Johnson, Sadie2019-03-132019-03-132018-12https://hdl.handle.net/11299/202183University of Minnesota Ph.D. dissertation.December 2018. Major: Chemical Engineering. Advisor: Benjamin Hackel. 1 computer file (PDF); xi, 129 pages.Molecular targeting is a powerful diagnostic and therapeutic tool for clinicians to combat disease. A vast majority of molecular targeting agents rely on a single ligand:target interaction to be both specific to the target of interest, specific to the diseased tissue, and have the desired potency or functional output. In this work we utilized multi-domain molecules to more efficiently detect and treat diseases based on their molecular profiles. We first addressed the issue of biomarker uniqueness, where biomarkers upregulated in cancer cells are often still present in healthy cells and cause myriad side effects or diagnostic false positives. We demonstrated that within a model system that the enhanced selectivity via monovalent affinity reduction can reduce high off-tumor tissue signal. We showed that yeast surface display could be used to engineer these moderate affinity ligands, but that many ligands used lacked modularity when expressed in the heterobivalent construct. We then showed in vitro that affinity modulations can confer improved specificity to EGFRhigh/CEAhigh cells over cells expressing only one of the two biomarkers. We applied this heterobivalent ligand concept to two distinct epitopes on a single target, via a protein — small molecule fusion, to enhance selectivity for homologous proteins. We engineered protein—small molecule fusions that would preferentially bind to a single isoform within the target protein class. We were able to evolve these selective fusions in a robust and efficient manner using a simple cysteine—maleimide conjugation strategy in the context of yeast surface display. To improve on non-invasive disease imaging technology, we partnered with the Ashkenazi lab to develop an activatable imaging probe using a novel imaging modality (photoacoustic lifetime imaging) and an enzyme-labile probe to create a probe that emits only in the presence of an enzymatic biomarker. We found that the contrast agent methylene blue could be efficiently dimerized by a hairpin peptide structure consisting a poly-glutamate and poly-arginine zipper sequence linked by a protease-cleavable site and activated by the MMP-2 enzyme. These molecular designs can improve selectivity and sensitivity of therapeutics and diagnostics, which is crucial for the next generation of cancer and disease treatment.enThesis or Dissertation