UNITED STATES FOOD AND DRUG ADMINISTRATION

	

	BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

	

	Bethesda, Maryland
	Friday, 13 July, 2001

	PARTICIPANTS:
	DR. DANIEL SALOMON
	Chair
	
	MS. GAIL DAPOLITO
	Executive Secretary
	
	DR. STEVE BAUER
	Center for Biologics Evaluation and Research
	
	DR. ESTUARDO AGUILAR‑CORDOVA
	Harvard University Medical School
	
	DR. BETH HUTCHINS
	Canji
	
	DR. RICHARD SUBLETT
	Introgen Therapeutics
	
	DR. PHYLLIS FLOMENBERG
	Thomas Jefferson University Medical School
	
	DR. ED SAUSVILLE
	National Cancer Institute
	
	DR. DAVID GAYLOR
	Sciences International
	
	DR. MAHRENDA RAO
	National Institute on Aging
	
	DR. BRUCE BLAZER
	University of Minnesota
	
	DR. RICHARD CHAMPLIN
	M.D. Anderson Cancer Center
	
	DR. JOANNE KUTZBERG
	Duke University

	PARTICIPANTS (CONT'D):
	MS. ALISON LAWSON
	Genezyme Corporation
	
	DR. DAN SALOMON
	Scripps Research Institute
	
	DR. RICHARD MULLIGAN
	Harvard Medical School
	
	DR. GARY KETNER
	Johns Hopkins University
	
	DR. MARSHALL HOROWITZ
	Albert Einstein College of Medicine
	
	DR. GENE ROSENTHAL
	National Institute of Health
	
	DR. JOYCE FREY
	Division of Cell and Gene Therapy
	
	DR. PHIL NOGUCHI
	Office of Therapeutics
	
	DR. EUGENE ROSENTHAL
	National Institute of Health
	
	MS. ALICE WOLFSON
	Consumer Representative

	*  *  *  *  *
	P R O C E E D I N G S
	DR. SALOMON:  Good morning everybody.  We've fulfilled my criteria for beginning a meeting.  Jay Siegel is seated. We're going to have to have a vote at the end of this meeting about what the criteria for the next meeting's going to be because Jay informed me that he wasn't going to be here, so ‑‑
	Anyway, I'd like to welcome all of you to Meeting Number 30 of the Biological Response Modifiers Advisory Committees.  So, 30, that means we've had 30 meetings, so when did that start?  When did ‑‑
	MR. SIEGEL:  Probably ────.
	DR. SALOMON:  And we've had 30 meetings in or 12 years?  I've been giving them a hard time for the last several meetings about not having more ‑‑ more titles, you know, good formal titles for these meetings.  And, apparently, there was a title here.  But it was taken off.  Maybe we'll get to see it later.
	Anyway, one of my pleasures this morning is ‑‑ in addition to welcoming you here to this meeting is to introduce four official new members of the BRMAC. Dr. Katherine High, who I believe is not here yet, but will join us a little bit later; Dr. Mahendra Rao, seated over there; Dr. Bruce Blazer; and Alison Lawton, seated to my left. And, again, the dedication and the amount of time and energy of the members of the BRMAC to the sort of process is something I think all of us involved in it really respect.
	I also see Abbey Meyers down there, which is a pleasure, welcome back.  We're never going to let you go, is that what it is?  Abbey's been a member of this committee off‑and‑on for as long as I've been a member and it's a real pleasure to see her back, as well.
	So, I'd like to go ahead and get started.  We have a one‑day meeting.  The next meeting of the BRMAC will be three days‑long, which, I guess, is proof that no good deed goes unpunished.  So just when you thought you were safe to come back to Washington, maybe not, but anyway, I think it will end up balancing out.
	This should be a very interesting meeting on adenovirus and return to my right‑hand person, Gail.
	MS. DAPOLITO:  I'll read the Conflict of Interest Statement.  This announcement is made part of the public record at this meeting of the Biological Response Modifiers Advisory Committee on July 13.  Pursuant to the authority granted under the Committee charter, the director of FDA's Center for Biologics Evaluation and Research has appointed Ms. Abbey Meyers, Doctors Estuardo Aguilar‑Cordova, David Gaylor, Marshall Horowitz, and Gary Ketner as temporary voting members.  To determine if any conflicts of interest existed, the agency reviewed the submitted agenda and all financial interests reported by the meeting participants.
	As a result of this review, the following disclosures are being made.  In accordance with 18 U.S.C. 208, doctors Katherine High, Estuardo Aguilar‑ Cordova, and Marshall Horowitz, have been granted a general matters waiver, which permits them to participate in the committee discussions.
	Doctors Blazer, Champlin, Kurtzberg, Mulligan, Salomon, Sausville, and Ms. Meyers have associations with firms that could be affected by the committee discussions.  However, in accordance with current statutes, it has been determined that none of these associations require the need for a waiver or an exclusion.   Ms. Alison Lawton is the non‑voting industry representative for the committee.  She is employed by Genzyme.  Genzyme has associations with various universities, investigators and research foundations that are involved in gene therapy.  Ms.  Lawton also has financial interests in several firms that could be affected by the committee discussions.
	In regard to FDA's invited guests, the agency has determined that the services of these guests are essential.  The following interests are being made public to allow meeting participants to objectively evaluate any presentation and/or comments made by the guests.  Dr. Beth Hutchins is employed by Canji, Inc., and has a financial interest in a firm that could be affected by the discussions.
	Dr. Eugene Rosenthal, is employed by the National Institutes of Health, Office of Biotechnology Activities.  Dr. Rosenthal is substituting today for Dr. Amy Patterson, who is unable to attend.
	Dr. Richard Sublett is employed by Introgen Therapeutics, and has a financial interest in a firm that could be affected by the discussions.
	In the event that the discussions involve other products or firms not already on the agenda, for which FDA's participants have a financial interest, the participants are aware of the need to exclude themselves from such involvement and 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 state your name, affiliation and address any current or financial previous involvement with any firm whose product you wish to comment upon.  A copy of the waiver's addressed in this announcement is available by written request under the Freedom of Information Act.  Thank you, Dr. Salomon.
	DR. SALOMON:  Okay, going on, I'd like to ‑‑ I always try and start the meeting with every body going around and just kind of giving a quick blurb on who they are.  I think this is particularly important that today, as we have a couple new members and we're also joined at the table by several additional experts,  Dr. Ketner, Dr. Aguilar, Dr. Horowitz.  And, again, we're very glad that you guys are here with us.  And so why don't we start with Abbey, just tell us a little bit about yourself and we'll just go around.
	MS. MEYERS:  Yes, I'm President of the National Organization for Rare Disorders, which is known as NORD and we're the orphan‑drug folks.  We helped to pass the law and we monitor the development of orphan drugs.  And I served on the Recombinant DNA Advisory Committee for many years, and the Biological Response Modifiers has me since then.
	DR. SAUSVILLE:  My name is Ed Sausville, and I'm the Associate Director for NCI's Developmental Therapeutics Program and we have an ‑‑ our major role is in the preclinincal evaluation and production of both drugs and biologics for use in clinical trials sponsored by NCI and by academic investigators with INDs.
	DR. GAYLOR:  I'm David Gaylor, I retired from the National Center for Toxicological Research of the FDA last year. I'm now working with a consulting firm, Sciences International.  My area is biostatistics and risk assessment.
	DR. RAO:  My name is Mahendra Rao, I'm a Section Chief at the National Institute of Aging.  My work is predominantly on stem cells, but I'm also interested in ────.
	MR. BLAZER:  My name is Bruce Blazer, in Bone‑marrow Transplantation Division at the University of Minnesota and I'm involved in the immunobiology of bone‑marrow transplantation and preclinincal and clinical studies.
	DR. CHAMPLIN:  Richard Champlin, I'm the Chairman of the Department of Blood and Marrow Transplantation at M.D. Anderson Cancer Center.
	DR. KURTZBERG:  Joanne Kurtzberg, I direct the Pediatric ──── program at Duke University and work with umbilical cord blood transplantation.
	MS. LAWTON:  Alison Lawton, I'm the industry rep.  I am senior Vice President of Regulatory Affairs for Genzyme Corporation and I chair the Cell and Gene Therapy Committee for the PhARMA Association.
	DR. SALOMON:  Dan Salomon, I'm in the Department of Molecular and Experimental Medicine at the Scripp's Research Institute, and my expertise and interest covers organ transplantation, gene therapy, tissue engineering and, more recently, angiogenesis. Thank you.
	MS. DAPOLITO:  Gail Dapolito, Center for Biologics, Executive Secretary for the Committee and Rosano Harvey, the Committee Management Specialist for the Committee.
	DR. MULLIGAN:  I'm Richard Mulligan from Harvard Medical School and we're involved in vector research and also stem cells.
	DR. KETNER:  I'm Gary Ketner, from the Department of Molecular Microbiology and Immunology at Johns Hopkins University School of Public Health ‑‑ excuse me, Johns Hopkins University, Bloomberg School of Public Health.  I'm an adenovirus biologist.
	DR. AGUILAR‑CORDOVA:  I'm Estuardo Aguilar‑ Cordova, I'm with the Harvard Gene Therapy Initiative, I'm involved in vector research and clinical trial applications of gene therapy.
	DR. HOROWITZ:  I'm Marshall Horowitz, I'm the Chairman of Microbiology and Immunology at the Albert Einstein College of Medicine and a member of their clinical and the pediatric infectious disease group. I work on adenoviruses, I work on their immunoregulatory genes.  In a sense, I'm coming home, in a way, I served four years on the FDA Vaccines and Related Products.  And at the time, which was about ten years ago, related products started to include gene therapy.  It's nice to see the expertise in the room this morning.  It was very hard to find it ten years ago, and I look forward to the discussion today.
	DR. ROSENTHAL:  I'm Gene Rosenthal, I'm a biotechnology program advisor in the Office Biotechnology Activities at NIH.  I'm substituting today for Amy Patterson.  One of the committees that we act as the support staff for is Recombinant DNA Advisory Committee.
	DR. BAUER:  My name is Steve Bauer, I'm a product reviewer in the Division of Cellular and Gene Therapies.
	DR. FREY:  Joyce Frey, Deputy Director of the Division of Cell and Gene Therapy.
	DR. NOGUCHI:  Phil Noguchi, Director of Cell and Gene Therapies in the Office of Therapeutics.
	MR. SIEGEL:  Jay Siegel, Director of Office of Therapeutics, CIBER (?), FDA.
	DR. SALOMON:  Well, please?
	MS. WOLFSON:  Alice Wolfson, I apologize for being late, I was driven to the wrong Holiday Inn.  I'm the consumer representative.
	DR. SALOMON:  Been there, done that.  And Katherine High hasn't joined us yet, so, that's okay.
	Well, I want to say it's a pleasure to be with real professionals.  All of you knew to push your button to speak, and then to turn it off, I'm impressed.  That bodes well for the rest of the morning.  Okay, Gail, are we ready to go?  Okay, thank you all very much, and I think we're ready to go.
	The first speaker today is Steve Bauer, our own Steve Bauer, Office of Therapeutics Research and Review and he's going to talk about Adenovirus Vector Titer Measurements and RCA Levels.  Steve and Joyce Frey‑Concells, who are there were sort of the lead FDA people on putting together today and the questions involved, so they'll be very much involved in the rest of the discussions this morning with Phil and Jay.
	DR. BAUER:  It's a pleasure to talk to you folks this morning and I'd like to express my gratitude to all of you for being here and being willing to participate in this deliberation this morning about adenovirus gene therapy products.
	My purpose is three‑fold:  I want to talk, first of all, just to update everybody on an initiative from the gene therapy community to develop a wild type adenovirus reference material that we can use to better or improve our ability to measure the amount of adenovirus product, in terms of viral particles and infectious particles that we administer to people.
	And the second purpose is to update the Committee and the gene therapy community and members of the public on recent changes in recommendations about some of the measurements of adenovirus products.
	And the third is to introduce some of the questions related to those measurements and how they impact the clinical trials.
	To put that in perspective, I have here a chart that shows current active gene transfer INDs and I wanted to point out a few things about adenovirus.  This is our second most common vector class, about 58 current active INDs in this area.  The majority of them, shown in green are direct in vivo injection of the vector by a variety of routes.  And then there are some, as well, that rely on ex vivo transduction.
	And also, there's a variety of patients that are treated with these different vectors, so in order to put in perspective the kinds of patients who would be exposed to RCA and would be administered these products, I put this chart together to show you that the majority of patients are cancer patients.  There are some coronary/vascular applications, some genetics and even some normals and then another thing that's relevant to some of the discussions we'll have later is how are these products actually administered, what is the rout of administration.  And I've broken those out. So they go from injection directly into the lesion or the tumors.  I mentioned ex vivo transduction of cells, but they're also intravascular, intraperitoneal, oral, directly into muscle and down the respiratory tract.
	So, there's a variety of routes of administration; a variety of kinds of patients.  We'll be discussing later and I think it's an important issue:  What is the status of the patients in terms of their ability to respond to either infectious virus or replication‑defective adenovirus?  A total of 58 current INDs, so that give you a perspective on what are the activity in this area.
	So, the first thing that I'm going to do is tell you about this development of an adenovirus reference material.  When it comes to looking at an adenovirus preparation final product, we measure several important parameters of what is actually in that product.  And I diagram this here.  There are a non‑infectious particles and, actually, this is a challenge to measure infectivity and Dr. Aguilar‑ Cordova will talk about some of the technical challenges that accompany that later.
	And one of the important questions is what is our ability to measure infectious versus infectious particles, but I diagram here in yellow that actually the majority of particles as we measure them currently, or most people do, are non‑infectious after purification.
	Then there are infectious units, which will infect permissive cells they are replication defective.  These are the ones that would actually deliver and produce the therapeutic trans gene.
	Another component, and this is the one that's of concern is the replication competent adenovirus, and with the most commonly used production methodology nowadays, this is an unavoidable consequence of manufacture.  And there will be discussions later of how replication competent adenoviruses arise in the current production methods.  But these would be infectious particles and could, potentially, could establish an infection in a recipient.
	And then we also measure all these particles together and I'll discuss a little bit more how that's done.  But we'll be talking about the technical challenges of these measurements and how we can improve them and the reference material, I think, will help the community at large to better measure these things.
	The vector particle measurement, which is looking at all the different particles is a physical/chemical measurement. The most common methodology is to lyse the particles, measure the amount of DNA and correlate that genome number with the particle number.  We'll hear a little bit more about that later.
	The infectious activity is a biological assay and, therefore, inherently, more variable.  There are a variety of ways to do this, including measuring plaques on a lawn of permissive cells.  These are cells that will allow replication of the otherwise replication‑defective particle.  There are other ways through immunological detection of some of the virus proteins.
	Then, finally, the replication competent adenovirus is a biological assay, where you measure the infection on cells that don't complement the vector, that are not permissive for replication of the vector, the intended product, but will reveal the presence of a recombinant replication competent adenovirus.
	These are all the measurements that I talked about are very important in terms of safety, efficacy and, also, in terms of an assessment of how well the production process is; how reproducible it is in manufacture of these vector products.
	Of course the safety issue is pretty obvious, if you administer a replication competent virus, what are the consequences you're exposing to that?  And we'll have a lot of discussion about that later.  The other issue is that exposure to viral proteins can cause some toxicity, itself, so knowing how many viral particles that you're putting into the patient is an important parameter and if there's a sharp elbow in the curve between dose and toxicity, having precise measurements of this is important.
	The other thing is inefficacy.  Of course, if there are a lot of nontransducing particles in a preparation, you're going to minimize your chance of actually having a benefit of expression of the intended gene. And as I mentioned before, this is a good way to assess a production process.
	Well, all of these things, of course, have been recognized very well by the adenovirus gene therapy community for quite sometime.  Starting in '93, adenovirus vectors were first used in cystic fibrosis protocols.  And even back then some of the technical challenges of measuring reproducibly, reliably, and across studies were recognized and there were recommendations at that time that are reference standard for vector being produced. There was a long interim during which there was quite significant development in this field, but nobody had really stepped forward to produce this kind of standard.
	In 1999, with the unfortunate death of Jesse Gelsinger, there was a RAC safety symposium and there was a renewed call for standards.  And there have been other calls for standards along the way, but I'm happy to say that this has gone forward rapidly since that point.
	The goals of producing a standard are to have more consistent, safer vectors. As it stands now, particle counts, itself, is subject to inter‑ assay variability, probably the best assays are 10 percent.  Infectious units assay is even more variable, but there's nothing, really, to hang our hats on, in terms of comparing studies that are done in different preclinical settings, different manufacturers and even different clinical studies.  So it's anticipated that our ability to relate measurements much more precisely to one another will really improve the safety profile and comparability of this vector product class.
	It also will help us to develop regulatory policy with more solid data, and we can make our recommendations based on measurements that we have more comparability between different clinical trials across those trials.
	So there's been formation of a group called the Adenovirus Reference Material Working Group.  This is a partnership between government, industry, academia ‑‑ it's going forward under the coordination of the Williamsburg Bioprocessing Foundation.  And there's a lot of good information on what the activities of this group are at this Web site.  There's also some at the FDA website mentioned here.
	But a lot of the activities that were planned are going forward.  This has been very gratifying to those of us who've been involved to see the spirit of cooperativity and volunteerism that has gone into this.  A master cell bank, has already been donated; adenovirus wild‑type virus stock has been donated; master viral seed stock has been made.  The participants who will produce and formulate the bulk virus have been identified and the initial characterization and provisional titer of that preparation ‑‑ these participants have been identified; vialing, the people who will serve as repository and distribution, these people have already been identified.  And there's an ongoing effort right now to solicit contributions to look at particle infectivity.  So, it's very active.  I think by the end of this year, we'll actually have a standard to distribute.
	And Dr. Beth Hutchins, Estuardo Aguilar, myself are some of the people and Steph Simek, who've been involved in this. And I think it's really a nice contribution to the field.  We're very happy and gratified it's going forward.  And I think this will have a big impact on our ability to look at these products.
	So, now, I'm going to change to a discussion of or to a discussion of our recent changes and recommendations.  And this is directly related to the information that we gathered from the March 6 letter that went out to all our sponsors of adenovirus gene therapy, as well as other gene therapies.
	I'll discuss our change in recommendation on the particle to infectious unit ratio and the recommendation on RCA limits.
	But to put that in perspective, I just wanted to introduce this idea.  There's a couple different ways to look at risks associated with different products and I borrowed this term from the people in the field of radiation safety.  But I think this is the way we basically looked at exposure to replication competent adenovirus.  And that is to say that we want it to be as low as reasonably achievable.
	So the recommendations that we've made were based on looking at the information in the March 6 letter responses and looking at the community's production data.  We actually solicited data on all production lots, whether they were used or not, why they were not used in the clinic?  Why they failed, in other words, a lot release.  And based on that kind of information, we reformulated our recommendations.
	And as the field has progressed, as people have gotten better and better at making virus or vector.  And as our ability to measure that has gotten better, these recommendations have changed several times over the years.
	The other thing to discuss is that, as we currently use these recommendations, we apply them to all product lots, regardless of their intended clinical use.  And I wanted to have discussion of the appropriateness of that.  That is shifting paradigms to a more risk‑based paradigm and the question is what kind of information do we have, what do we need if we want to reconsider this kind of approach, where we just say we want it ‑‑ the exposure to be as low as reasonably achievable.
	So these recommendations of the particle‑to‑ infectious unit ratio, the reason for this is, as I showed you earlier, that one diagram of what's in a purified adenovirus particle.  It's a mixture of noninfectious and infectious particles and the infectious ones can either be of a desired type that will deliver the therapeutic gene or replication competent. But in the past, we've asked that there be per infectious units, less than 100 total viral particles.  So this is, basically, you could express this as saying you want at least 1 percent of your product to be able to transduce or deliver the gene.
	This previous recommendation, again, had been based on review of production lots in earlier days of gene therapy.  The March 6 letter came in, that data was looked at and we decided in the spirit of trying to increase the efficacy or the ability to transduce to change this ratio.  And the recommendation now is that per infectious unit there be less than 30 total viral particles.  So another way to look at that is to say that you want more than 3.3 percent of your preparation to be infectious.
	Now, recognize that infectivity assays are problematic, and we'll have more discussion of that later.  Keep that in mind, but that's ‑‑ that was the rationale for this change.
	The other change was in the limit of how much RCA should be present in clinical lots.  And the previous recommendation was that there be less than RCA and 10 to the 9th infectious units.  And, as I've said several times, with recognizing that this is a problematic assay compared to the assay for vector particle, we've changed that.  And the new recommendation is based on particle number, and we also dose on particle number, so I think this is also in harmony with that. I think there's better precision in this measurement and this way of expressing it.
	So the current recommendation is now there be less than 1 RCA and 3 times 10 to the 10th vector particles.  And this was derived ‑‑ multiplying this number by that factor of 30, so that's how we came about getting that number.
	Finally, I'd like to discuss application of this recommendation.  As I said, this is currently recommended for all adenovirus vector lots regardless of the clinical use.  And in a trial where there doses of 3 times 10 to the 13th vector particles, this recommendation would say that the potential exposure was up to 1,000 RCA. Now, you'll hear data later that there have been substantially higher potential exposures to RCA based on our previous recommendations and, as I said, this field has been going forward, our recommendations have been changing as the technology's changed and improved, but I think that it's important to realize that this would be the current level and in the past, the levels have been higher.
	So, if we wanted to think about changing from this overall type of risk minimization paradigm of as low as reasonably achievable to a risk‑based recommendation, we need to consider what kind of information do we have and there's some literature on this. There's clinical experience with wild‑type ad infection and Dr. Phyllis Flomenberg will give us a talk later about that.
	At the last BRMAC, Dr. Steven Channick gave us a nice talk about that.  And I provided for the committee members the transcripts and slides from his presentation last time, as well.
	There's clinical experience with gene transfer studies and I've asked Beth Hutchins and Dick Sublett to come and tell us about that and to relate to us what the status of the patients was in terms of their immunity and their potential exposure to replication competent adenovirus.
	There's also some notable adverse events.  I already mentioned the one, you know, the death, the unfortunate death of Jesse Gelsinger.  And also an early notable adverse event in Ron Crystal's cystic fibrosis trial.  And these still remain somewhat mysterious, but there have been questions whether or not they could related to RCA, but I think retrospective analysis of some of the lots using a standard will help us understand that sort of thing.
	But the other thing is, what information would we like to see.  If we would like to say, for instance, that for certain routes of administration, certain patient populations and so on, we can try to make more a risk‑based recommendation than this universal application of our current recommendations to all clinical lots.
	And some, if you look at the data that came in with the March 6 letter, there are, actually, a wide variety of experience of how many clinical lots were made and how many had to be discarded because RCA.  Some manufacturers had as high as 40 percent lot rejection, some had zero.  And there's a lot of adenovirus vector biology that is involved in that.  But if we have an ability, a better ability to quantify RCA and have some confidence that different clinical applications can have different standards, we could, perhaps, not be so wasteful in our adenovirus lot utilization.
	So, I wanted to just spend a few minutes talking about what Dr. Steve Channick's talk ‑‑ well, since he's not here, found his talk very informative, last time. But I mentioned that some of the things that we can use to look at risks of RCA exposure are what happens with the wild‑type adenovirus infection and in different patient populations.  And the previous information is focused ‑‑ the best studies, I think, are in the arena of bone‑marrow transplantation. And in that setting, adenovirus, including types 2 and 5, which are used for gene therapy vectors can be a significant cause of morbidity and mortality in bone‑marrow transplant.
	And I think one of the take‑home lessons is it's important to consider immune status in terms of infection or reactivation of a previous infection, and latency of viruses is an important issue.
	Some of the lessons that have been learned by the field and that were communicated to us by Dr. Channick include, these:  The neonatal adenovirus pneumonia is significant.  There are sporadic, severe, localized outbreaks.  If you look, these aren't really immunodeficient people, but they're immunonative.  If you look at SCID population where there's a severe immunodeficiency, they're at high risk for adenovirus.  If you look at a syndrome like DiGeorge, where there's immune defect, there have been case reports of fatal hepatic necrosis.
	In the realm of solid organ transplant, adenovirus infection of the transplanted organ has been recognized as a problem and perhaps been tied, recently, to rejection of cardiac transplants.  The source of that could be reactivation in the recipient or the donor.
	Then in AIDS patients, there's been some recognition that adenovirus can cause some problems.
	So in contrast to that, there has been a lot of clinical experience with adenovirus and we'll hear about that, as well, or with adenovirus gene therapy products with relatively good safety profile.
	So, with those kinds of considerations in mind, we're going to later discuss whether or not recommendations regarding acceptable levels of RCA should be the same for all different clinical uses.  I explained these two different ways of looking at risk, either minimizing it to the greatest extent possibility or look at risk‑based. And then, I think, I pointed out that the status of the patient in terms of their immunity is an important consideration.
	The other thing that's not on here, but I think is important, will be discussed later is route of administration.  With the exception that we will discuss ex vivo transduce cells and whether or not RCA measurements should be done on those as well.
	And then if we would like to move towards a different recommendation for RCA exposure, what kind of information should be gathering.  And I think having a standard will help us look retrospectively at what's been given and correlate clinical outcomes there and we'll also be able to increase out precision and accuracy of future lots.  So I don't think we necessarily have all the data that we want to do this now.  That's a point of discussion, but I think we could think in terms of, you know, gathering it and what we need to do that.
	An with that, I'll conclude, thank you for your attention and I'll take any questions.
	DR. SALOMON:  Thank you, Steve.  I had a couple questions and there may be some questions from the rest of the group.  The first one is, are we going to come ‑‑ are we going to get some more information a little later in the day about going back to the standard reference set and exactly how that's going to be used?  Because, if not, I think that would be something important to  just to give a little more detail on.
	DR. BAUER:  Well, Dr. Aguilar will talk a little bit more about that in his discussion, so, if you haven't gotten the answer to whatever questions you have at that point, you know, bring it back up.
	DR. SALOMON:  A second thing is, are our speakers today going to also talk about ‑‑ there are some developments in the vectorology of the adeno, as well as some new strategies for producer cell lines, and other production techniques that could significantly alter, for example, the ration of replication‑competent virus and also, hopefully, increase the efficiency of production of infectious viral units.  And that also could have, I think, significant bearing on our ‑‑
	DR. BAUER:  Absolutely ‑‑
	DR. SALOMON:  Discussions.
	DR. BAUER:  Absolutely, there are developed and developing alternative ways to produce the vector that are much less prone to this recombination event, that ends up in RCA.  And you'll hear from our speakers what that kind of recombination event is and how to avoid it.  So, we'll get some information on that later.
	DR. SALOMON:  And the last question, I'm sort of playing a role now, as chair, but, you know, clearly the field has been under the shadow of the Gelsinger case, publicly, if not also within the experts involved.  The obvious question here, of course, is when that's driven so much of the concern over the safety of the adenovirus in the last two years, until recently, I understand, that for legal reasons there were ‑‑ there's been not a lot of information that's been able to have been shared.  At this point, are we going to have any information on the replication competent retroviral titer and particle titer, et cetera, of those preparations that were used in that case?
	DR. BAUER:  To my knowledge, the status hasn't changed and we're not able to disclose everything ‑‑ I don't know they'll ‑‑ any of my FDA colleagues have a different answer for that.
	DR. SALOMON:  Well, that may be something that other members on the committee may want to comment later but, again, sort of just playing a role his as chair, I would say that the discussions and the recommendations of the committee will all have to officially be taken in the context of if we don't know what exactly happened in that case, and I'm not, you know, saying that right now there aren't good reasons why we're not getting full disclosure on it ‑‑ I was just saying, without knowing that, at the same time all of us around the table knowing that, you know, there was a patient death involved, I think we have to make sure that officially, at least my view of this is, that that has to modify anything that we come to conclusion today.  Any questions from anyone else, Mahendra?
	DR. RAO:  I had a quick question about the monitoring on the patient side. How do you distinguish between the activated virus versus donor virus that you've put in to the patient if the seratype is the same, I mean, from wild‑type?
	DR. BAUER:  Yes, that's a good question.  There are hallmarks, molecular hallmarks of recombinant virus that you can look at.  And we have asked people if they have indication of a replication competent adenovirus arising in a patient to look at the molecular structure of that.  So you could distinguish that between a reactivation of a latent virus that had already been there and a replication competent adenovirus.
	DR. SAUSVILLE:  So, there are two potential, as I see it, components to the issues that we will be discussing.  One is a set of problems, such as the generation of replication competent that may occur at a given frequency, but affect a very special patient subset.  And then there's the more general types of toxicity that may reflect the range of normal, quote/unquote "host responses" to adenovirus co‑ proteins, et cetera.
	Does the agency have a perspective as to which of these components is most important or should dominant the discussion, as it were, because I think they lead us in somewhat different directions.
	DR. BAUER:  I think they're hard to separate those out, and I think that both are important, but I hadn't really thought this through before, but thinking about the replication competent adenovirus as an established infection is probably the area that I would like most feedback on at this point.  I think it is unclear and there, you know, there is some information on adverse events with ‑‑ in preclinical models saying that innate immunity and an immediate or very quick response to the adenovirus co‑ proteins is an important toxicity.  But the relationship between that and the adverse events in the Gelsinger case are probably ‑‑ that ‑‑ since that was a very quick relatively fast event, that probably reflects the kind of response that would be innate immunity and not so much an infectious event, but I think we do want to discuss infectivity of adenovirus products.  Does that help.
	DR. SALOMON:  Abbey?  I'm just going to let Dr. Mulligan respond, because I think he's going to respond specifically on this, and then go to you.
	DR. MULLIGAN:  I would echo the importance of Ed's point.  If you look at the Gelsinger case, I would think that it's not going to be because there is replication competent adenovirus that there is difficulties.  But it may be there is just more adenovirus than a human has ever seen. And so, I'm struck by the issue on the focus on the replication competents as opposed to other things that track with real recombinant adenovirus particle.  So, for instance, if there was proteins that are associated with the virus particle because of the cell type in which you propagated the vector, could that account for differences between the toxicities that you might see.  This is exactly the kind of thing I would think in the Gelsinger case could be an issue.  That is, you're not only putting in many, many particles but, you know, could it be that there's something in those particles, some contaminating host protein?
	Certainly, I know about retroviruses and in the case of retroviruses, it's very, very clearcut depending on how you grow the retroviruses, you get different host proteins that incorporate in the code.  Now it's a very different system than adeno, but I think that's an issue, perhaps, not for, you know, this conversation, but I think it's key.
	While we're on the replication competent question, I'm curious whether you went into the very old literature that looks at the helper, the e1‑like capacities of different cells, too?  One of the questions I was thinking, as you were talking about how you calculate the amount of RCA is, you know, is there a possibility that there are human tissues uniquely, a pocket of pituitary cells or something, that actually have e1‑like function and, therefore, you would have replication competent adenovirus vector in vivo, uniquely in those cells?  And Marshall, you know, might have something to say about this.  But that would be another way to look at this where all of a sudden the issue's not an issue of having a hundred or a thousand replication competent adenos, but 10 to the 6th or 10 to the 7th depending on what tissue you hit.  So, I'd like to hear if there is something, you know, do we really know that that can't happen is there any precedent for it?
	DR. KETNER:  Yes, there is data. In the teratoma cell line, there are complementing e1a functions that allow the virus to grow.  That's old data from ‑‑ it's quite old data ‑‑ state of the cells and I don't remember exactly which way it went, but one of the two states of differentiation of teratoma, human teratoma, did complement the e1a functions.
	DR. MULLIGAN:  That's interesting, from the tumor application, of course, you know, where, you know, if you had some undifferentiated strange tumor type and you were trying to direct infection, would it be possible that you would have replication, so it just calls into question, you know, how you'll actually measure and whether it is relevant to try to see whether there's pockets of tissues where you'll get replication competents.
	DR. SALOMON:  Well, I think that's very interesting.  Part of what you're saying, though is not that replication competent adenovirus wouldn't be an important thing to measure, but you're actually suggesting it ought to be measured also after infusion of the viral vector, perhaps at several different times.  And that's not something that was mentioned.
	DR. MULLIGAN:  More just the appreciation that their might be an interest in trying to push towards looking at this question of compensation.  And the other issue is, whether or not, unfortunately that complicates potential regulatory guidelines where, maybe the status of the vector becomes much more important.  Is it doubly, you know, how many different complementing events in the cell would you have to have to actually give something that would look like replication compromise.
	DR. BAUER:  Well, one thing, I can say is that you'll hear some data later about monitoring for virus shedding.  Or in the peripheral blood and looking at what is there and how long it persists.  So, the model that you're putting forth that you have complementation by e1‑like activity in the cells, I think we would probably see that by that kind of monitoring.  If you look at a virus that is increasing in titer or that there's a lot of shedding or unusual persistence, and you say that, then you could look at what is the structure of that virus. Is it what you put in that replicating for some reason?  To my knowledge, there isn't any evidence that that sort of thing's going on, but I think that is something to keep in mind, and it might be affected by, as Dr. Horowitz said, different kinds of cancer.
	DR. MULLIGAN:  The other thing is that in the simplest case, the virus that is essentially replicating in particular tissue, when assayed in culture, would not be replication competent, so ‑‑
	DR. BAUER:  Right.
	DR. MULLIGAN:  You might have a local effect of having replication competents, but you'd never really see it as such.
	DR. BAUER:  Right.
	DR. SALOMON:  Abbey, you've been put off a little bit, I'm sorry.
	MS. MEYERS:  Patient.  If there were something that happened in the Gelsinger case that is important for the purpose of discussion today, and especially would help us in the public health mission that we're supposed to have, I don't understand why we can't talk about it.  Now, if there's proprietary information, can we just meet in private without the public to discuss what we know?  And if it's not proprietary and there's some other reason, what is the reason?
	DR. NOGUCHI:  Well, it is a good question, I think on a practical basis, Dr. Wilson has actually recently published or is in press a couple of more‑ detailed analyses of the events surrounding Mr. Gelsinger's death.  In fairness, none of that really seems to shed any great light or insight as to what to really look for in terms of questions of whether it's RCA, whether it's an immune function, whether any of those are absolutely critical.  I think the available data would suggest it's really a set of immune, cascading immune responses to the massive viral load that really is most closely associated with his death and the spiraling events that we saw.  But that's really not anything that I think is any different than what was discussed almost a year and a‑ half ago.
	So I, your point is well taken, but I don't think we have anything that we feel would add substantially to our discussion here on that.  That's part of the issue.  The numbers of adverse events that are critical in terms of being much more than expected are relatively rare, and they're in a few different cases.  And that's part of the difficulty that we're facing here in that we don't have enough information, even on those cases, in order to be able to compare.
	I think that's one of the reasons that we have been focusing on creating a standard, which should have been available, probably, earlier, but now, by focusing on the standards, by focusing on all the different events that can occur and all the considerations, such as packaging of novel proteins, I think we can do a better job in the future.  But right now, we just, unfortunately, don't have a lot more that either privately or publicly we can add on the Jesse Gelsinger case, vis‑a‑vis leading us to a new approach or a better approach.
	MS. MEYERS:  So, for the purpose of today's discussion, then, the Gelsinger case, there's nothing being held back that we don't know about?
	DR. NOGUCHI:  That's correct.
	DR. SALOMON:  Phil, one of the concerns I have is, if we go forward, let's say, and at the end and get into discussions of sort of a maximal viral particle load that could be administered in a clinical trial ‑‑ which is one of the questions, indirectly here ‑‑ do we know what Jesse Gelsinger got, in terms of number of particles?  I know ‑‑ I've ‑‑ do we know what ‑‑ did we really know what he got?  In other words, was there an error?  Did he get more than we think?  I mean, that's the kind of thing, I guess, that my initial comments were aimed at, that I just see that as coloring our discussions somewhat.  Not necessarily implying that anyone's holding anything back, but just not know exactly what happened there is an issue for me.
	DR. NOGUCHI:  Well, that point is well taken and I don't have those figures right on hand, but it's clearly of the nature of close to 10 to the 13th particles, that's total particles and the transducing units was ‑‑ Steve, do you remember exactly what that way?  It's an ‑‑ it's an excellent question, we just didn't prepare for the ‑‑
	DR. SALOMON:  Right.
	DR. NOGUCHI:  I would like to suggest that a lot of these questions are of a more general nature and we'd like to make sure that we get all the prepared talks in here, some of the questions will be answered, many will not be, but that's part of what the more general discussion for the afternoon is about.
	DR. BAUER:  I just had one more comment in response to Dr. Sausville's earlier question and thinking about ‑‑ seeing the discussion that's going on now and where do we want to go?  I would like to focus more on the relationship between, you know, the risk for RCA infectivity and exposure in patients, rather than toxicity.
	I think the story that the viral proteins have toxicity, you know, is pretty well acknowledged and that the acute ‑‑ very acute toxicity is related to that dose. There might be a range of responses that we don't appreciate how to, you know, predict that when we first, you know, administer the product.  But I think if we could focus ‑‑ it's ‑‑ it's hard to say these are totally separate, of course, but we can focus more on the infectivity risks.
	DR. SALOMON:  Last question, Dr. Kurtzberg.
	DR. KURTZBERG:  Is there any data regarding the state of the patient whose receiving the adenoviral vector, in terms of their innate immunity or previous exposure to wild‑type adenovirus or whether or not they're shedding adenovirus at the time of the gene therapy?
	DR. BAUER:  When patients are enrolled, they're ‑‑ most of them, in the past, have been assessed for their immune status with regard to antibodies and to adenovirus and they're also looked at in terms of their ‑‑ whether or not they have a current infection, by adenovirus or other viruses, so those patients aren't ‑‑ these products aren't administered to those patients.  So you don't want to give something to a patient where there's a helper virus already in the patient, for instance, so.
	And you'll hear some data later about what was immune status and what was the outcome of the immune system response in certain patient populations, to the vectors.
	DR. SALOMON:  Thank you very much, Steve.
	DR. BAUER:  Mm‑hmm.
	DR. SALOMON:  So just to put this into context, then, our first obligation as a committee by the end of this afternoon is going to be to respond to the FDA's staff's specific questions.  And those are outlined in your book ‑‑ your panel.  And that's ‑‑ that, at some time we're going to have to just focus on answering those questions because that's what the job of this committee is to do.
	However, as is evident from the discussions that we just had, there are other issues and it's not necessarily meant to imply that those issues aren't as important in thinking about adenovirus.  So, to the extent that we can separate questions that we specifically have to answer from more general discussions of topics that are not on the docket today, then that would be extra value for the ‑‑ for the FDA staff.
	So, at some point here, I don't find any particular problem with the idea that there may be alternative things to talk about.  If you'll forgive me, ever‑so‑often focusing us down on that.  I think we'll have a fairly reasonable day, in terms of time, but that's maybe famous last words, so ‑‑
	MR. SIEGEL:  I just want to clarify on that, actually, we would much appreciate, and will benefit, time permitting from any discussions on any topics related to adenoviral safety.  And I think what Phil was talking about in terms of general questions was, simply, that we think it might be useful to have questions for clarifications to the speaker‑specific points after each speaker. But in terms of some of the general discussions we're getting into, it might be best to have all the speakers first, because I think they'll inform of those discussions.
	DR. SALOMON:  Yes, absolutely. Okay.  Well, then, it's my pleasure to introduce a new friend.  Dr. Estuardo Aguilar‑Cordova from the Harvard Gene Therapy Initiative, whose going to talk to us about history and overview of adenoviral vectors.
	DR. AGUILAR‑CORDOVA:  So, I was asked to just give a general overview of some of the adenoviral characteristics, which I think will be pertinent to the discussions and understanding.  I know it will be very repetitious for some of you.
	Also, then, to give a little bit of the nuts and bolts of what it means when people talk about titers for replication competent detection.  And, finally, to give you a little bit of background on the standard that is being developed by a working group with great participation by Beth Hutchins and Steve and Stephanie and others.
	So, historically, the adenoviruses were identified in the early 50s from an adenoid and they're an etiological agent for some of common cold‑ like symptoms and other inflammatory responses in various tissues, depending on the seratype, but they have not been identified as an agent of any tumerogenic potential in human tissues.
	They are a linear double‑stranded DNA encapsulated in a protein shell.  There's over 100 in the adenoviral group, two different characterized adenoviruses.  And the wild‑type has been used as a vaccine in military recruits, actually, of seratype 4N7 (?), and I think we'll hear a little bit more of that indicating some of the safety profile that is known in this type of viruses, at least when interaclly (?) delivered.  It's important to understand a little bit about the characterization ‑‑ physical characterization of the adenoviruses and it's an icoshedral.  And this is really quite critical:  13 percent DAN and 87 percent protein and that's how, back in the early sixties it was determined that 1‑OD (?) of adenovirus is roughly equivalent to 1.1 times 10 to the 12th viral particles, by comparing the protein characterization and then the OD readings and so, and it has been borne out by other types of analysis in the future that in fact that does hold true and that's a fairly accurate quantitative measurement.  The gene structure and organization of adenoviruses are ‑‑ has two ITRs and two origins of replication.  The transcriptional units are 5 "early" genes; 2 "delayed early" genes and one major late transcript.  What's important is that these are what has been primarily lated in the vector constructions and I'll go through that a little bit in a minute.
	They were able to be manipulated with vectors with two critical characteristics of the virus:  That it can package up to 105 percent of it's genome size, so if one can take a little bit off, one can also put a little bit back in.  And, also very important, it can be manipulated as a circular form, put back into the cell and it'll go into the linear transcriptional unit.
	So, the early antiviral vectors were considered replication deficient.  One could package up to about 8 kilobases (?) of foreign DNA by doing E3 deletions and E1 deletions.  It's relatively easy to produce it in high titer, it can infect a wide variety of tissues and it has high expression in non‑replicating tissues.
	So there's been an evolution of adenoviral vectors and, again, this will be, perhaps, important in our discussion of what the importance may be of RCAs and such. Original vectors, one would stick a gene of interest in the E1 region and that would be replaced, and it could have various deletions in the E3 region.  The E1 region, as I mentioned earlier, is a transcriptional activator that would then lead to expression of E3 for the other early transcripts and then, ultimately, to a cascade that would start the transcription of the late transcripts, most of which are structural proteins that would serve towards creating new viriants (?).
	However, as mentioned by Dr. Mulligan a little bit ago, there are multiple cellular proteins that have been described that are able to basically pinch hit for the E1 functions, including IL6 is one of the critical ones.  And that can, in fact, transactivate some of these early genes, which could then be toxic to the cell. We know that the E2 product and the E4 products, some of the E2 and some of the E4 products, are toxic to the cells and, in fact, there's an E3 product, which would be transactivatable because there are enough papa B and AP1 sites in that promoter, which could be upregulated and in different cell types.  And there's a protein that are called the adenovirus death protein, which serves towards lysis.
	So there are multiple ways in which a vector not being replication competent can still cause significant tissue damage and it can be observed in almost all cell types in vitro, where, if we put sufficient concentrations of replication deficient vectors, we will observe cytopathic effect.
	Now, the first‑generation vectors that I've been mentioning here are E1 deleted and they can be either E3 deleted, partially deleted or not deleted at all.  So‑called second‑generation vectors, went farther and deleted either the E2 or the E4 regions and transcomplimented in the production cell.  I will show you some data that may or may not have any effect on their toxicity.
	And more interesting, we get in the gray area which, what I'm calling here Generation X, I just ‑‑ because it doesn't really matter, but these are prototyped by the Onyx virus, which is, in fact an E1A positive cell, E1B minus, and that supposedly is effective only in P53 negative cells because the E1B would, then, bind the P53 under normal circumstances and absorb it out, so letting the cell go through the replication cycle.  So, putatively, when there's a P53 minus cell, the E1B would not be necessary in that case and this virus would then replicate more efficiently in those cells and it can, again, be E3 plus or minus.
	This is very close to a wild‑type virus and this is what's been used in all the Onyx trials.  There are Generation X.1, which are where they put either the E1A or the E1B with tissue‑specific promoters and then, theoretically, this would only replicate in the tissue where the promoter is active.  But as we all know, many of these promoters can be leaky, especially when they are outside of the genome context.  So, again, very gray zone as to whether these products are, in fact, replication competent adenoviruses and their measurement of what is an RCA in these would be a complicated issue.
	And this, perhaps, should have been called Generation Y, because it's completely different.  And these are the helper dependent vectors and the helper dependent vectors are those in which, basically all of the genome of the adenovirus has been taken out and only the ITRs and the packaging sequence remain.
	So, as I was mentioning in the sort of Second‑generation vectors, some data that was by O'Neal, {IOn}et al{IOff}., in human gene therapy back in '98, and what we can see here, this is platelet count and it's been a fairly repetitive observation in vivo, in humans that there is at least a transient thrombocytopenia that's observed and in some cases a consumptive thrombocytopenia after adenoviral vector delivery.  And we can see here that the deletion of an E4 or an E2 doesn't seem to generate much of an advantage with regard to the causation of thrombocytopenias.
	Also, when we see here the elevation of ALT, what we can see is this is 1 times 10 to the 11th, 1 times 10 to the 12th, all three of these experiments were done at 3 times 10 to the 12th and this is 1 times 10 to the 13th.  Down at the bottom are doses, the effects were not noticeable, either with a first‑ generation or second‑generation, only in the 3 times 10 to the 12th window was there a differential of any statistical significance, but once one gets to the 1 times 10 to the 13th, again, that significance is gone.
	And what's also of interest to note is that the difference between almost no effect to a complete, very significant rise is only a ten‑fold difference in as far as the quantity that was delivered to the ‑‑ to the animals.
	That's not to say that all of these things make absolutely no difference.  Here we have what I call the Generation X.2, or the helper dependent vectors and what we see in the liver enzymes in this case.  This is the first‑generation vector, one can see as the dose increase, there's an increase in the liver enzymes that then drops down, so it's self‑ limiting.  And that was not seen with this helper dependence, even at the same number of particles.
	So these are all equivalent particles, you can see the level of ‑‑ the duration of expression from this first‑generation vectors was very low, whereas the duration of expression from this later helper dependent vectors was much longer lasting, this is 8 weeks in this case. And ‑‑ but, also, that's not to say that this is always the case.  We have seen, in some of our monkey studies, where occasionally a monkey would develop immunity against the transgene and limit the effect at all.  But this does start to address a little bit on the ‑‑ at least at these low doses of incoming viral proteins that maybe it's not the low‑dose of incoming viral proteins that's causing that acute increase in liver enzymes, as here we have the same number of viral particles and we can see with the first‑generation there is some more acute toxicity.  It may have to do with the early gene expression that we see from these vectors.
	So, from this first part of it, we can see that adenoviruses can be converted into efficient transfer vehicles.  They're not inherently dangerous as a class of vectors or vehicles.  Not all adenoviral vectors have equivalent toxicity profiles, but some of the safety mechanisms that we might have heard or read about may not be that significant.  The dose of vector is related to the toxicity of serve, that is ‑‑ that's very clear and reproducible, the more we give, the more toxicity we observe.
	And I will now show you that standardization of dose specification is necessary and that it hasn't been there to date.
	So, characterization of viral vectors are two things, generally speaking: Purity and strength.  And lack of contamination by advantageous agents, including replication competent virus as part of the purity issue.  And the strength is the active concentration for toxicity and efficacy, just very loosely defined.  And I'll go through some of the things that we do here.
	Physical determination, as I've mentioned is the most common method utilized at this point, is absorption measurements and, as I said, from the early 60s it's been known that 1OD 260 is about equivalent to 1.1 times 10 to the 12th viral particles.  This has been confirmed by other methodologies, as well.  So, that's a very quantitative measurement.
	The next is the biological determination, that is the infectious units. And we've heard a little bit from Dr. Bauer here about how, right now, we're content to get 3.3 percent activity, that's ‑‑ the other way of looking at that is that there's 96.7 percent nonactive in each one of these preps.
	And how we determined this ‑‑ it has some physical characteristics and I'll go through some of these.  But the key issue here is the likelihood of the vector and cell ever meeting in your detection system, so that you could ever see it.  And there's some functional characteristics of the system whether the cell that you're testing it on has good receptors for the adenovirus and it's detected.  Most people are using either HeLa A549s or 293 cells for detection systems, so I won't go through these.  All of these are highly susceptible to adenoviral transduction.  But I will go through some of the physical characteristics.
	Typical titer set up has a culture dish, some cells at the bottom, adherent cells, and then one puts a mixture of virus on top of those cells.  The collision between the virus and the detectors cells here is mandated by Brownian motion of the virus, a concentration gradient towards the cells or external forces, and I'll go through each of these.
	But Brownian motion is random and for small molecules within a liquid at this temperature, it's basically negligible.  What that means is that, by Brownian motion, those viruses will never move from where they are, and these cells will mostly just see the viruses that are right here next to them, but they'll never see anything that's up here.
	And with the ‑‑ four or five years ago some experiments to show this and that is by putting different volumes of the same virus dilution onto some plates, we could observe the number of positive ‑‑ this is betagal, so we could see little blue cells, and these are averages over many, many wells. And what you can see is that it doesn't matter how much volume we put on top of it, the number of positives of serve was the same, which is what we predicted by just physical characteristics of the system, which, if we translate that to titers, we can see there's a significant difference in calculated titer, so how one sets up the assay will have a significant impact of what titer is calculated.
	We then did some centrifugation experiments calculating the displacement of the virus, if we were to centrifuge the plate, and that was just calculated as a distance between the pressures, RCFs put in, the time and the sedimentation coefficient. And by doing that, what we can see is that now we've brought down by spinning at 90 minutes for 1,000 RCF, we calculated that the distance traveled by any viriant would be .4 centimeters and we knew the depth of each one of these wells.
	So, at 50 microliters per well, all of the vectors should have come down and the same is true at 100 microliters per well. But at 200 microliters per well, the depth was greater than the distance traveled.  So we didn't expect all of the vectors to come down.  And, in fact, that's what we saw. These are the static conditions and the vector particle‑to‑ infectious unit ratio went from 22 to 82 under static conditions.
	When we centrifuged the vectors, we observed that there was exactly a two‑fold difference between 50 and 100 microliters, as expected and not a two‑fold between 100 and 200, again, as predicted, the titers are much higher, as you can see, we're now at 1.5 times 10 to the 12th versus a low of 9.8 times 10 to the 10th, and if you remember where the toxicity thresholds, a ten‑fold difference is very significant in whether there will be toxicity or not.  And the viral particle‑to‑infectious unit now are between 5 and 9.
	And I won't go through what all these calculations mean, but this is using the fixed law of diffusion and Stokes' equations and by that we can then calculate what is the maximum expected observed hits and what would be the true value behind that. And by doing that we come up with what we call the normalized and standard titer.
	And if we calculate it whether using the static conditions or using the centrifuge conditions, we come to very much the same number of titers and we see that the true value of the viral particle‑to‑infectious unit ratio in that preparation was really closer to 1.3 to 2.4 and 1.3 to 1.9, very, very tight numbers.
	And based on that, we conclude that the majority of the particles that are being produced in these high‑quality productions are, in fact, infectious.  And it is only a consequence of the detection system that we come up with the empty viral particles or non‑infectious viral particles that are often talked about.
	There's one other little bit of information that's critical within this and that is that there's an original titer and there's a clinical titer.  And it's important for people to keep track of the titer, not only at the point of production but, also, at the point of the clinical distribution.  This was a little experiment, it was published back in "Nature Medicine," back in 1999.  And what it showed was that when these vectors were being transported to the clinic in dry ice, even during a short period of time, the PH of the buffer that they were being transported in would drop, the virus would precipitate and one could lose seven logs of titer in a ‑‑ in a very short period of time, within hours.
	So that shows some of the problems that may be attached to quantifying these viruses, and also some ‑‑ why it might be very important and now then, why do we need to standardize and what is being done for the standardization?
	We need to standardize so that we have something to measure it against and we can all be talking about the same things. People talk about platforming units, CPE units, all kinds of different units and we in our laboratory, when I was back at Baylor, we had made a production and sent it to six antiviral laboratories to be titered, and there was a two‑log differential between the lowest and the highest titer that we received back from the people that had experience in titering adenoviruses.
	It's very important to have something that really ‑‑ is meaningful.  And when there's a threshold of toxicity between patients, and one group may base their starting dose or their continuing dose, based on another group's toxicity observed, if that has no correlation to each other's titer, then it really, it's very meaningless.  So, it's crucial for managing the manufacturing process but, also crucial for maintaining consistent, quality controls and dose escalation studies.  And, ultimately, of course, from the producer's standpoint, it will be critical for ever having a true product.
	And, lastly, I'd like to say that the standard doesn't need to be perfect at this point.  Since I've already pointed out to you how there is a lot of difficulties in where and how to measure these things.  And that, hopefully, will continue to evolve, but if we have a fixed point, then everything else that's around it can be related to that fixed point.  And if that fixed point is there as we talk about this square, we know that it's one down and one to the left, when we talk about this square, it's two right and two to the ‑‑ two down and two right.  That, also let's us measure what's the difference between this point and this point.  So that will be an equivalence so that we can all be talking about at least comparable units.
	This is the last slide and, unfortunately, one can't tell very much.  But there's a moral in here.  And this is just a quick note about talking of multiplicity of infection.  It's a fairly meaningless term and we're all very used to it.  It's a classic virology term, but as you can see here in these two diagrammatic wells, they're both a multiplicity of infection of one, we have the viruses here.  I guess it would be .3, but this is much more likely to reach the cells than this one is.  And, yet, they're the same MOI.  And when one goes fishing, one numbers the number of Marlins probably a multiplicity of infection of 10 to the 16th and 1 and there's 10 to the 16th of them, it's very hard for me to ever find one of those so, I would say that using the terminology of multiplicity of infection is not very useful in characterizing these viruses.  Thank you.
	(Applause)
	DR. SALOMON:  That was excellent, Estuardo.  So, one of the things that just occurs to me is that I found it very striking that there was such a difference in the titer when one did something as simple as move the virus from a production facility to the bedside.  And that's a concern, actually, we've had in our own laboratory with retroviral vectors, that many of them fall apart at rather small PH changes, and it sounds like the adenovirus is prone to the same thing.
	So how do we ‑‑ number one, have they figured out a way to address that issue, since that would be dramatic?  And, secondly, related to that, how does that fit into how one would manage the standard, because it could be quite a problem, if at different times, you standard was changing because of the conditions.
	DR. AGUILAR‑CORDOVA:  Yes, and so, the first answer is, yes, that the way to manage that has been figured out.  And it's fairly simple, you know, one just doesn't ‑‑ one protects it from dropping the PH in this case, either by a physical barrier or different packaging, formulation makes a difference and such.  And, you know, our industrial colleagues, in industry they have a lot of experience in this.  And what they do is, they usually validate shipping conditions and they validate that the product at the end has the same characteristics as the product in the beginning.  A lot of us in academia had that kind of experience in the past, so we weren't quite as aware of these things.
	And for the standard, it has been done vialing and it was an issue that's come up in our many discussions ‑‑ the vialing system, the formulation of it and the shipping ‑‑ storage and shipping, to validate that it has the same characteristics.
	DR. SALOMON:  Dr. Mulligan.
	DR. MULLIGAN:  Could you walk us through the exact context where the standardization would be helpful?  I can definitely see for detection of RCA how helpful having this would be, because you're looking at truly presumably the same thing, wild‑type versus a wild‑type.  In the case where you have ‑‑ people have vectors that are different ‑‑ that are not growable on, you know, 293 cells, so doubly deleted or more sophisticated things.
	I'm not sure I see how this would help guide standardizing the dose given to patients because, you know, the characteristics of the growth of these things in different cells is different.  How would you see that this would be helpful for that application, which is a very important application to make sure that some people aren't giving, you know, 1,000 times more virus than we know to be safe?
	DR. AGUILAR‑CORDOVA:  So, from the standard of particles, if one is working with an antiviral particle, a particle is a particle, so it doesn't matter how it grows within a cell.  So it still will standardize the measurement of viral particles.  In addition, to that, if one is working with what the majority of vectors are today, which are simple, that will grow in 293 cells and such, or whatever production cell line one is using, they will usually call a CPEF, a cytopathic effect on those cells.  And so one does not need to be measuring, necessarily, whatever the transgene is but, rather, the physical effect on those cells.
	And at least one has ‑‑ and the replication competent will cause the same issue.  So, at least one has the ability to quantitate it with regards to something else. It's unlikely that one would have a vector that would be that much more efficient than a wild‑type in causing the cytopathic effect.
	Now, if you get farther towards, say, a helper dependent vector, in that case, the standard would probably only be useful in as far as the particle number.
	DR. SALOMON:  So, getting back ‑‑ the standard is going to be wild‑type adeno?
	DR. AGUILAR‑CORDOVA:  This first standard is going to be wild‑type adeno.
	DR. SALOMON:  And it's going to be adeno‑5?
	DR. AGUILAR‑CORDOVA:  It is an adeno‑5, yes.
	DR. SALOMON:  Right.  So, how big a problem will that be for vectors based on adeno‑2 or 4?
	DR. AGUILAR‑CORDOVA:  For vectors based on adeno‑2, it'll be almost no problem at all because they're very similar viruses.
	DR. SALOMON:  Right now, are the criteria for the clinical trials that are ongoing as specific as would be suggested by some of your discussions?  Such as, when you do the titer counts, you have to centrifuge at so many RCF for so long in such a volume to do the sort of particle counts?  I mean, how much standardization do we have today on that?  And that may not be ‑‑ let me direct it first to you Estuardo, but then, perhaps, to the FDA staff.
	DR. AGUILAR‑CORDOVA:  To my knowledge, there is no standard protocol and, in fact, there is this system, including the physical calculation which we term NAS titer, or normalized adjusted standardized titer, that was proposed a few years ago, but, at this point, I think that there is no standardized protocol, and that's why a lot of the numbers, even that were received in the March 6 letter, which is now, one has to do the best that one can to interpret those, but a lot of those numbers are just not comparable.
	DR. SALOMON:  Steve, Joyce, do you have comments?
	DR. BAUER:  Yes, I have a comment about that.  It might be true that if you looked at different vectors from different people and, you know, gave each other your vectors, you'd get different titers, but we're also looking at the fact that it's one manufacturer whose done one set of preclinical studies and they're dosing ‑‑ in their preclinical and clinical studies based on their own measurements.  So, at least within their study, they are, you know, achieving a fair degree of reproducibility.
	The question is, how would that compare across studies and that's an important thing for us to make some of these recommendations and considerations about toxicity but also, I think that the dose that you start with in the clinic is always based on a preclinical study with your particular vector preparation, so that the comment that Estuardo made about basing doses, I think that's true to the extent that you might look at other studies to base your starting dose in the preclinical model, but then you would use that dose and your own experience in a preclinincal with your own product to determine what your dose range in the patients will be.
	DR. SALOMON:  And we won't get into it today but, of course, preclinical models are either small rodents or large animal models.  And I don't want to say the obvious, but for the record, you know, going from a dose in a nonhuman model to a human model has got it's difficulties.  And most clinical trial designs are done as dose‑escalation studies, particularly at this stage in gene delivery, so, you know, at some point here this dose issue is really ‑‑ is going to be critical.
	DR. BAUER:  I think, also, one of the benefits of having the standard is it will, in a way, bring people to use the same methodology, you were asking about that, but I think that's going to be an important outcome.
	DR. SALOMON:  I think that's great, yeah.  That's why I wanted to make sure that we got enough discussion of that.  Dr. Lawton and then Dr. Mulligan.
	DR. LAWTON:  I was actually following up on the comment that you just made and one of my questions was, from a practical perspective in the future, once the standard has been set and the methods have been set, are you assuming that everybody will, indeed, be working to that so that you have that measure, and that that will be a requirement that those methods are used so that you understand it across all of the different companies that are doing the work?
	DR. BAUER:  I think we haven't really seen what the outcome will be once we have a standard or ‑‑ let me just say one thing, we refer to it as a reference material, because the word standard in regulatory parlance has a specific meaning, so ‑‑ when the reference material is available, we haven't said that we're going to require everybody to do the assay the same way, but my belief is that, in effect, that's what will happen because there will be a titer assigned by a consortium of laboratories an agreed‑upon standard and I think, given the kinds of consideration that have just been pointed out.  I think people are going to have to use the same methodology, but if it ‑‑ if that doesn't work out, you know, we might go to saying, well, you need to do it this way so we have confidence that we can compare titers.
	DR. SALOMON:  Dr. Mulligan, do you have a comment?
	DR. MULLIGAN:  Yes, you raised this interesting question of the E1A plus E1B deficient vectors.  How has the FDA looked at those in terms of RCA?  I would have thought from what you see in papers that, you know, you can get virus titer, you know, in normal 293 cells in some of these.  There's certainly some controversy about this but there's certainly a number of conditional adenovirus vectors where you will see, although it's reduced ‑‑ that is, things that have a preference for tumor cells or whatever, you will see some growth.  How would you possibly get virus that passes even the old tests?
	DR. BAUER:  I think that's a good question.  The approach has been that we realize these replication selective viruses, in fact, it's a selective, it's not an absolute barrier, as you pointed out, but the approach is to say we asked to develop better assays to measure the degree of selectivity. And the other thing is from the preclinincal and clinical studies, we do keep in mind that these vectors are a different class, in effect, and, therefore, ask for increased clinical monitoring and that sort of thing. So ‑‑
	DR. MULLIGAN:  That's interesting because depending on how much you believe in how well they work, you might have a lot of ‑‑
	You know, they're shooting in a lot of these virus particles.  So, I mean, basically, they're as exempt from the normal standards for traditional vectors, is that right, in terms of helper.
	DR. BAUER:  We don't have a good handle on ways to measure it, that's true, but we respond to that by different clinical monitorings and different considerations for the preclinical studies.
	DR. SALOMON:  Jay, did you have a comment?
	MR. SIEGEL:  I have a question for Dr. Aguilar‑Cordova.  So, you show data suggesting that the sensitivity of an assay to infectious particles can vary vastly, depending on how the assay is done.  As you know, we've set a limit at that 3 percent ‑‑ 3.3 percent of ‑‑ at least, of particles should be infectious, I guess that was tightened up from 1 percent.  And that was based on what seems achievable in current experience.
	Is it your conception that, actually, a much higher level is typically being achieved and that those numbers reflect insensitive assays for infectious particles?
	DR. AGUILAR‑CORDOVA:  I can only speak to some of the data from our own productions and, in those cases, the majority of them are achieving that and better.  Now, having said that ‑‑
	DR. SALOMON:  The majority are achieving 3.3 percent or are you saying you're achieving ‑‑ because in one of your numbers, it looked like you were getting over 50 percent in one case?
	DR. AGUILAR‑CORDOVA:  Well, not, let's see, I think I showed data that showed that the Vp‑to‑iu ratio was in the neighborhood of 1.9, 1.3 so up close to that, I think.  And, again, I believe that some of that is just due to detection ability.  But having said that, there are some constructs in which ‑‑ even with our own system and, as Steve mentioned, very reproducible system within house.  Some constructs have a little higher Vp‑to‑iu ratio than others and we haven't quite figured out why yet?
	DR. SALOMON:  But, Estuardo, I also was confused by that, only in the context of what Steve had presented and what you presented.  It's a ratio of 1.3 ‑‑ I ──── mess up the math, particularly, when I'm up here not thinking straight, but 1.3 would be almost 75 percent, wouldn't it?
	DR. AGUILAR‑CORDOVA:  It would be about 50 percent ‑‑ close to 50 percent.
	DR. SALOMON:  Yes, so ‑‑
	DR. AGUILAR‑CORDOVA:  1‑to‑1 would be 50.
	DR. SALOMON:  So, that's a whole lot more than 3 percent.
	DR. AGUILAR‑CORDOVA:  Right.
	DR. SALOMON:  So, I'm having trouble with ‑‑
	DR. AGUILAR‑CORDOVA:  Remember, though, the data that I showed you ‑‑ what it also described is that from the very same virus vector preparation, we were able to have titers, I believe it was from 9 times 10 to the 12th to ‑‑ I mean, 9 times 10 to the 10th, to 6 times 10 to the 12th, depending on how we set up the titer assay.  And so, in one instance we're almost 100‑to‑1 and in the other we're at 1.3‑to‑1.  It's the same vector preparation, it's just detected in a different way.  And so, that just emphasizes the actual point, that it may not be an intrinsic characteristic of the vector preparation, but rather a consequence of your detection system.
	DR. SALOMON:  I can't think of any better way of articulating why were here today.  Marshall.
	DR. HOROWITZ:  Do you take off the input virus after a period of absorption?
	DR. AGUILAR‑CORDOVA:  We have tried that, yes.  And under static conditions, we basically see no difference in titer, if the virus is taken out of the well and placed on a different set of wells 12 hours later and, I think it was 24 hours later, as well, and the titer did not change.  Indicating that the majority of the virus was still in the supernatant and also indicating something about the stability of that virus.
	DR. HOROWITZ:  Yes, so what you did is the supernatant, replated, has the same titer the second time around as the first time around?
	DR. AGUILAR‑CORDOVA:  Correct.
	DR. HOROWITZ:  Saying that the extraction is relatively low, because that's what I was going to suggest.  And that's another way, although more tedious to really see what's there, until titer and exhaust, I should say, the supernatant.  By repeating titers, you obviously realize there's more virus there than you're scoring the first time?
	DR. AGUILAR‑CORDOVA:  Right.
	DR. SALOMON:  Dr. Sausville?
	DR. SAUSVILLE:  So, I think this discussion, though, illustrates a point that Alison Lawton was leading us to in the sense that, it's one thing to describe a reference standard, but it's ‑‑ but I think we're going to have to go a lot farther than that. There's going to have to be a, I think, a consensus, potentially, at some point as to how the assay's done, what the cell types, et cetera, so it's more than just a standard, because what this discussion is showing is that the concept of titer is really very context and assay dependent, and so it seems that without defining those things, you know that a standard is going to be relatively meaningless, actually.
	MR. SIEGEL:  On the other hand, it's clear that a standard is the first step toward evaluating what's the best assay. And, often, standards are sent to multiple labs for evaluation is where you determine what assays are sensitive and what are not.
	DR. SAUSVILLE:  Right, so I certainly take your point.  And the standard is the first step.  I just would certainly hope that we don't regard it as the last step.
	DR. SALOMON:  Yes, that's a key point, I think a couple of us have made it in different ways, so, that's great.  I mean, we owe that to Estuardo for focusing us on that. Any other comments or questions at this point?  I always, and I apologize to the audience, not having earlier said that we ‑‑ you're part of this discussion, as well, that's the purpose of these public meetings.
	So there is a microphone there. All I ask is that you identify yourself for the purposes of the record, but I would welcome any of you at anytime during this place to get up and make a comment.
	DR. HUTCHINS:  Hi, Beth Hutchins of Canji.  As far as the reference material goes, the real purpose of this reference material is actually to define a unit.  We're actually going to put the markings on the ruler and then later we'll figure out what's the best way to measure those ‑‑ measure ‑‑ how to use the ruler basically.  And it goes back to what Estuardo was saying in terms of, we're creating, in essence, an arbitrary unit.  The method is not so critical right now.  In terms of the reference material, we are, in fact, including the diffusion calculation correction.  That's being incorporated into getting that into the initial number for the reference material.
	And down the line, then we can look at the issue of methodology.  But we're creating a unit is what we're really doing. We're defining that unit right now, and that's why, in the end, laboratories, different companies, whatever, can use this reference material to validate their method. Their method may be different, but the unit will be defined and as long as they can report back units that mean the same thing, then we'll all be able to reference relative to each other regardless of the methodology that they're using.  In the end, it may mean that people move towards very similar methods, but it doesn't force people to do that automatically.
	That's also true for not just the infectious unit, which varies a lot but, also, for the particle, the physical particle number itself.  Because even though people do OD260 SDS methods, those methods, depending upon exactly how you do that, how you lyse the material and make the measurement vary also and I think the FDA has commented that they see variability even in that approach.
	So, even though you would think the physical measurement should be the same for everybody, in point of fact, a lot of us recognize that there are definite differences there, and so the reference material will also define the physical unit, which I think will also be very useful to the field.  So I just want to point out those two issues that we're defining units, not so much how you measure the unit.
	DR. SALOMON:  Yes, good points ‑‑ still, however, you have to admit that the definition of a unit, then, would allow us if we set, then, standards based on units, that would be very useful.  Yet, how one handles the reference material when it gets to the different labs in the field will also have a lot to do with how accurate the unit determinations are.
	So there's still going to have to be a significant amount of specification on exactly how the assay's done and that gets back to Dr. Sausville's point.
	Is there another comment, and then we'll go on to the next talk?
	MR. MURPHY:  Chris Murphy with Genzyme.  I just wanted to kind of clarify: The change in the particle to iu ratio that's being proposed ‑‑ is that going to fall in line with the addition and, I guess, correction with, you know, the calculation with Fick's Law?  And the reason that I ask is, you know, currently it's not uncommon to get a particle‑to‑iu ratio using a Spearman‑Carber calculation without correcting for the diffusion of virus up around 30 or 40.  If I correct for diffusion of virus, I can bring my particle‑to‑iu down to 1‑to‑5, that sort of thing.  I guess what I want to kind of verify is that is this change already going to be implemented now regardless of coming to a consensus on the titer assay?
	DR. BAUER:  I can respond to that. I think the methodology ‑‑ we have not, in the past told our sponsors the way that they need to do these measurements, but, of course we look at how they do the measurements.  So, if you have a measurement and a system for calculating the infectious particles that, you know, we have looked at and accept, so I think, in effect, we will be applying those correction factors.  I think that was it.
	DR. SALOMON:  Okay, then the next speaker I'd like to introduce is Dr. Beth Hutchins, Director of Process Sciences for Canji.  The title of her talk is Replication Competent Adenovirus Assays and Clinical Data for rAd‑p53 in Cancer Patients.
	DR. HUTCHINS:  Can everybody hear me?  All right.  I'm going to give a little bit of an overview of how RCA assays are typically done to just give you some better feel for, actually, the variation in that area and then talk, specifically, about adenovirus p53 vector and our methodology and then data from patients relating to RCA shedding and et cetera.
	There really are two sources for RCA:  One is that it can be created during the actual construction of the vector and this can happen with the most common methodology and really it was the traditional methodology until more recently.  And that is the large fragment method of recombinant adenovirus construction.  And that's where the recombination takes place in the production cell line.  And it does not matter what type of production cell line you're using.  It doesn't matter if it's a 293 cell oops ‑‑ did something just cut out or, okay, or PER.C6 or any of the more truncated E1A complementing cell lines that are now becoming available ‑‑ you want to adjust something?  In any case, if you allow the recombination event to occur in the production side and then try to select out viral plaques from that, what you'll find is whether you do serial plaquing or not, you don't eliminate multiple things in the construct.
	The newer E. coli recombination methods, where you do all your plasmid (?) recombination and then you select a then you select a single plasmid from the bacteria and use only that single plasmid to do your transduction into the production cell line can eliminate this as a source of RCA.  Most of the constructs, though, that are in the clinic these days were still made by the old large fragment method of production or construction.
	The other source of RCA is ‑‑ during production that is recombination frequency between an overlap in the E1 region of the cell line versus the vector backbone itself.  And the thing is, the frequency of that recombination isn't really known and it, actually, has to be estimated for every combination; that is for the overlaps that exist between your specific vector backbone and your specific production cell line.  Now the newer cell lines try to address that by specifically eliminating this ‑‑ these types of overlaps.
	In the field today, RCA testing is a bioassay involving either one or two cell lines.  The indicator cell line is most commonly the A549 cell line or a lung carcinoma.  And detection can be by a variety of methods:  Either cytopathic effect or immunostaining or PCR, as the end thing that you're measuring.  When you rely on cytopathec effect, generally, the assays are set up with a confirmatory step to show that the specificity is not something that ‑‑ some infection event of some other virus that just happened to occur during the assay that is, in fact, an RCA.
	There's no guidance on, you know, which to do this and you'll find that there's a variety of methods out there.  I think FDA can comment on that for the committee.
	Nonetheless, all of these assays are set up and qualified to be sensitive to 1 pfu or IU or infectious unit of RCA at some confidence level, hopefully, at the 95 percent confidence level.  And, in essence, what you're doing is a plus/minus or quantal assay.  And how you get quantitation then is based on the sample size that you test.  So, you don't get out from the way these assays are typically run out is I have X‑number of RCA in my material, what you get out is I have one or I have less than one in whatever amount I test.
	And this goes through an example of this type of method of quantification.  So, you test at different amounts of sample.  5 times 10 to the 8th; 1 times 10 to the 9th; 5 times 10 to the 9th, 1 times 10 to the 10th vector particles.  And you get negative results, no positives detected at the two lower levels, but you get positive results at the two higher levels of particles assayed. And what you then estimate is that the amount of RCA is less than 1 in 10 to the 9th or greater than 1 in 5 times 10 to the 9th vector particles, which, if the clinical dose is 10 to the 12th vector particles, then it does contain somewhere between 200 and 1,000 pfu of RCA is what we estimate.  There's no exact number that's coming out of this, it's a range.
	Part of that is the 1 pfu detection is also, of course, limited by the distribution of virus in a sample.  This is where the amounts of replicates of the RCA bioassay can become quite critical.  This last calculation is corrected based on your handout, somehow my math was not that good that day.
	Now, the construct and the data that I'm going to talk about more specifically relates to recombinant adenovirus that expresses the wild‑type human p53 gene.  The backbone of this vector is an E1a, E1b, protein IX deleted cassette with also a partial E3 deletion that completely inactivates the E3 expression.
	These deletions were created to allow the insertion of the p53 expression cassette in the E1 region and we purposely put the protein IX deletion in to decrease the overlap between the vector backbone and the 293 production cell line, which is the cell line that we use for production of this vector.  That decreases it, actually, from about 1,000 base pairs down to 200 base pairs, just for background information.
	We had ‑‑ we've done pretty much of the routes of administration that  Dr. Bauer showed earlier.  The data that I'm going to talk about relates to these trials and these patients.  We've done intratumoral injection, with 72 different subjects; intrahepatic artery infusion, with 50 different subjects, with doses up to 7.5 times 10 to the 13th particles, though most of ‑‑ a good majority of those patients got 2.5 times 10 to the 13th particles; and also by the intraperitoneal route, with 54 patients and the dose ranged in that case up to 7.5 times 5 to the 13th particles; and the majority of those patients got 7.5 times 10 to the 13th particles in a dose.
	So, the bioassay for RCA that Schering Plough (?) uses to test our production lots of the p53 adenovirus is, again, one of these quantal bioassays, and this one has a CPE readout.  We use a two cell line method because the p53 gene has a particular effect on the A549 cells that we wanted to avoid and so the indicator cell line, though, is still the A549 cell line. It's a four‑week ‑‑ typical four‑ week assay. We use PCR to confirm any positives so that we know that we are detecting RCA, if we do detect it.  And it is sensitive to one pfu of RCA.
	Now, it's sensitive to one pfu RCA, but because of plasone (?) distribution, when you want to validate the assay and show that you have 95 percent confidence level for detection of something, when you do triplicate tests of ‑‑ or you do an N of 3 on testing your material, we can, with 95 percent confidence, detect 4 pfu of the control spike.  If we wanted to have 95 percent confidence in one pfu detection, the end jumps up quite dramatically and it's not practical to do that.  And the only RCAs we've ever been able to detect, either through a process ‑‑ a validation study or that come up in actually assaying production lots are, really, the dl327 backbone, that is the p53 expression cassette's been kicked out, the E1 gene looks normal, the E1 region looks normal again, but it still has the E3 deletion, that is part of the parental backbone from which the vector was originally derived.
	The specification for the 58500 p53 adenovirus clinical product is less than 40 pfu of RCA in 7.5 times 10 to the 10th viral particles.  Each batch is tested either in triplicate at 2 times 10 to the 9th particles, which is, actually, the way it's been done for the ‑‑ about the last 2 years. Or it's tested earlier the first years that we were doing this, it was tested and not at one but at three different levels.
	Based on the assay confident levels for the triplicate test at just 2 times 10 to the 9th particle, if we find ‑‑ if there are no positives in ‑‑ of the three tests, or there's 1 of the 3 is positive, then the batch meets the specification.  If two or three of the tests come up positive then the batch fails the specification because there would be greater than or equal to 40 pfu in that 7.5 times 10 to the 10th particle amount.
	And this data just represents the summary of the validation data, looking at the confidence levels relating to this triplicate testing.  And so, you can see that we have 95 or better than 95 percent confidence in testing ‑‑ we can detect the 40 pfu in 7.5 times 10 to the 10th or, because we're testing at 10 to the 9th for pfu detection limit.  And that this confidence declines dramatically, which is why zero or 1 can meet the specification but more than that, obviously fails the specification.
	Now, if you look at or as we look at our protection lots over a period of a number of year, what we find is that a certain percentage, roughly 10 percent consistently fail the specification.  And ‑‑ which means that they would have greater than the amount of 40 pfu in the 7.5 times 10 to the 10th vector particles; and about half, based on testing at the three different levels come in right on this ‑‑ right where you end up in ‑‑ if you look at the 7.5 times 10 to the 13th vector particle dose, you would have about 4,000 RCA pfu maximally. And about, the other half would have ten‑fold more than that.  So at our highest dose levels, we could have put ‑‑ we could have dosed a patient in a single dose with as much as 40,000 pfu of RCA.  Now, we can't say exactly how much we dosed with except that it's in a particular range, just because of the way the assays are set up, so you need to keep that in line.  But it is, obviously, more than the numbers that Steve was talking about earlier.
	Now, I'm also going to just briefly the type of methodologies that we were using to monitor the patients clinically and to look at various biological samples from them and these methods included both specific and nonspecific methods.  So, one nonspecific method is an ELISA that detects the hexon antigen.  This does not distinguish intact virus from just parts of the virus, and it also does not distinguish product from RCA or wild‑type.
	Another type is an infectious assay on 293 cells.  In this case, we're using fluorescence ‑‑ a flurocytometer and fluorescence to detect infectious adenovirus. Again, this does not distinguish the type of adenovirus that is an RCA a product or a wild‑type infection, but it does say that it is an intact infectious virus and not just pieces of virus that are in the biological sample.
	We also had a variation of this assay where the sample was placed onto A549 cells.  And, again, this detects infectious virus, but it only detects either RCA or wild‑type and does not detect product because the product cannot replicate on an A549 cell.
	And, finally, we had PCR assays that you could use those to specify what you were detecting, that is, you could specify that it was RCA or wild‑type adenovirus or the product, but one caveat of that is that you're detecting DNA and it does not tell you that that DNA represents an in tact virian. So, these methods detect different things, but they do allow you to get certain valuable pieces of information, depending on the method you're using.
	The types of biological samples I'm talking about are either urine, feces, sputum, saliva, any variety of things and, also, several of the methods have been used to monitor serum samples, blood samples.
	Of the studies that I mentioned earlier and the patient numbers, here listed by the various routes of administration and then the four different methods.  These are the ‑‑ just looking at a variety of biological samples but just now looking at the issue of shedding.  You'll see the only method that specifically you can look at RCA, these 2 methods, actually, but in this assay, we found, in 63 different samples we ‑‑ or from 63 different patients, we did not detect any shed RCA.  This one positive here is product, not RCA.  And these ‑‑ these were all product‑related positives here.  Now, you can see, also, it's a very low rate of shedding, but no RCA's been detected in any of the samples we've analyzed.
	So, what are the types of populations we're studying?  This vector has actually been developed for ‑‑ was being developed for specific cancers.  And so all the patients were cancer patients.  All these subjects were initially selected to be antiadenovirus positive prior to entry onto the studies and, of course, not to have any evidence of an active adenovirus infection and what we did know that the neutralizing capacity of these antiadenovirus titers before they received any of the adenoviral p53 vector varied considerably in the population.  But always, consistently, increased and increased dramatically with dosing with ‑‑ after administration of the vector.
	And for the subjects were ‑‑ the very few subjects we had were adenoviral shedding was detected, we were not able to identify any adverse clinical sequeli.
	This is just to give you an idea of the type of information you can get from monitoring patients looking at their ──── antibody response, this is now for patients enrolled in the ovarian interperitoneal administration study and this is a multiple dosing phase of ‑‑ portion of that phase 1 study, and in this case the patients we all receiving 2.5 times 10 to the 13th particles or 7.5 times 10 to the 13th particles, I think about half and half in this case.  They also did, at the same time receive chemotherapy.
	So ‑‑ and the way the drug was dosed is there are five days in a row where the drug is administered each day at that does 7.5 times 10 to the 13th particles, and then 4 weeks later another cycle of 5 days of dosing occurs.  So, this is referring to one cycle of dosing, another cycle of dosing and another cycle of dosing.  And you can see that this is now average data, and I have to say that the arrow bars are not ‑‑ are fairly wide, but it gives you a sense that you do see some drop in the hemoral response but, of course, memory comes back and you see that rise again and it continues to go up and up and by the time you're out past cycle three in later studies we did up to five cycles, of course the neutralizing capacity is quite dramatic.
	Now we do also have evidence, though, that we're still getting transgene delivery and expression at cycle three and we had very limited samples that told us the same thing for out to cycle five.  So, even though there's this very large neutralizing hemoral capacity in the body, we were still getting delivery of our vector.
	Just to get to the summary points, the bioassays that are used are quantal, but they are sensitive and they can reliably detect RCA, at least in replication deficient vector products.
	The more precise quantitation than this quantal method where you get, sort of, this range of RCA that you can estimate in your material is impractical in this bioassay mode, because of the amount of testing that was required.  The amount of cell culture that would be required is what becomes impractical.
	If the desire is to have a specific quantitative number come out of a particular method, you would have to consider real time PCR as an option, looking for quantitating more specifically.  No one's doing that right now, routinely, but that would be another way to look at the RCA issue.
	We were not able to detect any shedding of replication competent adenovirus in our clinical subjects.  There was not PCR positive for RCA and no infectious assay‑detected RCA.  And where we did find shedding, there were no adverse clinical sequeli identified of the shedding's really very low amount and less than, you know, about maybe 1 percent by the infectious titer assay that was used to look at that.  And I think that's basically it.  So, I'd be happy to entertain questions from the committee.
	DR. SALOMON:  Thank you, Beth.  I'm remiss in not having allowed the panel a break, so ‑‑ one option here, would be to do some questions, I think, while it's fresh in our mind and then have a short 15‑ minute break if that's okay with everybody.
	I had a couple questions.  Some of them may come up with others, so let me just ask one of them and then we'll see how things come along.  One of the things I find very interesting is this whole idea of what are the molecular mechanisms that generate the recombinant ‑‑ the replication competent adenovirus through recombination in these models.  So you have some evidence here that you gave that the RCAs were rising by replacing the p53 transgene cassette with E1, did I get that correct?
	DR. HUTCHINS:  Well, the RCAs that we've been able to detect their structure is all that the E1 ‑ the p53 expression cassettes not there and the normal E1 region is there, but the rest of the viral backbone which has this E3 deletion, that's backbone on which the vector is based is the same. So, you don't have a substitution just at the place where, in fact, the large fragment recombination was supposed to be taking place.
	DR. SALOMON:  Right, so, again, I'm not an adenovirus ‑‑ so this could really be stupid, but what ‑‑ so what I'm trying to understand is when you constructed the vector, you did a number of different things; you deleted E1a, you deleted E1b, you deleted E3, and you made changes in protein IX to reduce the homology (?) right?
	DR. HUTCHINS:  Right, from a practical point of view, what's done is or what was done in this case is the dl327 adenovirus, which has the E3 deletion in it already was the starting point.  And then the expression cassette was constructed through bacterial plasma technology.  And so, you have sort of the front‑end of the virus constructed in bacteria and you have what really is going to become the back‑end of the virus be from a virus from the adenovirus dl327.  That virus is digested using specific restriction enzymes, claw‑1 (?) is most frequently used to get thee large fragment. And, basically, is what it does is you end up with the adenovirus but from the end of E1 on, and what you're doing is asking these things to recombine, now you're really, just the part of the virus that has the expression cassette and the front‑end with the ITR to recombine in the producer cell line.
	This is the most traditional way that this is done, regardless of whether you have an Ad2 backbone and Ad5 backbone and what other elements you're putting together, that's what's traditionally done.  It gets more complicated when people have additional deletions, like in the E4 region, so they change what the back‑end is.
	DR. SALOMON:  So, I guess the question I'm asking is, in this process of engineering the vector ‑‑ I mean, part of my thinking here is, if we could figure out the nature of creation of recombinant adenovirus, I mean, replication competent adenovirus through these events, we could suggest that that be part of the criteria upon which one would accept a clinical vector in a trial.
	So, you made all these changes, you reduced it to 200 base pairs instead of 1,000, yet, you still got recombination with E1, so is that telling us that these are not ‑‑
	DR. HUTCHINS:  We think it was there right in the beginning ‑‑
	DR. SALOMON:  ────
	DR. HUTCHINS:  The virus material because of the way we did ‑‑ we created it, that it was always there and no matter how hard we tried to, basically, subclone it out by serial plaquing, we didn't ‑‑ we don't achieve that, at least not in ten rounds of serial plaquing.
	DR. SALOMON:  Okay.  So there were ‑‑ there was already the backbones to create the replication competent adenovirus in the process of engineering the original transgene?
	DR. HUTCHINS:  That's our hypothesis for this particular vector, and we think it's a pretty common experience.  Now, at the time that we were using that technology in the earlier nineties, we didn't really recognize that that could be a consequence of what we were doing.  As we have more data and really begun to understand better what ‑‑ the consequences of what we were doing, we now moved to a method where we do everything in E. coli, select the specific thing that's the intact vector and that's what goes to create the intact viral ‑‑ the viriant, so ‑‑ and now that source, would be limited, and now the only way you could get RCA is through recombination of events that occurred during production.
	DR. SALOMON:  Okay, well, that would explain something Estuardo said, then, too.  Good.  Ed.
	DR. SAUSVILLE:  But to pursue that direction of thinking, we, in past meetings, have set out the general idea that vectors of certain sizes should be sequenced and there should be precise definition of what goes into a product.  And we stated that for higher molecular weight, or higher sized genomes, larger genomes, there can be a point of ambiguity in relation to the gene of interest as opposed to the background.
	So, what this leads to is that, if recombination frequency is going to be the major determinant of this in the next generation, do we need to firm up the sequence definition, flanking the proposed target gene and try and use that for a basis of hedging our bets as to what the recombination frequency would be?  I place that on the table.  I mean, I'm not an adenovirus expert, either, but it seemed that might go part of the way to dealing with this.
	DR. SALOMON:  And that may be something that we want to take up right after lunch when we start going into the questions. But I certainly would welcome any comments.
	DR. HUTCHINS:  Well I have one, and we're one of the groups that fully sequenced our virus early on and submitted that information to the FDA and the ‑‑ there's limits of detection to what you're going to be able to see in there if you have a low‑level variant when you're doing this sequencing, both strands, full length.  To detect very small quantities of some other molecular variant, you would have to do a different type of analysis and study than you do when you're just trying to sequence the material and just say this is the sequence of my product.
	So, sequencing of your vector, the requirement that I think Dr. Sausville's referring to in terms of your earlier discussions and recommendations, is not, by itself, the answer to address this issue.
	You're talking about some other type of molecular analyses where you have to really look at and I hope the committee does not recommend this today, but you would have to look at, you know, a large number of clonal isolates of your vector from some kind of production lot or a number of production lots and then try to analyze those and that would be, you know ‑‑ one of the issues, I think, that's being raised today is, well, okay, we could maybe collect that type of data, but if the risk of that material in your product isn't very high to begin with, what is the necessity to do that.  You know, what's the benefit that's gained from that.
	DR. SALOMON:  I would say that's exactly what you need to be telling us today. That's exactly the kind of reality check from people doing this that I want to hear and that the committee wants to hear and the FDA wants to hear.  Dr. Ketner and then Dr. Siegel.
	DR. KETNER:  You mentioned that you constructed your E1 replacement to minimize the overlap between 293 cells and the recombinant.  So, there are cell lines which are engineered to have no overlap, I mean, PER.C6, I think, is intended to be that way. I wonder whether you've had experience growing your viruses on PER.C6 and whether, in fact, the RCA level drops to zero, as in principle, perhaps it ought?
	DR. HUTCHINS:  Your vector backbone still has to match the PER.C6 cell line construction.  So if I took my current p53 vector and grew that in PER.C6, assuming I even had ‑‑ okay, the way ‑‑ I could still, potentially, get recombination because my vector backbone does not, at least the way it's currently set up, does not currently match the way the PER.C6 construct is.  So, what Crew Cell (?) will tell you is, if you want to use their PER.C6 system, your vector backbone ‑‑ they'll give you the ‑‑ or they'll offer to build it for you ‑‑ the specifics that you need to know so that you don't have that overlap.  You actually need to know what the E1 region is in your producer cell line, so then you can make that match‑ up.
	But then, again, if you still do large‑ fragment recombination as the way to create that vector and you make it in a PER.C6 cell line, you're still going to end up with variant molecular variance in your material.  You would still have to create that change, that matched backbone, outside of the producer cell system, such as through an E. coli bacterial system, and then put the selected thing in to not have RCA.  And there are other cell lines that are designed the same way.
	DR. KETNER:  Given that, I mean, given that this is in principle a possibility, maybe we ought to at least discuss the notion that the RCA levels be reduced to, I mean, to zero by choice of appropriate sub strains.
	DR. HUTCHINS:  I would argue that that's the point of your discussion today, what the risk is and what is the necessity.
	DR. KETNER:  Yes.
	DR. HUTCHINS:  In countries where that's the requirement, they don't have clinical trials going on.
	DR. SALOMON:  Well, then that's ‑‑ that's good.  I mean, we ‑‑ that's what we need to discuss, I agree.  Dr. Siegel.
	MR. SIEGEL:  In discussing the sensitivity of your assay and the ability to use the virologic the viral culture for precise quantitation, you referred to issues of practicality and number of replicants. With the quantal assay, if you have a single hit, kinetics then ‑‑ with enough replicates if you, and you have a plasone distribution, you an get a rather precise quantitative in most systems using, you know, the, say, 96 cell plates or whatever there's not that much practical limitation to getting reasonably precise ‑‑ I wonder if you would speak, since I also don't work with adenoviruses, what are the limits?  How many cultures can be done with how much effort?  Is there a problem doing larger numbers?  You said you did three replicates ‑‑
	DR. HUTCHINS:  Yes.
	MR. SIEGEL:  Because more would be hard?
	DR. HUTCHINS:  Yes, typically, when people are testing near the level of the FDA guidance in that 10 to the 9th, 10 to the 10th particle range, because there are ‑‑ as Estuardo mentioned earlier, the more ‑‑ or the higher the concentrational particles that you're putting onto your culture, the more likely you'll see toxicities that will actually affect the assay.  This is, you know, this is just a byproduct of using cell lines and high concentrations of the material.
	So, this means that in order to test, say I wanted ‑‑ decided that I wanted to test 10 to the 11th particles each time I did my assay.  In order to ‑‑ and also still kept my pfu sensitivity to 1 ‑‑ and I would have to use a larger number of cells or more wells or roller bottles or flasks in order just to look at that 10 to the 11th particles, and now, if I want to do that with a certain percentage confidence level in my ability to detect that 1 pfu, I've got to do that same number of flasks x‑times each time for each lot.
	And, you know, I have to say that I'm very happy to be affiliated with a corporation where our lot sizes were quite large, but I know that many institutions, the lot sizes aren't that big, and so that ‑‑ that can be a real burden.
	The other thing is that because it's a quantal assay, the quantitation comes out of the amount you test, not ‑‑ because you're only checking one or none, basically, right?  So, it's yes or no.  And so the amount you test drives the quantitation in this bioassay method.  So, that's the other reason why most people only test to about 10 to the 10th, because the amount of cell cultures that's involved.
	And the other point that I should point out is that when you do this test, you don't just do the material by itself.  You do the material by itself, the material with a spike, so, I mean, you already have these other controls built in that just in a single assay, you have other cell culture, not just the culture involved with the actual replicate numbers of the unspiked product that you're testing.
	DR. RAO:  We talked about a reference standard earlier.  So if you were to use the reference standard here would you just use it as a spike of the wild‑type instead of what you used, or would you have some other method in your system?  Or would you use the reference standard?
	DR. HUTCHINS:  Well, I would not be using the reference material directly, because I don't want you to use up all the stuff we produce.  What I would like people to do, and what we're going to be recommending is that people create an internal reference material that they tie to this material that they'll be able to use. But what the reference material will do is define the unit, the infectious unit, so now when I say I'm spiking or I'm making sure my RCA assay can detect one infectious unit of RCA, it'll be the unit's that's defined by this adenoreference material.  And I'll make sure that my RCA assay is validated based on that ‑‑ the ability to detect that unit.  So, does that clarify that point for you?
	DR. SALOMON:  Dr. High.
	DR. HIGH:  With respect to some of the questions that we are asked to answer this afternoon, I wonder if you could tell us something about this group of cancer patients that you presented data on?  You have about 200 people with non ──── and head, neck and colon cancer and ovarian cancer and so forth. Are there requirements that these are all people who are relatively early‑stage?  Do you know anything about their ‑‑
	DR. HUTCHINS:  They were all ‑‑
	DR. HIGH:  Immunologic status ‑‑
	DR. HUTCHINS:  They were all relatively late stage, heavily pretreated, I mean, these were phase 1 studies, so this was definitely ‑‑ this was not a neoadjutant type of situation, this was, you know, they've already exhausted a lot of their other options.  Nonetheless, they had to have relatively good performance status, so that we could distinguish disease‑related effects from, presumably, product‑ related adverse effects.
	So, but they are, you know, treated ‑‑ on the other hand, we have evidence that at least many aspects of their immune capacity were quite well in tact.  Hemural immunity as well as cellular immunity from cytokine profiles and some other data that I'm not presenting today.
	DR. SALOMON:  Marshall.
	DR. HOROWITZ:  Is it appropriate to ask about the 50 patients that had intrahepatic artery infusion with an average of 2.5 times 10 to the 13th ‑‑
	DR. HUTCHINS:  Thirteenth.
	DR. HOROWITZ:  Times the 13th, yes. Would it be appropriate to ask about toxicity and chemokine and other measurements and expression in liver, is that expanding too much?
	DR. HUTCHINS:  It is appropriate to ask that ‑‑ we have reported, for instance in the December '99, I guess it was RAC Safety Symposium, we reported in some detail our safety data ‑‑ clinical data, and also preclinincal studies related to that and that route of administration and dosing.  We, in fact, did see dose limiting toxicity.  You notice that in our ovarian trials we heavily focused ‑‑ ended up with the 7.5 times 10 to the 13th particles.
	Now that's by a different route of administration, that's tolerated quite well in patients.  But at 7.5 times 10 to the 13th particles, by the intrahepatic arterial route, that is not tolerated in patients. But the effect was on ‑‑ was a cardiac toxicity, in fact, not a hepatic toxicity. And we went down then to being able to establish that 2.5 times 10 to the 13th viral particles was a safe dose.
	One of the things that ‑‑ we did not reinitiate our IHA protocols after the Gelsinger death, although we could have.  We had permission to do so.  But for a variety of reasons, including our own concern about how close were we to, you know ‑‑ the difference between 2.5 and 7.5 isn't that big.  And while we felt very confident that we were defining our particle dosing consistently and, you know, accurately, we just felt that it wasn't worth the risk at the moment until we had more information.  So we started to focus more on the ovarian IP route of administration after that.
	DR. HOROWITZ:  Did you measure chemokines circulating at that time?
	DR. HUTCHINS:  Actually, in the IHA studies we did and I'm hoping, very soon, that manuscript will actually be published, but I think Dr. Bob Warren has actually presented that type of data at several forums, including at the Recombinant DNA Advisory Committee.  We do see cytokines IL6 (?) and such in both in serum as well as in tissue, we've looked at that quite extensively.  I mean, there's definitely a local immune reaction in liver and there's a systemic immune reaction, but there's no evidence to us that RCA is related to that. I think that gets back to general ‑‑
	DR. HOROWITZ:  Right ‑‑
	DR. HUTCHINS:  Viral toxicity.
	DR. HOROWITZ:  These questions are not so much for RCA, but for the total dose ‑‑
	DR. HUTCHINS:  Right.
	DR. HOROWITZ:  And if I may ask, were any liver biopsies done and evidence of expression of the transgene in the liver?
	DR. HUTCHINS:  Yes, yes.  And there was, consistent ‑‑ once we got above a certain minimum dose, we had consistent transgene expression in both tumor and non‑tumor samples from the liver.
	DR. HOROWITZ:  Thank you.
	DR. SALOMON:  One question that I had, let me see, is just a simple one, you know, from my calculations and these, again, are flawed when I'm up here, I'm not usually thinking as straight as I should be, but I think it means that about 50 percent of your production runs would not be acceptable if we adopt the new guidelines that FDA staff has suggested?
	DR. HUTCHINS:  Well, if you read the guidance, there's always the ‑‑ there's always the ability to collect data to support a different specification and then discuss that with the agency and determine if they agree that your data supports that different specification.  That is, in fact, the type of thing that we did ‑‑ to have a different specification than the less‑than 1 in 10 to the 9th that was in the original guidance. So, we have preclinical studies that support ‑‑
	DR. SALOMON:  Well, my question though are you okay that this is going cut ‑‑ is this going to cut out 50 percent of your production runs, and is that okay?  I mean, I have no idea what that means.
	DR. HUTCHINS:  If it becomes an absolute rule, yes, it would, but I ‑‑ I think the FDA needs to comment here on how they would apply that.
	DR. SALOMON:  I'm sorry, Alison, did I get that wrong?
	DR. LAWTON:  Can you just clarify that, Beth?  If it becomes an absolute rule, my understanding is, yes, it would cut out 50 percent of the lots ‑‑
	DR. HUTCHINS:  That's correct.
	DR. LAWTON:  And the question is, is that acceptable?
	DR. HUTCHINS:  No.
	DR. LAWTON:  Right, thank you.
	DR. HUTCHINS:  You need to understand we're pro ‑‑ we produced the vector ‑‑
	DR. SIEGEL:  But ‑‑
	DR. HUTCHINS:  At a fairly large scale.  You're talking about vector runs where we had 10 to the 16th particles produced at a time, not that we would make purification batches on that same scale, but, I mean, and the RCA level would be consistent in that entire, you know, viral culture batch, so that would not be acceptable.
	MR. SIEGEL:  I think, though ‑‑
	DR. SALOMON:  And that's what I was trying to get you to say.
	MR. SIEGEL:  There needs to be clarification.  We've not proposed a rule, we're proposing a guidance.
	DR. SALOMON:  Right, exactly.  And, I mean, I just wanted some feedback on, you know, what the field thought of that, you know, guidance before we get into it this afternoon.  Dr. Flomenberg and then Dr. Sausville.
	DR. FLOMENBERG:  Phyllis Flomenberg, Thomas Jefferson University.  You mentioned that you prescreened all of your patients for antibody to adenovirus, was that sera‑type‑specific antibody?
	DR. HUTCHINS:  The test was ‑‑ the assay was designed, you know, the adenovirus type 5, but how specific are ‑‑ we did not then go back and look at whether those antiadenovirus titers that we measured were only ‑‑
	DR. FLOMENBERG:  So, it wasn't a neutralized assay?
	DR. HUTCHINS:  To ad‑5 versus to any other adenovirus isotype.
	DR. FLOMENBERG:  So ‑‑
	DR. HUTCHINS:  We wouldn't care, actually.  That wasn't what we ‑‑ the question we were asking ‑‑ we were only looking at the ad‑5, but to say that it was only ‑‑ those responses were specific to ad‑5, I can't say that.
	DR. FLOMENBERG:  Was it a neutralizing assay?
	DR. HUTCHINS:  We did both kinds of assays, the requirement for entry did not discuss neutralizing capacity, just that they had to be seropositive.
	DR. SALOMON:  Dr. Sausville.
	DR. SAUSVILLE:  So a question that the introduction of the clinical trials data that you alluded to in the cancer patients and also in your response to the question about viral replication in both liver and tumor, do you routinely quantitate the expression of coxsackie adenovirus receptor in tumors and does that influence or could that influence the potential perception of infectability of a given lot ‑‑ given ‑‑ depending on the use of the material?
	DR. HUTCHINS:  We did look at that in a number of our Phase I studies. Actually, the IHA study and the ovarian IP study.  In the IHA study, there was a relationship between CAR level and expression.  In the IP study, there was not in tumor tissue.  Why that is I can't say, but besides the fact that there's CAR and, of course, the secondary integren receptors are required for internalization, there are still undefined receptors that most of us believe exist and we don't know what they are, and so there can be other mechanisms for entry of adenovirus into tissues.
	And just to correct a point:  When I was talking about detecting expression of the p‑53 gene, we don't believe that the vector was replicating in those tissues.  You were saying, just to clarify ‑‑
	DR. SAUSVILLE:  But, I guess it ultimately raises the question, when one uses a standardization procedure that's based, presumably, on a given report or cell type it has a certain receptor and entry mechanism. Is it clear that that's the same receptor and entry mechanism that might either mediate a toxic or therapeutic outcome in the various contexts that the virus would be used.
	DR. HUTCHINS:  I'm sorry, you're asking in terms of an infectious titer assay?
	DR. SAUSVILLE:  Right.
	DR. HUTCHINS:  You ‑‑ it would not really be feasible to set up an infectious titer assay to represent the human condition in terms of the variety of tissues and, you know, depending on the indication you were interested in, the route of administration, whether this was a direct in vivo administration or an {IOn}ex vivo{IOff} situation, I don't think the intention is to say that infectious titer assays should reflect all aspects of what you're going to be running into, that's not going to be possible.
	I think it comes back to what is the ‑‑ what is the practical outcome of a reference material?  If you ‑‑ if we define infectious unit and it's going to be based now on the wild‑type virus that we have the most clinical information about from a safety perspective in terms of what's known in terms of the natural infections.  That ‑‑ it goes back to the point Estuardo made that arbitrary unit will help us determine the relative activity of everything else.  I don't expect my p‑53 adenovirus to be as infective from that unit perspective, regardless of how I assay it, as ad‑5 wild‑type.  It ‑‑ I don't expect that.
	And there are a couple or reasons for that:  One, the gene of interest can have an impact on infectivity and cytopathic effect or expression of a gene measured by immunostain, however you decided to do your end‑point measurement for infectivity.  Many of us are well aware of the affects of different genes that we put in the expression cassettes on infectivity measurements, which can impact or is not accounted for in the diffusion corrected calculation.
	The other thing is that it's somewhat crippled in terms of its ability to replicate even in the context of a complintation cell line like the 293 cell because we cut out some other things that an enhance replication of the virus.  So, the relative IU unit, however that ends up being defined by the reference material will assist us from looking at these things across the field, but one, I don't think you should expect that my recombinant vector A and my recombinant vector B actually all have the same infectivities.  And, in fact, the issue, then, of the vector particle‑to‑infectivity ration will probably have to be revisited after this, if you start looking at the complications of that issue.  I mean, that's not a subject for today, but it's ‑‑ potential outcome of having this reference material defining infectious unit for us.
	DR. SALOMON:  I think that's a good place to be, I mean, you're defining that the task today is to look at sort of advice on standards for the preparation.  And what Dr. Sausville's pointing out is another issue that's going to have to be dealt with is what happens after you give it to the patient. We've kind of touched on that, already, before.  Last Comment?
	DR. MULLIGAN:  Let me go back to the method you used to make the original vector.  I'm not sure I see the reasoning ‑‑ is there any data that suggests your contention that that's what accounts for the increased helper?
	DR. HUTCHINS:  I'm sorry, you're talking about the large‑fragment methodology?
	DR. MULLIGAN:  Yes ‑‑
	DR. HUTCHINS:  Of ‑‑ I would contend that a viral plaque does not ‑‑ I know this is really against traditional virology ‑‑ but I would contend that a viral plaque does not necessarily contain just one viral entity and that's what we're relying on to pull out of what you've produced during construction, the thing that you then want to ultimately amplify.
	DR. MULLIGAN:  Well, I mean, I assume most people would do a multiple plaque purifications.
	DR. HUTCHINS:  And they have and ‑‑
	DR. MULLIGAN:  Yes, I mean, I would just make the comment, I doubt that's the case and the alternative is that, in fact, the vector you have for whatever reason has a different recombigenic potential than other things.  And I think that it's very hard, I mean, I don't think there's any good ‑‑ that very directly attributes the recombination potential to an adenovector's ‑‑ in a linear fashion to the amount of overlap, it's more complicated.  But there's a sense that that has to be the case that there's going to be trend towards more recombination.  But I think it's equally likely that different precise, unusual contexts of foreign sequences juxtaposed to vector sequences will lead to different amounts of recombination, that's why ──── question ‑‑
	DR. HUTCHINS:  I didn't say that that wasn't necessarily true and I didn't present data today supporting why we think there are two possible sources of RCA.  The fact remains that you could have recombination during production.  And you would have to examine that very specifically for the vector you're looking at and the production cell line you're looking, okay. So, in that sense, you are correct.  But there is data to support the other ‑‑ the other way of creating a source of RCA and I'm not presenting that today.  That's all I can say.  And, obviously, no, I have not made that data available to you and we hope to do that soon, but not today.
	DR. MULLIGAN:  But this is key because Ed was asking questions if we every want to think about giving input on how one goes about making original adenovirus construct, which I probably don't think we'll do, this is important to have a sense of how likely is it that that really is an issue. And it's also important if there will be constructs that, in fact, tend to have a greater propensity for recombination, even when they're put into PER.C6 or other kinds of fancy patching cells.
	DR. HUTCHINS:  I guess, in respect to your ‑‑
	DR. SALOMON:  Let me interject ‑‑
	DR. HUTCHINS:  Mission today, though ‑‑
	DR. SALOMON:  Yeah.
	DR. HUTCHINS:  I don't think the mission today is to discuss how to eliminate RCA or sources of RCA.  I think the mission today is to discuss what is the risk when you have RCA, because the reality is, regardless of what means we might have to not have it in the future, it ‑‑ we do have it in today's clinical lots, every single one of those 58 active INDs, you could be sure there's RCA there.
	DR. SALOMON:  What I was ‑‑ let me interject ‑‑ I think that it's important to also protect the speaker in the sense that, you know, the deal here is that Beth and Canji agreed to present a certain set of data and you've done that.  And I'm not saying that the line you're taking now isn't relevant to the overall field, but, you know, she's not going to be able to show us that data and she never agreed to show it to us.
	DR. MULLIGAN:  Yes, the issue, I think, is that in the briefing document, the FDA indicated that when they looked at the answers to the March 6 letter, they had a sense that, I think the term was, "routinely" people obtained less than such and such and, therefore, the FDA made, I think, a probably reasonable argument that that's a reasonable limit ──── and so, I mean, maybe there should be some discussion on the reasoning behind that argument.  I mean, it seems straight forward that if most people can attain that, maybe that is reasonable.
	Is that, indeed, the argument, that you are making?  Because it will be very relevant in how you set that and we need to know precisely how you came to the recommendation, and if it is precisely because there was, in your look‑see, there was a routine capacity to get that level ‑‑ that low level of RCA, then I assume that means that you think that that's a reasonable basis for setting that as the limit.  And that's relevant, then, to people happen to be not routine.
	DR. BAUER:  No, that's precisely the way this recommendation came about was looking at the data that we had and saying, I'll use your terminology, routinely people could achieve that level, but I did mention in my talk there was a range where some manufacturers had zero, some have as many as 40 percent of their lots rejected.
	And the other point that's relevant is this is a recommendation that can have, you know, qualifying considerations that are applied to it.  So ‑‑
	DR. MULLIGAN:  That means that, I hate to harp on this, but I think it's very key ‑‑ that means that the feeling of the FDA is it's not unreasonable that if the state‑of‑the‑art ‑‑ if the common state‑ of‑the‑art can achieve something, that that should be the standard.
	DR. SIEGEL:  Absolutely, we ‑‑ you know, for any of a number of types of contaminants, you know, there's a long history in biotech products of setting different standards, say, like, for e‑coli dna present or for LPS present, you know, there are two approaches to setting a standard and I think Steve outlined both and both have good reasonable logic.  One is to say it's reasonable and feasible to achieve a certain level and you should achieve that level.  Another is to associate a certain level with a risk and ensure that the level is set below the risk.
	The reason one often goes to a ‑‑ there are two reasons one might choose the first to set a standard based on ‑‑ one might be one simply does not know what level there's risk at, where the other might be the assumption, as with radiation, that there is a ‑‑ there is no cut point that risk exists at any level, it's just proportional to level and it should be kept lower.
	And, I guess, one of the things we hope to get out in discussion here is whether we should be moving more toward risk‑based levels, as opposed to achievability‑based levels.  A risk‑bask level, one might imagine, would be different in different populations, one might imagine would be more based not on the per‑‑total‑virus particle, but per‑dose given, for example.  And there might be times where we want to combine the two and allow ‑‑ we're working with guidance ‑‑ and allow, in certain populations or in certain dosing regimens, a different limit might be appropriate and that's ‑‑ I think that's the gist of what we hope the discussion will guide us toward.
	DR. SALOMON:  Okay, I think that this is a good time to break, and this is ‑‑ what I know that some you don't know is that there's no public comment that's been requested.  Now, we will call for public comment anyway, as part of procedure, but we have an hour that, you know, we're taking up with some of this discussion, which I think's been good.
	So, what I'd like to do is take a ten‑minute break now, with the idea that we all know 10 minutes will turn into 15, no matter what I say.  And be back here and then Dr. Flomenberg will give us her talk.
	(Recess)
	DR. SALOMON:  If we can kind of bring everybody in.  Cognizant of the fact that oftentimes some of the most interesting things get done during these breaks.  And I certainly learned a couple things that will come up later.  So, I don't see anything bad about a little longer break, but I'm going to get in trouble with time soon, so ‑‑
	Couple of just real quick things, and I mean really quick:  One, I'd like to welcome Dr. High to the table.  She, unfortunately, had some transit problems, again, we've all been there and don that, but anyway, welcome to the Committee as a new member.  Also, Dr. Simek?
	DR. SIMMICK:  Simmick.
	DR. SALOMON:  Simmick, has joined us.  Dr. Noguchi had something that he had to do and Dr. Simmick has been involved actively in the adenoviral working group within the FDA, so now we have three of the FDA staffers directly involved in and we'll take advantage of them later.
	Okay.  And I made one egregious ‑‑ yeah, well that's your job, you know, you get paid the big bucks for this.  I made one egregious mistake and I apologize to Dr. Sublett.  I just missed it, there was Ad‑5CMV‑p53 in the title of two things and my brain skipped over them.  So, anyway, the next speaker, before Dr. Flomenberg is Dr. Richard Sublett, Director of Quality Systems for Introgen.  And the topic of his talk is ‑‑ where is Dr. Sublett?  Oh, sorry. Absence of RCA Isolated from Patients Treated with INGN 201 and Ad5CMV‑p53 Construct, Dr. Sublett.
	DR. SUBLETT:  I'd like to thank the Advisory Committee for inviting me to speak today and talk about Introgen's experience with RCA and our Ad5CMV construct.
	Very briefly, we're going to discuss replication competent adenovirus levels in INGN 201.  We have data from a semi‑quantitative assay and also from a qualitative assay and we're going to compare those and establish the levels of RCA that we believe we have in our product.  We're going to talk about patients who have treated with INGN 201, the numbers of patients, doses, and essentially give an estimation of the RCA exposure to these patients.
	Then I'll talk about our attempts to isolate RCA from patient samples, and I'll very briefly discuss the immune status of these patients.
	INGN 201 is another ad5‑p53 construct.  It is an E1 substituted adenovirus also, and E3 partially deleted construct.  And within the E1 region, there is a p53 expression cassette.  It is produced in the 293 cells that we've been talking about quite a bit today.  And you can get double homologous recombination events between the flanking regions of the 293 host cell and the expression cassette, which result in low levels of RCA in the product.
	We've already talked about this in great detail, but this is just a cartoon showing how you can get the double homologous recombination.  We've used two assays over the past several years.  Most of the clinical lots that we have used were released using a qualitative roller‑bottle CPE assay, or cytopathic effect assay.  Lately, we have started working with a semi‑quantitative plaque‑like assay to get a better handle on the actual levels.  Both of these assays were looking at 3e10 viral particles and, in general, we believe we have approximately one RCA and three E10s, we're testing right at the limit of detection.
	Back to the cartoon ‑‑ this is a kind of a silly cartoon but, essentially, this box represents a vial of our product. There are a few RCA in our product.  And our ability to detect those, largely depends on how big our net is.  For logistic reasons, our net is about 3e10 viral particles.  So, if we take one swipe through this box, we may or may not pick up one of these red balls. And that's a very simple‑ minded way of looking at it.  But to address  Dr. Siegel's question of, well, can we just ‑‑ we're testing 3e10 viral particles, what about 3e11?  Already the assay we're working with is 40 tissue culture plates, and to increase that to several hundred tissue culture plates is logistically a nightmare.  And, in fact, most of your QC assays would become invalid because of one reason or another with that size of an assay.
	The consequence of testing at the limit of detection, now, is lots with the identical RCA level may test as positive or negative.  So, you're not going to get good consistency.  Also, if you do multiple tests of the same lot, you may get inconsistent results.
	This is a comparison of the two assays.  In the first column, well that's just the lots that we've prepared.  The second column is the number of RAC plaques that we identified in assays on these lots. And we have compared that with the CPE data that we obtained on those same lots.  The level of sensitivity for both these assays is quite similar, with the semi‑quantitative assay we had 8 out of the 14 were positive with either 1 or more RCA plaques.  With the qualitative assay, 6 of the 14 were positive. What you see highlighted in yellow is where you have disagreement.  We had, in both columns, positives that were negative with the alternative assay.
	Also, please note this is a little bit of our problem lot B2029701.  This lot was released using a CPE assay where it tested as negative and, in fact, this is the highest level of RCA plaques that we've ever seen.  So, these results really just are not consistent.  And, again, I want to emphasize this is a consequence of us testing at the limit of detection.  I would like to say we've used this lot extensively in the clinic.  It was one of the lots we used a great deal.  That's because this assay was not available at the time the material was produced and released for clinical use.
	We have tested a total of 35 lots of INGN 201 at the 3e10 viral particle level. These are the results of the plaque titer assay.  And about half our lots test with zero plaques.  And then we've also seen 1, 2, 3, and 4 and then we've had that single lot where we saw the 7 RCA plaques.  Again, what I want to emphasize is, I don't think these data argue strongly that these lots really have fewer RCA than these lots.  The assay's not that sensitive.  And, again, that's why we're calling this a semi‑quantitative assay. We just aren't testing enough viral particles to make this a quantitative assay.
	Our conclusion is that all of our lots contain some RCA and the level is consistently low, approximately 1‑to‑2 RCA and 3e10 viral particles.  One think I should emphasize here, when I ‑‑ we're talking about viral particles tested, but this is an infectious titer assay, so these are, essentially, pfus or infectious units.  So the number of viral particles is greater than that and that goes back to talks we've heard earlier today.  This is just to emphasize, this is a very small number of RCA we're talking about.
	Using these data, we can come up with an estimate of the number of RCA that we have exposed patients to.  Through June of last year, we had treated 406 patients. We've treated about 100 patients since that time, so these numbers are out of date. About ‑‑ over 2,800 doses, and we have given a little over 3 times 10 to the 15th total viral particles to these patients.  The RCA just says vp, but that really should be pfu administered is probably on the order of 2 times 10 to the 5th.  So, the total RCA exposure in these patients is low, but it is definitely there and we don't want to pretend that some patients were not exposed to RCA and others were.
	This is more of the same type of information.  The maximum dose that we have given in our studies was 3e12 viral particles.  This would be an RCA exposure of about 150, again this say pfus and not vps. One patient had received ‑‑ had gone through multiple cycles, he had received a total of 4.6 e13 viral particles, so over 2,000 RCAs have been administered to that patient.
	So now that we've discussed that, I want to move on to our clinical experience. In Phase I clinical trials, we've done ‑‑ I don't want to talk in great detail, because there's been a variety of Phase I clinical trials.  Most of these were cancer of the head and neck.  As a rule, most patients were treated by intratumoral injection.  The dosages ranged wildly or not wildly, but, widely, since this is a Phase I clinical trial ‑‑ we were in control every time we dosed a patient, it just varied.
	The doses were, essentially, 2.5 e12, up to 1e12 viral particles and in these patients, essentially, we looked at urine and plasma and, also, some oral rinse samples. And these were tested for the presence of RCA by replication on the A549 cells.  These cells are not permissive for the ‑‑ our construct, which is E1 deleted.  So, this requires ‑‑ this is specifically looking for the RCA.  We looked at acute samples and chronic.  The chronic would actually go out to close to 12 months after treatment was concluded.  And the assay sensitivity in these studies was less than 10 pfu in a 0.5 mL sample.  And in 3,200 patient samples, we no evidence for RCA in any of these samples.
	We also have data from three Phase II clinical trials, looking at plasma, urine, feces, and oral rinses from these patients. These patients were all cancer of the head and neck.  To address an earlier question, these were all late‑stage patients, so they were all quite ill.
	We did PCR for Hexon and p53 sequences and we also looked for CPE.  It gets a little complex:  The plasma samples were all prescreened by PCR before the CPE testing was performed.  All the other samples were tested by both PCR and CPE.  And I'm focusing on the CPE results, again, going back to Beth Hutchin's talk, the PCR, certainly, can detect the presence of DNA, but we do not know if that's from intact virus or just a DNA fragment, so I'm focusing on the CPE data here.
	The CP test was performed, initially, on the 293 cells, which would be permissive for the E1 deleted adenovirus, our product.  If it was positive on the 293 cells, we then passaged that material onto the A549 cells, which are not permissive for the E1 deleted adenovirus.
	Okay the three studies ‑‑ the first one was T201, again, had neck cancer.  The dosing was 4e11 to 2e12 viral particles. There were two treatment cycles; one, was treatment on days 1, 2, and 3, the alternate cycle was 1, 3, 5, 8, 10 and 12.  We collected the samples for biodistribution studies pretreatment and on day 28.  So, in this case, if it was this treatment cycle, it would be 25 days post‑ treatment; on this one, it would be 16 days post‑ treatment. This trial treated 107 patients.
	202, similar subject profile.  The dosing was lower in this study it was 1e10 to 4e10.  Treatment was on days 1, 2, and 3. Again, the samples ‑‑ the biodistribution samples were collected either pretreatment or on day 28, 47 patients were treated.
	And then, finally, we had study T207, this one, daily dosing was 1ea2 viral particles, they all received the same treatment.  They were dosed either on day only or days 1, 3, 5, 8, and 12.  The biodistribution samples were more extensive in this study.  They were collected every other day following the last treatment of the cycle, through 15 days post‑ treatment.  I also want to say that this is a treatment cycle, but the patients did go through multiple treatment cycles, or they could go through multiple treatment cycles.  And most of them did go through multiple treatment cycles.  And that's true for all the studies. Would you go back, I'm not quite through with that slide.
	Thirty‑six patients were treated. Also, in this study, we looked at the immune response, adenobody response to the virus. We also evaluated at least one household member from each patient's family that was treated in the study.
	Just looking at the CPE data for these three trials:  T201, we had 459 samples were tested for CPE, 8 of these were positive on 293 cells, this is ‑‑ would be our construct or permissive for our construct; none of them were positive on the A549; 201 a similar results, 175 samples were tested; 3 positives on the permissive cell line; none on the nonpermissive cell line.  And then, on T207, we had 880 patient samples, not ‑‑ this does not include the household member samples, 18 positive samples.  We did isolate two CPE positives on the A549s, but both of these were sera type 11 adenovirus, it was not related to our construct.  Before we move in, all of these samples were tested by southern blot analysis to look for genetic rearrangements of the virus and there was no evidence of any genetic rearrangement in any of these.  They all appeared to be identical to the original construct.
	So the results is, we could isolate virus or its DNA in body fluids up to four weeks post‑ treatment.  This includes the CPE treatment.  We, I think day 25 or so, we actually could get a CPE positive on the 293 cells in at least one or two cases.  The only CP positives on the A549s were not related to our construct at all.  And while I did not show the data in the household members, there was no evidence of horizontal transmission of the virus.
	The immune response ‑‑ this slide might be a little complicated to follow. This was only on Study T207.  We had data from 29 patients pretreatment and then 22 after 1 cycle and 9 patients after cycle 2; there are also some data after cycle 3, but this shows for the point I'm trying to make.
	The ELISA is an antihexon.  ELISA, the neutralizing is actually just the ability to inhibit a plaque formation or I should say CPE in an assay.  We did have a number of patients had low titers at the beginning of the study, about half of them had no titers at all.  Somewhat fewer had neutralizing titers.  Two patients actually had very high titers at the beginning of the study, this was not an enrollment criteria.  And one of these patients here, is in fact this patient right here, so not only did he have very high ‑‑ I say, he, I don't know if it's a he or a she ‑‑ had very high antihexon titers but, also, moderately high neutralizing titers as well.
	After the first cycle of treatment, which could be either one or multiple doses, most of these patients were starting to mount an immune response and this was both the ELISA and the neutralizing titer.  And then, after two cycles, only one of nine didn't respond.  This patient is the same patient, so one out of the nine patients did not respond with either an antibody titer against hexon or a neutralizing antibody titer.
	So, our conclusions is INGN 201 does contain RCA; in our assay that is 1‑2 RCA in 3e10 viral particles.  This is very near the limit of detection so this number is fairly soft, but based on looking at a number of lots, we're fairly comfortable at this level.  We think the levels are fairly consistent from lot to lot, generally only a single lot tested with seen, they've always been less than 10 RCA.
	As a consequence, patients treated INGN 201 have been exposed to low levels of RCA.  We have not been able to isolate RCA that had been derived from INGN 201 in patients or household members and we've looked fairly extensively.  We've see no adverse events that appear to be related to RCA in these patients.  Most patients treated with INGN 201 developed antiadenovirus antibody and this is both antihexon and neutralizing titers.
	And, finally, we are not comfortable with the current guideline of 1 RCA in 3e10 viral particles, we would like to see that reconsidered based on topics that have been discussed earlier, and these are largely risk factors.  So, we think the patient population, the indication of the product, and the available safety data should be taken into account.  Thank you very much. (applause)
	DR. SALOMON:  That was excellent. I had one quick question.  And that is, if we know molecularly that replication competent adenovirus would require reinserting the E1A ‑‑
	DR. SUBLETT:  Mm‑hmm.
	DR. SALOMON:  ‑‑ or the E1 segment, rather, couldn't we ‑‑ wouldn't it be simple to design an assay using quantitative real‑time PCR for the E1 segment and so, obviously, there's something stupid about this question because you guys would have done this, so, you need to educate the chair.
	DR. SUBLETT:  Well, I'm not sure I can answer it well.  One concern that's been raised, and this may be discussed in the next talk, although I'm not certain, is many of these patients, there are latent adenovirus that, as the patients proceed are started to be shed and looking for the E1 region and we have a concern that looking for the E1 region may not be due to our construct at all, but may be due to these latent adenoviruses that have become activated.
	DR. SALOMON:  I'm assuming you know the E1 sequence in your producer cell line, or you can go back to the lot and do this?
	DR. SUBLETT:  There's some overlap, although I'm not sure that I'm qualified to address how much it is.  We have discussed this assay, but I think there is merit in that argument.
	DR. SAUSVILLE:  The other issue is that PCR wouldn't tell you anything about the function of the product that that came from so ‑‑
	DR. SALOMON:  I guess what I'm saying, Ed, is that if ‑‑ if the molecular mechanism of creating a replication adeno is now recombination allowing insertion of this, then, if you could quantify the number of particles that have this inserted element, that would actually relate directly to the number of RCA in the prep.
	DR. SAUSVILLE:  But it would not only be the number of nucleic acid events that you could detect by PCR but the functionality of them, because, supposing, you could detect that, but they were nonfunctional viruses, it wouldn't be ‑‑ it wouldn't be an effective virus.
	DR. AGUILAR‑CORDOVA:  That is part of it but in addition, during the process of production, you have the ‑‑ that sequence coming from the cellular DNA and it can be stuck or carried with the virus.  So, at the early stages, because there is small pieces of DNA that are on the outside of the virus or maybe they're on the inside, as well.  So, at the early stages of ‑‑ or with the very actual product, that's a difficult one to decide where the cut‑off is.  However, through a cycle of amplification and most of these tests do go through a biological amplification, as well as the ‑‑ or can go through the biological amplification as well as the molecular amplification ‑‑ and they can become a little bit more feasible.  And such assays do exist and people do run them.
	DR. MULLIGAN:  I don't think that was a dumb question.  If you had PCR primers across the junction, you know, obviously, you could generate something that would show that, and I think it would be useful, even though, of course, you're not showing it as functional, but you're directly ‑‑ as directly as possible measuring the frequency of the event and you might be interested.
	DR. SALOMON:  That's what I was thinking, where you'd have primers that would be above the junction in the 293 indigrant (?), which would tell you that it was DNA that came from the producer cell line and others that were crossing the junction in, you know, so you'd have ‑‑ you're have your viral construct and you'd see where it got reinserted, but ‑‑
	DR. AGUILAR‑CORDOVA:  In the producer cell line, you would have exactly the same sequence that you would have in the recombinant because of the crossover, but once it goes through one noncontaining cell lot, then it's ‑‑
	DR. SALOMON:  Dr. Rao.
	DR. RAO:  I just had the same question that we had for the earlier speaker. If you adopted the new standards of the recommendations, am I right that all the lots that you have would be then ‑‑
	DR. SUBLETT:  Approximately half the lots that we've tested would have been negative at that level, but first of all, we find rejecting half the lots commercially unacceptable and this is a commercial enterprise, although we certainly ‑‑ many of us are motivated from trying to do a little bit of good here.  But, also, and this is one thing that testing at the limit of detection means is the lots that we rejected, in our view, have the same RCA level as the lots that are acceptable and in some ways, it's become very artificial.
	DR. RAO:  Can I extend that question?  So, taking, presumably, what are higher levels of RCA than what are current proposed guidelines ‑‑ in any of your immune‑compromised patients that have been used in this trial, have you seen anything ‑‑ I mean, you presented three studies with patients, which suggested that there was no evidence of any effect of RCA?
	DR. SUBLETT:  That is one of the things, I was trying to point out in the last slide, and maybe I didn't do so well.  I think you do need to take the patient population into account.  There have been reports on the severely immunocompromised patients and, perhaps, those should be excluded from using adenovirus constructs with these types of RCA issues.  But the patients that we're looking at do not appear to be that severely immunocompromised that we have any evidence problems to date.
	DR. SALOMON:  One of the things that came up in the break was a question that, have there been any documented cases of replication competent adenovirus in any patient group causing a disaster?  I mean in the sense that a lot of us in the retroviral field have been sensitized by work in non human primates and a couple patients where we had RCR?
	DR. SUBLETT:  Not to my knowledge, that hasn't happened.  It does get muddy, in all honesty, because both Beth Hutchins and I are really looking at similar patient populations, they're similar constructions, they are late‑stage cancer patients and, in fact, the death rate is fairly high in these patients.  But it has been associated or believed to be associated with the underlying disease that may make seeing some of these things hard to see, but I have not heard of any case where anyone really felt that RCA was a contributing factor in any of the adverse events.
	DR. SALOMON:  I think that's an important thing for the Committee to keep in mind.  Are there any comments from anyone else on the Committee on that, in terms of the literature?  Yes.
	MS. MEYERS:  Do you know how many of these patients are still alive?
	DR. SUBLETT:  I do not know the number, but I do know it's fairly few from the ‑‑ certainly, the Phase I trials, it's very few.  There are a few from the Phase II trials, but these patients, typically, were having life expectancies of 3 to 6 months when they entered the trials.  And, in fact, most of them have passed on.
	MS. MEYERS:  So, it's clear, then, that these patients really don't live long enough for you to see what the long‑term effects would be?
	DR. SUBLETT:  Although in one patient, we followed him for ‑‑ or more than one patient, we followed them for a year and we haven't see evidence of RCA.  But the number of patients that we've followed for that long is fairly small.
	MS. MEYERS:  Mm‑hmm, thank you.
	DR. SALOMON:  Would the sensitivity of your assay be significantly improved if you went to 10 to the 11th or 10 to the 12th viral particles, instead of 10 to the 10th? I think this is a question that came up to Dr. Hutchins, as well, and so I just want a reality check, also, on whether that would be, yes, but it would be difficult to do commercially?
	DR. SUBLETT:  With our assay, I mean, the answer's clearly, yes.  The standard deviation on ours, essentially, we averaged about 1.3 RCA per assay, and if you look at the standard deviation, it's 1, so we have, essentially, 100 percent error in this assay.  That would ‑‑ if we increased the assay size ten‑fold, which would be difficult, that standard deviation would probably drop but, in fact, if we average ten RCA in the assay or if we tested 3e11, I guess it would be 13, you would still have a sizeable standard deviation, because that's still a small number of plaques to get a good statistical number.  But it would, clearly, be better.
	DR. SALOMON:  So, just so, again, just so we have a clear reality fix here, the problem with these assays is that you're tending to go to the borderline of their sensitivity, even really beyond them, frankly, because it's just a ridiculous amount of supernatant to be able to do the assay right?
	DR. SUBLETT:  Well, more ‑‑ no, it's actually more complex, even than that. Putting on more virus on the same number of cells is not so much a volume issue ‑‑ is that you run into toxicity from the virus that is not related to replication.  And so, if you go under ‑‑ in the presence of Estuardo, I had to use the word MOI, but at the concentrations ‑‑ at the concentrations we use, and the exposure times that we use, and the cell number, and the virus number, if you're over 200 MOI, you start seeing toxic effects that interfere with the detection of the RCAs, so the, you know, the really easy solution is just to throw more material on and it doesn't work.  You have to scale it up.  And that's when it gets to be hard to handle.
	DR. SALOMON:  Point made.  Any other comments?  Estuardo?
	DR. AGUILAR‑CORDOVA:  I think another thing to keep in mind besides you could just add more volume, I mean, get rid of your toxicity with that MOI.  But the other point to make is that all of these are RCA assays that we're hearing about are based on the wild‑type that's been titered in the laboratory that's reporting them.  So if they're based only on the sensitivity of whatever their assay to detect RCA, so it's sort of a circular argument and we really don't know, and that's where the standard comes in.  We really don't know how that relates to anything else from anybody else.
	DR. SALOMON:  Thank you very much. Then we go to the last, but not least talk of the session, which is from Dr. Flomenberg on discussion epidemiology of adenovirus and its impact in certain patient populations.
	So, by the way, just to give sort of a plan, I thought we would finish with Dr. Flomenberg, have a brief discussion, break for lunch, and then come back and tackle the questions and end the session.
	DR. FLOMENBERG:  Good morning ‑‑ good afternoon, by now.  It's an honor to be here today.  Can you hear me?
	I was asked to talk about adenovirus infections in immunocompromised hosts and also talk about the issue of persistence and reactivation of infection.  I think the main points that need to be understood about adenoviruses is that they are truly ubiquitous.  Nearly everyone's exposed to these viruses in early childhood and nearly all adults have evidence of prior infection with the endemic seratypes and I would say that just about everyone in this room has antibodies to adenoviruses truly ubiquitous infection.
	As was implied, the clinical manifestations do vary according to the age of a patient and the immune status of the host and, as we know, certain seratypes are associated with distinct syndromes.
	As we can imply from the fact that adenoviruses are associated with epidemics, they are ‑‑ they do seem to be easily transmissible to susceptible individuals, which would be individuals without neutralizing antibodies to that seratype, which should be protective.  The seratypes that are associated with the epidemics are seratypes that are the nonendemic types: Ad3, Ad7, Ad8.  And these can be spread relatively easily amongst susceptible individuals.  We really don't have any information, however, what the minimum infectious dose is.
	In healthy individuals, I'd like to just point out, adenovirus pneumonia may occur, primarily in children.  It causes about 10 percent of the pneumonias.  The disease is more severe in younger children and infants, and it can on occasion cause fatal infections in healthy children, rarely healthy adults.  It also is associated with extrapulmonary symptoms that usually do not correlate with any viral‑ specific histopathology.  And things that have been seen in these patients include meningoencephalitis, hepatitis, myocarditis, nephritis, neutropenia and DIC.  And the mechanism for some of these extrapulmonary symptoms is not entirely clear.
	People have questioned whether it was toxin mediated.  From what we now know about some of the acute reactions to the gene therapy vectors, maybe some of this is an immune‑mediated phenomenon.
	Let's move to adenovirus infections in immunocompromised hosts.  These can, clearly, range widely from asymptomatic shedding, as has been mentioned, to fatal invasive or disseminated disease.  Disease may result from several different mechanisms. These patients may acquire a primary infection, maybe the result of reactivation of endogenous infection in the patient, as well as transmission of infection from the donor organ in the case of solid organ transplants.
	One of the groups that's been the best studied is the bone marrow transplant patients.  These are, certainly, the most immunocompromised patients that we deal with. And there, again, is a wide range of clinical syndromes ranging from pneumonia to gastroenteritis, hepatitis, hemorrhagic cystitis, nephritis, encephalitis, and myocarditis.
	It is clear from a number of studies that the incidence of infection is significantly higher in the pediatric population compared to the adult population.
	The mortality of invasive disease varies, but is in the range of 50 to 60 percent in the bone marrow transplants patients.  The risk factors for invasive disease include, clearly, the allogeneic transplants are at much higher than the autologous and, in particular, as the field has evolved, patients who have T‑cell depleted transplants seem to be at even higher risk, as you'll see, when we talk about some of these studies.  Essentially, these patients have a ‑‑ they're naive to adenovirus.
	Other risk factors include the presence of graft versus host disease and two or more culture‑ positive sites.
	This was one of the earlier studies, done in 1985 from Seattle.  They reviewed over 1,000 patients who were bone marrow transplants recipients.  At this pint of time most of these patients had unmodified grafts from related match donors.  It's not clear what proportion of children were in this study.  The overall incidence of adenovirus infection was 5 percent and the incidence of disease was 1 percent.  And in the individuals that developed disease, the mortality, again, was rather high, 50 percent.
	So, this was about the status of adenovirus infections back in the mid‑eighties.  As transplanters became more aggressive and we ‑‑ they started to use transplant more high‑risk patients and used T‑cell depletion, what we have seen is an increase incidence of adenovirus infections.
	This is the study that I was involved with out of Milwaukee, published in 1994.  This was a smaller group of patients, but look at the difference in the patient characteristics.  Virtually all of these patients had T‑cell depleted grafts.  These were naive patients, essentially.  Half of the patients had these high‑risk, unrelated or partially‑matched grafts and the other thing that is unique is 40 percent of these patients were pediatric patients.  We reported a much higher incidence, both of adenovirus infection, 21 percent and disease, 6.5 percent and the mortality was about comparable.
	So, in this very different patient population, the risk was higher.  But, again, the converse is two‑thirds of these patients developed shedding or infection without evidence of disease and did well.
	There was a much higher incidence of adenovirus infections in children compared to adults, 31 percent versus 14 percent. There was a much earlier time of onset in the children, as well, in both the endemic types as well as the AD11, 34, 35 were the most common seratypes isolated and I think that's been a general trend and it's not entirely clear why we're seeing a lot of these groupie seratypes in these patients.
	Similarly, a 1999 study out of Kentucky reviewed a similar patient population where they had 40 percent of their transplants were T‑cell depleted, not as high an incidence of children and their incidence of infection was 12 percent, again, very consistent in all these studies, the incidence of infection's higher in children. Their incidence of disease was relatively high, 7 percent with an overall mortality of 73 percent, it was hard to sort out in this study, what was attributable to adenovirus. So, again, in a higher‑risk patient population, we are seeing more adenovirus infections, as well as disease.
	This was the other study in your handouts from the U.K.  It's a little harder to evaluate.  They evaluated over 500 bone marrow transplant recipients, again, about half had T‑cell depleted grafts and unrelated donors, but there was a much higher proportion of children in this group.  They documented a much higher incidence of adenovirus infection, 17 percent, but I think I would have some differences of opinion in terms of their definition of disease versus shedding and the mortality was lower.  I think, again, that ‑‑ I don't think they distinguished very clearly the difference between shedding versus disease.  So, I would say in most of this, the more recent studies about a third of the patients go on to develop disease.
	Moving on to solid organ transplantation, adenovirus infections, typically involve the donor organ.  They have been the cause of hepatitis in approximately 3 percent of the pediatric liver transplants. That incidence is a lot lower in the adults. They cause pneumonia in about 1 percent of the lung transplants and hemorrhagic cystitis in about 1 percent of the renal transplants, and the mortality is a lot lower in that patient population.
	Congenital immunodeficiency syndromes, as was mentioned ‑‑  patients with severe combined immunideficiency disease can develop very severe infections, these are primary infections, the mortality is quite high and pneumonia and hepatitis are most frequently associated with these.
	In contrast, I think, adenovirus infection in AIDS patients we see a lot of asymptomatic shedding if you look for it, the incidence of disease is quite low.  I mean, all we have in the literature are case reports of documented invasive infections. But I've never seen a serious adenovirus infection.  I've seen lot of AIDS patients, so the incidence is quite low in comparison.
	And, similarly, in the cancer patients, again, adenovirus infections are a lot more common in the pediatric patients than the adults and there are only case reports in the literature.  Again, much less common than in the solid transplants and the bone marrow transplants.  So most of these patients do not have a problem with severe disease.
	We have no specific treatment for adenovirus.  In the case of transplant patients, if possible, we try to discontinue any possible immunosuppressive therapy.  We do not have any antiviral that's been documented to be of benefit, although Cidofovir has the best in vitro activity.
	This is a very broad spectrum nucleoside monophosphate analog.  It is active against many other viruses.  It has some in vitro activity against adenovirus, however, they've also documented the development of resistance in vitro.  It is active in a rabbit eye model of adenovirus infection.
	And there are only case reports of responses coincident with the administration of these antivirals, including case reports of hemorrhagic cystitis in the bone marrow transplant patients that have responded to IV ribavirin or vidarabine.  There's a case report of colitis in an unrelated bone marrow transplant recipient who did not have a response to IV ribavirin, but whose symptoms resolved on cidofovir within two weeks. There is also a case report of adenovirus colitis and cholecystitis in an AIDS patient that responded coincidentally with the administration of cidofovir.
	So, I think that it is somewhat encouraging, but these are only anecdotal reports, so as things stand, we really do not have an effective antiviral therapy for this adenovirus.
	Immunotherapy ‑‑ probably plays a limited role.  I will give IV immunoglobulin to patients if I am concerned about adenovirus infections.  They do have very good titers of neutralizing antibodies to the endemic seratypes not, for instance, the Group B seratypes that some of these patients may have run into problems with.  There is also a case report using donor lymphocytes in a bone marrow transplant patient with a good response.
	What do we know about the immune responses to adenoviruses?  Based on our clinical experience, certainly, we can presume that cell‑mediated immunity is quite important because most of these severe infections occur in hosts with cellular immune defects.  As I mentioned, neutralizing antibodies are felt to be protective against reinfection with the same seratypes, so there are seratype‑specific, and that may be one thing to look at in patients pretreatment with gene therapy vectors and, as I said earlier, by age ten most individuals do have evidence of antibodies to the common sera‑types.
	Looking at healthy adults, just about everyone also has detectable memory T‑cell responses both helper and cytotoxic. Two adenoviruses and what's been shown is that unlike the neutralizing antibodies that are seratype specific, the T‑cell responses seem to be targeted, in part, to epitypes that are conserved across different seratypes.
	What is the pathogenesis of adenovirus infection?  Clearly, it's a lytic virus.  It can directly kill susceptible cells.  Is there a component of immunopathology?  We don't know.  There is evidence for a persistence that I will present.
	Adenoviruses were originally isolated from tonsillar tissue in asymptomatic individuals and we do know, also, that they can be shed in stool for weeks to month, post‑infection.  There are also cases of transmission documented from donor organs that I'll mention and, also, clear cases of reactivation in bone marrow transplant patients.
	There are handfuls of cases like this.  Renal transplant patients with hemorrhagic cystitis where they are antibody‑negative pretransplant and they seraconvert.  Consistent with transmission from the donor kidney or, less likely, a primary infection.  There are cases of pediatric liver transplant patients with ‑‑ who have developed Ad5 hepatitis where a majority of these patients are seronegative pre‑ transplant and the donors are antibody positive and with the early time of onset, again, points to a probably transmission from the donor organ.
	In regard to reactivation in the bone marrow transplant patients, there are also handfuls of cases reported ‑‑ patients with Ad5 hepatitis where there was Ad5‑specific neutralizing antibody present in pre‑ transplant sera, which, again, would be consistent with reactivation of endogenous virus.  I also have some data with some of the Ad35 cases from the Milwaukee study, where all the adult patients had neutralizing antibody pre‑transplant, again, consistent with reactivation.
	What are the mechanisms of persistence?  We don't really know.  Do they remain episomal in long‑ lived cells, such as lymphocytes?  It's possible. It's of interest that the seratypes 11, 34, and 35, which we see in the bone marrow transplant patients to reactivate.  Is it possible that they can establish persistence more readily?  There is some recent data that they seem to infect hematopoietic cells more efficiently compared to other seratypes.  Is there a low‑level control replication in tissue?  There's another possibility and do adenoviruses integrate?
	There was a very limited discussion about the adenovirus early region 3, earlier. This region is deleted from most of the vector constructs, but it's a very interesting region that codes for programs that actually downregulate the host immune responses by a number of different mechanisms.  And may act to reduce immunogenicity in natural infection and/or facilitate persistence.
	What are these reservoirs?  Again, lymphoid tissue, we mentioned tonsils, maybe they're ‑‑ the lymphoid tissue in the gut may be a reservoir.  No one's really looked at that.  They're ‑‑ the kidney and liver, based on the cases of probable transmission from organs in transplant patients.  And then there's also some PCR data in tissue such as the lung and brain.
	Are lymphocytes a reservoir?  There was old data that PBMCs from most individuals were strongly positive for Ad2 by Southern blot hybridization.  I looked at a large number of PBMCs from children and adults using a nested primer PCR for Ad2 and I did not detect this ‑‑ 72 out of 73 were negative using primers to both E1A and hexon.
	There are handfuls of reports such as this looking at E1A ‑‑ detecting E1A by PCR in lung tissue, such as in this case in this report where it was detected in 20 out of 20 biopsies from lung cancer patients.  In comparison, E1A was detected by {IOn}in sito{IOff} hybridization in only two of these patients. They detected the E3 region DNA in half of the patients and the author suggested that this was evidence that, perhaps, E1A might be integrated into the host DNA.  There's also one report in brain tissue, where they detected E1A in microglial cells in seven out of seven patients.  One of the problem with these studies is they don't really have good negative controls.
	As was mentioned, we have an experience using a live wild‑type ‑‑ type 4 and type 7 vaccine in the military.  It's enteric‑coated, it's given orally.  It's been shown to be highly safe and effective, so this is an example of the safety of administration of wild‑type adenovirus, but the caveats are it was given by a specific route of administration, orally.  And it was given to healthy military individuals.
	Issues of RCA in gene therapy vector preparations include the fact that we really don't know what the minimum infectious dose is.  It's likely dependent on multiple factors, including in particular, route of administration and the presence or absence of seratype‑specific antibody.  I would be somewhat concerned about giving RCA to a naive individual, say, a child, a four‑year‑old‑child, who may not have been exposed to adenovirus or, in particular, Ad5 and does not have any neutralizing antibody. I think that's different than giving it to an adult whose been exposed.  The severity of disease is also likely dependent on the route of administration we don't have any information about.
	I mean, it's different than a natural infection, which is acquired orally or respiratory ‑‑ by the respiratory tract. We're giving the vectors perennially, we're injecting them into the liver.  We really don't know anything about how RCA would affect the severity of disease given by these other routes of administration.  Clearly, the status of the cellular immune system is a factor and also the seratype.
	As we talked about, there are techniques now to truly significantly reduce levels of RCA from Ee1 deleted vectors using altered cell lines that may reduce recombination events.  Also, I think there's going to be a lot more interest in using the gutted and helper‑dependent vectors and these, obviously, need to be purified away from the E1 deleted helper adenovirus and whatever RCA is contained in them.  And, as was implied, all of these preparations still have input viral proteins, the coat proteins and that does not address the significant issue of the acute reactions, which I think you will see in all of these vectors.
	Then the issue of recombination: It's certainly theoretically possible that this could occur in vivo, as well, as has been suggested.  And this could occur with persistent adenovirus, as well as duly acquired adenoviruses after the treatment with vector therapy.  It's probably not of great clinical significance and, presumably, these patients will have been immunized or boosted and will have a very vigorous immune response to adenovirus by the time this occurs, but it's still a theoretical possibility.  And that's it.
	(Applause)
	DR. SALOMON:  Thank you very much for a really nice presentation.  If I had that many slides, we'd be here tomorrow.  So I think you did very well.  Are there specific comments, Abbey?
	MS. MEYERS:  Well, I just want to ask, if a person has a gene therapy treatment, as an outpatient, and goes home and maybe they get no symptoms from the adenovirus or maybe they get a little cold, some kind of respiratory thing, but they have a kid in the house who has asthma, who is taking steroids or his wife is taking something for a normal immune disease.  Does it put the other people at risk, the rest of the family?
	DR. FLOMENBERG:  Well, I ‑‑ I would just refer to the earlier talks that were presented.  We ‑‑ there have not been examples where they have found wild‑type adenovirus shedding after treatment with the gene therapy vectors.  So I would not think there would be a risk to the other family members.
	DR. CHAMPLIN:  Although adenovirus can cause severe disease in the most profoundly immunocompromised patients, most ‑‑ even bone marrow transplant patients handle this infection very well, and it's only the ones that have failing grafts or mismatches that are most profoundly immunocompromised that we see these kind of overwhelming infections.  And so that, on the question list, you know, the disorder is sort of listed in the, you know, intense immunosuppressive area, I think probably are, perhaps, overly conservative, because most solid‑tumor patients or most people on steroids with autoimmune disease are not coming down, that is naturally, with fatal adenovirus infections.  And it's really the, you know, the most critically immunosuppressed transplant recipients.
	And even in the organ transplant area, perhaps, other can speak to this better than me, it's more of an issue of the graft being affected and possibly sensitized for rejection as opposed to systemic adenovirus infections causing pneumonia or other tissue infections.
	So, it's the rare patient that really has the overwhelming infection and they have to be profoundly immunosuppressed.
	MS. DAPOLITO:
	DR. FLOMENBERG:  Yes, I would agree with that.
	DR. BLAZER:  I think an interesting point in the presentation was that the children were at higher risk after transplantation, at least early.  And if one tries to put this together, children, in fact, make T‑cells more readily than adults and yet they don't have, necessarily, antibodies ‑‑ high antibody titers going into transplant.  So, one could potentially envision three phases of response:  The initial antibody response that helps to clear the virus; then an innate immune system response, like, natural killer cells, which adults and children make very rapidly after transplant within two to four weeks; and maybe that's why the disease ‑‑ invasive disease risk is low; and then a final phase of a T‑cell response, which happens later and puts the adults at greatest risk because of the persistence of this virus and their inability to produce new T‑cells as rapidly as children.
	So, as we look at the immune response and identify risk patterns, I think, part of this is asking how much virus do they have to respond to and when do they have to clear it and what are the multiple ‑‑ if there are three different mechanisms of clearing the virus, then there are three stop‑gaps in preventing a disease.  And you may have to have a disability of all three, depending on the viral load to really be susceptible to an invasive disease from this virus.
	DR. SALOMON:  Just following‑up on that, I mean, that certainly would go along with the correlation with GVHD and with T‑cell therapy, that would fit that.  Is there evidence that ‑‑ actually ‑‑ so an hypothesis that would come out of what Bruce just said is that the time of onset of adenoviral disease would be different, perhaps, in the children, which might occur early, versus the adults, which might occur before they develop a T‑cell immune response. Is there any evidence that the timing of onset was different in these populations?
	DR. FLOMENBERG:  I'm a little confused about what you're postulating, now. I mean, clearly, in our Milwaukee study, the children ‑‑ the time of onset was a lot earlier for the children than the adults.  Do they reconstitute their immune response quicker, is that what you're ‑‑
	DR. BLAZER:  I mean, the children will make new T‑cells that are thymic derived earlier than more robust than adults ‑‑
	DR. FLOMENBERG:  So you think it's immune mediated?
	DR. BLAZER:  So the dichotomy is the fact that the children were more susceptible than the adults and the time of onset was earlier than the adults and the children aren't making as much antibodies because they haven't had as much exposure. So there may be this issue of this initial wave of an antibody response requirement and then depending on that ‑‑ and K cells come back quickly, and maybe that's why most people don't get disease.
	And then the fact that GVHD T‑cell depletion in adults are susceptible, particularly for late onset disease means that the T‑cells have to have some immune response later to completely wipe out the disease or to protect against continual exposures.
	DR. FLOMENBERG:  I would probably look at it a little differently.  I would suspect in the children that they may just have higher viral loads of persistent virus and/or some of it could be primary infection, that may be why we're seeing it a lot earlier, they have higher residual ‑‑ they've been exposed to it earlier ‑‑ closer in time and they may have more residual virus that may reactivate earlier.  I doubt that within 30 days there's that much difference in the immune reconstitution compared ‑‑ in the children compared to the adults.
	DR. BLAZER:  So, that brings up is there ‑‑ have there been any good correlations between antibody neutralizing titer pretransplant and risk of reactivation in the first 30 days post‑transplant?
	DR. FLOMENBERG:  I don't think it's been specifically looked at.  As I said, in the few cases that have been reported, a fair number of the bone marrow transplant patients do have evidence of neutralizing ‑‑ seratype‑specific antibody pretransplant more consistent with a reactivation of an endogenous virus.
	DR. SALOMON:  Estuardo?  Oh, I'm sorry, yes.
	MS. MEYERS:  I just want to ‑‑ are you saying that the only people who are at risk from the adenovirus are transplant patients?
	DR. FLOMENBERG:  No, you can ‑‑ a healthy individual whose naive can develop a serious adenovirus infection.  I mean there are fatal cases of pneumonia in healthy infants and children.  It's rare, most people handle these well and they have a self‑limited illness.  The majority of the severe infections occur in the highly‑immunocompromised patients.
	MS. MEYERS:  But there's no danger of contracting it from a person who has had some kind of gene therapy treatment, it's not ‑‑
	DR. SALOMON:  Abbey, we can't answer that question.  We ‑‑ we've been given data today suggesting that the danger is low and it's not been measured yet.
	MS. MEYERS:  Mm‑hmm ‑‑ okay ‑‑
	DR. SALOMON:  They've looked for it.
	MS. MEYERS:  The cystic fibrosis cases, where they had an overwhelming reaction to the adenovirus, is that applicable here?
	DR. FLOMENBERG:  Again, yeah, I think most people would agree that a lot of the acute responses are due to reactions to the input co‑proteins.
	DR. SALOMON:  Estuardo.
	DR. AGUILAR‑CORDOVA:  I was wondering if there was any data on ‑‑ I know you said that there was no data on the infectious dose.  But once these patients that have been documented that have disease caused by systemic or by localized adenovirus, is there any data on the kind of viral load, be it from fluids or from tissue that may be correlated with that disease onset?
	DR. FLOMENBERG:  I am not aware of data, Marshall, do you know?
	DR. SIEGEL:  In the cases where livers or lungs or kidneys have been thought to actually transmit the infection, is there any information on the quantitative viral load in the transplanted organ?
	DR. FLOMENBERG:  No, no one's really looked at that.  It would be interesting to look at some of these reservoirs.
	DR. HOROWITZ:  There's probably very little.  Even when you look at a fatal case of adenovirus hepatitis, if you look at viral inclusions, for example, there are relatively few cells, it's ‑‑ it looks like it's not an overwhelming viral infection, it's the host response to a relatively small amount of virus, so I wouldn't think there would be a lot, but I mean, I don't ‑‑ it's not been measured.
	DR. HIGH:  Since the numbers do seem to be an important consideration here, I was wondering, is there ‑‑ is there any data about size of inoculum?  I mean, for example, in respiratory droplets or any, I mean, is there any information that would give us numbers?
	DR. FLOMENBERG:  I'm not aware of that.
	DR. AGUILAR‑CORDOVA:  What about on the size of inoculum that had been used, albeit in an oral dose, but the size of inoculum that's been used in vaccination protocols?  I also think there was some intranasal vaccination that was done in the early seventies, as well.  Do you know what kind of doses those were?
	DR. FLOMENBERG:  No, I don't know. But, yeah, we could certainly get that information, in terms of the vaccine inoculum.  But, again, I mean, I guess my concern is you're giving it in different routes.
	DR. AGUILAR‑CORDOVA:  But also ‑‑
	DR. FLOMENBERG:  An inoculum, you know, we talk about numbers of particles, numbers of RCA may mean something different when you're giving it orally versus intravenously or intrahepatically, but we just have no information.
	DR. AGUILAR‑CORDOVA:  Yes, I was wondering, in some of these children that do come down with it, though, when you take just blood or serum, one can culture that from there is that correct?
	DR. FLOMENBERG:  It can ‑‑ rarely, I mean, people don't routinely do it, but there have ‑‑ you can, occasionally culture it during an infection.  So, it probably does cause a viremia, people haven't really looked closely for it.
	DR. HOROWITZ:  Well, your one patient that was positive in your PCS study of lymphocytes, was a patient who had active disease ‑‑
	DR. FLOMENBERG:  No, these were healthy donors.  I did also, I did find it in ‑‑ I looked at two patients with invasive disease and they were strongly positive ‑‑
	DR. HOROWITZ:  Right, right.
	DR. FLOMENBERG:  In PBMCs, but amongst the healthy donors, most of them were negative.
	DR. SALOMON:  Okay, I think then that what I'd like to do is break for lunch. Last time that we were here, it took less than a half hour to eat downstairs, they're rather efficient.  So, what I'd like to do is try and meet back up here in 30 minutes, you know and, obviously, I'll be pragmatic about it if it seems like it took us longer.
	(Whereupon a luncheon recess was taken.)
	A F T E R N O O N  S E S S I O N
	(1:45 p.m.)
	DR. SALOMON:  I want to thank everybody for being very efficient about lunch and joining us back up here, that's good.  A couple members of the Committee and it may also apply to others that I haven't talked to are going to need to leave within the next 45 minutes to an hour.  So, I think one of my jobs here is going to be to begin and to have a very focused discussion the specific questions and then once we've addressed those, we can relax a little bit as the time goes on and talk about some of the broader issues that came up during this morning's conversations.
	I'd like to welcome Dr. Flomenberg to the table and thank her for that ‑‑ the microphone's still open there, so our other speaker's and anyone else in the public who wants to make a comment at this point are welcome.  This is the ‑‑ what we call an open public section of the meeting.  We've not had any specific requests for anyone, but it's still part of the procedure here to make that offer open.  So.  Gail tells me that the offer has been open, so we're okay, then, I managed to.  Okay.  All right, guys.
	I think it was really clear from the discussions this morning that there are several different layers of discussion that we should have and nobody's trying to say that there is any particular relative value or merit to those levels, but we are going to answer these specific questions.  Because, otherwise, I get all kinds of grief, appropriately.
	So, let's just first, answer these questions, and then I will back off and the conversations can go in a, you know, more in a natural way.  So, I apologize to everyone for a short period of time, but.
	So, question number 1 is:  Should recommendations regarding acceptable levels of RCA in the adenovirus gene transfer products be the same for all clinical uses?
	And in thinking about that question, let's consider the following patient populations as they might differ in their relative risks.  So, we're talking about different levels of immunocompromised patients and this now picks up on the themes that we were discussing as triggered by Dr. Flomenberg's comments, as well as themes that were touched on by Dr. Sublett and Dr. Hutchins.
	So, yeah, I think everybody gets it, so what does the Committee think ‑‑ Dr. Rao.
	DR. RAO:  Shouldn't it be even more focused and say just the two choices that we really have before the Committee, right? It's 30‑fold different or remains the same, right, in terms of RCA levels, right?
	DR. SALOMON:  So, what kind of discussion do you want to have?  Richard?
	DR. MULLIGAN:  I'd like to ‑‑ before we get to this, get a definition of recommendation.  I'd like to hear from the FDA what ‑‑ what a recommendation really is, because I think that will turn out to be very important here, rather than people arguing about whether there is or isn't a different risk associated with something.  As I understand a recommendation, it is a general guideline to guide the development of the production method and testing and so forth. If we take it as that loose a definition, then it may be easier to give general principles.  I think a general principle might be, is there enough risk assessment information to make weighing risks a valuable part of the criteria that's one thing.  But if you say it's a recommendation, you know, you can go in and make the case, then what you really want to hear is the arguments that people will make when they come to the FDA, that is, they'll say, yeah, I know, I have this wrong ratio or helper content, but this is in a very, you know, safe population, immune competent group.
	DR. SALOMON:  So, Richard, let me tell you how I'm thinking about this, and we can see whether we're on the same page.  To me that's more question 2 than question 1. So, question 1 is saying, is there going to be a difference in the risks of replication competent adenovirus by patient population? Question 2 is asking what kind of experiments or data would you do to set that risk and in context of that would be the question of do we have enough data, which I think is what you're asking?
	DR. CHAMPLIN:  Well, I don't think so because I think the first one is saying should it be based on just setting a common guideline or should it be based in risk fashion?
	MR. SIEGEL:  Let me try to clarify that.  What we would expect and let me ask the review staff to correct me if I'm wrong ‑‑ but we would expect all manufacturers to set a specification for, you know, a test specification for RCA testing and usually that would be a release specification where when they ‑‑ if they fail to meet the specification the lot would not be releasable.  Now, we provide a guidance, not a rule, but a guidance in ‑‑ as to what that specification should be.
	At the present time, the guidance is a single ‑‑ at a single level that is independent of what the clinical use is. Whether we have more than one guidance, depending on clinical uses or whether we have one guidance, which as with all guidance ‑‑ all things of its nature can be modified as appropriate for clinical use, I think we'd come to the same end.
	So, the question before us is, more or less, as I think you characterized in your second option, that industry or manufacturers are indicating and, I think appropriately, that the levels we're setting do place some considerable burden on both testing and also on production lost.  And in some cases might seek and find useful a more ‑‑ a different standard or a looser standard and then the question comes ‑‑ and they can propose any standard, and they might well, as you've suggested, propose a different specification based on the fact that it's ‑‑ that there's low‑ perceived risk because of the nature of the target population.  And, in order to deal with those ‑‑ whether it's dealing with those requests or setting different specifications, the guidance that this Committee provides and the expertise regarding whether that should be done and how it should be done is what we're seeking.
	DR. SALOMON:  So, can we, I mean, I ‑‑ okay, if you're not totally satisfied, then that's fine, but I think what I'd like to hear is the opinions of the group based on what, obviously, the expertise you bring to the table and what we've heard today, whether you think that there is a higher or lower risk of RCA administration in different kinds of patients, the immunocompromised patients, bone marrow patients, cancer patients, children, et cetera.  I think we ‑‑ we have some data and, you know, people should have a comment on that.  And then, secondly, whether or not we ‑‑ based on that presumption of overall risk in any patient population with RCA, because we've been given data on that, whether we should be, you know, how stiff and how flexible we ought to be in setting criteria.  Which I think gets ‑‑ segues to where you're going Richard, right?  So, can we just start with, sort of, the first concept, what's your impressions now, what's your expertise on whether or not ‑‑ what's the risk here ‑‑ is this a big deal, a little deal, no deal?
	MS. MEYERS:  From what I heard this morning, and I'm just a layman so all I can do is interpret what I heard is that there seems to be a higher risk for bone marrow transplant patients, a higher risk for children in general.  And there's a lower risk, but there is no population where there is no risk.  So, in some people who are perfectly healthy, they can still get a life‑threatening infection from the adenovirus.  That's what I heard this morning.
	So, in looking at this rule and saying why is there a need to change it, seems to come down to financial.  I mean, some people, companies would like to save money by not throwing away so much of their sample, and I don't think that the financial reason is enough reason to change the rule.
	DR. SALOMON:  Well, I mean, I agree with everything you said, that's a good ‑‑ you made your point.  But the problem here is that the last conclusion you made that the only thing driving it is financial, is probably not fair.  In that if there is little or no risk, then putting a gigantic financial and practical burden on the companies isn't justified.  So that's ‑‑
	MS. MEYERS:  Right.
	DR. SALOMON:  I mean, so that's what we need to discuss right now.
	MS. MEYERS:  But ‑‑
	DR. SALOMON:  If we decide that there's a really high risk or that there's a really high risk in a specific patient population, then we can go on to the next part of it, which is what Dr. Mulligan was saying is, where should we set that limit, realizing in a real world that that's going to have it's implications on the whole field.
	MS. MEYERS:  See, I would think that the only place that there is no risk ‑‑ if we could find a population where there's not risk and you could guarantee there's no risk, I'd say go ahead and change the rule. But we can't do that.
	DR. SALOMON:  So, let's ‑‑ let's continue discussing it from around the group to what extent do you think there's risk? Alison and then ────
	DR. LAWTON:  Let me just throw out something and see what reaction I get, because just in general, from the presentations this morning, I would say that the proposed limit of the 1 RCA per 3 times 10 to the 10, is too overly tight for certainly category C and potentially category B of patients, given the information that we've seen with regards to the risk around that type of level.
	DR. SALOMON:  So, you've put B and C, just for everyone else, you're referring to mildly immunosuppressed patients and patients with genetic defects, right?
	DR. LAWTON:  Yes, sorry, I'm looking at an old version of the questions, that have B and C actually written on them.
	DR. CHAMPLIN:  The, you know, adult patients who do not have one of these major transplant issues going on, the risk, almost, is zero.  I mean, you know, there's never anything that's truly zero, but it's, I'm unaware of adult ‑‑ normal adult patients having severe infections from adenovirus. And, in fact, it's the opposite.
	That's the problem; you have immune response that limits the ‑‑ your retreatment of patients with adenoviral vectors because of a vigorous immune response.  So, I'm not sure we have a problem.
	You know, as we talked about this morning, the toxicities that have been observed are probably to the total viral particles, probably from something related to the proteins on the virus and not the recombinant virus, per se.  And so, one is reacting to a theoretical problem that, at least, has not been documented to have occurred in any patient.
	So I would support the concept of not being overly restrictive.  You would like to, you know, reduce contaminants in a product as much as is reasonable, but you wouldn't want to be throwing out half of your lots for no reason in the situation where we haven't see a symptomatic case.  So, certainly for the B and C, the immunocompetent categories of patients, I would see no reason to change from the current, you know, standard of what ‑‑ 100 or whatever the units were to a more rigorous standard that would really impede the development of the field.
	DR. SALOMON:  So, we'll go Estuardo, Richard and Joanne.
	DR. AGUILAR‑CORDOVA:  I think that, you know, part of the discussion is, of course, always based on the cost risk benefit type of analysis and what's the risk is somewhat indeterminate and I don't think that even though we have data, what the risk is not.  It's the majority of people that get exposed to adenovirus ‑‑ wild‑type adenoviruses on a daily basis do not come down with fulminant viremias that cause any disease.
	So, I think we have a fairly good impression for immunocompetent things.  We also have some data to show that there are severely immunocompromised people may have no way to keep a check on viremia and so, I think we can't really analyze what the risk would be ‑‑ at what level an infectious dose would come.
	So the first issue would be that there is no data to know what the risk would be and how to evaluate that.  And as if that weren't enough, really, when we're talking about these ratios and these new specifications, they really would be based on somewhat floaty and dicey characterization methods.  And so, to say that they're unachievable or too costly, they are, if one uses one method, then they may not be, if one uses another method.
	So, it really becomes almost a circular argument.  So, it is difficult with the amount of data presently available to make any strong conclusion.  I think the only strong conclusion that I could possibly make is that with the levels of contaminants that are possibly there today, there hasn't been any significant disease.  There have been some case reports of significant adenovirus related to disease in severely immunocompromised patients or neonates or infants in ‑‑ that are not immunocompromised but not in adults and the preclinical data that is available, shows that not only the age of the animals but the route of administration and the total doses of viriants are related to toxicity.
	I think those are the data on which we can base an answer that says it depends on the patient population and depends on the route of administration.
	DR. MULLIGAN:  You know, I think I may be saying the same thing as what you just heard, but let me try it differently.  I think that there's very little at all, if any sense of the absolute risk in any of these cases.  And that's the difficulty.
	The relative risk is what we're championing and trying to have a heavy discussion about, but I think that's obvious that, you know, the more immunosuppressed everyone's going to say there's more of a risk.  But I think that that just ‑‑ the issue of whether there's an absolute risk cannot be addressed.  So I think that because you can't address that, I would use the relative risk as a modifier when people come to the FDA to break the guidelines.
	And on the guidelines itself, I find amusing that the new recommendations.
	(Interruption)
	So the way, as I understand the recommendation comes about is looking at what is routinely achievable, okay?  And I still think that's very reasonable.  But, in fact, based on all the discussion we had, we don't actually know what that number is, because, in fact, we know people calculate it differently.  So, those in industry that say, you know, that 50 percent of our lots wouldn't pass, have no idea, because they don't really know what that number is.
	And I think the spirit, as I understand it of the FDA, is to make it to set that guideline at a level that is reasonably obtainable.  And I think that it's a very good thing to set a high technological standard ‑‑ I think that's the purpose, in fact, of having such a guideline to actually get people to push towards having a more pure product.
	But at the end of the day there's no ‑‑ no sense, I think, at all that there's any biological reason for why the level has been set, it's completely and totally arbitrary and as far as I can tell, so was the number before.  So, I would just push for getting to a sense, you know, with the better reference standards and, you know, a more unified test of what is the state of the art, what is doable by people and try to set it at that point, and leave the risk as a modifier for particular cases because of the ‑‑ really, I think the lack of any real sense of how important the absolute risk is.
	DR. SALOMON:  Okay, Joanne.
	DR. KURTZBERG:  What I was going to say, has actually been said, but I just don't think the numbers we have right now are meaningful.  And so, I agree with what Richard said.
	DR. SALOMON:  What do you think, Joanne, with your experience, what do you think of this whole idea that our ‑‑ do you agree that there is some patient group‑specific risks higher in the young children, higher in the immunocompromised bone marrow transplant patients?
	DR. KURTZBERG:  I mean, intuitively, with what we know about the wild‑virus, you would say that, but we have no data to know if that's going to apply to, you know, modified virus, but intuitively you would identify those patient populations as a higher risk, yeah.
	DR. SALOMON:  Ed?
	DR. SAUSVILLE:  I mean, along those lines, I mean, although everyone has bought into the idea that there's more risk with the younger ‑‑ with immunocompromised patients, none of the clinical experiences that we've actually see so far where, I guess, some of the populations could be characterized as immunocompromised has there been the suggestion that that has translated into some actual increased risk of disease.
	So, I would even go so far as to say that this is the sort of thing that you have to trade off what the potential benefit or value of he scientific exercise is, versus some theoretical risk and it should be the sort of thing that should be part of the consenting process and I ‑‑ and at one level, I think this is something where the patients are going to have a voice in what they would see as their risk.
	DR. SALOMON:  One thing, again, I believe it's correct, at least I've heard no data in given today or in my own reading where they gave adenovirus to patients with bone marrow transplants.  At least none of the data that was presented today.  Are there any studies guys that they gave them to that patient population?  Because the patient populations we've heard a lot about today have been patients with metabolic disorders and those with a series of different kinds of cancers, which I think we've sort of all come up thinking are relatively low‑risk populations.
	And certainly I should add, wearing my hat as an organ transplanter for 20 years, I, you know, we don't think much about adenovirus and I immunosuppress the hell out of everybody with anti T‑cells antibodies and cyclosporin and prednisone and cellcept (?) and other drugs, so.
	DR. SAUSVILLE:  I guess, I'm a little bit, I mean, again, this isn't a question that's there, but I'm a little bit more concerned about the issue raised by the metabolic abnormality patients.  I don't think we have a very good idea of what drives the inflammatory or quote/unquote "immunnu" or whatever response is.  I think that's going to be far more telling as a safety issue than anything having to do with recombinant viruses.
	DR. SALOMON:  I think we agree with that, Ed.  And we're just focusing right now on this first question.
	DR. HOROWITZ:  I guess it's sort of almost like a vote to sort of repeat some of the things that have been said.  I mean, I think we have enough data for B and C to know through experience that there are no problems.  And I think the difference between 1 and 10 to the 9th or 3 and 10 to the 10th are probably not ‑‑ certainly not significant.
	So we really ought to concentrate on A and decide when and what help we can be to the FDA in terms of suggesting steps of caution along the way.  After all some of the questions that some of us here feel comfortable with now, we were very uncomfortable with a few years ago, but experience has taught us that we can ‑‑ that this is not so much of a problem.
	Just for the record, you know there were two deaths reported in young men in the military presumably from adenoviruses, although the cases were not as well, perhaps, documented.  They were reported in the MMWR, I think, last week.  These were young people in the military who were not immunized because the vaccine is no longer available, at least currently not available, and they died during an adenovirus epidemic of respiratory disease.
	Now, those were type either four or seven, which are not being considered today for vector considerations, which are mostly two or five.  But I want to just ‑‑ I don't want the group to feel that that we can have an absolute no‑risk situation with whatever decision we make and there are children that will develop pneumonia even with type five or sometimes with type two, where occasionally it will die.  So, you know, there's no trade‑off with 100 percent certainty, but as physicians and others we deal with relative levels of certainty, which make me feel B and D are fine and ‑‑ B, D, and C are fine and we really ought to try and help by 2 and 3, we ought to help in trying to reach some steps so that we can help the FDA with that big category ‑‑
	DR. CHAMPLIN:  Now, I think the A category, you know, the profoundly immunosuppressed, I'm not sure a 30 percent or 50 percent reduction that we're talking about, you know, in terms of technology to limit the recombinant adenovirus is going to make a difference there.  Because you've got a proliferative virus in a permissive host, where there's no effective immune response. And I'm not sure there's going to be a safe level of recombinant adenovirus if there's any, you know.
	So, in that group, I would be real cautious about doing the treatment at all. And would want to be sure that the risk‑benefit relationship of the proposed study would, in fact, justify it's going forward.
	DR. SALOMON:  So, let me try and, I think that I hear what sound likes somewhat of a consensus, but let me try it out.
	So I think we all agree that, well, there is a consensus, it would appear, that there is a higher risk in this A, this first group of patients with severely immunocompromised and in very young children, who probably haven't have a historical experience with adenovirus.  And that, otherwise, in other groups, it's certainly not zero, Abbey, Marshall, we hear that.  But it just doesn't seem to be very high.  Anyone agree with that?
	DR. CHAMPLIN:  And then within the A category, it's clearly the allogeneic bone marrow transplants that are T‑cell depleted and mismatched are the highest risk.  The organ transplants and autologous (?) bone marrow, blood stem‑cell transplants seem to be low‑risk, and I would actually probably put them in the next category, myself.
	And the ‑‑
	DR. SALOMON:  That's an excellent point.
	DR. CHAMPLIN:  And even the HIV patients as has been discussed seem to have a relatively low‑ risk of serious infection.
	DR. SALOMON:  And I would agree also for organ transplant patients ‑‑ solid organ transplant patients.  Bruce.
	DR. BLAZER:  Just another quick point.  I think after the bone marrow transplant patients reconstitute and if they're off immunosuppression, we immunize them, they do make responses.  So I don't think it should be a moratorium on high‑risk bone marrow transplant patients, but it should be until their immune system has other evidences of normal responses.
	DR. SALOMON:  Right, I'll actually return to that in a second because I want to pick up something that Ed Sausville made a point of.  I guess part of it is that I'm also hearing, and I think Ed captured it, that even if there is a higher risk acknowledged in these settings and both Bruce and Richard have refined that for us.  That a lot of that could be handled within an appropriate informed consent procedure.  And doesn't require any sort of moratorium based on the risk or the risk to the public around them.  Now, that's I'm just putting that out for further comment.
	MR. SIEGEL:  But still, if we're talking about relaxing the standard for product testing, are you then suggesting that the consent form would say to the patient, if you want to be frank, are you willing to take a ‑‑ to receive a product that isn't quite as clean of infectious virus as it could have been because we didn't want to have to deal with that, is that how you're suggesting we deal with in consent?
	DR. SALOMON:  I mean technically I don't know if I was quite going there, yet, I ‑‑ that was my next ‑‑ that was where I was going next, though.  But, I mean, yeah, kind of, if you want to talk about it that way in the sense that ‑‑ the sense that if we're ‑‑ if the Committee's grappling with the idea of what's the risk to begin with and then, based on some consensus based on what we feel the risk is, how, you know, tight, and how obsessed do we want to be within, you know, the quality of ‑‑ the exact number of RCA particles per, you know, total.
	So, yeah, I mean, I suppose you could put ‑‑ that's the kind of thing that would ‑‑ irrelevant to how tight you made it, unless you made it zero, you know, it's zero RCA, which I don't think anybody's suggesting for lots of reasons.  Unless you made it zero the informed consent at some point would have to say that, wouldn't it Jay?
	MR. SIEGEL:  Well, zero, is sort of out of the question because of the technology.  I think at some point we thought we had a zero standard when people weren't able to produce doses as high as they were able to test.  At this point, you can produce a lot more than is feasible to test.  And all we can do is exclude certain amounts and it looks like, from the data, that we're nowhere near to achieving zero virus going into patients.
	And in that regard, I want to say that based on ‑‑ aside from whether to loosen or tighten the standards, there seems to be a little attention that we need to take based on this morning's discussion to, perhaps, rationalizing the standard because we set thee standard at what a test result shows rather than ‑‑ without accounting for the confidence intervals of that test result and we're doing tests that are at their limits. You know, if you find nothing in 3 times 10 to the 10th that tells you probably have less than 1 particle per 1 times 10 to the 10th, is your 95 percent confidence interval and you can get aberrant results if you test a little more or a little less. You can wind up approving unsafe products and holding back safer products.
	So I think we'll pay a little more attention to trying to rationalize how that standard is.  But that's an independent question about whether it should be a variable standard or a tight standard.
	DR. SALOMON:  Well, it's exactly where I want to go next.  But I wanted to just sort of finish this because I made a statement for discussion and I don't believe that we reached any kind of consensus on it. So, the question I had said, again, was picking up Ed's point is to what extent can we be comfortable that even in a ‑‑ even in what we recognize as a high‑ risk population, that a lot of that can be a part of the standard consent procedure.
	I mean, you just say, you're here you are we're doing it in a T‑cell depleted allogeneic ‑‑ you know bone marrow transplant, we're going to give adenoviral vector to you, there's going to be some replication competent adenovirus in your preparation and you are going to have this increased risk and we can't quantify it.  I mean, I'm comfortable with that, but I'd like to hear what my, you know, other members of the Committee say.  Richard.
	DR. MULLIGAN:  I would just state it differently which is that there wouldn't be any difference in criteria for the riskier thing.  I mean, in a way you're saying that. You're saying you're going to set some limit and you've maybe going to vary how you devise the clinical protocol or what you're going to tell the patients, but you're really ‑‑ you're really just saying, I think that you would not change the criteria or that's another way of looking at it to not change it.
	DR. SALOMON:  Yes, I mean, we'll get to that, but I guess I'm just trying to make sure that we're all comfortable with the concept that, in the absence of ‑‑ I mean, so one idea here is we know the exact answer, we set this limit, we hold the manufacturers to it and everything's great.  And I'm saying I don't think we're going to come to that.  We all know that.
	So, if you don't have that, then what you do is ‑‑ well, we don't know the limit so you're going to ‑‑ we're just going to do reasonable informed consent and that's an appropriate place to be today in this field.
	DR. MULLIGAN:  The only problem that I have with this is that once we go through the risk and then we go to how we set the dosage, and it's totally arbitrary, I mean, I don't think anyone would disagree with the fact that there's no biological basis for thinking that three‑fold difference is going to make any difference, other than, you know, less is better, right?
	DR. SALOMON:  That's my point.
	DR. MULLIGAN:  So ‑‑
	DR. SALOMON:  Yes, no, we're agreeing.
	DR. MULLIGAN:  So, I'm just trying to get us to focus on practically ‑‑ we're going to get eventually to whether the one number is the right number or the second number's the right number, and I would say, it's pretty ‑‑ the concept probably ought to be what's a doable number?  And that's about it, and I wouldn't make it complicated by all these risk assessments.
	DR. SALOMON:  Okay, yeah, I mean that's the kind of discussion ‑‑
	DR. GAYLOR:  There was a bit of a discussion this morning about the guideline should be based on RCA per dose rather than RCA per 10 to the 10th or whatever.  It's really the dose of RCA that's important.  So, maybe we should have some discussion about that rather than about ‑‑ it's the number of RCA that's apparently important and it's not 10 to the 9th or 10 to the 10th, it depends on the dose.  So, makes it more complicated, but that's more relevant it seems.
	DR. SALOMON:  Okay, Abbey and then Joanne and Ed.
	MS. MEYERS:  I just want to say handling it through the informed consent document is absolutely not acceptable, because it would come out sounding just the way Jay said, you know, you will have a more contaminated product than the guy in the next room.  And it's not right, so ‑‑ and it's a scientific concept, I think it's going to be impossible for consumers to understand.
	DR. SALOMON:  That's an interesting point, I wasn't thinking that there would be different standards just that you would inform that patient group differently but, yes, I can see the idea that different patients would get different amounts of RCA would be problematic.
	MS. MEYERS:  Yes, the RCA might be higher in people with genetic diseases than in people with bone marrow transplants and that ‑‑ it's not right, there's got to be one standard.
	DR. SALOMON:  Joanne.
	DR. KURTZBERG:  Two things.  I don't see where we have any data that says a higher number of RCAs is riskier.  And nobody's shown data to even say people measure it the same way.  But even if you had that, there's no data that says we know what's risky or that a higher number is risky.  So, we're making one assumption and then we're making rules about other things based on an assumption to begin with and I think we need the data.  And we don't have it yet, so we ought to get it.
	I also think that, you know, one orphan population that might theoretically come to therapy with this kids with inborn errors who undergo algeneic transplant, but also need gene therapy because the bone marrow transplant doesn't reach all the organs that need correction.  And in that population, you could consent that family, very easily and weigh the relative risks of your child's IQ will be that much lower or their bones will be that much more deformed and they would be able to weigh the risk of maybe they might get a virus versus they get gene therapy at a time when those organs are developing and they may not be the ‑‑ as injured by whatever the underlying disease is.
	I think you can consent that kind of ‑‑ I think you can have that kind of discussion and get ‑‑ give informed consent for that kind of therapy.  You're underestimating the depth of the knowledge of the families that might have to make that kind of decision.
	MS. MEYERS:  I have to disagree with you because a parent in that position will do anything to save a child and, in fact, the Belmont Report talks about parents of dying children as being a particularly vulnerable population.  And so, the concept, at that point, when they're standing there reading an informed consent document, talking to their child's doctor, they're going to sign just about anything.  And for them to truly understand that there's going to be more of a possibly dangerous virus in that product, then in the child next door who is getting it for cancer, he's going to have the idea that it might be more dangerous than what the kid in the next room is getting is ‑‑ it's not digestible to a family.
	DR. KURTZBERG:  Well, number 1 I think it is.  And number 2, when you're getting that kind of informed consent, you're also taking a true mortality risk with the procedure, which is greater, by far, anything, any theoretical risk you might take with the recombinant virus.  I mean, you know, you have ‑‑ in some of these procedures, you have a 20, 30 percent mortality risk from one or another organ's failing just from the procedure you're doing. And I do think people can be informed.
	I understand what you're saying about them being vulnerable but, nonetheless, they're usually well educated about their child's disease, well informed about the options and they really spend a lot of time weighing all of these things.
	DR. HOROWITZ:  Well, in a sense, we've already been there.  Because in the dose escalation, some patients will get more RCA and beginning patients will get less. So, I mean, in those studies, it wasn't said that you will get a more contaminated product because you'll have a bigger does.  I mean, there was a single informed consent, I assume at the various dose level.  So, in a sense, it's already been dealt with because of these dose escalation studies and not been a problem.  At least in that aspect of informed consent.  It depends ‑‑
	DR. SALOMON:  The other thing to ‑‑
	DR. HOROWITZ:  I mean, if you actually use the word contamination, you know, obviously there's going to be a reaction, but if you use it with more neutral words and just describe it accurately, in a sense, we've been there already.
	DR. SALOMON:  The other point is that we've also set ample precedent in other clinical trials like that.  Not just dose escalations but, for example, just different immunosuppressive drugs, I use them in liver transplant patients, heart transplant patients, kidney transplant patients and kidney/pancreas patients at all different dosages and different strategies.  And I'm, you know, I don't have any problem explaining that, even though the risks vary, you know, substantially so, I think those things can be done.
	MR. SIEGEL:  But we've never really asked people to consent about the standard for the quality of the product.  That strikes me as a different issue to consent on.
	I want to make a couple comments that might help put things in perspective and also to summarize and also to summarize in part what I'm hearing.  First, I just want to say that with almost all new technologies, it seems like we work through these same sorts of issues of theoretical risks that either become of less concern, such as, say, murine (?) retroviruses with monoco (?) antibodies and E. coli DNA with recombinant protein. And sometimes we come up more concerned.  And interestingly, there's always this tension because as they become less concerned and you think you might want to lower the standard, you get better technologies to where you can actually lower the levels and you realize that you're not just looking at safety, you're looking at quality control, and consistency control.
	And it's important to note in that regard that every drug you take, every product you take, certainly everyone I've been involved in improving ‑‑ approving, undergoes a lot of testing for maybe residual of every solvent that's been used for contaminants of all sorts of types that are potentially there.  The limits in many of those cases are not set on the basis of safety determinations, they're set on the basis of achievability both because less is almost always safer, but also because going lower because it is an issue of quality control ‑‑ you know, even if you know a certain level of LPS of endotoxin in a product is safe, if one batch has ten times as much endotoxin as all the other batches did, it should raise your eyebrow and make you wonder what happened in that manufacturing.
	And so based on what this Committee has said, it sounds like there is a broad consensus that there's no data on which to set a risk‑based limit, as far as any data we have, except in certain populations the risk appears to be limited and we have no quantitative data about risk at all.  And so, we're going to set a limit based on feasibility or achievability.  Now, one of the implications of that is ‑‑ well, there's a couple things to say about that.  One is that, unfortunately, achievability limits are going to be based on particles per total particle, not on RCA per total particle not on per dose, because what's achievable is a function of manufacturing and independent of whether you're going to give a lot or a little to a patient, even though the risk may be a function per dose.
	But as we've heard and as it sounds like, when we set a limit, if we set a limit based on achievability, we have a lot of options.  We can set a very rigid limit that can be achieved only at, you know, at great attention and then, if you're lucky, or you can set a looser limit, where as long as you're doing a good job, you're going to achieve it, you know, the large majority of the time.  And where we fall in that spectrum will depend on, in part, on the sense of how critical a factor this is.  If there's a broad consensus that, boy, the risks of adenovirus preparations are huge compared to the contributory risk of RCA whether it's 10 to the 9th or 10 to the 10th, the real risk are, you know, it's a small part of the total risk, that might feed into that.
	DR. SALOMON:  But I think ‑‑
	MR. SIEGEL:  But also, the nature of the population ‑‑
	DR. SALOMON:  Right ‑‑
	MR. SIEGEL:  Might feed into that to where, for certain populations, we may want to go one step further.
	Now, as to ‑‑ just one more comment, which is does it make sense to go one step further for certain populations than others?  Can we say to somebody, well, we're giving you a cleaner preparation, or if we don't say to them than the person in the next room.  I would say this, chances are that ‑‑ if you look at those two children, one of whom is in this first group and one who isn't, for example, in the hospital, we're already giving the one whose in that first group a different air environment, probably there's more gowning and gloving going on, you know, there may be filtered air, there may be ‑‑ there may be different foods, we're already exposing certain people ‑‑ you know, there are risks that every body's exposed to every day that we don't expose to severely immunosuppressed people to.
	So, I'm not sure it's irrational to say, you know, a certain amount of adenovirus is something that is reasonable to have in a product, but not in a certain special population.
	DR. SALOMON:  I think what I want to see us go to right now is we're not ‑‑ we're not ‑‑ we haven't lost sight of the fact that we understand that ‑‑ there has to be some kind of standard because we have to have something, I think, as Richard pointed out, I mean, you have to set some sort of standard and even in some ways one could argue a little bit of a higher standard, I think as long as it's not an absolute but, rather, one for the technology to evolve toward.  And also, so that when a complication occurs, whether we thought it was likely or not, that there is a track that we can come back to that we've moved the field forward in terms of knowing.
	So the question here is not whether or not we agree with the basic premise that some sort of standard could be, but I think what everyone said and, again, let's pick this up for discussion ‑‑ what everyone said is, these numbers are just not very valid, I mean, we don't have any confidence in the current assays that have been done, you know, at the very levels of detection of these assays for technical reasons and we haven't seen, really the true correlations between multiple laboratories with the new reference standards.  And I think that I hear from the Committee that I don't think we would want to give you advice on, you know, x‑number of RCA per thousand particles, at this point.
	Now, I mean, that was not meant to ──── discussion.
	MR. SIEGEL:  We didn't specifically ask for a number, none of these questions asked ‑‑ they asked you to tell us how to go about determining what this ‑‑
	DR. SALOMON:  And I think ‑‑ so, I'd like to hear some discussion on that, Ed, do you want to ‑‑
	DR. SAUSVILLE:  Well, actually, I didn't so much want to address, I did want to follow‑up on this discussion that, again, seems to be ‑‑ or could be inferred as meaning that we're going to accept cleaner, dirtier, contaminated, noncontaminated ‑‑ I mean, I don't ‑‑ I'm a little troubled with that is that we heard this morning that some levels of standard might cause up to some 50 percent of batches to be disqualified by more than one company.
	And I guess I'm a little concerned that, again, recognizing that each of these entities ‑‑ these viruses are unique that we could, potentially, be creating a situation by having this notion that we're going to have one standard that's going to ensure all batches or all production lots have the same degree of quote/unquote, "cleanness" that we might disincentivize the creation of certain constructs which for some reason or another are difficult to get to that standard.
	And I return to the fact that, again, this is a nuance that can be addressed in the informed consent process.  And it's not a question that a more or less contaminated it's a question of what the achievable biology is.
	DR. SIEGEL:  That's a good point, nobody's ‑‑ if I understand your point, that there's not a proposal on the table that you make a bunch of lots and you give the cleaner ones to some patients and the dirtier ones to another.  The proposal is that a given manufacturer can achieve a given level and, depending on where they're going whether that's acceptable may ‑‑
	DR. SAUSVILLE:  And that's going to be, to a certain extent, driven by what they're trying to do, actually.
	DR. SALOMON:  Phyllis.
	DR. FLOMENBERG:  I agree with what has been said about there being a rather low‑risk for toxicity from RCA in most cases. I would again, just like to bring up the situation of a naive patient.  I mean, I have a concern about giving 5,000 RCA to a naive child whose never seen that 5 and do we want to consider some type of prescreening of patients before ‑‑
	DR. SALOMON:  Let me ask ‑‑ I thought about that.  So, let me ask you a question, Phyllis, if ‑‑ and, of course, to anyone else, if you had documentable neutralizing antibodies to seratype 5 using a 5‑based viral vector, would that satisfy you ‑‑ would that be diagnostic and protective?
	DR. FLOMENBERG:  Yes, in most cases, other than the severely immunocompromised patient, I'd feel more comfortable having that information.
	DR. SALOMON:  And we have to keep in mind the point that Joanne made, vis‑a‑vis one of the target populations for this kind of the future could very much be a young or young child getting an autologous or an allogeneic bone marrow with a metabolic disorder.
	MR. SIEGEL:  Just as a point of information, most of our sponsors, I think, almost all are doing serologic testing and most of them are excluding seronegative patients, but some are not, and I think we heard from who are not, and I guess that's valuable information to include as a consideration.
	DR. SALOMON:  So, let's turn back to the question of does anyone ‑‑ so my impression right now is that the field deserves tremendous credit for picking up the mantel in 1999 and in less than, you know, in two years that's fantastic, to have developed a reference standard that'll be shipped within a year.
	So, I think that's great.  And until that's done and until the data is really there to discuss and even retrospectively to go back on some of your frozen lots that were given to these patients and get a real sense ‑‑ to look at the Gelsinger case, for example with the reference lot, because now you know, you can see how it could have been 10 to the 12th, Gelsinger could have gotten 10 to the 14th r 10 to the 15th, it's possible.
	I mean, I'm not saying anything like that happened, but until that happens, my sense is that, I don't think the Committee wants to go there with specific numbers. That's open for discussion.
	DR. KURTZBERG:  I just want to comment again on the babies with in‑born errors.  And I think in ‑‑ number 1 no matter what you can control in the product, you can't control the exposure of the patient. And that could, theoretically be much ‑‑ a much greater risk for recombination in vivo because the patient gets wild‑type virus, which happens in kids.  And I think that the relative risk of that versus the relative benefit of whatever the therapy is has to be weighed and that, there are going to be times in those populations where the therapy still carries more promise than the risk in that you have to inform the family and the parents, but you might still go ahead with therapy and I would hate to see something put in stone that restricted the availability of that kind of therapy to that population.
	DR. SALOMON:  Steve, Joyce, Dr. Semmick, I mean, what do you guys think. I'm concerned that we've now dodged your number issue, and I want you guys to comment on that because if you're not comfortable with that, you need to tell us that.
	DR. FLOMENBERG:  Well, I think if you look at the questions, we never really asked you to discuss a number.  It was what's the type of data that people need to be collecting in order to make the risk assessment analysis.  It's not what is the number.  That, I think, clearly, like Dr. Siegel was saying is based on what is achievable of the manufacturing record, it's more, but is there a risk or not a risk?  And if there's not a risk, then it's dealing more with, like you say, process, validation, you know.
	DR. SALOMON:  So, I think that you know, I think that what we're telling you is that the process that ought to happen now is this reference standard ‑‑ and it's happening, you didn't need our advice for this, I mean, what you're doing is right, the reference standard's going to get distributed, there's going to be retrospective as well as prospective studies based on the reference standard.  And that I think the Committee's comfortable with that going forward.
	We also accept the fact that the FDA does have to set standards for a product, that's a given and I think Abbey you should be, you know, I think that's the point you were making and I didn't want to give the impression that we disagreed with the concept of a standard.
	What we were saying is that special‑risk groups would get an informed consent saying, even within that standard, you may have a differential risk.  We're comfortable with that part, but not that we make all kinds of different preparations or different standards.
	MS. MEYERS:  I would feel very comfortable with the suggestion that people would be given some antibody tests to see if they have any kind of a ‑‑ will have an immune response to the virus.  I think that's an excellent suggestion.
	DR. SALOMON:  That was noted and let's see what else?  Yes.
	DR. BLAZER:  Let me ask you, even with the antibody test, there still may be, clearly, patients that you'd still want to consider this for whether you give them immunoglobulin for a period of time or ‑‑ you know, I think, I go back to Joanne's point. For the transplant population, and particularly in children, the risk benefit ratios are very long discussions but we're testing in children for their benefit the therapies that they may be the first ones that are receiving that, where we don't really know an outcome, but there's potential high benefit and there ‑‑ aside from immunoglobulin infusions, even there may be other strategies that would still allow us to provide benefit to these children and adults that would make the risk acceptable.
	DR. SALOMON:  Yes, I think that's a really important point, Bruce, I think we all agree that nothing that we're saying here should be an absolute prescription on anything, but it should be ‑‑ they're all contributing to relative risk.
	I think the point that you are well aware of, is that you could measure the titer in a young child before, and it could be positive and then what you do is you go ahead and totally wipe out their bone marrow and you and I both know in two weeks the ‑‑ you know, the antibody's cleared if even that long, right?  And the new B cells aren't making antibody so effectively that was all irrelevant so, I mean, I agree with you that in that population there are special considerations.  But those of us doing those kinds of transplants are aware of them.
	DR. HOROWITZ:  But in terms of the gene therapy after autologous bone marrow transplantation, we have already learned and should be careful in the future that trials not be approved where we know the expression of the gene is going to be short‑lived when we know the need for the gene is going to be life‑ long, I mean, that issue came up with the OTC trial.
	Now, there are two sides of the story, I mean if that therapy were given to tide over a two‑ day‑old baby for three weeks, until that child could be metabolically balanced by diet, there'd be a different consideration then if a manufacturer came in and said we are proposing long‑term therapy with a nonintegrating virus.
	So, with the current technology ‑‑ so I think, I mean, clearly the FDA will be responsible for assessing those risks, but I just think we should note there are differences of risk based on short‑ or long‑term therapy.
	DR. KURTZBERG:  I agree but in the in born error kids when you use bone marrow transplant, you have a 5‑, 6‑, 8‑month period before cells get to some tissues from the transplant and so you might be in a temporary situation, but where you're preventing damage until the more permanent therapy takes effect.
	DR. SALOMON:  Okay ‑‑
	DR. KURTZBERG:  Also, I just have to mentioned wearing my transplant hat, that whatever you set is going to have huge implications in terms of what insurers are going to cover.  And so if you say, well it's really okay, you know for you to treat these in born error kids because we understand, but your package insert says something different, then that makes a huge public health problem or a small public health problem, but a huge problem for that population, because that caveat is in there.
	MR. SIEGEL:  At this point, of course, we're not close to writing package inserts, that would come.
	DR. SALOMON:  So let's, I mean, I think that sort of covers question 1 and a lot of questions 2.  Please discuss the sort of experiments or data that you've used to set acceptable limits for RCA exposure. Joyce, you're looking concerned.
	DR. FREY:  I think we've all agreed that question is probably, we've got plenty of guidance from the Committee.  It's ‑‑ we definitely want discussion, though, on number 3.
	DR. CHAMPLIN:  I hope we're collecting data on the ongoing trials on the dose of recombinant adenovirus that people are, in fact, getting, and being able, then to draw some conclusions on the safety of various levels of every components of every component of the infusion, because there's a lot of data out there from the hundreds of patients that we've seen in terms of real‑life experience.
	DR. FREY:  Well, we are, but I think you have to understand it's also in the caveat of the ability of the assays for detection of RCA and that's why some of it, with the reference material to be able to retrospectively go back and more definitively measure that is one of the things that we plan to do.
	DR. GAYLOR:  I'd like to make a suggestion for the FDA.  I think we're collecting ‑‑ generating the right kind of data and particularly getting a better measure now of RCA.  But it's very difficult if you're looking at data from, say, a clinical trial in advanced cancer patients, where half of them are expected to die within the next three to six months.  It's very difficult to tell if you're doing any harm. And what I'd suggest, and I assume FDA's going to do this, just analyzing data from individual trials, it's going to be very difficult, but I hope you're going to put together data from two or three dozen of these trials, and not just look at incidents of disease, which is a pretty crude measure, but look at survival, not necessarily time to death, but look at time to disease or pneumonia‑free days or survival type analyses that are more powerful than just looking at crude incidents.
	So I think the right kind of data is being generated, what I haven't heard is whether the right analyses of these data are being planned.  I assume they are.
	DR. SALOMON:  I think they are.
	MR. SIEGEL:  Right absolutely.
	DR. SALOMON:  I think that if you hang in there that the next meeting will be on one of the things we're going to talk about is long‑term follow‑ up ‑‑
	MR. SIEGEL:  Right.
	DR. SALOMON:  The databases that are being developed with collaboration between the OBA, Recombinant Advisory Committee and the FDA on just those things. I think that's really important.
	MR. SIEGEL:  For the record, though, one of our highest priorities is work with the NIH at building a database that will allow or facilitate those sorts of analyses as we get more experience and larger numbers. The analyses are being done now, but on a less formal basis and, of course, as you've pointed out correctly, working on the policies for long‑term follow‑up it can be very tricky to make sure that you get the right information at a high reliability and that we will be discussing that.
	DR. CHAMPLIN:  When, you know, considering that the problem that we're talking about today is infectious recombinant adenoviruses, you know, these can be easily cultured, so, needless to say the patient should be frequently cultured in terms of trying to detect that virus directly as opposed inferring things from survival.
	DR. SALOMON:  Yes, I think I agree with that and I think the Introgen trial, specifically, set a pretty good example for that looking at different times.  I think sometimes you're only looking at 28 days, but in a couple of your studies, you look more frequently and I think that was, you know, that's the kind of thing that needs to be done.
	Okay, so, when adenovirus is used for {IOn}ex vivo{IOff} transduction of target cells, ex vivo now, we're shifting gears a little, should RCA measurements be performed on the transduce cells, before you infuse them back in?  Now, remember, we do that routinely for retrovirally ex vivo transduction of cells with retroviral vectors, you always RCR in the transduce cells, even though you also have to show that there's no RCR in the initial suit so ‑‑ the initial infectious suit.  Now, what do you ‑‑ in this case, though, we are going to put in some RCA, so what do you think?
	DR. LAWTON:  Maybe I can just, I mean, we've just been talking about the methods.  If we're having difficult measuring it in what we're adding, how are we going to actually measure it in the actual cells?
	DR. SALOMON:  Are you suggesting, Alison, are you suggesting that you don't have confidence that the current detection sensitivities are low enough that if you do 10 to the 10th T‑cells or something ex vivo that you're not going to know whether you got it or not?
	DR. LAWTON:  I mean, obviously, it's a question, yeah.  And until we have better understanding of the methods and the detection levels, et cetera, I'm not sure whether you're going to get anything extra from testing those cells before you put them in.  That's just an observation.
	DR. BAUER:  I think the thinking here was that there would be amplification if it was an RCA, so it would be a relatively sensitive method.
	DR. SALOMON:  If you put ‑‑ this is, again, I just don't know this, I mean, there are people here who know the answer to this ‑‑ if you put wild‑type adenovirus on T‑cells or hematopoietic stem cells, which would be two logical populations for this sort of thing ex vivo, do you get replication and is it detectable?
	DR. FLOMENBERG:  It's very inefficient except, as I mentioned some of the group B serum types seem to be able to bind better to hematopoietic cells.  But, in general ‑‑
	DR. SALOMON:  Bind or actually become productive infections, right?  I mean, I'm sorry I'm a retrovirologist.
	DR. FLOMENBERG:  Not really know they get in better, but yeah, it's not entirely clear to me, but you get very little ‑‑ you don't really get ‑‑ you get very little replication in hematopoietic with adenovirus.  And there are probably several steps that are blocked, including binding, internalization and then expression.
	DR. HOROWITZ:  Well, that's true, Phyllis, you do have a ‑‑ produce a B cell line, right, that makes adenovirus?  I think, in general her answer's correct.  I mean, most lines that the adenovirus will enter will not replicate the virus and produce either any or very little progeny, but there is this EBV transform line that right, that produces adenovirus?
	DR. FLOMENBERG:  We isolated a BE cell line from a patient, a bone marrow transplant patient, both transformed with EBV and also had a productive adenovirus infection.  But, in some cell lines, some transform cell lines, T‑cell lines, B‑cell lines, you can get like a jercaps (?) are relatively ‑‑ you can get some replication. But primary cells, I think is very limited in a number of steps.
	DR. KETNER:  But this is really the question, isn't it, I mean, you take A patient cells, treat it with a gene therapy vector and, you know, most, you know, maybe it won't replicate it in most cases, but maybe it will in some and so I'll need a test and see whether it did in those.  I mean, I agree, I think there would be an amplification, so I think it's easier in looking for RCA in the inoculum.  So, I guess I vote yeah.
	DR. BLAZER:  Can I just ask, how long does it take to replicate and how many cells would you need to study in order to pick up anything in the time frame after which you've added the virus and before you're going to infuse the cells?
	DR. HOROWITZ:  Well, the minimum replication cycle is probably about 16 hours but, as a practical thing, probably about 24 hours in the permissive lines that we use in the laboratory.  In some of the less permissive cells, I mean, that don't replicate as well, I mean you might have to wait two or three, you know, up to three ‑‑ two or three days to do this assay.  But definitely the data should be obtained as I think because very small amounts, as has been pointed out already could be amplified quite significantly.
	Those cells that would amplify virus most likely will die and the virus would then be slowly shed extracellularly. It could be dealt with in other ways, I mean, those cells could be treated with neutralizing antibodies to reduce the risk of RCA extracellularly, but definitely the data should obtained so ‑‑
	DR. BLAZER:  So, if you know how many RCAs you're putting in and you know how many cells you can actually assay and you figure out the time frame, if something's even 100 percent permissive, would you be able to pick it up given the aloquata (?) cells that you'd be able to measure, would you use that as a lot‑release criteria? There's a difference between getting retrospective data and saying that you can't infuse the product without that information? I'm just asking as a question, I don't know the answer.
	DR. HOROWITZ:  It would be hard to do it quickly, I guess.  Although you could do it by real time PCR to look at the amount of virus that was released.  I mean, the problem is compounded a bit because productive adenovirus infections, the virus remains cell bound, so it might not be out in the supernatant to assay for even a few more days beyond what I've mentioned.  I mean, one of the things that the manufacturers know and we all know who work with it that the virus does remain, even completed virus in cells that ultimately would die will remain cell associated for a number of days, so, yeah, I ‑‑ the time frame of speed would be somewhat compromised in terms of our ability to give an answer within two or three days.  How long do you think from a cell‑point of view ‑‑ how long do you think one would have to keep the cells for transfusing?
	DR. BLAZER:  Most people, when they culture cells will do it in a matter of days to a week to ten days, there are some that are going three weeks, but people are trying to shorten culture periods to seven days ten days or less ‑‑
	DR. SALOMON:  Well, in hematopoietic stem cells, I mean I wouldn't want to go over 72 hours.
	DR. BLAZER:  And you can't for those, but I was even thinking of T‑cells to take the extreme, most people are trying seven to ten day cultures.  So you're thinking of, when you expose the cells to the virus, and then you're going to have to take cells at when you would infuse it, hold those cells, take several days to do the assays, keep the cells in culture and then release it, you're only measuring part of the time period anyway and if the statistics are such that you're really not going to have any kind of likelihood of holding up the infusion of those cells, then I'd say get it retrospectively on the cells being infused.
	But if the statistics are that you have a significant risk that you're trying to avoid, and you pick a time period before infusion and you have a sensitive enough assay, then that would ‑‑ then you could build that into a lot‑release criteria.
	DR. HOROWITZ:  Well, of course, this experiment could be just done ‑‑ I mean it could be done, I mean, on cells that were not going to be transfused to infect them, and I don't know if anyone in the room has done those experiments could help us.
	DR. SALOMON:  Marshall, Beth had a point did ‑‑
	DR. HUTCHINS:  Yes, along those lines, we don't have that kind of data but the fact is, I'm also on the U.S. expert committee on cell tissue and gene therapy and we've discussed this issue, actually, in terms of looking at prospective lot release and what real‑life situations actually occur. And the fact is that you would probably be forced, even with your longer‑term, well, maybe not with three weeks, but if you're really only talking seven to ten days at best before that gap that's your time frame that you've got to do to deal with things, that's probably not, maybe, that's on the cusp of not being realistic at all.  Because with an amplification step, even if you used PCR as your read out to get very specific information and they're sensitive right off the bat, you're going to need to allow a couple of days of amplification.
	I mean, most people do a minimum of three to five days as a first amplification for RCAs and the sampling amount that you're going to be able to take, just because the number of cells you're going to have is limited and you don't want to take, you don't want to use it all up just to do this one test, you're also going to be doing other analytical methods as well, again to ensure that you knew something about what you were doing prospectively, not just collected retrospectively.  I think you would actually be forced into a retrospective analysis situation most of the time.
	I'm not saying you shouldn't necessarily get that data, but I'm not sure you could do it on a lot‑ release basis, just practical aspects of it.
	DR. LAWTON:  One of the things that you could do prospectively is, actually, I think somebody else mentioned it earlier is to actually look at the cell type being transfused and see whether it's permissive ──── virus.  At least understand that.
	MR. SIEGEL:  Let's take as a given, we have a log history of regulating cell therapies and, you know, this issue always comes up in a cell therapy, you know, it takes three weeks to do a fungal culture. Should you require a fungal culture before you give the cells?  We've never required the results of a test that can't be done in a manner consistent with the manufacturing of a test.
	So, let's just take that as a given, but there are times, we have some products where the cells are transduced and frozen before they're administered and you can keep them as long as you need to and so, let's just, please look at the question. Assuming, you know, and some tests can be done quicker and some cells are long enough. If it's feasible should it be required prior to release?  And if not, should it be required as part of a data gathering tool for further understanding retrospectively.
	DR. SALOMON:  So, I think what I've heard, so far, again, trying to seek some sort of consensus is, if you have ‑‑ if you can prove definitively that your target cell population will not support productive virus in any setting, and that could be, you know, that would be something that you'd have to be convinced that had been proved appropriately by the sponsor, but then it probably would be okay to exclude it.
	If you have a procedure that you can't do that, therefore, there is a possibility of amplification, then I think to pick up what Gary said is that if you can do it, then you should do it.  And then that really just kind of feeds into what everybody else said, including Bruce's and my points, and that is, if you can do it.  And if you can do it prospectively, fine, that's you know an added safety.  Why shouldn't we support that?  If you can't do it prospectively, then it should be done retrospectively and, again, that should be a decision based on the protocol being presented.  Does every ‑‑ Gary.
	DR. KETNER:  Let me just emphasize again, that we don't have a clue what the risk of injecting any number of adenoviruses are, so, I mean, I guess this is data we sort of collect and then wonder about later until we learn what the infectious dose is.  So ‑‑
	DR. SALOMON:  Right, the question on hand now isn't would we not allow delivery of the product if we found replication competent, that's your point and it's well taken.
	DR. FREY:  I think you also have to keep in mind, when it comes to cell therapies, I think particularly, like T‑cells, CD34 cells per, you know, cell populations isolated from the peripheral blood, rarely do we see purified cell populations.  They are mixtures and so, to say that you look at it and say that it's nonpermissive, I think you have to be very careful in saying that, because, like I say, rarely do we have purified cell populations when we deal with this.
	DR. SALOMON:  Yes, so, but let me point out the way I would think about it. So, and I proposed a gene therapy with T‑cells.  Well, we'll do leukoferresis and we purify away the DC34 and we take what's left as T‑cell enriched, and that's our target. That's the kind of thing that one could specify for any given study, you know, whatever the product was that we were going to use to study on.  Even if it's a mixture of cells.  You know, you're point is right. You could still reasonably give you ten, I mean, I could, theoretically, get ten patients samples together and do the study, and demonstrate one way or the other whether they were permissive.
	DR. HOROWITZ:  Well, I would I assume from what I know about peripheral blood cells that ‑‑ stem cells, that they will not be very permissive, but it seems to be before these products should go into patients, that someone should get some preliminary data on cells that will not be transfused but just be obtained to see whether you produce 10 to the 13th, which I doubt, or nothing or, you know, a hundred RCA.  I ‑‑
	DR. SALOMON:  You're saying just, that's something the field should do ‑‑
	DR. HOROWITZ:  The field should do it before ‑‑
	DR. SALOMON:  ‑‑ to increase the overall safety profile ‑‑
	DR. HOROWITZ:  Exactly ‑‑ exactly.
	DR. SALOMON:  Well that's an interesting ‑‑
	DR. HOROWITZ:  And then, once we have the data, we could decide how to proceed, I mean there doesn't seen to be the data in the room.  I would guess that it's not going to be a problem, but I don't want my guess to set a standard for the field, but the experiment should be done.
	DR. SALOMON:  Well, Marshall has an interesting point, i mean he is pointing out an interesting irony in that we've been infusing these things directly into the blood and there isn't data that we've heard, specifically, saying that if you took a leukoferresis pack, for example, that he wouldn't get this tremendous amplification of RCA.  So, I think that's a very interesting point that hasn't come up yet.
	MR. SIEGEL:  Well, of particular interest to me is I'm told that probably the single most common application we're seeing of ex vivo cell transduction is tumor cells in the manufacture of potential tumor vaccines, and it would seem to me given what we heard this morning about, the fact they say teratoma lines might support even replication of noncompetent adenovirus and given that any given tumor line you don't really know what genes are on and off and we don't know which ones are the critical ones, that it would see that, like, both in general in terms of reproduction in tumor lines but also more feasible in the specific cases that it would make sense to get some information so we know what.
	DR. SALOMON:  So, I think that as far as I'm concerned now, we've answered the three questions that the staff has asked us. And now there are a couple other things I'd like to throw out that in the next ten or fifteen minutes is that I mean what's the Committee's with right now.  Is everyone going to run off right now to a plane or can we have another 15 or 20 minutes of your time to raise one or two other issues?  Can I get a little bit of feedback here?  Okay.  One question, just to put this into context, is that there's another class of adenoviral vector that is intended to be replication competent.  And so, I think that you know, I'd like to just throw that out, because I don't think our conversation's quite complete unless we just think for a second that there are people proposing adenoviral trials with vectors that are designed to be replication competent or certainly to be driven by promoters so that you get a relative increase in production, let's say in a tumor cell line, but we all know how leaky promoter systems are in that activated cells and other cells in growing areas, are going to be turning this on to lesser extents but still real, so I mean, does the ‑‑ do you want maybe the FDA staff give us some sense of where that fits into the conversations we've had all day?
	DR. BAUER:  I think that one of the perspectives we have is that with those kinds of indications or those kinds of vectors we're looking very closely at what the indications are.  Most of them have been in cancer patients so far, and then the other thing is that we have an increased level of concern reflected in preclinical studies and clinical monitoring for those vectors right now.  But I think it is a very difficult and challenging task to try to separate out replication competent recombinants from a replication selective preparation.  I think that's a difficult task.
	One possibility that is being explored is PCR, but I think we've heard some discussion that the limitations of that and the caveats that come along with that, you don't know if you're looking at just a piece of DNA or something that's really a biological event.
	DR. SALOMON:  Yes, though this PCR thing still is ‑‑ there may be some sensitivity issues, but in some of the newer tack ──── allow you to do rather long pieces and so it wouldn't be impossible to generate a quantitative PCR assay where you had a up ‑‑ your downstream primer was in your transgene and your up‑stream primer ‑‑ your sense primer was up in the above the or in the first part of the E1 region and argue that if you got, you know, you got the right‑size construct ‑‑ a reasonable construct there and sequenced a little bit of it then that's a replication competent retrovirus ‑‑ I mean, adenovirus sorry.  I did pretty good today, that's the first time I did that.
	DR. BAUER:  I think we would agree that that's, you know, there's just some assay development that's needed there, but that's, perhaps, the most promising avenue is PCR, at this point.
	DR. SALOMON:  Do we agree, though, that for the group for the sponsors that are thinking about going forward, I guess you called them replication‑ selective adenoviruses, that you wouldn't hold these ‑‑ you couldn't hold the same criteria, obviously, for RCA levels, right.
	DR. BAUER:  Yes, that's correct, we acknowledge it is not a reasonable way to measure them in a biological assay.
	DR. HOROWITZ:  Well, I was just going to say, I mean, the experiments are already going on, of course, with the onyx 015 which one of us believe is replication competent in so many situations that, in a sense, the data that's being obtained should be very helpful in this regard.
	DR. BAUER:  But also I didn't say, again, that we are looking at PCR data, such as it is, to make sure that the replication competent recombinents are looked for.
	DR. SALOMON:  Another question I had was, you know, we've talked about replication competent adenovirus and all this in terms of a context of safety and we go back to a reference standard that's based on a wild‑type adeno, so our risks are in the context of what the risks of replication of a wild‑ type adeno.  Do we need to be concerned about the additional risks of replicating a construct that has a transgene it in?  I mean, it's one thing to have a wild‑type adeno replicating in the patient, but it's another, you know, delivering, let's say an anti‑A poptosis or a pro‑A poptosis gene into multiple cells and to what extent is that a risk factor that we haven't discussed at all today, relevant?
	DR. BAUER:  I could make one ‑‑
	DR. SALOMON:  Beginning to look, like, don't go there.
	DR. BAUER:  I can make one comment that most of the events with the vectors that are currently used that result in a replication competent virus, eliminate the transgene.
	DR. HOROWITZ:  ────
	DR. BAUER:  It's the part of the genome that needs to be replaced in the recombination event.
	DR. HOROWITZ:  That would be my answer, always and ‑‑
	DR. SALOMON:  That's a good answer, I mean that ‑‑
	DR. HOROWITZ:  Yes ‑‑
	DR. SALOMON:  That would raise the safety quality, a bit.
	DR. HOROWITZ:  For most of those considerations that's exactly correct and the answer I give when people worry about working with recombination will eliminate the transgene.  There are some  ways that you could think of getting around it, but in general that would be the most common thing.
	DR. RAO:  It's just a question for the FDA though, is, do you have, right now, in a standard, what is the absolute limit of wild particles that you can infuse in a patient in any of these trials?  So there is no standard?  So it's impossible ‑‑
	MR. SIEGEL:  The standard we have, as I understand it is based solely on the proportion, if you will, of the total, that is RCA, not on the total that would be infused.  Now, of course this is what happens in clinical research is that one does dose escalation from levels that one has a lot of information about gradually into levels that one has less information about.  So, on wouldn't suddenly push the boundaries tremendously, but on the other hand there is no specific top limit of what could be given set at this point.
	DR. SALOMON:  I think what we talked about before, and it's just beyond the agenda we set for this meeting, but it is a good message, I think, Mahendra, that the probably the bigger risk of ‑‑ in terms of to the patient, not a public risk, but a personal patient risk is the effect of these different viral proteins and the immune reactions and the cytokine release.  And that is probably going to be a function of the total dose given at any one time and the total dose given over by the protocol, though I'd be more concerned about the total dose given in one shot than I would be over, you know, ten shots of a relatively small amount over a period of time because of the antibody data that we've seen from the sponsors, but. Okay, any comments, last questions from the FDA staff?
	DR. BAUER:  I'd just like to say thank you very much for these deliberations. They're going to be very helpful and I don't have anymore questions for you folks.
	DR. SALOMON:  Okay, well, if, anyone else on the Committee have anything or public?  No?  Well then thank you all very much for a good job done and see you guys in a few months.
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