I am interested in developing therapeutic antibodies as potential treatments for Alzheimer’s disease (AD) as current drugs are limited to treating the symptoms and do not affect the underlying disease process.
There are two characteristic neuropathological features of AD, neurofibriallary tangles made of hyperphosphorylated tau and extracellular senile plaques made primarily of ß-amyloid (Aß), a 40-43 amino acid peptide produced from amyloid precursor protein (APP). An increase in Aß levels (especially Aß42) is thought to be crucial for the pathogenesis of AD. Aß is produced from APP by the amyloidogenic route involving the enzymes beta- and gama-secretase which cleave APP sequentially to liberate Aß. Since the discovery of ß-secretase, an aspartic protease called beta-site APP-cleaving enzyme (BACE1) 1999, there has been much interest in developing inhibitors but there are no commercial drugs yet and there are a number of problems (e.g. multiple substrates, specificity).
We have devised a different approach which could prevent or halt the build-up of toxic Aß. We have made a monoclonal antibody which should inhibit ß-secretase by steric hindrance as it binds in the vicinity of the ß-secretase cleavage site in APP (Fig. 1).
Aims of Project
We hypothesised that the antibody will bind to APP when it is at the cell surface and then be internalised with APP. 2B12 recognises APP using ELISAs, Western blotting on cell lysates and immunocytochemistry. We used two human cell lines, neuroblastoma SH-SY5Y and astrocytoma MOG-G-UVW (MOG), constitutively expressing APP. We have also shown that 2B12 can only very weakly detect Ab1-40 in an ELISA and not at all in Western blotting. However, the antibody does bind, albeit weakly, to the C99 peptide including the first amino acid before the N-terminus of Abusing Western blotting and an ELISA. These results all suggest that the antibody binds to a specific epitope spanning the b-secretase cleavage site. To test whether the antibody could inhibit Ab1-40 production, we incubated SH-SY5Y and MOG cells with increasing concentrations of purified antibody for 4 days. 2B12 produced a significant concentration-dependent inhibition of Ab1-40 secretion from both cell lines (Fig. 2). No inhibition was seen with control murine IgGs.
This inhibition appeared within 6 hours of incubation and was sustained for up to 6 days. Neither 2B12 nor the irrelevant IgGs had any significant effect on cell number over the concentration range used so the data cannot be explained by toxicity or the presence of a large molecular weight protein. In all our studies, the reduction in Ab observed in the presence of 2B12 reached a maximum inhibition of approximately 50 % implying that, if the antibody is working in the manner that we propose, a significant proportion of APP does not come to the surface but is cleaved directly after synthesis, agreeing with work suggesting that APP can be processed in different compartments. Interestingly, the rapid reduction in Ab detected after 6 hours of antibody treatment suggests a rapid turnover of Ab and a rapid cycling of APP to the cell membrane. As far as we are aware, we have demonstrated for the first time that it is possible to inhibit the action of b-secretase by blocking its cleavage site with a whole IgG molecule in cells that constitutively express APP. Overall, our results suggest that it may be possible to use 2B12 to prevent or slow the development of AD. In conjunction with other strategies to reduce plaque load, the use of such an antibody might potentially revolutionise the currently available treatments for this neurodegenerative disease.
Currently, we are carrying out studies to prove that 2B12 does indeed inhibit b-secretase activity. We are also investigating how the antibody enters cells and the trafficking of APP inside cells in various subcellular compartments. We have shown time-dependent entry of the antibody into cells suggesting that our hypothesis that 2B12 is internalised with APP is correct (Figure 3).