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.

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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

Malignant melanoma is an immunogenic tumour capable of inducing a humoral immune response, as shown by tumour-reactive serum antibody in patients. Lack of effective chemotherapy in association with the immunogenic nature of the malignancy, has stimulated interest in the immunological management of the malignancy by antibody. Many mouse monoclonal antibodies against melanoma antigens have been developed, and some have been shown to induce tumour regression. However, a limitation on the use of mouse monoclonal antibodies in patients is the induction of an immune response against the immunising xenogeneic protein. The employment of human monoclonal antibodies, may be expected to reduce the patient's immune response against the allogeneic protein. Although more difficult to produce than mouse monoclonal antibodies, several human monoclonal antibodies have been established which induce tumour regression. Here I describe the establishment of mouse/human heterohybridomas producing human monoclonal antibody, from tumour-draining lymph nodes. A series of novel assay systems, initially developed and characterised using melanoma reactive mouse monoclonal antibodies, were sequentially employed for the selection of human antibody exhibiting high tumour specificity. Several clones producing melanoma reactive human antibody were established. Clone MDT. 1 was selected for further characterisation, because of its highly selective reactivity against viable melanoma and other neuroectodermal cell lines, but lack of reactivity against other common malignant and non-malignant cell lines. Such restricted cell reactivity is characteristic of reactivity with class 2 tumour associated antigens. MDT. 1 was shown, in ELISA, to exhibit reactivity to ganglioside antigens GD3, GD2, GD1b, GM3 and GM2. These antigens are commonly associatedw ith the malignant transformation of melanocytes and other neuroectodermal cells. Enzymatic modification of GM3, with neuraminidase, identified the reactive minimal essential epitope as Neua2- 3Galß1-4GIc-. Reactivity with rat monoclonal antibody 9G4 and molecular analysis showed MDT. 1 is encoded by the highly conserved VH4 gene, VH4-21. Like other VH4-21 encoded autoantibodies MDT. 1 exhibits reactivity with the cold agglutinin T. Analysis of the structures of `i' and sialogangliosides has identified similar structural epitopes, which may confer MDT. 1 reactivity. VH4-21 encoded autoantibody 216 exhibits similar reactivity with tumour associated ganglioside antigens as MDT. 1. Sialo-ganglioside/`i' reactive VH4-21 encoded antibodies, could therefore represent an important aspect of autoantibodies in the overall host immune response to tumour.