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dc.contributor.advisorK. Dane Wittrup.en_US
dc.contributor.authorHackel, Benjamin Josephen_US
dc.contributor.otherMassachusetts Institute of Technology. Dept. of Chemical Engineering.en_US
dc.date.accessioned2010-08-30T14:44:59Z
dc.date.available2010-08-30T14:44:59Z
dc.date.copyright2009en_US
dc.date.issued2009en_US
dc.identifier.urihttp://hdl.handle.net/1721.1/57701
dc.descriptionThesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009.en_US
dc.descriptionVita. Cataloged from PDF version of thesis.en_US
dc.descriptionIncludes bibliographical references.en_US
dc.description.abstractMolecular recognition reagents are a critical component of targeted therapeutics, in vivo and in vitro diagnostics, and biotechnology applications such as purification, detection, and crystallization. Antibodies have served as the gold standard binding molecule because of their high affinity and specificity and, historically, because of their ability to be generated by immunization. However, antibodies suffer from several shortcomings that hinder their production and reduce their efficacy in a breadth of applications. The tenth type III domain of human fibronectin provides a small, stable, single-domain, cysteine-free protein scaffold upon which molecular recognition capability can be engineered. In the current work, we provide substantial improvements in each phase of protein engineering through directed evolution and develop a complete platform for engineering high affinity binders based on the fibronectin domain. Synthetic combinatorial library design is substantially enhanced through extension of diversity to include three peptide loops with inclusion of loop length diversity. The efficiency of sequence space search is improved by library focusing with tailored diversity for structural bias and binding capacity. Evolution of lead clones was substantially improved through development of recursive dual mutagenesis in which each fibronectin gene is subtly mutated or the binding loops are aggressively mutated and shuffled. This engineering platform enables robust generation of high affinity binders to a multitude of targets. Moreover, the development of this technology is directly applicable to other protein engineering campaigns and advances the scientific understanding of molecular recognition. Binders were engineered to tumor targets carcinoembryonic antigen, CD276, and epidermal growth factor receptor as well as biotechnology targets human serum albumin and goat, mouse, and rabbit immunoglobulin G. Binders have demonstrated utility in affinity purification, laboratory detection, and cellular labeling and delivery. Of particular interest, a panel of domains was engineered that bind multiple epitopes of epidermal growth factor receptor. Select non-competitive heterobivalent combinations of binders effectively downregulate receptor in a non-agonistic manner in multiple cell types. These agents inhibit proliferation and migration and provide a novel potential cancer therapy.en_US
dc.description.statementofresponsibilityby Benjamin Joseph Hackel.en_US
dc.format.extent185 p.en_US
dc.language.isoengen_US
dc.publisherMassachusetts Institute of Technologyen_US
dc.rightsM.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.en_US
dc.rights.urihttp://dspace.mit.edu/handle/1721.1/7582en_US
dc.subjectChemical Engineering.en_US
dc.titleFibronectin domain engineeringen_US
dc.typeThesisen_US
dc.description.degreePh.D.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Dept. of Chemical Engineering.en_US
dc.identifier.oclc654110473en_US


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