Csizmar, Clifford2018-11-282018-11-282018-08https://hdl.handle.net/11299/201057University of Minnesota Ph.D. dissertation. 2018. Major: Medicinal Chemistry. Advisor: Carston Wagner. 1 computer file (PDF); 199 pages.The ability to direct cell-cell interactions has tremendous value in several therapeutic fields. While genetically-encoded artificial receptors have proven efficacious, their scope is limited by the genetic engineering that underlies the approach. To circumvent some of these limitations, our group has developed a non-genetic method to modify any cell surface with a targeted protein scaffold. First, we engineered a protein ligand based upon the human tenth type III fibronectin domain (Fn3) that binds to epithelial cell adhesion molecule (EpCAM), an overexpressed tumor antigen. Using yeast surface display, mammalian cell panning, and a novel titratable avidity-reduction selection technique, we evolved Fn3 clones exhibiting high affinity and robust selectivity for cellular EpCAM. We then incorporated these Fn3s into a multivalent chemically self-assembled nanoring (CSAN). EpCAM-targeted CSANs were anchored to cell membranes through the hydrophobic insertion of phospholipids into the lipid bilayer. The targeting elements were subsequently removed from the cell surface by disassembling the CSAN with the antibiotic, trimethoprim. Using this system, we successfully directed and reversed targeted intercellular interactions in vitro. Finally, the modular CSANs were used to study how avidity impacts the apparent affinity of a multivalent scaffold. By tuning the number of Fn3 domains on the CSAN, we quantitatively described how the apparent affinity changes as a function of ligand affinity, domain valency, and antigen expression density. These results informed the development of a CSAN capable of discriminating between cells expressing different quantities of EpCAM both in vitro and in vivo. In conclusion, we developed a diverse toolkit for directing and studying cell-cell interactions. The CSAN platform is applicable to several therapeutic arenas and, by balancing affinity and avidity, may offer advantages over current cell-directing methods.enAvidityCell-Cell InteractionsCell Membrane EngineeringNanotechnologyProtein EngineeringTargeted TherapeuticsThesis or Dissertation