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Edgington first encountered TF almost 20 years ago, when he was pioneering research into blood coagulation, thrombosis, and the connections between the immune system and the vasculature. In the process, he first cloned the gene for TF in 1987, and subsequently worked out the structure and how TF works.

TF is the primary molecule that initiates the cascade of reactions in thrombosis, which involves about 30 interacting proteins, and ultimately results in the processing of fibrinogen molecules in the bloodstream to form the sticky clot-forming fibrin.

As a cell surface receptor TF is highly efficient, binding to its target substrate with picomolar affinity. "One molecule of TF running 100 percent can produce in one minute over a billion molecules of fibrin," says Edgington.

Because of this efficiency, TF is effective in very small quantities. In fact, its concentration in tissues is estimated at only three parts per million or less. The search for the protein responsible for the function of TF was 50 years from first description to isolation and cloning.

"TF had been the missing element in the coagulation system," says Edgington. "On paper it had to exist, but nobody could isolate it."

In 1986, Edgington and associates were the first group to sequence TF and clone it after two years of dedicated effort. "We started in 1984, working full time, six to seven days a week," says Edgington.

They eventually succeeded in isolating the elusive TF molecule by reducing 500 fresh human placentas, which, taken together gave them enough protein to isolate the trace TF protein and with a new type of amino acid analyzer that he designed and built it was possible to determine the amino acid sequence of the minute amounts of TF that could be isolated.

In the years since, Edgington and Associate Professors Wolfram Ruf and Nigel Mackman have directed much of their efforts towards characterizing TF, its gene and its regulation, the protein's structure and mechanisms of action, and the complicated cascade of physiological reactions that TF directs in hemostasis, thrombosis, inflammation, certain immune reactions, and even in tumor biology.

Hitting the Target

In a paper published this month in the journal Cancer Research, Edgington and his colleagues report that they have found a way to deliver molecules such as TF to specifically target only those vessels that are supplying blood to tumors and leave the rest of the vasculature alone. To do this, they have employed a small part of a protein called vascular endothelial growth factor (VEGF), a growth factor that regulates the growth of new blood vessels.

Certain forms of VEGF have a particular stretch of amino acids, called the heparin binding domain, that when properly folded binds to a number of sugars decorating proteins on the surface of cells. And one of the sugars it binds to seems to be only on the surface of endothelial cells local to cancer tumors.

Edgington and his colleagues used a truncated 24-amino acid stretch of this heparin binding domain and showed that when injected into the blood stream it can find and anchor a viral phage particle to the blood vessels only of a tumor.

"This really shows that you can [use the truncated part of heparin binding domain to] deliver molecules or even particles selectively to the tumor vasculature and thus to a tumor," says Edgington.

Edgington attached the heparin binding domain to an additional copy of a phage gene that codes for a coat protein displayed on the surface of a phage particle—a virus that infects bacteria. Then he carefully controlled the number of this additional gene and its heparin binding element expressed on the surface of the phage so that of the 2,000-plus proteins on the surface of the phage only one to seven will have the heparin binding domain. This low copy number is important because Edgington wants to find a targeting molecule that could strongly anchor to the tumor vasculature.

In experiments described in the paper, Edgington and his colleagues injected the modified phage particles into an in vivo cancer model, a mouse with a large solid tumor. Normally the large phage particles will circulate through the bloodstream and their levels will drop as they are progressively cleared by the body. But if the phage binds tightly to some part of the body, like the cells lining tumor vasculature, then it will remain even after the rest of the phage is cleared from the bloodstream.

Edgington found that even a single molecule of heparin binding domain on the surface of the large phage particle will localize and anchor the phage. The concentration of phage in the tumor vessels increased as the levels of phage in the bloodstream dropped. By looking for those particular heparin–phage constructs that were present in the tumors when all the phage was washed out of the bloodstream, Edginton and his colleagues were able to identify the constructs that tightly bound to the tumors selectively. Then they could recover these phage particles for analysis.

"You can anchor the phage with only one copy [of heparin binding domain]," says Edgington, "but the protein must be specific for the tumor vasculature if you are to only recover it from tumor and no other tissues."

Edgington has several hopes for this technology. The ability of heparin binding domain to target tumors may be used as the basis of a diagnostic to image the tumor vasculature—a technology that could help surgeons see the exact size and locations of tumors that could then be surgically removed.

Also, the targeting potential of the heparin binding domain might be used to direct molecules like TF to the tumor vasculature, where they could block the flow of blood and kill tumor cells.

The beauty of Edgington's technique is that it targets those cells that line the vasculature, which means that any potential therapeutic that would be derived from it would have easy access to the targets. Unlike the tumor cells, which readily mutate to resist treatment, the endothelial cells are not prone to mutations and therefore represent a more stationary target common to all solid tumors independent of the type of cancer.



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A Tumor Targeting Molecule.