The immune system is a complex set of cells in the body that work to prevent disease or infection by recognizing and eliminating foreign material or unhealthy cells. One essential type of immune cell that is part of the innate immune response are Natural Killer (NK) cells. These cytotoxic effector lymphocytes can detect certain unhealthy cells, such as cancer cells, that normal B and T cells would miss. For example, many tumor cells have evolved to bypass immune system surveillance by not expressing major histocompatibility complex class I molecules (MHC class I), on their surface, reducing recognition by cytotoxic T cells. However, NK cells are activated when they detect low to no MHC class I on cells and lyse them by secreting toxic perforins and granzymes. NK cells are currently being developed as an adoptive cell therapy for treatment of cancer. However, because these cells only make up 5-10% of the lymphocytes, novel methods are being developed to expand these NK cells outside the body while also enhancing their cytotoxic properties. One method for NK expansion has been developed by the Copik Lab utilizing plasma membrane particles. These particles are derived from a K562 Leukemia cell line that expresses IL21 and 41BBL on the plasma membrane (CSTX-002) that have been found to enhance NK cell proliferation while maintaining their cytotoxic abilities. The Copik Lab is now developing a novel methodology to expand and/or modify NK cells using engineered exosomes. Exosomes are nanovesicles secreted by many types of cells, such as cancer cells, to transport genetic information and to communicate with other neighboring cells. Preliminary studies have shown that exosomes secreted from the CSTX-002 cells can expand NK cells. This study will develop methods to characterize exosomes from a new cell line engineered in the Copik lab, CL-001, that specifically target IL21 and 41BBL to the exosome surface and determine if they can expand functional NK cells. Methods for characterizing tThe exosomes will be characterized for size, quantity, and protein expression will be developed. Their ability to expand functional NK cells will be determined by culturing NK cells in the presence of the exosome and testing the cytotoxicity of the exosome-expanded NK cells. This project will provide the foundation for new studies in the Copik lab developing novel methods for ex vivo expansion and/or modification of NK cells using exosomes.

Immunotherapy with checkpoint blockade has revolutionized cancer treatment, sparking the search for other immune cell inhibitory receptors as novel immunotherapeutic targets. Among those, TIGIT has been the most promising based on strong initial results with multiple therapeutic antibodies under clinical development. Yet, first results of two phase III trials of anti-TIGIT, tiragolumab, in combination with anti-PD-L1 in lung cancer were disappointing, prompting a search for answers. Tiragolumab and many other candidate antibodies include an Fc-active domain, which binds to Fc receptors on effector cells, including to FcγRIII on Natural Killer (NK) cells. This induces killing of opsonized TIGIT+-cells by antibody-dependent cellular cytotoxicity (ADCC). The Fc-active domain of anti-TIGIT is associated with enhanced efficacy over Fc-silent versions due to ADCC-based clearance of unwanted TIGIT+ exhausted effector lymphocytes and Tregs. Yet, TIGIT is also expressed on NK cells, which potentially could result in their depletion through fratricide and negatively affect treatment outcomes. NK cells are innate immune cells that upon activation directly kill cancer and coordinate anti-tumor immunity and are being pursued as adoptive cell therapy. Prior studies in mice showed that NK cells play an essential role in the efficacy of anti-TIGIT, while TIGIT expression on NK cells was associated with exhaustion. Despite this, TIGIT expression and signaling on human, activated and expanded NK cells and the effect of Fc-active vs. silent anti-TIGIT in the context of NK cell function and fratricide have not been studied and are the subject of this dissertation. This study found that activation of NK cells upregulates TIGIT, and in fact, TIGIT+ NK cells had superior anti-tumor function compared to TIGIT- NK cells. However, chronic TIGIT engagement by its ligand PVR during tumor exposure induced dysfunction in NK cells. TIGIT blockade with Fc-silent anti-TIGIT restored NK cell effector function while Fc-active anti-TIGIT resulted in NK cell depletion via fratricide and reduced killing compared to Fc-silent. To overcome this, NK cells were expanded with clinically relevant PM21-particle method and engineered using CRISPR/Cas-9 to generate TIGIT knockout (KO) PM21-NK cells. TIGIT KO not only improved PM21-NK metabolic fitness and cytotoxicity during long-term tumor exposure, importantly, it prevented PM21-NK cell depletion and decrease in cytotoxicity when combined with Fc-active anti-TIGIT. Overall, these findings suggest that Fc-active anti-TIGIT therapeutics can deplete activated TIGIT+ NK cells. This study also demonstrated the therapeutic potential of fratricide-resistant expanded TIGIT KO PM21-NK cells alone or in combination with Fc-active anti-TIGIT to improve treatment outcomes.

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Fumagillin is the natural product isolated from fungus Aspergillus fumigatus, and is recognized as a potent anti-angiogenic compound. Substantial investigation has been focused on the anti-tumor activities of fumagillin and its analogues, some of which have been adopted in pre-clinical and clinical studies. However, investigation on the immunomodulating activities of this class of compounds is limited and results have been controversial. As there is intense interest in elucidating the interrelation between immune modulation and tumor development, novel immunopharmacological properties of chemotherapeutic agents have recently been explored for their therapeutic potentials in clinical applications. As a combination to both these research topics, fumagillin and its synthetic analogues were firstly investigated on different types of immune cells, such as T lymphocytes, dendritic cells and macrophages. F23, a fumagillin analogue with potent immunological activities, was further examined in three different murine cancer models, EL4 lymphoma, CT26 colon carcinoma and 4T1 mammary carcinoma, and their anti-tumor activities and intrinsic immunomodulatory effects were explored. Fumagillin and its analogues exert diversified functions in different types of immune cells. For example, they showed inhibitory effects on cell proliferation and cytokine production of T lymphocytes upon polyclonal stimulation, stimulatory effects on dendritic cells by inducing a highly-matured population, which contributed to induction of syngeneic and allogeneic lymphocyte proliferation and a preference to Th1 polarization, and multiple effects on macrophages based on phenotypic and cytokine analyses. Studies in murine cancer models showed that the fumagillin analogue F23 caused substantial inhibition of tumor development in three cancer models to different extents, with pronounced inhibitory effects on the expansion and functions of myeloid-derived suppressor cells (MDSCs), the signature cell population responsible for tumor progression and refractoriness to chemotherapeutic and immunotherapeutic agents, thereby suggesting the novel immunopharmacological properties of fumagillin and its analogues contributed to tumor suppression. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Malignant mesothelioma is an aggressive cancer with increasing incidence worldwide. Exposure to asbestos is believed to be the main mechanistic basis of malignant transformation of mesothelial cells. Despite decades of efforts, treatment options for this malignancy are still limited to traditional surgery and chemotherapy, which do not provide significant survival benefits, highlighting the importance of finding novel therapeutic and preventive approaches to fight mesothelioma. For this reason, we aimed to examine the efficacy of immunotherapy strategy using DNA vaccines targeting tumor-expressing antigens. Immunotherapy targeting tumor associated self-antigen WT1 with conventional and PD1-based DNA vaccines was unable to induce tumor regression or improved survival in a quantitative mouse malignant mesothelioma model due to insufficient levels of antigen-specific immune responses being elicited. While why PD1-based DNA vaccine does not improve self-antigen WT1-specific immune responses remains to be investigated, it becomes important to define the level of vaccine-elicited immune responses for protection. To date, the immune correlates of vaccine-elicited immunity remains poorly understood for the prevention and eradication of malignant mesothelioma. With the development of a malignant mesothelioma mouse model stably expressing HIV-1 GAG model antigen, we utilized the remarkably enhanced antigen-specific T cell responses elicited from our PD1-based HIV-1 GAG p24 vaccine to define antitumor responses. It has been demonstrated in this study that vaccine-elicited host immunity not only achieved complete and long-lasting protection against murine mesothelioma cell challenges but also resulted in therapeutic eradication of pre-existing mesothelioma after four consecutive DNA vaccinations. Vaccine-elicited 〖CD8〗^+ T cells attributed primarily and dose-dependently to the protective efficacy in both preventive and therapeutic settings. Moreover, the consecutive vaccinations activated polyfunctional 〖CD8〗^+ T effector cells via T-bet and Eomes-mediated pathways, leading to the rejection of mesothelioma by releasing inflammatory IFN-γ and TNF-α in the vicinity of target cells and by triggering the TRAIL induced apoptosis. Importantly, the vaccination not only activated 〖CD8〗^+ T cells and maintained their effector function but also overcame immunosuppressive networks by downregulating inhibitory PD1 and Tim-3 molecule expression on 〖CD8〗^+ cells and reducing suppressor cells such as myeloid-derived suppressor cells (MDSCs) and Treg, leading to the shift of tumor immune oediting from progression to elimination. Taken together, the generation of malignant mesothelioma mouse models in our study can enable targeting immunotherapy strategies to be evaluated in a quantitative way. Our data suggested that high frequency of vaccine-elicited 〖CD8〗^+ T cells could prevent and eradicate malignant mesothelioma. The activation of quantitatively and qualitatively enhanced CD8+ T cells caneliminate theimmune suppressive network contributing to the complete tumor rejection. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy