Cationized Antibodies: Imaging Tool for Cerebral Amyloid

The diag nosis of Alzheimer’s disease (AD) during the life of th e patient is still made clinically mainly by exclu sio n of other p ossible causes of dementia . This is afflicted with some degree of uncertainty, and the diagnosis can o nl y be made a t a relatively late stage of the disease. Both studies into the natural course of AD, and a potential therapy, which seems most promising at an early disease stage, would greatly benefit from a reliable and sensitive diagnostic test. There is mounting evidence, that the abnormal brain deposition of a specific protein, ca ll ed beta amyloid , is causally related to AD. Presumably, the deposition of beta amyloid starts many years (d ecades) before the ftrst clinical signs of AD deve lop. Therefore, a noninvasive method to detect beta amyloid co uld be a promising diagnostic approach. However, currently the se beta amyloid de posits can only be detected at autopsy or with brain biopsy , which is a highly invasive neurosurgical procedure and is unusable on a broad scale. This proposal evaluates the use of a monoclonal antibody, which is able to specifically recognize beta amyloid protein, as a diagnostic tool in an animal model of AD , namely old dogs. Following peripheral intravenous administration , accumulation of this antibody in brain tissue would indicate the presence of amyloid deposits. There are at least two problems, which have to be solved, before this idea can be tested. (i) Antibodies are large protein molecules. The mammalian brain posseses a tight barrier preventing the free passage of proteins and many other substances from blood to brain , called the blood-brain barrier. (ii ) The antibody needs to be detected, once it has entered the brain. Problem (i) can be solved by chemical modification of the antibody in the form of cationization. This process increases the number of positive charges attached to a molecule. It has been demonstrated , that such cationic proteins can penetrate the blood-brain barrier. The second problem can be solved by radioactive labeling of the antibody. Usage of a suitable label allows then the application of modern imaging techniques such as single photon emission computed to mography (SPECT), which is similar to the CAT scan, to noninvasively detect and localize the radioactive m olecule inside the body. It is hypothesized that the presence of beta amyloid in the brain results in a stronger signal compared to normal brain tissue. The specific aims of the project will evaluate the feasibility of the proposed diagnostic approach in the following steps:           1) The specific monoclonal antibody and a suitable nonspecific control antibody are produced and purified in sufficient amounts . Then, the antibodies are cationized and radiolabeled . The integrity of the proteins has to be checked at each steps with appropriate analytical methods .           2) An in vitro model of the blood-brain barrier is used as a screening procedure for the expected brain uptake of the antibodies in the subsequent animal experiments .           3) The disposition and tissue accumulation of the radiolabeled antibodies will be evaluated in mice. This experiment is proposed in anticipation of mouse mode ls of AD , which are currently being developed by genetic engineering. Obviously, mice would be the preferred animal model over dogs or nonhuman primates.           4) Analogous studies of the disposition from blood of the radioactive labeled antibodies are performed in dogs in preparation of the following imaging studies in this species .          5) Brain imaging with SPECT will be performed in young control dogs and old ( > 10 years of age) dogs. More than 50 % of the latter can be expected to show brain amyloid deposits , which resemble the alterations seen in human AD brain, making aged canines a valid model for the structural consequences of AD . In these experiments , the brain accumulation of the nonspecific antibody and the specific antibody recognizing beta amyloid will be compared within the same animal. It is expected that in the young dogs and in the old dogs without amyloid deposits (normal brain) there is no difference between the two antibodies. In contrast, dogs with brain a myloid should demonstrate a longer retention of the specific antibody in brain tissue , because it binds to beta amyloid , and is therefore eliminated more slowly.           The long-term goal is the application of this technique as a diagnostic method in humans. There is another point to be addressed in this transfer , which is the source of the monoclonal antibody. Typically, these monoclonal antibodies are of mouse origin . The administration of mouse proteins to humans may provoke an immune response. However, methods have been developed to “humanize” such antibodies, i.e . to replace most of the mouse molecule with its human counterpart. This should diminish the risk of immune reactions. Finally, there is also a potential therapeutic significance of the proposed work. The development of future AD therapeutics will most likely be directed towards the processes which culminate in the abnormal beta amyloid deposition . An antibody which has been enabled to cross the blood-brain barrier could be used as a carrier to target drugs directly to the site of amyloid deposition .