Targeting isoaspartate-modified A[beta] rescues behavioral deficits in transgenic mice with Alzheimer's disease-like pathology

Background Amyloid [beta] (A[beta])-directed immunotherapy has shown promising results in preclinical and early clinical Alzheimer's disease (AD) trials, but successful translation to late clinics has failed so far. Compelling evidence suggests that post-translationally modified A[beta] peptides might play a decisive role in onset and progression of AD and first clinical trials targeting such A[beta] variants have been initiated. Modified A[beta] represents a small fraction of deposited material in plaques compared to pan-A[beta] epitopes, opening up pathways for tailored approaches of immunotherapy. Here, we generated the first monoclonal antibodies that recognize l-isoaspartate-modified A[beta] (isoD7-A[beta]) and tested a lead antibody molecule in 5xFAD mice. Methods This work comprises a combination of chemical and biochemical techniques as well as behavioral analyses. A[beta] peptides, containing l-isoaspartate at position 7, were chemically synthesized and used for immunization of mice and antibody screening methods. Biochemical methods included anti-isoD7-A[beta] monoclonal antibody characterization by surface plasmon resonance, immunohistochemical staining of human and transgenic mouse brain, and the development and application of isoD7-A[beta] ELISA as well as different non-modified A[beta] ELISA. For antibody treatment studies, 12 mg/kg anti-isoD7-A[beta] antibody K11_IgG2a was applied intraperitoneally to 5xFAD mice for 38 weeks. Treatment controls implemented were IgG2a isotype as negative and 3D6_IgG2a, the parent molecule of bapineuzumab, as positive control antibodies. Behavioral studies included elevated plus maze, pole test, and Morris water maze. Results Our advanced antibody K11 showed a K.sub.D in the low nM range and > 400fold selectivity for isoD7-A[beta] compared to other A[beta] variants. By using this antibody, we demonstrated that formation of isoD7-A[beta] may occur after formation of aggregates; hence, the presence of the isoD7-modification differentiates aged A[beta] from newly formed peptides. Importantly, we also show that the Tottori mutation responsible for early-onset AD in a Japanese pedigree is characterized by massively accelerated formation of isoD7-A[beta] in cell culture. The presence of isoD7-A[beta] was verified by K11 in post mortem human cortex and 5xFAD mouse brain tissue. Passive immunization of 5xFAD mice resulted in a significant reduction of isoD7-A[beta] and total A[beta] in brain. Amelioration of cognitive