PHARMACOLOGICAL CHARACTERIZATION OF BAN2401-MEDIATED Aβ PROTOFIBRIL CLEARANCE BY MICROGLIA

Abstract

Amyloid β (Aβ) peptides aggregate into soluble, large molecular weight species (protofibrils) that eventually form the insoluble fibrils deposited in plaques. Aβ protofibrils are the dominant form of soluble Aβ in Alzheimer disease (AD) brains and are thought to play a role in the pathogenesis of AD through their deleterious effects on a number of physiological processes. BAN2401 is a humanized monoclonal IgG1 antibody with high selectivity for Aβ protofibrils that is currently in clinical development for early AD. mAb158, the mouse precursor, has previously been shown to remove insoluble amyloid plaque in mice, neutralize large soluble Aβ aggregates in vivo/in vitro and facilitate their clearance via a shift toward FcγR-dependent microglial phagocytosis. The present work aims to further characterize the mechanism of action of BAN2401-mediated Aβ protofibril clearance. Protofibril uptake was assessed in cultured human primary microglia prepared from AD brain by visualizing fluorescent tagged antibody under high power microscopy. Protofibril uptake was also assessed in an EOC-20 microglia cell antibody dependent cellular phagocytosis (ADCP) assay by measuring intracellular Aβ via ELISA in the absence or presence of FcγR blocker or F(ab’)2 fragments. Aβ protofibrils were readily taken up in human AD-derived microglia in an FcγR-independent manner, while BAN2401 facilitated further uptake of protofibrils in FcγR-mediated fashion. BAN2401 facilitated protofibril uptake in EOC-20 microglia cells was blocked with an FcγR blocker or F(ab’)2 fragments (EC50=257±61 ng/mL; corrected for background uptake). Aβ can be taken up by microglia via a non-specific pattern-recognizing receptor pathway or through an FcγR-mediated process. Previous work has demonstrated that BAN2401 facilitates protofibril clearance, and this clearance was inhibited by an FcγR blocker to levels lower than those seen in the absence of BAN2401, suggesting a shift from non-specific uptake toward microglial activation and FcγR-dependent microglial phagocytosis. The present studies isolate the FcγR-dependent component of BAN2401-mediated protofibril uptake and provide further support for antibody dependent cellular phagocytosis of Aβ protofibrils as the primary mechanism of action for BAN2401.