McKnight, Brandi2024-07-242024-07-242024-05https://hdl.handle.net/11299/264330University of Minnesota Ph.D. dissertation. May 2024. Major: Medicinal Chemistry. Advisor: Carston Wagner. 1 computer file (PDF); xviii, 211 pages.The field of bispecific therapeutics has experienced significant growth in recent decades. With their ability to target two target antigens, bispecific therapeutics have facilitated cell-to-cell interactions and found applications in various therapeutic domains such as cancer immunotherapy, inflammation, and angiogenesis, as well as in drug delivery and medical imaging. Bispecific therapeutics are available in various formats, ranging from full antibodies to smaller antibody-derived fragments and non-antibody-derived formats known as alternative proteins. (Chapter 1). The Wagner lab has contributed to this field with a unique protein scaffold, termed chemically self-assembled nanorings (CSANs), which are not only bispecific but also multivalent, enabling cell-to-cell interactions. CSANs have broad utility in multiple biomedical applications such as targeted drug delivery, PET/CT imaging, and CSAN-assisted cargo transfer. This dissertation delved into two additional applications of CSANs: cancer immunotherapy and cell delivery. In previous studies, CSANs were employed to redirect T cells to target cancer cells by use of bispecific CSANs consisting of two monomeric proteins, one targeting a cancer antigen and the other containing a T cell-targeting scFv (αCD3). In Chapter 2, we aimed to condense the two monomeric proteins into a single protein scaffold named E1-DHFR2-CD3. This scaffold comprises two targeting ligands, one a fibronectin that targets EGFR (αE1) and the other a single chain variable fragment that targets T cells (αCD3). We designed, expressed, and characterized E1-DHFR2-CD3, demonstrating its ability to form CSANs in the presence of bis-MTX. Further testing showed that E1-DHFR2-CD3 effectively induces T cell-mediated cytotoxicity, both in its monomeric and CSAN form across multiple protein concentrations and effector-to-target (E:T) ratios. In later experiments, the valency of the αE1 fibronectin and αCD3 scFv in E1-DHFR2-CD3 CSANs was modified by altering the ratio of E1-DHFR2-CD3 incubated with the αE1 and αCD3 monomers. However, cytotoxicity experiments revealed that changes in valency did not significantly affect treatment outcomes across different treatment groups. Building on the successful use of E1-DHFR2-CD3 to induce T cell-mediated cytotoxicity, Chapter 3 details our use of E1-DHFR2-CD3 to modify glass-supported lipid bilayers, which have historically been used to investigate T cell signaling. We employed CSAN-modified bilayers to assess T cell behavior as they land and spread on the CSAN-modified surface using single localization microscopy. We confirmed that SLBs could be modified with CSANs and observed the mobility of CSANs on the bilayer. Furthermore, we determined the stoichiometry of our CSANs using single step photobleaching, which aligned with previously acquired data. We further investigated if the presence of the CSANs on SLBs impacted T cell landing and spreading by measuring their maximal contact area, finding no significant difference across various CSAN densities. In the final part of this dissertation (Chapter 4), we explored using CSANs to deliver hematopoietic stem cells (HSCs) to the blood-brain barrier (BBB) to treat Hurler Syndrome, a rare lysosomal storage disorder. After our initial method employing αVEGFR2 CSANs proved unsuccessful, we pursued a secondary strategy that involved using αVEGFR2 prenylated CSANs to modify the surface of HSCs without reliance on antigen expression and direct HSCs towards VEGFR2+ brain endothelium. We designed, produced, and characterized two αVEGFR2 monomeric biparatopic proteins and confirmed that the αVEGFR2 prenylated CSANs could bind to two different cell lines that do not express VEGFR2. Lastly, in Chapter 5, we discussed potential future applications of the proteins discussed throughout this dissertation.enThesis or Dissertation