Contributions of a Highly Conserved V H/V L Hydrogen Bonding Interaction to scFv Folding Stability and Refolding Efficiency

Abstract

The assembly of single-chain Fv (scFv) antibody fragments, consisting of an interconnected variable heavy chain (V H) and variable light chain (V L), is a cooperative process that requires coupled folding and domain association. We report here an initial investigation of V H/V L domain-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 κ V L (Glx in 95% of all λ 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 Gln 44 were constructed to first investigate their contributions to thermodynamic folding stability, second to investigate whether stability can be improved, and third to determine whether refolding efficiencies are affected by mutations at these positions. The results demonstrate that the Gln 168-Gln 44 interaction is not a key determinant of S5 scFv folding stability, as sequential modification to alanine has no significant effect on the free energy of folding. Several mutations that alter the glutamines to methionine or charged amino acids significantly increase the thermodynamic stability by increasing the m g associated with the unfolding isotherm. These effects are hypothesized to arise largely from an increase in the V H/V L association free energy that leads to tighter coupling between domain-domain association and folding. All of the mutants also display a reduced antigen binding affinity. Single and double methionine mutants also displayed significant increases in refolding efficiency of 2.4- to 3-fold over the native scFv, whereas the double alanine/methionine mutants displayed moderate 1.9- to 2.4-fold enhancement. The results suggest that reengineering the V H/V L interface could be useful in improving the stability of single-chain antibodies, as Ala/Met mutations at these conserved positions increase the free energy of folding by 46% while minimally perturbing binding affinity. They also could be useful in improving scFv recovery from inclusion bodies as the mutations increase the refolding efficiency by more than twofold.