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Fibronectin domain engineering

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dc.contributor.advisor K. Dane Wittrup. en_US Hackel, Benjamin Joseph en_US
dc.contributor.other Massachusetts Institute of Technology. Dept. of Chemical Engineering. en_US 2010-08-30T14:44:59Z 2010-08-30T14:44:59Z 2009 en_US 2009 en_US
dc.description Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009. en_US
dc.description Vita. Cataloged from PDF version of thesis. en_US
dc.description Includes bibliographical references. en_US
dc.description.abstract Molecular 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.statementofresponsibility by Benjamin Joseph Hackel. en_US
dc.format.extent 185 p. en_US
dc.language.iso eng en_US
dc.publisher Massachusetts Institute of Technology en_US
dc.rights M.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.uri en_US
dc.subject Chemical Engineering. en_US
dc.title Fibronectin domain engineering en_US
dc.type Thesis en_US Ph.D. en_US
dc.contributor.department Massachusetts Institute of Technology. Dept. of Chemical Engineering. en_US
dc.identifier.oclc 654110473 en_US

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