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

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (leaves 98-110).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.(cont.) The antibody D44.1 was improved 140-fold to 30 pM, and the FDA-approved antibody cetuximab (Erbitux) was improved 10-fold to 52 pM, with an experimental success rate of greater than 60% for single mutations designed to remove undersatisfied polar groups or improve misbalanced electrostatic interactions. Finally, a physics-based improvement to the calculation of the nonpolar component of solvation free energy was implemented and parameterized to address the over-prediction of large amino acids. These results demonstrate novel computational capabilities and indicate their applicability for enhancing and accelerating development of reagents and therapeutics.by Shaun Matthew Lippow.Ph.D

Creation of a type IIS restriction endonuclease with a long recognition sequence

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases