The UCSF-led research is being published on line the week of August 5 by The Proceedings of the National Academy of Sciences.
Botulinum toxin, naturally produced by a soil bacterium, is the most poisonous substance known. A gram of the toxin, if evenly dispersed and inhaled, could kill a million people, according to a recent study. Yet no anti-botulism drugs are available which could be produced in the quantities needed for treatment or prevention if the toxin were used for bioterror, the new study points out. The new drug potently neutralizes botulinum toxin and can be readily mass-produced, the researchers report.
The drug was developed by expanding the technique now used to produce monoclonal antibodies against pathogens or other molecular targets. Scientists isolated and identified three antibodies against botulinum toxin and combined them. Each antibody is capable of binding to a different part of the toxin molecule. When administered together, they bind the toxin much more tightly and block far more of the toxin surface than a single antibody could, the scientists report. As a result, in animal studies, the antibody "cocktail" neutralizes much more of the toxin than observed for single antibodies.
Treatment for botulinum poisoning usually requires many weeks of intensive-care hospitalization, and exposure of even a small number of people would seriously disrupt health care delivery in any major city, according to a recent assessment. A vaccine has been developed, but widespread use is not currently being considered, the researchers say, since the likelihood of exposure is uncertain. Also, vaccination would block accepted treatments for a number of overactive muscle conditions, including dystonias, which respond to the toxin when administered in very small doses.
"This approach has allowed us to develop a drug consisting of only a few antibodies which neutralizes toxin better than the most potent natural immune response," said James D. Marks, MD, PhD, UCSF professor of anesthesiology and pharmaceutical chemistry and senior author on the paper. Marks directs his laboratory at the UCSF-affiliated San Francisco General Hospital Medical Center.
"The procedure could be scaled up to mass produce and stockpile the drug to be used to prevent or treat botulism." The long half life of human antibodies means that a single dose could protect people at risk for six months, Marks said. Its high potency suggests the cost per dose would be relatively low. And unlike vaccines, antibodies allow immediate treatment or protection.
The drug development approach, selecting a small number of antibodies to work I combination, could be applied to produce drugs against other deadly agents where antibody has been shown to have neutralizing activity, such as anthrax, smallpox, plague or hemorrhagic fever viruses, the scientists conclude. UCSF has applied for a patent on the drug.
The paper reports on three antibodies, one fully human and two that derive from both mice and humans. Although such "chimeras" have been approved for human use, Marks says the research team wants to make all three antibodies human.
Botulinum toxin can enter the blood stream when a person either swallow or inhales it. When the toxin reaches the junction of a nerve and muscle – the presynaptic motor neuron – it binds to receptors on the surface of the motor neurons. After entering the cell, a portion of the toxin chops up proteins and prevents the neurotransmitter acetylcholine from reaching the muscle. As a result, muscles cannot contract, leading to paralysis. Progressive paralysis of the breathing muscles is fatal without mechanical ventilation.
The researchers refer to the technique used to make the drug as neutralization of the botulinum toxin by "recombinant oligoclonal antibodies." Recombinant refers to the fact that the antibody genes are cloned into a manufacturing cell line, so unlimited quantities of the antibodies can be produced by the cultured cells. Oligoclonal means that the drug involves a combination of a precise, small number of antibodies. Although ten monoclonal antibody drugs are approved as treatments, no approved drug employs multiple antibodies, according to the scientists.
One of the keys to the new technique is finding the right antibodies of the highest affinity that can bind to the toxin simultaneously without interfering with each other. This requires generating and analyzing a large number of antibodies. To generate the best antibody candidates, Marks's team used a technique his lab has developed over the past decade called antibody phage display. The scientists first isolated the immune system's B cells from either mice or human volunteers who had been immunized with an inactivated version of the botulinum toxin, known as a toxoid. They used the highly efficient polymerase chain reaction, or PCR, to amplify the genes encoding the thousands of different antibodies comprising the immune response to toxin.
The antibody genes were then expressed on the surface of viruses that infect bacteria (antibody phage display). Antibodies binding the toxin (and the genes encoding them) were then recovered and characterized further to find the highest affinity antibodies that could bind simultaneously to the toxin. Since the screening procedure uses only the binding portion of each antibody, they then had to generate full length antibodies to determine whether the antibodies could neutralize toxin in mice.
Lead author on the PNAS paper is Agnes Nowakowski, BS, staff research associate in anesthesia and pharmaceutical chemistry at UCSF. Co-authors are Caili Wang, PhD; David Powers, PhD; and Peter Amersdorfer, PhD, all visiting postdoctoral fellows in anesthesiology and pharmaceutical chemistry at UCSF.
Also: Theresa Smith, MS and Vicki Montgomery, both microbiologists; and Leonard Smith, PhD, chief of immunology and molecular biology, all in the Toxinology and Aerobiology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD; Robert Sheridan, PhD, research physiologist, Neurotoxicology Branch, Pathophysiology Division, U. S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD; and Robert Blake, PhD, professor and chair, basic pharmaceutical sciences, Xavier University, New Orleans.
The research was funded by the Department of Defense.
Journal
Proceedings of the National Academy of Sciences