FOOD AND DRUG ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
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BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE
MEETING #35 - TELECONFERENCE
OPEN SESSION
Monday, June 9, 2003
1:30 p.m.
National Institutes of Health
Building 29A, Room 1A09
Rockville, Maryland
ATTENDANCE
Gail Dapolito, Executive Secretary
Rosanna L. Harvey, Committee Management Specialist
Committee Members:
Mahendra Rao, M.D., Ph.D., Chair
Jonathan Allan, D.V.M.
Bruce R. Blazar, M.D.
David Harlan, M.D.
Katherine High, M.D.
Joanne Kurtzberg, M.D.
Richard C. Mulligan, Ph.D.
Anastasios A. Tsiatis, Ph.D.
FDA Participants:
Steven Bauer, Ph.D.
Kathryn M. Carbone, M.D.
Suzanne Epstein, Ph.D.
William Freas, Advisory Committee Office
Deborah Hursh, Ph.D.
Malcolm Moos, M.D., Ph.D.
Philip Noguchi, M.D.
Raj K. Puri, M.D., Ph.D.
A G E N D A
Welcome and Introductions
Mahendra Rao, M.D.,
Ph.D., Acting Chair 3
Conflict of Interest Meeting Statement
Gail Dapolito, Executive
Secretary 7
FDA Introductory Remarks
Philip Noguchi, M.D. 8
Laboratory Presentations
Laboratory of Stem Cell Biology
Steven Bauer, Ph.D. 9
Laboratory of Immunology and Developmental Biology
Deborah Hursh, Ph.D. 18
Malcolm Moos, M.D.,
Ph.D. 27
Suzanne Epstein, Ph.D. 31
Open Public Hearing 37
Adjourn 40
P R O C E E D
I N G S
DR.
RAO: Hi to everyone. And everyone is here, except Dr. Harlan,
right, who needs to be away for a couple of minutes?
MS.
DAPOLITO: Except I'm not sure if Dr.
Mulligan and Dr. Blazar have come on yet.
DR.
RAO: I don't think we really need
introductions, right? But perhaps we
may want to just briefly go around and just make sure we know who's there.
MS.
DAPOLITO: On our end?
DR.
RAO: Yes.
MS.
DAPOLITO: Absolutely. I'll start with me. Gail Dapolito, the executive secretary. We'll go around our table here.
DR.
EPSTEIN: Okay. Suzanne Epstein, Division of Cellular and
Gene Therapies.
DR.
NOGUCHI: Phil Noguchi, Office of
Cellular, Tissue, and Gene Therapies.
DR.
PURI: Raj Puri, Division of Cellular
and Gene Therapies.
DR.
CARBONE: Kathy Carbone, Office of
Director.
MR.
FREAS: Bill Freas from the Advisory
Committee Office.
DR.
BAUER: Steve Bauer, Division of Cell
and Gene Therapies.
DR.
MOOS: Malcolm Moos, Cellular and Gene
Therapies.
DR.
HURSH: Deb Hursh, Cellular and Gene
Therapies.
MS.
HARVEY: Rosanna Harvey, BRM Advisory
Committee.
MS.
DAPOLITO: Can everyone hear us okay on
this end? Okay. And we have one member of the public, sort
of, a guest student from our Office of Special Health Issues here today.
MR.
FREAS: Can we do a roll call of who's
on the line?
MS.
DAPOLITO: Yes, Dr. Rao will do
that. Okay, Dr. Rao, do you want to
call the roll anyway?
DR.
RAO: I'll do that.
MS.
DAPOLITO: Okay.
DR.
RAO: Maybe the easiest way will be to
just do it in turn. Does everybody have
the BRMAC participant sheet in front of them?
Well, then why don't I call out that list, and then we can just go down
that as a roll call?
So
I'm here. I'm Mahendra Rao, and I'll be
acting as the chair for this meeting.
Is Dr. Allan there?
DR.
ALLAN: Yes, I am.
DR.
RAO: Dr. Blazar?
MS.
DAPOLITO: I think he's not on the line.
DR.
RAO: Dr. High? I think I heard her sign on.
DR.
HIGH: Yes. I'm here.
DR.
RAO: Okay. Dr. Kurtzberg?
DR.
KURTZBERG: I'm here.
DR.
RAO: Alison Lawton?
MS.
DAPOLITO: No, Alison won't be joining
us.
DR.
RAO: Dr. Mulligan?
MS.
DAPOLITO: He's not on yet.
DR.
RAO: Dr. Tsiatis?
DR.
TSIATIS: Yes, I'm here.
DR.
RAO: Dr. Harlan?
DR.
HARLAN: I'm here.
DR.
RAO: And is the consumer rep, Alice
Wolfson, here?
MS.
DAPOLITO: No, she won't be joining us.
DR.
RAO: I guess we have a roll, Gail?
MS.
DAPOLITO: Okay, thanks.
DR.
RAO: You need to make a statement?
MS.
DAPOLITO: Yes, I'll read the conflict
of interest meeting statement. The following
announcement addresses conflict of interest issues associated with this meeting
of the Biological Response Modifiers Advisory Committee on June 9, 2003.
Based
on the agenda made available, it has been determined that the committee
discussions present no potential for a conflict of interest. In the event that the discussions involve
specific products or firms not on the agenda for which members have a financial
interest, the members are aware of the need to exclude themselves from the
discussion. Their exclusion will be
noted for the public record.
With
respect to all other meeting participants, we ask in the interest of fairness
that you address any current or previous financial involvement with any firm
whose products you wish to comment upon.
I
also have a couple of just administrative items. The teleconference is being recorded by a transcriber. So if the committee members would identify
yourselves before you make a comment, that would be a great help.
It
doesn't look like we have had any requests to address the committee during the
open public hearing. So we won't use
that portion of the meeting, unless someone walks in between now and then from
outside. So we'll go directly to the
closed session after we hear from the speakers--the FDA speakers.
We'll
just take a minute, though, in between to clear the room on our end before the
committee goes into the closed session.
And then we'll turn it back over to Dr. High and Dr. Rao and Dr. Harlan.
Thanks,
Dr. Rao. I'm done, Dr. Rao.
DR.
RAO: Did Dr. Noguchi need to make any
introductory statement or any remarks?
DR.
NOGUCHI: Yes, I'll just take a minute
here. You can tell Malcolm and I seem
to have the same hoarseness. But this
Office of Cellular, Tissue, and Gene Therapies has just been formed officially
in October of 2002. And actually, the
site visit that we're going to be reviewing was the first official site visit
of the office of the division under that as part of the new office.
I
just want to, first, thank all the members of the site committee team, who
actually put together the report, spent a lot of time and effort with this, and
really would be pleased to now present the--in brief the scientists who were
reviewed at that time.
So
I really don't want to take very much more time than that because there's a lot
to cover. And it's not really our show,
it's the scientists' show that we're reviewing. Thank you very much.
DR.
RAO: Do we want Dr. Bauer to go first?
DR.
NOGUCHI: Yes, that would be fine. He's--
DR.
BAUER: I'm here, and I'm ready. Shall I go ahead? Okay.
I
have been acting lab chief of the newly--well, a new position for me, the
Laboratory of Stem Cell Biology, since October 2002. And prior to that time, I was a member of the Laboratory of
Immunology and Developmental Biology.
So most of the time covered by the site visit, I was a member of that
laboratory.
In
addition to the research program report, the site visit package that I think
you folks have a copy of contains a summary of my lab resources, including
staff, budget, other funding, and other CBER research support
infrastructure. There is also a little
bit of information on my role in regulatory activities, including review of
INDs in the area of gene therapy, cellular therapy, xenotransplantation products,
as well as development and presentation of policy in these areas, and public
outreach at scientific and regulatory meetings.
And
finally, there's a list showing--or a diagram showing where the lab is in the
Division of Cell and Gene Therapies and a list of other personnel in that lab.
My
research program examines cell-cell interactions that govern development of
lymphoid lineage cells and play a role in transformation of these cells. And I'm going to briefly describe two
projects that investigate these two types of cell-cell interactions.
The
first project is on the role of a molecule called dlk in stroma pre-B
interactions. One of the challenges
facing the field of stem cell biology is to determine what molecules and
signals are exchanged between self-renewing stem cells and other cells in their
microenvironment in which the stem cells proliferate and differentiate.
And
to study this process, we use a model system consisting of normal,
self-renewing, precursor B cells that grow on a stromal layer of mesenchymal
origin. And in the presence of the
growth factor, IL-7, these precursor B cells self-renew and remain
undifferentiated.
However,
you can get them to differentiate by removing IL-7, at which point the
precursor B cells turn into mature B cells and then undergo apoptosis. The precursor B cells can be administered to
immunodeficient mice and can reconstitute part of the B-cell lineage. So this model mimics the clinical use of a
variety of stem cells in which there is in vitro propagation before
administration.
Now
our previous work has shown that this molecule I mentioned earlier, dlk, is
expressed on stromal cells and that it influences the response of pre-B cells
to IL-7. And the key observation was
that downregulation of dlk on stroma allows precursor B cells to self-renew
without IL-7 and to forgo differentiation and apoptosis normally caused by
removal of IL-7. And that observation
has led to a lot of subsequent work in my laboratory, both in in vitro systems
and in a knockout animal.
What
we have seen is that adaptation of pre-B cells on these stroma with low amounts
of dlk on their cell surface involves loss of the requirement for IL-7. And to summarize a lot of work, the major
finding we've seen to explain that is that there is a change in Notch-3
receptor expression on the pre-B cells under these conditions. So, basically, it increases.
We've
also further examined the role of dlk using a knockout mouse model. So dlk has been knocked out, and we're
characterizing lymphoid cell populations in those cells and studying functional
perturbations, including differences in antibody responses. And we're also deriving knockout--or a
variety of cell lines from the knockout animals to further characterize the
role of dlk in those cell lines.
Overall,
the knockout mice have some developmental defects, including growth
retardation, fatty change in the liver or liver steatosis, obesity, and they do
have some perturbations in B-cell development, which mimics our results from
the in vitro culture system.
The
B-cell changes that we've seen include the following. There are increased B cells in the bone marrow. There are immature B cells in the spleen. There are increases in marginal zone B
cells. And functionally, there are
changes in serum immunoglobulin G levels, and there is a decreased secondary
antibody response to T-dependent antigens.
So both development and function of B cells have been changed in this
knockout animal.
So
in the future we continue to look at the role of dlk and how it might affect
the Notch pathway in a variety of cells through the use of microarray analysis,
RT-PCR, et cetera. And we intend to
develop more stem cells from these knockout mice to look at potential roles for
dlk and its modulation of Notch signaling in a variety of different important
tissue types that could be of clinical significance, including bone marrow
derived, multi-potent adult progenitor cells, hematopoietic stem cells,
neuronal stem cells, pancreatic stem cells, and perhaps others as well.
And
of course, using the knockout animals, we will continue to characterize the
lymphoid lineage cell function and subpopulations in the knockout animals.
And
finally, we can use these mice to cross to other interesting knockout strains,
such as IL-7 knockout, or Notch knockout, or knockouts of other members of the
Notch/Delta family that might represent a sensitized genetic background that
can further elucidate the role of dlk in those systems.
Now
I'll go on to a second project, which was to look at the effects of TGF-beta on
precursor B cells and its role in pre-B cell leukemagenesis.
DR.
RAO: Steve, can I interrupt you for a
second here?
DR.
BAUER: Absolutely.
DR.
RAO: I just wanted to poll the
committee and ask them if they'd like to ask questions on this project before
Steve moves on to a second because, given we're doing this by phone, it may be
hard to keep track of all of these things.
Does
anybody on the committee have any questions?
Maybe they can identify themselves first and then--
[No
response.]
DR.
RAO: No one? I have one quick question, Steve, and that's have you considered
deriving ES cell lines from this in occult mice, studying one cell line? I mean, studying somatic stem cells, you can
also study ES cells?
DR.
BAUER: Well, we do--we have the
knockout line that was used to, you know, make the knockout animals, and we
have considered doing in vitro differentiation studies using those lines.
We
also would potentially be able to do that kind of study in human embryonic stem
cells or stem cells of a variety of different tissues from human cell lines
that are commercially available or available through other sources.
DR.
RAO: And do you have access to
presenilin knockout mice?
DR.
BAUER: I could probably get--through
some of the people here on the NIH campus, I could consider getting those
animals as well.
DR.
RAO: Thank you, Steve.
DR.
BAUER: Okay. All right. So I'll
briefly go into the second project.
I'll start by saying just a little background. TGF-beta has been shown to influence B-cell development, and loss
of TGF-beta sensitivity has been associated with B-cell tumorigenesis in
several different B-lineage tumors.
So
we've been looking at the effects of TGF-beta on these normal mouse precursor B
cells, which we are able to culture, and also its potential role in mouse pre-B
cell leukemagenesis.
There's
previously been little data available that shows exactly what stages of normal
early B-cell development in mouse could be susceptible to TGF-beta and very
little data about the role of TGF-beta in tumorigenesis in early B cells in
mouse.
So
just to summarize our results, we've shown that these normal precursor B cells
that we grow in culture express the TGF-beta receptor and are sensitive to
inhibition of IL-7 induced proliferation.
TGF-beta can inhibit pre-B cell proliferation by inducing G1 cell cycle
arrest. And normal pre-B cell lines, as
well as their malignant progeny--those are cells that we've transformed in
vitro by putting in dual oncogene-containing viruses--they express the TGF-beta
receptor as well. However, they've lost
sensitivity to TGF-beta.
Now
the retroviruses we use all have the myc oncogene in them, and we have derived
pre-B cell lines from myc oncogene expressing transgenic animals. And they are not sensitive--or they remain
sensitive to TGF-beta, even when there are tumors.
So
all of this argues that there are multiple pathways for transformation of pre-B
cells, and our ability to use the system and to introduce oncogenes, either
singly or in combination, could provide a very nice methodological approach to
the dissection of molecular pathway of TGF-beta signal transduction and how
these pathways are abrogated during transformation.
And
of course, the Division of Cell and Gene Therapies is responsible for oversight
of safe and effective gene therapies, including approaches that involve vectors
that insert into cellular DNA. And the
prospect of cellular transformation subsequent to insertional mutagenesis is a
safety concern for gene therapy. So
understanding of molecular mechanisms of transformation is an important pursuit
in the FDA.
And
I think with that, I'll stop my presentation and welcome any more questions.
DR.
RAO: Thank you, Steve. Are there any questions?
DR.
HIGH: Steve, this is Kathy High. I just wanted to ask one question about is
that--is the description of the dlk knockout mice published yet?
DR.
BAUER: No, it's not. We're working on that.
DR.
HIGH: Okay. Okay, just want to make sure I hadn't missed it.
DR.
BAUER: Yes, but there was another group
who had made one. And we see very
similar things in terms of some of the effects on fat metabolism, obesity, and
so on. But we're the only ones that I
know of looking at the effects on the development of the immune system.
DR.
HIGH: Okay.
DR.
RAO: If there are no more questions,
then we can move on.
DR.
HURSH: Okay. So this is Deborah Hursh.
I'm from the Laboratory of Immunology and Developmental Biology. And like Dr. Bauer said, my regulatory
contributions are in the book that--the site visit book that you all should
have. My regulatory contributions are
in the areas of cell therapy, gene therapy, and assisted reproduction.
And
now, what I'd like to do is just give a brief overview of my research
program. My research program studies
the role of intercellular communication pathways in pattern formation and
morphogenesis. The model system is the
fruitfly Drosophila melanogaster. The
major focus of the laboratory is on the bone morphogenesis protein, or BMP, signaling
pathway, particularly the Drosophila homolog of BMP 2 and 4, which is the
product of the decapentaplegic, or dpp, gene.
BMP
2 and dpp are essentially identical.
And dpp and BMP 4 are 90 percent homologous. The BMP pathway, like other extracellular signaling pathways, is
highly conserved among all metazoans.
So data derived from Drosophila are applicable to mammals.
This
research program is significant to CBER's mission for several reasons. Currently, both TGF-betas and BMPs are being
studied directly for clinical use and are part of the manufacture of cellular
products.
In
addition, the entire area of signal transduction is of critical importance, as
the development of cell therapies and engineered tissue products will require
in-depth knowledge and manipulation of extracellular signaling pathways. The behavior of stem cells in particular is
governed by signal transduction, including the BMP, Hedgehog, Wnt, and Notch
pathways.
My
research program is taking an integrated genetic and molecular biological
approach to study the action of the Drosophila BMP, dpp, in a specific event in
development--the formation of the adult head.
Our goal is to understand the biochemical pathways that a TGF-beta
protein utilizes in the construction of a complex integrated structure like the
fruitfly head.
This
approach is based on my isolation of a specific class of dpp mutations whose
effect is limited to the adult head.
These recessive mutations reside in the 5' cis regulatory region of the
gene and cause a syndrome of alterations of the adult head that include eye
reduction, sensory structure eliminations, and duplications.
The
research program is divided into three areas.
The first is a genetic and molecular analysis of head capsule mutations,
where our aim is to identify cis-acting sequences and trans-acting factors
required for dpp's role in head capsule formation. The second area is to use the dpp head capsule mutations to carry
out a genome-wide genetic screen to identify other participants in BMP-mediated
adult had formation, and the third area is to understand the role of dpp in the
formation of the adult head.
I
will briefly discuss the first two projects.
Project 1. I isolated mutations
in dpp that specifically altered the adult head of the fly. These mutations reside in 5' cis regulatory
DNA and do not alter the protein coding region of the dpp protein. Our starting hypothesis was that these
mutations caused their mutant phenotype by disrupting dpp expression in a
critical region of the head primordia.
In flies, this structure is the eye-antennal imaginal disk.
We
used these mutations to map the exact location of head determining
cis-regulatory material within the dpp gene's 75 kb of cis regulatory DNA. Using these data, we have created a series
of beta-galactosidase reporter constructs from DNA in the region identified by
our mutations and introduced them into flies transgenically.
These
constructs all express in the primordia of the adult head, the eye-antennal
disk, thus we have identified a 10-kb region that contributes to dpp expression
in the head. The most critical of our
mutations was a 17 bp deficiency.
Within this deficiency are sites predicted to bind several homeodomain
transcription factors of the HOX class and also the TALE class.
The
HOX proteins deformed and labial and the TALE class proteins extradenticle and
homothorax are expressed in the eye-antennal disk. These two groups of homeodomain proteins are also thought to work
coordinately together, further supporting our observation that dpp's head
expression may, indeed, be regulated by homeodomain transcription factors.
We
tested the sufficiency of the 17 bp mutation to alter dpp expression in the
eye-antennal disk by creating a beta-galactosidase reporter construct from mutant
DNA. These constructs fail to express
in the eye-antennal disk, while an equivalent construct from wild-type flies
expresses as expected in the eye-antennal disk. These data support the idea that the head capsule phenotype is
caused by loss of dpp expression in the head primordia and that homeodomain
transcription factors are implicated in the regulation of dpp in this
developmental event.
We
are currently in the process of identifying the exact homeodomain transcription
factors that interact with this DNA and assessing their contributions to dpp
expression in the head.
And
if there are any questions on Project 1, I'd be happy to take them now.
[No
response.]
DR.
HURSH: Okay. Project 2. My initial
interest in the head capsule phenotype stemmed from the observation that the
number of flies within a class that actually displayed the mutant phenotype,
which geneticists call penetrance, correlated with the level of dpp protein
function. When head capsule mutations
are put in transheterozygous combinations with a class of dpp mutations that
reduce but do not eliminate the activity of the dpp protein by altering the
protein coding region, the penetrance of the head capsule phenotype increases
with the severity of the activity-reducing allele.
These
data indicated that the head capsule phenotype was sensitive to BMP pathway
signaling activity. I reasoned that the
head capsule defect would serve as a sensitized background to recover mutations
in genes that acted with dpp in head development.
The
sensitized dpp background, in concert with a second unlinked mutation, would
reduce the level of the TGF-beta/BMP signaling to a level where a mutant
phenotype would be revealed. Such
mutations could then be recovered and identified. This type of dominant enhancer screen has been used with great
facility to identify other members of genetic pathways.
We
have carried out several genetic screens using this idea to search for genes
that act with dpp in this pathway to form the adult fruitfly head. This approach has identified approximately
20 second site mutations that interact with dpp to create head capsule
defects. These genes encode
transcription factors from various classes, participants in signal transduction
pathways, and a variety of other known proteins, including cyclin A and type 4
collagen. Several proteins of unknown
function have also been identified.
We
have focused to date on one very strong interaction with the product of the
Drosophila odd-paired gene. Odd-paired
encodes a zinc finger transcription factor of the conserved "zinc finger
of the cerebellum," or ZIC family.
This family is involved in neural, specifically brain development in
frogs, zebra, fish, and mice.
We
have analyzed the post embryonic requirements for odd-paired and find it is
expressed in the eye-antennal disk and is required for proper head
development. We are focusing on
understanding how dpp and odd-paired interact in head development. And the current--the future focus of my lab
will be identifying and understanding the interactions of dpp with these genes
we have identified.
And
I'll be happy to take questions now.
Thank you.
DR.
RAO: Deborah, this is Mahendra Rao.
DR.
HURSH: Yes?
DR.
RAO: You had good success with doing
these sort of dominant enhancer or suppresser screens, right?
DR.
HURSH: Yes.
DR.
RAO: You do it the reverse way,
too? Then looking for dominant, you
look for things that make the phenotype work.
Did you also look for things that skip the phenotype?
DR.
HURSH: No. That would be a dominant suppresser screen, and we haven't quite
figured out a way to do that yet. That
has been done with great facility in flies, but my phenotype is very subtle,
and it's easier to make it worse than it is to make it better. To do a good dominant suppresser screen, you
need to have a very, very strong phenotype that you can make better and that
it's easy to see that it's been made better.
And
this has been done with, for example, mutations that severely alter the eye,
and then they get--they look better. I
think it would be hard to do this kind of thing with the type of phenotype I'm
working with. Though if I could do it,
I would because it's a very powerful screen.
DR.
RAO: My second question was, you know,
you mentioned right in the beginning that you were going to do a genome-wide
sort of scan to look at candidate binding sites in the upstream region?
DR.
HURSH: Well, not a genome-wide
scan. We actually were very lucky in
that one of our mutations is a 17 bp deficiency.
DR.
RAO: Right.
DR.
HURSH: And within that 17 bps, there
are HOX sites and two different TALE protein sites. So that really brought our attention to those particular proteins
as perhaps interacting with this DNA.
And so, within a 600 base region around that 17 bp deficiency, there are
also multiple other sites that would also bind to HOX and TALE proteins. That's really focused our attention on this.
But
we're not going any further to look for that, just within this very small
region.
DR.
RAO: Okay. Anyone else?
[No
response.]
DR.
RAO: Thank you, Deborah.
DR.
MOOS: Are you ready? This is Malcolm Moos. With respect to regulatory endeavors, what I
do is very similar to what Dr. Hursh and Dr. Bauer do. Also I think it's fair to mention, I've been
involved with certain miscellaneous activities that Dr. Noguchi may wish to
share with you in closed session and some projects from the center director's
office involved with comparability, impurities, and specifications, the ICH
document.
Our
primary goal in our research endeavors has been to study critical processes
controlling cell fate that will be of likely importance in the design of future
improved cell and tissue-based products.
Primary among these are the bone morphogenetic proteins, or BMPs, and
the Wnts, and the factors that elaborate with them in these processes,
including many potential unknown factors.
To
do this, we've used xenopus laevis and, more recently, xenopus tropicalis,
which are vertebrate model systems with several advantages. A few of these are the capability to do both
gain and loss of function experiments relatively quickly and easily; the
multiple readouts, which include morphology and histology; analysis of gene and
protein level; analysis of gene and protein distribution, including the ability
to look at the entire organism globally, both for gene expression level and
distribution and protein expression and distribution.
In
addition, xenopus is uniquely suited for analysis of the proteon in that it's
possible to generate very large amounts of material for biochemical studies.
This
approach has allowed the identification of a number of novel molecular
entities, which have included the cartilage-derived morphogenetic proteins, the
CDMPs 1, 2, and 3, which are TGF-beta family members involved in skeletal
patterning; anti-dorsalizing morphogenetic protein, which is important in
specification of the body axis; and FRZB, which is the first of the class of
secreted inhibitors of the Wnt growth factor oncogenes.
In
the interest of time, in case there are some questions, I'm going to discuss
further studies with just one of these, CDMP 1.
Our
original functional studies revealed an apparent very stringent requirement for
proteolytic processing of this molecule, and it was immediately apparent that a
potential explanation for this was that it shared a consensus proteolysis site
with one other TGF-beta family member, known as Vg1. This protein is also known to have a stringent requirement for
proteolysis.
We,
at the site visit, presented data suggesting that a point mutagenesis relieves
this stringency by allowing bioactivity to be released and that this effect
could be mimicked by co-administering a combination of two proteolytic enzymes
belonging to the subtilisin-like proprotein convertase family, or SPC family. And indeed, with the combination that we
tried, we could recover an activity that was comparable to the point mutant.
Since
the site visit, we have confirmed this data and tested all other known SPCs and
found that the combination of furin and SPC-6 was, indeed, optimal and
essentially matched what could be obtained with the point mutant.
Of
particular interest to us and also to members of the review committee were
analogous experiments on the Vg1 protein.
We have done the biological experiments and have confirmed that a point
mutagenesis of Vg1 indeed releases its activity, and it appears on the
basis--and this is just one preliminary experiment--that the synergistic effect
of furin and SPC-6 may also obtain.
But
that needs to be repeated, and we probably need to tweak our bioassay a little
bit. It needs to be a slightly
different bioassay than is needed for CDMP 1.
We've
also used an oversight model to confirm the cleavage of these proteins
biochemically. And again, preliminary
data suggests that both the point mutant and the co-administration of SPCs with
the wild type causes detectable cleavage of these molecules that you can't see
with the wild type.
What's
left, to complete the picture, is to perform the immunoblot analyses on the Vg1
protein and also to localize the SPCs by hybridization in situ, both in the
limb, to analyze the situation with CDMP 1, where we feel the patterning
is--where we feel its influence on patterning is critical, and in the gastrula
with Vg1 because this would resolve an important outstanding question in the
specification of the vertebrate body axis.
We
think we could have this done so that a manuscript can be submitted by
July. I'm happy to field questions on
this project or anything else that's in the book that I didn't talk about
today.
[No
response.]
DR.
MOOS: Do we still have you?
MS.
DAPOLITO: Yes.
DR.
NOGUCHI: Okay. Shall we move on to Dr. Epstein?
DR.
RAO: I think so.
DR.
EPSTEIN: Okay. This is Suzanne Epstein, and an overview of
the Laboratory of Immunology and Developmental Biology was in the book. So I won't review that now. In terms of regulatory activities, that's
summarized also, and I mainly review gene therapy INDs, master files for vector
production, and then serve in a policy working group.
Since
the site visit, I was asked to and have agreed to serve as informal unofficial
science advocate for OCTGT. To review
my research program, first of all, its relevance to these regulatory
responsibilities.
Viruses
and plasmids have potential uses in human health care as vaccines and also as
gene therapy vectors. Development of
safe and effective products in both these categories requires an understanding
of the immunity they induce and its consequences. Can you hear me?
Okay. They're moving the microphone. The vaccine must contain the right antigens
and induce the right immune effectors with adequate potency if it is to prevent
disease. And in gene therapy, immune
responses to viral vector components or to transgene products are usually
undesirable. They can be a limiting
factor, blocking efficacy upon repeat administration and, in some cases,
causing pathology due to cross-reactions on self components.
My
lab studies immune responses in the influenza system. Influenza is still a major public health problem, and the current
approach to vaccination is not optimal.
Also the risk of a pandemic due to spread of a new subtype into humans
makes it important that we develop new means of immunization.
In
addition, our findings are relevant to the design of vaccines for other viruses
with unpredictable strain variation--that would include HIV, for example--or
unpredictable outbreaks, which would include families of potential bioterrorism
agents, and also to the consequences of gene therapy.
Thus,
the results can inform regulatory decisions about safety and efficacy of viral
vaccines and gene therapy vectors, and the choice of endpoints to monitor in
preclinical and clinical studies.
Now
our studies involve inducing immunity with live virus and also plasmid DNA
constructs expressing individual influenza virus proteins in the mouse model,
which has a number of advantages. The
report you received described four project areas. In one, we studied mechanisms of heterosubtypic immunity, which
is broad cross-immunity to influenza that protects against infection with
strains of viral subtypes differing from the immunizing subtype.
We
have studied the roles of T cells and the antibodies in cross-protection,
whether induced by virus exposure or DNA vaccination. We showed that T cells provide some cross-protection against
challenge in knockout mice lacking all antibodies and B cells.
We
also studied knockout mice lacking IgA, selectively, and showed that they have
heterosubtypic immunity. However, we
saw some impairment in their ability to clear primary infection and in their
responses to challenge under some conditions.
The role of IgA at mucosal surfaces will be further investigated in this
model.
In
the next project, we showed that DNA vaccination with conserved components
nucleoprotein and matrix of an H1N1 influenza A virus could control infection
with H5N1 chicken flu viruses from the lethal outbreak in 1997 in Hong Kong.
The
vaccination protected against lethal challenge with a moderately virulent
strain and protected partially against challenge even with the most extremely
virulent strain. In a situation with a
strain-matched vaccine not available, DNA vaccination with conserved components
like these might provide partial defense against a pandemic virus.
These
findings are also relevant to other families of viruses in which unexpected
emerging strains must be controlled, even if imperfectly, before matched
vaccines become available. So that
would include by analogy agents like hanta viruses and arena viruses.
Another
project addressed the question of whether humans have heterosubtypic immunity
to influenza as seen in the animal models.
I examined archival records from the Cleveland family study before and
during the 1957 pandemic. Many children
who had culture-proven influenza in earlier study years became infected again
in 1957, but almost no adults did.
While
some further analysis is still pending, the greater resistance of adults after
prior infection suggests accumulated immunity and suggests that human
heterosubtypic immunity may have been sufficient to alter susceptibility to the
1957 Asian influenza virus.
The
last project described involves approaches to improving vaccination with
conserved components of influenza. We
are studying M2, or matrix 2, which is already known to provide broad
cross-protection, and PA, or acidic polymerase, which we identified by
screening of genes by DNA vaccination.
That was a screen of all the genes of influenza A. PA DNA, followed by PA vaccinia boosting,
protected against lethal challenge in BALB/c strain of mice.
It
induced a CD8 T cell response that was readily detectable in B6 but not BALB/c
mice by intracytoplasmic staining for interferon production. But in contrast, it protected against lethal
challenge in BALB/c mice and not B6 mice.
So the readily detectable staining response does not correlate with
protection.
We
have now made new constructs with murine optimized codon usage to enhance the
potency of this vaccination, and they will be used to further investigate these
findings.
Since
the time of the site visit, we have succeeded in mapping a T cell epitopes in
BALB/c mice of the PA antigen using ELISPOT analysis instead of
intracytoplasmic staining for interferon, and we are now investigating the
functional role of these peptides.
In
order to alter immunodominance patterns and enhance the contribution of various
antigens to protective immunity, we plan additional studies of DNA prime-viral
boost regimens using both adenovirus and vaccinia recombinants. We're now making some adenoconstructs.
Simultaneous
versus sequential immunization with different influenza antigens will be
tested. And then the most effective
prime and boost regimens, as demonstrated with our usual lab challenge strains,
will be subjected to challenge with model pandemic systems H5 and H9 in
collaboration with Terrence Tumpey of USDA.
Overall,
we will be continuing to explore protective immunity against influenza
infection and the contribution of conserved components to it. I'm happy to answer any questions, either
about the research program or the Lab of Immunology and Developmental Biology.
DR.
RAO: I have a quick question on your
project. You know, you mentioned the
difference in the immune response or susceptibility of children versus adults?
DR.
EPSTEIN: Yes.
DR.
RAO: Any speculation other than
accumulating immunity as to why this could be true?
DR.
EPSTEIN: Well, one, of course, might
speculate at first that children behave differently, for example, putting
things in their mouths and so on. But
if you look at the data for 1950, '51, and '53, the susceptibility of children
and adults was fairly similar. That was
within the era of the H1N1 subtype.
When
H2N2 arrived for the first time, so that all people were naive
serologically--they didn't have neutralizing antibodies to that hemagglutin or
neuraminidase--suddenly, the spike of difference between children and adults
occurs. So it's something unique to
that situation. It's not a general
behavioral matter.
Also
in that family study, all the adults were living in households with young
children. So it's not exposure--an
exposure issue that the children are exposed at school and adults are not. Frankly, I think immunity makes the most
sense.
DR.
RAO: It could be a really nice handle
into the systems. It might be nice.
DR.
EPSTEIN: Oh, absolutely. And I don't know how to duplicate that body
of data except to, if there were a surveillance study going on in the future at
the time of another pandemic.
What's
retrievable from those archives is limited, and I welcome your ideas, if you
have any. But it will not be possible
to prove that that's what occurred, and we, of course, can't go back and do
serological or T cell assays now. It
was not considered at the time of the study.
DR.
HIGH: Suzanne, this is Kathy. What's available from that study is only
data, no samples?
DR.
EPSTEIN: That's correct. There was a serum archive sent to Yale by
Case Western. But I corresponded with
Yale University, and at the time a certain person retired, it was all thrown
away. It had mostly been used up by his
studies. What was left was dregs. But when he retired, he cleaned out the
freezer.
It
wouldn't have been probably enough to use.
But that's not as tragic as it might seem. What I'm interested in would not be present in serum. The serum would have shown neutralizing
antibody titers, and the publications from the '50s have an extensive analysis
of the antibodies by HAI and complement fixation.
That's
not what mediates broad cross-protection, as we've shown in our other studies,
or probably not. If it is antibody
mediated at all, it's probably mucosal IgA, but probably not serum antibody. So the T cell responses could not have been
tested from that anyway.
DR.
HIGH: Right, because they were just
serum samples.
DR.
EPSTEIN: Right. What's left is case report forms and charts
of various kinds. I have to return to
Cleveland this summer to track down--to try to track down which individuals may
have received an experimental vaccine late in the observation period and that
was not very effective, but to try and see if that changes anything.
DR.
RAO: Any further questions?
[No
response.]
DR.
RAO: Thank you, Susan.
MS.
DAPOLITO: Dr. Rao, this is Gail. Could you give us a couple minutes to get
things sorted out on our end? Clear the
room, okay?
[Whereupon,
at 2:15 p.m., the open session was adjourned, to reconvene in closed session,
this same day.]
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