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Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design

Biogen Idec, Inc., Cambridge, Massachusetts 02142, USA

(RECEIVED December 12, 2005; FINAL REVISION February 13, 2006; ACCEPTED February 16, 2006)

Improving the affinity of a high-affinity protein–protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure-based computational methods to optimize the binding affinity of an antibody fragment to the I-domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd 7 nM) and the moderate resolution (2.8 Å) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high-affinity quadruple mutant complex at 2.2 Å. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher-affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single-mutant success rate. Our results indicate that structure-based computational design can be successfully applied to further improve the binding of high-affinity antibodies.

Keywords: antibody; affinity maturation; computational protein design; protein–protein interactions; binding energy prediction

Reprint requests to: Louis A. Clark, Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA; e-mail: louie{at}alumni.northwestern.edu; fax: (617) 679-4998.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.052030506.

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