Early in this new millennium, Mad Cow Disease was set to devastate the UK with contaminated meat from infected cows entering the human food chain and causing significant risk to the UK population. At the peak of the epidemic, nearly 200,000 cows were diagnosed with the disease. At the time, it was unknown how many people had become infected from eating infected meat and how many of these would go on to develop vCJD, the human form of Mad Cow Disease. Consequently, it was also unknown what the risk was from blood transfusion. Without a test people could not be diagnosed and the blood supply could not be safeguarded.
Against this backdrop, Microsens was established to develop a test for Mad Cow Disease. The biggest issue in developing such a test was that the protein that caused the disease, the prion protein is the same protein in normal uninfected people and in infected people. The prion protein comes in two forms; a normal non-infectious form that everyone has and an abnormal infectious form. The infectious form of the protein has the same identical amino acid sequence to the normal protein but is folded differently. This different folding, causes aggregation of the protein and these aggregates can recruit the normal protein causing it to change to the abnormal infectious form. A hallmark of the diseases in infected animals and people is large clusters of these aggregates in the brain that cause inflammation and brain cell death. The fact that the pathogenic infectious protein is identical to the normal protein, except for the way it is folded, made it difficult to develop a test through the normal route of raising an antibody that would only detect the abnormal infectious protein.
Some tests that could be used on brain material were developed as it turned out that the large lumps of aggregated proteins were resistant to enzyme degradation by proteases. To test a brain sample, the sample could be treated with protease to digest the normal prion protein, leaving the indigestible lumps of infectious protein behind to be detected by an antibody that recognised the prion protein. Using this approach, the antibody did not have to differentiate the abnormal infectious protein from the normal one; that work was done by the protease.
The problem with the protease treatment was that eventually, the large lumps of infectious protein would also be digested away with time so the test was hard to standardise. Also, as research was carried out it was realised that the infectious prion protein in the blood was not in the form of protease resistant lumps (the lumps were far too small) and even in some forms of Mad Cow Disease, the lumps in the brain were not protease resistant either.
This is where the technology developed by Microsens came in. Microsens developed a technology, Seprion that avoided the use of protease altogether. To do this, they developed a chemical that only stuck to the lumps of infectious prion protein and not to the normal protein. Microsens likens the chemical to a long piece of string that wraps itself in a tangle around the lumps of protein, capturing them. Single normal prion protein does not get tangled in the chemical string and can easily fall off so is not captured. It also turned out that the chemical bound to the infectious protein whether it was in large lumps such as found in the brain or very small aggregates such as those found in the blood. This meant that the test could be used in all forma of the disease and even in blood too.
A simple test was developed in which the chemical was coated onto plastic plates in wells. Brain samples are mashed up and placed in the wells. If aggregated prion protein is present, these bind to the chemical and after washing, to remove normal prion protein, any remaining abnormal prion protein is detected by an antibody. The test was licensed to Idexx Laboratories and was so successful that since development it has been used to screen millions of cows before their meat entered the human food chain.
Since its use in screening for Mad Cow disease, Microsens has broadened the applications of Seprion. Mad Cow Disease is just one of a growing number of protein aggregation diseases. There is long list of diseases in which protein aggregates are implicated. For example, in Huntington’s disease a protein called huntingtin forms aggregates; in Parkinson’s Disease it is alpha synuclein and in Alzheimer’s Disease it is tau and beta-amyloid. In Alzheimer’s Disease, it is the aggregates of beta-amyloid that cause neuronal cell death, perhaps by causing inflammation, and this loss of neurones is devastating to those affected by the disease. In all of these diseases it has been found that Seprion can bind the aggregated protein in the same way that it binds aggregated prion protein. As such, Seprion has been used by research groups to further the understanding of protein aggregation diseases and to help develop therapeutics. For example, a group at the University of London have an animal model of Huntington’s Disease and in their drug screening programme they are using Seprion as an easy way to monitor the effect of drug treatment of the animals. Another group used Seprion in Alzheimer’s Disease to screen existing drugs that were known to penetrate into the brain (after all, this is where the pathology occurs and a drug that didn’t find its way into the brain would be of no use) for prevention of aggregation of beta-amyloid. Two such drugs were found; trimipramine and fluphenazine that were being used to treat psychosis at the time.
This ability of Seprion to bind to protein aggregates but not bind to the unaggregated protein has also been used in the manufacture of therapeutic proteins. Anti-cancer drugs are often proteins such as monoclonal antibodies. These antibodies are produced by growing the cells that secrete the antibodies which then have to be extensively purified before use in patients. This often complex procedure can cause stresses to the antibody protein that can cause it to aggregate. Such aggregates have the potential to reduce the effectiveness of the antibody or even to generate toxic effects in the patient. Manufacturers of therapeutics spend much time and money designing manufacturing processes that reduce the tendency for aggregation and also in monitoring the extent of aggregation throughout the manufacturing process and even after manufacture when the therapeutic is stored. Seprion is one method by which such aggregates can be detected and monitored.
One of the recent applications of Microsens’s Seprion technology is perhaps one of the most exciting. Cancer is often caused by the malfunction of one of the most important tumour suppressor proteins, p53. p53 is an essential protein that directs the cell to suicide (apoptosis) in response to genetic damage or other cancer-promoting events. People born with defective p53 have much higher risk of cancer. In recent years it has been discovered that many cancers are associated with aggregated p53. Once aggregated, the p53 cannot fulfil its function of guiding cancer prone cells to suicide. It has also been suggested that cancers with aggregated p53 may be less treatable by common platinum-based anti-cancer drugs. Microsens sees a role for its Seprion technology in research into aggregated p53. It has already been shown that Seprion binds to the aggregated form of p53. It is envisaged that Seprion could find use as a research tool, or as the basis of a test that predicts therapeutic effect of anti-cancer drugs or even as a blood screen to detect cancers earlier thus improving survival times.
It is clear that the field of protein aggregation has grown in importance from the infamous epidemic of Mad Cow Disease to a major issue in diseases such as Alzheimer’s Disease and cancer. In Seprion, Microsens has a unique tool that can be used for research, screening and drug development.