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

The hypothesis that amyloid beta peptides (Aβ) are central to the pathogenesis of sporadic Alzheimer’s disease (AD) is still hotly debated. Although several monoclonal antibodies (mAbs) against Aβ have failed in therapeutic clinical trials, two conformation-selective, anti-Aβ mAbs continue to show promise. A significant challenge has been to discover mAbs that preferentially target Aβ 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 Aβ protofibrils. An immunization campaign and subsequent screening funnel identified a panel of mAbs with high-affinity to Aβ. Two mAbs in particular, 1A8 and 7C8, displayed significant conformation sensitivity and preferentially bound Aβ protofibrils over monomers. Furthermore, 1A8 delayed Aβ 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 Aβ protofibrils for use in immunization campaigns. We hypothesize that the stabilized protofibrils retain the neoepitopes of the Aβ protofibril and the aggregates found in mouse models of disease and post-mortem AD brain tissue.