P2-351: Exceptional catalytic hydrolysis of amyloid-β peptide by immunoglobulin light chain variable domain constructs

Abstract

Immunoglobulins (Igs) that bind amyloid-β peptides (Aβ) are under consideration for passive immunotherapy of Alzheimer disease (AD). Catalytic Igs that hydrolyze Aβ are candidate second generation therapeutic agents by virtue of their ability to inactivate multiple Aβ molecules per Ig molecule. We have described Aβ hydrolysis via a nucleophilic mechanism by intact Igs and recombinant Ig fragments. However, homogeneous Igs with catalytic rate constants sufficient for therapy have not been generated until now. In this study, we identified single chain Ig variable domain constructs (IgVs) with catalytic activity sufficient for AD therapy from a human IgV library composed of single chain Fv clones (scFvs; these constructs mimic the VL-VH paired structure of physiological Igs) along with a few unusual IgVs generated by cloning errors. Aβ hydrolysis by the IgVs was monitored by radioassay and mass spectrometry. Mutations (4–6/IgV molecule) were introduced by error prone replication. Specific nucleophilic IgVs displayed on phages were selected by covalent binding to an electrophilic Aβ analog. Screening and phage selection procedures identified rare catalystic IgVs with exceptional hydrolytic activity directed at the His14-Gln15 bond of Aβ. Irrelevant proteins were not hydrolyzed. The activities were sufficient to ensure hydrolysis of 105 Aβ molecules/catalyst molecule over 10 days, corresponding to the approximate half-life of full-length IgG in circulation. The IgVs had unusual structures containing heterodimeric VL segments (IgVL2) or an unpaired, intact VL segment linked to a truncated VH peptide (IgVL). Repair of the aberrant VH segment in an IgVL clone yielded scFv constructs with substantially reduced Aβ hydrolyzing activity. Random mutagenesis of an IgVL2 construct followed by covalent phage selection permitted isolation of clones with ∼40-fold improved Aβ hydrolyzing activity. An electrophilic hapten reacted irreversibly with the IgVs, suggesting that the Aβ hydrolysis reaction occurs via a nucleophilic catalytic mechanism. Molecular modeling suggested the presence of serine protease-like catalytic sites in the VL domains. Unlike physiological Igs, the IgVL scaffold supports exceptional Aβ hydrolytic activity and represents a new molecular format suitable for development of catalytic AD immunotherapy.