Cleavage of amyloid precursor protein (APP) by – and -secretases results in the production of amyloid- (A) in Alzheimers disease (AD). to their antigen, suggesting the antibodies will remain bound to APP in the acidic endosomes where -secretase cleavage probably happens. Only 2B3 and 2B12, but not control antibodies, inhibited the cleavage of sAPP by -secretase inside a cell-free assay where effects of antibody internalisation and intracellular degradation were excluded. 2B3 virtually abolished this A 740003 cleavage. In addition, levels of C-terminal APP fragments, CTF, generated following -secretase cleavage, were significantly reduced in cells after incubation with 2B3. These results strongly suggest that anti-cleavage site antibodies can generically reduce A levels via inhibition of -secretase by steric hindrance and may provide a novel option therapy for AD. system any effects of APP internalisation A 740003 or enzyme/substrate colocalisation are eliminated. In conjunction with the observed decrease in CTF levels, results from the cell-free assay system suggest that the mode of action of our cleavage site antibodies is most likely via steric hindrance. Comparable effects on A levels were obtained by Arbel et al. [35] who produced monoclonal antibodies using a peptide made up of part of the Rabbit polyclonal to FLT3 (Biotin) Swedish mutation at the -secretase cleavage site. They also exhibited a reduction in both extracellular and intracellular A levels but in cell lines over-expressing APP. These antibodies have also been shown to improve cognition in the Tg2576 Swedish mutation mouse model of AD pathology [40] and reduce A levels in the V717I London mutation mouse model [41]. We believe that our use of model cell lines which do not over-express APP is very important as the majority of cases of AD occur in people with much lower levels of APP than those associated with transfected cells. As far as we are aware, we have exhibited for the first time that the most likely mode of action for such antibodies is usually via steric hindrance. Immunotherapy for AD remains an exciting prospect, despite the failure of the AN1792 clinical trial [36]. Passive immunisation with -secretase cleavage site antibodies might alleviate some of the problems associated with this trial such as the T-lymphocyte meningoencephalitis and cerebral micro-haemorrhages [37, 38]. These antibodies would not bind to existing A and would not therefore stimulate the T-lymphocyte response or lead to the excessive match activation that some believe would be a problem with A antibodies [39]. In conjunction with other immunotherapeutic strategies to reduce plaque load, such antibodies may have a considerable impact on the development of disease-modifying treatments for AD. MATERIALS AND METHODS Materials and cell culture All chemicals and reagents were purchased from Sigma-Aldrich, Poole, U.K. or Fisher Scientific, Leicester, U.K. and all reactions were performed at room heat unless normally specified. Astrocytoma cells, MOG-G-UVW (ECACC, Porton Down, U.K.) were cultured in a 1:1 mix of Hams F10 and Dulbeccos Modified Eagles Medium supplemented with 10% foetal bovine serum (Perbio Science U.K. Ltd, Cramlington, U.K.) and 2mM L-glutamine. Antibody production and isotyping Full details of the immunisation protocol and hybridoma development are detailed elsewhere [25]. Antibodies (2B12 and 2B3) were raised to a 15-mer peptide spanning the A 740003 -secretase cleavage site on APP, EEISEVKMDAEFRHD, termed Ka. Both antibodies were concentrated from culture medium using Amicon Centriplus YM-100 filters (Millipore, Watford, U.K.) with a nominal molecular excess weight cut off of 100kDa and the isotype decided using the Isostrip mouse monoclonal antibody isotyping kit (Serotec, Oxford, U.K.). Western Blotting was performed using standard methods. Briefly, samples were resolved on 10% polyacrylamide gels, transferred on to 0.2m nitrocellulose membranes (Amersham Biosciences, Little Chalfont, U.K.), incubated with the relevant antibody and detected as previously explained [25]. Determination of antibody epitopes The.