Computational analysis, design, and experimental validation of antibody binding affinity improvements beyond in vivo maturation

This thesis presents novel methods for the analysis and design of high-affinity protein interactions using a combination of high-resolution structural data and physics-based molecular models. First, computational analysis was used to investigate the molecular basis for the affinity improvement of over 1000-fold of the fluorescein-binding antibody variant 4M5.3, engineered previously from the antibody 4-4-20 using directed evolution. Electrostatic calculations revealed mechanistic hypotheses for the role of four mutations in a portion of the improvement, subsequently validated by separate biochemical experiments. Next, methods were developed to computationally redesign protein interactions in order to rationally improve binding affinity. In the anti-lysozyme model antibody D1.3, modest binding improvements were achieved, with the results indicating potentially increased sucesss using predictions that emphasize electrostatics, as well as the need to address the over-prediction of large amino acids. New methods, taking advantage of the computed electrostatics of binding, yielded robust and significant improvements for both model and therapeutic antibodies.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2007

Author dc:creator

• Lippow, Shaun Matthew

Advisor dc:contributor.advisor

• Bruce Tidor and K. Dane Wittrup.

• Chemical Engineering.

Statement 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.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/38886

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/38886