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Biophysical characterization of high affinity engineered single chain Fv antibody fragments

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dc.contributor.advisor K. Dane Wittrup. en_US Midelfort, Katarina Senn en_US
dc.contributor.other Massachusetts Institute of Technology. Biological Engineering Division. en_US 2006-03-24T18:15:07Z 2006-03-24T18:15:07Z 2004 en_US 2004 en_US
dc.description Thesis (Ph. D. in Molecular Systems Toxicology and Pharmacology)--Massachusetts Institute of Technology, Biological Engineering Division, 2004. en_US
dc.description Vita. en_US
dc.description Includes bibliographical references. en_US
dc.description.abstract High affinity antibody binding interactions are important for both pharmaceutical and biotechnological uses. However, designing higher affinity interactions has remained difficult. Both high affinity interactions from nature and the results from directed evolution affinity maturation processes may yield clues about the important structural and energetic contributions to attain these tight associations. In this Thesis, we investigate affinity maturation of antibodies for very high affinity binding. Two single chain antigen-binding fragment (scFv) antibody systems that were engineered to obtain higher affinity interactions through directed evolution were probed using biophysical techniques to illuminate affinity modulation in proteins. First, anti-c-erbB-2 antibodies and their binding partner, the extracellular domain of the glycoprotein tumor antigen c-erbB-2, were examined. Thermodynamic studies were carried out on the originally identified human scFv and three higher affinity mutants. Although the first two steps included either entropic or enthalpic gains to affinity, the third improvement came from both types. This study demonstrates that a single energetic component is not generally responsible for the increased affinity within a given protein-protein affinity maturation pathway. Second, a family of anti-fluorescein antibodies and their binding to the small molecule fluorescein-biotin were explored. The femtomolar affinity matured anti-fluorescein antibody, 4M5.3, was compared to its wildtype high affinity precursor, 4-4-20. Affinity, thermodynamic, kinetic, and structural characterization of the binding identified 4M5.3 as one of the highest engineered affinity protein binding interactions known and en_US
dc.description.abstract (cont.) illuminated how subtle structural changes can lead to large consequences for the kinetics and free energy of binding. The affinity mechanisms were further studied by the creation of a series of partial mutants. Context dependent and independent mutational effects on binding affinity indicated the extent of complexity in higher affinity mechanisms attained through directed evolution affinity maturation processes. These studies emphasize the importance of a large number of residues working in concert to create a very high affinity binding molecule. Based on these results, both rational design and directed evolution studies will need to allow for mutations in a spatially broad range around the binding site and involve many biophysical contributions to the binding free energy to reach very high antigen binding affinities. en_US
dc.description.statementofresponsibility by Katarina S. Midelfort. en_US
dc.format.extent 171 leaves en_US
dc.format.extent 6186854 bytes
dc.format.extent 6186661 bytes
dc.format.mimetype application/pdf
dc.format.mimetype application/pdf
dc.language.iso eng en_US
dc.publisher Massachusetts Institute of Technology en_US
dc.rights MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission. en_US
dc.subject Biological Engineering Division. en_US
dc.title Biophysical characterization of high affinity engineered single chain Fv antibody fragments en_US
dc.type Thesis en_US Molecular Systems Toxicology and Pharmacology en_US
dc.contributor.department Massachusetts Institute of Technology. Biological Engineering Division. en_US
dc.identifier.oclc 55591149 en_US

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