Oncolytic viruses (OVs) are novel biological agents that selectively infect and kill malignant cells. OVs can also generate anti-cancer immunity. Our lab exploited this phenomenon and developed an in vitro vaccine with infected leukemia cells with oncolytic virus vaccine – and named immunotherapy by leukemia-oncolytic virus (iLOV) – that provided in vivo protection in a murine model for acute lymphoblastic leukemia. This work further characterizes iLOV biology and the interaction of its immune responses. An in vitro immune response assay was optimized to detect and quantify the in vivo anti-leukemia immunity generated by iLOV. Anti-viral immunity is an obstacle for OV therapy. Although iLOV created anti-viral antibodies towards itself, these neutralizing antibodies did not hinder the vaccine’s ability to initiate complement or dendritic cell activation. We envision personalized versions of iLOV for leukemia patients in remission to prevent the possibility of relapse. This work highlights new advantages for infected cell vaccines and supports the progress of iLOV toward clinical testing.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22694号 / 医博第4638号 / 新制||医||1045(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 生田 宏一, 教授 竹内 理, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23105号 / 医博第4732号 / 新制||医||1050(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 竹内 理, 教授 上野 英樹, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The first reports of bacteria as a cancer therapy date back to the pioneering work of Dr. William Coley–now widely regarded as the father of immunotherapy. As far back as 1891, Coley demonstrated that the intratumoral injection of live and later heat-killed isolates of Streptococcus pyogenes and Serratia marcescens could induce durable remission in patients with bone and soft tissue sarcoma. While this therapy was deemed to unsafe at the time, Coley’s findings have formed the basis for our modern understanding of immunology and cancer immunotherapy. Over the past two decades, the advent of synthetic biology is driving a new era of medicine through the genetic programming of living cells. This transformative approach enables the creation of engineered systems that sense and respond to diverse environments, permitting safe and effective targeted delivery of therapeutic payloads within disease sites. In this thesis, I seek to utilize principles from synthetic biology and immunology to engineer bacteria for immunotherapeutic delivery. I have developed multiple strains of non-pathogenic E. coli capable of colonizing solid tumors and delivering diverse immunotherapeutic payloads specifically within the tumor. This local delivery approach enables the utilization of therapeutic agents that may be otherwise systemically toxic. In one instance, we engineered an encoded nanobody antagonist of CD47 (CD47nb), an anti-phagocytic receptor commonly overexpressed in several human cancers. We show that delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in a syngeneic tumor model. Thus, engineered bacteria may be used for safe and local delivery of diverseimmunotherapeutic payloads leading to systemic antitumor immunity.

Genetically engineered oncolytic viruses (OVs) have been proven to be effective anti-cancer agents. However, the heterogeneity of tumours and obligate attenuation of OVs to achieve safety can limit their efficacy. Our lab has previously shown that diverse small molecules, which we have termed “Viral Sensitizers”, used in combination with OVs can potentiate the infection of cancer cells by OVs over 1000-fold in some cases, resulting in cancer-specific killing in both in vitro and in vivo tumour models. We observed that a subset of viral sensitizer compounds ultimately acts by reducing the expression of IFNb, thereby inhibiting antiviral signaling. Here, we aimed to further refine the mechanism of action of this class of compounds. Our results suggest that VSe1 and more stable analogs such as VSe1-28 inhibit nuclear accumulation of NF-kB p65 and expression of various antiviral cytokines including, TNFa, IL-6, IFITM1, and MX2 in multiple oncolytic VSV-resistant cancer cell lines but not in normal cells. This was also observed in vivo in CT26wt immune-competent mouse tumour models, where our group has already demonstrated the therapeutic benefit of combining VSe1-28 with oncolytic VSV. Using various biochemical methods, we have determined that VSe1 and its analog VSe1-28 lead to these effects at least in part through covalent modification of NF-kB p65. In sum, this study provides a new understanding of how these novel viral sensitizers work at the molecular level. This new understanding will not only aid in the discovery and development of improved molecules but also their clinical translation in combination with oncolytic viruses.

No description available.

T cells can fight cancer, but an immunosuppressive tumor microenvironment (TME) disallows them from carrying out their function over time. Upregulation of inhibitory checkpoint molecules such as programmed cell death protein 1 (PD1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) can lead to such an immunosuppressive TME. Despite their widespread use, immune checkpoint blockade (ICB) antibodies targeting checkpoint molecules remain ineffective in most cancer patients. We do not understand why some patients respond to ICB better than others. To understand the heterogeneity of ICB response, we must understand the heterogeneity of the T cell subsets acted upon by such therapies. Here, we ask how T cell subsets change in the presence and absence of ICB. We track T cell clones through their T cell receptor sequences and link phenotypes with T cell receptor specificities. Through multiplexed single cell TCR sequencing, single cell RNA sequencing, and the use of cell- surface CITE-seq antibodies, coupled with surgical biopsy, we longitudinally tracked the fate of individual T cell clones within tumors at baseline and in response to ICB in an immunogenic mouse tumor model. Furthermore, computational clustering of T cells solely based on their gene expression profiles may ignore upstream regulatory mechanisms that control T cell gene expression. Hence, we employed Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis to cluster CD8+ and CD4+ T cell phenotypes. VIPER leverages inference of gene regulatory networks to allow full quantitative characterization of protein activity for transcription factors, co-factors, and signaling molecules by assessing the enrichment of their transcriptional targets cell-by-cell among expressed genes. This gave us a window into the transcriptional states and their inferred protein activity. We next developed a computational analysis toolkit to study TCR clonality incorporating sub-sampling of TCR clonotypes, forward and back tracing of shared clones between timepoints, and in turn, inferred shared clonal evolution. We employed the above workflow to MC38 tumor-infiltrating and tumor-draining lymph node-derived CD8+ and CD4+ T cells. We found that T cell phenotypes are highly dynamic within tumors at baseline, in the absence of ICB, particularly within the window that they are responsive to therapy. In the absence of ICB, effector phenotype of CD8+ T cells diminished, while the exhaustion phenotype was enhanced as tumors progressed. Within the CD4+ population, a heterogenous subset of regulatory CD4+ T cells (Tregs) changed phenotype over time, and CD4+ Th1 like effectors, along with stem like progenitor CD4+ showed distinct dynamism. Next, by analyzing responses to therapy within his context, we found that both anti-PD1 and anti-CTLA4 act through distinct mechanisms on CD8+ and CD4+ T cells. Anti-PD1 acted upon intra-tumoral effector CD8+ T cells to slow their progression to terminally differentiated exhausted cells, i.e., increased their persistence within tumors. Anti-CTLA4 therapy increased recruitment of novel effector CD8+ T cell clones to tumors from lymph nodes while diminishing tumor-infiltrating Tregs. ICB also potentiated CD4+ Th1 like phenotype. These results uncovered a behavior pattern of CD8+ and CD4+ T cells within tumors at baseline tumor progression, and then in the presence of ICB. We believe these findings have added to our understanding of the subtleties of T cell phenotypes in tumors, specifically in response to ICB. This will provide a practical framework for designing and validating novel checkpoint blockade therapies in the future.

Pancreatic cancer is the 10th most common cancer diagnosis and 4th most common cause of cancer mortality in the United States, highlighting a disparity between disease prevalence and outcome. Ineffective drug delivery to these tumors contributes to the poor prognosis for this disease, as intravenous drug delivery is hampered by poor vascularity within these tumors. Bacterial therapy, or the use of bacterial components to treat disease, is thought to be able to overcome such drug delivery challenges; through a combination of tumor homing and long-term colonization, bacteria can be utilized to produce anti-cancer molecules directly within the cores of tumors. As such, here, we interrogate the feasibility of bacterial cancer therapy for pancreatic ductal adenocarcinoma (PDAC). Before delving too deeply into bacterial therapy design, it was important to first address one major limitation in therapeutic screening models. As a therapeutic should be effective against the entirety of the tumor, without a specific emphasis on the malignant epithelia, we developed and characterized a novel protocol for culturing ex vivo (explant) murine PDAC tissue with a corresponding protocol for human PDAC tissue. We demonstrated that these tumor slice explants retain the complex cellular architecture and population complexity throughout culture, making them a useful resource for not only therapeutic screens, but also paracrine interactions, which are infeasible to explore with in vitro and in vivo models. Use of these murine and human PDAC explant models assisted in the selection of a potent, bacterial-derived cytotoxin, theta toxin, as a potential therapeutic candidate for PDAC, in both bacteria lysate and live bacteria contexts. Ultimately, we employed a strain of a probiotic bacteria, E. coli Nissle 1917, as a ‘living drug’ to selectively produce theta toxin within the confines of a PDAC tumor in a mouse model of pancreatic cancer. In in vivo studies, we demonstrated that live bacteria preferentially colonize tumor tissue following a single, direct, intratumoral injection into the primary PDAC tumor. We found that not only did the bacteria colonize the injected tumor, but also translocated to distant regions of metastasis and secondary tumors such as anogenital papillomas. However, the long-term efficacy of this strategy is in question, as bacterial colonization and therapeutic capability waned after several weeks. Despite the limited time scale of the bacterial colonization, treatment with a single dose of live, theta toxin-producing bacteria provided a nearly 3-fold improvement in overall survival compared to vehicle and standard of care chemotherapy (gemcitabine) treatment arms. Preliminary evidence suggests that this improvement is due to a combination of the direct cytotoxic effect of the theta toxin and an inherently immunostimulatory capacity of these bacteria, resulting in an influx of anti-tumor immune cells and an overall reduction in immunosuppression phenotype markers. These findings suggest that bacterial therapy could be a useful tool for the treatment of pancreatic cancer, not solely due to the direct cytotoxic effect on the tumor, but with the potential for a combination treatment with immunotherapies.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24139号 / 医博第4879号 / 新制||医||1060(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 森信 暁雄, 教授 上野 英樹, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cancer immunotherapy is emerging as a powerful tool in the treatment of cancer. Multiple clinical trials have established that infusion of tumour-specific T cells can cause regression of advanced tumours and prevent tumour relapse. While tumour-specific T cells are typically rare, engineering methods have been developed to introduce tumour-specific receptors into T cells and engender peripheral blood T cells with the ability to kill tumour cells. These engineering successes notwithstanding, tumour cells demonstrate variable sensitivity to T cell attack. Therefore, to maximize the impact of the engineered T cells, it is necessary to develop therapeutic strategies that render tumour cells sensitive to immune attack. For my thesis research, I sought to develop a high throughput screening assay that would allow me to screen chemical libraries for agents that sensitize tumour cells to T cell attack. My ultimate goal is to define chemical agents that effectively sensitize tumour cells to T cell attack but display a better toxicity profile than existing chemotherapies. To this end, I developed a screen where resistant tumour cells were exposed to T cells engineered with chimeric antigen receptors and positive hits were defined as agents that could enhance killing of the tumour cells. My work explored both murine and human systems and I ultimately decided to use human cells for my screen. Multiple methods for measuring tumour cell killing were evaluated, many tumour lines were screened and I optimized the conditions for generating large numbers of engineered T cells for the screen. The net result of my thesis work is a miniaturized assay that is ready for high throughput screening. / Thesis / Master of Science in Medical Sciences (MSMS)