O1–06–05: Monoclonal antibodies selective for Aβ protofibrils reduce plaque burden in transgenic mice models of Alzheimer's disease

In recent years there has been an increasing interest in soluble Aβ oligomers as the pathogenic species in Alzheimer's disease (AD). Different types of Aβ oligomers have been described: low molecular weight Aβ oligomers, Aβ derived diffusible ligands (ADDLs), amylospheroids and large soluble Aβ oligomers, so called protofibrils. We are focusing on the protofibrils, since the Arctic mutation (Aβ E22G) causes AD due to an enhanced propensity of Arctic Aβ peptides to form protofibrils. Enable measurement of Aβ protofibrils in biological tissues through novel antibody–based assays using protofibril selective monoclonal IgGs. In addition, to evaluate these antibodies for immunotherapy in cell culture and transgenic mice models. Protofibrils were generated of synthetic Aβ and analyzed by HPLC and non–denaturing gels. Monoclonal antibodies were raised against the protofibril preparations and characterized with ELISA and Biacore. Protofibril selective antibodies were used in immunoassays to measure protofibrils in APP transgenic mouse brain homogenates. Finally, the ability of the antbodies to inhibit protofibril–induced neurotoxicity was evaluated in vitro using PC12 cells, and the therapeutic efficacy was analyzed in vivo using our APP–ArcSwe transgenic mouse model, which display intraneuronal Aβ before deposition of senile plaques. The size of in vitro generated Aβ protofibrils was determined to be 250–500 kDa according to both Blue Native Gel electrophoresis and size exclusion chromatography. Using protofibrils as antigen, we developed protofibril selective monoclonal IgG2a antibodies. One of these, mAb8, had a hundred–fold higher affinity for protofibrils than for monomers. By using mAb8 in immunoassays, Aβ protofibrils could be detected at low pM levels, which enabled measurement in brain homogenates from transgenic mice. In immunotherapy experiments, mAb8 significantly reduced protofibril toxicity in PC12 cells and reduced Aβ plaque burden in transgenic mice brains. Protofibril selective monoclonal IgG antibodies have been developed and used to measure Aβ protofibrils in transgenic mouse brain homogenates. Measurements of protofibrils in human tissues are ongoing. The reduced protofibril toxicity in PC12 cells and diminished plaque burden in transgenic mice brain after administration of anti–protofibril antibodies indicate that these antibodies could be candidates for passive immunization in the treatment of AD.