Single-Chain Fv of Anti-idiotype 11-1G10 Antibody Interacts with Antibody NC41 Single-Chain Fv with a Higher Affinity than the Affinity for the Interaction of the Parent Fab Fragments

Abstract

A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining V_H and V_L domains with a (Gly₄Ser)₃ linker, with a pelB leader sequence, and two C-terminal FLAG^™ tag sequences, and expressed in E. coli (10 mg/L). The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 μg/ml) was stable for 7 days at 21°C and 30 days at 4°C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomer–dimer equilibrium mixture after 30 days at 4°C. The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of M _r ≍ 156,000. Binding affinities, determined in solution using a BIAcore^™ biosensor, showed that the affinity for the interaction of 11-1G10 scFv monomer with NC41 scFv monomer was five- to six-fold higher than the interaction of the parent Fab pair. This is the first example of an scFv derived from a monoclonal antibody with a higher affinity than its parent Fab.