Good morning. Please standby for realtime transcript. We will reconvene in a moment. I'm just waiting for the slides to be loaded. Good morning everyone. We're going to reconvene the December 2005 RAC meeting. Good morning. I have two announcements this morning. Quiet. Please sign up for transportation to the airport if you would like transportation at the registration desk outside the doors. If you drove to the meeting today, please go to the registration desk for your complimenttary parking vouchers. On that note we will open this mornings's session with the transduction of upper airway ep they'll yum air ways in human placental Dr. Dusty Miller will present. Dr. Miller? >> Is this on? Yes. >> Thank you I didn't see you behind me. >> Moira L. Aitken is also here. >> Will Dr. Moira L. Aitken be presenting this morning? >> Yes I'll start out with the vector presentation and biosafety data and Moira will follow-up with clinical presentation. This is recfor--i'm going to review a little bit so everybody's on the same page. It's a very similar AV recfor that has ACDA there are no other genes and then it has the AAV terminal repeats. This is packaged--this is basically an AV2 vector much like what you've seep in the past. It's packaged using AV two rep the only difference between the ones in people is it has a AV6 capsid. Why we are excited is the lung ducktion that's we put anywhere from 100 to 200 micro liters into the 2 nose inhaled spoontaneously. It was AP positive cells. You can see a lot of transduction in the airway. A closeup up here shows many are transduced in the airway the major in the lung. There are a few blood vessels here, here and here and prangca ma is down there. This is where we want to be for CF. The nose of these animals is transduced all this black staining is AP positive cells in the nose. They get it by naisele insulation by this end we can show good nazele transduction. This is experiments that we did with human naisele polyps. Here I show AV2 with con pair son the trabs transduction with AV6 is better in the human samples. To back up a little bit we have proposed an AV2 trial and now we're proposing an AV6. One of ideas would be to compare these in the humans. We would like to know which vector works best if either at owl in comparison what we've seen in mice and rats. We've done a preclinical safety study with AV6. These are the details here. Wee used rats. And the reason we used rats instead of mice we you used them both in the first study with AV2 the rats give normal levels of serum parameters and are generally healthy. We had problems with the mice in the last study where the liver enzymes bobbed up and down. It want related to the vector administration but you couldn't tell. We used rats 150 grams when we start. Administered these doses her 0, 3.3 times 10 to the 9th. 2.2 times 10 of the 8th vector gene Nomes per gram. CF subjects are quite a bit lighter than normals. We're 3 planning to do 0, 10 to 11th in the first group and 10 to the 12th vector genomes. 2 times 7 vector genomes to the gram tenfold less the max there. We examined them day 6 day 60. We looked at DNA the lung, blood and go nads and did historiology. All of these were uneventful. The serum chemistry was find expect one parameter and the CBC was normal range. Histology I already sent that to reviewers it was fairly complicate but nothing that suggested a toxic vector response. There was sign usite tus and things like that. I don't have times to present all of that. This was one area where we had a serum chemistry fortunately in the right direction for us. Here's the date for the serum chemistry in the blood. You can see in in group everything was in normal range. No difference between the different controls. What I'll showing is control, control male, this is a low does male group, high does male group, control female low and high dose. There is no effective dose towards higher enzyme levels here and they're. That's day 3. Day 60 we had one group a bit high control males showed high ADAST. None of the vectors showed normal level over that. that was the only variable that we had that was significantly different between the groups. As far as the DNA I have to explain the chart. We looked at lung, blood and gonad and the des sick Nateors--designateors is the high dose female 1 through 5 in the animals. Here's the high dose male and we looked at high dose for genome 4 transfers. You can see high levels of the DNA in the lung. It's variable with the section of the lung where it winds up. Sometimes you get a low darkly stained sometimes not. You see high mass DNA in the lung. If you look at the blood then, two animals were positive at purple bars. The rest of animals were at the background levels I indicated that as 14 copies per microgram DNA that was limited detection. In gonad all gonads were negative we were below the limited detection of SA14 copies per micrograms in DNA. In the day 60 animals, the lungs were positive, less so than day 3 as you would expect for DNA and all of the woods and gonads were negative. There was no signal above the copy per microgram protection of as say. Given we're going if the lung very little escapes in the blood and we can't detect anything in the gonads. That's it for the biosafety vector design and Moira was going to present on clinical aspect of the disease. >> Thank you very much for your review of this protocol. I'm in the department of medicine in the division of critical care medicine. So unlike your other protocols that you are reviewing at this meeting Cystic Fibrosis sis is not a is--epithelium cells of the organs but the vast majority patients die of the respiratory disease in this sued we'll use the sinuses as the lungs because of easy of access and it's been used in other gene therapy trails. As I said, the survival of Cystic Fibrosis is improving with treatments of anti-botics and patients survival remain 35 year-old and 5 the need for novel therapies in cystic fib brose sis. So or ren at a time the protocol will be presented in the next three slides. Our objective was to determine the safety, transduction efficiency and emmun response associated with the delivery. A double blinded placebo control randomized trails. The escalation to the second level will only occur if if there no safety concerns at any stage during the first dose level. The sample ceise there are two dose cohort per subject cohort the placebo 3 will perceive the vector and total of 8 subjects if both levels are study. The subjects are patients with Cystic Fibrosis if will be dealt until 50 years old and subsequent can cohort 10 to the 12th vector genomes. Our outcomes from the study are primarily safety. Secondly looking at the serum neutral lizing anti-bodies and he have efficacy outcome is--subject inclusion criteria aged Cystic Fibrosis aged 18 to 50 years, their lung function has to be relatively good. We're taking 40% predicted lung function at screening. Their oxygen saturation has to be above 90% predicted and serum negative for the A section and use contraception for the studied that's 190 days and have to give written, informed consent. I haven't outlined the exclusion criteria which is longer. In the preliminary work-up towards the study we looked at how many people are serum negative or positive. 70% of adults patient are serum negative of AV. This f study of almost 200 patients. The first three have Cystic 6 Fibrosis and we looked at a group of normal adult. As you can see in the Cystic Fibrosis adults which is the this population the pink or purple darker purple are AV6 coming here about 25% and AV2 is more like 27%. We're being conservative in saying the catch of the patients will be about 70% of the adult patients. We are planning to deliver the vector as we have historically with other vectors to the in fearer ter banant this is showing the mild. The cat der is placed 2 to 5 back from the noztral we know it's expressed in that area. 100 to 300 micrograms is delivered over a 30-minute period where the patient either upright or in a semi-recome bant position to that they're comfortable. An issue that came up that there may be spillage up here to the ol facry never up here--there is no AP expression in the brain in rats. Again in preparations towards this trial we wanted to make sure we could get adequate biopsy samples and sampled the in fearier ture banant for five patients for other reasons this good sample tissue was obtained I'm grateful for these samples from the University of Washington. You can see the epithelium is shown here and no in dodgeious staining. We feel we will get it for our measure N. response to some of the questions that came up from the committee, the sample size justification. This is a proof of concept study to determine if AP can be delivered to the upper airway. We think it's a pilot study to guide the AVCSTR trails, future trails. It's a 7 machine nisic studied nod powered to produce a statistically result. The reason for this AV6AP is not a therapeutic vector and wanted to limit the subjects in this precious patient population we use. In summary the number we chose is a compromise to ob taint enough scientific data but to minutenize the number of the subjects and my patients with non-therapeutic vector. AP expression, we predict that expression of AP will increase over time. We're doing our biopsies at day 7, day 28 and day 112. We predict day 28 and 112 will be similar but more than day 7. We predict that the transduction rate will depend on the vector dose, a higher dose will have for transduction. So the transduction rates will be at the higher dose level. AP expression, we're going to primary outcome measure is a percentage of AP positive epithelium cells. We have designed a strongly positive response. If any of the three naisele biopsies taken at each of the study days will be greater than 10% of the epithelium cells A. dose will not extend on the lower dose transduction rates. So if we had a relatively high transduction rate of 50% we would want G on to the higher dose. We do have a proposed amendment if 2 of 32 study subjects are strongly positive, in other words they have more than 10% transduction, the study will be considered a success and no further patients will be enrolled. However it no subject or one subject is strongly positive at the higher dose then 8 we wish to recruit three additional subjects, that's a total of six we wish be able not just study three with one placebo but have three at that higher dose. We would like to the committee's permission to recruit more patients with a equivocal dosing. Our future studies with AV6 is we would like to procedure to AV6AP in the lower airway recognizing the upper airway may not be a good surrogate. We would like to procedure with AP6TFR with nazele and ultimate goal is AV6CFTR into lung as a potential therapeutic measure in patients of Cystic Fibrosis. That's what I have to say and Dr. Dusty Miller and I have happy to try and answer any questions you may have. >> Thank you both very much. We're going to go through our formal review process and begin with Dr. Dewhurst. >> I would like to thank you both for a very informative presentation and also to the responses to the questions we raised. I want to preface my comments by saying this is a very different trial than some of the ones we heard yesterday whether there was a lot of discussion of potential benefit and risk. Obviously in the case the benefit is very indirect benefit. There is no potential benefit to any of the study participants here. And the other aspect of the study that's unusual is your initial choice for the surrogate tissue for the one you're interested in down the line. It's some of those areas I would like to discuss I think. And some of the future plans to get a better feel. 9 Some of your responses indicate requirement for more information from regulatory bodies. I think it would be helpful for us to understand a little bit more where these studies are at and how compareable those data sets are in this particular study in terms of design and that sort of thing. For the record I had a number of questions many of which you have answered very satisfactorily. And the first one is you eluded to in the presentation related to the potential of transduction of cells in the ol facry bulb and transduction of cells in the central nervous system. I think the responses were adequate and concerns resolved. A second question and general interest about the AAV6 receptor and you indicated there's nothing known about that and that's perfectly acceptable. I had a question about focusing exclusively on the nazele route of delivery in your response you indicated that you're considered at some level that the nazele epithelium is all the regulatory bodies will allow you to examine. I'm curious if you can elaborate on that specific term. Clearly your long-term goal is to go in the lung. One of my concerns is what you eluded to Dr. Moira L. Aitken is the precious population you have one of the potential risk is these individuals my convert to AAV6 and a consequence where your vector prove in the future were they exposed to the AV vector may find themselves at a disadvantage down the line. I guess I wanted sort of some feedback from the both of you. So there's some historical 10 context to that. We initially propose to the norms and look at transduction rait in the nazele epithelium and lower airway. The tissue is more relevant to our ultimate goal. You can argue both ways. We had the same problems with CF patients immunizing them against A6. It's a subjective position and basically the RAC says we do CF patients that's why we're here with CT patients again. That's a reasonable argument I agree for you the down side for the patient is not what we'd like. >> In terms of--you know one of the issues at the heart of protocol is the extent to which the nazle epithelium is valid surrogate for lower respiratory tissue. At least in the human it sounds more or less an unresolved question. Is that accurate to say. >> It's a epithelium and looks like air ways there have been a lot of studies done in the nose with add know viruses. You could measure from there to here and we can go through a successful trial and measure a change in the epithelium. It's a reasonable epithelium. The problem with the lower air ways it's more invasive and harder to sample the tissue and quantitate what you have da duced. Maybe you would like to follow up on that. >> Absolutely I agree with dusty. I agree with him completely. The issue with sampling the lower airway did the that the patient have to undergo bron cose Topi and requires seddation. We have shown and phase 1 trial of that many of the serious adverse events were thought to be 11 secondary to the bron cose pea procedure itself rather than delivery. I actually think using the nose as a surrogate is a very good first step toward that. and regarding the serra conversion which I also had an issue with, the consent form as proposed puts that in there but I feel strongly that we would like to procedure with this and--precede with this and speaking for my own patients I feel they would like to proceed better for some to convert than for us not to proceed at all. I'm not belittling that issue. It has led to hours of discussion internally in our own group. >> You may remember too targeted genetics are conduct target 2CF in humans. We don't know in the transto transduce cells. We no very little from the study as far as why it didn't work. CF disease is highly variable and hard to get an efficacy measure that you can use on limited patients. We would like to know it it work first and then transfer our measure to CFDR. >> All right, thank you. I had a question for plans direct comparison for AEB2 that's contingent as you indicated upon resolution of the questions from the FDA with respect to animal studies on that. I wonder if you would add on that. >> We're in the sticking point with the FDA in terms they want an additional animal study which we don't have the money to pay for right now. It's a huge studied takes a lot of effort and all the controls and laboratory procedures and so on. Our position is sort of AV2 has been used in people to a large 12 extent over 100 people why you would want for animal studies. It showed no bad efforts but the problem was we had variable levels of--they bounds around with the mice it was a problem too. >> I would like to kind of make a comment here. I think there's a number of issues involved here so I'll try to point out maybe 1 from the FDA's point of view is that although--i'm not going to go into discussion about animal models for AAV2. I think one of problems the FDA had is if you're doing a direct comparison you do exact protocol where you look at them and the issue will come back up again with a lot of the comments I've read it looks like you have to two separate protocols if you're comparing them why you aren't comparing them together. >> This is a new one for me. Doing them together would be I think time tense difficulty because of where we're at. If we can't get one protocol approved it's hard to do two comparisons. I think one of the concerns of FDA they felt that our data suggested AV6 was better vector and why do AV2. Let me finish. On the one hand we have the scientific question of which one really works better in people. On the other hand we have it that AV6 works better. I think the argument could be made to with AV6 and see what the result are. >> That's fine if that's what you feel go forward with the AV6. I have a question while I've got the floor. Again it looks like you're going to do there in sort of the multi-steps. First go in the 13 nazle epithelium to look for the transduction efficiency and not tell you if it's hitting the target tissue in the lung. The next step is the lung. Now you have the two separate patient populations to have potential to convert and not until the third trial that you go in with the therapeutic gene. Is there a reason why with all safety, safety studies is there a reason why you wouldn't go forward with the actual therapeutic gene. >> Part of reasons for not moving quickly is does not fit well in the AV vector it's too big. Tough do something to solve that issue. The way we approached it it so use two vectors to allow a combination to give the full length CRT gene are promoter. It was never clear in my mind whether that worked at all well in subjects. So we got a little bit of problem with CFTR approach. We're working on that. We've it going well in mice and better in rats. We're doing the CFTR studies. May be the best approach would be to take the dual vector approach and put if the nose and show it works in the people. What you're asking for was the original RAC review why don't you do the whole thing from the start. I think we have to to take a step-wise approach. >> If the actual product changes dramatically or enough that the FDA things it has changed considerably you would have to do animal studies to do know I'm thinking to udefense--why to the develope that system first? >> If we wanted to do CFTR we have the two-vector system. We can bed with that if we get in 14 the patients we can't so the affect we don't not what the problem is we don't know if Idaho a blocked poor CFTR expression. It's a measured approach. We may not go to the lower airway with the AVPA vector. I think it depends on the results of nothing whether the nose is transduceable with this vector. It's epithelium makes CFTR and a reasonable circuit for the airway. We know in the mice and rats we get more transduction in the nose with higher doses and more transduction in the lung. We may be surprised. I mean if the results were negative in the nose, who it suggest we ought to go to the lung? We don't know until we get more results. If we don't take that first step, it'll never happen. I mean have you arguing we should to with CFTR right into the nose. >> I'm not arguing anything it's your trial I'm asking questions why this approach. And my concern is what you see in the nose may not have any relevance in what happened in the lung because you know with the most of the case with AAC2 there was not transduction in the lung so even if you see it in the nose it doesn't actually say it will be transduced in the lung. If you get positive that doesn't necessarily say you will get positive in the lung. I guess I'm going in circle here. I would like you to think about how you are designing this experiment doesn't mean you will get what you are. >> Just to show transfer. One of things I approached with targeted genetics why not put an AE2 vector at the same time of 15 the other vector to express AP. If we could show AV2AP is safe in the nose we might think of that as a marker for the actual gene transfer. Do you see what I'm saying? If we had a CFGR vector we put the lower airway and spike a ten fold lower dose and get a transfer of that. >> I don't want to turn this into a an argument. This is clearly a discussion between you and FDA. It just wanted to bring it out because I had questions and I hope you'll be able to help answer it. >> I certainly appreciate the discussion because it highlights the issues in a protocol and that's what we're here to discuss. Dr. Dewhurst I'm turning to you as first pry primary reviewer. >> That summarize the comments I had wren and the patient enrollment and numbers many we have a number of members in the committee here who may have enput on that I'm not an expert on that area but I would be interested in hearing what my colleagues have to say. I don't know that I have remaining questions or concerns myself but I am interested in sort of hear what the group feels on the broader issues of the nazle target, the use of the vector that is not the end game versus CFTR those broad issues. The broad issues as you eluded to as a combination-based strategy which is different than the strategy you're piloting at present. At least in my mind it seems reasonable to want to validate the activity of the AAV6 capsid transduction and go to the strategy. It's a reasonable approach and I'm 16 interested to hear what others have to say. >> Doctor Helen Heslop. >> I would like to thank the investigatingors for their response. My first couple of questions were regarding the the clinical study did he seen and I asked for om rationality in choice of sample size and discussed in more detail how the study would be used to guide future investigation. I think that was done in the supplement they provided in the presentation this morning. I asked for more justification of the choice of study design between the C bell and study agency and eng that was addressed in the presentation. My next question was biodistribution after nazle distribution that also I think was addressed in the presentation. My final question was same one raised by other doctors about whether there was preclinical data to suggest the result with the nazle delivery can be extrap lated for future studies and I suspect there's nothing else to add. >> Thank you Dr. Helen Heslop. Our third reviewer is Ms., quan. >> I think a lot of the questions I had was raised by the previous two reviewers. My concentration was on trying to get these technical explanationses that were ch changed at the table into the language that is understandable by potential participants in particular because as the discussion showed it's possible that subjects that would agree to participate in this kind of trial which has no possibility of a therapeutic benefit, 17 whether they could understand it is position they're being placed in with the potential of not being able to benefit from a treatment later developed. And I think with patients of this type where it is not an immediately fatal disease but certainly with a very difficult outcome, a lot of the potential subject population might have a very al true ristic view of participation and I think it requires more really careful explanation of just where this particular experiment fits in a long-term model for trying to find a real therapy for this disease and to really ensure that the person is able to wage weighing the farther force before agreeing to participate. In looking at the informed consent document I really don't think it does enough to really layout those things in terms that a layperson could understand. And as we have had other forums here regarding informed consent, I think probably there also needs to be instructions that are delivered orally and with films and or with other things that are not just confined to a paper document to explain this. But I think the discussion here at the table really raises an issue that speaks to a role for the RAC an evolving role for the RAC. I think at this point we really need to look at the possibility of the RAC's role in developing study design models so that the statistics and the ethics can be kind of summarize and put into sum guidance documents similar to the informed consent guidance that the RAC did within the last five 18 years so that each of these kinds of po sal--propo sals especially combines the risk of the participant not being able to benefit from the future therapy that these kinds of things really would best be handled if there were some concentrated focus on that to bring together and provide the entire community with consolidated document or guidance to help people make these disution decisions in a more informed manner. Part of my recommendation is not just for this particular study team but to really urge strongly the study design work group really be staffed and assisted in doing some very important work that I think will help the entire community. And I would like to hear a little bit more discussion here at the table with regard to ethical considerations for this particular study in light of statistical weakness and the potential for hindering the participants from further benefit. >> So the ethical question seems to me to be how--in the context of a study which is in ways uncertain, then how does one consent a subject--now this subject is an adult not a child, so the consent, the entire consent the ethics of consent are different but how does one do that when it is very likely the that the participation in the current study is diagnose to exclude the subject from what we all hope will be effective and efficient future studies that's really the quandary I think. >> Yes, and the further dimension being that eats not 19 clear there's sufficient statistical power to make the study move the idea forward either. >> So Ms., Kwan, would an informed consent document satisfy the quandary or dilemma for you? >> I think a more thorough discussion that's what is currently in the informed consent document bold also strongly suggest that there also be a plan in face to have face to face discussions in addition to the written consent document. >> Thank you. >> A couple of comments with regard to that. >> Please. >> I keep on hearing this statistical power is weak in therefor the study is not worth doing. How would you have us design the study in light of limited patient resources and valuableness? It's a test bodies like this want it test you if you don't have the answer you're in trouble. If you can tell me how you would like to power this study to we can get statistically meaningful results without damaging more human subjects than we need to, I'd be glad to consider it. Especially if you're thinking about writing a white paper saying this is way you design a trial how would you redesign our trial to get a statistically meaningful result. >> Dr. Miller I'm not sure we can provide a specific answer to your question Dr. Steven Piantadosi asked to speak. >> Let me answer our question with a couple of questions. You state in the procall the primary purpose is safety. I'm going to put aside the considerable 20 issues about context of the studied subjects sur rendering and their eventual possibility of having a therapeutic vector administered. Let's suppose there is a safety concern that is yet unobserved, I mean that's the purpose of doing the trial. What would be an unacceptably high frequency of such an event? >> Well we design it in phase 3 but Moira could address that better than I can. >> What frequency would be high for this yet unobserved safety event? >> So as--as this committee may or may not be aware the Cystic Fibrosis patients because of their multi-system disease have many adverse events. I believe the number for phase 1 study was 242 adverse events. On a shorter term study. I think when we're looking at this small number of subjects, we're really looking at serious adverse event three or four toxic cities and this would will immediately reported. >> You are referring to the adverse events that's a consequence of the underlying disease. I'm asking about intervention, the gene. If you atribute a particular unobserved event to that gene what would be an unacceptable high frequency of the event? >> It defends on if the event is death it's 0. >> Okay, let's take death. What's an sun unacceptable high frequency of death. >> Patients with Cystic Fibrosis do die. >> They do not die from this gene. >> They do not die from this gene. I think it's so often 21 with experimental treatments not so request gene therapy. The University of Washington is one of the factor development centers for a multitude of experimental treatments. And in shows studies sometimes the protocol design allows the site principle investigatingor to determine if an adverse event is likely due possibly due to the therapeutic intervention. So I think that as a site PI I am extremely cop servetive about that in my possible or probable. I think with any especially with this particular step toward a therapeutic adventure, we would be, I would be extraordinarily conservative in my approach as an adverse event. Not only for the reg tribodies to--regis tree bodies and report back to the five regulatory bodies but individually one would be very conservative about any adverse events that one thought were possibly due to that or even a relevance. >> I accept that but you're answering a different question than I'm asking and I can't let you off the hook because it gets to the fundamental principle about a study design. You're saying what you'll do if you observe thing and the attribution of the events. What I'm asking is if you have a solid attribution of serious adverse event of the nature that might terminate the development of this vector and you attribute that to the vector reliably there is some frequency that you would observe that that would be unacceptable clinically if 25% of the subjects died for example from a cause attributable to the vector anaphylaxis or 22 overwhelming systemic in fession or a mucous sinus attributeable to that. How frequently would you have to to see a disasterious event is a high frequency 10%, 50% of the subjects? What would that answer be? >> I think it's interesting because if we're talking about four subjects of whom three are getting the vector. We're talking about three with a dose. A statistical answer--this is a machinistic study. I don't understand your question. If you're asking about statistics . >> Let me ask if T about another way. Forget N equals 3. What if you had a thousand individuals treated this way so you had a very large number. Just do the a experiment in your mind. If you had very large number subject and you observed the certain frequency of adverse event. >> Inflammation in the nose? >> I'm talking about you would see as unacceptable developing term Nateing event 10%, 20%, 30%? What do you think it would be? >> I'm going to have to ask for a clarification. You mean of this particular study? Or in future studies? >> I'm talking about this product, this vector. There is some unacceptable rate of complications from this that would terminate development. That's my point. >> I understand. Let me help Dr. Moira L. Aitken just a minute, just a little bit. In this with this particular product with the AP gene, there is really no hope for any benefit; therefor, at least if I 23 were answering the question I would say I wouldn't accept very many if more than one serious adverse event directly attributable to the gene and gene product because there's no possibility of benefit. On the other hand if I put myself out five years and there's another transgene out there that has a high likelihood of cure, I might be a little--I would have very different answer. But if Dr. Steven Piantadosi is asking about this gene product to the upper airway, then would you be able to handle or tolerate as the hands-on physician, more than one? >> I think that's an excellent response to that. I'm just thinking ahead. A serious adverse event might be a hospitalation of a patient and--for example, in doing these who were performed by an Oto largeologyist would not accept a nosebleed. I could imagine there could be serious adverse events. Should we stop the trails because of that? That-- >> You're free to define adverse event anyway you want. After all that's the objective of the trial and safety. >> It's clearly outlined. >> I understand. I think the point operative point from my prospective of dope--dean sign your tolerance of serious adverse events is low you have a particularly low threshold. And whatever the complication is that you haven't yet seen you don't want to see very many of them because of the context, right? >> That's right. >> But the point about study design is that the study doesn't 24 permit you to rule out relatively high frequencies of side effect. For example if all three subjects are treated absolutely safely, you only rule out frequencies as high as 70%. 70% of your subjects could in fact in reality in that larger 1,000 patient study that I high hype sides. 70% could see it and 70% of them could die. >> That's true. >> And you could get three that don't. >> That's correct. >> That's the problem with this study design. That's the problem. >> But phase 1 trails are often like that. >> I understand that they are. But it did you want help that phase 1 trails are lousy designs. It doesn't help. >> But I think, if I could interrupt here too, I think there's a corollary in the other direction because with only three subjects that actually received treatment you could have one adverse event resulting in the person dieing and it might represent really a very, very low frequency. You could throw out the entire results and say no, this is unacceptable we don't go forward with this and it actually could be the road map to an efficacy shus therapy. The things that could go wrong you could get a death among the three that actually received it. It could actually represent a much lower incidence than apparent. You can stop the entire study because of one death and lose a promising therapy. That's the other part of it. >> Of course we can always be 25 victimized by chance in either direction. We need to plan the study that we are not victimized to a great extent which was any concern with the small sample sizes. Don't get me wrong I'm not suggesting that you have an early developmental study that has dozens or thousands of individuals on it. What I'm suggesting is the difficulty in the investigatingors in thinking this way has led them as is typical to use a design that is an off the shelf design that was never intended to answer the basic safety questions at play here and it's made worst by the fact that the subjects who participate in the study are probably giving up their ability to derive a benefit from a similar therapy in the future should everything work out exactly as we hope. So what I would look for and the way I would turn around the question that you asked me about study design is that the protocol needs to reflect some of this kind of thinking and if all the more critical in this context. It needs to reflect your ideas about what you might miss because that's the error that's going to be made here is that we're going to miss something important based on successful application of a treatment in three subjects and how we interpret the absence of information when we have a denominator of three is exactly what the problem is and it's exactly the flaw in almost every subject--every study that's reviewed by this committee. >> Can I respond to that briefly? >> Briefly. 26 >> If I see where the problem is and I understand where you're coming from we listed safety as primary outcome. I think the reason we listed it that way we want to sort of state that primarily we are concerned about the patient's safety we don't want to allow adverse events. Our primary goal here is not to compromise the patients but really the primary scientific goal is to figure out transduction rate of AV6 vector in epithelium. I think that's our primary goal I think with three subjects in each group get that information. That's what we're after we like what it does in mice or rats and show if it works or doesn't. If we find safety problems. >> Stop. >> Okay. >> Go ahead I don't mean you stop I was trying to figure out what I was going to write. Go ahead. >> Really primary outcome measure the reason we're doing the trial is to figure out if AV6 works in epithelium. >> I understand that and I'll be quiet after this comment. You can't get off the hook in a direction either. Because if you have three out of three successes then you only ruled out success rates in excess of 30%. That may or may not be appropriate but it cuts both ways. >> But there we're talking about strongly positive is greater than 10% I expect we'll see some transduction rate if all subjects. That's what we see in the animals. We get a quantitative number. >> I gave you three out of 27 three. >> Dr. Steven Piantadosi I would like to reaffirm Ms., Kwan's comment is that this trial is another example, for me at least, where the current construct for phase 1 and phase 12 studies doesn't work. We desperately need for a field like gene transfer a new construct that's one of charges to the RAC to develope such new construct. I would just put it out to our entire group. We've been talking about it now for the last two sessions. We've had two out of four studies this time. We're at directly applies and we need to move forward so that we can provide our investigatingors with answers to questions like the 1 Dr. Dusty Miller proposed to us. There are other questions Dr. Strom you had hur hand raised earlier. >> Thank you let me state for the record I'm not an experiment in the CF transfer in this area. But I'm an oat to largeologyist. Having said that I think it's unfair and wrong to discount the import of the epithelium on this disease as an intern price in and of IT. Folks with Cystic Fibrosis one of the most devastating things that happen to them is their nazele systems and often difficult to control and disease. If you can help their symptomology you can help them whether through better irrigation, exposure etcetera. Look at the nazle epithelium I would encourage you to not say it's a certain mark but perhaps I'm biased a target point in and of itself. If anybody has biopsied a turban Nate, you get a certain amount of bleeding. It bleeds like crazy in CF. I 28 would say that it should be a limiting factor because you'll exclude everybody. You're going to get bleeding. It's eas to control and I would just stop with that and know that it's going to happen. >> Dr. Albellda. >> I'll put on this hat instead of cancer hat. I think the nazle epithelium is the good target to start with. Eng there's lots of data. You mentioned some of it. They are sillyated epithelium. If you can't affect the nazele epithelium you won't have success with the lung. Eng it's a reasonable place to start and probably easier than a bron cose top pea. I would like you to think about and person opinion to discuss the idea of using normal subjects rather than CF patients. I got some sighs, I'll bring it up. There was very good point that you sensetizing a rare population group that's not eligible to do it. I think when you're looking lung gene transfer in CF patterns it may be different than normal patients. The nazle epithelium I don't think is so different. If you can't transduce normal patients I think it will tell you right away you better find another serum type or something else. It is a big controversial. There could be big side effects. Is there are another way besides biopsies to get reasonable data? Maybe Dr. Strom can answer that. You do want to look at the surface epithelium and I think you can get a decent amount of cells if you do scraping N. normal patient I would imagine a lot less bleeding and 29 inflammation. It's difficult to recruit CF patients. They're sicker. You're going to have much--as you mentioned the adverse event is nightmare because they have million things going on. I personally don't like the randomized then with one placebo patient and three treatment patients the statistical power is 0. Why bother doing it? May be with the CF patients with lots of side effects with one placebo I don't see how it helps you and puts the other patient the risk for bleeding and complications and especially CT patients without benefit. We can discuss this. The FDA may have strong feelings about this. I don't know. This is ideal study in in my opinion that is strong trial. There hasn't been any type of complications. I will view this as very low risk and low enough risk that I think normal volunteers would be a reasonable group. Now people may disagree but it's something to talk about. >> Dr. Strom what about scrapings. >> You can do brush buy ob op sees--biopsies in the nose. I'm not familiar with how much epithelium you would need in order to get your answer. I can't comment in the terms of sheer volumes of you can get epitheliums with the nose brush biopsies. >> It's nice with the sample you can see what percent of the surface epithelium what cells are involved with AP you can tell what cells are positive. With brushings I imagine you get a mix of cells. >> The bleeding is usually very 30 easy to control. I mean it's not a--it's usually not a problem. So I think you can do it. You are going to get bleeding whenever you take a biopsy in that area but it's reasonably easy to control if most situations. >> You get about with brushes I've even done it to myself as well as others you get to about 10 to 15 cells we can get a costain with sigh careton. If we are going to do with limited number of patient we would to get the ideal--you can see it clear but the others would lose--the idea with the biopsies is to get more specific which cells are being transduced in supposed stem cell out of the basele cells or is a termly differentiated cells. That was the idea behind the buy ob sees. I personally would still prefer them. >> I don't think it's unreasonable. The other problem you will have CF patients are nazele problem and trying to find the turban Nate is not a trivial task. You can get pollps all over the nose and identifying the turbannate is different. Your going to have a hard time. >> With delivery with one of the half dozen centers in the United States that can do the difference measurement so we're--we're familiar with that area. And then the Otto lar renologyist is who you use and we're getting the most expertise in that area. >> Doctor are you comfortable with the response? >> I would like to hear comments are the panel what do they think about using normal patients. 31 >> No, I was going there next. I meant to the response to issue of brush biopsy? >> Yes. >> Thank you, let's go next to the Dr. Arrestbellda's second comment was that prepares the investigators should reconsider conducting this study in normal people rather than in patient with CF. >> Can I ask a question? I should be more aware of that. Investigators said they came to the RAC and the RAC were the people who we said that you should not use normal subjects. I actually. >> That is correct. >> So I actually applaud this trial. I think the problems with gene therapy is we don't know when something doesn't work, why it doesn't work. It this seems to me a very good step-wise approach to try to work out are we getting transduction, it's not in the best tissue and so on. Then we go on from that stage to the next stage and so on. Could I ask the investigators if you had the choice, would you use normal people or CF? It sounds like you wanted to use-- >> I think we're right in the middle actually. We argued a lot--we talked about this a lot before coming here. That is somebody could argue well when we you'd the normal we haven't got the right target epithelium if you cop pair AV6 to AV2. May be AV 2 work better with something under stress. If you take from the stained standpoint that AV6 works we can't do trails and take the best horse and ride with it. It would go best with norms and say does 32 this work well and move on from there. >> At the risk of the RAC is pushing you from here to here and killing you on both issues. >> Patient accrual would be much faster we wouldn't do CF patients there's a lost of pluses going with normal. Human biology is human biology and receptors that are there are probably not going to be that difference. >> I'm not expert in the normal epithelium and CF in anyway and I can't address that. It does seem to me that Dr. Albellda's point is a good 1 you can tran duce the cells the issue of normal subjects. >> To the RAC's point the last time I apologize that I didn't review the transcript. But there I think the main issue is an ethical one which I actually personally did I agreed with. But just to revisit that so that the viewpoint was that ultimately if we succeed with this venture that it would benefit that patient population and therefor that patient population should provide the study subjects. Not--to my argument was of course that 1 in 25 patients are carriers so actually you know I am a study subject because of you know my ethnicity. But that was--so that was the argument, the ethical viewpoint of RAC last time. >> I do apologize I should have this in the memory and being on RAC. >> You weren't here this is four years ago. >> Oh, good that get me off the hook. Can I ask at that meeting was the ethical issue of what's 33 coming up now if you have patient who is enrolled on this trial with no benefit then excluding them from any future therapeutic benefit was that considered by the RAC at the time? >> It was. And so the other argument had taken precedent over that. So Dr. Miller and I at that time wanted to go with norms and we put that in our protocol and we were instructed that could not happen. >> May be a good way to procedure is you consider us using normal. We don't know how the rest of regulatory agencies might behave. >> It's just suggest. >> Could I offer a potential compromise? You know the debate about about normals might lead you to say the study should include both and safety information gathered in normals and small cohort CF patient as well. >> Start with perform and move on? >> --normal and move on? >> That's a possibility it would meet both of objectives and of course you would get double the criticism probably. >> Let's go quickly to Dr. Powers. >> Ms., Shapiro and I were thinking through the framework here. Mar of what's at stake I think if we can try to lay this out as this, when you have a non-therapeutic or something that's not going to benefit the subject population in adults, you either then got to have some generalizeable knowledge and all the things raised become very important and as Ms., Kwanponed out you have to ask if some of 34 the basic powerings not really going to be there is there something coming forward that you can describe as generalizeable knowledge? The more problems you get in this clinical design the weaker our case is on the benefit side. That means all the kinds of risk on the other side you can tolerate far less. You know the scales get tipped. Weak generalizeable knowledge, weak case for going forward. Less ability to tolerate downsides. In this case you now have a down side for you know it's may be speculative because it depends on what the success downstream at least some groups of people in the CF population are under a new disability. They are persons who will no longer. They will sacrifice their ability to take advantage of whatever benefits might come downstream. A bit speculative non-the less a--none the less a real worry for them. If you were choosing between normal subjects and those with CF you would first of all ask yourself the question in the choice of those two populations do I loss or gain anything in terms of any capacity to generate generalizeable knowledge? If it turns out for example that the in choice of two populations you didn't have any difference in generalizeable knowledge then it might seem reasonable to ask normal volunteers to participate in this and then you certainly don't have that added down side. I don't know the answer to that question. But it seems to me that's the framework for answering it so that if the CF patient population still 35 provides you some prepares weekly general riseable knowledge not nearly as good as we would hoped for as committed on in term of safety and benefit. That therein lies the frame work and Ms., Shapiro you may want to pop in there as well. We were mumbling among ourselves. >> There has to be a way from FDA just a brief--what would be yours--in this is a first take. >> I'm not making any statements about this. What I'm going to say for the record the FDA entertains all sound well designed clinical trails. If you have a justification, a rational that is is found for why you're going to go forward whether it's with whatever patient population and I think the most important thing here which you know I didn't state but I think everyone understands is that many ways we can get around some of the safety issues is by doing good animal studies which will give us a better feel up front. I know there's not always great models but that helps us make decisions when we have uncertainty like this. So the questions I ask today are not meant to state that the FDA doesn't like this protocol. That's not what I mean here. I based on your presentation I had questions I wanted to ask. I will reiterate we will intern tab taken--entertain any well-designed clinical trial. >> Ms., Shapiro. >> I entirely agree with Dr. Powers eloquent explanation and even one-step further. Not having been a part of the RAC--I guess part of it now. [ LAUGHTER ] 36 >> We'd still like you to comment. >> Okay. I'm not bound by the institutional need for the institutional memory however it's generally sometimes said when you have research that offers to prospect of birth benefit--benefit that you should use the affected condition in other words you should enroll those who are affected with the condition. I myself have big problems with that approach in terms of justice and terms of autonomy. In terms of justice they are burdened and sick. In this case we go beyond that because they are going to perhaps be precluded from something beneficial down the line. I think the therapeutic misconception istively in the population in that they may be reaching out for anything that's benefit in research. This case the exact opposite is true I worry following up on Ms., Kwan's comments if they really understand what they're giving up. I have a problem with that approach. >> Two comments another rationality you--really dusty and I talk about the issues over normals versus CF. The issues to do CF patient is we don't know about the transduction--inflammation, infection and mucus there. The normals may not be a perfect substitute because of that. Us that one thing for that. And then how we would obtain consent has come up a couple of times and as we've been through three regis tree bodies I can't see what it would be. The normal we do many. I guess we have 20 activity RIB studies currently. 37 The normal is going away from the principle investigating for to obtain consent. We have a research coordinators a nurse to try to meet unbiassed consent. Having kind of overheard him we retry to verbally go through it, they let people take their time and with prior gene therapy trails not all of them but some of them we have consent monitoring. Again it was a nurse, a different nurse calling back in three to five days and making sure the patients seemed to understand. I'm not sure how effective that was. We actually--I didn't try Dr. Robinson at Harvard tried to study that. Those two--actually for any study we don't have the PI we try to have an unbiassed person as we can ask for--to get permission because a lot of these you know FDA you know approved trails may not be therapeutic. As you know most of them don't come to therapeutic intervention. >> We're going to have to move on very quickly. Dr. Naomi Rosenberg. >> I wanted to ask briefly. You mentioned one reason in your last response as to really there are scientific reasons on which you might base the need to use patients as opposed to normal subjects. You did mention one. But if you could just say a little bit more about that aspect. >> So--well, so the AV receptor is the basele lateral part of the cell--AV2. And the AV6 is the at the surface. With AV2 if you look at the area epithelium in CF biopsies it is quite disrupted and you can see the membrane. AV2 does get in 38 there. However there is a inflamation in the CF airway Dr. Strom pointed out with the enzymes there and mucus theoretically stopping transduction. It didn't in the in vitro studies of the pollps that Dr. Miller pointed out. With AV2 it seems that the CF patient actually may be a better model with AV6 because of where the receptor is normal may be a better--you know a better suetable but it's not--suitable but we still have the airway inflammation issues. As Dr. Strom pointed out almost all CT patients have very abnormal upper air ways. In the small cross section I did all of them had very abnormal CT scans of their upper air ways. It's a very unusual CF patient you you want very mild genome-types that don't have upper airway involvement. >> You are next in line. I'm going to ask everyone for very ters comment. >> The advantage of doing norms would be the ability to find out to extra extent preexisting immunity precludes administration of this spector. Is really known how much the preexisting immune response prevents administration of the virus. >> We have done that the study in the mice and administer one marker and come back with another and ask what the transduction rate and with AV6 we got 50% transduction although developed some anti-bodies against the virus. It's not known in the people. Those would be nice additional studies. It would be great to come back in some of these 39 individuals a year later and ask if you could readminister the rye rye Russ--virus to the nose. >> What's the duration? >> More than a year. AV is quite nice and gives a long-term expression. >> Preexisting immunity does that affect the duration? >> No we haven't looked at that. We only looked a month after the first dose we haven't looked a year later. >> The point I'm make something we're--this sounds like there is on a assumption that treating CT patients who lack immunity is going to come pry mice their--compromise their-- >> We got a strong response you couldn't administer AV 2 there was no transduction in the second round. We haven't looked at all those 78ables. Maybe AV8 would be good for the patients that immune to AV6. I am there are others to try. >> Dr. Weber you've had your hand up. >> Just a quick question. The study will be terminated as I read it correctly if off greater three toxicity is that your stopping rule is that correct? >> Due to the. >> Due to the vector rules yes. >> Dr. Howard Federoff. >> A tiny point. Isly any reason to believe that the enzyme that you choose to express will be active in the respiratory epithelium and might alter the function of those cells. >> We are transfer heat Foss so tase it's express and adult tissue. It's also true if you don't heat and activate the lung there's massive AP staining. It was hard to get the good 40 conditions to see the heat. We think the small amount of additional AP is not going to be an issue. >> Other burning comments? Okay, comments, questions from the public? I'm smiling. We have a private joke. All right, I will precede then and read what I have gleaned from this extraordinarily productive discussion. And to reassure the presenttors you are presenting a study trial at a time when the RAC has been fairly intensively considering clinical design issues your presentation entersecond with obviously some topics we are considering. The discussion was not meant to thoroughly question your study design but rather to raise questions that we all feel are incredibly important in 2005. I guess I should also add because the RAC said one thing four or five years ago I hope that does not mean that we will consistently be held to what may be an old standard. We're trying desperately to move forward especially with issues of trial design and risk-benefit analysis. So I really appreciate your participation. Let me read. General, the unusual aspects of this trial is written included the absence of direct benefit to the subjects, normal subjects, and the use of a quote surrogate and end quote nazle epithelium rare lower air ways. The investigators plan a sequentially approach to the trial of the gene with an effective vector in a lower airway. The RAC asks the investigators to reconsider the enrollment of normal subjects rather than adults with CF to 41 this trial in order to alleviate the likelihood that adults with CF who enrolled this a study would be excluded from future studies. Ethical contract should balance the risk of participation in future studies for CF patients in the and the possibility of therapeutic misconception with the possibility that patients rather than normal subject will provide the most scientific data. As an aside I don't think we should instruct based on the discussion we had--I don't believe we should instruct the investigators to do one then or the other but rather raise issues for reconsideration and this is all up for rewording. This is possible that the same study could initially enroll snowstorm subjects and if the vector proves effective progress in adults with CF. I tried to summarize what where I believe the discussion led us. Would you prefer to reword this section or would you prefer me to read the remainder. Read the remainder. I have nothing under preclinical. Under clinical. I repeat myself here whether the nazle epithelium in man is adequate surrogate marker in lower air ways with CF I put unclear. The RAC remains concerned cat role of the proposed trial and planned sequence of tude for patient with CF is somewhat unclear. RAC endorse the proposed amendment. We did not say this but I want to bring it up for consideration endorses proposed amendment to allow the addition of the three-subject cohort at the highest dose level should the only one in initial cohort 42 in the nazle mu cos sa. That was a amendment that Dr. Miller put up on his slide that would introduce the additional power in terms of real goal of that study. To Dr. Steven Piantadosi's comment that the current design allows 70% of larger group of the subject although none of the three proposed--the RAC requests that the investigators reconsider the study design in light of unique characteristics of this trial. Please interpret the quote absence of information end quote likely to result from this trial. That is all I have. I have nothing on the IC. Dr. Powers. >> Just a thought that may be we, I don't want to keep the researchers getting bounced around too much. >> I don't either. >> May be the language would be something more than reconsider it sounds more directive to an outcome. >> give me some words. >> I would say we encourage the investigators to undertake a careful reevaluation of the risk benefit ratio with an eye toward subject selection predicated upon what we yield the greatest generallyized knowledge. I can only speak at the rate in which I think. >> And I can only write. >> We could encourage the investigators to undertake a careful evaluation of the potential for generalizeable knowledge. Based upon the subject population--the subject selection criteria choice between normal subjects and CF-affected subjects and to consider on the other side the 43 adverse consequences associated with--i'm not sure what the right word is--making it difficult for future subjects to take advantage of future benefits. Just something a little-- >> I can do that. >> Something a little weaker than the urge to reconsider sounds like we're being for directive than we really are. We are at less I think a clear point of view. >> Agree. >> Dr. Vial. >> Could you reread the bit where the RAC is concerned about the step-wise progression whether the nazle epithelium is good place to start sort of thing? Maybe. >> No, I know your question. Unclinical my first comment was whether the nazle epithelium in man is an adequate surrogate marker in lower air ways with subjects in the CF remains unclear. RAC remain concerned that the role of the proposed trial in the planned scenes sequence. >> I see what you're asking. I don't agree with it. >> I would like to say and applaud that is step-wise to build it up earn that what we're concerned. >> I think I can just delete it because I did applaud in the first paragraph which the much straighter. I tried to record the nuances of the discussion and this does not make sense. Thank you. Okay so the first few sentences remain as is. I'll try to modify the next to begin with the RAC encounseling the investigators to undertake a careful evaluation of the 44 potential for generalizeable-- >> Generallyizeable knowledge. >> Based upon the subject selection cry tear ya--that is the chance between normal subjects and CF affected suggests and rolls forward from there. So we have taken out the language that says we're concerned by we want you to redo and I'm much--if you all agree I'm much more comfortable with this. All right. I'd like to ask for a motion for approval then Dr. Powers a second, Dr. Dewhurst and we will take a vote. We'll begin with Dr. Naomi Rosenberg. >> Aye,. >> Aye. >> Aye. >> Aye. >> Aye. >> Aye. >> Aye. >> Aye. >> Aye. >> Aye. >> Aye. Thank you all very much especially thank you both for coming and for participating as actively and as willing as you both did. We hope it will be useful. We'll take a break for 15 precise minutes. >> We are not in quorum. But we are below quorum. the only person who I am aware of that won't be joining us is Dr. Heslop. So, I think we are good enough to go now. The second protocol for discussion this morning is 727 clinical translation of a mammaglobin A DNA vaccine for breast cancer prevention and therapy. Dr. Gillander, Dr. Gillander from Wash U will present. >> We are having a technical issue here. >> I understand. We are on pause. >> All right. We are off pause. &%FO &%FO. >> Thank you for your introduction. I am William Gillanders, 2 associate professor of surgery at Washington University and I am a breast cancer surgeon and I am background in tumors and I would like to present today our proposed protocol of a mammaglobin A DNA vaccine. And just, I already found the reviews of the RAC very helpful and made some changes to the protocol and to give an overview, before I give a background of mammaglobin A, we are doing a phase of mammaglobin A vaccine. And the broad objective of the study is to identify a safe and immunological dose of the DNA vaccine that can be used in future studies. The mammaglobin A gene was first identified at Washington University School of Medicine by members of our group using a differential screening approach 3 directed at the isolation of novel, human breast cancer associated genes. And mammaglobin A enclosed lipoprotein and is a member of the globin family. And forms the B. To characterize the tissue expression of this novel gene, we performed northern BLAT and fetal human tissues and these analyses demonstrate that mammaglobin A is for breast cancer. This is a northern blot analysis showing an expression of a mammaglobin A in breast cancer and to a lesser extent in human breasts. And this is conventional RTCPR analysis. Again demonstrating tissue specificity of mammaglobin expression with expression in breast cancer and normal breasts and no expression in 4 the other tissues indicated. To study mammaglobin protein expression. Control for cellar, and between tumors specimens that could be used for routine immuno analyses. We synthesized the peptide to the carboxy terminal and sequence and generated rapid polyclonal antibodies. After rigorously examining this agency. We examined metastatic breast cancer specimens and demonstrated that over primary breast cancer specimenses were positive for the mammaglobin A and for normal breast epithelium. Rare and epithelial cells are seen. And then in contrast, on the right, strong cytoplasmic for the protein is seen in the 5 primary invasive carcinoma. Then of particular note in this preliminary survey of over 100 primary breast cancers, strong cytoplasmic staining was seen in all the cases of carcinoma in situ and appeared -- In situ and was of the specimens analyzed and differentiated breast cancer. So among moderately differentiated, we saw 79% and poorly differentiated breast cancers 87%. Since the identification of mammaglobin A, the molecule has been the focus of over 100 publications in the medical literature. The exquisite tissue made this an informative molecular marker for the micro metastatic breast cancer and lymph nodes as we have recently demonstrated in a cohort study. 6 And also appears to be very informative for the detection of breast cancer in peripheral blood. To summarize these initial studies. Mammaglobin A is a novel breast cancer that has several unique characteristics that make it attractive for immune targeting. To characterize further, the mammaglobin A as an intervention, we generated CDAC lines and in vitro that was specific for mammaglobin A. We had blood from breast cancer from breast cancer patients and stimulated them with mammaglobin A. And the line that we were able to generate showed significant cytotoxic activity against breast cancer cell lines that are H2 positive and mammaglobin 7 A positive and there was no cytotoxicity that were negative or mammaglobin A negative demonstrating the immuno specificity of this cell line. And these results clearly demonstrated that mammaglobin A is processed and presented by breast cancer cells and can be recognized by the immune system. And then to determine if there is any evidence of mammaglobin reactive T cells in breast cancer patients, we performed analyses and mammaglobin reactive T cells in seven breast cancer patients and seven healthy volunteers. As shown in this figure, the reactive of the cells in the peripheral of the breast cancer patients was significantly higher than in the healthy female volunteers. 8 Just to include this, crystal structure of the MHC class one and trimeric structure to emphasize that we have now performed additional studies to study this immune response more in depth at the molecular level by identification of the peptide epitopes that bind A two and three and study the response at the epitope level. So initially we identified HCLA two and three peptides from mammaglobin A using the class one binding program from the National Institutes of Health and shown here, mammaglobin A derived peptides through A 3 but we also performed similar analyses for HCLA 2. And subsequently, we confirmed the ability of these peptides to bind the molecules in cell membranes stabilization using the efficient cell line T 2 A 9 3 and we demonstrate here mammaglobin A .35. A .37 bind to the molecule with high affinity and we have also designed which peptide epitopes bind HCLA two and three are the dominant epitopes. To further, in breast cancer patients to mammaglobin A at the epitope level, we performed the spot analyses and as shown here, there is evidence of an immune response to A and breast cancer patients and no evidence of response in healthy controls. So just to summarize these experiments. We have demonstrated that CDA and specific for mammaglobin A can be developed in vitro from the peripheral blood confirming that the immune system can recognize the antigen and from 10 breast cancer patients, they do have evidence of a higher frequency of mammaglobin A T cells. And so to target mammaglobin A, we decided on a DNA vaccination approach and these are some of the reasons why we think that a DNA vaccination approach is an attractive approach. And I will say this. That one of the main reasons why we decided on this approach is that because vaccination with the full length C.N.A. avoid the restrictions. So, there is increasing evidence that cancer vaccines may be most successful if they are applied when patients either have minimal residual disease or if they are applied in a setting of disease prevention. So one of the attractive things 11 about mammaglobin is because it is expressed near universally breast cancers, it seems it would be a very attractive antigen for prevention and this is in contrast to other antigens and it has been targeted by immune strategies which is only expressed or over expressed in about 30% of patients with breast cancer or primary breast cancers. The other -- so, if you use a DNA vaccine approach, you produce the entire protein and then it is processed and presented in the context of the vaccinated patients individual MHC molecules and this prevents the necessity to either use multiple peptide approach or identify peptide epitopes from every class of molecule. So, in preliminary experiments of this DNA vaccine, we used a 12 double transgenic model and this is transgenic for A two and transgenic for human CDA, so, these -- this is for mammaglobin in the class of human class one molecules. These mice have expression on all of their tissues and they can recognize human HLA, A2 restricted epitopes. So, we in initial studies, we vaccinated the mice by IM vaccination every week for three vaccinations and performed immune analyses and were able to demonstrate that T cells from the mice are able to HCLA two positive and mammaglobin positive breast cancer cell lines but they are unable to recognize A 2 negative or mammaglobin A negative, again the specificity of the immune response. We used a model to determine if 13 the T cells were able to mediate any antitumor immunities. So in this model, we challenged skid mice with mammaglobin A or mammaglobin B and we adopted a transfer of cells from vaccinated mice and then we measured the tumor size by calipers. And as shown here. HBL100 is an A two positive mammaglobin A positive cell line and then the M.D. A M B, 231 is a mammaglobin A negative cell line. And these are the vaccinations with empty vector and this is vaccination with the mammaglobin vector and you have tumor growth regardless of what factor you use. So this shows the specificity of the immune response. We propose a phase I dose 14 ranging vaccine safety trial of the mammaglobin A DNA vaccine and the broad objective of the study is to identify a safe and immunologically safe dose of the DNA vaccine that could be used in future studies. The primary objective is to evaluate the safety of the DNA vaccine and other objectives include assessing the immune response induced by the mammaglobin DNA vaccine by evaluation of the CDC and RAD immune responses and also, I am sure everybody is aware when we initially proposed this study, we proposed the study in patients who were, they were AJCC stage two A, Three B or breast cancer patients who had no evidence of disease. And after considering the reviews by the RAC, we decided 15 to amend the protocols. So we are looking at stage four breast cancer patients who appear to be stable either following chemotherapy or who are on hormonal therapy and are stable and these are new objectives, but since we are targeting stage four breast cancer patients in the initial safety study, we will, you know, assess the impact of mammaglobin A and vaccination on breast cancer tumor markers and analyze tumor cells and also look at molecular evidence of disease using some of our molecular essays and also evaluate the patients for times and disease progression following vaccination with the DNA vaccine. >> So again,--so, again, our new criteria, patients with stage four breast cancer will 16 be eligible for enrollment and have metastatic disease stable, follow disease stable following chemotherapy or hormonal therapy and our goal is not to identify the maximum tolerated dose. But the dose escalation is appropriate. This is the first time this agent will be used in humans. So we will start at a low dose and increase the dose. There is some evidence in the literature that there is a dose response. So there have been several publications that have shown no response at the lowest levels of DNA vaccination. But evidence of a response at higher levels of the DNA vaccine. Again, a dose escalation will only incur if we demonstrated 17 safety of the vaccine at lower dose levels. We plan to perform, I have shown you data from assays that we have done and we will perform intracellular and analyses for HLA two and three patients who identified the immuno peptides and for the sub set of patients we will be able to use HC peptide and analyses and then for the other patients we will use LE spot and expression assays. We, like I said, the advantage of a DNA approach is DNA vaccines are considered to be very safe. I think there is two issues that in terms of the safety and I think, if you read the review of the RAC and one issue is just whether or not our vaccine construct will be safe. And if there is any acute 18 toxicity from the vaccine and we don't expect acute toxicity from the vaccine construct. If you read, most of the phase I and clinical trials of DNA vaccine have shown acute toxicity is limited to site reactions and other reactions that have been described which may not necessarily even be related to the DNA vaccine and include hypoglycemia, Myalgia. Chills. --myalgia. And chills and safety which is the major concern here is what the potential is for autoimmune toxicity related to the DNA vaccine and one of the reasons why ultimately we chose to do the study in stage four breast cancer patients is that although we don't believe that there is a significant risk of 19 autoimmune toxicity, there is no animal models that we can use to test that hypothesis. So, the animal, there is no home log of DNA, and to study the immune response to the mammaglobin A DNA vaccine cannot be used to study immune toxicity. Although the tissue expression of mammaglobin A, it is, it seems to be that it is expressed. It is dramatically expressed in breast cancers and very low levels in normal breast epithelium and no other tissues. Although it suggests it is very low since we can't formally assess that in animal model. We have chosen to look at stage four breast cancer patients for this initial safety study. We define safety as using the 20 National Institutes of Health toxicity criteria. So grade three or greater toxicity and we also define the optimal dose as a dose of the mammaglobin A DNA vaccine that it is safe and the one that is associated with maximal immune response and if we see two or more doses are equivalent in terms of immune response, we define optimal dose that is the lowest dose that gives the maximum immune response. And there is one, I wanted, one other concern that I wanted to address. We have changed the vector and we have been working closely with investigators at Sloane Kettering and one of the investigators on the study is post doctoral fellow in Allan's will be and involved in the creation of the parent vector 21 which has now been used successfully in a number of human clinical trials. So we thought that and that Dr. Howt ton in Memorial Sloane Kettering is willing to share this with us and one of the reasons we chose the parent vector. It had been used safely already in human clinical trials. This vector is very similar to the vector that we used in our animal studies that have been published and using the same CMP promoter and small differences it has a resistant gene for bacterial resistance gene where as the other one. The PCI neo had an ampicillin resistance gene and we are currently testing the ping using the same animal model we used before. So, thank you and I am glad to 22 answer any questions about that study. >> Dr. Gillanders, thank you very much for a nice presentation. I would appreciate if you would stay at the podium so you can interact with members of the RAC as we ask our questions. I have one just to clarify the background for the moment. What is a life expectancy of a woman with stage four breast cancer? To put this in perspective for everyone. >> I think there was a nice study where they were looking at in the New England Journal of Medicine recently where they were enrolling patients in looking at circulating tumor cells and they all had stage four breast cancer and they were initiating a new regiment 23 of chemotherapy and it was 18 to 24 months for those women. >> Somewhere between 18 months and two years. Thank you. >> That was the median. You know, breast cancer, some live longer and some shorter, obviously. One of the, you know, initially we hoped to do the study in minimal residual disease. --residual disease and residual and this is most likely to be successful. They may not have normal immune function. There are advantages and disadvantages of, you know, each approach and then the main reason why we selected stage four breast cancer patients is because the primary concern obviously is safety and so, we want to start there. 24 Let's begin our reviews with Dr. Vile, if that's all right and then I wanted to move to Dr. Piantadosi and then Dr. Powers,. >> Would like to thank him for my replies to my written questions. Just so that I can read the questions into the record. My first comment was really that this is a long term interest of the group and they clearly identified a molecule which appears to be very selectively expressed on breast cancer. Little bit expressed on the breast normal tissue and very poorly or undetectably detailed elsewhere to make it a true antigen. I guess the second point is the outstanding issue with all of this is related to the toxicity 25 of potential, whether that is a big potential issue is unclear. And biological function of the protein remains elusive and I was wondering if there is further insight into the function of the protein in terms of this exquisite impression and indeed in terms of what toxicity and the immune reaction might be. >> That is obviously an area of research interest for our group and unfortunately, we haven't made a lot of progress in identifying the function of this protein. I mean, there is a number of speculative ideas about, but, I don't think there is really any data that we can-- >> So,. >> We don't know. >> Just for the record, you know, if you are vaccinating 26 against a protein that you don't know what it is doing on the normal tissue, you know, there remains concerns, but you expressed those in your discussion. >> Dr. Vile, how concerned are you by the strategy? >> You know, given the exquisite expression, it is only seen on normal breasts apparently and breast cancer tissue. >> Right. And I think it is important to remember, that it is dramatically overexpressed in breast cancer. >> Your RPCR, that showed normal levels. >> That is conventional. Not quantitative. I think it is, and also, the immuno chemistry is pretty dramatic. 27 >> So, I guess, my, you know, the concern is whether there are tissues that, so the concern is if this vaccination strategy is effective, essentially. >> What the investigator is trying to do is raise autoimmunity against the antigen and the consequences against any tissue that does express it, perhaps is not being detected in your studies or what ever. These are theoretical concerns which I am voicing for the record. And if there was an autoimmune activity against normal breast, I am not a clinician, I assume it would not be the end of the world. >> I did a pretty extensive literature search trying to address this. 28 >> There is no well defined, you know, natural autoimmune diseases of the breast, but my guess would be, if you had a significant autoimmune reaction regarding the normal breast epithelium and you would get swelling of the breast. Pain of the breast. You know, and you might also, end up with a premenopausal women, you might destroy the ability to produce milk in lactating women. It would be easy to diagnose because it would be the clinical symptoms would be obvious and it would be confirmed with the minimally invasive procedure such as a core biopsy of the breast. But that's what I believe and that's one of the reasons why we have chosen to document the safety in stage four cancer 29 patients is that we won't know until. >> I appreciate that sort of response. You say there is no hem globe -- mammaglobin A, are there other, does the dog have mammaglobin A? >> Well, there is no published data in any other species other than humans. There are other, in the mouth, there are other members of the secretive globin family and we are studying the members of the secretive globin family in the mouth. There is no hemology at the sequence level. But as of right, but as of right now, since we haven't identified any members of the family that is specifically expressed in the breast. I don't know if they exist or 30 not. >> Just to take it to the sort of -- >> Not that I am aware of, there is really no, there has been no formal study of the tissue expression of secretive globins in other species other than man. >> Have there been attempting to make. >> In the mouse. >> So, you know, for the record, my outstanding issues remain that this is molecule all of which is not understood and the expression of which, there is no model for autoimmune reactivity but with the published data on the vaccinations, I am sorry. With the published data on other DNA vaccines and other melanoma and the restricted expression, I am not sure I 31 would be any more overly concerned at this point. My next comment was-- >> Can I, in the response to that concern, the investigator has chosen to enroll stage four patients, does that help to-- >> Yes, express the concern. >> Yes, it does. >> Yes, it do. >> Okay. >> So my next comment was that in the protocol, the investigator described, we see no evidence of chronic or acute toxicity other than the development of autoimmune and I believe that in the, in that section, there was discussion of other trials mainly in the melanoma field, but I think a patient or somebody else reading that would get the idea that you get the idea of with the mammaglobin vaccine. 32 >> I acknowledge that probably should be revised to clarify. >> Right. And indeed, so the response was that this section has been revised extensively. I haven't seen the revision and that is something that should be done. Because the reference is all set to melanoma studies and that could be confusing. >> Right. >> My next comment was sort of, 180-degree turn, having argued about the problems of autoimmune disease. I just for more interest wondered what the status of this added would be to make it stronger and the response of the investigator was very fair. He argued that they thought about a -- >> We are interested in 33 integrating an adjuvants into the trials. Now that we have decideed to do the initial study in stage four patients. We may revisit that issue in the future. And also adjuvant of --adjuvants or DNA plasmic or other strategies to after an adjuvant into the study, it might document the safety of the mammaglobin DNA vaccine. We thought it would be, that was one of the main reasons we chose not to integrate an adjuvant into the--adjuvant into the study. >> I am satisfied with that. That's a reasonable response. So the next issue has been covered very well. About what would autoimmune manifestations of the breast, what would that be and the 34 investigator covered that very satisfactorily. And the next comment was the change of the plasmic and those -- Plasmic and the reply very extensive. Again, sort of more interest, you show clearly there are T cells in breast cancer patients. If there are T cells which are circulating and this is general immunology question. Why are they not effected. >> There is a lot of evidence that and in these patients with stage four breast cancer that have a significant disease burden. That there is an increased prevalence of regulatory T cells. Our group has published on that subject and now, there has been a number of other follow up publications that have 35 confirmed that observation, so that may be one reason is that although these cells are circulating. They are neutralized by regulatory T cells. And other studies that we have considered doing is studying breast cancer patients with minimal residual disease in an effort to determine if an increased frequency of mammaglobin A T cells is associated with better outcome. But we haven't done, we don't have any data to report at the present time. >> Okay. Thank you. These were all of my questions. >> Dr. Vile, are you comfortable with that response? >> Yes, absolutely. >> With the transgenic mice models and autoimmunity if it 36 could develop, that would be interesting for future that is more of a scientific. >> I hope your colleagues on the study sections will agree with me. >> Thank you. >> Dr. Piantadosi. >> I don't know that I will ask a lot of additional questions. But I will have some comments and maybe Dr. Gillanders will want to inject comments of his own. My initial concern on the study related mostly to design. I found myself comfortable with the biological setting in the motivation for the trial. However, in reading the protocol, there was not a clear articulation as to the actual purpose of the investigation. It was variably described and I think the investigators have 37 cleaned this up cosmetically and they appear to have chosen safety as the primary purpose of the study and it is mentioned twice in their response. One of the things that they did was to quote a chapter of my book, and I don't know whether they were using that against me or not. What I said was my perspective on the coast finding was at play, has been heavily influenced if not damaged, and I will emphasize that. If not damaged by phase I oncology studies. And because the dose versus safety for cytotoxic agents are rather artificial and simpler than others in questions, but many clinical investigators have difficulty breaking the phase I paradigm in, for me, 38 that's a very heart felt comment, I think it is the ubiquitous comment is that the RAC faces here is a very old fashioned study designed that is barely adequate for its purposes and forcing many of these gene transfer questions into the paradigm and they have avoided the and noted the consistencies in the first comment. Which I think is fine. But that's a cosmetic fix up and I think some of the really structural issues about whether this is the right design persists and I will say a bit more about that in a minute. The study design relies heavily on the maximum tolerated dose which is a, I tried to point out and I think the investigators have agreed. Is really not relevant, at none 39 of the proposed doses was there expected to be seen any dose limiting toxicity. And in fact, the background indicated with other DNA vaccines in other context, there had never been any significant toxicity and that is good evidence for the safety of this but it removes from consideration the notion of a maximum tolerated dose as the underpinning of the design. And investigators seem to agree with that. And I do think there is room for doing some sort of dose titration if that's an important question. But you have to titrate and of course the typical phase I study is not intended to do that. It titrates, so it is not a appropriate outcome I would 40 agree and I think the investigators have deemphasized it. However, they tend to insist on the structure of the dose escalation. But it wasn't clear to me what the surrogate for clinical toxicity would be. That is, how far--how are you going to pick which of the four doses is the dose optimum. So there should be some definition to offer there. Maybe I will ask a question at this point. You proposed doses ranging from 155 micrograms to 5-milligrams. What is the optimum definition of the dose? What are you hoping from the set? >> We will be measuring the immune response to the mammaglobin A DNA vaccine and 41 will be using the measures which you talked about briefly and we will measure the immune responses. Using the spot analyses. And using intracellular cyto kind and and by peptide and tetra analyses. And tetra and we will be able to with the T cells and we will define the optimal dose as the dose that results in the maximal and we may not have powered that adequately to differentiate between subtle differences and the immune response. So, it is not a -- immune response and we will be able to measure the immune response and determine you know, if it is, if there is an immune response. >> But, if you are going to use the immune response as your measured end point for 42 determining optimal dose, wouldn't it be wise to put into the study exactly into your trial design, exactly how you are going to utilize it? Because you mentioned in your protocol, looking at T regular, and L.A. spot, you have a lot of end points and how are you going to use all of those to come up with the best immune response? >> Well, I didn't measuring the T reg response is, I think that's more of an exploratory end point and then basically the other, the L.A. spot and peptide tetra and they are all looking at frequency and functional frequency in the case of the LA spot analysis of mammaglobin A T cells. I think we will be able to clarify that in the protocol that the dose of the vaccine, 43 that results in the maximal immune response to what we consider optimal. We are here to make it better. I am open to suggestions by members of the RAC and we have chosen this design. Because we believe dose escalation is appropriate. This is the first time it has been used in humans and we think dose acceleration is appropriate and we are open to suggestions from the members of the RAC and. >> Clearly the way to make it better. Is to have more subjects. Because of your statistical power goes up. Each patient costs a lot of money to do all the analyses. Hard to recruit the patients. There is usually a limited amount of time. 44 So we are in the terrible dilemma of we want to use as few patients as we can. Especially when the doses are safe. And missing and having enough statistical power to say anything. Having done the clinical trials, there is a lot of variability in the patients. They are not inbread mice. It is very difficult to get anything. That's a dilemma we face. >> And in this particular case also, there is a real desire to move beyond the safety testing so that we can start using this free agent and most appropriate context and also, you know potentially building on the vaccine in a modular fashion with the immune adjuvants that Dr. Vile mentioned and in my 45 enhanced efficacy of the vaccine approach further. So I think there is that -- >> Well, I am appreciative of the dilemma and the issue about the scarcity of subjects and I certainly would not propose a design that was wasteful of such a valuable resource and trust. But the scarcity of subjects and the desire for efficiency is not an excuse for asking the wrong study question just because it leads to a smaller sample size and that's really what I am arguing. Dr. Gillanders indicated earlier that he read chapter 10 in my book and I guess I would respond and say he should have read chapter nine. >> I read that chapter too. >> Thank you. >> Because you are a surgeon, I 46 hope you will read chapter four also. But in any case. Seriously, Dr. Wara's point was where I was going and that's it it is exception in these kind of studies to define what you mean by the optimum in a very simple and explicit way. After all, that's what the traditional side of toxic phase one does. It is a pretty lame definition But at least it is a definition. The dose optimum is characterized by the various clinical features and then you know when you have reached it. You decided on a dose optimum that is devised on my laboratory base or biological characteristics but you must define it as simply as possible and then you have to ask the 47 question. Whether the particular design that you have chosen is likely to locate that optimum reliably and efficiently and all I am asking here, is maybe this design doesn't do it. And I am pretty sure it doesn't do it in many cases. And let's not forget the primary question here which is you said safety was the primary objective of the study. Not dose optimization. And those two are only connected very artificially in the classic cytotoxic setting F you are not in the cytotoxic setting the question between dose and safety is, we know there is a dose between toxic and safety. They have a weak relationship between dose and safety so to use a design that is informing 48 about poisons that is very safe, it is probably not the right way to go. Any way, I have made that point. One other word about safety, you said on your slide that safety is the absence of severe toxicity. That's the slide that had the typo on it. Well, if that's true, then you have to provide through your investigation reasonable evidence that such toxicities are reliably absent and I don't think the small cohort sizes you propose especially at the dose optimum is going to provide that information. Now, if you knew the right dose and you put all 15 or so subjects op that dose, you might have a chance of generating reliable evidence 49 that severe toxicity is in fact absent. Couple of other points on my review. Originally safety and feasibility were mentioned as possible study out comes. We--outcomes. Outcomes and we know feasibility seems to have gone away. You said basically you took feasibility out. That's fine. Again, I think it is more cosmetic but I think I am okay with that. I did have a comment for which it has been corrected and I think the investigators response on that point was fine. And my final comment was really a summary indicating that I didn't feel able to suggest 50 firm directions for the study design until the ambiguity and the purpose of the trial had been resolved. The investigators pointed out something that I am sensitive to but only a little bit and that is that there are many other studies that have been approved by the RAC that have used a design similar to the one that they propose. And the polite answer to that is I wasn't around when they were reviewed, and they may have been right or wrong. I think it is largely irrelevant. What I am trying to do is get you to think in a new pattern. Think in a better way and design a study that really meets the objectives that you have set out. So thank you for your 51 consideration of my review and I hope that the comments that I have offered you helped to improve the study. >> And, thank you, very much. Miss KWAN. >> No, Dr. Powers. >> My only comment or query had to do with what is in the consent documents. Those comments were prompted in the initial round of review in the RAC and wondering about the theoretical risk particularly in autoimmunity and the like. And ching of vector and the patient, research subject criteria. I would say that since your own document says that risk of autoimmunity cannot be discluded. It would be to include the document to that effect. Although I think everyone 52 agreed it is up nope and difficult to ascertain. But probably either in this population. Just, it would be prudent to just add that small phrase and that is really my own further suggestion. >> I would like to open this up to discussion by other members of the RAC. >> Yes, I have three comments. The first one is that it is a minor comment. Which used HMAC, throughout the change. They are ditch and need to be changed. Major concern which is, I didn't see addressed, you have an equal likelihood of generating tolerance. What do I mean by that? Well, you have patient population where you are giving 53 a drug not necessarily targeted to the cells and if it is targeted to the tentretic cells. They could potentially induce a T cell response which could be negative. What precautions are there in the protocol to look for tolerance? I think we do more than we think. If you could comment on that, I would appreciate it. >> Well, as the protocol stands, currently, I don't think there is any formal way of assessing that kind of in a real time, in a real time basis and we do propose to measure regulatory T cells and the frequency of regulatory T cells and a lot of people believe now that may be one of the mechanisms of which tolerance 54 is induced. So you know, but we don't, not currently planning to measure that kind of in real time. >> Because if you transfactor and put it into cells and it is clear you get tolerance. That is shown by multiple groups. Now, you are take ago vector and giving it to cells where you may be expressing class one and don't necessarily have molecules on the surface and essentially you are reproducing a state where you have breast cancer cells expressing the molecules. What makes you think in humans, it is going to be any different than the potential that already exists in breast cancer patients. The likelihood is that it is not. The lookly -- likelihood than 55 you to have inducing immunity and you need to look at that in your Kirk lat circulating T cell responses looking for delesion of T cells and potential for the stimulation of site toe kinds which. And the last one is a comment. Which is, that I would look at pick one marker for your trial. Which is probably the L E spot and look at totally cellular response to that as a parameter and address Dr. Piantadosi's question. It is going to be possible to determine what is toxicity and what are the criteria for dose escalation. I will stop. >> Other comments? Dr.? >> I do, even though we like to use small numbers, if you don't have enough people to answer 56 the question, you shouldn't be doing the study because we won't gape anything from it. How many of that number is. I am not sure. But it is difficult for each study. Just a very brief comment. I would echo what Dr. Vile said, it is going to be and it is related to the last comment. It is imperative to get an adjuvant and just the vaccine alone is looked at for many years and it could be optimized and you pushed yourself to the stage four breast cancer patients. But for safety reasons it is improvement. But for the immune response, it is not. And I would suggest and let me ask you, are you planning to do control vaccinations with 57 influenza or C M V or Canada to show that those patients are able to generate an immune response. I think obviously if they can't generate an immune response, you are aren't going to have toxicity but you may falsely say, this isn't working in the patients. I would ask you to consider having some sort of control flu or something where you could measure TETRA responses where they could respond. >> Another surrogate for that, that is commonly performed in these patients with advanced stage cancer is to do some type of immune testing prior to the initiation of the vaccination--vaccination strategy dike the response and knowing we obviously have to make a number of changes to the 58 protocol. And there is one of the things, now that we are looking at advance advanced stage of breast cancer patients, we likely will. Miss guan? >> I would like to respond to some of the conversation around the table. As well as a comment regarding previous protocols that the rack has approved there is a few of us who have been around for three or four years now and the whole issue of using the three cohort dose escalation model from cytotoxic cancer treatments has been raised every time the model is used at this table. >> It is important to remember that the RACneither approves or disapproves of models but tries to raise questions that are 59 appropriate for the PI to consider further and for the general research community to consider. And I think the key question that has been raised now multiple times over the years that I have been here is the use, the almost blind use of the three cohort cytotoxic cancer treatment model for the design of human gene transfer protocols and I think from the conversation that has evolved around the table today, it is clear that by simply saying, okay. It is a phase I and therefore, it is maximum tolerated dose, that that really isn't the appropriate question to be answering. In a study such as this and really a lot more thought has to be given in the real 60 questions that would create a protocol that answers some serious questions about putting patients at risk in order to push forward a model that might result in in new therapies and that it is inappropriate to say we are going to keep escalating with a few people exposed as possible until we see negative effects. The questions that have emerged here by the scientific members have certainly raised different questions than the classical cancer treatment question. >> Could I interject there, I very much support that idea. And I just like to point out that the three patient per cohort. Phase I design is not even the best design in for cytotoxic agencies and some how of a mystery to me how it became 61 popular. It uses relatively few subjects and Dr. Alvada has seen how important to say. The investigators get to pick the doses and they come out of the air. The cohort size comes out of the air. The decision rules about when to up and down. Who couldn't love the design? It gives you a fantastic amount of freedom. The problem is that it doesn't necessarily address the questions that should be asked as you have already pointed out. Thank you for doing that. >> Other comments by either member of the RAC or the public. Doctor? >> >> I have a couple of comments on the informed concept 62 document. One is the ever present comment that the language is rather complex and probably wouldn't be understood by most subjects. Another comment is that although in most places in the informed consent document, you refer to the gene transfer product as the DNA injection which I think is appropriate. In a couple of places the word vaccinated was used or immunization referring to the product or the injection of the product and you are trying to see whether or not it is a vaccine or immunization. And using the words in that way may imply it works or is going to work. I would recommend sticking with either DNA injection or saying experimental vaccination or experimental immunization. 63 >> Any other comments? >> Thank you all. I am thank you very much Mr. Gillanders for your willingness to interact with this sometimes rather frisky group. I am going to go ahead and read my comments. It is true. The comments are exclusively clinical in nature. I didn't hear any substantial preclinical concerns for the DNA vaccine. The RAC remains concerned that the vaccine strategy is directed against a molecule mammaglobin A for which the function is not defined. If the vaccination strategy is effective. Then an autoimmune response will be used against an antigenin normal breast tissue. Although there are no currently 64 defined autoimmune disorders of the breast, this remains a possibility and perhaps should be evaluated in the future in the transgenic model. >> Can I stop you for one minute. >> Yes, please. >> So, I am not sure that I didn't like the word autoimmune reaction will be induced. I think it may be more appropriate. >> May be induced. >> You are correct. >> Happy to change it. >> Thank you. >> The investigators chose to alter the protocol to enroll stage four patients to address this concern. Now, I will need help with the next section which I hope expresses Dr. Piantadosi's and other's trial design. 65 The traditional phase I trial design remains inappropriate. Now, I chose that strong a word on purpose. For a study where no dose limiting toxicity is anticipated. Because I heard and it certainly has been the experience that with the DNA vaccine, it really or where little DNA-- >> Let me ask you this. So, does the RAC believe then this agent which has never been used in humans can be given at a dose. >> No, if you will wait, let me-- >> All right. >> Let me continue through my list of issues and then I will ask you for comments. >> Okay. The RAC recommends reconsideration of the design 66 use an inpoint. Such as maximum immune response for dose escalation and to designate how the choice of an optimal dose will be made. It is important to clarify the precise definition of a maximum immune response. Don't know how else to say that. Subsequently the trial design should be reconsidered. I tried to get to the point that I thought you all were discussing which is this is not a traditional, DNA vaccine study. Perhaps should not be considered in the framework of a phase I trial design because there is no substantial toxicity anticipated. Rather, the dose escalation should be based on the positive obviously with input pay the 67 figurative. But on the positive which would be a biological response. One has to define the biological response in precise enough terms so it could be utilized. >> I thought your wording captured what my essential concern about the dose question was and that is that if there is a dose question. It real relates to the question of dose and biological efficacy and surrogate as measured by the immune--and surrogate and that is actually not the stated primary purpose of the trial. Investigators have said that safety is the primary purpose of the trial and so the design shouldn't be designed to investigate the dose question when, in fact, the primary purpose is something different. 68 There is still a substantial amount of homework left about how they are going to address the safety concern which they define as the absence of severe toxicity and shed some light on the relationship between dose and immune response. >> Is it fair to say ambiguity exists regarding the primary objective? >> Just the comment, I mean there could be acute toxicity at the site of the injection of the expression of the gene where it doesn't belong. --expression of the gene where it doesn't belong. >> I think the reason why we are still proposing the dose escalation, in some form, and you know, maybe it doesn't need to be three patients per cohort. Maybe weaken roll more patients 69 in the doses we think will be effective. At some level, the reason we are proposing the dose escalation is this is a knew construct that has--new construct that has never been tested in humans and I feel strongly we should test it at a low dose initially and then with that, with that base, we can proceed to test it at a dose that we feel would be likely to be effective. Now, I guess, if we break the mold of a traditional dose escalation. It gives us the opportunity to enroll more patients at the higher doses and some other changes. >> Well, Dr. Gillanders-- >> Maybe it is appropriate. And maybe I am just naive. >> No, you are not at all. 70 And the comment as I have and we will endorse or not constructed it, is not directive. It uses words like reconsider. Think about. >> Also uses the word and says the current trial design is inappropriate. >> It does. >> So, that's a pretty strong directive. >> I can modify that. Optimal, controversial. >> May not be appropriate would be okay with me. >> Or you could use, we encourage the investigator to consider a trial design that may be more effective than the current trial design. I don't know. >> But you know, Dr. Gillanders, you are the one who broke the mold. 71 Not we. You said safety was the primary outcome and that the dose question would be decided not on cytotoxic outcomes so you broke the mold of the dose escalation. All I am suggesting is fine. Do something about it. >> In defense here, and I understand what you are saying, the issue here is phase I trials are FDA's main goal is safety trial. But phase I trials usually also consider dose escalation. That's how you you are going to get a feel for what you are going to do in your later trials and moving towards your confirming trials. It is like an exploratory trial and that's why you add dose escalation. It doesn't get away from your 72 issue of numbers and safety and the reason why it is traditional, that's what, besides safety, which is always important, that is what also these trials, you are trying to get to. >> Are you tell me the FDA is done with the-- >> No, I am not telling you that. I am trying to explain why you are always seeing that. This. And okay. So the paradigm exists because of the way trials are going forward. If a single dose. So I am not a clinician. You have to take it with a grain of salt. If you have one dose and you don't see any response. You don't and you also don't 73 see any safety issues. It doesn't, I mean, what does it mean? >> I don't think the issue was. >> You are listening. >> Whether to escalate the dose, but for what signs are you looking for as a result of this escalating the dose. And so I rewrote this. >> Can I make one more comment? It might help too if we could come up with a, well, it would not-- >> We are not able to do that. >> Make any recommendations. >> The recommendations for reconsideration of trial design are based on a general concern by members of the RAC that the--that the current standard design for phase I of three patient, three subject per cohort dose escalation trial are based on the use of this 74 design for cytotoxic drugs in cancer studies and that the design may not be appropriate when different questions are being asked. Our wording is fairly gentle and I have changed it to read, and I will read this one more time, the use of da da da, may not be appropriate for a study. I took out the stronger language where minimal or little DLC is anticipated. The RAC suggests reconsideration of the trial design. It is important to clarify the, and then the end of that sentence. The precise definition of a max member immune response and ambiguity exists with safety and biological response and the trial design should be reconsidered based op a balance 75 between these two potential objectives. Does that, that's pretty soft language and it merely says, that we as a group are concerned by the use of a trial design meant for one purpose but extrapolated to another. Now I need some help from everyone. Am I overstating? >> This is the first time in subject, so, you know, based on, so you don't know that there isn't some toxicity. >> I don't know. >> And I am playing devil's advocate. To go with just. Again, I am not saying this to go on either side. I am being devil's advocate. You don't know, what is safety. One dose with 10 subjects that gives you an idea that 10 76 subjects that is safe but you don't really search see much of an immune response. But then you have to do another study with that many patients at another dose to see if you have safety. See what I am saying here. Maybe you can increase the number per cohort but the dose escalation is to get you to a point. Where. The objective is to see what the toxic dose is. You may not reach that. But. >> I understand what you are saying. But we always design studies designed on what we expect to see. And I didn't make this up. The investigators said they don't expect to see any 77 toxicity and when I asked them. They said it again. They don't expect to see toxicity. >> That may be correct on that part. >> I don't know. We agree there are tensions here for things that are unexpected. It is already pointed out there could be injection site problems and the history of vaccines is full of that kind of stuff. But the point is that what they are expecting to do is run right up to the highest dose. Because it is very likely that the highest dose will produce the maximal response within the scope of the doses of what they have chosen. >> Be that as it may, they are testing a new gene and I think 78 the people are very uncomfort, using this as an example of our arguments for changing protocol designs. >> Right. >> I am touched too. I am trying to express what I have heard so, Dr. Help me, would you eliminate this concern completely? Are there other used words that can be used that may not be appropriate? Because I have used the softest English that I can come up with short of eliminating the concern. >> Would you be happier if they add added more subject per cohorts and still with the dose escalation. >> I am not arguing against the dose escalation. >> nor am I. >> I am arguing a rational for 79 what is--rationale. >> You are saying their trial design is not adequate, that they shouldn't be-- >> No, no, no. Thank you. Because that's not what my intent was. Let me, I know how to fix this. The use of a traditional phase I trial design may not be appropriate. >> Jeez. I okay. Because I still think dose escalation is exception. It is just that and help me everybody. I can't do this alone. It is too important. >> This is not the place to rewrite trial designs. You want to put in language that you have a concern. You mention what your concern 80 is, and this isn't the time to change trial. It is something if the RAC, wants to talk about. Get a committee, that's your prerogative. >> So, does someone else, Dr. Powers, could you take a crack at what we should say? >> I am definitely scared to jump into this. It is something to do about an emerging and continuing concern on the part of the RAC about the problem of the mismatch and stated objectives and that we encourage the researchers to think carefully about will it is safety and the other issues that Dr. Mentioned and primary objectives and whether the trial design and whether the out come measures are appropriate match to achieve the status and objectives that 81 are central. >> Something in the neighborhood. But, does that soften it or does it make the mess out of your concern? >> I think it is closer. Mismatch between the primary objectives and the outcome measures really. I am getting no help. Dr.? >> >> Well, I was okay. With the earlier wording. But only because I sort of understood in my own mind what the problem was. But, the basic design that we see over and over again was meant to provide a fairly crude information about the relationship between a predictable serious frequently occurring clinical toxicity and 82 dose. That's the setting in which that design was constructed and applied. And it does an okay job at that but by no means a good job at it. And it was meant to be applied prior to the acquisition of safetyive at a later -- safety at a later stage of development. >> I think what the folks on this side of the table are saying. That is a real issue in this case. Do you agree with that? You want to know about the toxicity of this particular gene. And the protocol that you just said. It is adequate. Not barely adequate but we are 83 uncomfortable with telling the investigator that now, change directions. I agree, he has another another stated goal. Which is he wants to get some information about efficacy and which he is going to get T cells developed and hopes to see and it is an issue of whether he gets tolerance which is not nearly addressed. He cannot enCorp. rate these into the protocol other than to get some information. >> So I am not disagreeing and I under your discomfort. You are saying you are saying you are loosely in the same context of wanting to investigate the occurrence of side affects. I am simply reacting to the investigator's statement that they don't expect to see any. 84 And if they don't expect to see any. Then I am sorry this isn't the right design. >> But we do. We may. >> I think he said he doesn't. Because it is probably true. Never the unless he doesn't know. >> So, let me try this for everyone. >> RAC has an ongoing miss match between the phase I trial steins. That's neutral. Fair. Jump two. Ambiguity exists regarding the primary objectives. Safety and biological response-and the it should be reconsidered based on a balance between the two potential objectives. 85 Isn't that motherhood and apple pie. >> It doesn't say change it from dose escalation. It says bring things into balance. It doesn't direct anyone. It just says rethink. Okay. >> Dr. Federoff. >> It needs to be discuss a different venue and great detailed discussion and this investigator should not be the product of an ad hoc and the second is, I think the anticipated potential toxicity maybe underestimated and better defined so that the design whether it is truly adequate or minimally adequate but it could address the question. So if the investigator was willing to acknowledge that there could be at some dose a 86 toxicity that is measurable. Quantifiable. Then there is an opportunity to preserve the design and actually achieve an end point that results to safety -- relates to safety. >> Well, I think that it is, I don't expect that we will see toxicity here. But obviously the whole point of the trial is that there is a very real possibility that there may be toxicity. I can't predict the future. And nor can anybody in the room, I think. But, I agree. I think if we, the reason why we are very excited about this, we think it will be very safe and there is a significant potential for generating a beneficial immune response. And first things first. 87 We need to document the safety of the biological. >> Okay. >> There are other biologicals similar to this that has proven to be safe. We can as assume this is safe. >> I recommend that we delete this entire paragraph and move it to a formal discussion out of the context of a specific protocol during the March meeting. Because we have spent an extraordinary amount of time discussing the issues during the last two days. >> Two years. >> Five years. >> So, let me delete this comment in its entirety. People comfortable with that? Gone. Next. The risk exists that rather 88 indicing a they are put particular response to mammaglobin A it will induce tolerance. This should be explored during the study. The RAC considered control immunizations with influenza and other vaccines to the stage four subject, a group with advanced stage cancer. >> Do you mind if we add. >> Okay. >> No. >> Is there any comment from members of the RAC. >> That is pub libbed data locking at breast cancer patients. That is published from the university of Washington and looking and immunotherapy trials. My recollection, I haven't looked at this for awhile. 89 They didn't find a difference between folks who had a recall response and they are not in the T cell response and breast cancer patients. I would look at at that data and reaffirm. Because that is published. So I don't know. I think you can site the references. >> Why don't I change consideration to review. >> Okay. Suggest review of control immunization with influenza and other vaccines. Thank you very much. It is not my area of expertise. Informed consent. Because the risk of autoimmunity cannot be ex clouded. This risk should be stated in the ICdocument and it should be 90 reviewed to review the language for simpler understanding and it should be reviewed to standardize the language for DNA regulation and which imply. I am going to put may imply. So we have a rather straightforward statement. Any comments for changes. >> Can you read the whole thing again. >> Yes, if I can make my computer work. >> Just a minute. It is directed against mammaglobin for which function is not yet defined if the vaccination strategy is effective then an auto immune response maybe induced in low levels in breast tissue. No currently defined autoimmune disorders of the breast. This remains a possibility and may be evaluated in the future 91 in the trance generalic mouse model. To enroll stage four patients to address the concern. The risk exists that rather than inducing a therapeutic response. The vaccine will induce tolerance. This should be explored during the study. The RAC suggests consideration. No, review. Of control immunizations with influenza and other vaccines by the stage four subjects. A group with advanced stage cancer. IC, you don't want me to reread these three comments. That what we have left. So there is no concern included about trial design. Dr. Powers. >> I, it may well be that we 92 want to include is modest. I am reluctant not to include the considerations coming forward and it is not a surprise or departure. Every investigator comes forward here and there are issues raised about ambiguity of the objectives and outcome. We make comments so they can think about them. Part of our job is to go on record for the public, not as a general measure. But with our best judgement of that. It would be a mistake to leave a complete absence of a reflection of the best judgement at this moment. Knowing it is a moving target. We have not left it out any time before. Nor should we. >> That's my sense of that. 93 >> How about the protocol remains ambiguous. >> But I deleted it. >> Can't you find it? >> I can't. It is far too back, folks. >> Let something fairly Bland. It is with regard to the primary objective and the end points and the trial design should be reconsidered in that context. The end. >> Good. >> Is that okay? >> All right. May I have someone make a motion. Dr. Federoff. We will now take a vote. Beginning with Dr. Rosenberg. >> Aye. >> Aye. >> Dr. Dewhurst. >> I do. 94 >> I do,. >> Aye, Pianadosi. >> Aye. >> Aye, Heslop. >> Thank you all very much. Please record carefully Dr. Pianadosi's negative vote. >> All right. It is 12:15. I would love to see people back in one hour at 1:15. So we can begin our afternoon review just a bit early. Thank you all. HUMAN GENE TRANSFER PROTOCOL 510-731OPEN-LABEL, DOSE-ESCALATION STUDY INVESTIGATING THE SAFETY OF A SINGLE ADMINISTRATION OF AN ADENOVIRAL VECTOR ENCODING HUMAN AQUAPORIN TO ONE PAROTID SALIVARY GLAND IN INDIVIDUALS WITH IRRADIATION INDUCED PAROTID SALIVARY HYPOFUNCTION DISBLD I'D LIKE TO FOCUS ON SALIVARY GLANDS. AND THEN TALK A LITTLE BIT MORE ABOUT OUR PRECLINICAL DATA. SO THIS IS SCHEMATIC DIAGRAM OF A SALIVARY GLAND. SALIVARY GLANDS ARE EPITHELIUM TISSUES MADE OF TWO DISTINCT TYPES OF EPITHELIUM. THE BUSINESS PART OF THE GLAND IS WATER PERMEABLE AND SODIUM CHLORIDE SECRETING. WHAT IT SECRETES IS CALLED A PRIMARY FLUID THAT IS ISOTONIC. >>PROBABLY 80% OF THE PROTEINS IN SALIVA. THAT FLUID ENTERS THE DUCT SYSTEM TO ENTER THE MOUTH. THE DUCTS ARE A VERY DIFFERENT KIND OF EPITHELIUM. >>THEY ABSORB -- THE FINAL SALIVA THAT ENTERS YOUR MOUTH IS HYPOTONIC. >>IF YOU THINK OF SALIVARY GLANDS LIKE A BUNCH OF GRAPES, AFTER RADIATION YOU LOSE ALL THE FRUIT AND RETAIN THE STEMS. THE STEMS ARE WATER PERMEABLE AS I SAID. PATIENTS SECRETE LITTLE TO KNOW SALIVA. WHAT DOES THAT DO? 2 THAT CONDITION OF SALIVARY HYPOFUNCTION LEADS TO CONSIDERABLE MORBIDITY. MOST PROBLEMATIC IS DYSPHAGIA. THEY HAVE DIFFICULTY FORMING A FOOD BOLUS ADEQUATELY AND TRANSFERRING THAT FOOD BOLUS. THEY CERTAINLY SUFFER FROM DRY SENSATION IN THE MOUTH. THEY SUFFER FROM MANY ORAL INFECTIONS, MOST NOTABLE -- OF THE MUCOSA AND RAMPANT DENTAL CARRIES. THEY HAVE REDUCED MY COASTAL WOUND HEALING IF THEY HAVE A CAN CANKER SCORE. MOST IMPORTANTLY THERE IS NO CONVENTIONAL THERAPY TO REMEDY THIS CONDITION. ANT ABOUT 15 YEARS AGO THESE TYPES OF PATIENTS THAT REALLY GAVE US THE IMPETUS TO EXPLORE GENE TRANSFER AS A POSSIBLE MOTIVE &%F0 &%F0 MODE OF TREATMENT. THE WAY WE TRANSFER GENES INTO SALIVARY GLANDS IS ACTUALLY QUITE EASY. THIS IS A RAT. AND WHAT YOU CAN SEE ARE TWO PLASTIC TUBES GOING INTO THE MUCOSA, INTO OPENINGS, THE ORIFICES OF THE GLAND, WAR TENS DUCT. FOR THIS PROCEDURE THE AND NEEDS TO BE ANESTHETIZED FOR RESTRAINT BUT THE IDEA OF DOING THIS COMES FROM ROUTINE RADIOGRAPHS WE TAKE OF PATIENTS OF THEIR SALIVARY GLANDS WHERE WE CANULATE THE DUCT ORIFICE, INFUSE A CONTRAST MEDIA AND TAKE A X-RAY. HERE OUT OF THE PLANE OF THE PICTURE WOULD BE A NEEDLE INTO THE CANULA, SIR SYRINGES INTO THE VECTOR AND SLOWLY INFUSE THE GLAND. SO FOR AN ANIMAL WE NEED ANESTHESIA FOR RESTRAINT. 3 NO ANESTHESIA IS REQUIRED FOR PATIENTS. THE REASON THIS WORKS SO WELL IS BECAUSE SALIVARY GLANDS ARE ACTUALLY ALMOST A MONOLAYER OF CELLS THAT LINE THE DUCT. SO IF THE MOUTH IS OVER HERE AND THIS IS THE MAIN EXCRETORY DUCT YOU HAVE THIS NETWORK OF DUCTS GOING BACK TO THE FURTHER ETH REACHES OF THE GLAND, THE SECRETORY END PIECE, THE AS PER REGION. ALMOST ALL THE EPITHELIUM CELLS IN THE GLAND LINE UP ALONG THAT DUCT LUMEN. SO ANY VECTOR INFUSED HAS DIRECT ACCESS IN THEORY TO THE API CAL MEMBRANE OF THOSE EPITHELIUM CELLS. >>IN 1997 WE PUBLISHED A PAPER, CHRISTINE DELPORT A FORMER FELLOW PUBLISHED A PAPER IN WHICH WE TESTED A REPAIR OF SALIVARY GLANDS. THISCARTOON DEPICTS IT. HERE IS A SALIVARY GLAND THAT'S BEEN IRRADIATED. YOU HAVE A DUCTAL EPITHELIUM CELLS LEFT THAT ARE WATER IMPERMEABLE AS I SAID. NO SALIVA SECRETEED. WE HYPOTHESIZED THAT WE COULD TRANSFER THE GENE FOR HUMAN WATER CHANNEL INTO THE DUCTAL CELLS, MAKE THEM WATER PERMEABLE AND SALIVA WOULD BE SECRETEED. AT A MECHANISTIC LEVEL, THIS IS THE MECHANISM THAT WE HYPOTHESIZED. THIS WAS ENTIRE PAPER PHYSIOLOGY THAT WE HYPOTHESIZED BACK THEN. ON THE TOP IS OUR WATER IMPERMEABLE DUCT CELL IN AN IRRADIATED GLAND. AND AT THE TIME, THIS WAS SORT OF THE COMPLIMENT OF ION CHANNELS AND TRANSPORTERS THAT WE KNEW EXISTED IN THESE DUCT CELLS. 4 AND THERE ARE RED X'S THROUGH THREE OF THESE. THE EPITHELIUM SODIUM CHANNEL, SODIUM PROTON ANTI PORTER AND A CHLORIDE CHANNEL, WHICH HAPPENS TO BE CFGR. THE REASON THESE HAVE RED X'S THROUGH THEM IS THAT IN THE ABSENCE OF THE SECRETION OF A PRIMARY FLUID, SO IN THE ABSENCE OF THIS SODIUM CHLORIDE RICH SECRETION COMING FROM THE ASNER REGION BECAUSE THEY WERE DESTROYED DUE TO RADIATION, THESE WOULD BE NOT OPERATING. AND THE OTHER TRANSPORTERS OR CHANNELS WERE THE ONLY ONES THAT WE THOUGHT WOULD BE OPERATIVE IN THIS CELL. >>IN THE LUMEN HERE OF THIS IRRADIATED GLAND, YOU WOULD STILL FIND A LITTLE BIT OF WATER. WATER MOVES TWO WAYS. WATER, THROUGH A WATER CHANNEL, IS WATER MOVEMENT THROUGH A FACILITATED PATHWAY THAT'S NECESSARY IN A CELL LIKE A SALIVARY GLAND, IT'S NECESSARY IN THE KIDNEY, ET CETERA. BUT ALL CELLS HAVE THE ABILITY FOR WATER TO MOVE ACROSS THE MEMBRANES IN A DIFFUSIVE FASHION. SO YOU HAVE A LITTLE BIT OF WATER IN THE LUMEN. CO 2 WOULD BE DISSOLVED IN THAT WATER. YOU COULD EXCHANGE THE PROTON FOR POTASSIUM AND YOU COULD HAVE A POTASSIUM BICARBONATE GRADIENT. WE REASONED THAT WHAT WAS LACKING WAS A WATER PERMEABILITY PATHWAY. THERE STILL HAS BEEN NONE IDENTIFIED AND WE OPTED TO TRANSFER IN HUMAN ACTION ACTION IN ONE BECAUSE IT'S NONPOLE ARIZED. 5 IT WILL BE DISTRIBUTED ALL AROUND THE PLASMA MEMBRANE OF THE CELL. >>THIS IS A SCHEMATIC DIAGRAM OF THE VECTOR THAT CHRISTINE DELPORT MADE. SIMPLE FIRST GENERATION ADENOVIRAL VIRUS. THE -- INITIAL TESTS WERE DONE IN VITRO. THIS SHOWS MDCK CELLS BECAUSE THAT'S A DOG KIDNEY LINE, A VERY COMMONLY STUDIED CELL TO LOOK AT SORTING OF MEMBRANE PROTEINS AND SECRETORY PROTEINS. IN THIS EXPERIMENT CELLS WERE GROWN ON A POLYCARBOHYDRATEIATE FILTER. THEN THE CELLS WERE INFECTED EITHER WITH A CONTROL VIRUS OR THE AQUAPORIN VIRUS. YOU SEE NOTHING ON THE TOP BECAUSE MDCK CELLS DO NOT EXPRESS ANY AQUAPORIN 1 IN THE BOTTOM. THE XY PLANE YOU SEE ALL AROUND THE CELLS. IN THE OTHER YOU CAN SEE IT'S ALSO AROUND THE CELL. SO IT DID JUST WHAT WE HOPED IT WOULD DO. >>THE NEXT THING WE DO AFTER SHOWING THAT THE VECTOR MEDIATES EXPRESSION OF THE TRANSGENE IS TEST TO SEE IF IT DOES FUNCTION AS A WATER CHANNEL. THE WAY WE AS SAY IT IS TO PUT IN THE CHAMBER A -- AND IN THE LOWER CHAMBER A -- AND THEN WATCH WATER FLOW INTO THE UPPER CHAMBER. >>THIS IS ANOTHER IN VITRO EXPERIMENT SHOWED WITH A CELL LINE WE DEVELOPED A LONG TIME AGO FROM RATS, SUB MANDIBULAR GLANDS. THAT'S A DUCTAL EPITHELIUM CELL LINE THAT FORMS A -- LAYER. YOU CAN SEE THE CONTROL EXPERIMENT WAS DONE WITH CELLS 6 INFECTED WITH AN ADENO VIRUS ENCODING HORMONE. THE EXPERIMENT WAS WITH INFECTION OF CELLS WITH THE AQUAPORIN FLOWING VIRUS. YOU CAN SEE A CONSIDERABLE INCREASE IN FLUID MOVEMENT ACROSS THIS MONOLAYER. SO THAT SHOWED THAT THE VECTOR WORKED AND THE TRANSGENE, THE PROTEIN ENCODER BY THE TRANSGENE WAS FUNCTIONAL. SO THE NEXT IMPORTANT THING WAS TO TEST IN VIVO. IN RATS THIS IS THE TIMELINE OF OUR INITIAL RAT STUDY. THERE WERE TWO COHORTS OF RATS. ONE THAT WAS IRRADIATED AT 17. 5 GRAY AND THEN TREATED WITH VECTORS AT 90 DAYS. ANOTHER THAT WAS TREATED WITH 21 GRAY RADIATION AND AT 120 DAYS TREATED WITH VECTOR. I'LL ONLY SHOW YOU DATA FROM THE LATTER. I'M BE HAPPY TO TALK ABOUT THE FORMER AS WELL. >>ONE OF TWO VECTORS WERE GIVEN, EITHER ADDL 312, WHICH IS ESSENTIALLY ADENO VIRUS SEROTYPE 5 WITHOUT ANY TRANSGENE OR THE AQUAPORIN AT -- AND THREE DAYS AFTER GIVING THESE VECTORS WE MEASURED SALIVA OUTPUT. THAT'S SHOWN IN THIS GRAPH. >>IF YOU LOOK AT THIS SIDE OF THE FIGURE THESE ARE ANIMALS THAT GOT THE CONTROL VIRUS. THESE RATS -- CAN BE TAKEN AS NORMAL SALIVARY FLOW. THESE RECEIVED 21 RADIATION. SO THE REDUCTION IN SALIVARY FLOW WAS ABOUT 60 TO 65%. >>AND JUST TO REPEAT, THESE ANIMALS, FOUR MONTHS AFTER THE IRRADIATION, GOT THIS CONTROL VIRUS, THREE DAYS LATER SALIVA WAS COLLECTED. >>ON THE RIGHT-HAND SIDE OF THIS FIGURE ARE SHOWN ANIMALS 7 THAT GOT THE AQUAPORIN VIRUS. THESE ARE THE SHAM IRRADIATED ANIMALS AND THESE ARE THE ANIMALS IRRADIATED WITH 21 GRAY. NO STATISTICAL DIFFERENCE BETWEEN THESE TWO AND THE SHAM IRRADIATED CONTROL VECTOR. SO THIS SHOWED THAT IN THIS IRRADIATED RAT MODEL, TRANSFER OF THE AQUAPORIN 1 GENE LED TO NEAR NORMAL LEVELS OF SALIVARY FLOW. >>I WANT TO POINT OUT TO YOU BECAUSE IT WILL COME UP LATER, EACH GLAND, EACH SUB MANDIBULAR GLAND GOT 5 TIMES 10 TO THE 9th PLATFORMING UNIT OF THIS VECTOR, ROUGHLY 100 TO 1 PARTICLES OF MOMING UNIT. ANT I WANT TO INTRODUCE THE CONCEPT OF MOI HERE, IN VIVO. WE'RE USED TO THINKING ABOUT -- IN TERMS OF IN VITRO TERM. >>IT'S HARD TO DETERMINE THE NUMBER OF CELLS OF TISSUE IN VEVO. THE WAY WE HAVE EXPRESSED MOI IS TO EXPRESS THE AMOUNT OF EITHER INFECTIOUS VIRUS OR PARTICLES OF VIRUS PER MICROLITER INFUSED INTO THE GLAND. THE FOLLOW OF THE INFUSATE IS PROPORTIONAL TO THE SIZE OF THE GLAND. WE MAKE AN ASSUMPTIONS THAT THE MASS OF CELLS IN ANY GLAND, WHETHER IT'S FROM A RAT, A MOUSE, A PRIMATE, A MINI PIG OR A HUMAN, THE MASS PER UNIT AREA IS THE SAME. SO THAT THIS TURNS OUT TO BE A CONVENIENT MEASURE OF THE RELATIVE AMOUNT OF VECTOR TO WHICH CELLS WOULD BE EXPOSED. SO WE COMPARE IN VIVO -- IN VIVO DATA USING THIS TERM. >>I'M GOING TO SHOW YOU DATA FROM OUR MINI PIG STUDY. VERY SIMILAR TYPE OF STUDY AS THE RAT STUDY. 8 ANIMALS IN WERE RADIATED TIMES 0. I'M SHOW YOU IN A MINUTE THEY EXPERIENCED A PROFOUND REDUCTION IN SALIVARY FLOW. THEREAFTER THEY GOT A CONTROL VECTOR OR THE AQUAPORIN ACTION VECTOR. >>AT TIMES 0 THE NUMBER SHOWN HERE 100% OF NORMAL PAROTID FLOW. TWO TIMES BEFORE THEY WERE IRRADIATED WE MEASURED PAROTID FLOW. >>ANIMALS WERE IRRADIATED. YOU CAN SEE THERE'S A 40% REDUCTION IN PAROTID FLOW. THERE AFTER THEY RECEIVED EITHER THE AQUAPORIN VIRUS OR THE CONTROL VIRUS ENCODING -- THE RED LINE IS THE AQUAPORIN VIRUS AND THREE DAYS LATER SALIVARY FLOW WAS INCREASED FROM ABOUT 20% TO OVER 80% OF NORMAL. AT 7 DAYS IT DECREASED SOMEWHAT, AND THEN BY 14 DAYS IT HAD DECREASED FURTHER. THERE WAS NO SIGNIFICANT CHANGE IN THE LEVEL OF PAROTID FLOW IN THE ANIMALS THAT RECEIVED THE -- VIRUS. I WANT TO POINT OUT TO YOU AGAIN THE DOSES THAT WE USE. IN THIS STUDY ANIMALS RECEIVED 10 TO THE 9th PLAQUE FORMING UNITS OF THE AQUAPORIN VECTOR OR THE -- VECTOR PER GLAND. THE SIZE OF A MINI PIG GLAND IS MUCH LARGER THAN A RAT. ACTUALLY WE CAN INFUSE 4,000 MICROLITERS INTO THE GLAND. SO THE MOI IS NOW 2.5 TIMES 10 TO THE 5th PLAQUE FORMING UNITS PER MICROLITER IN FUSE. THAT'S 1%, 100 TIMES LESS THAN WAS USED IN RATS. IMPORTANTLY, THIS IS ALSO A DOSE THAT SCALE THE BETWEEN MICE AND MINI PIGS. ANIMALS OF MICE GIVEN THIS DOSE 9 OF A REPORTER GENES AND MANY PIG MINI PIGS GIVEN THIS DOSE OF REPORTER GENE LEAD TO THE SAME LEVEL OF REPORTER GENE EXPRESSION. IRRESPECTIVE OF THE SIZE OF THE ANIMAL. >>SO OBVIOUSLY IN A MOUSE WE'RE INFUSING MUCH LESS. 50 MICROLITERS, IN A MINI PIG 4,000. BUT ON A PER MICROLITER BASIS IT'S SCALABLE. >>IS -- THIS SAFE? WE'VE CONDUCTED A NUMBER OF GOP AND NONGOP STUDIES. NONGOP RESULTS IN MINI PIGS AND HUMAN PRIMATE STUDIES. 100% OF SURVIVAL. NO PAROTID SWELLING. -- NO -- NORMAL FOOD CONSUMPTION. IN THE MINI PIG STUDY AS WELL AS IN THE PRIMATE STUDIES WE MEASURE NUMBERS OF CLINICAL LAB STORY PARAMETERS. THOSE IN BLACK WERE NORMAL. THOSE WERE BLUE SHOWED ELEVATIONS IN GLOWULENS AND WHITE CELLS. >>WE'VE ALSO CONDUCTED A NUMBER OF GOP STUDIES WITH FIRST GENERATION ADENOVIRAL VECTORS AFTER DELIVERY TO RATS SUB MANDIBULAR GLANDS. THEY'RE SHOWN HERE. >>IT'S UNAUDITED AT THIS TIME BUT A SUBSTANTIAL AMOUNT OF INFORMATION HAS ALREADY BEEN GLEANED FROM THAT STUDY. AND IN MY RESPONSE TO DOCTOR FEDEROFF'S QUESTION, ALL OF THESE FOOT NOTES ARE ANSWERED. I WON'T GO INTO IT NOW BUT I'LL BE HAPPY TO SPEAK ABOUT IT LATER. IN GENERAL, THESE THREE VECTORS WERE IDENTICAL EXCEPT FORT TRANSGENE ENCODED, ALL WITH THE SAME V PROMOTER, ALL WITH THE 10 SAME SV POLY DENTALATION SIGNAL. THE MAXIMUM DOSE IS ROUGHLY THE SAME. THIS WAS DONE IN THE LATE '90s AND WE WERE REFERRING TO VECTOR DOSAGES AND PLAQUE FORMING UNITS. AND THAT'S WHY AT SEVERAL TIMES DURING MY PRESENTATION USED PLAQUE FORMING UNITS SO THAT WE COULD RELATE TO EARLIER STUDIES. BUT AGAIN AT A RATIO OF ABOUT 100 TO 1 PARTICLES TO PLAQUE FORMING UNITS. THIS IS SIMILAR TO THIS. THIS ONE HAD TWICE THAT DOSE IN TERMS OF PARTICLES. >>WE'VE GONE OUT IN THE AQUAPORIN STUDIES 9 2 DAYS. THE RESULTS OF ALL OF THESE ARE PRETTY SIMILAR AND NO SERIOUS ADVERSE EFFECTS WERE NOTED. >>IN THIS STUDY THERE WERE 120 ANIMALS. IN THIS STUDY 144 ANIMALS. IN THE AQUAPORIN STUDY 200, EQUAL NUMBERS OF MALES AND FEMALES. PLEASE NOTE THAT THE MAXIMUM DOSE IN THIS STUDY, MAXIMUM TOTAL DOSE IS TWICE THE MAXIMUM TOTAL DOSE IN THE PROPOSED CLINICAL STUDY AND 10 FOLD THE MAXIMUM MOI IN PARTICLES PER MICROLITER FOR THE PROPOSED CLINICAL STUDY. >>THIS IS THE DOSAGE SCHEME FOR THE CLINICAL STUDY AND THIS IS THE LAST SLIDE OF MY PRESENTATION. AND I JUST WANT TO POINT OUT TWO THINGS. I MENTIONED IN THE COURSE OF MY PRESENTATION THAT A DOSE OF 2.5 TIMES 10 TO THE 5th PLAQUE FORMING UNITS PER MICROLITER. SO OUR WAY OF EXPRESSING MOI SCALED BETWEEN MOUSE AND MINI PIG. AND THIS IS THE SAME DOSE THAT 11 WAS EFFECTIVE IN THE MINI PIG STUDY. I'M ASSUMING FOR THIS AMOUNT OF PARTICLES, THE 15 TO 1 IN THE CLINICAL VECTOR, 15 TO 1 PARTICLE TO PUF RATIO AND APPROXIMATE 1,000 MICROLITER INFUSION VOLUME INTO PATIENTS. >>THE SECOND THING -- SO THE MIDDLE DOSE HERE IS THE DOSE THAT WAS EFFECTIVE IN MINI PIGS AND IT WAS A DOSE THAT SCALED BETWEEN MOUSE AND MINI PIG. THE HIGH DOSE WAS DIRECTED 10 TO THE 11th PARTICLES TOTAL DOSE, BECAUSE IN EXTENSIVE SERIES OF STUDIES EVALUATING THE SAFETY OF ADD 5 VECTORS IN HUMANS, ORIGINALLY PUBLISHED IN 2002 BY CRYSTAL AND COLLEAGUES ABOUT 90 PATIENTS AND SUBSEQUENT STUDIES BY OTHER INDIVIDUALS, THIS 10 TO THE 11th PARTICLES TOTAL DOSE TO HUMANS APPEARED TO BE WITHOUT SIGNIFICANT ADVERSE EFFECT. SO THAT'S WHY THAT WAS CHOSEN. AND THAT'S MY LAST SLIDE. I'LL BE HAPPY TO ANSWER ANY QUESTIONS. >>DOCTOR BAUM THANK YOU VERY MUCH FOR A LUCID AND VERY RESPONSE PRESENTATION TO RAC MEMBERS COMMENTS AND QUESTIONS. WE HAVE 5 REVIEWERS THIS AFTERNOON, TWO AD HOC REVIEWERS AND THREE RAC REVIEWERS. AND WITH YOUR PERMISSIONI'D LIKE TO REVERSE WHAT I HAD PROPOSED EARLIER TO THE RAC REVIEWERS AND ASK OUR AD HOC REVIEWERS TO GO FIRST. AND I'LL REMIND EVERY ONE THAT YOU SHOULD QUICKLY REVIEW ALL OF YOUR CONCERNS AND AT THE END OF EACH CONCERN, WHICH YOU NEED TO REVIEW PUBLICLY, STATE WHETHER YOUR CONCERN HAS BEEN ADDRESSED BY DOCTOR BAUM OR NOT, EITHER IN WRITING OR IN HIS ORAL PRESENTATION. 12 IF THERE'S RESIDUAL CONCERN THEN YOU COULD HELP ME BY CONCISELY STATING THAT SO THAT I CAN PUT IT INTO THE RECORD. DOCTOR SCOTT STROEM IS OUR FIRST REVIEWER. WE'RE VERY FORTUNATE TO HAVE HIM WITH US. AN EAR, NOSE AND THROAT SURGEON FROM THE UNIVERSITY OF MARRIED. DOCTOR STOCK MARKET. STROME. >>THANKS, DOCTOR BAUM THAT WAS A GREAT PRESENTATION. I ENJOYED READING YOUR PROTOCOL. JUST TO PUT THINGS IN CONTEXT FOR FOLKS WHO DON'T DEAL WITH HEAD AND NECK CANCER EVERY DAY, WITHIN THE LAST TWO YEARS THERE'S BEEN A NUMBER OF CLINICAL STUDIES WHICH HAVE SHOWED REALLY THE BENEFITS OF CHEMORADIATION FOR HEAD AND NECK CANCER. SO IT'S INCREASING IN PREFERENCE. PERHAPS THE MOST DEVASTATING THAT FOLKS HAVE FROM THIS IS DRYNESS OF THE MOUTH. THEY ALSO GET SIGNIFICANT PHARYNGITIS OBSTRUCTION. I PUT THAT IN CONTEXT TO SAY THIS IS A VERY IMPORTANT CLINICAL PROBLEM AND ONE THAT WE AS A COMMUNITY OF FOLKS THAT TAKES CARE OF THESE PATIENTS HAS FAILED TO ADDRESS. THERE'S BEEN A COUPLE STUDIES NOW USING -- AND I AGREE WITH DR. BAUM'S STATEMENTS, HAVE NOT ADEQUATELY CONQUERED THIS PROBLEM. AND IT REMAINS A STATE -- FOR THOSE OF US WHO SEE THESE FOLKS WHERE THEY'RE LITERALLY WALKING AROUND WITH WATER BOTTLES ALL THE TIME WHEN THEY COME INTO YOUR OFFICE. AND IT'S A DEVASTATING QUALITY OF LIFE ISSUE. 13 INDEED THE CRUSTING GETS SO BAD THAT THEY CAN OBSTRUCT THEIR AIRWAYS. SO IT'S A SIGNIFICANT PROBLEM. >>I THOUGHT THAT THE STUDY OVER ALL WAS VERY WELL DESIGNED AND BASED ON A LARGE AMOUNT OF PRELIMINARY DATA, MOST OF WHICH HAS BEEN PUBLISHED. IT SPANNED A LONG CAREER AND IS VERY CAREFULLY THOUGHT OUT. >>I'M GO THROUGH MY CONCERNS ONE BY ONE JUST FOR THE RECORD. THE FIRST CONCERN I HAD WAS THAT THE INFORMED CONSENT SHOULD BE MODIFIED TO INCLUDE THE RISKS WHICH I MENTIONED IN MY STATEMENT. AND THOSE RISKS SPECIFICALLY INCLUDED VASOVAGAL RESPONSES FROM THE MANIPULATION OF THE PAROTID DUCT AND INITIALLY THE POTENTIAL TO GET OBSTRUCTION FROM THE PAROTID DUCT AS A RESULT OF THE MANIPULATION. AND DR. BAUM AGREED. I ADDED EXCLUSION CRITERIA TO THOSE WHOSE DUCTS WERE NOT CLINICALLY ACCEPTABLE. DR. BAUM AGREED AND THAT WAS SATISFYTRY. >>DISTAL STENOSIS -- DR. BAUM AGREED WITH THAT AND THAT WAS SATISFACTORY. CRITERIA SHOULD BE EXPANDED TO INCLUDE PATIENTS WHO REQUIRE GEN ANESTHETIC -- AND DR. BAUM AGREED WITH THAT AND THAT WAS SATISFACTORY. >>MY PRIMARY CONCERN ON THIS SECTION WHICH I STILL HAVE SOME QUESTIONS ABOUT AND I'D LIKE YOU TO COMMENT IF YOU WOULDN'T MIND IS THAT APPROPRIATE TASK AT THIS POINT IS THAT WITH REGARD TO HOW YOU'RE GOING TO MEASURE OUTCOME ASIDE FROM THE FACT THAT YOU HAVE MANIPULATED THE DUCT. IN OTHER WORDS, IF YOU INSTILL YOUR VECTOR AND THE GOAL IS TO 14 INCREASE WATER FLOW THROUGH THE DUCT, AND BY SIMPLE MANIPULATION OF THE DUCT OR THE INFLAMMATORY RESPONSE WHICH WAS NOTED IN YOUR PAPERS WHICH IS PARTIALLY BUT NOT COMPLETELY ALLEVIATE ALLEVIATED WITH STEROIDS, HOW DO YOU SEPARATE THAT KIND OF OBSTRUCTION AND HOW DO YOU MEASURE THE WATER FLOW IN DUCTS THAT ARE POTENTIALLY OBSTRUCTED FROM YOUR MANIPULATION? IS IT FAIR TO EXCLUDE EVERYBODY WHO YOU DECREASED FLOW TO 0 BECAUSE THAT COULD BE THE RESULT OF THE VECTOR OR THE MANIPULATION? >>LET ME TRY TO ANSWER IT AS FOLLOWS. THE INCLUSION CRITERIA STATE THAT PATIENTS MUST HAVE SOME FLOW. SO THEY HAVE TO BE GREATER THAN 0 AND LESS THAN 0.2 MILLS PER -- SO ALL PATIENTS FOR THE TWO PREENTRY VISITS, THEY WILL BE TESTED FOR PAROTID SALIVARY FLOW. THERE SHOULD BE SOME LEVEL OF WHAT IS FOR THEM THEIR QUOTE NORMAL FLOW. THAT'S WHERE I SAY IT'S 0.1 MILL. THEY ENTER THE STUDY. WE DO SEVERAL MANEUVERS. WE DO CYALO GRAMS. MOST INVASIVE ENDOSCOPIC BIOPSY. WE WILL BE MAKING MEASUREMENTS OF SALIVARY FLOW QUITE FREQUENTLY. FIRST DAY, SIX-HOURS, 12 HOURS, 24 HOURS AFTER DELIVERY OF VECTOR. AND THEN AT SET TYPES. CALO ENDOSCOPIC BIOPSY ON DAY 2. AS I INDICATED TO YOU IN MY RESPONSE IN MY MIND THE WAY WE WOULD DISTINGUISH A MANEUVER ASSOCIATED REDUCTION IN FLOW, MANEUVER BEING DEFINED AS COULD 15 BE THE CALO GRAM BIOPSY OR THE DELIVERY OF VECTOR ITSELF WAS A CHANGE FROM THE PREENTRY SALIVARY FLOW. >>CORRECT ME IF I'M WRONG. WHEN I RED SOME OF YOUR STUDIES, IT SEEMED TO ME THAT YOUR INITIAL STUDIES OF THE VIRUS BEFORE YOU WERE GIVEN STEROIDS ALONG WITH IT YOU WERE GIVEN SIGNIFICANT INFLAMMATION OF THE DUCT AND THAT WAS REDUCED BUT NOT ELIMINATED WITH THE CONCOMITANT ADMINISTRATION OF STEROIDS. SO MY QUESTION IS, IF THAT'S THE CASE AND YOU'RE HAVING A STEROID WHICH CAN POTENTIALLY LEAD TO OBSTRUCTION, IS IT FAIR OR IS THERE A BETTER WAY PERHAPS THAN LOOKING AT COMPLETE ABSENCE OF SALIVARY FLOW BECAUSE THAT COULD BE RELATED EITHER TO DUCT MANIPULATION OR TO THE DRUG ITSELF? >>WELL, FOR ONE THING, WE DON'T INTEND TO GIVE THESE PATIENTS STEROIDS. FOR A SECOND THING THE MINI PIG STUDY WAS DONE WITHOUT STEROIDS. AND WHILE THERE IS ON A HISTOLOGICAL LEVEL THE DOSE THAT WAS EFFECTIVE, A HISTOLOGICAL LEVEL YOU CERTAINLY CAN SEE AN INCH FLAM INFLAMMATORY INFILTRATE. IT WAS NOT IN ANY WAY OBSTRUCTIVE AND THERE WAS AN INCREASE IN SALIVARY FLOW. WE DO NOT ANTICIPATE AT THE DOSES THAT WE ARE GIVING TO THE CONSIDER OR THAT WE PROPOSE TO GIVE TO THE PATIENTS TO SEE A REALLY MASSIVE INFLAMMATORY INFILTRATE SUCH THAT IT WOULD OBSTRUCT. >>DO YOU THINK IT MIGHT BE REASONABLE IN THAT SETTING JUST TO HAVE A WAY WHERE YOU COULD PUT A SMALL STENT IN THE DUCT 16 LIKE WE NORMALLY DO WHERE IF YOU GOT COMPLETE OBSTRUCTION -- >>ABSOLUTELY. >>IT WOULDN'T RUIN YOUR STUDY? >>ABSOLUTELY. >>MY CONCERN IS NOT SAYING THIS IS WRONG, IT'S JUST HOW ARE YOU GOING TO INTERPRET THE DATA ? THAT'S A GOOD POINT. THAT'S A GOOD SUGGESTION. >>THAT WOULD BE MY ONLY SUGGESTION. THE SECOND POINT I THINK WE DISCUSSED WITH THE IMRT AND THE MO PHOSPHATE. I THINK THAT'S DEBATABLE BUT I'M COMFORT ARM WITH YOUR RESPONSE. THE THIRD POINT REGARDING THE CHOICE OF SALIVARY GLANDS, TRADITIONALLY IT'S BEEN TAUGHT AT LEAST IN THE HEAD AND NECK LITERATURE AND I THINK SUB MANDIBULAR GLANDS ARE THE PRIMARY WORK HORSE SALIVA GLANDS WHERE THE PAROTID GLANDS ARE PRIMARILY DEPENDENT UPON STIMULATION. I SIMPLY ASK THE QUESTION, MIGHT THAT BE A BETTER MODEL. I THOUGHT THAT YOUR ANSWER WAS SATISFACTORY TO THAT QUESTION. >>THE FOURTH POINT THAT I RAISED WAS THE RADIO THERAPY DOSES SCHEMES ARE VERY DIFFERENT WHICH WE USE FROM PATIENT TO PATIENT. AND IF YOU'RE TRYING TO GET A STANDARDIZED SET OF RESULTS YOU OUGHT TO STANDARDIZE THE INCLUSION CRITERIA UP FRONT BY DELINEATING AT LEAST THE DOSE RANGE WHICH THE SALIVARY GLANDS ARE GOING TO RECEIVE. AND YOU AGREED WITH THAT AND I THOUGHT THAT WAS APPROPRIATE. >>MY OTHER CONCERNS WERE PRIMARILY DERIVED AT HELPING MAKE IT A BETTER STUDY. I DON'T KNOW IF YOU AGREED OR NOT BUT THEY WERE WHAT I HOPED. 17 I JUST WANTED YOU TO EXPAND YOUR INCLUSION CRITERIA. A LOT OF HEAD AND NECK CANCER PATIENTS ARE OLDER GENTLEMEN. IF WE LIVE LONG ENOUGH AS MEN WE'RE ALL PROBABLY GOING TO GET PROSTATE CANCER. SO I THOUGHT IT WOULD BE A REASONABLE THING TO ADD THAT INTO THE GROUP SINCE IT'S PROBABLY NOT GOING TO EFFECT -- IF YOU GUYS THINK YOU CAN GET ENOUGH PATIENTS WITHOUT THAT I THINK THAT'S REASONABLE. >>PET CT IS PRETTY MUCH THE BASELINE RIGHT NOW FOR STANDARD OF CARE FOR DIAGNOSING HEAD AND NECK CANCER IN THE SETTING OF RADIATION FIELDS. AND I THINK IF YOU'RE GOING TO LOOK FOR PRELIMINARY DATA ON MAKING SURE THAT YOU DON'T HAVE A MALIGNANCY UPFRONT YOU NEED TO CLUT PET CT AND YOU AGREED WITH THAT. AND THEN FINALLY IN THE STOPPING RULES, I WOULD QUALIFY DEATH. YOU SAID DEATH FROM MANY CAUSE. BUT REALISTICALLY AS WE KNOW FOLKS WITH HEAD AND NECK CANCER BY DEFINITION ARE IN A HIGH RISK GROUP FOR DYING OF MANY OTHER MORBIDITIES. AND I THINK IF YOU EXCLUDE THAT YOU MIGHT STOP YOUR STUDY VERY EARLY AND YOU AGREED WITH THAT AS WELL. SO THAT WAS THE END OF MY COMMENTS. (. >>THANK YOU, DOCTOR STROME. I HAVE ONE PERHAPS TWO REMAINING CONCERNS FOR YOU. THE FIRST WOULD BE THAT EITHER MANIPULATION OF THE DUCT OR INFLAMMATION FOLLOWING GENE TRANSFER MY CON FOUND THE EVENTUAL INTERRATION OF SALIVARY FLOW GENE TRANSFER EFFICACY. AND THEN I THOUGHT YOU MENTIONED 18 THAT IT IS POSSIBLE THAT STENTING THE DUCT MIGHT PREVENT THIS COMPLICATION. >>I THINK THAT'S ONE WAY TO TRY AND -- I WOULD JUST HATE TO SEE THE STUDY END WITH NO DATA SIMPLY BECAUSE OF AN INFLAMMATORY RESPONSE WHEN INDEED YOU'RE GETTING THE EFFECT YOU'RE ON SERVING. THAT WAS THE ONLY THING I WAS THINKING THAT MAY ALLOW YOU TO INTERPRET YOUR DATA COMPARING AFTER WILLS TO APPLES. >>AND YOU'RE SUGGESTING THAT THE STENT BE PLACED PRIOR TO THE GENE TRANSFER? >>NO. I WOULD LEAVE HOW DR. BAUM WANTS TO DO IT UP TO HIM AT THE TIME. I'M NOT SURE YOU NEED TO DO IT IN EVERY PATIENT. BUT IN PATIENTS WHERE YOU'RE STARTING TO SEE OBSTRUCTION THAT WILL ALLOW YOU TO POTENTIALLY SEE FLOW WHERE YOU OTHERWISE MIGHT NOW. >>THANKS YOU VERY MUCH. WE'RE GOING TO GO THEN TO DOCTOR POPPAS WHO IS A SPECIALIZED DENTIST AND INVESTIGATOR FROM TUFT UNIVERSITY. DOCTOR POPPAS. >>I WANTED TO MAKE A GENERAL COMMENT IN TERMS OF THE ORAL COMPLICATIONS OF RADIATION THERAPY. AND I AGREE WITH DOCTOR STROME THAT GIVEN THE NEW CHEMORADIATION PROTOCOL WE'RE SEEING MUCH MORE MORBIDITY IN OUR PATIENTS IN THE LAST TWO YEARS. AND I ALSO AGREE THAT WHAT WE HAVE NOW JUST ISN'T SUFFICIENT. YOU CAN GET SOME IMPROVEMENT WITH IMRT BUT CERTAINLY NOT THE KIND OF IMPROVEMENT THAT IMPROVES QUALITY OF LIFE. >> WITH THE LOSS OF SALIVA IT'S 19 NOT JUST WATER. I TELL MY PATIENTS IT'S LIKE LOSING THE BLOOD SUPPLY TO THE MOUTH. TEETH HAVE NO CONNECTION TO THE BODY EXCEPT THROUGH SALIVA. SO WHEN SALIVA IS LOST, THE TEETH SLOWLY DISINTEGRATE. THERE ARE ALL SORTS OF INFECTIONS THAT ARE PREVENTED BY PROTEINS IN SALIVA. HISTATINS OCCUR IN HUMANS OR IN PRIMATES AND ABOVE, THESE PREVENT FUNGAL INFECTIONS, ET CETERA. SO WITH THE LOSS OF SALIVA WE SEE OVERGROWTH OF BACTERIA THAT CAUSE BOTH PERIODONTAL DISEASE AND CARRIES AS WELL AS FUNGAL INFECTIONS IN THE MOUTH. >>THE PREVENTIVE PROTOCOLS THAT HAVE BEEN DEVELOPED ARE VERY COUPLE BETTERSOME TO THE PATIENT. AND UP TO 70% OF THE PEOPLE WILL STOP DOING THESE PROTOCOLS AFTER AWHILE. THIS LEADS TO RAMPANT CARRIES IN THE MOUTH, THE TEETH DISINTEGRATE, -- CAN OCCUR. SO IT IS AN IMPORTANT PROBLEM FOR THOSE OF US WHO TREAT THE COMPLICATIONS OF RADIATION THERAPY. AND I WILLING ON TO MY COMMENTS. >>THE FIRST ONE WAS MAKING SURE THAT THE DUCTS WERE OPEN. AND DR. BAUM RESPONDED TO THAT TO MY SATISFACTION. MY SECOND COME WAS ASKING IF HE WAS GOING TO ATTEMPT TO BIOPSY THE ASSIN AR Y CELLS AND HE RESPONDED THAT THEY WOULD TRY. IT MAY BE VERY DIFFICULT TO GET THAT FAR DOWN. I AGREED WITH DR. BAUM THAT THE PAROTID IS A GOOD PLACE TO START, ESPECIALLY SINCE THE PAROTID IS ENCAPSULATEED. AND I ALSO FOUND THAT THE USE OF 20 SALIVARY FUNCTION IS A GOOD WAY TO SELECT PATIENTS. >>I WANTED TO MAKE SURE THAT THE EXCLUSION CRITERIA INCLUDED AUTO IMMUNE DISEASES, DIABETES, ET CETERA, OTHER CAUSES OF SALIVA HYPOFUNCTION. AND DR. BAUM RESPONDED HE WOULD EXCLUDE THOSE PATIENTS. AND SINCE SMOKING AND ALCOHOL CONSUMPTION ARE USUALLY RISK FACTORS FOR HEAD AND NECK CANCER, SMOKING HAS BEEN SHOWN TO EFFECT SALIVARY FLOW. AND I WANTED TO MAKE SURE THAT THOSE PATIENTS WERE EXCLUDED. AND DR. BAUM AGREED TO THAT. >>SO HE DID RESPOND TO ALL MY CONCERNS. >>THANK YOU VERY MUCH. OUR NEXT REVIEWER WILL BE DOCTOR FEDEROFF. (FEDEROFF. >>I TOO WANT TO THANK DR. BAUM FOR A VERY THOUGHTFUL AND COMPREHENSIVE SET OF ANSWERS TO QUESTIONS RAISED BY MYSELF AND OTHER REVIEWERS. WHAT MAKES THIS PROTOCOL INTERESTING AT LEAST IN ONE RESPECT IS THE CHOICE OF USING A RELATIVELY SHORT LIFED GENE TRANSFER REAGENT TO EXPRESS A BIOLOGICALLY ACTIVE MOLECULE, IN THIS CASE A WATER CHANNEL IN INDIVIDUALS WHO PROBABLY HAVE A CHRONIC NEED FOR RESTORING SALIVA FLOW. AND I THINK THAT THAT ISSUE IS ONE THAT I'D LIKE TO COME BACK TO AT THE VERY END, BECAUSE I THINK YOU WENT TO GREAT LENGTH TO TRY TO JUSTIFY THE APPROACH THAT YOU'VE TAKEN AND I THINK IT WILL BE WELL WORTH HAVING YOU ELABORATE ON THAT IN THE CONTEXT OF THIS LARGER FORUM. >>I HAD A NUMBER OF QUESTIONS. AND THE FIRST ONE WAS RELATED TO A RELATIVELY SMALL SET OF 21 NONHUMAN PRIMATES THAT YOU HAD UNDERTAKEN A SERIES OF GENE TRANSFER STUDIES IN WHERE THE RESULTS WERE LESS THAN OPTIMAL. AND WE WERE INTERESTED IN WHETHER THE MODEL AND OR THE SUBJECT NUMBER WAS PERHAPS THE REASON FOR THE DATA BEING LESS THAN WHAT WOULD HAVE BEEN PREDICTED BASED ON YOUR RAT STUDY AND CERTAINLY THE MINI PIG STUDY DATA WHICH YOU PRESENTED. AND I THINK THAT YOU'VE RESPONDED WELL TO THAT, AND I THINK THAT WHILE YOU'VE ONLY DONE A LIMITED NUMBER OF NONHUMAN PRIMATES I THINK YOU'VE CONVINCED ME THAT THE MINI PIG, AS ANATOMY AND AS A MODEL FOR LOOKING AT POST RADIATION CHANGE TO THE PAROTID AND THE CONSEQUENT LOSS OF ASSINER CELLS MAKE IT A RELIABLE MODEL TO LOOK AT WITH RESTORING SALIVA CELL BY VIRTUE OF AQUAPORIN 1 GENE EXPRESSION. >>ONE OF THE THINGS THAT I ASKED WHICH WAS RELATED TO THE SUBJECT OF THE EXTENT OF FIBROSIS THAT MIGHT BE ANTICIPATED IN THE PATIENT POPULATION, AND AS SUCH THOSE INDIVIDUALS MAY BE FURTHER REMOVED FROM THEIR RADIATION THAN POSE ANIMAL PROTOCOLS THAT YOU'VE UNDERTAKEN. AND THAT STILL REMAINS AN ISSUE. YOU RESPONDED THOUGHTFULLY I THOUGHT IN THAT YOU ARE USING THE CYALO GRAM AS A MEANS OF ASCERTAINING WHETHER A SUBJECT WOULD BE INCLUDED OR NOT AND THAT THAT MAY ALONE BE SUFFICIENT TO IDENTIFY THOSE INDIVIDUALS WHO WOULD BE ENROLLED IN THE STUDY PROVIDING THAT THEY MEET ALL THE OTHER INCLUSION CRITERIA. >>IN YOUR PRESENTATION TODAY, ANOTHER THOUGHT CAME UP. 22 AND I'M JUST GOING TO ASK THIS AND YOU CAN TAKE TIME TO RESPOND TO IT. GIVEN THE NATURE BY WHICH THE CHANNEL GETS SEGREGATED TO A VARIETY OF DIFFERENT MEMBRANE SITES IN THE CELL, IS IT POSSIBLE THAT INTRASTITIAL WATER ACCUMULATION MIGHT ANTICIPATED AS A CONSEQUENCE OF HAVING THE CHANNEL BE PUT INTO A WAYS SO LATERAL AS OPPOSED TO AT THE ADLUMENAL SIDE? I KNOW YOUR DATA SUGGESTED THAT IN THE MINI PIGS YOU GET ALMOST A 2.5 TO THREE FOLD INCREASE IN SALIVARY FLOW. BUT IS THAT A LEGITIMATE CONCERN THAT ONE NEEDS TO THINK ABOUT? >>I THINK IT'S A VERY REASONABLE QUESTION BUT I DON'T THINK IT'S A REAL CONCERN. AGAIN IF THE ASSINER CELLS WERE FUNCTIONAL AND YOU HAD A SODIUM CHLORIDE RICH SECRETION, THE WATER CHANNEL IS NONPOLE ARIZED. SO IT'S ALL AROUND THE CELL. AND WATER CAN GO THIS WAY, WATER CAN GO THAT WAY. IT JUST DEPENDS ON WHICH SIDE THE OSMOTIC GRADIENT IS ON. IF THERE IS NO SODIUM CHLORIDE SECRETION WATER WON'T GO INTO THE INTERSPATIAL. >>THANK YOU. THE NEXT SET OF QUESTIONS WERE RELATED TO THE MINI PIG STUDIES THEMSELVES. AND ONE RELATED TO THE POINT THAT I MADE AT THE OUTSET REGARDING THE SHORT DURATION OF GENE EXPRESSION. PART OF IT WAS ASCRIBEED TO THE VIRAL VECTOR CONTEXT AND THE VIRAL PROMOTER IN THIS CASE, THE ACMV PROMOTER THAT'S BEING USED. BUT IN RESPONSE TO MY QUESTION YOU ALSO THOUGHT THAT THERE WAS A MULTIFACETED IMMUNE RESPONSE INVOLVING BOTH INNATE AND 23 PERHAPS EVEN OTHER PARTS OF THE IMMUNE SYSTEM BECOMING INVOLVED. COULD YOU ELABORATE A LITTLE BIT FURTHER ON THIS? BECAUSE I THINK THIS ALSO GOES TO IN WHAT WINDOW OF TIME YOU MAY ANTICIPATE SEEING CLINICAL RESPONSE TO THE GENE TRANSFER THAT YOU PROPOSE. >>SURE. THANK YOU. IN THE STUDIES THAT I'LL DESCRIBE WERE PRIMARILY STUDIES THAT HAVE BEEN DONE IN RATS. AND WE'VE DONE THEM IN SALIVARY GLANDS. MANY OTHER PEOPLE HAVE DONE THEM IN DIFFERENT TISSUES. BUT WHEN WE ADMINISTER AN ADENO VIRAL VECTOR IN ROUGHLY THIS DOSAGE RANGE, IMMEDIATE RESPONSE IN TERMS OF TRANSGENE EXPRESSION, TRANSDUCTION OF CELLS IS REALLY QUITE HIGH, PEAKING ABOUT DAY TWO OR THREE. WHAT YOU DON'T SEE IN THAT IS IN AN INNATE IMMUNE RESPONSE. AND THAT'S PROBABLY BEEN BEST STUDIED IN THE LIVER. WHERE HUGE AMOUNT OF VECTOR IS DESTROYED WITHIN PROBABLY 90% IN THE FIRST 24 HOURS. >>ANY WAY, AFTER ABOUT DAY THREE, THAT BEGINS TO -- THE TRANSGENE EXPRESSION BEGINS TO WAYNE. IF AT DAY 7 YOU THEN GIVE ANOTHER DOSE OF THE SAME VECTOR, SAME DOSE, SO NOT A HIGHER DOSE OR LOWER DOSE, EXACT SAME DOSE, YOU SEE SIMILAR KINETICS PEAK AROUND DAY TWO OR THREE BUT THE RESPONSE IS ABOUT ONE-THIRD OF THE INITIAL RESPONSE. LET THAT GO DOWN TO A WEEK, GIVE A THIRD DOSE, NOTHING. AND TO MAKE A LONG STORY SHORT, IT INVOLVES BOTH CELLULAR IMMUNITY DESTROYING TRANSDUCEED CELLS THAT ARE STILL EXPRESSING 24 ADENO VIRAL PROTEINS AS WELL AS AND BODIES THAT ARE FOUND IN THE SALIVA THAT WILL BLOCK TRANSDUCTION. >>SO JUST FINISH THAT OFF AND SAY SOMETHING ABOUT THE WINDOW. >>YES, SORRY. >>THE MINI PIG STUDY WAS FOR US A REALLY TRUE MODEL STUDY FOR WHAT WE EXPECT IN HUMANS. NO STEROIDS WERE GIVEN. THE CAVEAT IS AS YOU MENTIONED EARLIER THESE ANIMALS HAD ONLY BEEN IRRADIATED 16 WEEKS BEFORE. IT'S NOT FIVE YEARS OUT. SO FIBROSIS AND THE EXTENT OR DUCT WORK IS DIFFERENT. BUT OUR EXPECTATION IS THAT WE WOULD SEE A PEAK AT DAY TWO OR THREE AND THEN A SLOW DECLINE TO ABOUT SOMEWHERE TWO WEEKS TO FOUR WEEKS OUT WE WOULDN'T SEE ANY INCREASE IN FLOW. SO THE REAL WINDOW FOR US IS THE FIRST TWO WEEKS. >>THANK YOU. THAT IS A VERY SATISFYING ANSWER. >>SO DOCTOR FED, DO YOU REMAIN CONCERNED ABOUT THE SHORT LIVED GENE EXPRESSION? >>I'M GOING TO COME BACK TO THAT. THANK YOU FOR REMINDING ME. I'M ALMOST THERE. I HAD ONLY ONE OTHER ISSUE THAT REALLY RELATED TO SOME OF THE PRECLINICAL TOXICOLOGY AND NOW WITH -- I RECOGNIZE THAT IT'S UNAUDITED DATA. BUT WHAT YOU PRESENTED AND PROVIDED SUGGESTS THAT THERE IS ONLY ONE RELATIVELY RATHER -- AND I THOUGHT MAYBE YOU COULD ELABORATE ON IT FURTHER. IN THE FEMALE SUBJECTS THERE WERE A VARIETY OF ALTERATIONS NOT DESCRIBED IN DETAIL IN TERMS OF THE CBC, THE WHITE BLOOD COUNTS I GATHER WERE EL GATED. 25 YOU DIDN'T CHARACTERIZE THEM. BUT IT OCCURED AT SEVERAL DIFFERENT TIME POINTS INCLUDING LATE. >>INCLUDING LATE. >>YES,. >>EARLY WAS NOT AN ISSUE BUT INCLUDING LATE. >>COULD YOU COULD YOU TALK ABOUT THAT A BIT MORE? >>I DON'T KNOW IF YOU -- DID EVERYBODY SEE -- EVERYBODY GETS THE RESPONSES THAT I GAVE TO YOU? SO THERE WERE THREE THINGS THAT WE ON SER OBSERVED IN THIS GOP LEVEL STUDY WITH THE AQUAPORIN VECTOR IN FEMALES. ALL THREE THE FIRST TIME THAT WE HAD ON SEVER OBSERVED THAT IN A TOTAL OF THREE STUDIES. ONE HAD TO DO WITH BODY WEIGHT, ANOTHER FOOD CONSUMPTION. I'LL ADDRESS THAT FIRST. >>ANIMALS -- FEMALES THAT RECEIVED THE VECTOR -- AND THERE ARE THREE DOSES OF VECTOR. I'M JUST BLANKING. THE LOW DOSE IS SOMETHING I THINK 8 TIMES 10 TO THE 9 AND TWO TIMES 10 TO THE 11, COMPARED TO A GROUP THAT RECEIVED JUST THE BUFFER IN WHICH THE VIRUS WAS ADMINISTERED. THERE WAS A ROUGHLY 10% DECREASE IN FEMALE BODY WEIGHT FROM ABOUT DAY 22 ON. AND THE CURVE WAS EXACTLY THE SAME, BUT AT DAY 22 THERE WAS LIKE A LITTLE BLIP AND THEN IT JUST ALL THREE -- ALL THREE DOSES SUPERIMPOSABLE. THERE WASN'T A DOSE EFFECT. BUT THEY WERE JUST BENEATH THE CONTROL. AND THE FOOD CONSUMPTION DATA ROUGHLY CORRELATED WITH THAT. >>THE POINT THAT YOU MENTIONED THAT WAS REALLY INTERESTING AND SOMEWHAT SURPRISING WAS THE 26 STATISTICALLY SIGNIFICANT INCREASE IN WHITE BLOOD CELLS, NOT JUST THREE DAYS AFTER WE ADMINISTERED THE VECTOR BUT 10 TO 14 WEEKS OUT LATER TIME POINT. AND THAT OCCURED. IT DIDN'T OCCUR IN MALES. AND IT -- ALTHOUGH IT WAS A STATISTICALLY SIGNIFICANT INCREASE, THE VALUE THE WERE STILL WITHIN THE NORMAL RANGE FOR THAT SPECIES OF RAT. >>HOWEVER, ON BOTH OF THESE -- WELL, ALL THREE OBSERVATIONS, FOOD CONSUMPTION AND THE BODY WEIGHT GAIN AND THE WHITE BLOOD CELL INCREASE RELATIVELY LATE AFTER VECTOR DELIVERY, MAKES US QUITE AWARE OF SOMETHING THOUGH WATCH FOR FEMALE PATIENTS THAT WOULD BE ENROLLED IN THIS STUDY. >>WAS THE DIFFERENTIATION COUNT AT ALL HELPFUL IN TRYING TO UNDERSTAND THE NATURE OF THAT? >>SEGMENTED IN MONOSITES, AS I RECALL, WERE BOTH ELEVATED. >>WERE BOTH ELEVATED? >>YEAH. >>OKAY. THANK YOU FOR THE ELABORATION. NOW LET ME COME BACK TO THE LAST POINT. YES. >>GIVEN THE DISCUSSION, DOES THIS REMAIN A CONCERN FOR YOU? >>IT REMAINS A POINT TO MONITOR IN THE SUBJECTS WHO WOULD BE FEMALE AND WHO MIGHT HAVE A PERSISTENT PERHAPS REFLECTION OF SOME ONGOING INFLAMMATION. >>GREAT AGREED. >>AND PARENTHETICICALLY THE ESRS ARE NOT ELEVATED? >>WE DID NOT MONITOR THAT. >>THE LAST POINT I THINK WAS INTRIGUING FROM THE STANDPOINT OF THE PROTOCOL. I'D LIKE TO HAVE YOU LAY OUT IN A -- IN A TERSE BUT NONETHELESS 27 DIRECTED WAY THE RATIONALE FOR SELECTING THIS REAGENT IN THIS GROUP OF INDIVIDUALS. SO LET ME FRAME THE QUESTION CAREFULLY. GIVEN THE DURATION OF EXPRESSION THAT IS LIKELY TO BE MEDIATED BY THIS ADENOVIRAL VECTOR AND GIVEN THE CHRONICITY OF THE REQUIREMENT TO ESTABLISH WATER FLOW, TELL US HOW YOU HAVE DETERMINED THAT THIS IS THE APPROPRIATE GENE TRANSFER REAGENT FOR THIS POPULATION OF PATIENTS. (TERSE (. >>THE ISSUE IS PHYSIOLOGY -- VIRTUALLY I SAID IN MY RESPONSE TO YOU IN WRITING THAT WHEN WE DEVELOPED THIS STRATEGY LITERALLY ON PAPER, IT WAS BASED ON RODENT STUDIES. UNFORTUNATELY ALMOST ALL OF OUR INFORMATION ABOUT DUCT CELL PHYSIOLOGY IS IN RODENTS FOR AN OBVIOUS REASON. THEY'RE EASY TO STUDY AND INEXPENSIVE. WE HAVE ALMOST NO KNOWLEDGE OF SPECIFIC TRANSPORTERS IN CHANNELS AS THEY EXIST IN HIGHER MAMMALS, DUCT CELL MEMBRANES. >>ALL OF THESE ANIMALS HAVE THE SAME GENERAL STRUCTURE. AND THE WAY THE GLANDS OPERATE ARE GENERALLY THE SAME. THEY MAKE A PRIMARY SECRETION. ADMITTEDLY THE DATA TO SUPPORT WHAT I AM ABOUT TO SAY IS INFINITELY STRONGER IN RATS AND MICE. BUT EVEN CLINICAL STUDIES IN HUMANS SUGGEST THAT IT'S TRUE. THE ATHENO CELLS MAKE THIS ISOTONIC SKIING CREW. SECRETION. >>SODIUM BICARBONATE IS SECRETEED. HAVING SAID, THAT I CAN'T DRAW A PICTURE OF THE TRANSPORT 28 PHYSIOLOGY OF DUCT CELLS. SO WE HIGH THOT SIZED IN A RAT, AND IN A THE ANYONE MINI PIG THAT I SHOWEDOUT, THAT BY INSERTING A WATER CHANNEL THAT WOULD BE NONPOLARIZED AND GO ALL AROUND THE CELL, THAT CELL COULD GENERATE AN OSMOTIC GRADIENT IN WHICH LUMEN WAS GREATER THAN INTERSTITIA AND WATER FLOW WOULD GO OUT. THAT REMAINS CONJECTURE IN HUMANS. >>THE CHOICE OF USING AN ADENOVIRAL VECTOR COMES FROM A COUPLE OF THINGS. ONE AT THE DOSES THAT WE HAVE SELECTED UP TO 10 TO THE 11th THERE HAS BEEN NO ASSOCIATED SEVERE ADVERSE EFFECT. RELATED TO THE DEVELOPING VECTOR. SO THAT WE HYPOTHESIZEED THIS WOULD BE A SAFE VECTOR TO DELIVER THIS GENE. >>SOUGHT HUGH SO THE -- YES, IT'S A SHORT EXPRESSION. BUT A POSITIVE INCREASE IN SALIVARY FLOW WILL INDICATE THAT THE GENERAL HYPOTHESIS HOLDS. WITH AEV, ALL OF OUR EXPERIENCE WITH AEV AND SIMILAR -- AND I SHOULD SAY THAT ALMOST ALL OF OUR EXPERIENCE WITH AEV IS WITH AEV 2. WE HAVE A FAIR AMOUNT BUT LETTER WITH OTHER AEV SEROTYPES. WITH AEV 2, WHEN YOU ADMINISTER AN AEV 2 VECTOR TO A MOUSE SALIVARY GLAND, THE VECTOR REMAINS THERE AT FAIRLY REASONABLE LEVELS AND COMPLETELY FUNCTIONAL FOR AS LOOK AS THE ANIMAL IS ALIVE. AND HENCE WE'VE HAD MICE UP TO TWO YEARS. EXPRESSING TRANSGENES THAT BEGINNING ABOUT 12 WEEKS IT ALMOST DOESN'T CHANGE. >> FROM A SAFETY STANDPOINT. 29 THE RASH ALE FOR CHOOSING THIS VECTOR FROM A SAFETY STANDPOINT, IF THE PHYSIOLOGY OF THE HUMAN DUCT IS SUCH THAT IT WON'T ALLOW GRADIATION OF THIS INTERLUMEN GRADIENT, THEN I DIDN'T WANT OR I WOULDN'T WANT TO ADMINISTER TO SOMEONE A VECTOR THAT WOULD BE IN THE GLAND AND POTENTIALLY FUNCTIONAL FOR A LONG PERIOD OF TIME IF I KNEW IT HAD NO BENEFIT. SO THAT WAS THE INITIAL -- >>THANK YOU. SO IF I COULD JUST SUMMARIZE THAT SUCCINCTLY, IT WAS THE PHYSIOLOGY THAT COMPELLED THE CHOICE? >>YES. >>THE DEMONSTRATED PRECLINICAL DATA THAT PROVED FUNCTION AND THE CONCERN ABOUT SAFETY ABOUT DELIVERING A VIROLOGIC AGENT SUCH AS AEV 2 THAT MIGHT PER SIFT INDEFINITELY WIN THOSE SUBJECTSth SUBJECTS. >>RIGHT. I WOULD ADD, IF THIS CLINICAL STUDY SHOWS SOME EVIDENCE OF BIOLOGICAL EFFICACY, IT DOESN'T PRECLUDE GOING IN AND ADMINISTERING AN AEV 2 VECTOR. >>THANK YOU. >>THANK YOU. >>DOCTOR FEDEROFF, COULD YOU RETURN TO YOUR INCLUSION ABOUT CONCERN ABOUT THE INCLUSION AND THE CYALO GRAM. >>THE CONCERN WAS IN PEOPLE WHO MIGHT HAVE RECEIVED THEIR RADIATION AT A MUCH EARLIER TIME THAN THEIR POTENTIAL INCLUSION INTO THE STUDY, I WAS CONCERNED THAT THE EXTENT OF FIBROSIS MIGHT BE DIFFERENT THAN THAT IN ANY OF THE PRECLINICAL ANIMAL STUDIES. AND THE SOLUTION THAT DR. BAUM HAS RECOMMENDED AND WAS PART OF HIS ORIGINAL PROTOCOL AND CALLED 30 ATTENTION TO IT AGAIN WAS THAT THESE INDIVIDUALS WILL HAVE A CYALO GRAM WHICH WILL DEMONSTRATE THE ANATOMY THAT WILL ALLOW FOR THEM TO EITHER BE INCLUDED ON THAT BASIS OR EXCLUDED. >>AND WAS THAT SATISFACTORY OR NOT? >>YES, IT'S SATISFACTORY. >>GREAT. DOCTOR MUZYCZKA. >>MANY OF MY POINTS HAVE ALL BEEN COVERED. BUT FOR THE RECORD I ASKED DOCTOR BAUM FIRST ABOUT THE ISSUE THAT WE JUST DISCUSSED, WHICH IS WHY THE PARTICULAR ADD VECTOR WAS USED AS OPPOSED TO A LONGER LASTING VECTOR. AND I FOUND THE ANSWER SATISFACTORY WITH WHAT YOU JUST HEARD. THE SECOND POINT WAS, I ASKED DR. BAUM IF THERE IS ANY EVIDENCE THAT LONG-TERM EXPRESSION OF AQUAPORIN HAD ANY ADVERSE EFFECT. IN THAT CASE YOU FOCUSED PRIMARILY ON YOUR ADENOVIRAL DATA. BUT YOU MENTIONED IN PASSING AND JUST NOW ACTUALLY AGAIN THAT YOU HAVE SOME AEV DATA. I WASN'T CLEAR WHETHER YOU HAD -- >>NOT WITH AQUAPORIN1. >>OKAY. >>WITH AQUAPORIN 1 PROBABLY A MONTH. >>OKAY. SO YOU BASICALLY DON'T HAVE DATA ON THAT POINT. AND THEN THE THIRD QUESTION WAS ABOUT THE 4 -- I BELIEVE IT WAS 4 PRIMATES. 35 PRIMATES. USUALLY DATA, DOSE-ESCALATION DATA THERE. (5 (. >> I THINK YOUR RESPONSE WAS 31 QUITE STRAIS STRAIGHTFORWARD AND HONEST AND ESSENTIALLY AMOUNTS TO 5 PRIMATES IS WORSE THAN YOU'RE OUR AVERAGE DOSE HE IS ESCALATION WHICH REALLY COULDN'T TELL US MUCH OF ANYTHING. I HAVE SOME ADDITIONAL QUESTIONS, SO I'M SATISFIED WITH RESPECT TO YOUR ANSWERS. I THINK I UNDERSTAND YOUR PFU PER MICROLITER MOI DEFINITION AND WHY THAT WORKS. AND THAT'S A NICE WAY TO GO. BUT DO YOU HAVE ANY INFORMATION ON WHAT THE ROW REAL MOI MIGHT BE? YOU HAVE ESSENTIALLY AN EPITHELIUM LAYER IN THIS GLAND, I GUESS. DO YOU KNOW HOW MANY CELLS THERE ARE AND HOW MANY VIRUS PER CELLS THERE ARE? >>RIGHT NOW I CAN'T TELL YOU OFF THE TOP OF MY HEAD. I CAN CERTAINLY GET FROM ALL THE ANIMALS I CAN CALCULATE WHAT THE NUMBER OF CELLS ARE IN AN ANIMAL GLAND. I CAN MAKE ASSUMPTIONS AND THEN CALCULATE THAT FOR A HUMAN. >>BUT NO ONE HAS ACTUALLY -- >>IN THE LITERATURE THERE ARE NUMBERS ABOUT THE NUMBER OF CELLS IN A RAT SUB MANDIBULAR GLAND. THERE'S A LOT OF DATA ON THE AMOUNT OF DNA PER OLDER STUDIES THAT HAVE BEEN DONE. BUT WHAT WE'VE TRIED TO DO FOR EVERY SPECIES THAT WE STUDIED IS -- IT'S OBVIOUSLY VERY IMPORTANT THAT WE DON'T BLAST FLUID INTO THE GLAND AND BREAK THROUGH THE CAPSULE AND SPREAD IT ALL OVER THE PLACE. SO WE TYPICALLY WILL DO -- INITIALLY WE DID LIKE TRY CAM BLUE, SIMPLE STUDIES TO SEE HOW MUCH WE CAN INFUSE WITHOUT IT BREAKING AND THEN GOT A LITTLE 32 MORE SOPHISTICATED AND THEN DID EVEN IN RODENTS CYALO GRAMS. AND I THINK I CAN DO THE CALCULATION BUT WE'VE NEVER DONE IT. MOST PEOPLE ARE ACCEPTING OF THE ASSUMPTION THAT THE GLAND MASS, THE AMOUNT OF CELLS PER ANY UNIT GLAND MASS IS EQUIVALENT. SO WE HAVE NOT BOTHERED TO GO FURTHER. >>AND A FINAL QUESTION JUST OUT OF CURIOSITY, IF THE DUCT CELLS NOW BECOME WATER SECRETING, IS THE MATERIAL, THE FLUID THAT THEY'RE SECRETING, IS THAT REALLY SALIVA OR JUST WATER? >>NO. IT'S NOT WATER. IS IT THE SAME SALIVA THAT YOU HAD BEFORE YOU WERE IRRADIATED? NO. SO LET'S SAY 80% OF YOUR CELLS ARE ASSINER CELLS AND THEY MAKE 80% OF THE PROTEINS THAT ARE IN THE GLAND AND THEN YOU HAVE 20% DUCTAL CELLS IN 20% OF THE PROTEIN. SO TAKE THE MOST EXTREME CASE WHERE YOU HAVE 100% LOSS OF ASSINER CELLS AND ARE ONLY LEFT WITH DUCTAL CELLS. THERE'S A CERTAIN COMPLEMENT OF PROTEINS THAT YOU WOULD NOT GET. DUCTAL CELLS DO NOT EXPRESS AMY L ASE, FOR INSTANCE. EVERYBODY KNOWS THAT FROM JUNIOR HIGH BIOLOGY. ONE OF THE REASONS -- AND IT ACTUALLY GOES BACK TO DOCTOR FEDROFF'S STANDPOINT. FROM A PHYSIOLOGY AND FROM A GENE TRANSFER STANDPOINT, DEALING WITH PROTEIN SECRETION IS A LOT EASIER THAN GENERATING AN IM GRADIENT AND GETTING WATER FLOW. WE REASONED 15 YEARS AGO THAT THE PROBLEM PATIENTS HAD WAS IN HAVING ANY FLUID IN THEIR DUCTS. 33 SAY THEY HAVE A SMALL PERCENTAGE OF -- SAY 10% OF THE ASSINER CELLS ARE LEFT, WHICH IS QUITE REASONABLE. AND GETTING SOME PROTEIN SECRETION. THEY BITE INTO AN APPLE. THERE'S SOME EXOCYTOTIC PROCESS TAKES PLACE. BUT THEN IT SITS THERE IN THE GRAND, WILL OBSTRUCT THE GLAND AS YOU HEARD EARLIER THE DISTAL PART. IF YOU. >>IF YOU PROVIDE SOME LEFT OF FLUID THERE IS A POSSIBILITY THOSE PARTICLES CAN BE DISSOLVED. IF YOU HAVE A FLUID MOVEMENT IT'S NOT DIFFICULT TO PUT IN A SECRETORY -- THE DUCT AGO CELLS MAKE A LOT OF ANTI-MIKE MICROBIALES, ET CETERA. THE OTHER THING TO REMEMBER IS THESE PATIENTS, AFTER THEY ARE IRRADIATED, IN ADDITION TO PAROTID GLANDS, SUB MANDIBULAR GLANDS HAVE MANY MINOR SALIVARY GLANDS IN AND AROUND THEIR MOUTH. THOSE ARE MUCH LESS AFFECTED BY RADIATION AND MAKE A MUCH MORE VISCOUS SECRETION. SO PART OF THE DISCOMFORT THAT A PATIENT HAD FOR STICKY SECRETION IN THEIR MOUTH, YOU CUT THAT IF YOU WILL WITH A FLUID. YOU MIGHT HAVE A GRADIENT. LIKE OUR SALIVA HAS MORE SODIUM CHLORIDE. IT MIGHT BE MORE SODIUM BICARBONATE BUT WE THINK IT WILL BE EFFECTIVE. >>OKAY. THANK YOU. >>MISSISSIPPI KWAN MIDKW MS. KWAN. >>I HAD ONLY TWO COMMENTS ON THIS PROTOCOL. THE FIRST ONE WAS A GENERAL 34 COMMENT ABOUT THE INFORMED CONSENT DOCUMENT. I FOUND THAT THE LANGUAGE WAS REALLY QUITE COMPLEX FOR A LAY PERSON TO UNDERSTAND USING TERMS AND PHRASES SUCH AS TRANSFER OF VECTOR, REPLICATE, CLINICAL PROTOCOL, COMMON CLINICAL AND IMMUNE LOGICAL MEASURES, VECTOR MEDIATED TRANSFER. AND I HAD SUGGESTED THAT SOME SERIOUS EFFORT BE MADE TO SIMPLIFY THE LANGUAGE. ADDITIONALLY, SPECIFICALLY ON A CERTAIN SECTION OF THE DOCUMENT, IT WAS STATED THAT THERE MAY BE NO DIRECT BENEFIT AND THIS IS WITH REGARDS TO THE PARTICIPANT. AND I THINK IF YOU LOOK AT THE ENTIRE PROTOCOL IT WOULD BE MORE ACCURATE TO SAY THAT THERE WILL BE NO DIRECT BENEFIT BECAUSE IT IS A CHRONIC ISSUE. AND THE PROTOCOL IS SPECIFICALLY DESIGNED TO BE TEMPORARY. >>THE RESPONSE WAS THAT THEY WOULD PROVIDE THESE COMMENTS TO ANOTHER TEAM WHICH IS RESPONSIBLE FOR THE CONSENT DOCUMENTS. HOWEVER, THERE WAS NO REVISED DOCUMENTS SO I REALLY CAN'T COMMENT ANY FURTHER THAN TO SAY THAT I DO THINK THE CONSENT DOCUMENT SHOULD BE REWORKED AND SIMPLIFIED. >>MY SECOND COMMENT HAD TO DO WITH A FOLLOWUP OF THE ISSUE FIRST DISCUSSED WHICH WAS I THINK THERE WAS AMPLE JUSTIFICATION FOR WHY THIS VECTOR WAS SELECTED, WHICH WOULD ONLY PROVIDE A TEMPORARY RESPONSE. MY QUESTION WENT BEYOND THAT AND SAID, ASK THE QUESTION OF WHETHER THERE COULD BE A FAIRLY SIMPLE UNDERSTANDABLE EXPLANATION OF HOW SUCCESS WITH THIS PROTOCOL WOULD LEAD TO 35 EXPERIMENTS WHICH WOULD PROVIDE A LONG-TERM RESPONSE. AND I'M NOT SURE THAT I HEARD ONE THAT I UNDERSTOOD. >>CAN I RESPOND TO THAT? I APOLOGIZE FOR THAT. IN THE FIRST CASE, THE OTHER BODY THAT WILL DEAL WITH THE CONSENT FORM, THE NIH CENTER HAS A SEPARATE GROUP THAT TRIES TO GET THE LANGUAGE AT 8th GRADE LEVEL. THAT'S OUT OF MY CONTROL. I HAVE TO SEND IT TO THEM. IN THE SECOND CASE, I WAS ANSWERING NOT ONLY YOU BUT ANSWERING THE OTHER REVIEWERS. AND I APOLOGIZE FOR NOT MAKING THAT MORE CLEAR. >>SO WHERE DOES THE STRATEGY FIT WITHIN WHAT YOU MENTIONED? >>YES. THAT IF THIS SHOWS SOME LEVEL OF BIOLOGICAL EFFICACY, PHYSIOLOGICAL EFFICACY, THEN THERE IS ANOTHER GENE TRANSFER AGENT THAT WE CAN USE WITH THE SAME GENE THAT GIVES LONG LIFE EXPRESSION. >>THANK YOU. >>SO MY SECOND POINT IS SATISFIED. >>THIS PROTOCOL IS OPEN FOR FURTHER DISCUSSION BY MEMBERS OF THE RAC FIRST. ANY FURTHER THOUGHTS? DOCTOR -- >>SO I'D LIKE TO USE THE LOGIC AGAIN OF USING ADENOVIRAL. BECAUSE IF THE ADVERSE EVENTS THAT YOU SEE ARE ONLY APPARENT AFTER LONG-TERM EXPRESSION, THEN YOU WON'T SEE THEM WITH THE LNO BUT THE TRUER -- YOU WANT TO USE IS A -- I'M SORT OF CONCERNED THAT THIS MAY GIVE YOU A SORT OF AN INDICATION OF THE PHYSIOLOGY AND AT LEAST FOR THE RECEPTORS AND THAT SORT OF THING ARE PRESENT IN THINGS -- ANIMAL 36 MODEL. BUT FOR THE AEV, IF THE ADVERSE EVENTS ARE LONG-TERM, THEN THE ADDENDUM WILL NOT SHOW THEM TO YOU, ANYWAY. >>WELL, -- I'M SORRY. >>JUST ONE MORE QUESTION, JUST TO INFORM ME, WHAT IS THE LIFE EXPECTANCY OF THE PATIENTS THAT YOU ARE GOING TO TREAT? >>ALL OF THE PATIENTS. LET ME DO THE LAST ONE FIRST. ALL OF THE PATIENTS THAT WE WILL TREAT WILL HAVE SURVIVED FIVE YEARS ALREADY. SO THEIR LIFE EXPECTANCY -- DOCTOR STROME CAN ANSWER IT BETTER THAN ME -- THEIR LIFE EXPECTANCY IS A REASONABLE LIFE EXPECTANCY GIVEN THAT THEY HAVE SOME CONDITIONS LIKE FOR EXAMPLE CHRONIC ALCOHOLISM AND SMOKING. REGARDING THE FIRST QUESTION, THE AEV VECTOR WHICH LET'S SAY WOULD BE THE ULTIMATE GOAL, IF THIS STUDY SHOWS A POSITIVE RESULT -- AND WE OF COURSE HAVE TO REPEAT AN ANIMAL MODEL STUDY IN MINI PIGS WITH AN AEV VECTOR, WE HAVE TO REPEAT TOC STUDIES IN RATS WITH AN AEV VECTOR. WE TOOK A VERY CONSERVATIVE APPROACH. >>THE POTENTIAL ADVERSE EFFECTS THAT MIGHT BE DUE TO AN AEV VECTOR WE CAN'T SAY ANYTHING ABOUT RIGHT NOW. >>OTHER ISSUES FOR DISCUSSION. CONCERNS FOR DISCUSSION? I'D LIKE TO OPEN THIS UP TO MEMBERS OF THE PUBLIC. DOCTOR BOROES? >>I HAVE AN ADDITIONAL COMMENT ON THE INFORMED CONSENT DOCUMENT. THERE IS A STATEMENT IN SECONDS 5.9 THAT IS JUST NOT HELPFUL AT ALL. THE RISKS OF THE AND TEE VIRUS ITSELF -- ARE MORE ACCURATELY 37 DESCRIBED AS POSSIBLE RISKS. I THINK ALL RISKS ARE POSSIBLE RISKS. I THINK THAT A RISK IS A POTENTIAL FOR HARM TO OCCUR. SO THAT STATEMENT IS TOTALLY NOT HELPFUL AND I THINK IT SHOULD BE DELETED. THAT'S ALL I HAVE. >>THANK YOU. LET ME TRY THEN. HOPEFULLY I'LL BE MORE SUCCESSFUL THIS TIME THAN IN EARLIER TIMES TODAY TO PRESENT WHAT I'VE WRITTEN. GENERAL. THE PROTOCOL IS INTERESTING IN THAT IT INCLUDES GENE TRANSFER WITH SHORT LIVED GENE EXPRESSION FOR A DISEASE COMPLICATION, DECREASED SALIVARY FLOW INVOLVING IRRADIATION TO HEAD AND NECK TUMORS THAT IS LONG LIVED. EVEN GIVEN THE SHORT-LIVED GENE EXPRESSION AND INCREASE IN SALIVARY FLOW WILL HELP DEFINE THE PHYSIOLOGY OF THE HUMAN SALIVARY DUCT. THE RAC SUGGESTS THAT THE INVESTIGATOR STRATEGIZE A FUTURE STUDY WITH AN ALTERNATIVE VECTOR THAT MAY RESULT IN LONG-LIVED EXPRESSION. WOULD ANY ONE LIKE TO MAKE THAT A STRONGER STATEMENT, A DIFFERENT STATEMENT? >>I WOULD LEAF IT AS IT IS. >>I WOULD LIKE TO SAY THIS WAS JUSTIFIED BY -- INTERESTING THAT THEY WANT TO USE SHORT-TERM GENE TRANSFER. THIS WAS JUSTIFIED BY A FUTURE PLAN TO USE LONG-TERM GENE. >>CAPTIONS ARE TEMPORARILY UNAVAILABLE). >>THE PROTOCOL IS INTERESTING IN THAT IT USED -- THAT IS LONG-LIVED. 38 THIS STRATEGY IS JUSTIFIED BY PLANS FOR A FUTURE STUDY WITH AN ALTERNATIVE VECTOR WHICH MAY RESULT IN LONG-TERM EXPRESSION. OKAY. CLINICAL. >>CAN YOU JUST CHANGE DISEASE EXPRESSION TO DISEASE COMPLICATION TO TREATMENT RELATED COMPLICATION? IT'S ACTUALLY NOT A COMPLICATION OF THE DISEASE. >>YES, THAT'S ABSOLUTELY CORRECT. SORRY. THANK YOU YOACH. CLINICAL. CONCERN REMAINS THAT EITHER INFLAMMATION OF THE OF THE DUCT -- FOLLOWING GENE TRANSFER MAY CONFOUND THE EVENTUAL INTERPRETATION OF SALIVARY FLOW SLASH GENE TRANSFER EFFICACY. IT IS POSSIBLE THAT STENTING THE DUCT IF OBSERVED WILL CREATE A BETTER VALUATION OF FLOW. CONCERN REMAINS -- NO. THE NEXT ONE GOES -- BECAUSE I REPEATED MYSELF. PRECLINICAL. CONCERN REMAINS THAT THERE IS PRECLINICAL DATA IN FEMALE RATS RECEIVING GOP VECTOR AND SUGGESTING INFLAMMATION AT 10 TO 14 WEEKS THAT IS GREATER THAN THAT SEEN IN MALE RATS. FURTHER MORE, FEMALES HAD A GREATER DECREASE IN BODY WEIGHT (NOT A DOSE) AND CONSUMPTION. THE RAC SUGGESTS CLOSE -- IN THESE POTENTIALS IN WOMEN PATIENTS. THE LANGUAGE IN THE DOCUMENT SHOULD BE SIMPLIFIED THROUGHOUT. STATEMENTS REGARDING THE POSSIBILITY OF BENEFIT SHOULD BE REVISED TO STATE QUOTE THERE WILL BE NO BENEFIT END QUOTE REFLECTING THE LIKELY TEMPORARY NATURE OF TRANSGENE EXPRESSION. 39 >>I THINK THAT WAS NO DIRECT BENEFIT. >>THERE WILL BE NO -- AWE -- NO DIRECT BENEFIT. THANK YOU, MS. KWAN. IS THERE ANYTHING ELSE THAT PEOPLE WOULD LIKE TO ADD ADD? ALL RIGHT. IF NOT, MAY I HAVE A MOTION FOR APPROVAL? >>SO MOVED. >>THANK YOU, DR. MYZ -- THANK YOU. AND WE'LL TAKE A VOTE. BEGINNING WITH DOCTOR ROSENBERG? >>EYE. >>MYZ DID. >>I. >>DOCTOR -- >>(READING OF ROLL CALL ALL SAID AYE). >>OKAY. THANK YOU ALL VERY MUCH. ON THAT NOTE WE WILL CLOSE THE DECEMBER 2005 RAC MEETING. I WANT TO THANK EVERY ONE FOR THEIR ACTIVE PARTICIPATION THROUGHOUT THIS 2-DAY SESSION. I WILL CERTAINLY MAKE EVERY EFFORT TO CONVENE A CLINICAL TRIAL DESIGN WORKING GROUP TO ADDRESS SOME OF THE ISSUES WE'VE DISCUSSED DURING THE LAST 2 DAYS OUTSIDE OF THE CONTEXT OF A SPECIFIC TRIAL. BUT RATHER MORE GENERICALLY. GENERICALLY. AND I'M LOOKING FORWARD TO EVERY ONE IN THE GROUP WHO HAS PARTICIPATED SO ACTIVELY IN THE LAST COUPLE OF DAYS ALSO PARTICIPATING IN THE PLANNED WORKING GROUP. THANK YOU ALL.