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

Synthetic genes encoding single-chain variable fragments (scFvs) of NC10 an ti-neuraminidase antibody were constructed by joining the V-L and V-H domai ns 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 resulti ng proteins were characterized and compared with the previously characteriz ed NC10 scFv proteins assembled in V-H-V-L orientation. Size-exclusion chro matography and electron microscope images of complexes formed between vario us NC10 scFvs and anti-idiotype Fab' were used to analyse the oligomeric st atus 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 orien tation, the scFv-15 V-L-V-H protein existed mainly as a monomer whereas dim er (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) rat her than to an expected trimer (triabody), Furthermore, the transition betw een dimers and higher order oligomers was Mot as distinct as for V-H-V-L. T hus reducing the linker length in V-L-V-H from three to two residues did no t precisely dictate a transition between dimers and tetramers, Instead, two -residue as well as one-residue linked scFvs formed a mixture of dimers, tr imers and tetramers.