3-DIMENSIONAL STRUCTURES OF SINGLE-CHAIN FV-NEURAMINIDASE COMPLEXES

The structure of the complex between a recombinant single-chain Fv con struct of antibody NC10 with a five-residue peptide linker between V-H and V-L (termed scFv(5)), and its antigen, tetrameric neuraminidase f rom influenza virus (NA), has been determined and refined at 2.5 Angst rom resolution. The antibody-antigen binding interface is very similar to that of a similar NC10 scFv-NA complex in which the scFv has a 15- residue peptide linker (scFv(15)), and the NC10 Fab-NA complex. Howeve r, scFv(5) and scFv(15) have different stoichiometries in solution. Wh ile scFv(15) is predominantly monomeric in solution, scFv(5) forms dim ers exclusively, because the five-residue linker is not long enough to permit V-H and V-L domains from the same poly-peptide associating and forming an antigen-binding site. Upon forming a complex with NA, scFv (15) forms a similar to 300 kDa complex corresponding to one NA tetram er binding four scFv(15) monomers, while scFv(5) forms a similar to 59 0 kDa complex, corresponding to two NA tetramers crosslinked by four b ivalent scFv(5) dimers. However, the dimeric scFv(5) in the scFv(5)-NA crystals does not crosslink NA tetramers, and modelling studies indic ate that it is not possible to pack four dimeric and simultaneously bi valent scFvs between the NA tetramers with only a five-residue linker between V-H and V-L. The inability arises from the exacting requiremen t to orient the two antigen-binding surfaces to bind the tetrameric NA antigen while avoiding steric clashes with NC10 scFv(5) dimers bound to other sites on the NA tetramer. The utility of bivalent or bifuncti onal scFvs with short Linkers may therefore be restricted by the steri c constraints imposed by binding multivalent antigens. (C) 1998 Academ ic Press Limited.