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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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