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dc.contributor.advisorSiahaan, Teruna
dc.contributor.advisorChadwick, Jennifer
dc.contributor.authorMcNiff, Michaela L.
dc.date.accessioned2017-11-16T04:04:52Z
dc.date.available2017-11-16T04:04:52Z
dc.date.issued2017-08-31
dc.date.submitted2017
dc.identifier.otherhttp://dissertations.umi.com/ku:15401
dc.identifier.urihttp://hdl.handle.net/1808/25385
dc.description.abstractPeptide and protein therapeutics encounter proteases at every stage of delivery, beginning at the site of administration and ending in the intracellular lysosomal compartment. An intact, stable molecule is desired upon administration, yet release of the payload or activation of the prodrug is necessary at the target site. Successful payload release or prodrug activation can be accomplished using enzymatic recognition sequences specific to proteases expressed in the tumor environment or within endocytic vesicles of the target cell. Control of the proteolytic susceptibility of biologics is essential to deliver a safe and effective molecule. Although degradation and release of proteins is desired at the target site, stability in the systemic circulation is required for successful delivery. Proteins and peptides often succumb to proteolysis in the systemic circulation, causing short half-lives due to their small size resulting in rapid filtration by the kidney. Proteases in the systemic circulation reduce half-life further through fragmentation rendering the biologic inactive. Achieving stability in the systemic circulation would greatly improve the half-life and efficacy of delivered biologics. Considering the half-life of peptides and proteins is especially important when they are used as diagnostic imaging agents. Contrast agents need a sufficient amount of time to accumulate at the target site to generate a high-quality image. Current contrast agents or diagnostic tracers consist of large chelators bound to lanthanide metals and are chemically conjugated to proteins creating a heterogeneous, unstable and toxic molecule. A common lanthanide ion used for imaging applications is gadolinium, which is often released from the chelator upon dilution into the body and accumulates in the kidney, causing renal toxicity. An improvement to chemically conjugated diagnostic imaging agents is to use a metal-binding tripeptide known as the claMP Tag. The claMP Tag consists of the amino acid sequence Asn-Cys-Cys and binds transition metals tightly at basic pH. Biocompatible transition metals such as copper and cobalt can be used to create a safe and effective molecular imaging agent. The claMP Tag enables design of a site-specific, homogenous and safe molecule, none of which is possible using current lanthanide binding tags. The compatibility of the claMP Tag within a metalloproteinase was investigated. Herein is the first application where the claMP Tag was placed within a molecule with a structural or catalytic metal-binding site. One hypothesis was that the claMP Tag would be able to interact with the catalytic Zn active site inhibiting the protease, while release of the claMP Tag could activate the enzyme. The claMP Tag followed by an eight-amino acid linker was engineered to the N-terminus of matrix metalloproteinase-8 (MMP-8). MMP-8 is very difficult to produce because of its inherent function of degrading extra cellular matrix (ECM) proteins and concomitant autoproteolysis. Production of this enzyme proved difficult as several fusion constructs were created and determined to be unsuccessful. A fusion construct consisting of a thioredoxin and S Tag, as well as, a polyhistidine tag enabled expression and purification of active, soluble MMP-8 in high yield. Studies were completed both with and without the claMP Tag placed inline with MMP-8. The claMP Tag had no structural or functional implications on the production of MMP-8. Further investigation of the claMP Tag as a modulator of enzymatic activity is of interest. Another focus of this work was to investigate the proteolytic stability of claMP-Tagged proteins. To investigate the proteolytic susceptibility of a protein containing the claMP Tag, a proof of concept study was designed to investigate the ability of the claMP Tag to inhibit adjacent proteolysis by a serum protease. The stability of the metal-bound and apo claMP Tag were compared to determine the effect of metal-binding on proteolysis. The claMP Tag inhibits proteolysis 3-fold compared to the control and use of the claMP Tag in applications creates a more proteolytically stable molecule. Following the proof of concept study, a targeted imaging agent was designed using the claMP Tag and the proteolytic stability of this molecule was analyzed. Previously, the claMP Tag was placed inline with the targeting protein, epidermal growth factor (EGF), and the structure and function of the protein were determined to be unaltered. EGF is a small protein that binds to the receptor EGFR and can be used to target EGFR (+) tumor cells, making EGF ideal for diagnostic imaging. One limitation is EGF has a very short half-life of only 3-7 min. Thus, to improve the half-life, addition of a polymer of amino acids, known as XTEN, was engineered to the C-terminus of EGF. Three EGF-claMP- XTEN molecules were created and analyzed for the structural and functional implications of the addition of XTEN and use of a relevant imaging agent, in this case cobalt, within the claMP Tag.
dc.format.extent143 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsCopyright held by the author.
dc.subjectPharmaceutical sciences
dc.subjectBiochemistry
dc.subjectChemistry
dc.subjectbiologics
dc.subjectclaMP Tag
dc.subjectmolecular probe
dc.subjectproteolysis
dc.titleDevelopment of Biologics Using the claMP Tag: Influence of the claMP Tag on Half-life and Proteolytic Stability
dc.typeDissertation
dc.contributor.cmtememberSiahaan, Teruna
dc.contributor.cmtememberChadwick, Jennifer
dc.contributor.cmtememberLamb, Audrey
dc.contributor.cmtememberTolbert, Thomas
dc.contributor.cmtememberPicking, Wendy
dc.thesis.degreeDisciplinePharmaceutical Chemistry
dc.thesis.degreeLevelPh.D.
dc.identifier.orcid
dc.rights.accessrightsopenAccess


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