Different equilibrium stability behavior of ScFv fragments: identification, classification, and improvement by protein engineering.

A classification of scFv fragments concerning their unfolding/refolding equilibria is proposed. It is based on the analysis of different mutants of the levan-binding A48 scFv fragment and the HER-2 binding 4D5 scFv fragment as well as a "hybrid" scFv carrying the VL domain of 4D5 and the VH domain of an A48 mutant. The denaturant-induced unfolding curves of the corresponding scFv fragments were measured and, if necessary for the classification, compared with the denaturation of the isolated domains. Depending on the relative intrinsic stabilities of the domains and the stability of the interface, the different scFv fragments were grouped into different classes. We also demonstrate with several examples how such a classification can be used to improve the stability of a given scFv fragment, by concentrating engineering efforts on the "weak part" of the particular molecule, which may either be the intrinsic stability of VL, of VH, or the stability of the interface. One of the scFv fragments obtained by this kind of approach is extremely stable, starting denaturation only at about 7 M urea. We believe that such extremely stable frameworks may be very suitable recipients in CDR grafting experiments. In addition, the thermodynamic equilibrium stabilities of seven related A48 scFv mutants covering a broad range of stabilities in urea unfolding were shown to be well correlated with thermal aggregation properties measured by light scattering and analytical gel filtration.