ALLERGENIC PRODUCTS ADVISORY COMMITTEE

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

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ALLERGENIC PRODUCTS ADVISORY COMMITTEE MEETING

By Teleconference

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PUBLIC MEETING

Friday, April 2, 2004

This transcript has not been edited or corrected, but appears as
received from the commercial transcribing services. Accordingly, the
Food and Drug Administration makes no representation as to its accuracy.

The above-entitled Public Meeting was held at 1:00 p.m., in Conference
Rooms A and B, Building 29B, on the National Institutes of Health
campus, Bethesda, Maryland, Dr. Wesley A. Burks, Acting Chair,
presiding.

[PARTICIPANTS]{.underline}:

A. WESLEY BURKS, M.D., Acting Chair (via telephone)

JANE S. BROWN, Committee Management Specialist

WILLIAM FREAS, Ph.D., Executive Secretary

[COMMITTEE MEMBERS]{.underline}: (via telephone)

MELVIN BERGER, M.D.

LYNELLE C. GRANADY, M.D.

REBECCA S. GRUCHALLA, M.D., Ph.D.

PETER R. HAUCK

DOLORES C. LIBERA

HAROLD S. NELSON, M.D.

[PRESENTERS]{.underline}:

RONALD RABIN, M.D.

JAY SLATER, M.D.

A-G-E-N-D-A

[Page No.]{.underline}

Administrative Remarks 4

Opening Remarks, **WESLEY BURKS, M.D.,**, Acting 8

Committee Chair

[COMMITTEE UPDATES]{.underline}:

Laboratory of Immunobiochemistry: Personnel 10

and Regulatory Updates, **JAY E. SLATER, M.C.**,

**DBPAP**, Laboratory Chief

Research Summary, **JAY E. SLATER, M.D., DBPAP**, 13

Laboratory Chief, **RONALD RABIN, M.D., DBPAP**, 27

Senior Staff Fellow

Cockroach Allergen Standardization 42

**JAY E. SLATER, M.D., DBPAP**, Laboratory

Chief

Use of Microarray Technology in Allergen 60

Standardization, **JAY E. SLATER, M.D., DBPAP**,

Laboratory Chief

Open Public Hearing 81

Committee Discussion 82

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]{.underline} 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]{.underline} with Dick
Lockey and Bob Esch. Ron published a paper in the [Journal of
Immunology]{.underline} 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]{.underline}, 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 LTC~4~, 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]{.underline} just last year that demonstrates that MRP4 transports
the other arachidonic acid products, such as PGA~2~ and PGE~1~, 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]{.underline} 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.)