scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between V-H and V-L domains dictates precisely the transition between diabodies and triabodies

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptide linker to tether the V-H and V-L domains together. An scFv molecule with a linker 5-12 residues long cannot fold into a functional Fv domain and instead ass ociates with a second scFv molecule to form a bivalent dimer (diabody). Dir ect ligation of V-H and V-L domains further restricts association and force s three scFv molecules to associate into a trivalent trimer (triabody). We have defined the effect of linker length on scFv association by constructin g a series of scf;vs from anti-neuraminidase antibody NC10 in which the lin ker varied from one to four glycine residues. NC10 scFv molecules containin g linkers of three and four residues showed a strong preference for dimer f ormation (diabodies), whereas a linker length of one or two glycine residue s prevented the formation of diabodies and directed scFv association into t rimers (triabodies). The data suggest a relatively strict transition from d imer (diabody) to trimer (triabody) upon reduction of the linker length fro m three to two glycine residues. Modelling studies are consistent with thre e residues as the minimum linker length compatible with diabody formation. Electron microscope images of complexes formed between the NC10 scFv multim ers and an anti-idiotype Fab' showed that the dimer was bivalent for antige n binding and the trimer was trivalent.