CONTRIBUTIONS OF A HIGHLY CONSERVED VHNL HYDROGEN-BONDING INTERACTIONTO SCFV FOLDING STABILITY AND REFOLDING EFFICIENCY

The assembly of single-chain Fv (scFv) antibody fragments, consisting of an interconnected variable heavy chain V-H) and variable light chai n V-L, is a cooperative process that requires coupled folding and doma in association. We report here an initial investigation of V-H/V-L dom ain-domain assembly with a site-directed mutagenesis study that probes a highly conserved V-H/V-L hydrogen bonding interaction. Gln(168) of the S5 scFv (Kabat V-H 39) is absolutely conserved in 95% of all V-H, and Gln(44) (Kabat V-L 38) is found in 94% of all kappa V-L (Glx in 95 % of all lambda V-L. These side chains form two hydrogen bonds in head -to-tail alignment across the V-H/V-L interface. Double mutant cycles at Gln(168) and Gln44 were constructed to first investigate their cont ributions to thermodynamic folding stability, second to investigate wh ether stability can be improved, and third to determine whether refold ing efficiencies are affected by mutations at these positions. The res ults demonstrate that the Gln(168) -Gln(44) interaction is not a key d eterminant of S5 scFv folding stability, as sequential modification to alanine has no significant effect on the free energy of folding. Seve ral mutations that alter the glutamines to methionine or charged amino acids significantly increase the thermodynamic stability by increasin g the m(g) associated with the unfolding isotherm. These effects are h ypothesized to arise largely from an increase in the V-H/V-L associati on free energy that leads to tighter coupling between domain-domain as sociation and folding. All of the mutants also display a reduced antig en binding affinity. Single and double methionine mutants also display ed significant increases in refolding efficiency of 2.4- to 3-fold ove r the native scFv, whereas the double alanine/methionine mutants displ ayed moderate 1.9- to 2.4-fold enhancement. The results suggest that r eengineering the V-H/V-L interface could be useful in improving the st ability of single-chain antibodies, as Ala/Met mutations at these cons erved positions increase the free energy of folding by 46% while minim ally perturbing binding affinity. They also could be useful in improvi ng scFv recovery from inclusion bodies as the mutations increase the r efolding efficiency by more than twofold.