Anti-amyloid precursor protein immunoglobulins inhibit amyloid-beta production by steric hindrance

The cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases results in the production of amyloid-beta (A beta) in Alzheimer's disease. We raised two monoclonal antibodies, 2B3 and 2B12, that recognize the beta-secretase cleavage site on APP but not A beta. We hypothesized that these antibodies would reduce A beta levels via steric hindrance of beta-secretase. Both antibodies decreased extracellular A beta levels from astrocytoma cells, but 2B3 was more potent than 2B12. Levels of soluble sAPP alpha from the nonamyloidogenic alpha-secretase pathway and intracellular APP were not affected by either antibody nor were there any effects on cell viability. 2B3 exhibited a higher affinity for APP than 2B12 and its epitope appeared to span the cleavage site, whereas 2B12 bound slightly upstream. Both of these factors probably contribute to its greater effect on A beta levels. After 60 min incubation at pH 4.0, most 2B3 and 2B12 remained bound to their antigen, suggesting that the antibodies will remain bound to APP in the acidic endosomes where beta-secretase cleavage probably occurs. Only 2B3 and 2B12, but not control antibodies, inhibited the cleavage of sAPP alpha by beta-secretase in a cell-free assay where the effects of antibody internalization and intracellular degradation were excluded. 2B3 virtually abolished this cleavage. In addition, levels of C-terminal APP fragments, generated following beta-secretase cleavage (beta CTF), were significantly reduced in cells after incubation with 2B3. These results strongly suggest that anti-cleavage site IgGs can generically reduce A beta levels via inhibition of beta-secretase by steric hindrance and may provide a novel alternative therapy for Alzheimer's disease.