Kilic, Ozgun2020-05-042020-05-042020-01https://hdl.handle.net/11299/213080University of Minnesota Ph.D. dissertation. January 2020. Major: Medicinal Chemistry. Advisor: Carston Wagner. 1 computer file (PDF); xi, 166 pages.Alternative protein scaffolds, such as fibronectins and affibodies, have gained interest in recent years over antibody fragments due to their protein stability, ease of production and their malleable nature, allowing them to be engineered to bind to any target of interest. One such important target is Epidermal Growth Factor Receptor (EGFR). In this thesis we fused EGFR-targeting fibronectins on Chemically Self-Assembled Nanorings (CSANs) format to yield diverse set of therapeutic applications including receptor downregulation, immunotherapy and targeted drug delivery. While fibronectin fusion proteins, yielded monomeric proteins maintaining their cell binding ability, they were incapable of preventing cell proliferation. Additionally, affibodies targeting other ErBB members, HER2 and HER3, also generated stable, soluble fusion proteins but were also not potent enough on their own. Hence, another effective cancer therapy is taking advantage of patient’s own immune system with immunotherapy. In this work we examined the efficacy of rapidly internalizing bispecific CSANs targeting EGFR and also the prospect of using alternative protein scaffolds in this setting. Bispecific CSANs were successful in vitro, selectively lysing EGFR-overexpressing cancer cells. Similarly, bispecific CSANs also showed tumor eradication in vivo with a unique mechanism of both making the cancer cells more immune responsive by downregulating PD-L1 and also activating T cells for selective tumor lysis. Therapeutic potential of these CSANs will be investigated further in other cancer types and in combination with other antigen targeting scaffolds for long-term remission.enThesis or Dissertation