Entremed: Starving Cancer Into Submission
by William Wells
(Posted May 1, 1998 ú Issue 29)
Abstract
Cells that line blood vessels make a great anticancer
target for two reasons: they are vital to the survival
of cancer cells, but their genetic stability makes it
unlikely that they will mutate to drug resistance.
Cut off a tumor's blood
supply - its source of
nutrients and growth
factors - and the tumor should perish. EntreMed, Inc.,
of Rockville, Maryland, is one of many companies
trying to put this simple theory of anti-angiogenesis into
practice. By licensing anti-angiogenic molecules
discovered by Judah Folkman (Children's Hospital,
Boston), EntreMed has acquired perhaps the hottest
intellectual property in cancer research today, and
become the consummate middleman. The company does
the preclinical grunt work for which Folkman does not
have the time or resources, and provides the big
pharmaceutical companies with drug candidates they
cannot resist.
Twenty-five Years in the Wilderness
Attacking angiogenesis, the process by which blood
vessels sprout and invade new areas, was not always
so popular. "When Folkman first proposed attacking
cancer by inhibiting angiogenesis he was quite alone,"
says Robert Auerbach (University of Madison at
Wisconsin). "He recognized the importance of it and
made it a real research goal. In that sense he's a real
pioneer."
Folkman first put forth his
thesis in a 1971 article in
the New England Journal
of Medicine. If
angiogenesis is not switched on, said Folkman, a tumor
will grow to be only the size of a pea. This state is
maintained by balanced proliferation and cell death,
until a combination of hypoxia, inactivation of tumor
suppressor genes, and activation of tumor promoter
genes results in the production of angiogenic molecules
(candidates include basic fibroblast growth factor
(bFGF) and vascular endothelial growth factor
(VEGF)).
Folkman went on to provide evidence for the
importance of angiogenesis in tumor growth. But what
he needed was a potent inhibitor - both to prove his
theory and as a possible cancer cure. He characterized
numerous inhibitors, including one from cartilage, one
from the vitreous area of the eye, protamine, and
various steroids.
The most promising
early compound was a
rediscovered natural
product called
fumagillin (table 1). But
the first of Folkman's
potential drugs to attract the attention of EntreMed
came from a database. Folkman and postdoctoral
fellow Robert D'Amato thought about the possible side
effects of an anti-angiogenic drug and came up with
two: problems with menstruation and birth defects
(limb development may depend in part on the pathways
laid down by blood vessels). Only six drugs in their
database had both side effects; the standout was
thalidomide.
Thalidomide was first used in Germany in 1957 as a
sedative for mothers with morning sickness. In the next
four years it was used in over 40 countries, although it
was rejected in the United States in 1961 by the Food
and Drug Administration (FDA) because it also causes
mild peripheral neuropathy. In that same year other
countries began banning its use, but not before ~12,000
babies were born with deformations including
flipper-like arms and stumps for legs.
In the same year that he
reported on thalidomide's
activity, Folkman found
that 2-methoxyestradiol
and a protein called
angiostatin were anti-angiogenic. Also in 1994, David
Cheresh of Scripps Research Institute in La Jolla,
California, found that integrin alphavbeta3 was needed
to mediate a survival signal for endothelial cells
(which line blood vessels). Antagonists to alphavbeta3
caused tumor regression in vivo, and are now in
clinical trials ((table 1)).
Postdoctoral fellow Michael O'Reilly
waded through over 40 liters of mouse
urine to purify a few micrograms of
angiostatin, using the strategy depicted in
figure 1. Surgeons had known for many
years that removal of a primary tumor
can cause secondary metastases to suddenly vascularize
and enter a growth spurt. Folkman hypothesized that the
primary tumor was producing a circulating
anti-angiogenic factor, which would be found in the
serum, or urine, of mice with primary tumors. The
primary tumor might overcome the effect of the
inhibitor(s) by making locally active angiogenic
factors, which the metastases cannot yet produce.
Folkman's latest and most exciting inhibitor, endostatin,
was purified from an endothelial tumor cell line. Both
protein inhibitors are fragments of larger proteins:
angiostatin is derived from the blood-clotting protein
plasminogen, and endostatin from collagen XVIII, a
component of the extracellular matrix.
A Company for Hire
EntreMed is not called
AngioMed because in 1991, the
year it was founded, it was open
to any good concept. "The basis
for the company was that we would foster the growth of
promising technology discovered at academic
institutions," says John Holaday, chief executive
officer. Since 1994, the projects that EntreMed first
licensed have taken a back seat to angiogenesis,
although a cell permeation technology may enter phase I
trials in the next year. EntreMed researchers will
electroporate inositol hexaphosphate (IHP) into red
blood cells so that the IHP binds hemoglobin. The
altered hemoglobin can release three of its oxygen
molecules (rather than just one) when it reaches an area
low in oxygen, such as heart blood vessels during an
attack of angina.
The breakthrough for EntreMed came in October 1993,
when the company signed a contract with Folkman and
Children's Hospital for rights to Folkman's work in
return for royalties and $2 million per year of research
funding. At this stage Folkman had good evidence for
thalidomide's efficacy in mice, and was close to
isolating angiostatin. Hoffmann-La Roche had decided
against a collaboration with Folkman, but meanwhile
D'Amato (who did the thalidomide work) had been
talking to Holaday, his mentor from an earlier time at
Walter Reed Army Institute of Research in Washington,
D.C. "Life is a process of personal relationships, " says
Holaday, and this personal relationship gave his
company a focus.
A cash infusion came in late 1995
from Bristol-Myers Squibb, when
EntreMed had only 60 days of
money in hand. Thalidomide trials
sponsored by the two companies and the National
Cancer Institute began in April 1996, and on the
strength of this work EntreMed went public.
Interim results of the phase II thalidomide trials are
promising: a 50% response rate for glioblastomas,
gliomas, and Kaposi's sarcoma. Phase II trials of
thalidomide for age-related macular degeneration are
also underway, as this form of adult-onset blindness is
characterized by excessive blood vessel growth in the
retina. If thalidomide is effective, its approval process
may be reasonably straightforward. Stringent safety
guidelines have already been worked out by Celgene
Corporation (Warren, New Jersey), which is awaiting
final FDA approval of thalidomide for the treatment of
leprosy sores.
Bring on the Big Guns
Thalidomide may, however, be merely a warm-up for
the protein inhibitors angiostatin and endostatin.
EntreMed has now produced active human proteins
from cultures of the yeast Pichia pastoris, and trials
may begin in 1999.
The most spectacular
preclinical data have come
from endostatin. In the
November 27, 1997 issue of
Nature, Folkman reported that endostatin could
repeatedly shrink tumors. Once the tumors shrank,
Folkman stopped endostatin treatment, restarting only
when the tumors had regrown to 1-2% of total body
mass. Each round of shrinkage occurred at the same
rate, suggesting that the genetically stable endothelial
cells do not become drug resistant. After 2 to 6
repetitions, depending on the cancer type, the cancers
failed to grow back at all.
"We were pleasantly surprised [by these results]," says
Edward Gubish, EntreMed's vice president for
research. The high rate of endothelial cell turnover in
tumors may explain why endostatin can shrink tumors
rather than just halt their growth. "It could be the
Hayflick experiment in vivo," says Craig Crews of
Yale University."The endothelial cells may have
senesced. " Alternatively, regrowth of new cells may
be halted by increasing amounts of inhibitor, which may
accumulate in the matrix as the endothelial cells
remodel their surroundings.
EntreMed's weakness is that
it doesn't know the precise
mechanism of action for any
of its three leading drug
candidates, which makes the
FDA approval process and the design of analogs more
challenging. It also has many competitors, although
some of the drugs proposed by these companies suffer
from a lack of specificity (the broad spectrum matrix
metalloproteinase inhibitors) or the possibility of drug
resistance (tumors may make alternative angiogenic
molecules when others such as VEGF are blocked).
If Folkman's proteins can make the leap from mice to
humans his early vision will find ultimate vindication.
"Folkman was fighting against a whole line of thinking:
that you have to kill all the cancer cells directly," says
Cheresh. Instead, he proposed that killing the support
cells would work. "I think he is right," says Cheresh. "I
think it is possible."
William Wells, Ph.D., is a scientific
journalist with Biotext, Ltd. in San
Francisco.
The above illustration is from EntreMed's
Anti-angiogensis page.
Send us your comments and ideas for future articles.
Endlinks
Tumor Angiogenesis in Splendid Isolation - Meeting
Brief of the Thirteenth International Symposium on
Cellular Endocrinology, September 11-14, 1997.
Angiogenesis Factors in Cancer, AIDS, and Diabetes -
a scientific review of angiogenesis, with illustrations
and bibliography.
Special Project Angiogenesis - describes research on
angiogenesis, with extensive graphics and links.
Anti-Angiogenesis Agents - featuring research
summaries with a clinical focus and extensive
references, it is one of the featured topics in Ovarian
Cancer Research Notebook, a comprehensive source of
treatment information for advanced ovarian cancer.
Fighting Cancer by Attacking its Blood Supply -
Scientific American article by Judah Folkman,
September 1996.
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