Discovery of conformation-sensitive anti-amyloid protofibril monoclonal antibodies using an engineered chaperone-like amyloid-binding protein

Abstract

The hypothesis that amyloid beta peptides (A{beta}) are central to the pathogenesis of sporadic Alzheimers disease (AD) is still hotly debated. Although several monoclonal antibodies (mAbs) against A{beta} have failed in therapeutic clinical trials, two conformation-selective, anti-A{beta} mAbs continue to show promise. A significant challenge has been to discover mAbs that preferentially target A{beta} protofibrils over natively-folded monomeric peptides or amyloid plaques. We have engineered a novel chaperone-like amyloid-binding protein (CLABP), Nucleobindin 1 (NUCB1), which enables the stabilization of protofibrils, allowing them to be used as immunogens in mice to facilitate the generation of mAbs that recognize A{beta} protofibrils. An immunization campaign and subsequent screening funnel identified a panel of mAbs with high-affinity to A{beta}. Two mAbs in particular, 1A8 and 7C8, displayed significant conformation sensitivity and preferentially bound A{beta} protofibrils over monomers. Furthermore, 1A8 delayed A{beta} aggregation, but did not prevent eventual fibril formation, while 7C8 significantly and dose-dependently reduced fibril formation by inhibiting both primary and secondary nucleation. Both mAbs protected against protofibril-induced cytotoxicity in vitro, and showed distinctive staining patterns by immunohistochemistry in PS1/APP mice and in post-mortem AD brain tissue. In summary, we describe a novel method to stabilize soluble A{beta} protofibrils for use in immunization campaigns. We hypothesize that the stabilized protofibrils retain the neoepitopes of the A{beta} protofibril and the aggregates found in mouse models of disease and post-mortem AD brain tissue.