ScFv multimers of the anti-neuraminidase antibody NC10:: shortening of the linker in single-chain Fv fragment assembled in VL to VH orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers

Synthetic genes encoding single-chain variable fragments (scFvs) of NC10 anti-neuraminidase antibody were constructed by joining the V-L and V-H domains with linkers of fifteen, five, four, three, two, one and zero residues. These V-L-V-H constructs were expressed in Escherichia coli and the resulting proteins were characterized and compared with the previously characterized NC10 scFv proteins assembled in V-H-V-L orientation. Size-exclusion chromatography and electron microscope images of complexes formed between various NC10 scFvs and anti-idiotype Fab' were used to analyse the oligomeric status of these scFvs, The result showed that as the linker length between V-L and V-H was reduced, different patterns of oligomerization were observed compared with those with V-H-V-L isomers, As was the case for V-H-V-L orientation, the scFv-15 V-L-V-H protein existed mainly as a monomer whereas dimer (diabody) was a predominant conformation for the scFv-5, scFv-4 and scFv-3 V-L-V-H proteins. In contrast to the V-H-V-L isomer, direct ligation of V-L to V-H led to the formation of predominantly a tetramer (tetrabody) rather than to an expected trimer (triabody), Furthermore, the transition between dimers and higher order oligomers was Mot as distinct as for V-H-V-L. Thus reducing the linker length in V-L-V-H from three to two residues did not precisely dictate a transition between dimers and tetramers, Instead, two-residue as well as one-residue linked scFvs formed a mixture of dimers, trimers and tetramers.