A. Worn et A. Pluckthun, Different equilibrium stability behavior of ScFv fragments: Identification, classification, and improvement by protein engineering, BIOCHEM, 38(27), 1999, pp. 8739-8750
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.