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A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of ␤-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.

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.

A set of six mutants of the levan binding single-chain Fv (scFv) fragment A48 (ABPC48), which have the identical light chain but differ gradually in the stability of the heavy chain, was generated. This was achieved by introducing one or both of the stabilizing mutations H-K66R and H-N52S into the VH domain of the A48 wild-type protein, which is naturally missing the conserved disulfide bridge in VH, and into the cysteine-restored variant A48cys scFv. The stabilizing effects of these two mutations in VH, which had been selected in the context of a disulfide-free derivative of this scFv fragment [Proba, K., et al. (1998) J. Mol. Biol. 275, 245-253], were found to be additive and transferable to the cysteine-restored variant of the A48 scFv, thereby generating extremely stable VH domains. The equilibrium denaturation of these scFv fragments was compared with the corresponding isolated VL domain and two of the different isolated VH domains. In the scFv fragment, the VL domain was found to be stabilized by a more stable VH domain, and, conversely, the VH domain was stabilized by a more stable VL domain. A folding intermediate with nativelike VH and denatured VL was found at equilibrium, if VH was significantly more stable than VL. In all other cases, a cooperative unfolding of the scFv was observed. We explain this observation with different contributions of intrinsic domain stability and extrinsic stabilization provided by the partner domain in the single-chain antibodies.