Quantitative account of the enhanced affinity of two linked scFvs specific for different epitopes on the same antigen

Protein and other antigens typically have a number of different epitopes. This presents an opportunity for designing high-affinity antibodies by connecting via a flexible peptide linker two antibody fragments recognizing non-overlapping epitopes on the same antigen. The same strategy was employed in natural and designed DNA-binding proteins. According to a previous theory, the linking enhances the antigen-binding affinity over those of the individual antibody fragments (with association constants K-A and K-B) by p(d(0))K-B or p(d(0))K-A, where p(d(0)) = (3/4pil(p)bL)(3/2) X exp(-3d(0)(2) /4l(p)bL)(1 - 5l(p)/4bL + (...)) is the probability density for the end-to-end vector of the flexible linker with L residues to have a distance do. The predicted affinity enhancement is found to be actually approached by a bi-specific antibody against hen egg lysozyme consisting of scFv fragments of D1.3 and HyHEL-10. The wide applicability of the theory is demonstrated by diverse examples of protein-protein interactions constrained by flexible linkers. (C) 2003 Elsevier Science Ltd. All rights reserved.