Browsing by Subject "Combinatorial"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Engineering a 45-Amino Acid Protein Scaffold for Molecular Cancer Imaging
    (2017-12) Kruziki, Max
    Cancer is the second leading cause of death in the United States. Molecularly targeted cancer treatments, including monoclonal antibodies and kinase inhibitors, exhibit strong performance on a small subset of patients but are inconsistent due to tumor heterogeneity. Biopsy-based genetic and protein tumor characterization provide value but cannot address spatial or temporal variations in heterogeneity. Non-invasive methods, such as molecular imaging, to characterize cancer cells will allow for easier patient stratification and treatment monitoring. Currently, molecular imaging is limited by the modest availability of quality probes that efficiently distribute throughout the body and quantitatively localize at the site of the cancer biomarker. Engineering effective diagnostic molecular probes would provide a substantial advance in cancer characterization and personalized medicine. Protein scaffolds, which comprise a large stabilizing framework and a randomized region onto which binding interactions can be engineered, offer an efficient platform for probe engineering. More broadly, engineered binding proteins are useful in many aspects of biotechnology and medicine. In this thesis, we mined ~100,000 known protein topologies to identify candidate small protein scaffolds. We developed the 45-amino acid Gp2scaffold and evolved multiple Gp2 variants that strongly (as strong as 0.2 nM) and specifically (greater than 50:1 target:control) bind their respective target while also retaining high thermal stability (65-80 ºC thermal desaturation midpoint) . A Gp2 variant that was evolved to bind with strong affinity to epidermal growth factor receptor (EGFR), a cell surface biomarker overexpressed in multiple cancer types, was more thoroughly investigated in pre-clinical studies. This variant exhibited strong (18 nM), selective binding, and was passive on normal EGFR signaling pathways, which is important to reduce off-target side effects. PET imaging of subcutaneously xenografted tumors in mice revealed effective probe localization to EGFR-high tumors while low signal was observed in EGFR-low tumors and from non-targeted control Gp2. Gp2 evolution was studied by comparing the efficacy of different combinatorial library amino acid diversity based on high throughput sequencing data, natural Gp2 homologs, structural data, and computed stability. Multiple library designs elucidated amino acid diversity that was beneficial or detrimental in different sections of the Gp2 protein, and will aid future evolution and developability of Gp2. From these libraries, high affinity Gp2 variants targeting an additional clinically-relevant cancer biomarker, programmed death-ligand 1 (PD-L1), were evolved, isolated, and characterized. The advancements outlined here make important contributions towards improving both protein engineering tools and methodologies as well as diagnostic imaging tools.
  • Thumbnail Image
    Item
    Understanding cominatorial atomic layer deposition and chemical vapor deposition.
    (2009-01) Moersch, Tyler Leighton
    The transformation of molecular precursor to solid film begins with an understanding of molecular structure, proceeds through delivery of the molecule to the surface and ends with the decomposition of precursor on the surface to form a deposit. An understanding of the physical and chemical processes leading from molecule to film enables the utilization of chemical precursors in effective deposition processes. Single crystal X-ray crystallography was used to study the structure of [NO]0.5[NO2]0.5[Zr(NO3)5] and [NO]0.5[NO2]0.5[Hf(NO3)5]. Infrared spectroscopy was employed to identify the nature of the cation in the crystal structure, and characteristic absorptions of both nitronium and nitrosonium cations were observed. Fluidized bed reactor technology has been used to study the sublimation behavior of solid-state chemicals. Fluidization behavior, precursor mass transfer rates and delivery uniformity for aluminum trichloride were studied and the results reported. Mixed metal oxide nanolaminate films of hafnium oxide and zirconium oxide interspersed with layers of silicon oxide have been deposited on silicon substrates by a combinatorial atomic layer deposition (ALD) technique. Exposure of repeated cycles of co-dosed alkoxide precursors Hf[OC(CH3)3]4 and Zr[OC(CH3)3]4 with counter-reactant pulses of Si[OC(CH3)3]3(OH) formed films of uniform thickness (±5%) and uniform silicon oxide concentration (85% per total metals basis). The hafnium and zirconium concentrations exhibited smooth gradation across the film from 18% - 82% (per Hf and Zr metals basis). Self-limiting deposition rates of 1.5 nm / cycle were measured, and a linear relationship of film thickness to number of deposition cycles was observed, both consistent with a true ALD process. Rutherford backscattering spectrometry, ellipsometry and X-ray reflectivity results were used to map the composition and determine the film microstructure. Single precursor depositions have been performed and compared to computational models created using CFD-ACE in order to further the understanding of the interaction of fluid dynamics and chemistry in the combinatorial chemical vapor deposition process. The physical and chemical processes contributing to film growth in combinatorial chemical vapor deposition were evaluated.