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AMA Conference Backgrounder: Duke's Center for Genetic and Cellular Therapies

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Duke Health News 919-660-1306

He called it "The Cure." That's how confident senior
research analyst David Boczkowski was that he had discovered
how to get the immune system to attack cancer cells. One crisp
autumn day in the fall of 1995 he handed co-worker Smita Nair
the auspiciously labeled vial and told her to include this one
extra tube in her cancer vaccine experiments.

Amused by the label, Nair, an immunologist by training,
agreed to test the innocuous looking clear liquid with the
other compounds she was testing. The experiments were already
planned, and one more tube wouldn't be much more work.

Nair had done these experiments many times before. As a
member of Duke's Center for Genetic and Cellular Therapies,
Nair is part of an interdisciplinary team seeking new ways to
combat disease by combining laboratory science with clinical
medicine. The researchers want to develop entirely new ways of
treating diseases using the information gained from studying
how genes and proteins work on the cellular level.

This approach is very different from how medicines have been
discovered in the past. Doctors and researchers would typically
test thousands of chemical compounds hoping one would have a
therapeutic effect. It was a shotgun approach that sometimes
worked, but took many, many years of work.

"Basic scientists have long been adding to our knowledge of
how genes and cells operate and how, for example, cancer cells
are different from normal cells," says Eli Gilboa, center
director for basic research. "At the other end of the spectrum,
doctors have been waiting for more effective treatments for
cancer patients. For a long time, the people who evaluate
funding for research have neglected the middle. How do we
bridge basic research and clinically useful treatments? That's
where our center fits in. We are doing that important
translational research."

The key to bridging these two worlds – bench science and
patient treatment – is to keep an open mind, to look for
connections that might have been missed, and to find new ways
of seeing, Gilboa says.

So it wasn't much of a stretch for Nair to take a tube
labeled "The Cure" and add it to her planned experiments.
Besides, she had been struggling for months trying to locate
proteins found only in cancer cells that she could use to wake
up a slumbering immune system to the fact that cancer had
invaded the body.

It's a strategy that many scientists have tried before – but
with limited success. Cancer cells, it turns out, are very good
at hiding from the immune system's normal sentinels, those
cells whose job it is to find abnormal invaders like bacteria
and viruses and target them for destruction.

Gilboa had been tackling the problem himself for many years,
trying first to find ways to get the immune system to attack
HIV, the virus that causes AIDS, then turning to cancer.

He, Nair and a team of laboratory scientists were testing
proteins found only on cancer cells for their ability to serve
as antigens, activators that would trip a switch in the immune
system and allow it to rid the body of cancer cells. Nair
assumed that Boczkowski's "cure" was another protein
antigen.

She mixed the various antigens with dendritic cells, a rare
type of white blood cell whose function is to "present"
antigens to the immune system. In effect, the dendritic cell
serves as that sentinel, a Paul Revere sounding a trumpet that
the British are coming and everyone better take up arms or be
conquered.

Once the dendritic cells were primed with antigen, Nair
injected them into mice with cancerous tumors and looked to see
if the tumors stayed the same, shrank or metastasized,
spreading throughout the body.

To her amazement, the mice that had "The Cure" injected into
them were doing much better than any of her other experimental
animals. She immediately ran to Boczkowski and asked what was
in the tube. He told her it was not a protein, but RNA – the
genetic code that tells the cell which protein to make.

"No one had ever thought to use RNA as an antigen," says
Boczkowski, "because they thought it would be chewed up by the
cell. But I knew it would work." Boczkowski, who was trained as
an RNA biochemist, knew that RNA could work to prime the immune
system. It just took someone brave enough, or crazy enough, to
try.

The results were shocking, even to the Gilboa, the lab's
director.

"When Eli [Gilboa] looked at our results, he nearly fell out
of his chair," Nair says.

A Team Effort Pays Off It has now been three years since
"The Cure" vial was first tested, and the RNA cancer vaccine is
already being studied in cancer patients, a blindingly fast
rate of development compared to the usual research pace. Gilboa
attributes the speed of development to a tight camaraderie
among the research team he has assembled.

In 1993, when Gilboa left Sloan Kettering Center in New York
to create the center at Duke, he knew he had to work along
several fronts to make molecular and cellular medicine a
reality. But it wasn't easy to figure out how to do that,
explains Gilboa, because there was no precedent for it, no
model for how it should work.

Therefore, he adopted a straightforward philosophy: Put
together a team of innovative graduate and medical students,
physicians, and post-doctoral scientists -- smart people from
different disciplines all working together to move ideas from
the lab to the clinic. There are 60 people working at the
center. In addition to Gilboa, primary researchers are: Dr.
Clay Smith, a practicing physician and a specialist in
hematopoiesis, a fancy word for how the body creates new blood
cells.

Dr. H. Kim Lyerly, a surgeon who treats cancer patients,
does clinical research and runs a research lab that tests
promising new cancer therapies.

Bruce Sullenger, a PhD scientist who studied the properties
of RNA under Tom Cech, a Nobel-prize winning biochemist at the
University of Colorado.

Like Nair and Boczkowski, everyone on the team contributes a
unique perspective, a fresh vision that can help solve puzzles
that crop up when moving laboratory research to the
bedside.

Take the time Smith was analyzing the recipe for growing
mouse blood cells in the laboratory. This same recipe had been
tried in people by investigators at other institutions, and it
hadn't worked. Smith realized that the researchers had been
using cow serum to grow their cells. That was fine for mice,
but people's immune systems attacked the cow serum ingredients,
not the cancer cells they had hoped to target. So Smith
developed his own recipe using nutrients that would normally be
found in people. In retrospect it seems obvious to change to a
more human-friendly growth serum, but cow serum is a staple in
every laboratory that works with cell culture and no one had
given it a second thought.

"This is the kind of problem-solving that needs to be done
early in the development process," says Gilboa. "No one wants
to fund this kind of work. Yet, by carefully checking each step
along the way and making modifications as we move from animal
testing to people, we've saved time and moved things forward
more quickly."

After their initial discovery, Nair, Boczkowski and Gilboa
repeated their laboratory experiments with the RNA cancer
vaccine several times to be sure their results were real. They
published their work in the August 1996 issue of the Journal of
Experimental Medicine, showing the vaccine dramatically reduced
spread of lung cancer in mice and protected them from
developing new cancer. At that point Lyerly stepped in to help
bring the vaccine to his patients.

In early 1997, the Recombinant DNA Advisory Committee of the
National Institutes of Health and the federal Food and Drug
Administration both gave their approval for the RNA cancer
vaccine clinical trial to begin at Duke. The trial, involving
18 patients with breast, lung, or colorectal cancers, was the
first to use RNA as an antigen against cancer.

To make the vaccine, Lyerly and Smith devised a way to
produce mass quantities of dendritic cells from a small patient
sample as well as the RNA that contained instructions for the
dendritic cells to make the common tumor antigen, CEA
(carcinoembryonic antigen). Mixed together, the tumor RNA
produces everything the immune system needs to launch an attack
on the cancer.

"Traditionally, we have believed that tumor cells stimulated
killer T cells directly, but now we understand that the
dendritic cell is the vital intermediary player," Lyerly
said.

The theory is that cancer may spread from an initial tumor
because the immune system is not given a strong enough signal
to destroy it. In some cases, cancers are even able to turn off
the immune response.

But the Duke scientists think it is possible to override the
ability of cancer to spread. They have devised a way to
separate dendritic cells from a mouse (or a human's) blood or
bone marrow and grow them in large quantities in the
laboratory. They can then be shown bits of the antigen and
re-infused into the patient to "present" or announce this
antigen to the immune system.

Moreover, the researchers hope that the use of RNA may
eliminate the problem of having to tailor a vaccine for each
individual patient because of their specific immunity
"fingerprint," Lyerly said, referring to previous efforts to
make gene-modified tumor vaccines. Not only is that process
laborious, but many patients in remission do not have enough
"tumor load" from which to extract cancer cells.

Progress for Patients

The first phase of the trial has shown that the vaccine is
not only safe but can produce immune responses in cancer
patients. Under the leadership of Lyerly, the center will be
making vaccines for up to 100 additional patients to be
enrolled in the second phase of a clinical trial, testing the
vaccine for breast, lung and colorectal cancers.

Mass quantities of the vaccine will be produced for each
patient using a high-tech, industrial-quality, cell processing
facility financed in a joint venture between Duke and
Rhone-Poulenc Rorer, a French pharmaceutical company.

"Having a facility of this caliber associated with Duke
means that we can actually conduct clinical trials of
experimental cellular vaccines that would not normally be
possible in an academic setting," Lyerly says. "It will allow
us to prove and refine our ideas to the point they are ready
for large-scale national testing."

The team already has tailored the concept that led to that
first clinical trial. Some patients don't exhibit CEA antigens
in their tumors, so the researchers hit upon a new idea –
inject all the RNA from tumor cells into dendritic cells. That
way, each patient's tumors produce a unique set of antigens
that will induce potent immune responses to eradicate their
cancer, according to Nair. Preliminary results from this
research indicate that total RNA induced an even more potent
immune response than the CEA antigen alone.

And that's just the beginning, Lyerly says. "In the future,
new therapies will be tackling complex diseases that are in
many ways intertwined with specific levels of gene products and
proteins. We are going to have to be cognizant of giving the
right medicine at the right time and in the right dose. All
this is going to require intensive cycles of experimentation
and clinical application that are rapid and tied tightly
together. That's what we are working toward in all our
translational research."

Looking toward that goal, Lyerly has designed six cancer
vaccine trials that are variations of the dendritic cell
therapy. In addition to the CEA antigen trial, the trials
underway are:

The total tumor RNA-dendritic cell trial. Doctors extract
RNA from colon, breast, lung, or ovarian tumor cells that have
been removed by surgery. The total tumor RNA is mixed with
dendritic cells and re-infused over four sessions, every two
weeks. This trial will enroll up to 18 patients.

Patients with aggressive breast cancer who have had
radiation, chemotherapy and have responded to a bone marrow
transplant can participate in one of two trials depending on
their white blood cell antigen type. Up to 43 patients receive
one infusion per month for six months.

Patients who have had pancreatic cancer that has been
surgically removed and have no evidence of disease, if their
tumors express the CEA antigen, are eligible for dendritic cell
therapy. Up to 24 patients receive one infusion per month for
six months.

Patients who have had colon cancer that has spread to their
liver may be eligible for dendritic cell therapy, if after
surgery to remove the tumor from their liver, no additional
disease is found. Up to 18 patients receive four infusions,
every two weeks.

A new trial for breast, ovarian, prostate and lung cancer
patients whose tumors make a protein receptor called Her2/neu,
which has been shown to be a key to spreading cancer. Studies
have now shown that blocking the Her2/neu protein can slow down
or stop cancer. This trial will use dendritic cells and the
Her2/neu protein antigen to try to spur the immune system to
block the Her2/neu protein in 18 patients.

The Mother of Immune Cells As scientific director of Duke's
bone marrow transplant program, Clay Smith's job is to
manipulate stem cells, those rare and rich factories that
produce specialized immune system fighters. His self-imposed
mandate is to learn how to get stem cells to work right to help
correct disease.

It's a problem that has defied biomedical science. The
problem is that as soon as stem cells are extracted from the
usual sources -- bone marrow or adult blood -- they are already
changing into the next generation. But he has found that stem
cells taken from the blood of an umbilical cord seem to behave
differently, and now he is working with medical center
physicians Drs. Nelson Chao and Joanne Kurtzberg to expand the
number of stem cells that can be available from an infant's
cord blood. "The quest is to have a resource of stem cells that
can grow and divide but that are still stem cells," he
says.

Smith wants to then reprogram those stem cells to attack
disease, be it cancer, HIV or sickle cell, to name just a few.
In cancer, for example, the idea is that a patient would
receive high doses of chemotherapy or radiation to destroy his
or her cancer, and then would receive a bone marrow transplant
to restore the immune system. Along with the BMT, stem cells
would be engineered to recognize and eliminate any last
vestiges of cancer cells.

"A big problem with bone marrow transplantation is relapse
from the few cancer cells left in a person after treatment,"
Smith says. "The concept here is that as the immune system
comes back, it is directed to attack whatever cancer remains."
The RNA connection RNA isn't just getting attention as a cancer
treatment. It also is part of a unique project that combines
Sullenger's expertise with a special kind of RNA, and Smith's
experience and understanding of the body's blood-making
factory, the bone marrow.

Several years ago Sullenger came up with an idea to use a
type of RNA called catalytic RNA or ribozyme as a therapy to
modify genetic messages in the body. It is an entirely new type
of gene therapy, one that fits right in with the center's
philosophy of inventing new ways to treat disease.

In contrast to traditional gene therapy schemes, which try
to replace defective genes in the genome, Sullenger's ribozymes
correct defective RNA, the message copied from DNA. Many
genetic diseases are caused by defective genes that are made
into defective protein. Simply adding back a normal gene to
cells doesn't decrease the amount of the "bad" gene product,
because the defective copy would still be present and the added
gene would not be placed under the cell's precise regulatory
scheme.

Sullenger used bacteria to prove that his idea worked and
then set out to find a disease to try the new therapy on. He
didn't have to look very far. Right across the hall, Smith was
experimenting with modifying the bone marrow of patients with
sickle cell anemia, an inherited disease that is most common
among people whose ancestors come from Africa and the Middle
East. The red blood cells of people with sickle cell disease
contain an abnormal type of hemoglobin, the molecule that
carries oxygen throughout the body. The defect is caused by a
single tiny change in the protein portion of the hemoglobin
molecule. This single mutation distorts the red blood cells
into a sickle shape, making them fragile and easily destroyed,
leading to anemia.

Other scientists have shied away from tackling gene therapy
for sickle cell anemia because the cell's production of beta
globin is precisely regulated.

Sullenger, Smith and their colleagues immediately saw that
sickle cell anemia would be a perfect challenge for the new
ribozyme therapy because there is now no effective treatment
for the underlying sickle cell genetic defect and because the
gene is under complex genetic controls that make it unsuitable
for other types of gene replacement therapy.

"The fundamental role of RNA molecules in cells is to help
manage the use of genetic information," according to Sullenger.
"We believe the correcting RNA is the most promising strategy
for correcting many types of genetic defects."

Since Smith treats sickle cell patients, there was a
built-in source of patient blood to try their RNA therapy.
Smith collected the blood from both sickle cell patients and
from umbilical cord blood, the afterbirth of infants. From this
blood they isolated precursor red blood cells -- the cells that
produce mature red blood cells. They added ribozyme molecules
that carried the corrected globin genetic sequence into the
cells using slippery, fatty spheres called liposomes. Once
inside the cell, the ribozymes located the faulty RNA by
matching up letters of the genetic code to the defective globin
RNA. They then snipped off the defective piece and added in the
corrected sequence.

When the scientists broke apart the cells and read the
genetic sequence of the globin RNA, in each case the ribozyme
had spliced in the new sequence correctly.

Next, the researchers will test the procedure in an animal
model of sickle cell disease to see if the corrected globin
gene can prevent development of sickling. If this procedure
works, Sullenger and his colleagues would like to begin testing
the ribozyme therapy in sickle cell patients within two to
three years. Given their track record with cancer vaccines,
they are right on target.

The most likely candidates to test the ribozyme therapy
would be sickle cell patients who have developed a rare
condition called alloimmunization. Patients with severe sickle
cell disease often require blood transfusions to replenish
their supply of healthy red blood cells. Some of these people
develop antibodies to elements in transfused blood, making
further transfusions dangerous to the patient. Sullenger
envisions taking some of a patient's own blood, correcting the
sickle cell trait, and giving it back in a transfusion. Since
it is a patient's own blood, he or she would not develop
antibodies against it.

In addition, promising experiments Smith is conducting with
umbilical cord blood suggest this unique source of blood stem
cells may offer a permanent treatment for sickle cell
disease.

"If we could find a good method for integrating the
instructions for making the ribozyme into the blood stem cells,
that would be a permanent source of good, working copies of the
globin gene that could at a minimum lessen the symptoms of the
disease," Sullenger says. "We know that sickle cell patients
who make 10 to 20 percent fetal globin do much better. If we
could reach that level or better, we could perhaps eliminate
the most serious manifestations of the disease."

Sullenger also is working on a strategy to use ribozymes,
not to fix defective genetic messages, but to alter viral
messages. The idea is to use ribozymes to change the meaning of
HIV's messages to encode an antiviral agent, which would kill
the virus when it tries to multiply inside cells. This unique
strategy offers the advantage of turning HIV's own genetic
messages against itself. Thus, non-infected cells would not be
affected by the ribozymes.

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