Case, Brett2018-09-212018-09-212018-06http://hdl.handle.net/11299/200220University of Minnesota Ph.D. dissertation.June 2018. Major: Chemical Engineering. Advisor: Benjamin Hackel. 1 computer file (PDF); viii, 130 pages.Molecular imaging is a powerful, noninvasive technique for patient stratification and treatment monitoring via cancer biomarker characterization with reporter probes. Small scaffold proteins that combine stable frameworks with diversifiable paratope regions are well-suited as engineerable probes for molecular imaging and often benefit from efficient physiological transport. Probe efficacy depends not only on diseased region localization but also passive noninteraction with plasma molecules and healthy tissues. Precise relationships between protein charge, physicochemical robustness, and in vivo performance are poorly understood, but the appropriate manipulation of ionic charge can stabilize native protein folds and reduce nonspecific interactions. Therefore, the effective engineering of protein charge in diagnostic molecular probes would advance cancer characterization and personalized medicine. In this thesis work we enlisted affibody and Gp2 proteins as molecular imaging scaffolds, applied discrete protein engineering designs to modify charge density and distribution, and evaluated promising charge-modified variants as positron emission tomography (PET) imaging agents in preclinical studies against the cancer biomarker, epidermal growth factor receptor (EGFR). Natural and functional (synthetic) homolog frequencies guided the design of charge-modified consensus clones and combinatorial libraries based on a hybrid anti-EGFR affibody, EA68; a synthetic consensus design proved most robust by providing variants that retain parental recombinant yield and stability while improving EGFR affinity. Charge-mutated combinatorial Gp2 libraries were enriched for binding against three clinically or biotechnologically relevant targets and revealed target-dependent total, net, and site-specific charge preferences demonstrating the utility of multitarget screening to determine globally accepted framework mutations. In summary, hybrids of combinatorial library selection and sequence informatics-driven design have proven effective for charge engineering. Preclinical investigation of lead anti-EGFR affibody and Gp2 clones and their charge-varied homologs in mice bearing EGFRhigh and EGFRlow tumor xenografts exposed differences in clonal biodistribution, tumor targeting, and proteolytic stability. Among tested variants, the charge-reduced affibody ‘EA35S’ and Gp2 ‘GαE35’ achieved the best overall performance with high (12 ± 2 %ID/g and 5.5 ± 0.5%ID/g) and specific (tumor:muscle = 34 ± 5 and 35 ± 5; tumor:blood = 12 ± 3 and 9.7 ± 1.3) EGFRhigh tumor localization at 4 and 2 h post-injection. Collectively, this work provides a suite of tools to maintain and enhance protein function through ionic charge engineering and reveals two promising probes for cancer characterization through molecular recognition of EGFR.enThesis or Dissertation