Probing the stability-limiting regions of an antibody single-chain variable fragment: a molecular dynamics simulation study

Abstract

Antibody single-chain variable fragments (scFvs) offer particular advantages over the full-size antibodies, including easy expression, efficient local concentration and fast body clearance. However, scFvs typically show low thermal stability that limits their biomedical and biotechnological applications. In this study, we examined the thermal stability of the human and murine vascular endothelial growth factor antibody scFv fragment by molecular dynamics simulations. A consistent observation was the dissociation of the light-chain (VL) and heavy-chain (VH) domains and loss of the native structures of both domains in the simulations at the elevated temperatures. The stability-limiting structural elements in the protein were revealed from the detailed analyses on the native contacts. We found that dissociation of the VL-VH domains was the first event leading to the unfolding of the native structure of the protein and the disruption of the VL-VH interface was largely due to the break of the interfacial hydrophobic and aromatic interactions while the hydrogen-bonding interaction between Gln38 in VL and Gln39 in VH remained. Within the β-barrel structure of the VL and VH domains, β-strands β6, β2 and β11 appeared to be the least stable. In addition, we found that the VH domain was more thermally resistant than the VL domain. Based on these findings, we discussed potential strategies to improve the stability of this therapeutically important scFv fragment.

Advances in Antibody Design

“…Two are located at the V H –V L interface (V H P101D and V L S46L), which has been shown to be critical in determining the overall stability of Fv and scFv antibody fragments (37, 40, 43, 47, 48). The negatively charged mutation (V H P101D) forms a salt bridge with a neighboring residue (V H R98), and the hydrophobic mutation (V L S46L) increases van der Waals contacts and induces favorable structural changes in neighboring residues.…”

“…These methods have yielded significant improvements in stability, although in some cases they have resulted in reduced expression. In addition, much effort has focused on optimizing the V H –V L interfaces using noncysteine mutations (40, 43, 48). This is important to improve both thermodynamic and kinetic folding stability (especially for Fvs and scFvs) (30, 47), and to reduce the complexity of the resulting antibodies by avoiding additional disulfide bonds.…”

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

“…[14][15][16] Broad medical application of scFvs has been limited, however, due to numerous biophysical challenges, 17 including the tendency of the molecules to aggregate and their low stability, especially at high concentrations. Moreover, robust conjugation of scFvs to lipid molecules requires incubation at temperatures greater than 60 C, and so high thermal stability of scFvs is highly desirable for nanoparticle targeting.…”

A Single Mutation in Framework 2 of the Heavy Variable Domain Improves the Properties of a Diabody and a Related Single-Chain Antibody

“…28 It has been shown that the Q170(V L )/Q39(V H ) contact remains stable during high-temperature molecular dynamics simulations, however, after the hydrophobic interface was disturbed. 29 Our molecular dynamics analyses suggest that the G43 residue may be stabilizing this region in the V L /V H interface. Other residues in this region have been reported to display similar effects.…”