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

A classification of scFv fragments concerning their unfolding/refolding equ ilibria is proposed. It is based on the analysis of different mutants of th e levan-binding A48 scFv fragment and the HER-2 binding 4D5 scFv fragment a s well as a "hybrid" scFv carrying the V-L domain of 4D5 and the V-H domain of an A48 mutant. The denaturant-induced unfolding curves of the correspon ding scFv fragments were measured and, if necessary for the classification, compared with the denaturation of the isolated domains. Depending on the r elative intrinsic stabilities of the domains and the stability of the inter face, the different scFv fragments were grouped into different classes. We also demonstrate with several examples how such a classification can be use d to improve the stability of a given scFv fragment, by concentrating engin eering efforts on the "weak part" of the particular molecule, which may eit her be the intrinsic stability of V-L, Of V-H, or the stability of the inte rface. One of the scFv fragments obtained by this kind of approach is extre mely stable, starting denaturation only at about 7 M urea. We believe that such extremely stable frameworks may be very suitable recipients in CDR gra fting experiments. In addition, the thermodynamic equilibrium stabilities o f seven related A48 scFv mutants covering a broad range of stabilities in u rea unfolding were shown to be well correlated with thermal aggregation pro perties measured by light scattering and analytical gel filtration.