The Evolution of ONTAK

Dr. Jack Murphy, The Evolution of ONTAK
February 27, 2011Welcome to Yale Cancer Center Answers with doctors Francine
Foss and Lynn Wilson.  I am Bruce Barber.  Dr. Foss is a
Professor of Medical Oncology and Dermatology, specializing in the
treatment of lymphomas.  Dr. Wilson is a Professor of
Therapeutic Radiology and an expert in the use of radiation to
treat lung cancers and cutaneous lymphomas.  If you would like
to join the conversation, you can contact the doctors
directly.  The address is canceranswers@yale.edu and
the phone number is 1-888-234-4YCC.  This evening Francine
welcomes Dr. John Murphy.  Dr. Murphy is Professor of Medicine
and Chief of the Section of Molecular Medicine at Boston University
School of Medicine.  Dr. Murphy developed the first fusion
toxin protein approved for clinical use in 1999 for the treatment
of patients with T-cell lymphoma.  Here is Francine
Foss.Foss
 It is a tremendous pleasure to have Dr. Murphy here tonight to
talk with us.  It is not often that we actually get to talk to
a person who invented a whole new technology that is very important
for us in treating cancer patients.  Dr. Murphy, or Jack, I
will call you Jack, can you start off by explaining to us your work
in molecular medicine?  Can you explain what molecular
medicine is and how that term relates to the term that we often use
on this show, which is translational research?Murphy
 I would be happy to.  Molecular medicine is brings to the
field of medicine contemporary molecular genetics and
molecular biology.  In understanding the underlying pathways
that govern how cells grow and multiply.  It is through the
understanding of these pathways and systems that allow us greater
insight into cells that cause disease by understanding what the
molecular basis is for many diseases now.Foss
 Jack, you developed the first fusion toxin protein.  Can you
tell our audience what a fusion toxin is?Murphy
 A fusion toxin is really a fusion protein, and a fusion protein is
one molecular entity that is one protein molecule that might be
thought of as having two different parts.  In this instance,
part of the protein comes from a bacterial toxin and the other part
of the same protein that is assembled genetically comes from a
human source, and in the case of the drug Ontak, that human protein
is known as interleukin-2.  It is a single protein that is
part diphtheria toxin, part human interleukin-2.Foss
 So, you used genetic engineering to design this protein?Murphy
 That is correct.  We started out many many years ago with an
understanding of the basic biochemistry and mode of action of
diphtheria toxin and what was abundantly clear thirty years ago, is
that the first step in the intoxication process was when diphtheria
toxin binds to its unique receptor on the surface of eukaryotic
cells, or mammalian cells.  Recognizing that the first step in
the intoxication process was binding to the cell surface, many
years ago, we asked the fundamental question, could one take away
that portion of diphtheria toxin, which binds to its receptor,
replace it with a molecular protein that is known to bind to its
unique set of receptors, and in doing so bring the toxicity that is
intrinsic to the toxin molecule toward only those cells that
displayed the new targeted receptor? And the answer to that
question happily was yes.4:25 into mp3 file 
http://yalecancercenter.org/podcast/feb2711-cancer-answers-murphy.mp3Foss 
            
 That was really a ground-breaking concept at the time, to take a
molecule like diphtheria toxin and reengineer it effectively so
that you could use the toxin part of that molecule to kill, in this
case, cancer cells.Murphy
 That is correct, and while at the time, the work was considered to
be a ground breaking, it really stemmed from our basic
understanding of the science of diphtheria toxin as well as the
science of the interleukins, in this case interleukin-2.Foss
 You had spent a long time studying diphtheria toxin in the
lab.  Can you tell the audience a little bit about diphtheria
toxin?  I think we all are familiar with it because we get a
vaccine as children to prevent us from getting diphtheria, but I do
not think that most people really understand the toxin itself.Murphy
 Diphtheria toxin is a protein that can be thought of as divided up
into three different segments.  So, if you were to look at
your left hand and stick out your thumb, your thumb would be that
part of diphtheria toxin, which when delivered into the cell causes
inhibition of protein synthesis and actually eliminates or kills
that cell.  Your middle three fingers are that part of
diphtheria toxin that forms a core in the cell membrane allowing
your thumb to enter into the side of the cell.  And your
little finger, which is sticking out, represents the native
receptor binding domain.  So again, what we did many years ago
was, in effect, take away the little finger, and replace it
conceptually with a pen.  In this instance, the pen was
interleukin-2, so the fusion protein toxin, Ontak, then only goes
to cells with interleukin-2 receptors.  Again, the basic
science behind the technology is rooted in the understanding of the
basic biochemistry of the toxin itself.Foss
 There are plant toxins and animal toxins.  Are there are
other molecules or drugs that capitalize on this toxin
technology?Murphy
 There are several that are in development for the clinic. 
Very early on, there were three toxin molecules that were used in a
way to try to target cancer cells with a very high degree of
specificity and selectivity, and those three toxins were diphtheria
toxin, which has been the focus of our work, Pseudomonas exotoxin
A, another bacterial protein toxin and work with Pseudomonas
exotoxin A was largely centered in the laboratory of Dr. Ira Pastan
at the National Cancer Institute, and the third toxin that was used
was a plant toxin, Ricin, and the Ricin A chain was linked to
monoclonal antibodies to make what we know as immunotoxins, part
bacterial or plant toxin, part monoclonal antibody in the hopes,
again, to develop drugs and biologic drugs directed toward
particular forms of cancer.Foss
 So, there is a difference between an immunotoxin and a fusion
toxin.  Can you just go over that a little bit for us?Murphy
 An immunotoxin is really a conjugate, and by a conjugate I mean
that each of the components are purified separately and then they
are joined together chemically in the laboratory to make a8:28 into mp3 file 
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conjugate molecule, and that linkage is generally made through a
bond that is called a disulphide bond and one has to modify
chemically each component and then mix them together so the
disulphide bond will form between the two.  That would be an
immunotoxin because of the use of a monoclonal antibody for the
targeting aspect of the drug that is ultimately formed.  In
contrast to that, fusion protein toxins are designed in the
computer, assembled at the level of DNA, at the level of the gene,
and that gene is put into usually a bacteria such as E. coli. 
And E. coli then makes the fusion toxin rather than having to
assemble it outside of a living organism.  So E. coli makes a
recombinant protein that is the fusion protein toxin that then can
be purified and used for the development of a therapeutic.Foss
 Can you talk to our listeners a little bit about the whole concept
of giving this diphtheria toxin molecule to patients?  We know
that we're all immunized against diphtheria and that diphtheria is
obviously a bad thing.  So there is a little a bit of concern,
probably, on the part of some of our listeners in thinking about
the fact that we're actually using this and giving it to
patients.Murphy
 One has to step back for a moment and recognize that number one,
diphtheria was an important disease until the development of the
diphtheria vaccine, which is now given to virtually all children in
the United States except those who choose not to be immunized for
religious reasons.  So diphtheria as a disease was an
important disease up until the early 1940s into the beginning of
the 1950s when mass immunization began.  With the vaccine
against diphtheria, that disease has been largely eliminated within
the United States.  There are less than a handful, that is
less than 10 cases per year, and again most of the cases that are
seen are seen in individuals who for one reason or another have not
been immunized against the disease.  There is a vaccine
against diphtheria that is wonderfully effective.  What we
realized very early on was that the antibodies that are generated
upon vaccination that prevent the action of the drug, or the action
rather of diphtheria toxin itself, are antibodies that are directed
against the little finger that I talked about just a moment ago and
that is the native receptor binding domain, so neutralizing
antibodies will bind to that little finger, if you will, and block
the toxin from binding to its receptor, therefore neutralizing its
activity.  In the case of the fusion protein toxins, we have
taken away the little finger and we have replaced it with another
protein, usually a human protein, that is not recognized by the
human immune system in an avid way, that is, to generate antibodies
against it, and so as a result, anti-diphtheria toxin antibodies
that are present in serum do not bind to the IL-2 component and
therefore are unable to block the action of the fusion protein
toxin.  That allows us then to develop therapeutics that are
directed in a cell receptor specific way that can be used to treat
refractory human disease.Foss
 When you first came up with this concept to use diphtheria toxin
as a way of killing tumor cells, you picked the interleukin-2
receptor as one of your first targets.  Could you just go
through how you actually made that choice?Murphy
 The first fusion protein toxin that we assembled at the level of
the gene, we used a small peptide hormone called alpha melanocyte
stimulating hormone, as the targeting ligand.  When we put
that gene into E. coli, E. coli recognized sequences and destroyed
most of the protein that was made. 13:34 into mp3 file 
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We then went to the next largest molecule which was interleukin-2,
rather than being 13 immuno acids it was 133, that solved the
problem of proteolytic degradation.Foss
 That is a really interesting story and we are going to hear the
second half of that story when we come back after the medical
minute.  Please stay tuned to learn more about targeted
therapies for cancer from Dr. John Murphy.Foss
 Welcome back to Yale Cancer Center Answers.  This is Dr.
Francine Foss and I am joined today by my guest Dr. John
Murphy.  Today we are talking about fusion toxin therapy for
cancer.  Jack, we talked a little bit before the break about
the choice of the interleukin-2 receptor as your target for this
first fusion toxin that went into clinic.  Can you talk a
little bit about this whole process of bringing a molecule from
bench to bedside, what were the hurdles that you had to
overcome?Murphy
 It is an interesting question.  The hurdles were really quite
significant.  At the time that this early work was done it was
pioneering work and there were segments of the population who
believed it should never have been done in the first place. 
This was in the early days of genetic engineering and recombinant
DNA and so there was a lot of work that needed to be done in order
to convince the public, as well as the funding agencies that
supported the development of this work, that it was in fact
worthwhile to do.  The early experiments that were performed
were actually performed under so called, Biosafety Level 4
containment.  Remember, we were after all developing a brand
new toxin, a toxin that the world had never seen before through
recombinant DNA activity.  Because of that, and I think
prudently so, first experiments were done in maximum containment
laboratories which would ensure that there was no release of
organisms into the environment.  After 18 months of
investigation under maximum containment we then were able to
demonstrate conclusively that working with these molecules in the
laboratory strains of E coli did not, in fact, pose any threat to
either the environment or to people and as a result those
experiments were
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downgraded from Biosafety Level 4 to Biosafety Level 2, which is
the normal laboratory environment.Foss
 In your position at Boston University you were in the unique
position to actually be able to not only develop this molecule, but
actually see it go into clinical trials.  Can you talk to us a
little bit about that first clinical trial and how excited you were
to see your molecule actually being given to a patient?Murphy
 It was really quite humbling to be honest with you.  I
remember very well the first patient who was sent to our medical
center at Boston University from Massachusetts General
Hospital.  He was, at that time, a 53-year-old man with a five
year history of cutaneous T-cell lymphoma.  His disease
continued to progress despite many different kinds of intervention,
as you might imagine, of photopheresis, chemotherapy, and electron
beam therapy, to name just a few.  Because his disease
continued to progress, he was sent over to our medical center and
was one of the very first patients to be treated.  I remember
very well not only the excitement but also the anxiety of following
this first patient, not knowing what this new recombinant toxin
would do when given to a human.  The transformation and the
resolution of his disease was, in fact, remarkable.  After a
single injection once a day for five days he was sent home for a
21-day rest and when he came back his disease was already beginning
to resolve.  After his second course of therapy, he went
almost into complete clinical remission followed then by his third
and fourth courses of therapy where he achieved a complete clinical
response.  I followed this gentleman for the next 10 years and
he remained free of disease very happily playing golf in
Florida.Foss
 And that is a dramatic story.Murphy
 Very gratifying for the investigator.Foss
 In that early experience I understand you noticed that there was
some problems with the way the drugs was formulated in terms of the
aggregation of the drug in the solution and you went back to the
lab and you redesigned the drug.  Can you talk a little bit
about that?Murphy
 This is what we have done over the past several years now. 
One of the most concerning adverse effects that is associated with
the drug Ontak is the fact that some patients develop a vascular
leak syndrome upon being given the drug.  This can range from
very mild symptoms to life threatening pulmonary edema in these
patients.  So, we went back to the drawing board and
identified amino acid sequences within the drug itself that gave
rise to this vascular leak syndrome through again recombinant DNA
in site directed mutagenesis.  We have now been able to change
those amino acid sequences and we have what I would refer to as the
next generation of this first fusion protein toxin that we are
trying to bring into the clinic as we speak.  The hope is that
this new generation will be safer, will be better tolerated by
patients who receive it and best of all we will able to perhaps use
a little more of this drug in treating patients and see even better
outcomes in the future.21:48 into mp3 file 
http://yalecancercenter.org/podcast/feb2711-cancer-answers-murphy.mp3Foss             
 This vascular leak that we see with Ontak is not specific to this
particular molecule, it does occur with interleukin-2 as well.Murphy
 Yes, that is exactly right, and so there is a component that can
be ascribed to interleukin-2 and there is a component that can be
ascribed to the toxin component of it.  The amount of Ontak
that's given is generally much less than the amount of, for
example, recombinant interleukin-2 and so the interleukin-2
component of the vascular leak that is seen, I think is less then
that of the toxin component, and by changing those toxin sequences
we retain the same potency toward targeted cells and at least in
experimental animal we have seen markedly less adverse effects upon
administration of the new drug.Foss
 Just to let our audience know, we are talking about a very minimal
syndrome that we see in patients who develop some fluid retention,
some of those patients put on weight, many of these patients
tolerate that reasonably well with the existing drug, but what we
were looking toward is trying to improve that for the future to
significantly decrease some of these side effects.Murphy
 That is correct, and our hope is that if we are able to decrease
some of these side effects, this particular targeted agent could be
used in a variety of clinical circumstances, which continue to need
new advances for therapeutic intervention.Foss
 Jack, after you designed the first molecule, the Ontak molecule,
which was FDA approved back in 1999, have you thought about other
molecules that could be designed on this diphtheria toxin backbone
and where do you see that going?Murphy
 We have given a lot of thought to that.  There are two
molecules that we have been developing in the laboratory.  The
first is rather than using interleukin-2 as a targeting agent,
using interleukin-3 as a targeting agent.  And in this
instance, patients with refractory acute myelogenous leukemia might
be a population that could benefit from such an agent.  The
second generation, or the second molecule that we are working on in
addition to the IL-2 and the IL-3 molecules is using epidermal
growth factor as the targeting component.  And in this
instance, we envision that patient's with brain cancer,
particularly glioblastoma, might benefit from such an agent.Foss
 I understand there is also an expanded use of Ontak as a molecule
that targets the interleukin-2 receptors.  We have talked a
little bit about its use in cutaneous T-cell lymphoma, but there
are also potential uses in other diseases that are associated with
these activated T-cells and also perhaps in tumor immunity as
well.  Could you talk a little bit about that?Murphy
 Sure, the drug Ontak targets the high affinity IL-2 receptor on
the surface of cells.  We know that there are several types of
cells, T-cells, which have that receptor.  One type of T-cell
that displays these receptors are so called T regulatory cells and
T regulatory cells serve to, if you will, suppress an immune
response against a particular antigen.  It is the body's way
of maintaining a balance of the immune system.  In many
instances, solid tumors grow in patients because their own
immune26:12 into mp3 file 
http://yalecancercenter.org/podcast/feb2711-cancer-answers-murphy.mp3system no longer recognizes them as being foreign.  What
this means is that there are T suppressor cells preventing an
immune response against that solid tumor, thereby allowing it to
grow.  We have thought a great deal about using Ontak or this
next generation IL-2 receptor targeted drug that we're developing
to deplete or diminish the number of T regulatory cells with the
hope that doing so would then allow the host, that is the patient,
to develop their own immune response against a tumor, a solid
tumor. That approach coupled with active vaccination against that
tumor offers an exciting avenue of research for the future.Foss
 There has been some really exciting data recently looking at this
molecule in metastatic melanoma.  Can you talk a little bit
about that?Murphy
 There has been and the results that have been published are really
fueling our enthusiasm toward this approach.  A metastatic
melanoma, as you know, does not have the receptor for interleukin-2
on its surface and the use of Ontak then to eliminate or to deplete
T-regulatory cells has been shown to be a really quite encouraging
in that in early reports about 30% of the patient's that have been
treated have had a clinical response following administration of
Ontak through the development of their own antibodies against the
tumor and some of those patients have gone on to do extremely
well.Foss
 This story has come a very long way since that day in 1999 when
this was FDA approved.  My last question for you is if you
could just reflect on how you felt back then in 1999, where you
felt that this molecule was going and where it has gone today and
give us your thoughts about that.Murphy
 The development of this agent over the years has been slower in
fact than I would have hoped for.  The commercial entities
that took over the control initially were interested primarily in
using this agent to make revenues for their company, appropriately
so, but they did not seem to understand that drug development is an
iterative process.  One needs to look at results that are
generated in the clinic and go back to the laboratory, solve
problems that arise, and keep refining agents until one can have
the best possible agent to use clinically. Dr. John Murphy is Professor of Medicine and Chief of the
Section of Molecular Medicine at Boston University School of
Medicine.  Dr. Murphy developed the first fusion toxin protein
to be approved for clinical use in the treatment of patients with
T-cell lymphoma.  If you have questions or would like to share
your comments, visit yalecancercenter.org,
where you can also subscribe to our podcast and find written
transcripts of past programs.  I am Bruce Barber and you are
listening to the WNPR Health Forum on the Connecticut Public
Broadcasting Network.