FOOD AND DRUG ADMINISTRATION

> CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
>
> "This transcripts has not been edited or corrected, but appears as
> received from the commercial transcribing service. Accordingly the
> Food and Drug Administration makes no representation to its
> accuracy..........."
>
> 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]{.underline}
>
> Gail Dapolito, Executive Secretary
>
> Rosanna L. Harvey, Committee Management Specialist
>
> [Committee Members]{.underline}:
>
> 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]{.underline}:
>
> 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]{.underline} [G]{.underline} [E]{.underline} [N]{.underline}
> [D]{.underline} [A]{.underline}
>
> 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]{.underline} [R]{.underline} [O]{.underline} [C]{.underline}
> [E]{.underline} [E]{.underline} [D]{.underline} [I]{.underline}
> [N]{.underline} [G]{.underline} [S]{.underline}
>
> 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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