It was - ironically - on Sept. 11, 1978, that a Bulgarian Cold War dissident named Georgi Markov, who worked for the BBC in London, walked across Waterloo Bridge on his way home. As he waited at a bus stop, he felt a sharp jab in his thigh and saw a man behind him holding an umbrella. Four days later, Markov was dead of ricin poisoning. It took only a 0.2 mg dose of the ricin toxin, off the umbrella's tip, to kill him.
Just the other day, police in London arrested six men after finding a quantity of ricin toxin in their apartment. The virulent substance, twice as potent as cobra venom, has been linked to al Qaeda terrorists
"Ricin toxin is considered a bioweapon on par with anthrax," observed molecular biologist Joan Smallshaw at the University of Texas Southwestern Medical Center in Dallas. "As of six months or a year ago," she added, "ricin was considered a level-B threat, but given that small amounts of it were found in a London suburb, ricin is now regarded as a real threat and a bioterrorist agent."
A postdoctoral fellow at the University's Cancer Immunology Center, Smallshaw deals in ricin toxin, but to save lives, not take them. She is first author of an article in Nature Biotechnology, released online March 10, 2003, and titled: "Genetic engineering of an immunotoxin to eliminate pulmonary vascular leak in mice." Its senior author is molecular immunologist Ellen Vitetta, director of the Cancer Immunology Center.
"Specifically," Smallshaw said, "we were able to remove vascular leaks from a single ricin immunotoxin. Our hope," she told BioWorld Today, "is that this might apply to many other toxins as well, for example, interleukin-2, which also suffers from the same vascular leak syndrome when given to patients."
Vascular leak syndrome (VLS) is a dangerous side effect of an immunotoxin that selectively kills cancer cells. In humans, the condition can cause severe weight gain, pulmonary edema and perilously low blood pressure. It occurs when the immunotoxin - made with the enzymatically active A chain of the full-strength ricin toxin - damages the layer of endothelial cells that line the blood vessels.
Dose-Limiting VLS Gone From Mice
"We've been able to remove what is usually the dose-limiting toxicity in a ricin-based toxin," Smallshaw continued. "So that gives a lot of potential improvement as to how much you can give of the drug safely to patients. In prior Phase I studies ricin-based immunotoxins have been fairly limited as to how much - what kind of doses - we can administer safely. So our hope is it can be used at higher doses for more efficacy, and given to more patients as well, over a longer period.
"The natural virulent ricin toxin," she explained, "is a two-domain protein, A and B, connected by disulfide bonds. The A chain itself is the toxin. The B chain is the targeting moiety, but it targets very indiscriminately, so it's very toxic in people. But once the B chain is removed, the A chain has relatively low toxicity - much lower than the whole ricin molecule.
"Its mechanism of selectivity to cancer tumors is based on the idea that you can attach ricin to any antibody of choice," Smallshaw pointed out. "In the model that we presented in the journal paper, it was targeting the CD22 molecule, which is highly expressed on many B-cell lymphoma cell lines. But you can attach ricin to any targeting antibody you choose with similar curative effect - hopefully."
Genetically engineered versions of the cancer-fighting immunotoxin were administered to mice with human lymphoma cells. The co-authors identified one altered immunotoxin that did not cause VLS in mice, and more effectively destroyed cancer cells than earlier generations of the therapy.
"We reported two in vivo mouse models," Smallshaw recounted. "One is our model of vascular leak. In the mice it's pulmonary VL, so we measured the amount of leaks by using radiolabeled albumin after treatment. It correlated with VL in mice.
"Early on in the development of these immunotoxins, and in all the toxicity screening that was done, no severe toxicity was shown in mice," she noted. "But vascular leaks occurred in patients, which was quite unexpected. So this is a mouse model that is not exactly what happens in people. We believe it's the same mechanism. The second experiment was in the immune-compromised SCID mice. We had the human xenograft tumors growing in them in an immunotoxin dose for treatment."
Vaccine Against Weaponized Ricin Works In Vivo
"We're trying to get grant money together to do a Phase I human trial of the modified immunotoxin," Smallshaw allowed, "to see if what doesn't happen in mice is also not happening in human patients. Therapeutic potential exists for other drugs to be developed on this research," she went on. "Considering that we believe a similar motif exists on many other toxins to use for immunotoxins - such as diphtheria toxin and exotoxin - there's a potential to engineer those sites out of those toxins as well. IL-2 also shows this motif in peptide form, and we've shown that motif is responsible for VLS."
As a spinoff from their anticancer research, the Dallas team has constructed a vaccine against the full-toxin ricin bioweapon. "To do that," Smallshaw said, "we took the ricin A chain, which has much, much lower toxicity than ricin, but still a significant amount of it. We made two alterations to that molecule. One, changing a critical residue at the active site so it no longer had its normal toxicity. And two, we made a change in the vascular leak site so our model also lacks VLS.
"To test the vaccine, we immunized mice over a period of time, then challenged them with an intraperitoneal. injection of ricin, which we calculated to be 10 times the LD50 - 10 times the 50-percent lethal dose of mice. All of our control animals died at that dose, but none of our vaccinated mice were affected. The military has taken an interest in our ricin vaccine," she observed. "We're working on developing it, but it's unclear," Smallshaw concluded, "how that will all work out."