Written by David Fishlock, this is the text of an article in "Target MD", published Spring 1998 by the Muscular Dystrophy Group of Great Britain.

Making magic bullets work

Dubbed 'magic bullets' because of their precision in hitting the right disease target, monoclonal antibodies are now used to study and diagnose MD.

Monoclonal antibodies are one of the great recent inventions of medical science. How to make them earned Nobel prizes for two Cambridge scientists, in 1984.

By then, many scientists all over the world were using the new method of cell engineering Cesar Milstein and George Köehler had discovered for raising these pure antibodies in unlimited quantities.

One was Professor Glenn Morris of the North East Wales Institute (NEWI), now at Wrexham in Denbighshire. He has a catalogue of about 300 of these remarkable reagents built up since 1975 when the technique first appeared in the pages of the pre-eminent science journal Nature.
Of the 300, about half relate directly to the study and diagnosis of Duchenne muscular dystrophy and others to rarer dystrophies, Glenn estimates.
Perhaps more surprising, his catalogue of monoclonal antibodies has been compiled - and is still being expanded - by Glenn's wife. He married the Vietnam-born geneticist Nguyen thi Man in 1975 and she has been a key member of his team ever since.

"We met when I moved into muscle in 1970", recalls Glenn. Successful mammalian cell culture, the technique required to make monoclonal antibodies, is an art as well as a rigorous science. The faintest whiff of infection can kill a culture stone dead. "We never get any contamination", he testifies proudly to his wife's prowess. Man, as he calls her, does all the cell culture production and research for his Biotechnology Group at NEWI.

In demand

Man's monoclonal antibodies are in demand for research as far afield as New Zealand. In 1991, after the dystrophin gene involved in Duchenne MD had been identified, she made the first monoclonal antibody against utrophin, a protein that is similar in structure to dystrophin.

Glenn read natural sciences at Cambridge. "By the third year it was virtually all biochemistry". As an undergraduate in Cambridge in 1967 he read a laboratory notice saying that Asher Korner, a lecturer in biochemistry, had been appointed to a new chair at Sussex University and had six research studentships available. Korner offered him one. Professor Korner encouraged his students to pursue their own ideas rather than his, reckoning that the way biochemistry was developing their research would eventually assist his own work on growth hormone. It was an inspirational approach, Glenn believes. Initially, Glenn was working on diabetes and how blood glucose controls the way genes are switched on to make proteins - or not, in the case of muscular dystrophies. Others at Sussex were already working on muscle differentiation and muscular dystrophy. The protein that particularly interested Glenn is called creatine kinase. In the late-1970s, working with Man, he began to make monoclonal antibodies against this protein, with the idea that it might be a way of diagnosing muscular dystrophy. In fact, it led to a method of diagnosing damage in heart muscle after a heart attack. In 1984, Glenn and Man left Sussex for Deeside, North Wales, where NEWI was then located. He was drawn by the fact that the job involved no teaching. He was to build up the Biotechnology Group. But it took time. "For a long period there were just three of us, Man, me and a technician, and Man had no funds". But a couple of the new biotechnology start-up companies helped out with contracts to produce monoclonal antibodies.

Turning point

Identification of the gene involved in Duchenne muscular dystrophy in 1986 was a turning point for Glenn's little group. Researchers in many areas of neuromuscular medicine began to see the value of raising monoclonal antibodies as an investigatory tool.

Were they hampered by their relative isolation in North Wales? I asked Glenn. More so than when they were in Deeside, near Liverpool, from which the group moved in 1995. "It's a problem", he admits, "there's nothing like being in regular contact". But their location has not stopped Glenn and his team from building strong links with other research centres. "Our aim is to produce monoclonal antibodies as highly specific research tools for the analysis of molecular mechanisms in the pathogenesis (development) of human genetic disease, and for improved diagnosis", says Glenn. Each monoclonal antibody is specific to a given bacterium, virus or whatever else is the cause of the disease. In the case of MD it is a protein that is either missing, malformed or in short supply.

His group comprises three other researchers - including Man - two technicians and five students. "Our group has an extensive network of clinical and scientific collaborators in hospitals, universities and research institutes in the US, Canada, Japan, New Zealand, Vietnam, Israel, Europe and the UK".

The Muscular Dystrophy Group in the UK has long supported Glenn's group, most recently with a three-year grant of £160,000 for 1995-98. Last year, he also won a small grant from the Muscular Dystrophy Association of the US. In 1993, in a successful effort to expand his group, he persuaded the Welsh Office to fund the application of his experience with monoclonal antibodies for MD to other inborn diseases, including Huntington's disease, and also to hepatitis C and malaria.

The beauty of the monoclonal antibody is that it can tell the researcher exactly where and how it is binding to a given protein molecule. Researchers become more and more specific in what they ask of the reagent. "For example, we may want one that is specific to man or to the frog", Glenn says. The first step will be to screen the library to see if there's already a monoclonal antibody that meets the researcher's specification. He worked closely with Kay Davies, professor of genetics at Oxford University, who featured in the Winter 1997 issue of Target md. Kay provided him with the recombinant protein from which to culture the monoclonal antibody.

Diagnosis of Duchenne MD using the panels of monoclonal antibodies they made is "a good technique, fairly well established", says Glenn. They are used mainly in the Muscle Centres in the UK and other diagnostic centres around the world. It is useful to have a comprehensive library of antibodies which bind to different regions on a protein, particularly when it is as long as dystrophin. For example, a test using just one antibody might mislead you into thinking dystrophin is present in muscle, whereas, in fact half the protein is missing and the individual does have Duchenne md. Knowing exactly which region of dystrophin is missing in a patient can lead to interesting insights. Glenn found that if a particular area was missing from the tail end, dystrophin would be present in normal amounts, but wouldn't prevent Duchenne md.

Rare gene

In 1994, the gene for the rare Emery-Dreifuss MD was identified. EDMD is a neuromuscular condition with slowly progressive skeletal muscle-wasting of the shoulder girdle and distal leg muscles, early contractures of the elbows and Achilles tendons, and heart problems which may eventually cause death unless a pacemaker is inserted at the appropriate time. Patients with EDMD are believed to lack a protein called emerin. The group has developed a simple antibody test for EDMD families that shows clearly whether emerin is present or absent. This work was done with Dr Caroline Sewry and Dr Francesco Muntoni at the Hammersmith Hospital, London.

"It's a very exciting area", says Glenn, "still at the research stage. It's at the point Duchenne MD had reached by the early-1990s" Also exciting interest is the diagnosis of myotonic dystrophy, another rare dystrophy, characterised by an abnormal expansion of a stretch of DNA, as discovered by Keith Johnson, professor of genetics at Glasgow University. Glenn works closely with Professor Johnson on a particularly complex disorder where "it's not even clear yet which gene is responsible". Different research groups are getting conflicting results. But they agree that the answer they seek may come from raising a whole family of monclonal antibodies to help identify precisely where the condition begins.

Formerly science editor of the Financial Times, David Fishlock is publisher of R & D Efficiency.