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

A systematic study has been performed on the relationship betweenlinker length, relative orientation of variable domains, multimerizationbehaviour and antigen binding activity for single chain Fvs(scFvs) of the tumour-binding antibody B72.3. Thirteen scFvvariants with linkers comprising up to six repeats of the motifGly-Gly-Gry-Gly-Ser were studied. All these scFvs showed a tendencyto form dimers or higher molecular weight species, and thistendency decreased with increasing linker length. The dimersand higher molecular weight forms may arise from head to tailintermolecular association of V_H and V_L domains. For each linkerlength, scFvs with the organization VL-linker-VH showed greaterbinding activity than those with the organization VH-linker-VL.In fact, for the latter organization only the variant with a30 amino acid linker showed good binding activity, suggestingthat (0 for B72.3 the C-tenninus of VH or the N-tenninus ofVL makes a structural contribution to antigen binding, and (ii)shorter linkers interfere with this contribution. Antigen bindingstudies on scFvs should be interpreted with caution becauseof their tendency to multlmerize. Such multimerization can beminimized by using linkers longer than those in common use