UNITED STATES

DEPARTMENT OF HEALTH AND HUMAN SERVICES

PUBLIC HEALTH SERVICE

FOOD AND DRUG ADMINISTRATION

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

ALLERGENIC PRODUCTS ADVISORY COMMITTEE

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MEETING

(via Teleconference)

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Tuesday,

April 8, 2003

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

The Advisory Committee met, via teleconference at 1:00 p.m. in Room
4NN15, Building 29B, of the National Institutes of Health, 8800
Rockville Pike, Bethesda, Maryland, Samuel B. Lehrer, Ph.D., Chairman,
presiding.

[MEMBERS PRESENT:]{.underline}

SAMUEL B. LEHRER, Ph.D

MELVIN BERGER, M.D.

A. WESLEY BURKS, M.D.

LYNELLE C. GRANADY, M.D.

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

SUSAN M. MacDONALD, M.D.

HAROLD S. NELSON, M.D.

MARIA C. SOTO-AGUILAR, M.D.

[NON-VOTING INDUSTRY REPRESENTATIVE:]{.underline}

PETER R. HAUCK

**EXECUTIVE SECRETARY:**

WILLIAM FREAS, Ph.D.

[COMMITTEE MANAGEMENT SPECIALIST:]{.underline}

JANE S. BROWN

[ALSO PRESENT:]{.underline}

NORMAN BAYLOR, Ph.D.

JENNIFER BEIDGWATER

WILLIAM EGAN, Ph.D.

KAREN MIDTHUN, M.D.

RONALD RABIN, M.D.

RICHARD WALKER, Ph.D.

[PRESENTER:]{.underline}

JAY SLATER, M.D.

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

1:10 p.m.

EXECUTIVE SECRETARY FREAS: I would like to thank everybody for joining
us this afternoon, for this our 19th and most technically advanced
Allergenic Product Advisory Committee Meeting.

We are broadcasting from Room 4NN15 in Building 29 on the NIH campus. In
Conference A on the first floor of this building there is a speaker
phone reserved for the public. The public is welcome to participate in
the entire portion of the meeting, however they must participate through
the speak phone and the video machine down in Conference Room A.

Those people who have been on the line for the last 30 minutes realize
why this is necessary. Those people in the public cannot understand it.

Today\'s entire meeting, of course, is open to the public, as advertised
in the [Federal Register]{.underline}.

At this time I would like to go around and check with each of our video
stations first, and then I\'ll check with the audio stations to make
sure that we can both see the visual and hear your audio.

So I\'ll go around and introduce you. I will start with our Chairman,
Dr. Samuel Lehrer, research professor of medicine, Tulane University
Medical Center.

Dr. Lehrer, could you say good afternoon and count to ten slowly.

CHAIRMAN LEHRER: Good afternoon.

EXECUTIVE SECRETARY FREAS: That\'s good. You don\'t have to count ten.

Actually, we did not pick up a video. I\'m sorry, could you talk to ten?

CHAIRMAN LEHRER: So you\'re picking me up on video now?

EXECUTIVE SECRETARY FREAS: Your audio is loud and clear, but we do not
have a video. We\'re checking on it right now. And we\'ll go on to the
next committee member, with your permission.

CHAIRMAN LEHRER: Certainly.

EXECUTIVE SECRETARY FREAS: And see if we can pick Dr. Burks. Dr. Burks
is professor of pediatrics, University of Arkansas for Medical Sciences

Dr. Burks, can you count to ten for us?

DR. BURKS: Wesley Burks, and I can see you now but I couldn\'t see Sam
when he came on earlier.

EXECUTIVE SECRETARY FREAS: Okay. We appreciate you being there.

And, again, you know hopefully everybody has the audio number. If you
give up on the video, we do have this audio number. But, hopefully,
we\'re going to get this video to work but we\'re still trying to get
the bugs out of the system.

Dr. Rebecca Gruchalla, you were the first one who joined us this
morning. Thank you very much.

She\'s associate professor of internal medicine, University of Texas,
Southwestern Medical Center.

Can you say good afternoon, Dr. Gruchalla.

DR. GRUCHALLA: Good afternoon, Dr. Gruchalla. Sorry. Well, I just had to
do that.

EXECUTIVE SECRETARY FREAS: Oh, I appreciate a little levity. Okay.

PARTICIPANT: Like good say good night, please.

EXECUTIVE SECRETARY FREAS: Dr. Melvin Burger, are you there?

DR. BURGER: I\'m by telephone, but apparently I\'m not hooked up by
video at all.

EXECUTIVE SECRETARY FREAS: Okay. And, of course, Dr. Burger is professor
of pediatrics and pathology, Case Western Reserve School of Medicine.

Dr. Burger, I\'m sorry you weren\'t able to join us by video, but we
greatly appreciate you joining us by teleconference.

Dr. Susan MacDonald. I saw you earlier. She\'s associate professor of
medicine, Johns Hopkins University School of Medicine.

Dr. MacDonald, can you speak to us?

DR. MacDONALD: Good afternoon. I can\'t see you at all, but I can hear
you fine and you can see me.

EXECUTIVE SECRETARY FREAS: Yes. We\'re very fortunate. We hear you loud
and clear, and see you, and everything looks good. Thank you.

On the audio portion only, is there \-- Dr. Harold Nelson, have you
joined us?

DR. NELSON: Yes. Good morning.

EXECUTIVE SECRETARY FREAS: Good morning.

Dr. Nelson is senior staff physician, Department of Medicine, National
Jewish Medical Center.

Also on the line is Dr. Maria Soto-Aguilar, physician in private
practice in Hudson, Florida, specializing in allergy, rheumatology and
immunology.

Dr. Soto-Aguilar, can you hear us?

DR. SOTO-AGUILAR: Yes. Good morning to everybody.

EXECUTIVE SECRETARY FREAS: Thank you.

Next on the telephone our industry representative, Peter Hauck,
Executive Director for Scientific Affairs Allergen Products and
Manufacturers Association and works for ALK-Abello. Mr. Hauck?

MR. HAUCK: And good afternoon everybody.

EXECUTIVE SECRETARY FREAS: Good afternoon. Thank you for joining us.

EXECUTIVE SECRETARY FREAS: And next I would like to welcome a brand new
member to this committee, Dr. Lynelle Granady. She\'s an associate
physician with ENT and Allergy Associates in New York, New York.
Welcome, Dr. Granady.

DR. GRANADY: Good afternoon. Thank you.

EXECUTIVE SECRETARY FREAS: I\'m sorry to announce that our consumer
representative, unless I\'m mistaken has not joined us, and that\'s
Delores Libera. She called me this morning. She has a high fever and was
not feeling well and sends her disappointment that she could not join
us.

Again, hopefully you all have this telephone number. If you get dropped
from the video portion of the teleconference, you can dial in with the
\-- excuse me. The video portion you can dial in on the teleconference.

Now, I would like to just quickly go around and introduce the members in
the room, starting over here with Dr. Karen Midthun. Dr. Midthun, if you
would raise your hand once the camera gets to you.

Dr. Midthun is the new Director of the Office of Vaccines, Research and
Review. Thank you very much.

Sitting next to her is Dr. Jay Slate, Chief of Laboratory of Immunology,
Immuniobiochemistry.

Next is Dr. Richard Walker, Director, Division of Allergenic Products
and Parasitology.

Next is Dr. William Egan, Deputy Director, Office of Vaccines, Research
and Review.

In the corner over here is our technical chief.

I will start around here. WE have joining us Dr. Ronald Rabin, who is
Senior Fellow in the Laboratory of Immunochemistry.

Next we have Ms. Jennifer Beidgewater, Consumer Safety Officer.

And we also have Jane Brown, who is the Committee Management Specialist
responsible for getting this putting this meeting for us.

In the other corner with Dr. Robert Stumphey, our technical expert who
is helping us with all the audio and the sounds.

I have been asked to remind you that this video conference is voice
activated. And so please try not to all speak at the same time. And if
you just wait a second, it\'ll be your turn to talk. And whoever speaks,
the cameras will zoom in on them.

Now I\'m going to read the conflict of interest statement which is
required to be read for all advisory committee meetings.

The following announcement addresses the conflict of interest issues
associated with this meeting of the Allergic Products Advisory Committee
on April 8, 2003. To determine if any conflict of interest existed, the
agency reviews the submitted data and agenda. The Committee agenda
addresses update issues only. The Committee members were screened for
their financial involvements with firms that could be mentioned in this
updates.

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 ALK-Abello, Incorporated and
thus has a financial interest in his employer. Mr. Hauck also serves as
Executive Director for Scientific Affairs Allergen Products and
Manufacturers Association. In the interest of fairness, FDA disclosure
that his employer is a manufacturer of allergen extracts.

In the event the discussions involved specific products or firms not on
the agenda and for which members have a financial interest, the members
are reminded that they need to exclude themselves from such discussions.
Their exclusions will be noted for the public record.

With respect to all meeting participate, we ask in the interest of
fairness that they address any current or previous financial involvement
with any firm whose products they wish to comment upon.

Dr. Lehrer, that takes care of the administrative introduction to the
meeting. I would like to turn the microphone over to you.

CHAIRMAN LEHRER: Thank you very much, Bill.

I\'d like to welcome everyone to the Advisory Committee broadcast.

Earlier this year I spoke to Dr. Slater about the format of this
meeting. And since there were no regulatory issues that required the
Committee\'s formal advice, we thought it best to hold a video
conference to keep the Committee up to date on FDA\'s research
activities and related allergen standardization issues. And certainly if
you\'ll remember from the last meeting, allergen standardization was of
importance to the Committee.

So Dr. Slater will be making up the update presentation.

I\'d like to encourage of the Committee members to make comments on
these presentations. We will have time for questions at the end of each
presentations. And a time following all of the presentations for our
comments.

And it\'s important anytime you make a comment, please identify
yourself.

Another housekeeping issue, we would like to \--

DR. MacDONALD: Excuse me. I can\'t hear anything either.

(Off the record while adjusting audio equipment)

EXECUTIVE SECRETARY FREAS: Dr. Lehrer, why don\'t you just talk for one
second. And if you don\'t mind, I would just like to recap that you
welcomed the Committee members and possibly maybe we should turn the
host microphone over to Dr. Slater.

CHAIRMAN LEHRER: Yes.

EXECUTIVE SECRETARY FREAS: Would that be acceptable? Here I can only
apologize to you for the technical difficulties that we are
encountering. And if there\'s some way we could check and see what the
matter is to get your audio back, we will. And also, if you would check
in your local station to see if there\'s a problem down there that can
be correct. And if not, I guess I will try to relay what you say to the
rest of the Committee members. If that\'s agreeable with everybody?

Okay. So, at this time, Dr. Lehrer, I\'m going to turn the microphone
over to Jay Slater, who will be giving us the initial presentation.

Again, Dr. Slater is the Chief of the Laboratory of Immunochemistry.

DR. SLATER: Thank you very much.

First of all, again, I want to thank all of the Committee members for
putting up with all the technical issues here. And I want to thank
everybody for their patience in participating in the meeting in this
manner.

We do have some interesting things to talk about today. But before we
do, I just wanted to show this. This is a copy of today\'s newspaper.
This in case, you now, given the unusual times, there was any doubt in
anyone\'s mind that this is in fact a live broadcast. This is not being
videotaped at an undisclosed location. We are all here and we are ready
to go on.

DR. NELSON: For those of us not on the video, can you just tell us what
it is?

DR. MacDONALD: What paper is it?

DR. SLATER: It\'s April 8, 2003. That should be enough, right. Okay.

So let\'s go ahead and proceed.

I\'m going to start showing the slides. Now, we do have people that are
viewing this conference in several different technological formats.
Those of you who are viewing it on the monitors that\'s being generated
by us don\'t have to listen to anything that I\'m going to say, because
I\'ll be controlling the slides.

Those of you that are looking at it on your own PCs, please set
telepoint to slide show because there are some situations in which it
will appear differently in slide show than it will if you\'re just
looking at it in the normal view. So if you\'re looking at it on the PC,
hit F5 now so that you\'ll be at the beginning of the presentation.

If you\'re looking at it in hard copy, just bear with me. When I say
next slide you can advance visually to the next slide. If I say click,
stay on the same slide. It just means that there\'s an animation that
you\'re not going to see. There aren\'t too many of those, so bear with
me.

We\'re now on the second slide of the presentation in which I\'m just
giving the outline of what we\'re actually going to do today.

First we\'ll have a very brief lab overview. Then we\'ll talk about a
couple of interesting operational issues that have come up. And finally
in the longest portion of the presentation, I\'ll be giving you a
research and regulation update covering some of our recent endotoxin
studies and covering some of our work in our attempts to go forward with
cockroach allergen standardization.

Next slide, please.

So for the lab overview, we\'re going to talk about staffing issues, lot
release issues and reference replacement issues.

Next slide. You should be on a slide that shows that I\'m the lab chief.
The two principal investigators in this laboratory are myself and Dr.
Ron Rabin, who is starting his third year as a Senior Staff Fellow in
the laboratory.

We have two post-doctoral fellows, Jonny Finlay and Bo Chi. Jonny is
also starting his third year and Bo just started with us, since she\'s
working with Dr. Rabin, and he started earlier this year.

Next slide, please.

We have research technicians in the laboratory. Al Gam, Mona Febus, Marc
Alston, Cherry Valerio and Katia Dobrovlskaia. At this point if we were
all the same room, I would have them all stand up and bow. But you\'ll
understand that that\'s not possible in the format today.

Next slide, please.

This is a graphic that shows the staffing. This is the biologist
staffing in the laboratory. And each of the colored arrows at the bottom
covers a 12 month period. So what you can see from this is that after
sort of ups and downs of lab building, really until the last year and a
half we\'ve been at a fairly stable position with 5 biologists
performing both our research and regulatory work. This is a good number
for our purposes and we\'re very happy that we\'ve been stable at this
point.

Some of you may have noticed that we have had some shifts in our
staffing, though, that aren\'t reflected in this. Dr. Melissa has moved
to Cedar and the past year and Melissa Patterson, who is a biologist in
the lab for several years, left the lab to become a full time mother.

We maintained our full compliment by moving Katia Dobrovolskaia from a
predominately research position into the visiting associate position, in
which she can do both research and regulatory work.

Next slide.

Our \"routine\" regulatory activities, routine is in quotes because it
often is anything but routine, include lot release activities, reference
distribution and reference maintenance activities.

Next slide.

In the past year we have reviewed 357 protocols. One of those protocols
was withdrawn by the manufacturer. We have distributed nearly 2,000
vials of reference materials in over 100 shipments sent to the
manufacturers. To put that in context, please switch to the next slide.

This table shows the protocols that have been submitted in the past four
years. And you can see that this year is somewhat lower than previous
years, but my guess is this is just a normal year-to-year statistical
variation.

Next slide.

You\'re see the referenced distribution in numbers of vials over the
past four years. And you can see that that has varied as well with the
peak in 2000. But basically our level of activity in 2002 is roughly
where we have been for the last several years since I came here.

That ends the brief introduction to the overview of the lab\'s
activities. I\'ll be happy to entertain any questions on that short
section of the presentation.

If there are no questions, then we\'re going to proceed to the next
portion of the presentation, which is operational issues in the
Laboratory of Immuniobiochemistry.

Go to the next slide. You should be now one slide beyond the title slide
operational issues.

What we\'re going to cover in this is a presentation of some issues that
came up with the replacement of our cat and ragweed antisera. And we\'re
also going to talk about our transition to ISO compliance.

Next slide, please.

We use sheep antiserum to measure, M being one content of ragweed
allergen extract and one content of cat allergen extract. The antisera
are raised in sheep and are sent out to our manufacturers for them and
for us to use in the measurement of these allergen for lab release.

And this past year it became clear that both of our antisera needed to
be replaced. As to a cat serum, it was originally released in 1988. S6
ragweed serum was released in 20000. And we replacement programs for
both of these in the spring of 2002.

Next slide, please.

When we embarked on this, we thought this was a fairly standard
procedure that was going to go uneventfully, but as you\'ll see we had
some difficulties with it.

Our plan of what is after a prebleeding of the sheep that we were going
to immunize them with 25 micrograms of allergen. Then a month later we
boosted them with another 25 micrograms. We did a test bleed 3 weeks
later. Following the results of that bleed, we determined that we needed
to give another boost. And then the plan was to go ahead and
plasmapheresis them in September of last year.

The next slide gives you a graphic representation of that plan with the
three immunization doses followed by a plasmapheresis that we thought
would give us a good supply of useable plasma sometime in mid-October.

Switch to the next slide, please. Next slide, please.

What we learned, though, in August was that the farm where the sheep
were kept notified us that another sheep, not one of our two sheep, that
had arrived in the farm in mid-July developed neurological symptoms at
the end of July and it was sacrificed in the first week in August. But
the presumptive diagnoses was that sheep was scapies. If that wasn\'t
bad enough, next slide, please.

We were then notified about a week later that a sheep that had arrived
in the farm in late June also developed neurologic symptoms, was
sacrificed with a presumed diagnoses of scrapies. On autopsy both of
these animals were found to have that disease.

Next slide, please.

So now we\'re going to review the transmissible spongiform
encephalopathies, a topic for those of you are on this Committee 2 years
thought and hoped I\'m sure you\'d never hear from again. But let\'s
just review it very briefly.

There are several animal forms of the TSEs. Scrapie occurs in sheep and
goats. Chronic wasting disease appears in mule, deer and elk here in
North America. There\'s a transmissible mink encephalopathy. And, of
course, there\'s the bovine spongiform encephalopathy. The feline is
lightly considered to be derivative from the bovine form, contracted by
cats who eat contaminated bovine material.

Next slide, please.

Of course, there are several human forms of the transmissible
encephalopathy, and the one that\'s of greatest concern to us is, of
course, Jakob disease, especially in the new variant that\'s associated
with bovine TSE.

Next slide, please.

Scrapie is really one of the best described of the TSEs. It has been
described in Europe for over 250 years. And it first appeared in the
United States in 1947 when a sheep fogger imported sheep from Europe for
local use. It now infects at least 1,000 flocks in the United States and
is considered to be endemic.

It is transmitted both vertically and horizontally. Horizontal
transmission is presumed to be by contamination of animals with placenta
and blood during lambing season.

As with the other TSEs, there\'s typically a long incubation period and
there\'s been no evidence of human transmission at anytime.

Next slide, please.

Sheeps are of varying susceptibility to scrapie associated with
polymorphisms in their prions, especially at codon 171. The form of the
animals that are most susceptible are QQ or glutamine/glutamine at the
codon 171 site. The animals that are arginine/arginine at that site are
highly resistent. And the animals that are QR are the intermediate
resistance, although they are pretty resistent, they\'re not halfway
between susceptible and the resistent animal.

Next slide, please.

The USDA has a well established scrapie eradication program that
consists of three essential elements. The first is preclinical testing
and surveillance. This includes a live animal test. It turns out that a
third eye biopsy can reveal scrapie before neurologic symptoms appear.
However, even the third lid biopsy may take months to years to turn
positive in an affected animal.

The second key element is the tracking of infected and exposed animals.
And the third key elements is cleanup strategies which involves
identifying and genotype exposed animals. When there\'s an animal in a
flock that develops scrapie, all of the remaining animals are supposedly
genotype. The QQ animals are destroyed immediately. The RR and QR
animals that are exposed are tracked for the development of the disease,
but they may be slaughtered for human consumption.

Next slide, please.

Scrapie is not believed to pose any risk to humans. There\'s been no
recognized human transmission in three centuries of exposure in Europe.
And our sense is that these serum that we obtain from our animal, which
you\'ll recall are only in the same farm with the affected animals, our
feeling is that these sera was probably safe. Again, because there\'s no
documented transmission to humans. The contact of our sheep with the
affected sheep was limited. They were not in adjoining pens at anytime.
They were at least 30 feet apart. And finally, because normal BSL 2
precautions should be in place for all work with all animal sera. And
this should be certainly more than enough to give an extra element of
safety. However, it was also our sense that in order to maintain a serum
reagent that is as safe as possible, we were going to have a somewhat
different approach.

Next slide, please.

So what we began to do, is we began to immunize two new sheep. We went
ahead and processed the plasma from the two exposed sheep, and we
initiated an immediate process of conserving our current and existing
stocks of both of those previous antisera.

Next slide. Our approach is that if the new sera are available before we
run out of the old sera, then the sera from the exposed sheet will be
safe frozen for possible future use. However, if we have a shortfall,
then sera from the exposed sheep will be used until the new sera are
available.

Next slide, please.

The advantages of this approach is that we\'re offering the highest
degree possible of safety to the workers that are using the sera for
diagnostic purposes. And in addition, we\'re collecting what we think
are going to be vast supplies of these sera for the future that we
certainly will be able to use. The disadvantage was the possibility of
two serum switches in a relatively short time, and obviously the time
and expense.

That ends that segment of the presentation. Let me just tell you that we
actually are going to be able to go forward with the new ragweed serum
fairly soon. We\'ve tested that and we will be able to go forward with
that.

With the cat serum, we\'ve continued to have some technical problems
with even the new cat serum that we\'re using. And what we\'re in the
process of doing in order not to have a shortfall is we\'re actually
going back and retrieving some of the original sera that \-- the
components of the original sera that we had used in previous years.
We\'re testing that out and we\'re going to \-- we think we\'ll be able
to have about a year\'s supply of that that will give us some time to
work out some technical issues that we\'re having within the serum. So
we\'re hoping that we will be able to avoid \-- we certainly will be
able to avoid shortfalls with both of those new sera.

That ends that portion. If anyone has any questions, I\'d be happy to
answer.

MS. LEE: Jay, this is Dr. Lehrer. Could I ask a question about your
immunization?

DR. SLATER: Sure.

CHAIRMAN LEHRER: You used advance \-- 25 microgram doses and I think
there were 3 doses and complete/incomplete. It\'s just 25 micrograms
struck me as being somewhat as a low dose for a kilogram basis with such
a size animal. But I haven\'t had a whole lot of experience with sheep,
so I just wanted to ask you about that in terms of your choice of
antigen.

DR. SLATER: It\'s a good question. We actually got very, very good
titers with the exposed sheep and with the subsequent sheep. So I think
you\'re asking a good question, but we did end up getting very good
titers.These were some previous protocols that were used in the
laboratory.

CHAIRMAN LEHRER: I see. Thank you.

MR. HAUCK: A question, Jay.

DR. SLATER: Yes.

MR. HAUCK: This is Peter Hauck.

Maybe two questions. So you anticipate there would be no disruption of
protocol release or manufacturers being able to use these materials for
lot release purpose?

DR. SLATER: No. We\'re not anticipating any delay at this point. Both
the new ragweed serum and the retrieved old cat serum are in the process
of being lyophilized over the next couple of weeks. We\'ll then do some
brief testing and we should be able to send it out shortly.

MR. HAUCK: Okay. And, Jay, one last question. It doesn\'t sound like
you\'ll need to, but at least two manufacturers have their own antisera
for ragweed that they use for stability testing. In a pinch, could they
possibly be used for lot release it came down to that? That\'s kind of
speculation, but \--

DR. SLATER: Yes, that\'s speculation I\'d rather not get into to. I
think we\'d have to know more about those sera and we\'d have to really
be in a pinch. I think we\'ve avoided that.

MR. HAUCK: Okay.

DR. BURGER: Jay, this is Mel Burger. Maybe I\'m missing something, but
why not go over to Mil\'s monochronials?

DR. SLATER: You\'re not missing anything. That would involve designing a
new assay and then validating it. It\'s certainly something that can be
done, and it\'s certainly something that you sort of think about a lot
when you go through this process of raising a new polyclonial serum.

You know there are \-- we would have to make sure that it performed
comparably. When you make that kind of a big switch, you have to make
sure that we\'re not going to have a shift in what the unitage actually
ends up meaning. So it\'s something that certainly can be done, and I\'m
always looking for new and better ways to allergens. But you have to
understand, it\'s not a process that one can use in two to three months
to make up for a shortfall.

DR. BURGER: No, I wasn\'t advocating it as a shortfall. To make up for
the shortfall, I was wondering why \-- since we\'re beginning to focus
more and more on the single most important allergen, why we don\'t have
a long term program of shifting over to monchronial antibodies for
standardization?

DR. SLATER: Well, it\'s certainly something that we could consider. I
think it\'s a good suggestion. But we\'re going to have to make sure
that it actually performs the same way as our existing assays so that
the actual unitage doesn\'t really change.

DR. BURGER: You know, maybe that should be an experimental goal of the
laboratory to develop that for one antigen and see how it works.

DR. SLATER: I appreciate the suggestion. I think that\'s worth thinking
about.

Okay. Dr. Lehrer, if there are no further questions, with your
permission I\'m going to go on to the next discussion.

CHAIRMAN LEHRER: Yes. That\'s fine, Jay.

DR. SLATER: Thank you.

Let\'s go to the next slide.

I\'d like to talk briefly about CBER\'s Laboratory quality management
initiative. And I\'d like to acknowledge that in the audience down in
Conference A is Bill McCormick from OVRR. He is the office quality
manager. And he has provided me with the information that I\'m using to
make this brief presentation.

CBER has committed to becoming ISO-170235 compliant in its office
product testing. So for those of you who don\'t know what that means,
let\'s define a couple of terms here.

ISO is the abbreviation for the International Organization for
Standardization. This is a nongovernmental agency founded in 1947 and
based in Geneva, Switzerland.

ISO-17025 is a document that was generated by ISO in 1999 entitled
\"General Requirements for the Competence of Testing and Calibration
Laboratories.\" This is a set of guidelines for labs that do testing and
calibration to show operation of a quality system, to assure technical
competence and production of valid results.

Next slide.

CBER\'s laboratory quality management initiative has several components
to it. The first is to establish a policy with a center level quality
manual. The second to audit the laboratories within the center for
compliance with ISO-17025. And finally, to obtain test and laboratory
accreditation where appropriate. And with accreditation, of course, is
the consequence of a successful third party audit.

Next slide, please.

Why is CBER becoming ISO compliant? This is a complicated and expensive
process. I think that\'s a reasonable question as to why we\'re doing
this. And there are several reasons.

The first is to establish recognized competence of our laboratories that
do testing. To assure the test and value of our data and our processes.

The second is, we require manufacturers\' labs to be i GMP compliance.
This is good manufacturing practices compliant. And although that
compliance is intended for manufacturers not for us, it certainly makes
sense that we would try to be compliant with ISO, especially since there
are international efforts of harmonization that are attempting to equate
GMP and ISO requirements.

The third is that ISO is an internationally recognized standard. And,
again, this is a way of assuring the value and trust of the results that
we obtain.

Next slide.

We are attempting to become ISO compliant in order to implement
laboratory quality management policies and practices for all the
official testing activities, to document a high level of training,
competence and proficiency and to establish a consistent product testing
process.

Next slide.

But are the elements of ISO compliance? ISO compliance consists of the
management of 4 different features of any laboratory testing system.
People, equipment, documents and processes. Let\'s go through those one
at a time now.

Next slide.

The management of people basically covers all the people that would be
involved in the laboratory testing of these products. All the people in
the lab need to have defined roles. They need to have training that\'s
appropriate for their roles in the process. They need to be demonstrated
to be proficient in what they\'re supposed to, and that\'s both
demonstration of proficiency at the beginning, initial demonstration of
proficiency, and ongoing on a regular basis.

And furthermore, there needs to be an authorization process that assures
that people are doing what they\'re supposed to be doing, what they were
trained to do and that their training is ongoing.

The next slide, please.

The management of equipment follows from the same kind of reasoning. All
of the equipment that\'s used in the testing has to be appropriate for
the testing. It has to be appropriately calibrated. It has to
maintained. There has to be a process in place to assure that only
calibrated and maintained equipment is used. And there has to be
assurances of measurement traceability to accepted standards.

Next slide, please.

ISO also requires that documents be \-- and those documents include
policies, procedures, specifications, equipment manuals and
certifications. These documents all have to be reviewed and issued and
controlled and they have to be approved and issued before they are used.

Next slide.

And finally, processes have to be managed. Those processes include the
approval of testing materials, environmental specifications and
monitoring, handing of samples, validation and suitability, the handling
of data including non-conforming data, corrective action, preventive
action systems, internal audits, management review and finally, although
we never have complaints, the recording and handling of complaints.

The next slide, please.

CBER has committed to the implementation of this lab quality system.
CBER has established a quality board. We\'ve developed a quality
assurance structure within CBER with the appointment of a center level
quality manager, with the hiring of office quality manager. Bill
McCormick, as I mentioned before in our case. The appointment and hiring
of division quality coordinators. With the preparation of a CBER wide
quality manual and with the purchase of an integrated quality management
computer software system.

So what will this mean to my laboratory? My laboratory actually has
fairly extensive written protocols for almost everything that we do. So
there\'s really not going to be extensive substantive protocol revisions
for our processes. There will be changes in terms of the documentation.
There will be formatting changes to make the formatting of our documents
consistent across the center. And there will be some substantive changes
as well.

This will probably require a more formal separation of research
regulatory equipment that we\'ve had in the past, simply because the
regulatory equipment will have more extensive record keeping and
maintenance than the research equipment, although certainly the research
equipment would benefit from the same maintenance and calibration as the
regulatory equipment.

There will certainly be a series of internal audits that we will subject
ourselves to as part of this process.

And then finally, in the near future, external audits as well as we
approach certification.

Next slide.

The time table for this, of course, is tentative. But the idea is for
implementation in stages of this process over the next 3 years with CBER
\-- with all labs seeking accreditation in the year 2005. The software
is becoming operational this year. The polices and quality manual is
being issued this year. We are beginning the initiation of compliance
audits. These are internal audits which, of course, is starting and will
be ongoing. And the training and process development is ongoing as well.

That ends this portion of the presentation. Dr. Lehrer, if there are any
questions, I would be happy to take them now.

CHAIRMAN LEHRER: Are there any questions?

Well, it seems to me, Jay, that you\'ve done an excellent job in spite
of trying circumstances. And also I\'m pleased to know that that is
really you and not your double.

DR. SLATER: Thank you. I appreciate it.

DR. BURGER: How do we know that?

CHAIRMAN LEHRER: We don\'t know that for sure. But I think it\'s
impossible to duplicate Jay, I believe. I mean that in the best of ways.

DR. SLATER: Well, thank you.

CHAIRMAN LEHRER: Next we\'re going to move the agenda to the open public
hearing. Bill, could you \--

DR. SLATER: Sam, we\'re not quite there yet. I think you\'re being
overly optimistic. We have some more talking.

CHAIRMAN LEHRER: I\'m sorry. I have my confused on there.

DR. SLATER: I apologize. We were going to be talking \-- we\'re going to
be doing the research and regulatory update.

CHAIRMAN LEHRER: Sorry about that.

DR. SLATER: No, I apologize. I wish I were faster, we could be done now,
but \--

CHAIRMAN LEHRER: Oh, that\'s fine. I think you\'ve done an excellent job
under trying circumstances.

So then the next presentation would be the research and regulatory?

DR. SLATER: Yes. Yes. Okay. Thank you.

So let\'s go to the next slide, please.

This is the research and regulatory update. And if you click to the next
slide, you\'ll see the active research projects within the laboratory.
Remember, there are two PIs in the laboratory, myself and Dr. Rabin.
Those of you that were participating in the meeting last year heard
about Dr. Rabin\'s project. We will not be talking about them at this
meeting.

In terms of my projects, I\'m going to focus on issues having to do with
cockroach allergen standardization and endotoxin in allergen vaccines.

Next slide.

But before we get to there, I\'m going to review some of our
publications from this year.

We had three primary research publications out of the laboratory. The
first and the third on your list are actually publications that you\'re
going to be hearing about today. The first is our cockroach work, which
appeared in [Clinic Experimental Allergy]{.underline} last year. The
last one is our endotoxin work that is going to appear in
[ACI]{.underline} within the next two to three months.

And the middle one is work that we did in collaboration with our
colleagues in Australia having to do with the latex allergen Hev b 5.
I\'m not going to be covering that work today.

Next slide, please.

And on the next slide you see that the chapter in [Meddleton\'s
Allergy]{.underline} textbook still has not been published yet. But,
hopefully, will be published sometime soon.

Dr. Rabin also authored a review article that is going to be appearing
in the [Encyclopedia of Hormones]{.underline} on CXCR receptors, which I
neglected to put on this slide.

And next slide, please.

The following are the four abstracts that we submitted to the Academy of
Allergy meeting this year, which cover the breadth of the work that
we\'ve been doing in the laboratory.

Next slide.

So before I start this presentation about the endotoxin and content of
allergen vaccines, I\'d actually like to thank the Committee for
encouraging this line of investigation last year. I think you\'ll all
recall that this was a topic of discussion last year and several
Committee members strongly encouraged that we expand our then fairly
small studies on endotoxin in allergen vaccines, and we did that. And we
came up with some results that we think are interesting. And I want to
thank the Committee for that extremely constructive input just about a
year ago.

As you probably are aware, allergenic extracts are not required to
undergo evaluation for the presence of pyrogens. And that is from
CFR610.13 paragraph (b). However, in prior studies done actually out of
this laboratory a confirmed variable endotoxin content in allergen
vaccines. Now, those studies were not done with standardized vaccines.
Largely they were done with house vaccines, but certainly there have
been studies before that have shown that allergen extracts do have
endotoxin content.

Next slide, please.

The products that are exempted in 21 CFR from pyrogen testing include
blood products, horse serum, bacterial, rickettsial and viral vaccines,
toxoids, toxins and allergenic extracts.

Next slide.

But we are interested and we were interested in looking at the endotoxin
content of allergen vaccines. And we used sort of the standard workhorse
method that\'s been used for the last 20 years in measuring endotoxin,
and that\'s the LAL gel-clot method. The slide that you have before you
has four pictures on it. On the left hand one is an imagine of the
underside of the horseshoe crab, Limulus Polyphem, which the hemolift
link, which is the basis for the LAL gel-clot method.

You see in the central panel a fisherman hauling in some horseshoe
crabs.

In the upper right hand panel you see beautiful Delaware sunset with
horseshoe crabs on the beach.

And in the lower right panel you see a worker bleeding horseshoe crabs
for their hemolift to be used.

You should note that this is a survival procedure. The horseshoe crabs
do survive the bleeding, although no one to my knowledge has asked how
they feel about the insertion of foreign components or biomedical
health.

On the next slide you see, if you look closely, a clot forming in the
bottom of the tube that has been inverted. This is what you\'re looking
for when you add endotoxin to the limulus emulase.

Now for those of you who are viewing the next slide on a hard copy,
there would be \-- there is a small animation that\'s not terribly
interesting, but it\'s there and you won\'t see it.

Next slide.

Those of us that are watching it animated are watching a wonderful
imagine of the cascade where endotoxin activates factor C, which in turn
activates factor D, which in turn activates a pro-clotting enzyme. And
finally generates the coagulant that forms the clot.

If that were the only mechanism by which this cascade could occur in the
limulus licate we would be in much better shape. But, unfortunately \--
and those of you that are doing this on your own PCs, please click now
\-- there\'s another route by which the clot can occur, and that is when
(1,3) ß-D-glucans activate factor G. Activated factor G can also active
the clotting enzyme and form the clot made out of coagulant.

Next slide, please.

So there are two possible ways that we can imagine that there might be
interference with the LAL gel-clot determination of endotoxin content in
allergen. And the first, of course, is that non-endotoxins, the glucan
content may induce clotting by an alternative pathway. And also we were
concerned since this is a proteolytic cascade that proteases especially
in cat and mite extracts, might also induce clotting.

Next slide.

So our approach would determine the endotoxin content using the standard
gel-clot method, but then independently to attempt to assess the
contribution of non-endotoxin components.

Next slide.

We started with only standardized allergen vaccines which we obtained
from all of the allergen manufacturers. We started out by just
performing LAL gel-clot assay. And then we attempted to control for
these confounding factors by selectively absorbing the allergen vaccines
with ENP-silica resin, followed by a repeat LAL assay.

ENP is endotoxin neutralizing protein. It\'s a 12 kDDa, cationic,
amphipathic protein that binds to and neutralizes the biological
activity of LPS.

Theoretically that adsorption would help us control for both glucans and
for the proteases, neither of which would be expected to bind to the
ENP. In addition, as an added control for protease activity, we
pretreated selected allergens at 95 degrees centigrade for 15 minutes,
and that treatment would be expected to inactivate any proteases. Of
course, that was followed by a repeat LAL assay.

Next slide, please.

On this first table you see 24 grass pollen extracts, so I\'m just going
to take you through this slowly since we have a few of these tables that
are like this.

There are 24 grass pollen extracts that were assayed. The manufacturers
are coded in the second column.

In the third column you see the total gel-clot activity, which is given
in a twofold range. And this was before any treatment or adsorption.

In the fourth column you see the depleted gel-clot activity, this is
after the ENP-silica adsorption.

And finally you see the corrected endotoxin content, which is actually
the difference between the geometric means of the first ones, and if
there was a range the geometric mean of the second one or if it was at
or below zero, it\'s essentially the geometric mean of the third column.

So the numbers you really need to look at the last numbers, but I will
direct your attention to the fact that there were really only a couple
of extracts in which a significant amount of the gel-clot activity
remained after ENP absorption. That was in ryegrass, the second ryegrass
pollen extract for manufacturer C where you could see that perhaps half
of the activity was not absorbed out by ENP. And in the last ryegrass
extract where perhaps 10 percent was not absorbed out.

What you can also see by looking at the right hand, far right hand
column is that the range was pretty variable. There were some that
really had very little or no endotoxin, and there were a few that had
certainly measurable and appreciable amounts.

Those of you that are working on your own PCs, if you would just click
twice now you\'ll see arrows that will appear indicating which of these
extracts are glycerinated, which one is in phenol. And it turns out
that\'s that one that had significant amount of non-endotoxin LAL
activity. And the GP stands for both glycerin and phenol.

The short answer is there really is no overall pattern in any of these
tables associating the solvent with the amount of endotoxin we were able
to measure.

Please go to the next slide.

This is a slide that shows 9 ragweed pollen extracts, two of them are
ragweed mix and 7 of them are ragweed \-- are short ragweed. Again, we
covered a range of manufacturers. Again, with some of them there was
some activity that was residual after depletion with ENP, but you can
see here that for the most part the lion\'s share of the activity seemed
to have been removed by ENP-silica indicating specific endotoxin. And,
again, in the form \-- you see that there is quite a bit of variability
among these different products.

If you go ahead and click three times, you will once again see which
ones are glycerinated, phenolated and which have glycerol and phenol.
And, again, no obvious pattern.

The next slide shows our result with four cat pelt extracts and with
seven cat hair extracts. And here you see a pattern that I think is
interesting, and will comment on it again later.

Again, on a percentage basis, there\'s almost no contribution by any
activity that doesn\'t get completely absorbed out by endotoxin
neutralizing proteins. So, again, the lion share of this seems to be
true endotoxin. But you\'re starting to see a difference in patterns
between the cat pelt and the cat hair that\'s suggests that the pelt
results are significantly higher than cat hair, and I\'ll show you those
data represented graphically in just a couple of minutes.

Those of you on PCs would go ahead and click twice now, you\'ll see
again which ones were in glycerol and which were in glycerol and phenol.
The other two were extracts.

Next slide, please.

And this slide shows perhaps our most surprising results. And this is
the result with 7 d.farinae extracts and 7 d.pteronyssinus extracts.
Again, covering the range of manufacturers. Again, very little
contribution from anything that appears to be other than endotoxin, true
endotoxin. But a real dichotomy between the results of d.farinae and
d.pteronyssinus.

If you\'ll go ahead and click twice, you will see that most of these
were glycerinated and one in each category had glycerin and phenol in
it. Again, no major differences could be observed.

Next slide.

This graph summarizes the \-- basically the percentage of LAL gel
productivity that was not adsorbed out by endotoxin neutralizing
protein. The ordinate is actually not quite accurate. It says gel-clot
activity from ß-D-glucans, but it really would be gel-clot activity from
any ß0glucans or proteases because that would be revealed in this
analysis.

And what you can basically see is that with the pelt, the hair and the
dust mites, there really is an insignificant amount of what\'s not
adsorbed out.

With the pollens, both the ragweed and the grass pollens are you saw in
the tables, there were some in which there was some percentage that was
not adsorbed by the endotoxin neutralizing protein. But in general we
think the LAL gel-clot assay is a pretty reliable assay and it doesn\'t
seem to be interfered with by these other things that we were concerned
about.

Next slide.

Shows our heat inactivation study. This is actually not all of our heat
inactivation studies. We\'ve done more since. But you can basically see
here that with the ryegrass pollen, short ragweed, a couple of cat pelt
extracts and four dust mite extracts the results before and after heat
inactivation are completely identical. And we\'ve actually confirmed
that with several other extracts that we\'ve studied since then.

PARTICIPANT: Jay, have you worked with mold extracts at all?

DR. SLATER: No, that\'s a very good question. This study was limited to
standardized extracts. I think you\'re raising a very good point. I
think we might actually come up with very different answers if you
looked at molds. But we haven\'t done that yet.

Let\'s go on to the next slide.

And the next slide, again for those of you \-- no actually, the next
slide graphically summarizes all of the corrected endotoxin data. Please
note that there are six different categories at the bottom. The grass
pollens, ragweed pollens, cat pelt, cat hair, d.farinae and
d.pteronyssinus.

Please note as well that the numbers are on a log scale, so these
differences are actually quite large. And if you\'ll go to the next
slide, you see the same data but with the significance analysis on the
top. This is based on a rank order sum test, which is the appropriate
test because these data are not normally distributed.

And what you can see is that cat pelt and cat hair are significantly
different with a p - 0.02. And it\'ll be no surprise having looked at
the table with the dust mites, just how significant the difference is
between D.farinae and D.pteronyssinus, the p value is .0003.

Next slide, please.

So, we have a series of conclusions and discussions.

The first conclusion is that we think that for the most part the
observed LAL gel-clot activity probably represents real endotoxin
content and not either the ß-glucan question or protease activity.

Next slide.

So the next obvious question is is this amount of endotoxin
physiologically significant? If you took all of the 58 extracts that we
studied and took them together into a single allergen immunotherapy mix,
he mean endotoxin content of all of those products is 1900 endotoxin
units per mL. Let\'s keep in mind that allergen immunotherapy doses can
range anywhere from 0.5 to 2.0 mL per month. In some cases even more.

Keep in mind as well that depending on the actual distribution of the
mix, that number could be much lower or it could be higher. Obviously, a
mix that consisted only of pollens would not contain nearly this much
endotoxin based on our serving. Likewise, if it contained
D.pteronyssinus, it would contain much, much less.

So the question is how significant is this dose and what does it
actually mean. And in order to find that out, you really have to go back
and dig. Because there aren\'t that many studies in which endotoxin has
been administered by injection to humans. Looking at the two papers that
are indicated

at the bottom of this slide from Wolff in 1973, which is really a review
of many years of previous work that Dr. Wolff had done and looking as
well at a paper by Dr. Michie that appeared [The New England
Journal]{.underline} in 1988, you can estimate that about 40 to 50 EU
per kilogram averaging to 2800 to 3500 EUs, when administered
intravenously can elicit a rise in temperature, heart rate and white
blood cell count.

Now, please note that this is when administered intravenously. There\'s
every reason to believe that the effects of endotoxin injected
subcutaneously would be much less. But there are no data on those
responses to endotoxins administered subcutaneously that I was able to
find.

Let\'s go to the next slide, please.

How are specific endotoxin limits set when they are set? Well, they are
product specific and they\'re generally based on the actual drug dose,
and on an overall limit of 5 endotoxin units per kilogram per dose.

Next slide please. This table just gives you some examples. This is not
an exhaustive survey by any means. Vaccines can have a wide range of
endotoxin content, mostly in the lower range of that range. But there
certainly can get somewhat higher.

The remaining four are drugs, not vaccines. And we determined those
numbers based on published USP limits at the estimated maximum
therapeutic doses. For example, Penicilloyl polylysine has a USP limit
of 5830 endotoxin units per mL, and a typical injected dose
intradermally would be about .01 mLs.

Doxorubicin we also calculated in a very similar way. Fentanyl and
insulin.

The purpose of this is really to give you an idea of what the range of
endotoxin is in other products.

Next slide, please.

The fact is that the clinical consequences of endotoxin and allergen
vaccines really have not been studied. There are no data that adverse
events from immunotherapy are associated with endotoxin level. And
furthermore, even though some people who have seen this data have raised
this possibility, there is no data to support a beneficial effect,
either, of endotoxins.

We do have some data that shows that the endotoxin content of allergen
vaccines doesn\'t effect their diagnostic performance. There have been
some skin testing studies that have indicated that it doesn\'t effect
the size of the immediate skin test reaction. But in terms of
immunotherapy there really are no data. And certainly we would think
that future studies in allergen immunotherapy should always be
controlled for endotoxin dose. This is something we now know is
potentially something that could effect immunotherapeutic responses and
should be monitored in future studies.

And finally, we think that, you know, the role of endotoxin and the
safety and efficacy of immunotherapy should certainly be assessed in
future studies.

Next slide.

What we conclude from these data in terms of descriptive results is
it\'s clear that the pollen extracts have less endotoxin in them than
the cat and mite extracts. It also appears that the cat hair extracts
have less endotoxin than cat pelt. And it appears quite clearly that the
D.pteronyssinus extracts have much less than the D.farinae.

We are in the middle of investigating why that is happening. There\'s
several different possibilities. One is that there really is a
difference in the bioburden between the two different kinds of bugs.
Another is that there\'s an endogenous heat-stable ENP-binding LAL
activator in D.farinae. That would be a first, but it\'s certainly a
possibility.

The other is actually a little bit more intriguing. Endotoxin
neutralizing protein is actually derived from the horseshoe crab. It\'s
certainly possible that there\'s an endogenous endotoxin neutralizing
protein in D-pteronyssinus that\'s confounding our measurements. But
these are all things that are addressable in studies, and we\'re
actually working with individuals who grow dust mites for a living and
trying to work with them to find out what the cause of this difference
is.

Next slide.

So our plan is to further expand our study of endotoxin content. We\'ll
be doing additional standardized and nonstandardized extracts.

We\'re also going to look at different methods. I still think the LAL
gel-clot method is a good method. I think we\'ve shown that it works and
that it\'s reliable in our hands. But there are other methods for
measuring LPS that are quite sensitive and actually can give you a
fingerprint of the kind of LPS in the product. In particular, we\'re
going to be working with colleagues here at CBER on using gas
chromatography, mass spec analysis. And that should be coming on board
within the next couple of months.

In addition, we\'re going to be investigating further the differences
between D.farinae and D.pteronyssinus.

Dr. Lehrer, that ends my discussion of endotoxins. I\'m planning on
going on next to talk about cockroach allergen standardization, but I\'d
be happy to take questions at this point.

CHAIRMAN LEHRER: Yes. I got a question. I think it\'s fascinating the
differences in endotoxin content with D.farinae and D.pteronyssinus. I
wondered if there\'s any differences in the way that these are grown or
harvested or any processing procedure?

DR. SLATER: Actually, interestingly enough that was my first question.
The media that they\'re grown on are absolutely identical. The
temperature and humidity conditions are identical as well.

There is some difference in how long they need to be grown to get to
saturation. But it\'s not that huge a difference. And I don\'t \--
honestly, I\'m sorry, I don\'t remember which one grows faster.

But I spoke to several people that are involved in growing dust mites
and we were really unable to find any obvious growth condition type of
situation that would explain this difference. It\'s a mystery.

The one thing I must tell you when I spoke to the entomologist, the only
entomologist that I found whose an expert on dust mites, is he did
assure me that these species really are quite different from each other.
That we tend to think of them as just sort of a variance of the same
thing, and they really are biologically very different.

So it wouldn\'t be terribly surprising, for instance, if one expressed a
protein that the other didn\'t that somehow interferes with the testing.

It also might be possible that one of them actually has a bioburden that
the other one doesn\'t, and we just don\'t know yet. We\'re working with
the microbiology department over in the Clinical Center, who have been
extremely helpful. And we\'re working with three different sources of
these raw material. And I think we are going to figure out what\'s going
on, but we haven\'t yet.

CHAIRMAN LEHRER: That might provide some interesting information just
about the basis of the assay just because these differences are so
dramatic.

Another question or comment about the assay that\'s used. I think you
touched upon biological significance of some of these measurements, or
tried to, these deal with that. But the \-- I think \-- I always bring
this issue up, and you\'ve touched upon it. In terms of the specificity
of the assay yourself, and I know that you\'re trying to control for
this in terms of the information that you presented. But there \-- one
could always say there may be other types of polysaccarides or \-- that
they cause reactions. And, indeed, the question concerning measurement
of molds I felt was rather interesting. Because I wondered how active
molds would be in such an assay.

And I know that it is accepted in the community. But I was wondering if
there was any good studies that have actually used some of the old time
endotoxin biological assays and showed a, you know, a good correlation?
When you measure endotoxin in crude material it\'s not measuring pure
endotoxin.

DR. SLATER: Are you talking about correlating this with a rabbit-pyrogen
test for instance?

CHAIRMAN LEHRER: Yes, something like that. Some type of classically
accepted endotoxin assay.

DR. SLATER: Well, you know, I must say I think now at this point the LAL
gel-clot assay is probably considered the classically accepted assay.
Certainly it\'s the USP standard now, although there are other tests
that are newer and are considered to be, you know, standard as well. I
don\'t think anybody uses the rabbit-pyrogen test as a standard anymore.

We actually very early in our study, we actually \--

CHAIRMAN LEHRER: No. I didn\'t mean for you to use another assay. I just
wondered if there was any correlations in the literature between these
classical assays and this assay?

DR. SLATER: Yes. There are correlations in the literature between the
LAL gel-clot assay and the rabbit-pyrogen test. I don\'t know whether
there are correlations between the GS mass spec assay and the
rabbit-pyrogen test. I\'m not aware that I\'ve ever seen that.

CHAIRMAN LEHRER: Okay. Are there any other questions from other members
of the Committee?

DR. SOTO-AGUILAR: Yes. I\'m Maria Soto-Aguilar. A question for Jay.

It seems like the D.pteronyssinus, which is maybe more protein genically
than the D.farinae, would you would you speculate or would you suggest
that the presence of endotoxin has something to do with it?

DR. SLATER: I\'m sorry, I didn\'t catch that. That it has something to
do with what?

DR. SOTO-AGUILAR: For potency between the matter D.pteronyssinus being
more potent (technical difficulties) that D.farinae when you\'re doing
an assay that before you may even give a little less dose of D.farinae
versus D.pteronyssinus for somebody whose reacted to both the species.
Would mean that maybe the presence of endotoxin is the one making one
less effective than the other with the endotoxin in the extract vial may
be effected (technical difficulties).

DR. SLATER: Well, you know, I think there are two different ways to ask
the question. If you\'re asking is it effecting the potency of the
allergens in skin testing, I think it\'s probably not. However, you know
obviously one of our questions is whether endotoxin might be functioning
as an averment, which certainly is not a thought that has originated
with me. And you know, that\'s certainly a possibility. But, again,
there really are absolutely no data, and believe me I\'ve looked, to
support either the argument that endotoxin is a problem in
immunotherapy, that is that it\'s associated with either safety problems
or decreased efficacy on the one hand, or on the other hand there\'s
certainly no evidence that it actually potentates immunotherapy.

MR. HAUCK: A question and a comment. This is Peter Hauck.

Jay, when you just slightly talked about mean endotoxins content as in
calculating the immunotherapy dose. You studied pretty much
concentrates, 10,000 and 100,000 PAU, so did you assume that people
would be getting between .5 and 2mLs of these concentrates per month?
Because that seems a little high.

DR. SLATER: Yes, probably is a little high That probably was a bit of an
over-estimate.

MR. HAUCK: Yes. Well, also with the cat and mite, it might \-- you know,
you could be approaching that dose, at least a .5 dose.

Secondly, I note some companies when they do their endotoxin testing on
their water or stoppers, what have you, their components, they use a
chromogenic assay for endotoxins. And I just wondered if \-- I don\'t
know the answer to this.

Number one, was it chromogenic because of the color of the extract be a
problem, the test was that the employee didn\'t do it. And, two, does it
suffer from the same maybe lack of specificity that the limulis test
has?

DR. SLATER: Well, the chromogenic assay is also based on the same
protein cascade, just generates a different endpoint, not a clot.

MR. HAUCK: Okay.

DR. SLATER: So, you know, I think \--

MR. HAUCK: But it gives the same results pretty much?

DR. SLATER: You would think so. We didn\'t actually direct \-- and I
don\'t know about the issue of the chromogenic assay. I don\'t think it
would be a problem in terms of the pigmentation of the product because
you have to dilute these products quite a bit.

MR. HAUCK: Okay.

DR. SLATER: My guess is it would not be a problem. But we didn\'t have
any experience with that.

MR. HAUCK: Okay. Thank you.

CHAIRMAN LEHRER: Are there any other comments, questions?

DR. MacDONALD: Jay, this is Susan MacDonald.

I enjoyed seeing your poster at the academy meeting. And I compliment
you for taking this on.

I also seem to remember that there was a poster from a guy from West
Virginia who had a mice model who gave \-- made the mouse allergic and
then gave endotoxin and showed that it switched to a quote/unquote TH1
type of \-- kind of cytokin response. So maybe \-- last year at this
meeting I was thinking that endotoxin was \-- after reading these toll
receptors and we were causing problems, that perhaps we\'re actually \--
we might be actually helping people if there are doses of endotoxin in
these extracts.

DR. SLATER: Or at least we\'re helping mice.

DR. MacDONALD: Oh, mice. That\'s right. And being from Hopkins, we
don\'t like mice.

DR. SLATER: It\'s a very good point, and I think it\'s a good
theoretical question. Again, I think it\'s actually critical that it be
looked at and certainly worldwide there are many immunotherapy studies
that are going on. I think it\'s very important. And, hopefully, by
disseminating this data and with the publication of studies like this,
we\'ll indicate to people that they really have to be very careful and
control the endotoxin content so we learn something.

DR. MacDONALD: I agree. Thank you.

CHAIRMAN LEHRER: Okay. I guess there are no more comments or questions,
and we can continue.

Jay, with the cockroach allergen standardization.

DR. SLATER: Terrific. Thank you very much.

And, again, thank you for your comments regarding the endotoxin.

I\'ve taken the liberty of saving the best for last. This is, I think, a
very encouraging talk the next one. If you think endotoxin was good,
this is better.

Next slide please.

We\'re going to talk now about cockroach allergen standardization. And
this isn\'t the first time you\'ve heard about standardization, those of
you that have been on this committee over the past several years. But I
actually think that we\'re sort of capitalizing on some of our previous
work, capitalizing on some very important collaborations. And I think
we\'re really going to make some good progress in a very important area.

I\'m going to talk about \-- there are a number of things that my lab is
doing involving the standardization of cockroach allergen extracts.
We\'re actually going to focus on the clinical studies and some of the
depletion studies. I\'m not going to talk about the IgE combinatorial
library work that Jonny Finley is doing my lab. And I\'m not really
going to talk much about the correlation studies. But those are,
obviously, important for our efforts.

The next slide.

Those of you who are on PC, go ahead and click twice and then click to
the next slide that\'s \"Current Standardized Allergens.\"

There\'s a danger in this part of the talk of preaching to the choir too
often, but these are the currently standardized allergens. There are 19
of them. And they\'re listed on this slide.

The purpose of allergen standardization.

Go to the next slide, please.

Is to establish for each of these allergen vaccines a U.S. standard and
to establish a testing procedure to measure the allergen content of each
of those allergen extracts. The manufacturers, as you know, may use the
established procedure or they may choose to develop an equivalent
procedure that they relate to the established procedure.

Next slide please.

The questions comes up as to which allergens should be standardized. And
the sort of trivial answer is well all of them should be standardized.
But we have to make choices and we have to do \-- we have to make
choices to which to do. And even if we were going to do all of them,
we\'d have to choose which ones to do first.

These criteria were discussed several years in a Advisory Committee
Meeting. And I really want to focus your attention on the second and the
last criteria, which I think are particularly relevant for today\'s
discussion.

The second point is that we should choose which allergens to
standardized based on the consistency of the currently marketed product.
The idea there is that if the currently marketed products are very
consistent and very high quality, should probably will not be that much
impact if we choose to standardize. And we might want to put that toward
the end of the list.

Conversely, if a product is very highly variable, then we might want to
move that product up. Especially if the last criterion is met, and that
is if there\'s a relatively high public health impact of the correct
diagnoses or of adequate treatment.

Next slide please.

Among the allergens that have been associated with asthma are several
indoor allergens, particularly dust mites, cat, more recently cockroach
mold and dog. And among the outdoor allergens, molds have been
associated with asthma as well.

The next slide \-- you knew if I was going to talk about cockroach, I
had to have a slide like this.

PARTICIPANT: Ugh.

DR. SLATER: Thank you. That\'s what I wanted to hear.

Now on the bottom you have panels with the standard, American, German
and oriental cockroaches. But I just have to tell you, those two roaches
that \-- this really is \-- this is not a \-- this is not a trick of the
digital age. Those cockroaches really are the size of a piece of
birthday cake. Those are hissing Madagascar cockroaches, and they really
do hiss. And they were actually on tour and they were here at the
Smithsonian several years ago. You could actually go down and handle
them. But we\'re not talking about that, so I\'m going to get off that
slide.

Next slide, please.

PARTICIPANT: Good.

CHAIRMAN LEHRER: Do they have cockroach allergy in Madagascar?

DR. SLATER: You know, I never asked the question. My guess is the
problem with those roaches is more significant than allergy.

CHAIRMAN LEHRER: I suspect that\'s the case.

DR. SLATER: It may be competition for food source.

PARTICIPANT: And birthday cake.

DR. SLATER: And birthday cake, right.

So why is cockroach allergy important? Well, we know that especially in
the urban environment cockroaches are ubiquitous. They\'re extremely
difficult to control. And they appear to be associated with asthma,
especially asthma in the inner city.

Next slide.

Why do we think that cockroach allergen standardization is important?
Well, I think it\'s important to the patient to provide for more
accurate diagnosis of cockroach allergy. And, hopefully, to provide for
safer and more effective immunotherapy for cockroach allergy.

To the physician/scientist, I think it\'s critical because, as I like to
say if you can\'t measure, you can\'t really study it. There are lots of
studies going on and we\'re just going to have better science if we have
a standardized cockroach allergen extract.

And, obviously, at the FDA our concern is to have safer and more
effective products.

Next slide, please.

So the first phase in standardizing an extract starts in the laboratory,
and that\'s work that we\'ve done to a great degree, to develop and
adapt methods for allergen determination and to compare the allergen
content of different lots of allergen.

Next slide, please.

And this is just the front page of that article from the [Clinical and
Experimental Allergy]{.underline} from last year. This is work that
Melissa Patterson and I did on looking at cockroach allergen extracts in
the United States.

Next slide.

The goal of that study, which I\'m going to review in the next few
slides, was to determine the consistency of available U.S. products in
protein content, in specific allergen content and in overall
allergenicity. And to determine the best lot release measures that might
be used.

Next slide.

In order to start at a starting point, we needed to use something as a
reference. And we chose to use as a reference extracts labeled E2-Cg and
E2-Ca. Cg is for German cockroach, Ca is for American cockroach.

These were both purchased by CBER from Holister Steer, which was then
Bayer Laboratories. These were lyophilized relatively high potency
extract that had been previously characterized by this laboratory.

There was some limited skin test data that we could use to try to relate
these to. And most importantly, it was lyophilized and it was available
in very large quantities.

Next slide, please.

We compared these extracts to 26 commercial extracts that we obtained
from all of the allergen extract manufacturers. Six of them were aqueous
\-- I\'m sorry. Did I put you on the next slide where the table is? Is
everybody on the table with the American and German cockroaches?

There were 6 aqueous and 20 glycerinated extracts.

Now let\'s go to the next slide, which should be titled \"Relative
Potency of All Cockroach Extracts As Determined by Competition ELISA.\"

For those of you that ar working on your own PCs, if you would do me a
favor and just go ahead and click three times. That\'ll highlight in red
the 3 numbers that I want you to focus on.

That column is the relative potency scaled to protein content. So the
third column, which is just the relative potency, is not controlled for
protein content, which was highly variable among these different
extracts.

What you can see by looking at the scaled relative potencies is that the
potency of the glycerinated American extracts was on average about
one-twentieth the potency of E2-Ca. What you can also see is that the
potency of the German cockroach glycerinated extracts was about
one-fifth of the potency of E2-Cg. And the aqueous extracts were
similarly quite low.

Now, a very reasonable next question would be well how potent were E2-Ca
and E2-Cg? And I don\'t have that data in this presentation, but based
on the limited skin testing data that had been done before, these were
both extracts that were less than 10,000 BAUs per mL. So they were not
very highly potent extracts. And conversely, the German and American
cockroach extracts that we tested, they were commercially available,
were much less potent than those.

Next slide.

At the time we really only had good ways to measure Bla g 1 and Bla g 2
in the German cockroach extracts. We didn\'t have the availability of a
Bla g 4 or 5 assay at the time.

And what you see here is a table that shows the mean and range Bla g 1
and Bla g 2 contents of the glycerinated German cockroach extracts. You
see at the bottom the E2-Cg content for Bla g 1 and Bla g 2.

And what you can see is that the commercial extract had Bla g 1 than
E2-Cg. And they actually had more Bla g 2 than E2-Cg. What you also see
is that the Bla g 2 content was really highly variable, ranging from 8
to 66 micrograms per mL. The bla g 1 content actually was fairly
constant, really only running over about a twofold range.

Next slide, please.

We did do some correlation analyses among the German cockroach extract
between protein concentration, relative potency, Bla g 2 and Bla g 1
levels. And in general they were fairly correlated. In fact, the weakest
correlation was between protein content and relative potency. But there
were actually good correlations amongst several of the others, notably
the Bla g 2 content and relative potency were fairly closely correlated.

Next slide, please.

So our conclusion from that study was that the commercially available
cockroach allergen extracts very widely in protein content, Bla g 2
content, SDS-PAGE banding pattern, which I didn\'t show you, and overall
allergenicity. And they appear to be less potent and contain Bla g 1
than the candidate reference extracts.

Next slide.

So our conclusion from this is that we think that from the point of view
of the variability of the products as well as the public health
importance of good cockroach allergen vaccines, our conclusion was that
cockroach allergen vaccines need to be standardized.

Next slide, please.

What we needed next was to develop a new set of cockroach references to
characterize those references to do ID~50~ testing, which I\'ll be
talking about shortly. And to proceed to the next phase.

Next slide.

So in terms of the clinical testing, skim testing is really the bedrock
upon which we can move forward with standardization.

Next slide, please.

And that skin testing is the ID~50~ testing or IDEAL testing that was
designed and promulgated here at CBER and was used to successfully
standardize grass pollen allergen vaccine. And there are four basic
steps in IDEAL testing.

First of all, this is a technically demanding test. The testers need to
be demonstrated in being proficient in the test. You need to recruit an
adequate number of study subjects. You need to actually do the testing
and then you need to analyze the testing.

The next slide in shows in very abbreviated form the essential elements
of that testing. That testing involves serial three-fold dilutions of
the extracts, performing multiple tests on a single individual. The
measurements in the bottom of orthogonal diameters of the erythema of
those test results, and finally the calculation of the D50, which is the
dilution at which a 50 millimeter sum of erythema result occurs.

Next slide, please.

The fundamental point of the inclusion criteria as you want to have
individuals who really are allergic to the product that you\'re testing.
You want to have individuals that have some clinical history that
indicates that they have an allergic disease, such as allergic rhinitis
related to exposure to the allergen of interest. And you also want to
have individuals that have a positive skin response to the allergen,
otherwise the plot that you\'re going to get for the previous slide will
just be flat.

We\'d like to get individuals who demonstrate at least a minimal
sensitivity of 30 millimeters of sigma E which is the sum of the
erthyema diameters to the allergen concentrate.

Next slide.

The exclusion criteria are largely aimed at safety issues as well as the
importance of being able to get valid reproducible data.

Next slide, please.

Those of you were at the Advisory Committee two years ago have seen a
version of this formula before. This is a formula that\'s based on the
two one-sided test analysis, designed by Schuirman several years ago.
This particular analysis is described in a paper that Ron Rabin wrote in
collaboration with Rich Pastor and Tony Lachenbruch here at CBER that is
going to appear in the proceedings of the most recent Paul Ehrlich
Institute.

From that analysis we see that n, the number of study subjects that we
need to study, is a function of sigma, which is the standard deviation
of the measurement in this case of the D50 measurement and delta, which
is the acceptable difference that you might not be able to detect if
your sensitivity was too low.

So number sigma is the standard deviation of the measurement. And delta
is the minimal difference that you absolutely want to make sure that
you\'re able to measure.

What you\'ll see here as well by looking at the above formula is that n
is a function of the square of the ratio of sigma and delta. So for
instance, so if the ratio of sigma to delta is one, at a beta of .05, n
is 26 or 26 study subjects.

If you double the ratio of sigma to delta, and you go through a sigma
delta ratio of 2, you don\'t double n, you actually quadruple n. You go
up four fold.

Now, if you\'re looking carefully at this slide, keep in mind that n is
always rounded up. So, for instance, where n is 18, it\'s actually 17.1,
which is why 60 as you\'ll note is really not four times 18. But these
numbers are rounded, that four fold calculation doesn\'t always work out
precisely. But that\'s because of the rounding.

So now we\'re going to talk about what sigma and delta are or should be
and how we\'re going to estimate the number of study subjects that we
need.

Next slide, please.

I\'m going to argue in this slide that delta for the D50 should be about
ten percent. Ten percent seems like a little bit of a tight measurement,
I suspect, to many of you. But I think we have no choice but to be able
to measure the D50 within 10 percent. And that\'s because the number
that we really want to come out with is the BAUs per mL of these
products. And the BAUs per mL is an expediential function of D50, as
given in the formula in the second line of this screen.

Just to give you an example of what ten percent difference is in D50
would yield, this table shows what ten percent differences in D50 would
give you in terms of your BAL/mL as changes.

If you had a fairly potent extract with a D50 of 14, this is in the top
row of the table, a ten percent decrease in the D50 would lead to an 80
percent decrease in the BAU measurement. Ten percent increase would be
nearly a four fold rise in the BAU measurement.

Now, if you go to less potent extracts with D50s of 10 and 8, those
differences are somewhat less, but you can see that you really don\'t
want to get much beyond a D50 difference of the percent if you want to
have anywhere near a good estimate of what your BAU/mL is going to be.

Next slide, please.

Those of you that have PCs, please go ahead and click one more time,
which will focus your attention on the numbers at the bottom of the
table.

To estimate what the sigma for D50 is going to be, we actually have some
previous data. And those data come from an article by Smith in the
[Anals of Allergy]{.underline} in 1995 in which they looked at three
different cockroach extracts in a number of allergic individuals. And
I\'m going to direct your attention to the bottom of the page where you
can see that the standard deviation for their mean sigma E measurements
were on the order of 12 to 20 percent. In the case of the German
extract, the standard deviation was 1.21 with a mean of 10.6. With the
American cockroach extract, it was 1.94 over a mean of 10.04.

Next slide.

We also have data from an earlier CBER study of German and American
cockroach extracts that was performed at three centers, Cincinnati,
Georgetown and Johns Hopkins. And I pulled together those data, and that
shows you roughly, again, of 20 percent sigma for these measurements.

Next slide, please.

So based on a ratio of about 1.5 between sigma and delta, and based on a
power of 80 percent, we estimate that for each of the extracts we would
need an n of about 39 or 40 study subjects.

Now, there are two different allergen extracts we want to study,
American and German cockroach. That would indicate 80 study subjects.

We also wished to have geographical diversity in our study. We know that
there can be some differences in allergen recognition in different parts
of the country. I\'m not sure that we would necessarily find it from
results in different parts of the country, but we do need to try to
obtain that. And, therefore, we\'re estimating that we\'ll need about
240 study subjects for this study.

Now, we may actually need fewer because it is possible that we will be
able to have some overlap between American and German cockroach allergic
subjects. That is, if the study subject allows us to test them to both
allergen. And if that\'s the case, we can anticipate that we may be able
to do this with between 150 and 200 study subjects.

So at this point we think we\'ve established that cockroach allergen
vaccines need to be standardized, and our next step is really to go
forward with this clinical study. And at this point I want to report on
a fairly happy collaboration that seems to be going forward very nicely.

Next slide, please.

Some months ago I approached the NIAID Inner City Asthma Consortium to
discuss the possibility of collaborating. I should note, even before you
go any farther, that we\'re fortunate that Dr. Gruchalla is not only on
this Advisory Committee, but she\'s also a member o the Inner City
Asthma Consortium steering committee, and has been involved in this
process for the last several months.

For those of you that don\'t know, this consortium 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. This consortium of basic scientists and clinical investigators
will conduct clinical studies to elucidate the immunopathogensis and the
natural history of asthma in this population.

Next slide, please.

This is a list of the members of the consortium steering committee, who
I must say were very hospitable to this project. I think the steering
committee really recognized that their ability to go forward and study
the role of cockroach as a causative agent in inner city asthma was
dependent on having good reagents and this was really a happy confluence
of interests between CBER and NIAID.

Next slide, please.

So, where are we? Well, the steering committee approved the process and
we\'re actually in the process \-- they are in the process of
identifying the study centers that will participate. You should
understand that the Inner City Asthma Consortium has in place study
centers in the inner city that are intended to perform studies on inner
city asthma. So they are simply finding out which ones will be willing
and able to participate in the study.

We need to, obviously, get many IRB approvals. We need to get IND
approval, which will be an interesting experience for me. We need to
order the extracts, distribute the material.

I think the proficiency testing should not be a problem. And then we\'re
going to proceed with the study.

As we proceed with the study, we\'ll be generating data. The inner city
has a consortium, has a consultant that will be analyzing the data. We
will also be obtaining the raw data and analyzing it ourselves. We\'ll
be doing correlative studies with in vitro tests. These studies will be
done both in my laboratory and in other participating laboratory from
the Asthma Consortium. I anticipate this is going to be a very, very
productive collaboration.

I should tell you that our time table now is aimed towards actually
starting the clinical part of the study by the end of the summer. Once
that is starts, it really should not take too long to generate the kind
of data that we all need to move forward.

Next slide.

CHAIRMAN LEHRER: Jay, could I just ask a quick question?

DR. SLATER: Absolutely. Sure. Yes.

CHAIRMAN LEHRER: That your citing this very important study. And this
kind of came to mind in the beginning when you were talking about the
variability of the commercial reagents and also the fact that they
seemed to have somewhat lower activity than expected. And it brought to
mind the studies of Bob Esch and others with the fact that cockroaches
with proteolytic enzymes and that they can essentially digest a lot of
the allergens.

What I\'m asking is that it\'s \-- your dealing with standardization of,
and particularly this kind of study which is kind of, as you say,
useful. Has stabilization been addressed at all prior to embarking on
this?

DR. SLATER: I think it\'s a very good question.

We really didn\'t see any evidence in our previous study that there was
tremendous instability in the products. We did look at several of the
products longitudinally to see if we noticed any inherent degradation.
In fact, interestingly enough, products from the same manufacturer even
of different ages were actually fairly consistent among themselves.

So we didn\'t see a lot of evidence of that.

I\'m not sure necessarily how active the proteases are in 50 percent
glycerol. Certainly what we\'re going to be using are going to be
glycerinated extracts at this point. And we are going to be looking at
stability issues as we move forward. But at this point what we\'re going
to do is we\'re going to obtain extracts that are glycerinated. We\'re
going to characterize them as well as we know how in the laboratory.
We\'re going to get them, do the clinical studies and then we\'re going
to see where we are with that.

CHAIRMAN LEHRER: I think that sounds good, particularly that they\'re
glycerinated. But something you might want to keep in mind, at least
discuss with your colleagues in the study, is that everything is done
the same way. For example, when extracts are brought out for skin
testing or whatever, sometimes some people leave them out for a couple
of hours, sometimes people leave them out for a whole day; those kinds
of things that if you can try to standardize in some way or just keep
them in the same time frame I think are important. Because they could
make a big difference in your results. And I think that this such an
important study, you want to minimize any of that.

DR. SLATER: Well, I think that raises a good point. The way we\'re
actually going to store the XEX, is they\'re going to be stored in
insulated racks which the investigators will keep in their
refrigerators, and then they\'ll take them out in those racks. So we
really should have fairly good temperature control, at least within the
constraints of the study. But you\'re raising a good point.

CHAIRMAN LEHRER: Good. Thank you.

DR. SLATER: So I think I already directed you to go to the next slide.

It\'s not going to be any surprise to any of you, given the previous
talk, that we\'re actually looking at the endotoxin content of cockroach
allergen vaccines. It\'s also not going to be any surprise to any of you
that there\'s a fair amount of endotoxin in these allergen vaccines,
although again I\'m often surprised at how variable it is. But some of
the levels are actually quite high, and we are going to be looking at
that as well.

Next slide, please.

So certainly the endotoxin issue needs to be studied in depth.

Dr. Lehrer, just to address a question I suspect is in your mind
already, we do heat inactivation on these, and we didn\'t really find
any evidence of heat inactivable activity causing the gel-clot to occur.
Likewise, when we did the endotoxin neutralizing protein absorptions, we
really found that virtually all of this was adsorbed out. So it appears
to be that this is real endotoxin that we\'re measuring. But, again, the
proteolytic enzymes could be there, they\'re just not contributing to
the LAL gel-clot measurement.

Next slide.

The next question that I\'d like to address as part of our cockroach
allergen standardization protocol, and this is actually the last topic
of the afternoon, is whether overall allergenicity measurements will be
sensitive to changes in specific allergen levels. This is a question
that really was first raised shortly after I came to the lab when we
were looking over mite stability data. These were data that appeared in
publication in JACI in 2000 looking at the stability of dust mite
preparations over long periods of time.

And one of the interesting things that we found that we really didn\'t
quite know how to interpret was that when we measured the relative
potency of these products over 12 months, we found that they were
absolutely rock stable, whether we stored them at minus 20 degrees or at
4 degrees. They were really quite impressively stable in terms of our
measure of overall allergenicity. But when we looked at specific
allergen, either by measuring group 1 and group 2 allergens in dust
mites or by looking at specific bands in the SDS-PAGE we found
significant loss of activity or of bands when the products were stored
at 4 degrees. Not minus 20 degrees, but at 4 degrees.

So it raised the question that I\'d like to address in the next couple
of slides.

Next slide, please.

When you have an assay in which you\'re measuring a particular protein
with a monospecific antiserum, this could either be a monochronal
antiserum antibody or it could be polychronial antibody that was raised
to a specific well defined protein. What you basically have is the
situation in this cartoon. In this cartoon the multi-colored smiley
faces on the right represent five different proteins in your allergen
mixture and the blue arrow to the left of them is the enzyme that you
use \-- is that the antibody that you\'re using to detect one of those
proteins. In this case, you\'re using the blue arrow to detect the blue
protein.

And you can easily imagine that if the amount of the blue protein begins
to drop out, that you\'re specifically be able to measure that dropout
of the blue smiley face. But the situation we have with the competition
ELISA is much more complicated.

Next slide.

With the competition ELISA we have a somewhat different picture. We\'re
measuring, let\'s say in this carton, five different proteins with five
different colored arrows, five different antibodies. So each one
represents its own smiley face.

Well, it\'s not entirely clear that if you lose one of those proteins,
you\'re actually going to pick up the signal. It\'s going to be
sensitive enough to detect it, and that\'s shown in the next slide where
you see that in the top part all five of the smiley faces are
represented and all five of their colors. But in the bottom one, those
of you that are looking closely, will see that the blue one is gone. And
there\'s a white space in its place.

So the question arises as to whether when you deplete a complex protein
mixture of a single protein and you\'re measuring with a mix of
different antibodies whether the signal is actually going to pick up
that loss of the specific protein.

Next slide.

We are proposing to address this question by doing a series of depletion
analysis of German cockroach extracts. We have already raised very high
titer specific antibodies to Bla g 1, 2, 4 and 5. We plan to selectively
adsorb the cockroach extracts that we have with each of these. We\'re
then going to test for specific allergen levels and then test for
overall allergenicity using the competition ELISA.

Next slide, please.

This is not a very impressive SDS-PAGE. We\'ve actually attempted this
with both Bla g 1 and Bla g 2. I\'m going to show you the Bla g 1 data,
and I\'m going to tell the Bla g 2 data, because that actually just came
to me yesterday.

This is the SDS-PAGE. And what you see here in the left most lane is
just the molecular weight standard. The next lane, which is labeled CR
is just the crude cockroach extract. We then have an empty lake. The
next lane that\'s entitled Sham was a cockroach extract that was
adsorbed with just normal rabbit serum. And finally the last one is the
one that was specifically adsorbed with the anti-Bla g 1 serum.

And what you can see, and really you guys will have to have an awful lot
of faith in me, and I wouldn\'t blame you if you didn\'t, the only band
that I could find that was really different between the sham and the
adsorbed one was that band all the way at the bottom that you can see
appears to be absent in the last lane and is not quite absent in the
sham lane. There\'s really no other difference that you can see on the
SDS-PAGE. And I\'m not even that sure that difference is highly
significant.

But let\'s go to the next slide.

What can you show, though, is that as measured by ELISA in our
laboratory in the upper left hand panel, there\'s a big difference in
the ELISA results with the product that was adsorbed with the anti-Bla g
1 as opposed to the one that was adsorbed with just the regular rabbit
serum.

In addition, when we sent these same samples to a contractor to have it
tested for Bla g 1, 2 and 5 content, you could see a fairly dramatic
drop in the Bla g 1 content from 149 unit per mL down to 14.8 units per
mL. The Bla g 2 content appeared to go up and it\'s probably not
significant. And the Bla g 5 content appeared to go down somewhat, but
not quite as dramatically as the Bla g 1.

So on the next slide we compare these two extracts with the competition
ELISA. And you can see that there\'s really no difference between those
two in this particular competition ELISA.

Now, let me just stop for a second. There are questions that we need to
answer about the competition ELISA as well. We do know that the serum
that we used, the serum pool that we used, does recognize Bla g 1, and I
think that\'s probably the most important question. But we need to go
back and actually qualify and perhaps retool the competition ELISA to
try to optimize it better than it\'s been optimized so far. And what I
mean is that these selectively adsorbed products that we\'re generating
could actually help us in our effort to optimize the competition ELISA
better. But it certainly does raise the question as to whether a
measurement of overall allergenicity that looks at 5, 6 or 7 allergens
really is capable of detecting a specific drop in a specific allergen of
interest.

Next slide.

So, the final conclusion that we have that we\'re going forward with is
we have to acknowledge the possibility that our best surrogate tests,
our best test for potency, may in fact not be the competition ELISA. And
it may not be the competition ELISA for two reasons.

There may be other tests that will simply correlate better. But in
addition, if we decide that one or two or three proteins are
particularly important, we need to make sure that changes in those
proteins are picked up in whatever assay or assays we choose.

Last slide.

So in conclusion I\'m very optimistic that we\'re going to be able to
standardize German and American cockroach allergen vaccines in the near
horizon, and this will facilitate definitive studies on the role of
cockroach allergens in inner city asthma, and on the best methods for
eradication and treatment, and it will make for safer nd more effective
products.

And I want to thank you all very much for your attention.

CHAIRMAN LEHRER: Thank you very much, Jay.

I have a quick question concerning reactivity with the issues that you
were addressing in your last several slides about the importance of
reactivity to particular allergens within the mixture and trying to get
at that looking at removal. And then when you started talking about your
reagents, I think that really hit the mark in terms of the serum that
you\'re using, and that\'s really going to in some ways define \-- or in
many ways define your results.

Do you think another way to look at this might be to just see what type
of reactivity the populations have? I think a lot of this has been done
already in terms of different allergens. Well, perhaps for the newer
allergens it hasn\'t been. Just to see that the variability? I mean, do
you have people that vary that differently in terms of these individual
allergens? The reactive. Because those are the ones that, you know,
could be most effected by using a single allergen or several allergens
to standardize the extract.

DR. SLATER: Right. Well, part of the study \-- I didn\'t really go
through it in all of its glory. But part of the study that we\'re
planning to sort of tag onto the IDEAL study, is we are going to be
getting blood samples from each of the study subjects. And we\'re
actually \-- one of my intentions is to actually use that to correlate
the skin test result with specific antigen reactivity in each of the
study subjects. And then it\'s determined whether it\'s even
theoretically possible to construct the serum pool that will detect each
of the allergen specifically.

Another alternative, of course, is instead of having a single
competition ELISA that measures overall allergenicity, to have a
multiple individual test and to set multiple individual limits. But that
has problems associated with it as well. But it is one of the objectives
of the collaborative study to try to answer some of these questions.

CHAIRMAN LEHRER: Good. I think it\'s particularly important to know the
variability of reactivity, and it may not be as variable as it could be.
But I think that will provide very useful information.

Very nice.

Now are there other comments or questions in the Committee?

MR. HAUCK: I have a couple of questions and comments.

Jay, did you notice or did any of the technicians that were doing the
assays with the commercial cockroach extract, did they notice sediment
or precipitate?

DR. SLATER: Yes.

MR. HAUCK: Yes, they did?

DR. SLATER: Well, in other words in our work with the commercial
cockroach extracts?

MR. HAUCK: Yes.

DR. SLATER: Yes. Yes, there were. And we made special effort to assay
them as soon as we could after we got them. But some of them were
actually precipitated upon arrival.

MR. HAUCK: It\'s a major practical problem that the manufacturers have,
even with the glycerinated cockroach extract. And we don\'t know what is
causing it.

Kind of the latest thing, you talked about comparing \--

CHAIRMAN LEHRER: Could I interrupt for a second, Peter?

MR. HAUCK: Sure.

CHAIRMAN LEHRER: Is there any information that the allergens are
actually in those precipitates or do the precipitates alter the
allergenicity?

MR. HAUCK: I\'m not aware of any data.

DR. SLATER: Well, we\'ve looked at it with several other allergens, and
we haven\'t really seen any evidence of that. We also looked at it
briefly with some of our cockroach extracts, and we didn\'t see any
evidence of that either.

But we certainly can\'t say that we\'ve done it on enough extracts to
draw a broad conclusion about that.

CHAIRMAN LEHRER: That would be important information.

DR. SLATER: Absolutely.

MR. HAUCK: May I continue?

CHAIRMAN LEHRER: Please.

MR. HAUCK: Sure. Thank you.

Another kind of related issue, when you compared the reference that you
had from Holister Speer and you compared relative potency based on
protein content between the two, the protein content found in the
commercial material and the protein content in the reference, correct?

DR. SLATER: Yes.

MR. HAUCK: Okay. Be aware that, I believe the reference material was
dialyzed and the dialyzed products, cockroach is very rich in urea,
which will assay as either an enheightened protein or protein nitrogen.

And as far as I know, the commercial extracts are not dialyzed, so
they\'re very rich in urea, which also may in fact be part of the
problem that we manufacturers have with the precipitating issue.

CHAIRMAN LEHRER: Peter, I\'m sorry. So the dialyzed product would not
have much urea in it, is that what you were saying?

MR. HAUCK: Right. So it would have a lower protein content and
therefore, if you compare it on an equal protein content, it\'s very
likely that the reference would have a lot more allergen activity per mL
protein.

Maybe another way to look at it would be if you could get milligrams of
starting material.

CHAIRMAN LEHRER: Yes.

MR. HAUCK: Jay, I could double check on that. But that \-- that\'s what
I understand. But it also could be a possibility that maybe that\'s the
way to manufacture the product, dialyzed.

DR. SLATER: I think we found that the E2-Cg and E2-Ca had a
substantially higher protein content than the others. So I\'m not really
sure. But what you\'re saying is that that protein was actually protein
whereas the protein that we were measuring in that cockroach \-- most
cockroach extracts was urea

MR. HAUCK: That\'s been my experience and my colleague\'s experiences.

DR. SLATER: Thank you.

MR. HAUCK: Okay. And then just a comment on the \-- you\'re to be
applauded on the sides of the clinical trial. And I just wondered if you
thought about, and maybe Dr. Lehrer can respond to this as well, the
American cockroach and the German cockroach don\'t necessarily hang out
together except for maybe New Orleans. But that\'s probably a good
reason to use a couple of different geographical areas.

I think you may have to demonstrate if you\'re going to use American and
German on the same patient, that you\'re not just measuring activity,
but you should demonstrate that those allergens from both species exist
in that patient\'s environment.

CHAIRMAN LEHRER: I think that\'s a very good suggestion. And you really
want to look at the diversity of areas in terms of reactivity.

DR. SLATER: Thank you.

CHAIRMAN LEHRER: And just a comment on New Orleans cockroaches. I think,
as any of you know who have visited our fair city, that New Orleans\'
cockroaches I think will hang about with just about anybody. And they do
that.

DR. SLATER: And they don\'t just eat birthday cake, right?

CHAIRMAN LEHRER: Not yet. Okay.

Any other comments or questions? Okay.

Jay, I wanted to thank you very much for an excellent presentation.

PARTICIPANT: Yes. Thank you, Jay.

CHAIRMAN LEHRER: Next on the agenda, I think I have it correct this
time, is the public hearing.

Bill, could you make the appropriate announcements regarding the open
public hearing on our agenda?

EXECUTIVE SECRETARY FREAS: Thank you, Dr. Lehrer. As part of the FDA
Advisory Committee meeting procedure, we hold open public hearings in
order to give anyone not on the agenda a chance to address the committee
and make a statement concerning matters pending before the committee.

In response to the [Federal Register]{.underline} announcement regarding
this meeting, I have not received any requests today to participate in
the open public hearing.

I would like to transfer to either Shiela Langford or Denise Louster
down in Conference A. And Shiela or Denise, could you activate the
microphones by saying good afternoon? It\'ll take a second. If you have
your mute button on, would you take the mute off.

MS. LANGFORD: Hello.

EXECUTIVE SECRETARY FREAS: Yes. Can you keep talking?

MS. LANGFORD: There\'s no one here.

EXECUTIVE SECRETARY FREAS: Pardon?

MS. LANGFORD: There\'s no one here for open public hearing.

EXECUTIVE SECRETARY FREAS: Okay. We do not have any open public hearing
at this time, Dr. Lehrer. We have a small group of people down in
Conference Room A.

We appreciate your participation in the meeting. And that closes our
open public hearing.

And Dr. Lehrer, I turn it over for you if there\'s additional Committee
discussion.

CHAIRMAN LEHRER: Thank you very much, Bill.

So in the concluding part of this meeting then, are there any other
comments or discussion concerning the information presented during this
meeting?

Any closing comments to be made by anyone?

EXECUTIVE SECRETARY FREAS: Yes, I just would like to thank all the
Committee.

First of all, I would like to thank you for chairing this meeting. And
thank all the Committee members for putting up with the difficulties
that we have encountered today.

I do apologize for the technical difficulties. I believe if you were
aware of the cold, wet, rotten rainy weather that we are having now in
Washington, D.C., you\'d be very appreciative of the fact that you were
able to attend by video conference and teleconference as opposed to
experiencing the climate down here.

But, again, my thanks to everybody for participating.

I would like to remind you that the next tentatively scheduled meeting
of the Allergenic Products Advisory Committee is November 19, 2003.
We\'ll be contacting you in September, as soon as we start formulating
any plans regarding the possibility of holding a meeting on that date.

So, again, my thanks and that\'s all.

CHAIRMAN LEHRER: Okay. I\'d like to thank the members of the Committee
for participating and also the staff of CBER for arranging the meeting
and the presentation.

If there are no further comments, then we can adjourn the meeting. Thank
you all very much.

(Whereupon, at 3:22 p.m. the meeting was adjourned).