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标 题: CANCER RESEARCH: KILLING TUMOR CELLS WITH ANTHRAX-BASED IMMUNOTOXIN
发信站: The unknown SPACE (Sat Jan 18 17:06:32 2003) WWW-POST

The following is from
http://www.nidcr.nih.gov/news/inside%5Fscoop%5Fcancer%5Frsch.asp

CANCER RESEARCH: KILLING TUMOR CELLS WITH ANTHRAX-BASED IMMUNOTOXIN

As the news broke last fall of anthrax-tainted letters, some reporters
eventually spun the story in a completely different direction. They
highlighted early research studies in which scientists use modified anthrax
proteins to fight human disease. Among the work mentioned prominently has been
studies at NIDCR to produce a recombinant immunotoxin that specifically
targets cancer cells. The Inside Scoop recently sat down with the two
scientists leading this research project to discuss how their immunotoxin
works. They are: Stephen H. Leppla, Ph.D., a scientist in the Oral Infection
and Immunity Branch, who has spent most of his career studying anthrax; and
Thomas H. Bugge, Ph.D., a researcher in the Oral and Pharyngeal Cancer Branch,
who investigates the role of enzymes, called proteases, in degrading and
remodeling connective tissue.

Q: What is a recombinant immunotoxin?

A: Leppla: A recombinant immunotoxin is a bioengineered antibody that has a
toxin fused to its surface. The antibody typically has been pared down in the
laboratory, making it smaller and more capable of infiltrating tumors. What
remains intact is the portion of the antibody that recognizes a short,
signature amino-acid sequence - or antigen - displayed on a protein receptor
that arises from the cell membrane.

As many laboratories already have demonstrated, the modified antibody binds to
the protein receptor that matches its antigen-recognition site, then it is
internalized into the cytoplasm. Enzymes inside the cell process the antibody,
much like the stomach digests food, cleaving the toxin free in the process. At
this point, the toxin becomes biologically active, disrupts the cell, and
eventually kills it. Unlike chemotherapy drugs, which kill chemically and
often require millions of molecules to saturate an individual cell, toxins
inactivate enzymes, the so-called workhorses of the cell, making these poisons
far more potent. One or a few molecules of these toxins usually does the
trick.

Q: But your treatment is somewhat different. Correct?

A: Bugge: That's right. Our strategy builds on these same basic concepts, but
it does so with a twist. Instead of an antibody, we use modified versions of
two proteins found in Bacillus anthracis, the same bacterium that causes
anthrax. One is called protective antigen. It has been designed by nature to
bind to a specific protein receptor that is present on the surface of many
cells, including most tumor cells.

A: Leppla: The other protein is called lethal factor. In nature, as a part of
the anthrax bacterium, this protein can be deadly. But in engineering our
immunotoxin, we use only a portion of lethal factor, and we fuse it to a
portion of another well characterized toxin called Pseudomonas exotoxin A.
Like grenades with the pins still inside them, both toxins don't turn deadly
until they are cleaved on the surface of the tumor cell.

Q: Can these proteins possibly cause an anthrax infection?

A: Leppla: No, they can't. We start with a thoroughly disabled anthrax strain,
and protective antigen and lethal factor are just two of thousands of proteins
that this bacterium makes. By themselves, they are incapable of causing
anthrax. We are simply interested in exploiting the natural properties of
these proteins, in hopes of efficiently delivering a deadly toxin directly to
tumor cells and killing them.

Q: What are those properties?

A: Leppla: Well, there are two critical properties. As mentioned, the first is
the ability of protective antigen to bind to a receptor that is present on
most tumor cells. In short, we know that our immunotoxin will target tumor
cells. The second property is what scientists call, "activation by protein
cleavage." That is, when protective antigen binds to its receptor, it must be
cut into two parts by an enzyme already present on the surface of our cells to
become biologically active.

Q: Why would the manipulation of this process be advantageous?

A: Bugge: The answer is a mouthful: urokinase-type plasminogen activator
(uPA). Most cancer cells actively secrete uPA, suggesting that this protein is
important to the cancer process. Indeed, that turns out to be the case. uPA is
an important link in a longer chain of enzymatic events that allows these
abnormal cells to degrade connective tissue and invade other parts of the
body, a process known as tumor metastasis..

This brings us back to protein cleavage. Like protective antigen in anthrax,
uPA must be cleaved while bound to its protein receptor to become biologically
active. Given this similarity in these two dissimilar proteins, our idea was
to create an immunotoxin that uses protective antigen to bind to tumor cells,
then tricks the enzymatic machinery of these tumor cells into cleaving
protective antigen, and internalizing the deadly immunotoxin.

Q: How exactly do you trick the enzymatic machinery?

A: Leppla: We remove the usual cleavage site on protective antigen and replace
it in the laboratory with the cleavage site for uPA. So, that gives us an
immunotoxin that has the binding specificity of protective antigen, but it
relies on tumor-associated enzymes to gain entry into the cancer cell and
introduce the toxin.

Q: How far along are your studies?

A: Bugge: We are currently testing the drug against mouse and human tumors,
the latter having been implanted in mice. So far, we have tested lung
carcinoma, melanoma, and oral cancer. We have seen a good effect in all three
tumor types, and we will continue the testing over time in a variety of other
tumor types. So, there is certainly many years of work ahead before we, or
anyone who takes up this project, can test it in people.

Yet, we hope the immunotoxin will be effective against most types of human
tumors. One of the remarkable things about malignant tumors is that almost
all, even from very diverse origins, express uPA. In fact, it is very
difficult to find a human malignant tumor that does not produce this
particular enzyme.

Q: What are the next steps in developing this immunotoxin?

A: Bugge: We need to do dose escalation and safety tests in larger animals. We
also are at a very early stage in understanding how these proteins circulate
throughout the body, where in the body they circulate, the routes by which
they could be most advantageously administered - those types of fundamental
questions.

A: Leppla: It's also important to remember that many things that look very
good in cell culture or in mice turn out to have unexpected toxicities.
Another major concern in developing an immunotoxin using a bacterial protein
like protective antigen is it will induce antibodies. So, it might be that you
cannot use this treatment over a long course of therapy. At the same time, we
are giving large doses in mice, which are clearly effective, and it may be
that giving ten-fold less may be equally effective, meaning we might be able
to give a longer course before getting antibody production.

A: Bugge: That's true. The good news, though, is we have a very thoroughly
investigated mechanism of action, and we know with considerable certainty why
this drug is so efficient at killing cancer cells. This is probably the real
strong point of our work. We know, more or less precisely, what happens from
the point of injecting the immunotoxin into the body until the tumor cell is
dead.

Q: So, this approach would fall under the heading of molecular medicine?

A: Bugge: Exactly. This was a designer drug that was basically drawn on the
blackboard. We said let's put this sequence here and this one in here, and it
should work as an anti-cancer drug. And, lo and behold, it worked.

A: Leppla: It was really amazing. It was one of the few projects that I've
worked on in which, as Thomas said, you sit down to design from first
principles, from the basic research knowledge that we have, and, bingo, it
works precisely as we predicted it would. It's just amazing.

Q: Your early success, then, is really a confluence of all of the information
that has been gathered over the years in two separate fields of research-

A: Bugge: Absolutely. From my perspective, our work builds on about 30 years
of research in our field to find out which enzymes are preferentially
expressed on the surface of tumor cells, how these enzymes are activated, how
they are regulated, how they interact on the cell surface. This is all
classic, basic biological research that was conducted without any immediate
clinical application. From Steve's side, his work with protective antigen and
lethal factor represents many, many years of research into the basic biology
of anthrax. Without this merging of the disciplines, this project would have
never been possible.

Q: Some have wondered why scientists at the dental institute would study
anthrax. But doesn't the institute have a longstanding tradition of studying
bacteria?

A: Leppla: This is a very important point. Periodontal, or gum, disease is one
of the major dental problems around the world. So, this institute has long
supported research on the bacterial pathogenesis of periodontal disease.
Anthrax is a classically studied pathogen and has played a role in the
development of the concepts of pathogenic bacteriology. So, it is certainly an
appropriate model system to study in association with other bacterial
pathogens.


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