Exceptional Amyloid β Peptide Hydrolyzing Activity of Nonphysiological Immunoglobulin Variable Domain Scaffolds

Nucleophilic sites in the paired variable domains of the light and heavy chains (V-L and V-H domains) of Ig can catalyze peptide bond hydrolysis. Amyloid beta(A beta)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for A beta-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological V-L-V-H-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage- displayed IgVs with an electrophilic A beta analog identified rare IgVs that hydrolyzed A beta mainly at His(14)-Gln(15). Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric VL (IgV(L2)-t) and single domain V-L clones with aberrant polypeptide tags (IgV(L)-t'). The IgVs hydrolyzed A beta at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain V-L with V-H or V-L domains resulted in reduced A beta hydrolysis, suggesting catalysis by the unpaired V-L domain. angstrom ngstrom level amino acid displacements evident in molecular models of the two domain and unpaired V-L domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the V-L domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs.