Washington state collaboration in basic research yields powerful tool in cancer fight
By Andy Koopmans
A collaboration that began several years ago among researchers at Fred Hutchinson Cancer Research Center, University of Washington and Washington State University has resulted in the engineering of a new “designer” protein enzyme developed by San Diego-based company Tocagen for use in a phase 1 clinical trial. The trial, conducted at University of California San Diego Medical Center, is testing the safety of the drug to treat malignant glioblastoma, a form of brain cancer.
Dr. Margaret Black, a professor in WSU’s School of Molecular Biosciences, initiated the protein engineering project in the early 2000s. The idea was to improve the properties of a yeast enzyme called cytosine deaminase (or yCD), which Black had studied for years because of its potential in cancer therapy. The trouble with the native form of yCD is that it prefers cool temperatures and becomes unstable at room or body temperature, impeding its use in human treatments. Black wanted to re-engineer the enzyme to be stable at higher temperatures without changing its basic activity.
Black brought the problem of re-engineering the yCD enzyme to Dr. Barry Stoddard of the Basic Sciences Division at Fred Hutch and his collaborator at UW, Dr. David Baker, a professor of Biochemistry. In Stoddard’s lab, Dr. Aaron Korkegian, then a doctoral student at UW doing graduate work at Fred Hutch, used computational protein engineering software called RosettaDesign to successfully endow the enzyme with increased stability and improved longevity at higher temperatures by replacing three amino acids within it.
The effort was considered a major breakthrough and Korkegian, Stoddard, Baker and Black published the research in a 2005 Science article, “Computational Thermostabilization of an Enzyme.” Stoddard gives credit to for the work to Korkegian, who now works in Seattle at the Center for Infectious Disease Research Institute. “It was one of the most impressive graduate student projects I’ve ever seen,” Stoddard said.
The researchers also re-engineered the enzyme’s specificity—that is, its ability to cause chemical conversions or reactions in other molecules. “Once we had the structure, we started to think about how we could manipulate it in a beneficial manner,” Stoddard said. They engineered the enzyme so that it has the ability to more efficiently convert a nontoxic, safe molecule called 5 fluorocytosine (5FC) into the potent cell-killing toxin 5 fluorouracil (5FU), which has been used for many years as a chemotherapy drug because it kills any proliferating cells it comes into contact with.
‘Suicide gene therapy’
This re-engineering makes it possible for scientists and physicians to introduce genes that express the enzyme into cells in the body and, if necessary, quickly and efficiently kill those cells by giving the patient a dose of 5FC, which is known as a pro drug. When 5FC comes in contact with cells that carry the gene expressing the yCD enzyme, it converts to 5FU and the cells immediately die without harming any other cells. Known variously as pro-drug gene therapy (PGT), or “suicide gene therapy,” this protocol has so far proven useful in two different applications.
The first application is as a precautionary measure in cancer immunotherapy in which T cells are removed and re-engineered so that they will attack cancer cells in the body. In such therapy there is often the risk that something will go wrong and the T cells will attack the wrong cells. If this happens, it can be very dangerous for the patient, but if those T cells have also been encoded to express the altered yCD protein, then the patient can take the pro-drug and the T cells die without doing more harm.
The second application, being tested by Tocagen in San Diego, is using a close approximation of the enzyme for its brain cancer trial. Tocagen has incorporated the DNA for the suicide gene into a virus which targets receptors that are overexpressed on the surface of glioblastoma cells. The virus, called Toca 511, is injected directly into the tumor bed during surgery while a mass is being removed. The virus is specifically designed to enter and inject its genetic material into those cells and will even replicate itself and invade other glioblastoma cells. The virus is a kind of Trojan horse; it contains the suicide gene which, when the pro-drug component is given to the patient, interacts with it and converts it into a toxin that kills the glioblastoma cells while leaving healthy brain cells unharmed.
A personal investment
Stoddard said that he is proud that the work in his laboratory has yielded such a potentially powerful, lifesaving protocol. “To me it demonstrates the importance of basic research for creating new therapies,” he said, adding that the new protocol being used in the San Diego trials has touched him and others on his team.
“My mom died of a glioblastoma, as did one of the founding members of our scientific division, Hal Weintraub, and many of us on the team have friends and family who’ve also died from brain cancer. On a personal level, I would be thrilled to hear years down the road that this new process either works or has provided insight into how to make it work.”