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 KA and KB) by p(d0)KB or p(d0)KA, where p(d0)=(3/4πlpbL)3/2 exp(−3d02/4lpbL)(1−5lp/4bL+⋯) is the probability density for the end-to-end vector of the flexible linker with L residues to have a distance d0. 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.