Characterisation of an anti-glycan monoclonal antibody against ovarian cancer

Abstract

Ovarian cancer is a deadly disease that has been associated with changes in glycans. Therefore, anti-glycan monoclonal antibodies (mAb) have the potential to selectively kill or retard chemoresistant tumours. However, the interactions between mAb and glycan epitopes are difficult to characterise due to the specialised tools and expertise required. Recently, our group discovered mAb-A4, a novel mAb found to bind and kill hESC and multiple cancer types including OC but not normal cell lines. Further analysis revealed that mAb-A4 binding was glycan dependent. Thus, it was hypothesised that mAb-A4 was targeting an oncofetal glycan antigen specific to stem and OC cells versus normal cells. In this thesis, the mAb-A4 glycan antigen in OC cell lines was investigated by advanced high-sensitivity mass spectrometry (MS), enzymatic digestion, glycan microarray and siRNA knockdown. Importantly, a novel adaptation allowed the detailed characterisation of polylactosamine termini from OC cell lines by MS. MS detected Type 1 and H Type 1 glycans on OC cells, and their soluble forms could also block mAb-A4 binding to cells in flow cytometry and Western blot. Furthermore, siRNA-mediated knockdown of B3GALT5 in SKOV3 demonstrated that the antigen was dependent on the Type 1 structure. It was concluded that mAb-A4 specifically recognises oncofetal Type 1 and H Type 1 on OC and hESC, and that this recognition was novel compared to other anti-H Type 1 mAbs such as anti-SSEA-5, 17-206, RAV12 and HESCA-2. Currently, the role of Type 1 and H Type 1 in OC is unknown, but these findings warrant further investigation into the purpose and prevalence of these glycan biomarkers in disease progression. The methods developed in this thesis enable the characterisation of additional anti-glycan mAbs and cancer cell lines, facilitating the discovery of novel biomarkers and therapies against cancer.