P-R-O-C-E-E-D-I-N-G-S

1:04 p.m.

DOCTOR FREAS: Let me just introduce myself to everybody. I am Bill Freas, I'm the Executive Secretary for today's advisory committee meeting, and this is the 20^th meeting of the Allergenic Products Advisory Committee.

We are holding this meeting in Conference Rooms A and B, in Building 29B, on the NIH campus. There's a speaker phone here for public participation, and, of course, the public is more than welcome to participate in our advisory committee meetings.

The entire meeting for today will be open to the public, as announced in the Federal Register on March 10, 2004.

At this time, for the people in the room and for the people on the phone lines, I would like to go around and introduce the committee members. They are our Acting Chair, Doctor Wesley Burks, Chief of Pediatric Allergy and Immunology, Duke University Medical Center. Doctor Burks can you say present?

ACTING DIRECTOR BURKS: Present.

DOCTOR FREAS: Doctor Rebecca Gruchalla is next, Associate Professor of Internal Medicine, University of Texas Southwestern Medical School.

DOCTOR GRUCHALLA: Present.

DOCTOR FREAS: Doctor Melvin Berger, Professor of Pediatrics and Pathology, Case Western Reserve School of Medicine.

DOCTOR BERGER: Present.

DOCTOR FREAS: Doctor Harold Nelson, Senior Staff Physician, National Jewish Medical Center.

DOCTOR NELSON: Present.

DOCTOR FREAS: Our non-voting industry representative, Peter Hauck, Executive Director for Scientific Affairs, Allergen Products Manufacturers Association.

MR. HAUCK: Present.

DOCTOR FREAS: Our acting consumer representative, Ms. Dolores Libera, Director of Publications, Allergy and Asthma Network, and Mothers of Asthmatics, Incorporated, Fairfax, Virginia.

MS. LIBERA: Present.

DOCTOR FREAS: And, we will soon be joined by Doctor Lynelle Granady, Associate Physician with ENT and Allergy Associates, New York, New York.

Unfortunately, Doctor Susan McDonald, from the Johns Hopkins University, could not join us today.

Now, should anybody get dropped from this teleconference, you all have the instructions of how to join us, but just to repeat, you dial 1-888-577-8990, enter a pass code of 25660, and push the # sign, and please rejoin us.

Now, as I said, there are about 20 people in the room. I will not introduce all of them, but there are several key FDA staff that I would like to introduce.

Oh, Doctor Granady, did you just join us?

DOCTOR GRANADY: Yes.

DOCTOR FREAS: Well, thank you very much. We have just been joined by Lynelle Granady, Associate Physician with ENT and Allergy Associates, New York, New York. Thanks for joining us. We are just going around now and introducing some of the key FDA people. There are about 25 people in the room now, I would guess, and I won't introduce everybody, but I would like to acknowledge Doctor Richard Walker, Director, Division of Bacterial, Parasitic and Allergenic Products. At the table is the organizer for this meeting here, Doctor Jay Slater, Chief of Laboratory and Immunobiochemistry. And, also at the table is Doctor Ronald Rabin, Senior Staff Fellow, Laboratory of Immunobiochemistry.

Now, we also have a transcriber here, and I must ask all committee members on the telephone lines to please identify yourselves each and every time you speak, because we are trying to transcribe an official record of this meeting, and we want to be able to attribute the comments to the appropriate individual. So, please identify yourself each and every time you speak.

Now, I'd like to read into the public record the Conflict of Interest Statement written for this meeting. "The following announcement addresses the conflict of interest issues associated with this meeting of the Allergenic Products Advisory Committee on April 2, 2004. The Director of the Center for Biologics Evaluation and Research has appointed Ms. Dolores Libera as a temporary voting member for this meeting. Based on the agenda, it has been determined that there are no specific products being approved at this meeting. The committee participants have been screened for their financial interests. To determine if any conflict of interest existed, the Agency reviewed the agenda and all relevant financial interests reported by the meeting participants. No waivers were required under 18 US Code 208. We would like to note for the record that Mr. Peter Hauck is participating in this meeting as a non-voting industry representative, acting on behalf of regulated industry. Mr. Hauck's appointment is not subject to 18 USC Code 208, he is employed by Alk Abello, Incorporated, and thus has financial interests in his employer. Mr. Hauck also serves as the Executive Director of Scientific Affairs, Allergen Products Manufacturers Association. In addition, in the interest of fairness, FDA is disclosing that his employer, Alk Abello, is a manufacturer of allergen extracts. In the event the discussions involve specific products or firms not on the agenda, for which members have a financial interest, members are reminded of the need to exclude themselves from these discussions. Their exclusion will be noted in the public record. With respect to all other meeting participants, we ask in the interest of fairness that you address any current or previous financial involvement with any firm whose products you wish to comment upon."

That ends the reading of the Conflict of Interest Statement.

Doctor Burks, I turn the meeting over to you.

ACTING DIRECTOR BURKS: I want to thank everybody for the conference call, and I especially thank Bill for allowing us to do the conference call, rather than having to come to Washington, and thank Jay for helping arrange that.

What we'll do for the next hour and a half or so is to listen to several presentations by Jay and Ron Rabin, and it's on the agenda that you have, and I think each of you have the slides. If you have questions as you are going along, if you'd just identify yourself and ask them, just try to stick to the topic that Jay is addressing at that time, and then he'll leave time for questions at the end of each section to see if we need to say anything else.

So, with that, Jay, we are ready to get started.

DOCTOR SLATER: Terrific, thank you very much, Wes, and thank you all for participating. You know, as you well know in the past this committee has been responsible for valuable contributions, and we look forward to this meeting every year.

Since I'm not in the room with you, I can't point to the slides. We are going to try, as we go along, to identify what slide we are on. All of the slides that we're using today are numbered, and we're going to identify them as we go along every once in a while by number, to make sure we are all literally on the same page.

We are now in the first ? these presentations are also numbered in order. There are five presentation files that you were sent, and it should be pretty easy to decide where we are but I will identify them as we go along ? we are now in the first presentation, the Lab Overview, and after the title slide you can go on to the second slide, please.

This is, basically, what we are going to cover today in our meeting. This is slide two out of ten. We'll start with the Lab Overview, in which we cover some staffing issues, lot release, reference, maintenance activities of our laboratory. That will be fairly brief. We will then get into the meat of the presentation, which involves a review by me and Ron broadly speaking of the research activities in the lab. Then I will focus on two specific areas of both research and regulatory interests. One is our activities in cockroach allergen standardization, and the other is our effort to develop novel methods of determining the potency of allergenic extract, in particular, our very recent and early work on using antibody micro arrays for this purpose.

Next slide, please.

So, quickly, we are going to go through the Lab Overview section, covering staffing, lot release and reference maintenance activities.

Let's go to slide four.

The two principal investigators in our laboratory are myself, I've been here for five years, and Ronald Rabin, who has been with us now for three years. We have three post doctoral fellows, Bo Chi, Jinsong Chang, Nicolette deVore.

Next slide, please.

Those of you that have been here for meetings know that I would normally ask people to stand up, but that would be a little silly under these circumstances, but they are all here and listening to the presentations.

Our research technicians, on slide five, Al Gam, Mona Febus, Marc Alston, Cherry Valeriod and Katia Dobrovolskaia.

Next slide, slide six. This is one of my favorite slides. This is a slide in which I give you an idea of where we are and where we have been, in terms of our staffing. What I include in this slide is only our research technicians, because those are the staff that really are in the forefront of our intersection with the allergen products manufacturers.

And, as you can see, after a couple of years in which our staffing fluctuated up and down we've really been in a very stable pattern for the last three and a half years, with about five research technicians. I think this has really helped us a lot, this stability, in terms of our ability to provide service and to effectively regulate the allergenic products.

Next slide, please.

The routine regulatory activities that our research technicians are involved in include lot release, reference distribution, reference maintenance activities. As you know, for the standardized allergenic extracts we maintain the U.S. Standards of Reference that are used both by us and by all of our manufacturers for effective measurement of the potency of their extracts. And so, we devote quite a lot of effort and expense into maintaining and distributing these references.

Next slide, please. We are now on slide eight.

In the past year, in calendar year 2003, we received 450 protocols which we reviewed prior to the release of Standardized Allergenic Extracts. Also, in the same calendar year we distributed 1,423 vials of reference standards in 96 shipments sent to the manufacturers.

On the next slide, slide nine, we give a little context to some of these numbers. You see here the lot release protocols that were submitted by year for the past five years, and what you can see is that 450 is more or less around the same mean where we have been for the last several years, a little bit higher than last year, but definitely in the same range.

The next slide, slide ten, we look at our reference distribution activities. The number of shipments, the number of vials, is down somewhat. It will probably be a couple of years before we know whether this is really a trend. If it is, then I'll be very grateful to our manufacturers. One of the things that we've been requesting for quite a while is that our manufacturers use the reference standards economically. But, this is a trend, and they've been heeding our request, and for that we can all be very thankful.

That's the end of the first presentation.

I'm happy to entertain any questions if there are any about that.

ACTING DIRECTOR BURKS: Does anyone have questions for Jay for this part?

Okay, you can go on to the next part, Jay.

DOCTOR SLATER: Thanks very much.

Okay. We are now in presentation number two, which is entitled, "Research Update." We can go now to the second slide.

The active research projects in the lab are detailed on this slide. Among the projects that I'm doing, we are investing a significant effort in our Cockroach Allergen Standardization Program, which I'll be talking to you about some more later.

Related to this, but not absolutely essential, but something that we consider to be an important contributor to this effort, is our effort to generate cockroach specific IgE and IgG combinatorial libraries. We've really been focusing on the IgG work recently, with an aim towards micro array analysis of allergenic extracts. And again, this work is very preliminary, but it's exciting nonetheless, and I'll be going into that in some more detail.

In addition, you are all familiar with some of the work that we've doing on endotoxins in allergen vaccines. We have some additional data to discuss with you at this ? in fact, in this presentation.

Ron Rabin has two major projects, the MDR proteins and their involvement in T cell activation, and RSV responses in human tonsilar tissue, and he'll be talking about the first of these projects in just a few minutes.

Next slide, please.

These are the publications that have come out of the lab this year. You can see them here. We published some of our first work with the endotoxin content of standardized allergen vaccines. I was also invited to co-author an article in Middleton's Textbook with Dick Lockey and Bob Esch. Ron published a paper in the Journal of Immunology on his work with the CXCR3 induction and CD4 T cell differentiation, and in addition he has a manuscript in preparation about his work which he'll be talking about a little bit more in a few minutes.

Next slide.

At this year's Academy of Allergy Meeting, this Laboratory contributed six abstracts, which were all presented at the meeting, and they are detailed on slide four.

Next slide, please.

Ron Rabin was invited to give two outside presentations, one at the SMI Conference in London in February, 2004, on the relationship between viral respiratory infections and asthma, and another at the most recent Academy of Allergy Meeting in March, on the FDA Food, Drug and Cosmetics Act as it applies to research studies.

Next slide, please.

And, I was invited to give a talk at the State of the Art Analytical Methods for The Characterization of Biological Products Meeting, in June, 2003, in which I talked about allergenic extracts, and I gave four presentations in workshops and courses at the most recent Academy meeting.

Next slide, please.

Ron and I both maintain outside collaborations. I've been collaborating with Larry Arlian, at Wright State University in Dayton, Ohio, as well as with Doctor Patrick Murray, who is the Chief of the Microbiology Service here at the NIH Clinical Center. Ron has active collaborations going on with Peter Collins at NIAID, as well as with Mario Roederer at the Vaccine Research Center, also at NIAID.

Next slide, please.

So, I'd like to spend just a few minutes talking about the endotoxin work, which I first presented to this committee about one year ago, and just to quickly summarize where we left it last year, we were embarked on an activity in which we were, basically, trying to update some very old work that had been done in this laboratory in collaboration with NIH back in the `70s, in which it was first observed that allergen vaccines, especially dust mite vaccine, contained a measurable amount of endotoxin.

Our work focused exclusively on standardized allergen vaccines, in which we found what's really summarized here on slide number eight, and that is that in general, although we were able to detect endotoxin in virtually all the allergen extracts that we examined, we found that the cat and mite extracts had more than the pollen extracts, that the cat pelt had more than cat hair, and what was most striking to us, and what really led us to the rest of what we're going to be presenting today, was our observation that the D. farinae extracts contained significantly more endotoxin than the D. pteronyssinus extracts.

As I noted last year, it's not clear that any of these endotoxin amounts are physiologically significant, but, nonetheless, we felt that we needed to examine this further to characterize the nature of this endotoxin content.

And, we wondered about several different possibilities. We wondered about the degree of bioburden that might be present. We wondered about whether there might be an endogenous heat-stable and debinding activator in D. farinae, and we wondered whether there was actually endogenous endotoxin neutralizing protein in D. pteronyssinus.

Just to very quickly go through what else we found, next slide please. The next slide is a graphical representation of the corrected endotoxin content of the different classes of vaccines. Note that this is on a log scale. The significant differences are shown, and this, basically, replicates what I had in the previous slide.

Next slide, please.

So, our plan was to investigate the differences between D. farinae and D. pteronyssinus and to attempt to identify some of these organisms.

Next slide, please. We are now on slide 11.

Our first approach, which consumed most of the year, was our attempt to actually culture organisms out of these different mite preparations. We were fortunate enough to be able to obtain fresh mite and egg materials from three different sources. These mites were all washed, free of media, using two different methods. One was sieving, in which the mites were caught on a fine mesh sieve, and the growth medium was washed off with sterile water. Another method we used was with sucrose gradients, and both of these were quite effective, at least visually, at removing all traces of contaminating material from the mites or the eggs. And then, we submitted the material for culture to Pat Murray's lab over at NIH.

Next slide, please.

This slide really summarizes several months' worth of culture work, and the bottom line is, we were able to culture organisms, both gram positive and gram negative, out of both of these sources, and there was no really big difference between the two, either quantitatively or qualitatively.

You'll note that we obtained E. cloacae, R. pickitii and acinetobacter from both species of dust mites. We also have some unidentified gram negative rods, but, basically, our culturing techniques really did not show us any large differences between the two species of mites.

Next slide, please.

Possible problems with this approach include the fact that the culture data were really non-quantitative, and that, in fact, the bioburdens between the two species might be very different. We are actually working on quantifying this now.

The culture techniques, we should note, of course, have been optimized for human pathogens, and it's not likely that what we are dealing with in these dust mites are, in fact, human pathogens, although that's a possibility.

The organisms may be in privileged sites, and the optimal conditions for extraction of the organisms may be uncertain. And, in addition, endosymbiotic organisms are notoriously difficult to culture.

So, we tried a different approach, even while we are continuing with the culturing at the same time.

Next slide, please.

Approach number two has been used by other investigators to identify endosymbiotic organisms, and that's actually to look for characteristic DNA of the bacteria from DAN that's extracted from the mites. In this approach, we extract genomic DNA from fresh washed mite material, and then amplify with 16S ribosomal RNA primers. As you know, 16 S ribosomal RNA is specific for bacteria, and the sequencing has been well identified. In fact, this approach of amplifying 16S ribosomal RNA is a well-established technique to try to track bacteria.

We identified individual clones with restriction fragment analyses. We sequenced and then we attempt ? the idea was to attempt to identify predominant organisms.

Slide 15, next slide, please.

What you can see here is just an acerose gel that shows that we were, in fact, successful at extracting what appeared to be reasonably good quality DNA from both species of mites. This was using standard techniques.

Next slide, please.

These are the 16S ribosomal primers that we used. The sequences were based on an article from 1991 by Weisburg in the Journal of Bacteriology, and these primers have, in fact, been used in multiple experiments by many investigators since that time. It's the upper case bases that are specific for the 16S RNA, the lower case segments of the primers are tails that include, as you can see, specific restriction enzyme sites for easy insertion and characterization.

Next slide, please.

On the next slide you can see that we were able to obtain 1.6 KB TCR fragments of DNA using this amplification method from both D. pteronyssinus and D. farinae. Now, you'll note that we used two different primer pairs. One primer pair, primer pair No. 1, really is relatively non-specific, and would be expected to generate results from most eubacteria, and primer pair No. 2 would allegedly be more specific for enterics.

But, you can see that we were able to obtain fragments from both organisms. Qualitatively, there appears to be somewhat more from D. farinae, but as you know without good internal controls it's impossible to quantify TCR results.

Next slide, please.

We then did a substantial amount of DNA sequencing, and these are the sequences that we recovered. Now, the fact is that from both organisms of dust mites the most predominant recovery is the first organisms ? the first two organisms that are indicated on this slide, Bartonella or Rochalimaea species, specifically, Bartonella henselae and Bartonella quintana. Just to refresh your memories, Bartonella organisms are responsible ? are the pathogens in cat scratch fever and in trench fever, which we don't normally see very much nowadays, although cat scratch fever we certainly see. Bartonella is also associated with infections in immune compromised individuals. There has been a series of reports of it causing endocarditis in immune compromised individuals, such as HIV positive patients and homeless individuals. It's also responsible for bacillary angiomatous in HIV positive patients.

Needless to say, if there were Bartonella in our dust mites they would not survive into the final extract. There's no evidence whatever of live Bartonella in our extracts at all. But, there is evidence, based on this very preliminary study, that Bartonella species may be in this endosymbiotic with some of our dust mite species.

Next slide, please.

Just a quick review, Bartonella endotoxin has, in fact, been fairly well described, both functionally and structurally. It's of interest from a scientific point of view, because it has a high degree of LAL reactivity, but in both human cells and in rat cells it has minimal inflammatory responses, and there's actually minimal activation of ? like receptors 2 and 4, and the two publications very recent are indicated in the bottom of this slide.

Next slide, please.

Obviously, this is a very early finding, and we are not ready yet to publish it in a peer review journal, because we really need to verify the source of the endotoxin. We need to do high-fidelity PCR and verify some of this sequencing data. We need to get some additional mite sources, and we need to attempt to profile the LPS structurally, which we actually can do with colleagues here at CBER.

We are also in the process of putting together internal standards so that we can quantify the DNA in our source materials. Obviously, if there's just as much signal from D. farinae and D. pteronyssinus, this may be of interest, but it certainly does not explain the finding of much more LAL reactive endotoxin in D. farinae extracts.

And, we are going to also need to look physiologically at the effects of this endotoxin on immune responses. These are all studies that are in progress.

That's the end of my presentation. We are going to go on next to Ron Rabin's research presentation, but I'd like to give you an opportunity to ask questions before we do that.

DOCTOR GRANADY: This is Lynelle Granady.

DOCTOR SLATER: Hi, Lynelle.

DOCTOR GRANADY: Hi. This is actually really very, very interesting. I have a quick question about the initial cultures, if you were able to culture the bacteria from the extracts or culture small amounts of bacteria from the extracts?

DOCTOR SLATER: No, no, I'm sorry, let me stop you. You can go on with the question afterwards, but we cultured nothing at all from the extracts. They are sterile. We cultured this from mites, live mites.

DOCTOR GRANADY: Oh, okay, from the live mites.

DOCTOR SLATER: Yes.

DOCTOR GRANADY: Now, is it possible to ? how do you determine which ? I don't know how to exactly ask the question ? but how ? is it possible to actually measure endotoxin from that same ? from the live mites, or not really?

DOCTOR SLATER: Yes, we've actually done that. Well, I mean, measuring the ? you have to sort of mulch up the mites to measure the endotoxins.

DOCTOR GRANADY: Right.

DOCTOR SLATER: You kill them in the process of doing it.

DOCTOR GRANADY: Right.

DOCTOR SLATER: But, we've done that. In our studies that we presented last year, we were able to show that endotoxin was not only present in the extract, but also in the source material, which is the live mites. So, it's present in both.

Are you asking whether it's possible to identify which organism the endotoxin comes from?

DOCTOR GRANADY: Yes.

DOCTOR SLATER: Yes, the answer is yes. We haven't really successfully done that yet, but there are a number of papers in which endotoxins can be profiled, both crudely on SDS-PAGE, and also much more sophisticated using HPLC and gas chromatography. We haven't done that yet, although we had intended to do that, we just sort of got sidetracked a little bit, but that's definitely something that we are interested in.

The problem, Lynelle, is that no one has really ever attempted to do this on a mixed culture, and that would be a little bit hard to sort out. The profiling activities, in general, are on pure cultures or vaccines, and my guess is that ? we are certainly planning on doing this, but my guess is that the data are going to be a little bit messy, just because they are mixed.

DOCTOR GRANADY: I see.

Thank you.

DOCTOR SLATER: Thank you.

ACTING DIRECTOR BURKS: Anybody else have questions for Jay?

All right, go ahead.

DOCTOR RABIN: Hi, this is Ron Rabin, and what I'm going to discuss with you is a project, and show you some data of a manuscript that's pretty much written. We are waiting for one experiment and then, you know, the final touches and getting it out.

If you'll to slide number two then. So, my interest in the multi-drug resistant proteins came from a serendipitous observation that I made while I was still a Fellow in Josh Farber's lab. And, as some of you may know, I have a reasonable amount of experience in flow cytometry, and spent some time in that laboratory looking at calcium flux in response to chemokines, and as a relationship to chemokine receptor expression on lymphocytes.

And, as sort of a side project I began to look at responses in ? in patients with Wegener's granulomatosis with a friend of mine. We were, basically, just playing, and in the process of looking at this I noticed that the patient cells had lost a lot ? or had a lot less of the calcium probe, which is ENDO 1, in their cells than cells from the healthy controls that we were using in the experiment. This was consistent from experiment to experiment, and so I wondered, I thought about that and wondered whether or not because these patients sort of had ? were in a state of inflammation, or activation, in a very general term, whether or not that affects this probe concentration, and the probe concentration then would reflect gene expression of some sort of a probe transporter, which might then modulate activation and give incite into a pharmacologic target. So, that was sort of the fantasy.

And, the cartoon that I'm showing you is drawing what I basically observed. So, when one does calcium studies on a flow cytometer with ENDO 1 you are looking at a ratio, which is essentially the slope of fluorescent's emission into violet on the Y axis or blue on the X axis, and you could see at normal the cells were kind of high up on the X/Y plot there, and the Wegener's granulomatosis patients that slope was down in the corner. But, the slope ? the lines were in different positions, but the slopes were the same, and it's the slope that reflects the calcium. So, it wasn't an issue of calcium, it was an issue of probe concentration, and that's really the bottom line.

And so, if you go to slide number three, that brought me to the MDR family, because the MDR family are proteins that transport substances across cellular membranes, against a concentration gradient, in an energy-dependent manner.

They are in a larger group of what are called ABC proteins, ATP Binding Cassette proteins, that contain distinctive nucleotide binding domains, and their genes are highly conserved across species. The first member that was identified was MDR1, which I'm sure many of you have also heard of identified as P-glycoprotein. And, its substrates are large hydrophobic cations, including such molecules as Doxorubicin and Ritonavir, and even the calcium channel blocker Verapamil.

A little bit later, the MDR-associated Resistant Protein-1 was identified, and now there are nine proteins that carry that prefix of MRP, and it has some homology to MDR, but it's actually more closely related to the Cystic Fibrosis transport protein.

MDR ? MRP1 transports organic anions and it also transports glutathione and glutathione conjugates, and it transports ? in fact, it is the mechanism by which cells export LTC₄, so it does have a physiologic function, and it is the transporter of many of these florescent molecules that we use as probes in cellular biology, because the molecules themselves are organic anions. So, my attention was then turned to MRP1, slide four.

Just to give you a little bit of context of these MDR family proteins, or the MRPs, you can see there's an identity amongst them or between them, the low, and they are widely distributed along the human genome. Many of them do transport conjugated glutathione, and some of them have known physiologic substrates and some of them don't, and there's various new level of distribution of expression, as you can see.

Slide five, please.

So, the first thing that we did was just to look at the basic ? if the basic hypothesis had any truth to it, then we would see two things. We might see a difference in expression of MRP1 in mammary cells versus naive T cells, and we should see a difference with activation. And, this 35 cycle RT-PCR on slide five demonstrates that that, in fact, is the case. So, if you look at the set of ? the area MRP1 there, you'll notice that the band is much brighter on CD4 memory versus naive, CD8 memory versus naive is also a little bit brighter, it seems to be expressed to some degree, and then K cells, B cells, and a little bit in monocytes.

To further drive home the preferential expression, these cells were ? the memory and naive cells were sorted by flow cytometry, using two markers in addition to CD4 and CD8, but we know for a fact that that isn't ? you don't get pure naive cells, and really one way to get absolutely pure naive cells is to use cord CD4 T cells. And there on the far right lanes you can see that there's no expression of MRP1 in cord CD4 T cells, and that after three days with anti-CD3 and anti-CD28 there is expressions regulated.

Now, I'm showing you a number of the members of the family for two reasons, one to call attention that this isn't just some sort of general thing, non-specific event, because MRP3, for example, is not expressed in any context, and MRP54 doesn't really appear to be affected by memory subsets in any event, but then there are some molecules that seem to have this same level or the same pattern of expression, such as MRP4 and MDR1.

Next slide, please.

So, we started ? we set about using a small molecule inhibitor of MRP1, that is well known in the literature, called MK-571. MK-571, as many of you might know, is sort of the granddaddy of singular or monteleukast, and so it is a leukotron, it is a ? LTR1 antagonist at very low concentrations, but I'm sure related to the fact that MRP1 is the exporter of LTC4 at higher concentrations MK-571 does block this pump. And so, we first just looked at ? we used the molecule TSST, the superantigen, to stimulate our T cells for, I think, all of the studies that I'm showing you here, because in certain times we thought we might want to look at those responding cells, and since TSST-1 is fairly specific for V beta 2, we can follow the responding cells if we wish.

And so, on the left these cells have been stimulated with the superantigen overnight, and you can see that they look like cells, they look as cells generally look when they've been stimulated overnight with some sort of superantigen, they tend to cluster and become aggregates. And, on your right, with the MK-571, they are just in a homogenous suspension.

They are mostly alive by the way.

Next slide.

So, slide number seven then shows some ? a little bit more specific characterizations of the inhibition here, and so these cells now were stimulated and we looked at them four hours after stimulation with TSST-1 in the presence of absence of this inhibitor MK-571, and on the far left you can see on the X axis that there's a subset of cells that express CD69 after activation and then a subset of those are staining positive for interferon gamma. In the presence of 50 micromolar, we've cut that down ? the MK-571, we've lost a lot of expression of the interferon gamma and CD69 both, and it's practically all ablated at 125 micromolar.

Next experiment.

To look at the cytokine profiles a little bit more closely, we did experiments such as demonstrated on slide number eight here, which shows interferon gamma on the X axis and IL-4 on the Y axis, and you'll see a very clear dose-dependent decrease in interferon gamma, as the dose of MK-571 goes up. What you don't see is that IL-4, or what you also see, I guess, is that IL-4 is similarly ? is not affected, okay, and that has struck us, you know, as very odd.

Now, we've tried to make sure that this is, in fact, the case, because, as you know, there's a lot of ? there's a lot more ? you know, the numbers are better for gamma than 4, and so we wanted to be sure we weren't playing with a number game, but, in fact, this does look like the case. It does look like at least in the context of these experiments MK-571 inhibiting MRP1 doesn't seem to affect IL-4. And, at the time I was a bit baffled by that, and I have a number of thoughts about that that I can share with you towards the end.

Next slide.

The specificity ? but, at any rate, to look at the gamma at least, this is ? we did a number of experiments now, so these are experiments similar to and including the one that I showed you on the previous slide, showing the dose response, and on the upper half of the panel there is the cumulative normalized response to interferon gamma of all these, and you can see that this is a statistically significant, dose-dependent decrease, and that this dose-dependent decrease in cellular expression of interferon gamma protein is at least ? is reflected by a decrease in gene expression, as shown by the TaqMan assay below.

Next slide, please.

We looked at a bunch of other cytokines using the BD biosciences cytokine bead assay, and so this is now secreted into the supernatant, and the interferon gamma, as you can see, is no surprise, it's reflected by supernatant concentration, as is TNF and IL-10, and to some degree IL-2. What's kind of interesting about IL-2, and it may give us some incite into the mechanism that this is working, is that you can see that the curves are a bit different, and it appears that IL-2 is not as easily affected by the MRP1 inhibition. But, it is affected, nonetheless.

And, this last point at 125 micromolar was excluded from the regression line, because a queue test demonstrated that it was clearly different than the previous five points.

Next slide, please.

Well, we wanted ? I was curious about whether or not this inhibition was cell specific, was T cell specific or not, and so one of the things that I did was, we looked at monocyte-derived macrophages, and we stimulated them with both SAC versus TNF in blue, SAC is red, and you could see that the MK-571 decreased TNF in response to the LPS, but not so much, and really not at all, in fact, with regard to the SAC.

So, while the effect occurs across a number of cell lines, it's clearly regulating something specific, in that certain cytokines are affected, certain cytokines are not affected, and the response may even be different depending upon signaling pathways.

And, the next slide just amplifies that point even more, because in the same supernatants we looked for IL-6, and we saw really no effect whatsoever. And so, this, you know, was comforting because, of course, the MDR family, people do think of them in terms of proteins that are simply there for self-survival, and that, you know, toxins build up, and are you just killing the cells, and this selectivity is one more demonstration that we're not.

And, we have done, I would state that I'm not showing you but we've done a number of cells looking at indicators of cell death ? blue, ? orange ethidium bromide. We've done stains for mitochondrial potential change for apoptosis, and we only ? we see some small changes above 75 micromolar, but not huge, and it's clearly not having a major role in what we are seeing here.

But, the paper that's being submitted is primarily descriptive, but we did want to look into mechanism a little bit, and one of the things that we looked at was to go back to the calcium flux, and so this is a calcium flux on T cells that have been stained with a bitintillated CD-3, and then cross linked with abadan, and on your left is just to demonstrate they are just overlapping histograms of CD-3 expression on peripheral blood lymphocytes to demonstrate that the MK-571 is not decreasing CD-3 expression at all. And, in the first calcium flux panel there, the multi-colored panel, you see time on the X axis and the ratio of fluorescence on the Y axis, and that's a pretty standard calcium flux response that one sees by flow cytometry after cross linking CD-3, and that's pretty much ablated after treatment with 25 micromolar of MK-571.

In addition, next slide, please, so slide number 14 also shows that the calcium flux in response to CXCL12, also known as SDF-1, is also ablated, and so that's kind of interesting. And then, on the histograms on the left, once again demonstrate that there's really no difference in expression of the receptor. So, this is an effect of signaling.

Next slide, please.

Well, if one is thinking about a pharmacologic target, and one is worried about whether or not you are killing the cells, you don't necessarily want to make an individual permanently inergic, you'd like to know whether or not this sort of a thing is reversible. And so, we did this kind of an experiment here, where we incubated the cells with MK-571 overnight, and then we washed out the MK-571 and stimulated them, you know, on day zero, day one, day two, day three, and the important thing is really demonstrated on the histogram on the top the line plot below being derived from the data above, simply normalized from the data on the top, which just shows that if you wash up the MK-571 compared to cells that had not been ever exposed to the MK-571 you regain most of your responsiveness with regard to interferon gamma expression the CD4 T cells, so it's reversible, and that's a good thing.

Next slide, please.

And then, just sort of coincident somewhat with the issue of the signaling studies that I showed you, we were curious as to how long before the MK simulation the MK-571 needed to be added. For the studies that I showed you, it was 30 minutes, it was 30 to 60 minutes, though here we demonstrate that, in fact, if we add it really with, or even somewhat slightly after the TSST-1 we still get inhibition, and it's only if we delay its addition 60 to 120 minutes after the superantigen does it lose its effect.

Next slide, please.

Well, one of the questions was, you know, the fact is, is that we are allowed this concern with allergens, allergy and allergic disease, and so while this is quite interesting it was a little bit disturbing that we didn't really see any response in the IL-4 side of the picture. And, while I was thinking about this, a paper was published in PNAS just last year that demonstrates that MRP4 transports the other arachidonic acid products, such as PGA₂ and PGE₁, and all those that you see listed, and as I'm sure that many of you know the prostaglanens have been shown to have a role in the Th1, Th2 paradigm.

Next slide, please.

And so, just to look at MRP1 versus MRP4 expression Th1 and Th2 cell lines, I did these TaqMan experiments and demonstrated that while MRP1 seems to be expressed fairly equally well in both, the MRP4 is somewhat preferentially expressed, this is gene expression now, in the Th2 versus the Th1 cell line.

Next slide, please.

And then finally, just to try to integrate my two projects to some degree, because my other fellow, Bo Chi, is working on, we are very interested in this issue of viral infections and their effect on asthma pathogenesis and episodes of wheezing, I was curious about whether or not RSV might increase MRP4 in dendritic cells, so we just did this one experiment which was somewhat promising, but needs to be repeated. It demonstrates that this, yes, does seem to be the case.

Next slide, please.

And so, what I've shown you is that MRP1 blockade abrogates a Type 1 T cell function, and blocks at least two signaling responses that we've looked at. MRP1 may have a very analogous function, MRP4 may regulate Th2 T cell responses analogous to this, and the ultimate objective of all this is not to suggest that a blockade of these pumps might be a good way to provide therapeutic immunomodulation, but, in fact, what we really want to do is, we want to define the mechanisms by which this works. We think that by the mechanisms by which this will work will provide us some drug targets for immunomodulation.

And, I think that's it.

Okay, so again, I want to acknowledge Jay Slater, of course, he supports this work, and Marc Alston has really done a lot of work, he's one of the biologists in the lab, Jinsong Zhang is the Fellow who is working on this particular project, and although I didn't show you any of Bo Chi's work, she's been very helpful in getting this project going.

So, thanks for your time, and I'll take any questions.

ACTING DIRECTOR BURKS: Does anybody have questions for Ron? Ron, that was good.

DOCTOR GRUCHALLA: This is Becky Gruchalla. I guess the only question I have is, and I think you've just shown all the data, it's very, very interesting, so it looks like it's specific, I guess I shouldn't use the word specific, for interferon, it can down regulate or decrease interferon production, does not touch IL-4, doesn't look like it touched IL-6 either, correct? Were there any other cytokines that you looked at, again, you've probably shown all the data.

DOCTOR RABIN: Yes, those were the only ones that I looked at. Do you have any ? well, we tried to look at IL-13, but, you know, it's really tough to get IL-13 responses in this kind of system.

But, you know, obviously, we are always keeping in mind that, at least to my way of thinking, that IL-13 is sort of the pathogenic, you know, Th2 cytokine. So, I always sort of keep it in mind.

Any other questions?

DOCTOR GRUCHALLA: That's all I have.

ACTING DIRECTOR BURKS: Okay, thank you, Ron.

DOCTOR SLATER: Okay. We're going to go now to presentation number four. This is the one entitled, "Cockroach Allergen Standardization."

Go to slide number two. I can't resist showing this slide. I supposed after a couple years I'm going to have to stop.

UNIDENTIFIED SPEAKER: Yes, you are.

DOCTOR SLATER: Okay, thank you. Well, all right. I have to reach a certain number of people to whom I've given nightmares before I stop this, but probably at the point I give this up we'll be able to animate the slides.

UNIDENTIFIED SPEAKER: Oh, no.

DOCTOR SLATER: Okay, let's go to ? Bill, let's go to slide number three quickly. Okay.

What we are going to talk about today is our progress with cockroach allergen standardization. The actual progress that I have to report has to do with the clinical studies that are actually underway.

We'll talk then briefly at the end of this about our efforts to develop an appropriate surrogate potency measure. I'm going to introduce the topic at the end of this presentation, which is presentation number four, and then in presentation number five will actually discuss some of the issues about appropriate surrogate measures in greater depth, and try to explain to you why, at least for the meantime, we are actually looking at this microarray technology as what we hope to be a new technique that will be very, very helpful.

Next slide, please. We are now on slide number four.

I know I'm reviewing things that this committee knows well, but let's just go through it quickly.

Cockroach allergy is important because roaches are ubiquitous. They are difficult to control, and cockroach allergen exposure has been associated in a number of studies with asthma, especially inner-city asthma.

We presented this work to this committee in February 2000, and outlined an approach to standardizing new allergen extracts, and at that time the standardization of cockroach allergen extracts, among others, was supported by the advisory committee on that date.

Next slide, please.

Cockroach allergen standardization is important to the patient, to the physician and scientist, and to the FDA. It's important to the patient because of the ability to make more accurate diagnoses, and, hopefully, safer and more effective allergen immunotherapy. To the physician and scientist, it's critical because without standardization you have a problem, in that the science is hampered by the fact that you can't measure what you've got with scientific experiments that are needed, our pathophysiology experiments, epidemiologic work, and environmental control. And, it's important to the FDA because our mandate is to make for safer and more effective products, and clearly this will lead to safer and more effective product.

Next slide, please.

Our initial studies, which we presented to this committee before, were in the laboratory, and those actually have been discussed before. We were comparing allergen content to different lots of U.S. manufactured materials.

Next slide, please.

That was reported in Clinical and Experimental Allergy in 2002, and on the next slide, which is slide number eight, our conclusions were that commercially-available cockroach allergen extracts in the U.S., vary widely in their protein content, in their Bla g 2 content, in the SDS-PAGE banding pattern, and in overall allergenicity, as best measures as we had at that time.

And, when we compared them to some candidate reference extracts which we had, they were less potent, uniformly less potent, and they contained less bla g 1.

Of note is that even among these highly-variable extracts, the amount of bla g 1 was actually fairly constant, although it was low. Our conclusion was that cockroach allergen vaccines should be standardized and based on both the public health implications and what we found in our laboratory studies, we wanted to go ahead and proceed with this as our next target.

Next slide, please.

So, the next stage is to do some clinical testing. And, in particular, based on models that we've used before, we wish to proceed with skin testing to help establish the biological unitage of the products and to try to establish ideal dosing ranges.

Next slide, please.

Our goal was to obtain valid clinical data on the biological potency of three commercial German cockroach allergen extracts, and to develop surrogate potency testing.

Next slide, please. Next slide. Let's go to slide number 12.

The clinical method that we used was the ideal method that was developed in the 1970s, both here at CBER and at Hopkins. You are all well familiar with this technique, which is based on serial dilutions, intradermal testing, the measurement of the erythema response, the erythema responses that are measured are plotted against the negative log of the serial dilutions, and from that a D50, which is the dilution at which a 50 millimeter erythema response occurs, is measured. The D50 is directly translatable by simple arithmetic into the bioequivalent allergy units, which is the standard that's used for grass pollen extracts and for cat extracts today.

Next slide, please.

The effort to do an appropriate, and well-designed, and well-controlled, and geographically-diverse ideal study for cockroach was a little bit daunting, but, fortunately, we were able to approach the NIAID Inner City Asthma Consortium, which as you can see in slide 13 was established in Fiscal Year 2002, to explore and evaluate promising new strategies for the treatment of asthma among minority children residing in the inner city.

Next slide, please.

The steering committee of the Inner City Asthma Consortium was enthusiastic in supporting the idea of using their resources to perform this study, and we proceeded on that basis.

You'll notice that Doctor Gruchalla, who is on this Advisory Committee, is also on the Consortium Steering Committee, and a number of individuals on that committee were highly supportive of our efforts in this regard, and we are very thankful to them, to the committee, and to the Consortium, for supporting this work.

Next slide, please.

So, out of the ten ICAC or Clinical Research Centers, four were designated to do this study. The centers in Baltimore, Chicago, Denver and Washington, D.C., are all on board to do the study.

Next slide, please.

Our recruitment, we are recruiting adult individuals with a history of allergic disease, such as allergic rhinitis, related to exposure of cockroaches, although with cockroach exposure this is certainly difficult to discern clinically, we also have a standard that on screening these individuals have a puncture sum of erythema response to the allergen concentrate greater than or equal to 30 millimeters in size.

Next slide, please.

Several exclusion criteria were indicated for both practical, scientific and safety reasons. We were not interested in recruiting individuals whose asthma was severe enough to require the use of systemic steroids in the past 12 months. We certainly did not want individuals whose flows were below 75 percent of predicted at the time of testing. For practical reasons, since we are measuring erythema, we needed individuals whose skin coloring or condition would not preclude the measurement of erythema responses, although that's a fairly uncommon exclusion criteria, even in the inner city. We've not had any problems with that.

Dermographism, dermographic subjects were excluded. Individuals who had received cockroach immunotherapy in the past were excluded, and individuals with current use of antihistamines, tricyclic antidepressants, MAO inhibitors or beta-blockers are excluded as well.

Next slide, please.

We then went about selecting three commercially-available German cockroach extracts. These are all FDA approved materials. We decided again, for practical reasons, to get 50 percent glycerinated materials, and we wanted three different manufacturers.

Next slide, please.

What you see in slide number 19 are some of our initial determinations on those three extracts that are designated A, B and C. In addition, at the bottom you have measurements on E2Cg. This is not so much a candidate reference material, but a highly-concentrated dialyzed and purified material that the FDA purchased some years ago and has been using for a number of studies. The tests that were done on it were bla g 1, bla g 2, and bla g 5 content. We actually did these tests with our own direct ELISA that we designed here in the laboratory, but the data that I'm showing you are from the two-site ELISA that's done by Indoor Biotech. These were concordant with our results, but I'm only reporting their results here.

In addition, we determined the relative potency of these extracts, again, compared to E2Cg, which may or may not be a fair comparator, by three different methods. In the first method, we used a pooled monospecific rabbit sera, in other words, we had injected rabbits and hyperimmunized them with bla g 1, bla g 2, bla g 4 and bla g 5, we were able to get good immune responses in all of the rabbits, and in RP1 we took a pooled sample of sera from those four animal sources.

In RP2, again, the same competition ELISA design, but this time we used serum from a single rabbit that was injected with German cockroach allergen extracts, and in RP3 we did a relative potency using a human serum pool that was created some years ago here against cockroach allergens. And, what you can see is that the bla g 1 content, again, of the three extracts is relatively constant, but again, much less than an E2Cg, again, I'm not sure how significant that is. We don't really know what content we are looking for, in terms of either diagnosis or immunotherapy of any of these allergens.

The bla g 2 content was somewhat more variable, but present in all three, again, much less than in E2Cg. Interestingly, bla g 5 was almost completely absent from extracts A and C, but was present in extract B in just about the same amount as in E2Cg. The potencies seemed to suggest in all three methods that extract B was the most potent, and that extract C and A were less potent. Again, I want to be cautious about not over interpreting the data on slide 19. We were not using these data to include or to exclude any extracts from our study, rather we are going to be using these data and other data to correlate with our skin testing data, which we hope will be coming fairly soon.

Next slide, please.

So, here we are with our time table for the clinical study, and, in fact, we are doing quite well. The steering committee approved it, the study centers were identified, we ordered the extracts, all of the IRBs approved the study. The IND submission to the FDA was approved. The materials have been distributed, and we are now at the stage of proficiency testing, which is a very important stage. In this stage, each of the study site testers are refining their ability with this fairly labor-intensive technique, using histamine and non-allergic individuals. That's in process, and we hope within a few weeks that we are going to be obtaining real data with the cockroach allergens from these sites.

Next slide, please.

So now, with the data that we hope and assume will be in our hands in a few months, the idea will be to develop surrogate potency tests. This is a critical stage in standardization. We certainly cannot ask our manufacturers to go out and recruit individuals to skin test with every new lot of material that they develop.

Next slide, please.

Now, the surrogate tests that we are planning on evaluating are competition ELISA, which is a technique that we use now, and we'll talk a little bit more about the pros and cons of these different techniques in a few minutes.

It's important to note that as part of the study, in addition to obtaining skin test data from these individuals, we will be obtaining about 30 to 40 mL of serum from each individual in the study. So, we are going to have a substantial amount of serum to study and to compare to the skin test data, and, hopefully, that will increase the power of our ability to develop good surrogate tests.

We are also going to be doing specific allergen testing, although as you know it is not clear what the major or immunodominant allergens in German cockroach are, but we will be doing some testing and, hopefully, again by correlating these data with the skin test data we may be able to come to some biologically meaningful conclusions. We are going to talk more about the antibody microarray profiles in a few minutes. You should note as well that Doctor Sampson at Mt. Sinai is going to be using the sera that we obtain in this study to look at sensitized mass cell lines and look at whether that is an appropriate surrogate measure as well.

Next slide, please.

So, in the next few slides, and in the first few slides of the next presentation, I'm going to be using a cartoon method that I've used quite a bit over the last few years. In this cartoon, I use arrows as the antibody or the detector system, and I use the antigen, I use the smiley face as the antigen and analyte. Color is used to indicate some specificity, so just as an example, a red arrow would be expected to be specific to a red smiley face.

Next slide, please.

So, the competition ELISA with pooled human allergic sera is really our workhorse assay that we used for dust mite allergens and for grass pollen allergens in the determination of their potency. The advantage of it is that it is quantitative. We have a great deal of experience with it. It reflects a broad spectrum of allergen recognition, and very importantly it does not require identification of relevant allergens. So, when you are approaching a new allergen to standardize, and you know very little about it, this is really our default position. This is a good method that works quite well in our hands, and has for many years.

The disadvantage of it is that it uses pooled sera which is hard to replicate when the serum runs out, and most importantly, and I'll discuss this in more detail in the next presentation, the effects of fluctuations in individual allergens is sometimes difficult to measure. Again, I'll come back to that point shortly.

Next slide, please.

Another approach is the ELISA with monospecific antiserum. This is a standard ELISA technique, and it's an extension of the radial immunodiffusion assay that we currently use. The advantage is that it's quantitative, it's monospecific, but the disadvantage, of course, is you need to know which allergens are important.

Again, as a consequence of this study, we may be able to do this, but you need to do that before you can actually decide which allergens are important.

Next slide, please.

Again, the two-site ELISA with monoclonal antibodies, the advantages of this are that it's highly specific. The disadvantage, in fact, is that it's highly specific. Again, you really need to know what you are measuring, and even with this specificity there is sometimes some degree of cross reactivity, and you do have to be cautious.

Next slide, please.

And finally, antibody microarray, which we'll talk about again in greater detail later, the advantages are that it's quantitative, it does reflect a broad spectrum of allergen recognition, it doesn't require identification of specific relevant allergens. The disadvantage is that it's a new technology, and the initial development is, in fact, labor intensive and expensive, but we hope it's worth it.

Again, I'm going to come back to that in just a couple of minutes. I want to wrap up on the cockroach standardization specifically with the last slide, and that is that standardized German and American cockroach allergen vaccine will facilitate definitive studies on the role of cockroach allergens in inner city asthma, and the best methods of eradication and treatment, and will make for safer and more effective product.

Just to clarify, we had intended and hoped to standardize American cockroach allergen vaccines, it was the opinion of the Consortium that we ought to start, at least, with German roach, which is probably more important pathogenically in inner city asthma, and we are happy to do this in a stepwise manner.

That's the end of the fourth presentation. I'm going to come back to some of the ? a lot of the issues that have to do with the assays themselves, but I really want to stop here and see if there are any questions or comments about the clinical studies, and then we'll go on and have a more thorough discussion of the assays.

DOCTOR NELSON: Jay, this is Hal Nelson.

What do you know about the protease activity in this cockroach extract in 50 percent glycerine? Is it inactivated, or are they inactivated, or is there still proteolytic activity?

DOCTOR SLATER: You know, it's a good question. I'm not really sure. My guess is that it's substantially inactivated. We don't see evidence of ? I really only have indirect evidence for this, Hal. When we looked at the allergens that we used in our 2002 study, and we looked at them over six months later, we really didn't see any substantial difference. In fact, we went back and replicated a large amount of our data, and really found that it was fairly stable.

In addition, E1Cg, which I haven't shown you any data on, is a glycerinated extract. It was made, oh gosh, at least ten years ago, it's potency and allergen content doesn't seem to have changed much in that time.

My guess is that ? well, you know well that there is a substantial amount of protease activity in roach extracts, but my guess is that the glycerol substantially inactivates it.

MR. HAUCK: Jay, this is Peter Hauck.

On slide 16, you talk about puncture sum of erythema equal to or greater than 30 millimeters. Maybe my memory is wrong, but I think when we did the mite, and the cat, and the grasses, I think the threshold was 40 millimeters. Did that change?

DOCTOR SLATER: Actually, you are wrong, but you are wrong in the wrong way. The original ? I'm sorry, you are right, but you are right in the wrong way, I'm sorry. The original proposal encourages us to look for individuals whose size of their screening test is 70 millimeters, but it provides for the possibility that that might be difficult to try.

When we looked at data from the mid-1990s, both in-house data and published data on testing with German roach extracts, we decided that it was really unlikely that we were going to be able to recruit individuals that had 50 to 70 millimeter responses.

The fact is, you can get fairly good data, even from less reactive individuals, and we are interested in the most reactive individuals that we can get. But, based on both the clinical data from the `90s, based on also our estimate of the allergen content, analogies with existing allergens, responses that we can predict, we decided to go with a lower cutoff.

The danger of that is that our data may come in more heterogenous than what we want, but our investigators were concerned that we were going to have difficulty recruiting, and that's why we lowered it.

MR. HAUCK: Okay.

DOCTOR BERGER: Jay, it's Mel Berger.

Can you just remind me what method is used now to standardize from lot to lot of something like grass which is standardized?

DOCTOR SLATER: Right, good question. The grass pollen allergen extracts were standardized, they were standardized in the `90s on the basis of this ideal testing.

DOCTOR BERGER: Yes, but what's going from lot to lot now?

DOCTOR SLATER: Yes, right, lot to lot we use the competition ELISA.

DOCTOR BERGER: Okay.

DOCTOR SLATER: Using a pooled human serum against grass pollens, with specific references for each of the different standardized grass species. The exception to that is Bermuda grass, which has its own serum pool.

DOCTOR BERGER: Thanks.

DOCTOR NELSON: Jay, Hal Nelson, just a quick question about slide number 19. What are the units, and are they all measuring the same unit, or are these different units, particularly, with the pooled rabbit sera and the, I guess, monoclonal antibody?

DOCTOR SLATER: Okay. So, in bla g 1, 2 and 5, bla g 1 and 5 are in arbitrary units that were defined by Martin Chapman in his work at Indoor Biotech. Bla g 2 is in, I believe, I'm not sure whether it's micrograms or nanograms per mil. I'm sorry to say I'm not sure, but that is in mass unitage.

RPs 1, 2 and 3 are relative potencies. Those are fractions relative to E2Cg. What you see if you look on the E2Cg line is the statistical variation inherent in this test. Normally, you would expect E2Cg to be exactly one. In fact, as our allergen manufacturers well know, even when you compare an extract to itself, based on the number of tests you do, there will be a variation around that mean of one, and that can be anywhere between .7 and 1.4.

DOCTOR NELSON: Great, thanks.

ACTING DIRECTOR BURKS: Any other questions for Jay?

DOCTOR SLATER: Okay.

ACTING DIRECTOR BURKS: Okay, Jay, do you want to go on to the next part?

DOCTOR SLATER: Okay, terrific, thank you.

So, we are now on our last presentation, and in that one I'm going to ? bear with me, because we are going to be at our most speculative work that we are doing, but we are very hopeful that this will help us introduce some new and very useful methods for measurement of potency of complex allergen mixtures.

Slide two, how do we measure potency? It depends on the extract, and we've already touched on this a bit already. There are some extracts, specifically, the hymenoptera extracts, in which we, basically, measure total protein. You are all aware that we measure the amount of hyaluronidase and phospholipaes activity in these extracts, but the potency itself is actually in mass unitage of total protein, and that's because it correlates quite well with the actual allergenic potency.

For other extracts, for the grass pollen extracts, there is a less close correlation between total protein and potency. On the other hand, we don't know exactly which allergens are the most important, and, therefore, we've used the pooled human antibody to determine overall allergenicity using the competition ELISA>

For the allergen extracts cat and ragweed, in which a specific allergen has been identified that's important, we measure that specific allergen. In this particular case, we used sheep antibody, using the radial immunodiffusion assay method, which is actually covered on the next slide.

Go to slide number three.

The RID method, using monospecific antiserum, is shown in this slide. Again, just to ? we are using a monospecific antibody, in this case it's polyclonal, but it's an antibody directed towards only one of those smiley faces in the lower right-hand corner. It's a blue arrow aimed at the blue smiley face.

But, the disadvantage of this approach, as we try to apply it to complex allergen mixtures, is we really need to know beforehand what allergens are important.

Next slide.

And again, this is our workhorse method, this is the competition ELISA, using pooled human allergic serum, and in this case we have multiple antibodies with multiple specificities, looking at a complex allergen mixture.

But, on the next slide we have the issue that dogs us about the competition ELISA. It's true that the allergens that we're measuring, the potency of the competition ELISA are ones in which we really are not sure which proteins are most important, but surely you'll all agree that the individual proteins probably are important, we just don't know what they are.

The question is, if we lost a single allergen in this complex mixture, would this method be sensitive enough to detect the difference. And, in fact, when we have looked at this, Al Gamm in my laboratory has specifically depleted cockroach allergens of bla g 1, 2 and 5, and in our hands we have ? although we've demonstrated using specific allergen measures that we've virtually completely eliminated the specific allergens from these complex mixtures, we've really been unable to detect this loss using the competition ELISA method.

Next slide, please.

So, the dilemma that we face with these potency measures is that, in order to measure specific allergens we need to know which allergens are relevant, but on the other hand if we measure overall allergenicity we are unable to detect the absence of specific and potentially important allergens.

Next slide, please.

So, it seemed to us that there were two possible solutions to this problem. The idea here is that we need to use multiple antibodies to look at multiple allergens, but we need to split the signal that we get. The problem with the competition ELISA is not the reagents that we use, but the fact that we come out with a single signal at the end of the assay, and we need to figure out a way to split the signal.

Now, the next slide, you know, I'm sure you all thought immediately that one specific way that scientists and investigators split the signal is by doing Western Blots. You separate the proteins out, you can see the smiley faces are now quite distinguished from each other, and if you come at them even with the serum pool you can detect which proteins are present and which are absent.

You all know the advantages. You also know the disadvantages. It's fundamentally a qualitative method. We are continuing to use pool sera that can be difficult to obtain and replace. There's no definitive identification of allergens specifically, and again, there's problems with cross reactivity, especially in allergens that contain proteases.

Next slide, please.

So, we wanted to look at this newer method, and this is antibody microarrays, and this is where we split the signal, instead of splitting it by dividing up the allergens, we are splitting it by dividing up the antibodies. It's a quantitative method. It reflects the full spectrum of allergen recognition. It doesn't require identification of relevant allergens, but again, it's a new technology and that's the major disadvantage that we see so far.

Let's go to the next slide. We are on slide ten now.

Our aims are to develop an antibody microarray method. We are profiling complex allergy mixtures, and our aim is also to apply this technique to our current endeavor in standardizing German cockroach allergen extract.

Next slide, please.

I just want to clarify what we are not talking about. We are not talking about allergen microarrays. You are all aware of really groundbreaking work that has gone on for several years now of putting allergens, specific allergens, cloned allergens, allergen epitopes, on microarrays, and using that to study the specific IgE responses of humans. That's not what we are doing here.

Next slide.

What we doing is, we are taking antibodies and putting them on the microarrays, and this is a technique that's actually been well developed, largely for the purpose of diagnostics, looking for specific antigens in the serum. One example is looking at prostate-specific antigens, by putting specific antibodies on the microarray. But, it's not been applied to the characterization of complex protein mixtures other than sera.

Next slide, please.

So, the idea here is, we have Nitrocellulose coated chips. We have an allergen mixture that we expose to the coated chips. The chips actually have clonal antibodies spotted onto them, and these clonal antibodies line to the specific allergens in the mixture.

Next slide.

To do this technique, we do need clonal antibodies, and those can be generated by the standard monoclonal antibody techniques, or we have preferred to switch over to a new method that many of you, I'm sure, are familiar with, phage display technology. In bare bones, the technique involves hyperimmunizing animals, and we'll talk about the choice of animals in a moment, taking ? extracting RNA from the spleens and bone marrows of these hyperimmunized animals, using RT-PCR to generate cDNA libraries with expression of antibody on phage surfaces, using the technique of phage panning, which I'll cover in a few minutes, to select for allergen specific cDNA, and then expressing the selected antibodies in either E. coli or yeast.

Next slide. We are now on slide 15.

The advantage of phase display technology is that with a single experiment you can theoretically clone the entire immunoglobulin repertoire of the animal, and you can go back repeatedly and probe that repertoire to look for allergen specificity of interest. You can generate large quantities of allergen-specific antibodies that can, of course, be used for structural studies, allergen purification, the definition of B-cell epitopes, and again, what we are doing, the analysis of complex protein mixtures by antibody microarray.

The retrieved antibodies can be expressed in E. coli or yeast, and the use of several different species to generate these libraries can mean the generation of fragments with considerably different allergen specificities.

Next slide, please.

Well, which animals and why? Clearly, mice have been the most constantly used going back for decades in the general of monoclonal antibodies, but in the development stage mice provide fairly small samples of serum, and most importantly for our purposes they have a large number of variable region families, which means that the construction of libraries using PCR involves multiple primer pairs and is very complex.

Rabbits, on the other hand, provide large quantities of serum during the hyperimmunization phase, and those sera, as you know, are very useful in development, and they have a relatively smaller set of V-region families. That makes it simpler to do these amplifications, and in addition they are a well-established model for recombinant antibody generation.

We focused on rabbits and chickens at this point in our studies, and the chickens are very useful. They have been found that others respond strongly to mammalian antigens that may generate weak responses in some mammals. Examples, of course, are cat and dog allergens, although you are all aware that it's not a problem eliciting strong responses, but we could expect that birds might give a stronger response.

A technical point of interest is that they don't require bleeding. These chickens lay eggs almost every day, and we can isolate milligram qualitites of IgY from the egg yolk using a relatively simple technique.

And, most importantly, the chicken genome encodes only two variable immunoglobulin domains. This means a very small primer set and a significant reduction of the serious problem of primer bias in library construction.

Next slide.

Just a review of Fabs versus scFvs. ScFvs are single chain fragment variable regions. Fabs are the fragment antibody binding regions. You know the structures of these. There are technical advantages and disadvantages of both, and it's our intention to develop both methods to have both kinds of reagents as our reagents for these assays.

The next few slides summarize the PCR methods that we are using. Basically, we isolate the RNA from the spleens and the bone marrow of these animals after they have an established immune response. We use standard Oligo dT priming to do RT-PCR, generate the cDNA, then through a series of PCR experiments we generate heavy chains and light chains.

Next slide, please.

Finally, constructing through an overlap PCR the product that you see in slide number 20, next slide, please, which is an overlap product that contains, in the case of Fab both variable regions, both constant regions, and some cloning markers at the ends.

Next slide, please. We are now on slide 21.

Once we've used PCR methods to develop our library, we then insert that library into a vector, in this case we used a pCOMB3X vector, which generates an M13 filamentous phage. The phage expresses on its surface the p3 code protein and if you place the gene fragments in the right location on the end of that p3 code protein it will express the Fab or the scFv that you've inserted ? that you've encoded and inserted.

Next slide, please.

And here, you see how we use this expression on the surface of the phage to pan for antibodies of interest. Remember, in this experiment, and I'll show you more in the next few slides, we are actually injecting the animals with multiple allergens at once. We extract the RNA and generate a library that reflects the entire immunoglobulin repertoire, using specific primers. We then insert this repertoire into the phage, and now we get to some specificity.

On the left side of the slide you see a phage with the specific scFv on its surface. We go to the upper panel, we then take that phage, put it into wells that have been coated with the specific allergen or allergens that we are interested in. We can easily wash the unbound phage off the surface. The only phage that then remain are the phage that are specific to the allergen of interest. We can use trypsin, now we are going to the lower right-hand corner of your slide, to release the phase. Notice that we leave the antibody behind, but that doesn't matter. What we are really after is the gene that's in the phage. We take that phage that's been cleaved, we infect E. coli, expand the phage overnight, isolate the phage, and then start the cycle all over again.

Next slide, please.

So, what we are doing now in LIB with antibody phage display technology is, we are trying to generate monospecific recombinant antibody fragments from rabbit and chicken, and we are doing two lines of experiments concurrently. On the one hand, we are looking for responses to allergens that we have pretty well characterized. We are looking for Fel d 1, Amb a 1, and the full yellow jacket venom responses, but in addition we are generating antibody fragments for the allergenic proteins of German and American roaches.

Next slide, please.

Here's a summary of the antibody animal libraries that we've built so far. On the left side you see the chicken libraries, middle column the rabbit libraries, and on the right column the allergens to which these animals were exposed.

Next slide.

We are focusing now on the C160 library. This is a chicken library, in which the animal is a single animal, by the way, was immunized with recombinant Fel d 1, with native Amb a 1, and with whole yellow jacket venom.

Next slide.

So, what I'm going to show in the next few slides is the kinds of antibodies that we could pull out of this single library, of this multi-immunized animal, using appropriate panning techniques. When we pan the C160 library with fel D 1, in other words, we put fel D 1 on the surface of the plastic that we use for the panning experiment, you can see that we develop scFv that's highly specific for fel D 1. In other words, we purified the scFv, we diluted it out, you see multiple log 10 dilutions, and we tested that scFv against yellow jacket, fel D 1, and Amb a 1, and what you can see is that we had a very good response against fel D 1 when we panned it this way, down to 10 to the -5 dilution, but we really have no significant response to either Amb a 1 or yellow jacket venom.

Next slide, please.

Same library, C160 library, and the same animal, but now we have panned with Amb a 1 on the panning surface, and you see here that we get a fairly specific response to Amb a 1. At the highest concentration, 10 to the -1 dilution, we do have a small signal from the ? with fel D 1, but really it doesn't last very long, again, good titers, out to 10 to the -4 or 10 to the -5.

Next slide, please.

And finally, when we pan with yellow jacket venom, again, crude yellow jacket venom material, we get a great deal of specificity for yellow jacket venom. Again, all from the same animal, same library, multi-immunized, but panned in three different ways.

Next slide, please.

And, what you can see here is that the scFvs that we obtained are fairly specific on Western Blot as well. There are four panels in front of you. They all have exactly the same proteins run on them. In each panel, the leftmost lane has molecular weight markers, the next lane has yellow jacket venom, the third lane has ragweed extract, not pure Amb a 1, true ragweed extract, and the fourth lane, the rightmost lane, has cat hair extract.

What you can see when we stain with coomasjie blue, this is in the left panel, is that again you see the markers perfectly well, the yellow jacket venom you see a very prominent albumin band. We used in this experiment, and certainly this was something that I'm revisiting, we should have done otherwise, but we used a yellow jacket venom extract that contained albumin, and you see that's really the prominent band on gel staining.

However, you can see, more faintly I hope, other bands as well in the yellow jacket venom extract. Next lane over you see the ragweed extract is quite complex, and finally the cat hair extract really doesn't show any bands on coomasjie blue staining. This is not unusual for this technique.

What you see in the next panel is that when we come back with the anti fel D 1 scFv, you pick up a single band, just around 10 kD in the cat hair extract, and you pick up some very faint bands in the other two, but not too much.

Likewise, when you come back to the third panel with anti Amb a 1, you get a very nice strong specific response to three bands in this complex ragweed mixture, presumably, Amb a 1, isoforms and, perhaps, proteolytic components at the bottom.

And finally, with the anti yellow jacket venom, scFv, again, predominant response to albumin, this is not a shock, but what was really reassuring is that we also seem to have good responses to multiple other bands, even bands that were visible in the coomasjie blue stained panel on the left.

Next slide, please.

So, our conclusion from this series of experiments is that we can raise chicken antibodies against multiple allergens from a single immune library, panning against multiple proteins at once can work effectively, antibodies raised against recombinant fel D 1 appear to recognize native fel D 1. That's not a surprise, but it is reassuring to see. We were very successful at raising these chicken scFvs in E. coli and having them secrete, and we used the HIS-TAG method to purify the scFv and we can use these scFvs effectively in both ELISAS and in Western Blot.

Next slide.

So again, let's go back. We have multiple other libraries that we're working with and are going to proceed with on a very active basis, but I wanted to highlight in the next slide what we are going to do with the microarray technology. There are really basically two approaches to use. One is the simple array, in which we put the scFv down on the plate, interact with the antigen, and then detect the antigen using some other antibody, either Fabs or polyclonal antibodies.

Alternatively, we could use the so-called dense array approach, in which the antigens are actually flourifor labeled. That maybe a direction in which we will go in the future, but at the moment we are really trying to prove a concept with what we are going to call the simple array technique.

Next slide.

And, let's just go over the experimental design of this simple array. The experimental design here is to apply the scFv against either Amb a 1 or against Fel d 1 onto the nitrocellulose slide, spot these on the slide, blot them with ovalbumin and then incubate with either ragweed extract, or cat hair extract, or negative control, which would just be ovalbumin.

We would then come back and detect the bound antigens with rabbit polyvalent serum R91. Remember, R91, like C160, was exposed to Amb a 1 and Fel d 1 and yellow jacket venom, so it's a multivalent reagent.

And then, of course, detect the rabbit serum with an anti rabbit conjugate.

Next slide, please.

So again, just to review, we are using the specific scFvs that we derived from C160 as our spots on the slide, but then detect ? we are using the crude polyvalent antiserum R91 that has been shown reacts with multiple allergens, including Fel d 1 and Amb a 1.

Let's look at the actual microarray. In the next slide, you see three different slides. They are all spotted the same way. They were spotted in the top two rows with anti Amb a 1, the bottom two rows with anti Fel d 1. The leftmost one is then exposed with cat hair extract 1 to 20 dilution. The middle one exposed to ragweed 1 to 20, and the right one to just 1 percent albumin. They were then all exposed to the same R91 serum. You can see in the leftmost one that only that Fel d 1 spots light up, in the middle one the ragweed spots light up, and in the right one none of the spots light up.

So, what we've been able to show in a very primitive and early way is that we are able to use these scFvs to detect, and separate, and distinguish specific allergens from each other.

Next slide.

So, our plan is to get considerably more complex at this point. What we want to do is, we want to develop a quantifiable fingerprint of complex allergen mixtures using clonal allergen specific scFvs and polyvalent sera.

Clearly, what we need to do at this point is advance the more complex allergens, and using the same reagents we can go forward and look at yellow jacket venom, which is, obviously, considerably more complex. But, we are also looking to develop microarrays to German roach and to American roach, and I can tell you, without showing you the data at this point, that we've been very successful at developing specific scFvs to German roach allergens.

How are we going to assure that our arrays recognize diverse allergens or epitopes? Well, we are going to use Bst01 analysis. This is a restriction enzyme, in which we can at least make sure that the gene products that were isolated from the different clones are different, and we'll, of course, use standard Western techniques to make sure that we are not just spotting the same antibodies over and over again in multiple addresses on the microarray chip.

We'll also be doing cluster analyses of the reactivity of allergens with the different spots on the microarray chips, and, obviously, if by cluster analysis we find that two, or three, or four, or five spots co-react 100 percent of the time, we will no longer use all five. We are looking for a diverse fingerprint of these allergen mixtures.

Next slide.

The key players in this work, which is exciting to us and very labor intensive, Jonny Finlay was my Post Doc. He has gone on to another position in Ireland at this point. Nicky deVore joined us over the summer, and she hit the ground running, and she's going to be taking it forward over the next couple of years.

Thank you very much, and I'll be happy to take questions on this presentation.

DOCTOR BERGER: Jay, it's Mel Berger. Is there any concern that the chicken may recognize a different epitope than humans would?

DOCTOR SLATER: Yes.

DOCTOR BERGER: Do you have any plan to do something like taking a good single chain sv, or a good serum and then do peptide mapping of the antigen?

DOCTOR SLATER: Well, we certainly can, and I think it's going to be absolutely irresistible to do that once we get the reagents. But, I think it's important to emphasize that one of the beauties of this technique is that it can be very effective, and yet at the same time be totally descriptive. In other words, what we really need to do here, and our objective here, is to use these fingerprints, which, again, we can quantify, and those techniques are out there very clearly, to use these fingerprints as a way of profiling the allergens. Toward that end, we really don't need to know exactly what each spot of antibody recognizes.

DOCTOR BERGER: I understand that, but it would be nice to have the reassurance that we didn't miss a major human ? major epitope that humans see.

DOCTOR SLATER: I think that's absolutely right, and once we get to the stage with roaches, with the roach extracts or any other extracts that we are studying, where we actually know what we are looking for, then it's going to be critical to go back and make sure that we're recognizing it. And, there's no doubt that we can do that.

DOCTOR BERGER: That would be a nice sort of icing on the cake.

DOCTOR SLATER: Thank you.

ACTING DIRECTOR BURKS: Anybody else have questions for Jay? That was good.

DOCTOR BERGER: And, it really is a tour de force.

UNIDENTIFIED SPEAKER: Absolutely.

DOCTOR SLATER: Thank you very much.

ACTING DIRECTOR BURKS: Jay or Bill, do we have other things that we need to talk about before we ask for any comments?

DOCTOR FREAS: I believe we're ready to move into the open public hearing session of this Advisory Committee meeting, with your permission, Doctor Burks.

ACTING DIRECTOR BURKS: Yes.

DOCTOR FREAS: And, let me just explain, this is a very important part of this Advisory Committee meeting, in the fact that this is how FDA gives the public an opportunity to participate in its Advisory Committee meetings. We welcome any member of the public to come and ? come up here and ask questions of either the presenters and/or the committee, and we're opening up the microphone at this time.

Would anyone like to address the committee?

Doctor Burks, I do not see anyone, so at this time we're going to close the open public hearing portion of this meeting, and see if there's any additional committee discussion.

ACTING DIRECTOR BURKS: Okay.

I think for the most part what Jay and Ron have done today is to try and inform us of what's been going on in their laboratories, both from a product and from a research standpoint.

So, does anybody have any other questions or comments that would be helpful to them?

If not, I think that really concludes our business.

Jay, Bill, do you have anymore comments?

DOCTOR FREAS: No, Doctor Burks, I want to thank you for helping us set up and plan for this meeting, and for being the chair for the meeting, and I want to thank all the committee members for giving us of their time, their comments. We do really appreciate your input as we move forward with our research and regular duties.

ACTING DIRECTOR BURKS: Okay, thank you, I think you did a really good job.

UNIDENTIFIED SPEAKER: Jay, you did an excellent job, thank you.

UNIDENTIFIED SPEAKER: Yes, thank you, Jay.

DOCTOR SLATER: Thank you.

UNIDENTIFIED SPEAKER: Okay, see you later.

UNIDENTIFIED SPEAKER: Bye-bye.

ACTING DIRECTOR BURKS: Bye.

(Whereupon, the above-entitled matter was concluded at 2:55 p.m.)