<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

Recombinant oncolytic virus (OV) vaccines that encode tumour-associated antigens are potent boosting agents for adoptive transfer of tumor-specific T cells (adoptive T cell therapy or ACT). Current strategies to exploit boosting vaccines for ACT rely on a priori knowledge of targetable tumour epitopes and isolation of matched epitope-specific T cells. Therefore, booster vaccines must be developed on a patient-by-patient basis, which severely limits clinical feasibility. To overcome the requirement for individualized pairing of vaccines and T cells, we propose a “universal” strategy for boosting tumor-specific T cells where the boost is provided through a synthetic receptor that can be engineered into any T cell and a matched vaccine. To this end, we are employing chimeric antigen receptors (CARs), which confer MHC-independent antigen specificity to engineered T cells, and a paired OV vaccine that encodes the CAR target. As proof-of-concept, we have developed and evaluated a model where murine TCR transgenic T cells are engineered with boosting CARs against a surrogate antigen for studies in immunocompetent hosts. In chapter 3, I optimized a murine CAR-T cell manufacturing protocol that allows for generation of highly-transduced T cells that maintain a predominantly central memory (Tcm) phenotype. This protocol leads to generation of highly functional CAR-T cell products that can be cryopreserved at the end of ex vivo culture for future use in adoptive transfer and vaccination studies. In chapter 4, I evaluated the in vivo boosting potential of our dual-specific CAR-T cells with paired OV vaccines. Adoptive transfer of these CAR-engineered tumor-specific T cells followed by vaccination with paired oncolytic vesicular stomatitis virus (VSV) vaccine leads to robust, but variable and transient, CAR-mediated expansion of tumour-specific CD8+ T-cells, resulting in delayed tumour progression in aggressive syngeneic tumour models. In chapter 5, I investigated the role of OV-induced type I interferon (IFN-I) responses on CAR-T cell boosting. I found that CAR-T cell expansion and anti-tumour function following OV vaccination is limited by the IFN-I response and can be further enhanced by blocking interferon alpha and beta receptor subunit 1 (IFNAR1). This IFN-I-mediated T cell suppression was found to be T cell-extrinsic and related to premature termination of OV infection and antigen expression in vivo. In chapter 6, I investigated the role of CD4+ T cell help in vaccine-mediated T cell boosting and evaluated different genetic engineering strategies to integrate pro-survival STAT5 signaling into the CAR-T cell product in an effort to improve persistence and long-term anti-tumour efficacy. The work presented herein describes a novel and clinically feasible approach to enhancing adoptive T cell therapies and contributes to the basic understanding of T cell biology in the context of CAR-engineering and cancer vaccination. / Thesis / Doctor of Philosophy (PhD) / Despite recent advances in cancer prevention, detection, and treatment, 2 in 5 Canadians are expected to be diagnosed with cancer in their lifetime and approximately 1 in 4 will succumb to their disease. New, more specific therapies are needed to improve responses to treatment and reduce therapy-related side effects. Cell therapy is a new way to treat cancer that uses the patient’s own immune cells as a living drug. The immune cells are taken from a patient’s blood or tumour, trained to attack cancer in the laboratory, and infused back into the patient where they will find and kill cancer cells. A major challenge with this strategy is that the trained immune cells do not always survive in the patient for long enough to get rid of the tumour. To “boost” the immune cells, we are developing a new strategy where the immune cells are genetically modified and combined with a vaccine to enhance their anti-tumor activity. Just like a vaccine against a bacteria or virus, this vaccine will tell the modified immune cells to turn on, make more of themselves, and to find and kill the cancer cells. By delivering this “go” signal through a vaccine, we think that the immune cells will be better able to survive and generate a stronger, longer-lasting immune response against the cancer. This thesis tests this approach in relevant mouse models of cancer and aims to understand how we can best design the immune cells and vaccine to work together in their tumour-killing activities.

Cancer immunotherapy has long been proposed as a powerful approach to curing tumors, based on the natural function of the immune system in protecting its host with specificity, thus holding the potential for developing long-term memory that prevents tumor recurrence. However, the immunosuppressive feature of the tumor microenvironment prevents the patients' own immune system from functioning normally in the fight against cancer. As one of the most potent cancer immunotherapies, immunostimulatory cytokines have been shown to elicit anti-tumor immune responses in preclinical studies, but their clinical application is limited by severe immune-related adverse events upon systemic administration. None of the current delivery strategies can fully address issues of toxicities and sustainably supply cytokines over the course of a few days without compromising cytokines' structural integrity. Herein, we have developed a novel formulation to anchor potent cytokine molecules to the surface of large-sized particles (1 µm) for local cancer treatment. The cytokines are confined in tumors and have minimal systemic exposure over a few days following intratumoral injection, thereby eliciting anti-tumor immunity while avoiding the systemic toxicities caused by the circulating cytokines. Such particle-anchored cytokines can be synergistic with other immunotherapies, including immune checkpoint blockade antibodies and tumor antigens, to safely promote tumor regressions in various syngeneic tumor models and genetically engineered murine tumor models. / Doctor of Philosophy / Cancer immunotherapy is a promising method to treat cancer by harnessing the power of the body's immune system, which naturally fights off diseases and can remember and prevent diseases from returning. Unfortunately, cancers create a hostile environment that weakens the immune system's ability to combat the disease effectively. Among the treatments explored, immunostimulatory cytokines (unique proteins that boost the immune system) have shown great promise in laboratory studies for their ability to fight cancer. However, when these proteins are administered to patients, they can cause severe side effects due to their systemic dissemination throughout the body. Herein, by attaching the potent cytokines to large-sized particles (1 µm), and injecting them directly into the tumor, their cancer-fighting abilities are focused precisely where they are most needed. This targeted delivery minimizes the cytokines' presence in the rest of the body, dramatically reducing the risk of side effects associated with their systemic dissemination. This method not only shows promise on its own but also enhances the effectiveness of other cancer treatments. Our findings suggest a new, safer way to encourage the body's defense system to fight cancer more effectively.

<p>Immunotherapy has significantly improved cancer treatment. However, many tumors are resistant to current immunotherapy due to the highly immunosuppressive tumor microenvironment (TME). Tumor cells can evade immune activation by externalizing phosphatidylserine (PS) on cell surface to trigger anti-inflammatory signals and induce immune tolerance. Recent studies show that PS is upregulated in TME and further increased after chemotherapy. For effective immunotherapy of tumors, the exposed PS needs to be blocked to relieve immunosuppressive TME and sensitize tumors to immune stimulants. </p> <p><br></p> <p>In this study, PS exposure level increased after the chemotherapy Doxil treatment on B16F10 melanoma cells, and the PS exposure reduced the response of antigen-presenting cells (APCs) to immune stimulants such as lipopolysaccharide. Dipicolylamine-Zn (DPA-Zn) shielded the PS exposure resulting from doxorubicin (DOX) treatment and reduced immunosuppressive interaction between tumors and APCs. The PS blockade by DPA-Zn improves the tumor response rate immune stimulants such as GM-CSF, STING agonist cyclic dinucleotides (CDN), anti-PD-L1 antibody. Among the combination at the tested doses, Doxil + DPA-Zn + CDN was the optimal combination that enhanced anti-tumor effect most significantly and prolonged the survival time in immune-cold B16F10 melanoma model. However, the anti-tumor efficacy was limited, which is attributed to poor tumor retention of CDN and DPA-Zn. </p> <p><br></p> <p>To prolong the intratumoral release of DPA-Zn and CDN and maximize the anti-tumor immunity, CDN was formulated as liposomes (CDN@lip), which significantly delayed the release of CDN in vitro and improved anti-tumor efficacy compared with free CDN formulation. Alginate hydrogel showed the potential to sustain release of DPA-Zn. DPA-Zn was loaded in the alginate hydrogel via electrostatic interaction, and the release rate was controlled by additional zinc gluconate. However, zinc caused detrimental effects on skin and can cause mice death at a high dose. To avoid the side effect of subcutaneously administered Zn, the dose of DPA-Zn in alginate hydrogel was readjusted based on the maximum tolerated dose study, and zinc gluconate was replaced with CaSO4.</p>

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25161号 / 医博第5047号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 小川 誠司, 教授 伊藤 能永 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In this PhD thesis the potential of T-cells as therapy for disease are explored. The applications of genetically modified T-cells for treatment of cancer and autoimmune disease; the functionality and optimal activation of T-cells are discussed. Successful treatment of cancer with T-cell receptor (TCR)-modified T-cells was first reported in 2006, and is based on recognition of a specific peptide by the TCR in the context of the MHC molecule. As antigen presentation in tumors is often defective and to avoid MHC-restriction, chimeric antigen receptors (CAR) molecules containing an antibody part for recognition of cell surface antigens and TCR and co-receptor signaling domains have been developed. Activated T-cells mount an efficient immune response resulting in the killing of the cancer cell and initiating T-cell proliferation. The rationale for using genetically modified T-cells instead of isolating tumor infiltrating lymphocytes from the tumor and expanding them (TIL therapy) is that it is often very difficult to obtain viable lymphocytes that are able to expand enough in order to use them for therapy. This thesis explores the possibility of using prostate-specific antigens to target T-cells towards prostate cancer. The prostate has many unique tissue antigens but most patients with metastatic prostate cancer have undergone prostatectomy and consequently have “prostate antigen” expression only in cancer cells. We targeted the prostate antigens TARP and PSCA with a HLA-A2 restricted TCR and a CAR respectively. In both cases the tumor-specific T-cells were able to generate potent proliferative and cytotoxic responses in vitro. The PSCA CAR-modified T-cells delayed subcutaneous tumor growth in vivo. It is evident from our in vivo experiments that the PSCA CAR T-cells were unable to completely cure the mice. Therefore, we aimed to improve the quality of the transferred T-cells and their resistance to the immunosuppressive tumor microenvironment. Stimulation with allogeneic lymphocyte-licensed DCs improved the resistance to oxidative stress and antitumor activity of the T-cells. We further investigated the potential of genetically modified regulatory T-cells (Tregs) to suppress effector cells in an antigen-specific manner. Using a strong TCR we hypothesize that the phenotype of the TCR-transduced Tregs may be affected by antigen activation of those cells. We found that the engineered Tregs produced cytokines consistent with Th1, Th2 and Treg phenotypes.

Dendritic cells (DCs) are promising mediators of anti-tumour immune responses. Unfortunately, a major hindrance to the development of highly effective DC vaccines is their short lifespan. Tumour antigen presentation may also not be optimal. We hypothesize that the introduction of exogenous survival factors (SFs) would prolong DC longevity and that modulation of TAA glycosylation will improve antigen presentation. To this end, we have constructed bicistronic lentivectors (LVs) encoding the xeno Tumour-Associated-Antigen (TAA), rHER-2/neu, and one of five candidate SFs. We demonstrated that our LVs can effectively protect transduced DCs from apoptosis when subjected to apoptosis-inducing conditions. TAA glycosylation has been proposed to obstruct the processing and presentation of peptides on MHC molecules. To address this second issue, we have engineered a LV that encodes a partially deglycosylated rHER-2/neu. Overall, we have generated the tools to alter intrinsic DC properties, which we believe will be integral to improving DC vaccine efficacy.

Dendritic cells (DCs) are promising mediators of anti-tumour immune responses. Unfortunately, a major hindrance to the development of highly effective DC vaccines is their short lifespan. Tumour antigen presentation may also not be optimal. We hypothesize that the introduction of exogenous survival factors (SFs) would prolong DC longevity and that modulation of TAA glycosylation will improve antigen presentation. To this end, we have constructed bicistronic lentivectors (LVs) encoding the xeno Tumour-Associated-Antigen (TAA), rHER-2/neu, and one of five candidate SFs. We demonstrated that our LVs can effectively protect transduced DCs from apoptosis when subjected to apoptosis-inducing conditions. TAA glycosylation has been proposed to obstruct the processing and presentation of peptides on MHC molecules. To address this second issue, we have engineered a LV that encodes a partially deglycosylated rHER-2/neu. Overall, we have generated the tools to alter intrinsic DC properties, which we believe will be integral to improving DC vaccine efficacy.

[Truncated abstract] The design of effective immunotherapies, using tumour antigens to stimulate a functional effector cytotoxic T cell (CTL) response in a tumour bearing host, requires an understanding of the 'real time' in vivo relationship between the host immune system and antigens expressed by the developing tumour. However, effector function of endogenous anti-tumour CTLs generated during tumour progression has largely been assessed by indirect ex vivo assays and often focused on a single antigen. Therefore, studies in this thesis evaluated the endogenous in vivo CTL response to multiple tumour antigenic epitopes in murine tumour models using Lewis lung carcinoma cells transfected with ovalbumin (an antigen that contains several intra-molecular MHC class I epitopes with a defined hierarchy) or a polyepitope (that contains a string of immunodominant MHC class I epitopes). Potent effector CTLs were generated to multiple dominant tumour antigenic epioptes early in tumour progression. However, in general, these CTL effectors only transiently retarded tumour growth, and at the later time points of tumour growth they were no longer generated in tumour draining lymph nodes. This coincided with diminished tumour antigen presentation in the same nodes which was found to be due to antigen loss. In both models antigen loss was the result of two processes; immuno-editing of the tumour by the host immune response and genetic instability resulting in antigen loss variants that could evade immune surveillance. A third model was generated that maintained low level tumour antigen expression throughout tumour progression. ... The impact of pre-existing endogenous dominant-epitope specific CTLs on tumour expressing the same epitope was also assessed, and resulted in a reduced tumour incidence and a CTL response restricted to a single antigen of the same MHC allele. Finally, the effects of two different immunotherapy regimens were examined. Intratumoural IL-2 treatment enhanced pre-existing CTL responses to the dominant epitopes leading to tumour regression. In addition, use of a multiple peptide vaccination regimen that avoided T cells competing for peptide-MHC complexes on APC was far more likely to be effective than one that did not. These results demonstrate that immunotherapies targeting tumours that express several dominant neo antigenic epitopes can be effective. The caveat for this approach is that it will only be effective in tumours that have generated an endogenous CTL response and must be used before antigen loss variants emerge.