strong> extensive CD4⁺ T cell epitope mapping experiments using overlapping peptide libraries. Interestingly, while we were able to identify “helper” epitopes within DCT that were required for maximal CD8⁺ T cell expansion, we were unable to identify “effector” epitopes responsible for tumor rejection. Further examination of the requirements for the generation of CD4⁺ T cell effector epitopes showed that post-translational modifications of the protein were involved.</p> <p>In <strong>Chapter 4</strong>, we investigated the modest efficacy afforded by DCT immunization in the context of established B16F10 melanomas. Using intratumoral transcriptional analysis, we demonstrated that the vaccine rapidly promoted an IFN-g-dependent immunosuppressive state inside the tumor. Concurrent treatment with the immunomodulatory antibodies anti-4-1BB and anti-PD-1 effectively counteracted this tumor immunosuppression, resulting in complete regression of tumors and long-term survival in 70% of the mice.</p> <p><strong>CONCLUSIONS: </strong>The research described in this thesis sheds new light into the mechanisms by which vaccine-mediated CD4⁺ T cell responses participate to tumor rejection and autoimmunity. Moreover, our findings indicate that cancer vaccine-induced tumor immunosuppression significantly limits tumor regression, emphasizing the requirement of combinatorial approaches for successful cancer immunotherapy. Overall, our research offers new insight for future vaccine development.</p> / Doctor of Philosophy (Medical Science)

<p>Cancer vaccine therapy aims at harnessing effective antigen specific immune responses to treat tumor. In particular, CD8+ T cells have the unique capacity to recognize and destroy tumor cell throughout the body. One potential approach to elicit high numbers of effector CD8+ T cells to control tumor growth is through repeated vaccination, a strategy called prime-boost vaccination. However, booster immunization is relatively inefficient during primary immune response because pre-activated effector T cells tend to impair robust antigen presentation. This phenomenon has been interpreted as a negative feedback mechanism where recently activated CD8+ T cells clear the antigen-bearing dendritic cells (DCs) and prevent memory T cells from the access of the boosting antigen. Interestingly, however, using in vitro activated B cell as a viral vector delivery system, we can boost T cell responses with the minimum viral input at a very short interval between immunizations. This B cell carrier is capable of delivering different viral vectors expressing different antigens, displaying a potential for broad application. The mechanisms behind B cell carrier-mediated efficient secondary responses are three fold: 1. Without the engagement of MHC molecules and antigen presentation, B cell carrying viral vector can bypass the killing by pre-existing effector T cells 2. B cells can delivery viruses to B cell follicles, a place separated from effector T cells, and mediate memory T cell expansion. 3. B cells can deliver antigen to both spleen and lymph node and induce antigen specific T cell expansion in multiple lymphoid organs. Our studies provide a novel boosting platform to accelerate CTL responses that has important clinical implications.</p> / Master of Science (MSc)

<p>Pre-clinical and clinical data strongly support the use of immunotherapies for cancer treatment. Cancer vaccines offer a promising approach, however, the outcomes of clinical vaccine trials have been largely disappointing, prompting a need for further investigation. Using the B16F10 murine melanoma, we have investigated the local events within growing tumors following recombinant adenovirus immunization. In chapter 2, we investigated the ability of a pre-clinical vaccine to elicit only transient tumor growth suppression. We observed that tumors were initially infiltrated by a small number of highly functional tumor-specific CD8+ T cells following vaccination that instigated a rapid adaptive response in the tumor that suppressed local immune activity. In chapter 3, we questioned whether increasing the rate and magnitude of early immune attack would result in more robust tumor attack prior to tumor adaptation. Increasing the rate of tumor-specific CD8+ T cell expansion following vaccination resulted in tumor regression and durable cures in approximately 65% of treated mice. Further analysis revealed that tumor regression correlated with an early burst in immune attack that outpaced tumor adaptation. In chapter 4, we explored whether the same vaccine could be improved when combined with immunomodulatory antibodies. Vaccination combined with anti 4-1BB and anti PD-1 resulted in complete tumor regression and durable cure of >70% of treated animals and was associated with increased local immune activity. Gene expression profiling revealed a unique gene signature associated with the curative treatment, which was also associated with positive outcome in human melanoma patients. The described research sheds new light on mechanisms that limit the efficacy of therapeutic cancer vaccines. Namely, rapid tumor adaptation, triggered by early vaccine-induced CD8+ T cells, acts to suppress the local immune response prior to maximal immune attack. Strategies to overcome these adaptive processes should therefore be considered in future vaccine design.</p> / Doctor of Philosophy (Medical Science)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23603号 / 医博第4790号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 上野 英樹, 教授 金子 新, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM