Using biophysical characterization to begin to understand the onset, diagnosis, and treatment of cancer
| dc.contributor.author | Krahn, Natalie Jean | |
| dc.contributor.examiningcommittee | McKenna, Sean (Chemistry) Butler, Michael (Microbiology) Lin, Francis (Physics and Astronomy) Reinhardt, Dieter (Anatomy and Cell Biology, McGill University) | en_US |
| dc.contributor.supervisor | Stetefeld, Jörg (Chemistry) | en_US |
| dc.date.accessioned | 2017-12-06T21:02:35Z | |
| dc.date.available | 2017-12-06T21:02:35Z | |
| dc.date.issued | 2017-01-01 | en_US |
| dc.degree.discipline | Chemistry | en_US |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | en_US |
| dc.description.abstract | Cancer is the leading cause of death among Canadians and therefore efforts are in place to develop novel or improved ways to diagnose and treat cancer to reduce mortality rates. This thesis focuses on the role of two important proteins in cancer: high mobility group AT-hook 2 (HMGA2) and a chimeric heavy-chain antibody (EG2-hFc). HMGA2 is a chromatin-bound transcription activating factor found in the nucleus. The overexpression of HMGA2 leads to increased cell proliferation, a crucial function in embryonic stem cells but which forms tumours in adult cells. HMGA2 is made up of three AT-hook binding domains which allows it to interact with dsDNA, replication forks (RFs), and Holliday junctions. The observed high cell proliferation is a result of HMGA2 stabilizing RFs. Recombinant HMGA2 was studied using a biophysical approach to investigate its unbound and bound behaviour in solution. I determined that HMGA2 is an intrinsically disordered monomer and binds in a side-by-side orientation to RF while maintaining its flexibility. From this information we can begin to elucidate the HMGA2:RF mechanism and understand its biological significance. EG2-hFc is a chimeric heavy-chain mAb raised against the epidermal growth factor receptor (EGFR). It was designed to be smaller in size than traditional mAb therapeutics for deeper tumour penetration. The composition and heterogeneity of the sugars attached in the Fc region can have an impact on the efficacy of mAbs. Therefore I implemented a glycoengineering approach to alter these sugars and analyze their effect on the mAb’s hydrodynamic and binding properties. Glycoprotein processing inhibitors added to the cell growth media generated mAbs with complex, high mannose (+/- glucose), or hybrid glycans. The secondary structure and hydrodynamic properties of EG2-hFc were unchanged upon glycan alteration. However, hybrid glycans reduced the binding affinity of EG2-hFc to FcγRI two-fold when compared to complex glycans while removal of the N-glycan drastically (>100-fold) reduced binding to a full-sized IgG1 but only marginally (~8-fold) reduced binding to EG2-hFc. These results suggested that the design of EG2-hFc stabilizes the Fc pocket to allow for complete sugar removal without abolishing its function, thus making it attractive as a potential therapeutic candidate. | en_US |
| dc.description.note | February 2018 | en_US |
| dc.identifier.citation | Krahn, N., Spearman, M., Meier, M., Dorion-Thibaudeau, J., McDougall, M., Patel, T. R., De Crescenzo, G., Durocher, Y., Stetefeld, J., and Butler, M. (2017) Inhibition of glycosylation on a camelid antibody uniquely affects its FcγRI binding activity. Eur. J. Pharm. Sci. 96, 428–439 | en_US |
| dc.identifier.uri | http://hdl.handle.net/1993/32712 | |
| dc.language.iso | eng | en_US |
| dc.publisher | European Journal of Pharmaceutical Sciences | en_US |
| dc.rights | open access | en_US |
| dc.subject | Biophysics | en_US |
| dc.subject | Biochemistry | en_US |
| dc.title | Using biophysical characterization to begin to understand the onset, diagnosis, and treatment of cancer | en_US |
| dc.type | doctoral thesis | en_US |