Multimerization behaviour of single chain Fv variants for the tumour-binding antibody B72.3

A systematic study has been performed on the relationship between linker length, relative orientation of variable domains, multimerization behaviour and antigen binding activity for single chain Fvs (scFvs) of the tumour-binding antibody B72.3. Thirteen scFv variants with linkers comprising up to six repeats of the motif Gly-Gly-Gly-Gly-Ser were studied. All these scFvs showed a tendency to form dimers or higher molecular weight species, and this tendency decreased with increasing linker length. The dimers and higher molecular weight forms may arise from head to tail intermolecular association of VH and VL domains. For each linker length, scFvs with the organization VL-linker-VH showed greater binding activity than those with the organization VH-linker-VL. In fact, for the latter organization only the variant with a 30 amino acid linker showed good binding activity, suggesting that (i) for B72.3 the C-terminus of VH or the N-terminus of VL makes a structural contribution to antigen binding, and (ii) shorter linkers interfere with this contribution. Antigen binding studies on scFvs should be interpreted with caution because of their tendency to multimerize. Such multimerization can be minimized by using linkers longer than those in common use.