Characterization of the IFITM1 signaling pathway in cancer
Sinclair, Elizabeth Hannah
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The aim of this thesis was to establish the therapeutic value of the IFITM1 monoclonal antibodies and to design and develop therapeutically valuable recombinant monoclonal antibodies so as to study the implication of these novel antibodies in cancer therapy. Cancer metastasis is one of the main interests that has given rise to the design and development of innovative strategies for cancer therapeutics. The Interferon Induced Transmembrane Protein 1(IFITM1), a notable member of the IFITM family of proteins has been identified as one of the most up-regulated trans-membrane proteins in metastatic breast cancer and cervical adenocarcinoma. This interferon-regulated protein is also involved in cell migration, invasion in glioma and squamous cancers. This PhD aimed to study IFITM1 as a pro-invasive cancer target by the use of IFITM1 monoclonal antibodies that were raised against the extracellular domain of the human IFITM1 gene. The epitope mapping of IFITM1 revealed the binding activity of the IFITM1 monoclonal antibody. This gave the opportunity to design and generate to new IFITM1-specific molecular tools, in the form of recombinant IFITM1 targeted murine scFv antibody, IFITM1-CPG2 yeast fusion protein antibody for potential application in ADEPT as well as a Mouse-Human Chimeric IFITM1 antibody secreting mammalian cell line. The immunohistochemical staining of IFITM1 in tissue micro array from breast, colon and oeosphegal cancer has revealed that the majority of these cancers produce this protein. However, IFITM1 is over produced in cervical cancer indicating it’s selective over expression in cervical cells. This PhD endeavored to investigate the expression of IFITM1 at a translational and transcriptional level and to study the clinical significance of IFITM1 in cervical cancer. The antibody dependent cell mediated cytotoxic activity of the chimeric IFITM1 antibody was found to be cytotoxic to SiHa cells in vitro. In the future these molecular tools could be used to regulate and further characterize the activity of this transmembrane protein antibody. In an effort to better understand the mechanisms that regulate the activity and the over production of the IFITM1 gene and its interacting proteins, a proteomic screen of cervical cancer cells was carried out using data-independent SWATH-MS on an AB SCIEX TripleTOF™ mass spectrometer. This Mass Spec analysis provided us with a host of IFITM1 biomarkers and revealed that the IFITM1 gene and its binding proteins also cross link with the IRF1 pathway. The data presented in this thesis, demonstrates that the IFITM1 gene can be targeted to either stimulate or inhibit IFITIM1 signaling to engage IFITM1 as a potential pro-invasive extracellular receptor as a target in antibody cancer therapy. In summary, this thesis aimed to confirm the activity and the binding specificity of the IFITM1 antibody. Additionally, this thesis demonstrated a promising application of the recombinant antibody in the ADEPT technology. Characterization of IFITM1 mAb effector functions indicated that the antibody was cytotoxic to cervical cancer cells. This highlights an important element in the immune suppressive tumour microenvironment. And finally, this thesis also provides the basis for the production of recombinant mouse human chimeric antibodies that are a part of a new group of immunotherapeutic molecules paving the way for cancer therapeutics.