Journal of Protein Chemistry, Vol. 17, No. 3, 1998 Single-Chain Fv of Anti-idiotype 11-1G10 Antibody Interacts with Antibody NC41 Single-Chain Fv with a Higher Affinity than the Affinity for the Interaction of the Parent Fab Fragments Peter Iliades,1 David A. Dougan,1 Geoffrey W. Oddie,1 Dennis W. Metzger,2 Peter J. Hudson,1 and Alexander A. Kortt1,3 1. INTRODUCTION Anti-idiotype antibodies bind to the variable region of antibody molecules and have been used to define the idiotype as an antigenic marker unique to the antibody (Pan et al., 1995). Of particular interest are antibodies that recognize idiotopes that are in contact with antigen, that is, they recognize residues in the CDR loops of an- tibody heavy and light chains important in determining idiotypic specificity (Davie et al., 1986; Goldman et al., 1 CSIRO, Division of Molecular Science, Parkville 3052, Victoria, Australia. 2 Department of Microbiology, Medical College of Ohio, Toledo, Ohio 43699. 3 To whom correspondence should be addressed at CSIRO, Division of Molecular Science, 343 Royal Parade, Parkville 3052, Victoria, Australia; e-mail: alex.kortt@molsci.csiro.au. Received July 31, 1997 A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining VH and VL domains with a (Gly4Ser)3 linker, with a pelB leader sequence, and two C-terminal FLAG® tag sequences, and expressed in E. coli (10 mg/L). The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 ug/ml) was stable for 7 days at 21°C and 30 days at 4°C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomer- dimer equilibrium mixture after 30 days at 4°C. The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of Mr « 156,000. Binding affinities, determined in solution using a BIAcore® biosensor, showed that the affinity for the interaction of ll-lGl0 scFv monomer with NC41 scFv monomer was five- to six-fold higher than the interaction of the parent Fab pair. This is the first example of an scFv derived from a monoclonal antibody with a higher affinity than its parent Fab. KEY WORDS: Antibody; anti-idiotype; single-chain Fvs; solution properties; complexes with antigens; bind- ing affinities. 1997; Marriuzza and Poljak, 1993). Anti-idiotype anti- bodies have attracted interest as potential therapeutic agents especially in their application as vaccines, as they can be used to elicit immune responses to pathogenic antigens such as viruses (Colman et al., 1987; Kenndy et al., 1986; Metzger and Webster, 1990). It has been suggested that anti-idiotype antibodies can mimic the image of the epitope (external antigen) (Pan et al., 1995; Nisonoff, 1991; Garcia et al., 1992). Such molecular mimicry can have homology either in primary or tertiary structure and is especially evident when the epitope is a linear sequence as evidenced by sequence similarity be- tween the CDR loops of the anti-idiotype antibody and the original antigen. In a study to map the antigenic surface of influenza virus N9-neuraminidase, monoclonal antibodies against influenza virus (NW strain, N9) were raised and found 0277-8033/98/0400-0245$15.00/0 C 1998 Plenum Publishing Corporation 245