1 
 
                 DEPARTMENT OF HEALTH AND HUMAN SERVICES 
 
                       FOOD AND DRUG ADMINISTRATION 
 
               CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

             BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE 
 
                           TWENTY‑SIXTH MEETING

                         OPEN SESSION ‑ VOLUME II

 
                         Tuesday, March 21, 2000 
 
                                8:00 a.m.

 
 
                           Holiday Inn Bethesda 
                          8120 Wisconsin Avenue 
                            Bethesda, Maryland 

                                                                  2

                               PARTICIPANTS 
 
       Daniel R. Salomon, M.D., Acting Chairperson 
       Gail Dapolito, Executive Secretary 
 
       MEMBERS 
 
                 Hugh Auchincloss, Jr., M.D. 
                 Richard E. Champlin, M.D. 
                 Carole B. Miller, M.D. 
                 Esperanza B. Papadopoulos, M.D. 
                 Edward A. Sausville, M.D., Ph.D. 
 
       TEMPORARY VOTING MEMBERS 
 
                 Jose Francisco Cara, M.D. (I) 
                 Joan Harmon, Ph.D. (I) 
                 Pamela Hartigan, Ph.D. 
                 Lynne L. Levitsky, M.D. (I) 
                 Robert S. Sherwin, M.D. (I) 
                 Alice J. Wolfson, J.D. 
 
       GUEST SPEAKERS 
 
                 Robert Goldstein, M.D. (I) 
                 Bernhard J. Hering, M.D. (I) 
                 Norma Sue Kenyon, Ph.D. (I) 
                 John O'Neil (I) 
                 Camillo Ricordi, M.D. (I) 
                 A.M. James Shapiro, MD, FRCS(C) (I) 
 
       GUESTS 
 
                 Jeffrey A. Bluestone, Ph.D. 
                 Jonathan Lakey, Ph.D. (I) 
 
       FDA 
 
                 Jay P. Siegel, M.D. 
                 Lauren E. Black, Ph.D.(I) 
                 Thomas L. Eggerman, M.D., Ph.D. (I) 
                 Neil D. Goldman, Ph.D. (III) 
                 Philip D. Noguchi, M.D. (III) 
                 Amy Rosenberg, M.D. (III) 
                 Karen Weiss, M.D. (I) 
                 Kathryn C. Zoon, Ph.D. (III) 

                                                                  3 
 
                             C O N T E N T S 
 
       BRMAC Administrative Update: Jay P. Siegel, M.D.           4 
 
                TOPIC III ‑ UPDATE CBER RESEARCH PROGRAMS 
 
       Division of Cellular and Gene Therapies 
 
                 Philip Noguchi, M.D.                            17 
                 Donald Fink, Ph.D.                              22 
 
       Division of Therapeutic Proteins 
 
                 Amy Rosenberg, M.D.                             31 
                 Gibbes Johnson, Ph.D.                           40 
                 Raymond Donnelly, Ph.D.                         44 
                 Kathryn Zoon, Ph.D.                             50 
 
                           TOPIC I (CONTINUED) 
             ISLET TRANSPLANTATION/PRECLINICAL ANIMAL MODELS 
 
       Welcome                                                   61 
 
       FDA Introduction: Karen Weiss, M.D.                       61 
 
       Open Public Hearing                                       62 
 
       Animal Models of Islet Therapy: Utilization 
       for Clinical Trial Design and Safety Assessment: 
                 Lauren Black, Ph.D.                             63 
 
       Animal Models of Islet Transplantation: 
                 John O'Neil                                     69 
 
       Non‑Human Primate Preclinical Models: 
                 Norma Sue Kenyon, Ph.D.                        109 
 
       FDA Perspective, Clinical Issues: 
                 Thomas L. Eggerman, M.D., Ph.D.                149 
 
       Committee Discussion                                     157 

                                                                  4 
 
   1                      P R O C E E D I N G S

   2             DR. SALOMON:  Good morning, everybody.  I guess we 
 
   3   are sort into the swing here; right?  There is nothing 
 
   4   formally I have to do this morning except begin by 
 
   5   introducing Jay Siegel for an admin update. 
 
   6                   BRMAC ADMINISTRATIVE UPDATE

   7             DR. SEIGEL:  Thanks.  I am not exactly sure what 
 
   8   an admin update is, but what I wanted to do was just have a 
 
   9   couple of minutes to follow up on a letter that I hope you 
 
  10   all received within the last couple of weeks to give you a 
 
  11   little bit of background about where we are heading with

  12   this committee. 
 
  13             We have some, I won't say directions, but a slight 
 
  14   shift in focus that I think is going to be really exciting.  
 
  15   Based on yesterday, in fact, I am quite sure that it will be 
 
  16   extremely beneficial to the agency.

  17             About a year ago, as a number of people, members 
 
  18   of the committee, rotated off, I was in a quandary about 
 
  19   exactly who to suggest to replace vacancies as they filled 
 
  20   because the committee has had a rather broad breadth of 
 
  21   topics it has been addressing ranging from issues with

  22   hematopoietic factors to hematopoietic stem cells, 
 
  23   transplantation therapy issues and so forth. 
 
  24             I consulted with Dr. Zoon and with the Division 
 
  25   Directors, Phil Noguchi and Karen Weiss who are here, and 

                                                                  5 
 
   1   others whom you have met at various meetings, about how

   2   would be the best way to utilize the expertise on the 
 
   3   committee and to utilize the committee and what sorts of 
 
   4   expertise best to emphasize on the committee. 
 
   5             Perhaps to understand where we were, if I could 
 
   6   take just a moment to mention historically that when this

   7   committee was started‑‑when would that be, the late '80's, 
 
   8   sometime‑‑its original focus, like virtually all other FDA 
 
   9   advisory committees, at least those for drugs and largely in 
 
  10   biologics, was on product approvals. 
 
  11             It was a reasonably broad spectrum of product

  12   applications, interferons for hairy‑cell leukemia and 
 
  13   Kaposi's sarcoma and hepatitis, interleukin‑2 for cancer.  
 
  14   Around the early '90's, we began to realize a couple of 
 
  15   products.  One was that while biological therapeutics had 
 
  16   been focused largely in the areas of cancer, we were seeing

  17   a great deal of application in sepsis and arthritis and 
 
  18   gastrointestinal disease and it seemed next to impossible to 
 
  19   try to have that sort of breadth of expertise on a single 
 
  20   advisory committee. 
 
  21             We were also aware that our sister center, the

  22   Center for Drugs, had advisory committees in each of those 
 
  23   clinical specialties.  Kathy and I and our colleagues 
 
  24   decided to move to increase our consultation with those 
 
  25   committees in most clinical areas and to retain the focus of 

                                                                  6 
 
   1   this committee in hematology, hematologic malignancies and

   2   some other areas of oncology. 
 
   3             With that, the numbers of meetings deceased by we 
 
   4   also started increasing, at that point in time, around the 
 
   5   mid‑'90's‑‑we always had some, but we started increasing the 
 
   6   number of meetings that were not focused on product approval

   7   but were focused on critical scientific issues and product 
 
   8   development. 
 
   9             We had a number of meetings on 
 
  10   xenotransplantation, in utero therapy with hematopoietic 
 
  11   cells, extracorporeal liver‑assist devices, use of PCR for

  12   hepatitis, and points most recently in that category, a 
 
  13   meeting that many of you were at that we all benefited 
 
  14   greatly from was a first of what I hope will be a series of 
 
  15   meetings on immunogenicity of biological therapeutics, what 
 
  16   should be studied during their development and how the

  17   products should be appropriately labeled. 
 
  18             So, with that as a background and with those many 
 
  19   very informative and successful meeting, as I talked again 
 
  20   with Dr. Zoon, Noguchi, Weiss and others, we realized that 
 
  21   those were extremely valuable, those meetings that really

  22   got at the heart of critical issues in new areas of 
 
  23   development generally early in product development before we 
 
  24   were faced with a large database that was pretty hard to 
 
  25   modify even if it didn't quite capture the endpoints or the 

                                                                  7 
 
   1   data that we most want to see.

   2             I think we have decided that there were a lot of 
 
   3   areas where we needed greater focus in product development 
 
   4   particularly in the fields of cellular and gene therapy, in 
 
   5   the areas of transplantation, tolerance inductions, a lot of 
 
   6   immunological areas, microbiology, product purity and safety

   7   from contaminating virologic agents, in particular; cell 
 
   8   biology‑‑assessing cellular functions and mechanisms. 
 
   9             When we started putting together a list of the 
 
  10   types of questions that we could bring to this committee, we 
 
  11   not only realized that, in these areas, there were a large

  12   number of critical questions but also realized that these 
 
  13   were not only areas in which we needed advice but in which 
 
  14   there was a need for a forum for public discussion because 
 
  15   we were developing approaches, scientific and regulatory 
 
  16   approaches, to new classes of products and there needed to

  17   be public airing and, where appropriate, public input on 
 
  18   more of these questions. 
 
  19             So, with that in mind, I would say it is not an 
 
  20   overhaul but a subtle shift in focus, but an important shift 
 
  21   in focus, of the committee.  We made a decision to call on

  22   your expertise more on the scientific issues critical to 
 
  23   product development in such fields.  So our having made that 
 
  24   decision, we are really pleased to welcome Dr. Bluestone as 
 
  25   our first appointment, having made that decision, and to 

                                                                  8 
 
   1   have yesterday's meeting as our first meeting with that

   2   focus in mind. 
 
   3             I think that yesterday's proceedings bear out, as 
 
   4   I said at the beginning, that this should be a very exciting 
 
   5   direction.  The other minor shift that will probably occur 
 
   6   in the committee is that, although the committee is

   7   chartered to have, what is it, up to thirteen or fourteen 
 
   8   members, I think we have realized that, even with this what 
 
   9   I am calling a focus, it is hardly really a focus in the 
 
  10   sense that there is such a broad spectrum of issues there, 
 
  11   we really have benefitted, and I think yesterday was, again,

  12   a good example of this, from bringing in significant numbers 
 
  13   of experts in the specific area under discussion. 
 
  14             I particularly enjoyed, as I hope you did, the 
 
  15   interplay between a group of you who have highly relevant 
 
  16   expertise in areas of development of cellular therapies,

  17   immunology, cell biology, microbiology and the knowledge 
 
  18   base of dealing with product development and FDA regulations 
 
  19   in advisory committee and interacting and combining and 
 
  20   synergizing with people who may not have some of those 
 
  21   expertises but, obviously, had tremendous expertise

  22   specifically in the area under discussion and I look forward 
 
  23   to today's discussion. 
 
  24             So I think the other thing that we will probably 
 
  25   be doing is keeping the number of the committee maybe two or 

                                                                  9 
 
   1   three people smaller than it had been, giving us a little

   2   more room to still have manageable discussions and invite 
 
   3   additional experts to interact. 
 
   4             So I am making this announcement both to keep you 
 
   5   informed, to find out if you have any questions and, also, 
 
   6   importantly to solicit from you, either now or any time you

   7   wish, by e‑mail or other mechanism, to the committee staff 
 
   8   or myself any suggestions you might have for topics you 
 
   9   think it would be wise for us to discuss in this forum at 
 
  10   future meetings. 
 
  11             Thank you.

  12             DR. AUCHINCLOSS:  Jay, I understand entirely and I 
 
  13   agree entirely and then I will give you a "but."  The "but" 
 
  14   has to do with maintaining the level of interest of your 
 
  15   committee members.  Part of excitement of serving on this 
 
  16   committee is that, at the end of the day, in many case,

  17   there is a real‑life decision, a vote yes/no. 
 
  18             There is a tension associated with that that 
 
  19   really kind of focusses both interest and, of course, the 
 
  20   specifics of the decision that is being made.  I understand 
 
  21   your interest in having broader discussions.  I just remind

  22   you that, from our point of view, to lose the sort of 
 
  23   case‑law kind of approach to things would be disappointing 
 
  24   if you gave it up entirely. 
 
  25             MR. SIEGEL:  I appreciate that.  I don't think we 

                                                                 10 
 
   1   intend to give it up entirely.  I think a couple of the

   2   meetings of this committee in the last couple of years have 
 
   3   focused on anti‑IL2‑receptor therapies and therapies coming 
 
   4   to approval for immunosuppression for transplantation. 
 
   5             We had a meeting‑‑well, it was of the xeno 
 
   6   advisory committee that focused, as you summarized, in

   7   Epicell.  I think that there will be a number of specific 
 
   8   product issues and product decisions that will come forward 
 
   9   as well many of which, as we use the Epicell case, as kind 
 
  10   of a case in point to set standards for other areas in 
 
  11   development.

  12             Hopefully, a lot of fields that we are focusing 
 
  13   this emphasis on, in the not too distant future, will be 
 
  14   leading toward product approvals. 
 
  15             DR. AUCHINCLOSS:  The Epicell case, I think, is a 
 
  16   case in point with part of what I am saying there where I

  17   think that if you look carefully at what the committee 
 
  18   recommended with regard to Epicell, I think it is actually 
 
  19   in conflict with its general principles under Topic I.  
 
  20   There is nothing like having the real thing in front of you 
 
  21   to get people to say what they really mean.

  22             DR. ZOON:  I can make a couple of comments on this 
 
  23   because I think it is very important to look in the context 
 
  24   of the broader issues of FDA advisory committees, I think 
 
  25   especially in biologics, looking at our TSE committees, 

                                                                 11 
 
   1   which is the Transmissible Spongeoform Encephalopathy

   2   Committees, the Vaccine Committees and our Blood Committees. 
 
   3             These committees will deal, like yourselves, with 
 
   4   product‑specific areas but, in many instances, their major 
 
   5   value in public health is actually looking at data early on, 
 
   6   identifying show stoppers early on, so when you do get to

   7   the point of looking at a product, there are no surprises, 
 
   8   not to the committee, not to the FDA, not to the 
 
   9   manufacturers, not to the public. 
 
  10             Our business here is to make an orderly transition 
 
  11   of science from the development of policy through product

  12   approval.  Now, you don't get rid of all surprises, but I 
 
  13   think, in the outside world, a consistency of scientific 
 
  14   soundness in framing policies and procedures as we go 
 
  15   forward in these complex areas of science, is critically 
 
  16   important and this committee plays an enormous role in

  17   helping us get there. 
 
  18             So I would say, while it may take away some of the 
 
  19   flash at the end, I don't think that is necessarily bad 
 
  20   because I think it is good public policy to have good 
 
  21   consistency and develop scientifically sound frameworks

  22   early on in product development. 
 
  23             DR. AUCHINCLOSS:  I understand entirely your point 
 
  24   of view and the other half of what I was talking about is 
 
  25   that you will lose some of the enthusiasm of your committee 

                                                                 12 
 
   1   if they come and talk early in the game and in the abstract. 

   2   It just isn't as‑‑you don't feel as important. 
 
   3             We got to NIH‑type advisory committees and future 
 
   4   task forces and all that kind of stuff and we usually feel 
 
   5   like, at the end of the day, we have done absolutely 
 
   6   nothing.  So it is just a practical matter.  To keep your

   7   committee as highly talented as you want it to be, involves 
 
   8   having them advise you on real‑life decisions sometimes. 
 
   9             But we are talking very much a matter of degree.  
 
  10   There is nothing that you are saying that I disagree with. 
 
  11             DR. SALOMON:  Just picking up on what Hugh is

  12   saying, when you decide, let's say, to do one of these 
 
  13   scientific‑‑like islet transplantation, for example, or 
 
  14   pluripotential stem cells of gene therapy which I think are 
 
  15   two topics, by the way, that ought to be on the list to come 
 
  16   now‑‑there are people who have come to you already for

  17   pre‑IND discussions if not actual IND discussions. 
 
  18             Why couldn't they be encouraged to come and 
 
  19   present?  I do agree with what you are saying in terms of 
 
  20   this focus.  Even the last meeting, the Epicell really did 
 
  21   provide a frame that focused the committee on some very

  22   specific sorts of topics. 
 
  23             I don't think we need to lose what you are talking 
 
  24   about, Kathy, in doing that. 
 
  25             DR. ZOON:  Usually, the way we do this is by 

                                                                 13 
 
   1   having a product as a case study and then the policy issues

   2   are discussed around a case study or a series of cases that 
 
   3   reflect the point.  It is not usually esoteric and in a 
 
   4   vacuum.  It is usually having something that is under 
 
   5   development of a series of products under development where 
 
   6   you would ask the sponsors to come and present.

   7             So it is related to real product and real‑product 
 
   8   issues.  I didn't mean to imply that that was not the case.  
 
   9   So most of these are linked to products, very specific 
 
  10   products and then those are used to discuss the more generic 
 
  11   issues surrounding the policy.

  12             DR. SALOMON:  But I guess what you and I are 
 
  13   saying is that when you actually confront the people, the 
 
  14   sponsors of these specific projects, they know they are on 
 
  15   the line.  They know that they have had to think it through.  
 
  16   If you grab any one of us on any day and say, "Let's talk

  17   about gene therapy.  Let's talk about islet 
 
  18   transplantation," yes; we have our opinions.  We have an 
 
  19   experience that we draw upon. 
 
  20             But we all know, and we would be the first to 
 
  21   admit, that we haven't nailed it.  We only nail it when we

  22   put ourselves on the line and put a protocol forward or 
 
  23   something like that.  So if you bring a group of sponsors 
 
  24   in, they really are going to say, "Okay; we have thought 
 
  25   exactly on how we are going to purify islets, exactly what 

                                                                 14 
 
   1   the room is going to be, exactly this, exactly that."

   2             I don't think you want to lose that. 
 
   3             DR. MILLER:  Do you intend to continue having 
 
   4   members of this committee, then, when the products actually 
 
   5   get to the other committees, be part of that committee?  I 
 
   6   was on a few ODAC.  There is not a whole lot of, unless they

   7   have changed, hematologic or bone‑marrow transplant 
 
   8   expertise on that. 
 
   9             I just think that if they are going to be, then, 
 
  10   doing all those, that either they need to pick up additional 
 
  11   hematopoietic‑malignancy people or biologic people.

  12             MR. SIEGEL:  Absolutely.  I am not sure that we 
 
  13   would want to take the hematopoietic replacement or 
 
  14   stem‑cell therapies over there.  I think yesterday was an 
 
  15   example, in fact, of how experience in that field, which is 
 
  16   a little bit more developed, was, actually, an interesting

  17   background and informative in terms of dealing with 
 
  18   pancreatic islet‑cell therapy. 
 
  19             So that specific decision has not been made, in 
 
  20   fact, but the answer would be, nonetheless, in general, yes.  
 
  21   I think, whether it is an Arthritis or an ODAC or a

  22   Gastrointestinal or Cardiorenal Committee‑‑those committees 
 
  23   have the clinical expertise, although I agree that the ODAC 
 
  24   does not have a great deal in hematologic stem‑cell therapy 
 
  25   but, often, not the kind of fundamental background in many 

                                                                 15 
 
   1   of the issues that we are facing in product‑approval time

   2   such as, say, the immunogenicity issue.  So we would intend 
 
   3   to continue. 
 
   4             DR. ZOON:  Just to highlight, we can supplement 
 
   5   this committee with experts from the CDER committees, or 
 
   6   vice versa, committees depending on where we think the

   7   expertise is mainly situated. 
 
   8             So that we look at as a combination and we look at 
 
   9   consistency of findings.  But we have the flexibility in 
 
  10   this to look at the situation and decide that the 
 
  11   committee‑‑we also can invite‑‑I was telling Dan earlier,

  12   once you are on an FDA advisory committee, you are there for 
 
  13   life as either a consultant or an invited guest. 
 
  14             We would very much look forward, as issues go 
 
  15   forward, even as things may cycle over the years, to invite 
 
  16   people back that have been involved to look at consistencies

  17   in these areas.  So we do have flexibilities in our 
 
  18   approach, depending on the nature of the condition and the 
 
  19   expertise. 
 
  20             DR. NOGUCHI:  I would just add, too, that one 
 
  21   thing Jay didn't mention but, with the current gene issues

  22   evolving, it is quite clear that the BRMAC committee can't 
 
  23   handle all the issues.  So we will be also bringing specific 
 
  24   gene therapy protocols and issues here as well. 
 
  25             One question, as an example, is for adenoviral 

                                                                 16 
 
   1   vectors.  We see that they have fairly high levels of

   2   toxicity that seems to be well tolerated in oncology 
 
   3   patients.  But in other patients with metabolic diseases, it 
 
   4   may or may not be the most appropriate choice. 
 
   5             We can bring questions such as, really, that 
 
   6   question, are adenoviral vectors, at any level, really

   7   appropriate for other than cancer.  So I think we can, 
 
   8   certainly, both bring specific cases as well as general 
 
   9   topics that will, in fact, shape and help to direct the 
 
  10   field in a safer and more efficacious manner. 
 
  11             DR. ZOON:  If I could just add one thing to that;

  12   I think we look forward to this committee getting very much 
 
  13   more involved in gene therapy.  As part of our proposal with 
 
  14   the department and NIH to address some of the recent 
 
  15   gene‑therapy issues, we have also proposed that some of the 
 
  16   gene‑therapy safety conferences be linked to the BRMAC.

  17             So we would be involved maybe having, one day, a 
 
  18   safety conference on an issue that we thought was important 
 
  19   in the safety of gene therapy and link that to the BRMAC's 
 
  20   normal committee schedule so that we can have those issues 
 
  21   discussed in a forum where we bring scientists in to discuss

  22   the issues and then, the next day, have our committee to 
 
  23   deal with specifics on issues. 
 
  24             So I think there is going to be a lot more 
 
  25   excitement and a lot more interest in some of these evolving 

                                                                 17 
 
   1   areas and we are looking forward to having the committee

   2   contribute to this. 
 
   3             DR. SALOMON:  My only other comment is that I hope 
 
   4   that we don't lose the component of hematopoietic stem‑cell 
 
   5   transplantation in the committee as it evolves because I 
 
   6   think that some of the future directions we are going in,

   7   whether it be stem‑cell therapy or gene therapy, and cell 
 
   8   therapy, after we get into the arguments of what is a tissue 
 
   9   and what is a cell, which I got into with Camillo yesterday, 
 
  10   I think it is going to be very important. 
 
  11             MR. SIEGEL:  There is no question a significant

  12   amount of the gene‑therapy work we are seeing is using 
 
  13   hematopoietic stem cells, manipulated, expanded or not, as a 
 
  14   vehicle for gene therapy.  There is also no question that 
 
  15   newer cell‑device, expansion devices, culturing pancreatic 
 
  16   cells, culturing neonatal cord blood cells is really part

  17   and parcel of the same class of issues.  I agree. 
 
  18             DR. SALOMON:  Okay, guys.  Good.  Thank you very 
 
  19   much, Jay. 
 
  20             Phil? 
 
  21            TOPIC III ‑ UPDATE CBER RESEARCH PROGRAMS

  22             Division of Cellular and Gene Therapies 
 
  23             DR. NOGUCHI:  Good morning. 
 
  24             [Slide.] 
 
  25             One of the very important extra duties that BRMAC 

                                                                 18 
 
   1   serves is a review of our scientific programs.  I would just

   2   like to, very briefly, go over some of the reorganization of 
 
   3   the Division of Cell and Gene Therapy and show you how our 
 
   4   newer programs in stem‑cell biology fits in. 
 
   5             [Slide.] 
 
   6             This committee has, in various aspects, reviewed

   7   the Laboratory of Cellular Immunology.  At the present time, 
 
   8   we have Dr. Ida Bloom, who has focused on some of the immune 
 
   9   processes involved in xenotransplantation and Carolyn 
 
  10   Wilson, who has been working on porcine endogenous 
 
  11   retrovirus.

  12             To augment this area, including that to focus on 
 
  13   gene therapy, Paris Byrd left recently and we are now 
 
  14   recruiting for a tenure‑track person with expertise in 
 
  15   adenoviral vectors. 
 
  16             The Laboratory of Molecular and Developmental

  17   Biology, in a way, splits some of its work between areas of 
 
  18   immunity of plasmid vectors, both where you wanted immune 
 
  19   response and where you don't want immune response, some 
 
  20   developmental programs of Malcolm Moos.  Deborah Hirsh is a 
 
  21   new tenure‑track person.  Steve Bauer has been looking at

  22   the interaction of stromal cells with hematopoietic cells 
 
  23   and their development. 
 
  24             [Slide.] 
 
  25             Dr. Raj Puri directs a program of tumor‑cell 

                                                                 19 
 
   1   biology and is responsible for the cellular‑tumor vaccines

   2   that we regulate.  Tom Eggerman has been working on some 
 
   3   very clever approaches to delivery of antisense compounds 
 
   4   through liposomes that is finally starting to emerge as a 
 
   5   major field. 
 
   6             We have also non‑lab‑based reviewers.  Dr. Joyce

   7   Frey heads up this particular endeavor and also serves as my 
 
   8   deputy for cell and gene therapy. 
 
   9             [Slide.] 
 
  10             The newest and last area is in the area of 
 
  11   stem‑cell biology.  Dr. Liana Harvath has recently joined

  12   our group moving from the Office of Blood.  But, as she has 
 
  13   moved over, she is also bringing over all the issues and the 
 
  14   work that she has done in hematopoietic stem cells. 
 
  15             Here is where we hope to extend some of the types 
 
  16   of approaches being used in hematopoietic stem cells to

  17   things like islet cell transplants, umbrella INDs, 
 
  18   development of external standards, heavy and very 
 
  19   interactive industry and academic involvement with the 
 
  20   process. 
 
  21             Dr. Gerry Marti has had decades of flow cytometry

  22   standardization and experience and a research program in 
 
  23   chronic lymphocytic leukemia.  Don Fink is our newest 
 
  24   addition to this who came from the old Division of Cytokine 
 
  25   Biology. 

                                                                 20 
 
   1             While the focus of the researchers that we

   2   currently have in stem cell biology has been mainly on 
 
   3   hematopoietic cells, we know that there is going to be a 
 
   4   very large and emerging area of nonhematopoietic stem cell 
 
   5   biology.  In fact, the next planned BRMAC meeting will be on 
 
   6   the area of neural stem cells and whether or not they may

   7   prove to be useful therapeutic modalities. 
 
   8             I think you will find that particular next meeting 
 
   9   to be quite interesting 
 
  10             I think, at this point, I would like to have, 
 
  11   then, Don Fink just briefly review some of his recent

  12   efforts. 
 
  13             DR. CHAMPLIN:  While you are fixing the projector, 
 
  14   Phil, I was just going to ask, with Liana Harvath coming 
 
  15   over, changing areas, does that change the regulatory 
 
  16   framework for hematopoietic transplantation in any way?

  17             DR. NOGUCHI:  No.  The approach that has been 
 
  18   outlined and that Liana has been presenting and working with 
 
  19   you and others will remain the same.  It is our intent to 
 
  20   continue that. 
 
  21             MR. SIEGEL:  Liana is now integrated into the

  22   group and the team, including Phil and myself and Kathy.  As 
 
  23   of a few months ago, we were hoping to renew an effort to 
 
  24   really finish, or at least advance, the regulatory framework 
 
  25   in a way that made sense and provided further clarity to the 

                                                                 21 
 
   1   field.

   2             I must say, many of us have been somewhat 
 
   3   distracted by events in the gene‑therapy field and has that 
 
   4   the work back a little bit, but we are meeting later this 
 
   5   month to continue that effort and her transfer into this 
 
   6   division was significantly motivated by a desire to be able

   7   to coordinate development of regulatory policy and strategy. 
 
   8             DR. CHAMPLIN:  Does that mean that will be sort of 
 
   9   in your office as opposed to the Blood Products group?  Or 
 
  10   is that an oversimplification? 
 
  11             MR. SIEGEL:  Most stem‑cell products have been in

  12   the Office of Therapeutics‑‑I shouldn't say that; not most, 
 
  13   necessarily that are in use, but many that have involved use 
 
  14   of growth factors, use of devices to select against tumor 
 
  15   cells, select for CD34 cells and whatever. 
 
  16             It is likely that‑‑perhaps Kathy can speak to this

  17   more directly, but it is likely that more of the stem‑cell 
 
  18   issues will be dealt with in my office; yes.  I don't 
 
  19   remember exactly where the rules are, but more of the less 
 
  20   manipulated stem cells are moving into my office with Liana. 
 
  21             DR. ZOON:  I think if you are interested in that,

  22   that is something maybe we can give you an update on at one 
 
  23   of the future meetings of its status and how that is 
 
  24   organized because I think it is an important question with 
 
  25   some personnel switches. 

                                                                 22 
 
   1             If the committee would like an update on how the

   2   center is managing stem cells, we would be happy to do that. 
 
   3             DR. FINK:  Good morning, committee members.  
 
   4   8 o'clock is a nice time.  I am glad to see you all awake. 
 
   5             [Slide.] 
 
   6             I am going to give you a Reader's Digest synopsis,

   7   really, of what I presented at my site visit back in 
 
   8   September of which Dr. Hugh Auchincloss was the chair.  Up 
 
   9   front, I would say, and he asked me this morning, I, 
 
  10   personally, have sent a letter to each of you, I believe, 
 
  11   who are committee members which had information that I felt

  12   was not in the briefing package that might be useful in your 
 
  13   deliberations. 
 
  14             Dr. Noguchi is aware of that.  Dr. Seigel has seen 
 
  15   that comment.  So you are free to talk about it.  It is not 
 
  16   out of bounds.  Those issues are quite fine.  So I have no

  17   trouble with that.  I will come back to that at the end to 
 
  18   tell you that some of those actions have actually been 
 
  19   implemented which was a positive outcome of all of this. 
 
  20             What I presented‑‑at that time, I had spent most 
 
  21   of my tenure here in the Division of Cytokine Biology and,

  22   as you know, cytokines are therapeutic proteins and 
 
  23   recombinant proteins.  My area of expertise and regulatory 
 
  24   oversight was in neurotrophic factors in the treatment of 
 
  25   neurodegenerative diseases. 

                                                                 23 
 
   1             That basis was how my research program had been

   2   developing.  So what I am presenting to you in terms of that 
 
   3   is what had been done in the lab up to that point. 
 
   4             The model that we are using in the laboratory is a 
 
   5   cell‑culture model to look at neuronal differentiation.  The 
 
   6   cell line is a PC12 cell line, which is derived from a

   7   fetochromocytoma.  I would consider this to be something 
 
   8   akin to a neuroprogenitor.  It works nicely in that you can 
 
   9   add a trophic factor such as nerve‑growth factor and it 
 
  10   differentiates and becomes a functional neuron. 
 
  11             Also used were various variants, mutant cell

  12   lines, which have dominant negative expressions of 
 
  13   signal‑transduction molecules to verify certain observations 
 
  14   that were made pharmacologically. 
 
  15             For the purpose of comparison, I will simply state 
 
  16   that my expertise had been in nerve‑growth factor for much

  17   of my training and then I have become interested in 
 
  18   pituitary adenylate cyclase‑activating polypeptide.  So I 
 
  19   run these things in parallel to find similarities and 
 
  20   differences.  They work in quite different fashions. 
 
  21             So what I am going to do‑‑I am just going to do

  22   overheads that I have to show you just to summarize the 
 
  23   bullet points for you so you will be familiar with the 
 
  24   findings.  Then I will tell you a little bit about what I 
 
  25   have done to progress the research in the last couple of 

                                                                 24 
 
   1   months orally and then any questions that you may have, in

   2   particular, I will entertain at the end. 
 
   3             Basically, in terms of the research findings, 
 
   4   using this cell model to look at neural differentiation, we 
 
   5   are measuring neuritic outgrowth.  We found in the 
 
   6   laboratory that the PACAP, which works through a

   7   seven‑trans‑membrane protein through g‑coupling mechanisms, 
 
   8   was able to elicit a neuritic outgrowth response similar to 
 
   9   what you would see if you added the neurotrophin 
 
  10   nerve‑growth factor. 
 
  11             We were able to deduce that these are independent

  12   phenomena and that the PACAP does not activate in crosstalk 
 
  13   into systems that somehow activate Trk A, thus resulting in 
 
  14   a borrowing of pathways in that manner. 
 
  15             In contrast to what we know about nerve‑growth 
 
  16   factor, if we use dominant negative expression systems and,

  17   number 2 at the bottom src and ras, which we know are 
 
  18   involved in the signalling of nerve‑growth factor, when we 
 
  19   use our dominant negative cell models, we find that PACAP is 
 
  20   able to work independent of those and is able to drive 
 
  21   significant signalling through the cell in the absence of

  22   activation of those particular molecules, which makes it 
 
  23   clearly distinct from nerve‑growth factor in that regard. 
 
  24             At this point, we decided to use varieties of 
 
  25   pharmacological antagonists to probe this in greater detail 

                                                                 25 
 
   1   and, in particular, looking at protein kinases A and protein

   2   kinases C which are known to be activated by PACAP and to 
 
   3   see whether or not we could inhibit, in any way, the 
 
   4   responses that we had been observing. 
 
   5             [Slide.] 
 
   6             On the top bullet point, you can see that, by

   7   using various pharmacologic inhibitors selective for the 
 
   8   various pathways, we found that, in particular, if we use 
 
   9   protein‑kinase‑C antagonism that we were able to inhibit a 
 
  10   downstream signalling molecule called Erk which is, we know, 
 
  11   to be very important to nerve‑growth‑factor activity in

  12   terms of inducing the neuritic response. 
 
  13             Similarly, when we antagonized protein kinase C 
 
  14   and inhibited Erk, we also found that PACA was blunted.  So 
 
  15   now we have kind of found a focus where we can coalesce and 
 
  16   we are converging on a point in this cascade where NGF and

  17   PACAP might be coalescing their efforts to result in a final 
 
  18   outcome which was neuritic outgrowth. 
 
  19             Continuing those studies, we were able to observe 
 
  20   that, in addition to the Erk and PKC dependence that protein 
 
  21   kinase A was not involved, thus eliminating one arm, at

  22   least, of potential signalling for PACAP as being involved 
 
  23   in this morphologic neurodifferentiative response that we 
 
  24   had observed. 
 
  25             Finally, the area that we are continuing our most 

                                                                 26 
 
   1   active study is really in the upregulation of a Trk A

   2   receptor.  Trk A is important for neuronal differentiation 
 
   3   and survival of various nerve‑type cells.  In the model, 
 
   4   PC12‑6.24 cells, we have a system where we have 
 
   5   overexpressed human Trk A. 
 
   6             What we have found has been very striking and very

   7   entertaining, actually, is that we have maybe a potential 
 
   8   area to look at regulation of this neurotrophin receptor 
 
   9   that has not been discovered or discussed extensively in the 
 
  10   literature.  We have found that PACAP treatment will 
 
  11   remarkably upregulate the expression of the Trk A at the

  12   protein level as well as its phosphorylation status, which 
 
  13   is indicative of its activation. 
 
  14             Now, 624 may be something akin to using sort of an 
 
  15   induced expression system because we find that with PACAP, 
 
  16   we from a basal level to over 200‑fold, 300‑fold, increase

  17   in this protein level.  So it is quite robust, but it also 
 
  18   has some interesting features. 
 
  19             It appears that receptor has been sequestered.  It 
 
  20   is not available to surface because the increase in overall 
 
  21   binding of NGF, for example, to surface is only about

  22   three‑fold to four‑fold.  So while there is probably some 
 
  23   endogenous separation there, it is quite interesting to use 
 
  24   as a marker. 
 
  25             We have extended those studies.  We did a time a 

                                                                 27 
 
   1   course and we find that‑‑we started cutting the time to

   2   exposure.  If you only expose for 15 minutes to PACAP, do a 
 
   3   wash‑out and come back 24 hours later, you still have the 
 
   4   robust signal.  So it is almost like there is an on‑switch 
 
   5   and it is very intriguing. 
 
   6             So that really summarizes the gist of what I had

   7   presented at that time.  Since then, I can say that, on the 
 
   8   basis of the site‑visit report, that we have been able to 
 
   9   actually do a little novel kind of experiment within the 
 
  10   purviews of research.  I am now associated with the 
 
  11   laboratory of Dr. Kathy Carbone who is in another office but

  12   works with neurotrophic viruses and has a laboratory that is 
 
  13   quite extensively focused on, fortunately, the cerebellum. 
 
  14             The cerebellum, it turns out, is where you have 
 
  15   the neurons, the cerebellar granule neurons which are 
 
  16   PACAP‑dependent early on in development.  Her model is using

  17   a bornavirus which, interestingly, affects the cerebellum 
 
  18   resulting in the death of cerebellar neurons following 
 
  19   neonatal treatment injection of the virus into the CNS. 
 
  20             So we are developing‑‑this is a new evolution, but 
 
  21   we are developing a strategy to look at what is actually

  22   happening in terms of expression of PACAP‑‑they have some 
 
  23   suggestion that it is modulated by the virus and that may be 
 
  24   representing a compensatory mechanism to try to protect 
 
  25   these neurons‑‑and then look for expression of various 

                                                                 28 
 
   1   neurotropic‑factor receptors such as Trk B.

   2             I also have a collaboration going with a colleague 
 
   3   in Israel where we have an extensive program looking at in 
 
   4   vitro modeling with the PC12 cell line at hypoxia and 
 
   5   glucose deprivation representing a stroke model.  We have 
 
   6   some very interesting data which suggests that PACAP, by

   7   itself, is able to protect PC12 cells in this deprivation 
 
   8   model from apoptosis by about 20 percent. 
 
   9             NGF is only about 35 percent protected, but the 
 
  10   two of them together, it is almost complete protection 
 
  11   against hypoxia and the glucose deprivation.  So there is

  12   some nice signalling crosstalk that we can look at in that 
 
  13   model. 
 
  14             Finally, I have a collaboration now with Dr. Ann 
 
  15   Marini.  She is at Walter Reed, very close by.  She does 
 
  16   work with primary cerebellar granule neurons and is very

  17   intrigued by the possibility that, A, in glutamate toxicity, 
 
  18   she can use PACAP as a protectant and, B, that I am in 
 
  19   interested in that because we can use PACAP in her model 
 
  20   which is primary neurons to look now at upregulation but 
 
  21   second neurotrophic receptor, which would be Trk B.

  22             Finally, let me just bring this all to a focus in 
 
  23   terms of stem‑cell biology and the recent presentation at 
 
  24   CBER by Dr. Catherine Verfaillie at the University of 
 
  25   Minnesota who is developing adult stem cells. 

                                                                 29 
 
   1             One of the important features of that model for

   2   getting these cells to replicate in culture prior to 
 
   3   differentiation is to use forskolin.  If you recognize 
 
   4   forskolin as a cyclic‑A‑and‑P activator, you can begin to 
 
   5   see how PACAP could be a physiologic modulator if, in fact, 
 
   6   these cells express receptors for it and are coupled through

   7   adenylate cyclase. 
 
   8             We anticipate the possibility of obtaining some of 
 
   9   these cells from her in order to investigate this particular 
 
  10   approach.  Within the laboratory of stem‑cell biology, along 
 
  11   with Dr. Liana Harvath, we are currently working together to

  12   try to look at the issue of cellular migration which she is 
 
  13   an expert in and which is a very important feature for what 
 
  14   will happen following the implantation of stem cells, 
 
  15   particularly neural stem cells because they will, in fact, 
 
  16   need to migrate to their proper location in order to

  17   establish connections. 
 
  18             So this could be a very fruitful and productive 
 
  19   interaction.  With that, I will conclude.  If anyone has any 
 
  20   particular questions they would like to ask, I would‑‑yes? 
 
  21             DR. SAUSVILLE:  In relation to that last point,

  22   your PC12 cells‑‑you say PACAP is PKA independent?  And yet, 
 
  23   in these stem cells that you just alluded to, you state that 
 
  24   they are stimulated by forskolin and you implicate it in the 
 
  25   potential role of PACAP. 

                                                                 30 
 
   1             So, does that imply that in different cellular

   2   contexts, PACAP could either be protein kinase‑A or cyclic‑A 
 
   3   and‑P‑directed and, in another cellular context, independent 
 
   4   of that? 
 
   5             DR. FINK:  That is absolutely correct. 
 
   6             DR. SAUSVILLE:  How do you see that coming

   7   together from a unitary mechanism of PACAP? 
 
   8             DR. FINK:  Let me just try to confuse you a little 
 
   9   further on that regard because we have tried to probe this 
 
  10   specifically using our upregulation of the receptor model as 
 
  11   indices.  There are aspects where, in certain cellular‑‑let

  12   me backtrack‑‑cellular contexts where the PACAP is mitogenic 
 
  13   and, with cyclic‑A‑and‑P activation, does, in fact, drive 
 
  14   mitogenesis. 
 
  15             However, in those cellular contexts, it is not 
 
  16   coupled efficiently, if at all, through protein kinase C. 

  17   So, it may be that you have a preponderance of activation of 
 
  18   one system that is, in the absence of another input from a 
 
  19   secondary cascade such as protein kinase C, allows it to 
 
  20   over‑‑not overexpress but to predominantly express that 
 
  21   particular phenomenon.

  22             In PC12 cells, and this is also the case with NGF 
 
  23   versus EGF, you have molecules that seem to activate similar 
 
  24   cascades and yet lead to, really, diametrically opposed or 
 
  25   different phenomenon. 

                                                                 31 
 
   1             All the players in that are not identified but it

   2   is clear that there are now capacities to activate distinct 
 
   3   molecules in different discrete cascades that have crosstalk 
 
   4   inhibitory effects.  So, in that context of cyclic A and P 
 
   5   that you suggested, in this case, it appears that, while the 
 
   6   cyclic‑A‑and‑P event is there, in terms of the neuritic

   7   outgrowth response, that is being driven more predominantly 
 
   8   by other players, presumably protein kinase C and Erk 
 
   9   activation at this time. 
 
  10             DR. SALOMON:  Thank you, Donald. 
 
  11                 Division of Therapeutic Proteins

  12             DR. ROSENBERG:  Good morning, everybody. 
 
  13             I would like to start out by showing you the 
 
  14   organization of our division and where the people who have 
 
  15   been site‑visited fit into the division.  I would like to 
 
  16   then tell you about some of their regulatory activities so

  17   that you can understand how their scientific endeavors 
 
  18   benefit the regulatory mission of the agency. 
 
  19             [Slide.] 
 
  20             We are the Division of Therapeutic Proteins.  This 
 
  21   division was constructed or generated last year, in October. 

  22   It is composed of the former Division of Hematologic 
 
  23   Products and some personnel from the Division of Cytokine 
 
  24   Biology. 
 
  25             I am the Deputy Director who is the Acting 

                                                                 32 
 
   1   Director.   There are four laboratories within our division. 

   2   First there is the Laboratory of Immunology.  Drs. Donnelly 
 
   3   and Petricoin who were reviewed are in this laboratory; the 
 
   4   Laboratory of Gene Regulation lead by Dr. Ed Max and Dr. 
 
   5   Gibbes Johnson is located within the laboratory.  Dr. Kathy 
 
   6   Zoon is located in the Laboratory of Chemistry that is led

   7   by Blair Frazier. 
 
   8             [Slide.] 
 
   9             What products do we have and what scientific 
 
  10   programs support the regulation of these products?  We have 
 
  11   an amazingly wide variety of products.  Starting with the

  12   cytokines, Dr. Donnelly has been responsible for regulating 
 
  13   IL1.  Recently, he has taken on regulation of IL2 as well as 
 
  14   IL2 fusion toxins.  He has had long‑term responsibility for 
 
  15   regulation of IL4, IL10 and IL12. 
 
  16             Regarding the interferons, there is a wide variety

  17   of interferons and many groups are involved in their 
 
  18   regulation.  So we have Dr. Zoon's group and, primarily, 
 
  19   within that group, Mr. Joe Bekicz has been responsible for 
 
  20   some of the interferon alphas.  Dr. Chip Petricoin, as well, 
 
  21   has been responsible for the regulation of interferons and

  22   it is Dr. Zoon's abiding interest in interferons that 
 
  23   qualify her laboratory as regulators of these products. 
 
  24             Dr. Petricoin has had a long‑standing interest in 
 
  25   interferon signalling although his direction has changed 

                                                                 33 
 
   1   recently.  The interferon gamma is also regulated by Ray

   2   Donnelly who has primary responsibility for it. 
 
   3             Interferon beta is handled by Dr. Gibbes Johnson 
 
   4   who has a long‑standing interest in signalling by growth 
 
   5   factors in general.  Dr. Chip Petricoin also has regulation 
 
   6   of interferon omega and interferon tau.

   7             [Slide.] 
 
   8             In terms of receptor antagonists, of which we are 
 
   9   seeing increasing numbers, Dr. Petricoin is responsible for 
 
  10   antagonist to TNF.  Ray Donnelly is responsible for receptor 
 
  11   antagonists to IL1 and IL4.  Regarding the lists of enzymes

  12   that you see here, which we recently acquired since the 
 
  13   reorganization of the divisions, Dr. Johnson is responsible 
 
  14   for the uricase. 
 
  15             There is a group of miscellaneous products which 
 
  16   are difficult to classify.  Dr. Petricoin is responsible for

  17   lactoferrin and relaxin, Dr. Johnson for ICAM‑1. 
 
  18             [Slide.] 
 
  19             Regarding tissue growth factors, Dr. Gibbes 
 
  20   Johnson has primarily responsibility for platelet‑derived 
 
  21   growth factors, vascular endothelial growth factor and

  22   hepatocyte growth factor.  There are others in the division 
 
  23   who are responsible for the remaining factors. 
 
  24             Regarding the growth inhibitors, Dr. Donnelly is 
 
  25   responsible for mammastatin. 

                                                                 34 
 
   1             [Slide.]

   2             So, having shown you what their regulatory aegises 
 
   3   are, I think that I would just like to briefly touch on the 
 
   4   nature of their laboratory programs so that you can see, in 
 
   5   a nutshell, how their laboratory programs address regulatory 
 
   6   issues.  So we will start with the laboratory program of Dr.

   7   Donnelly. 
 
   8             [Slide.] 
 
   9             The scope of his research really focuses on the 
 
  10   mechanisms by which cytokines cross‑regulate signalling by 
 
  11   other cytokines.  He uses a monocyte model in this regard. 

  12   He has most recently focused on the role of the SOCS 
 
  13   proteins.  These are suppressors of cytokine signalling.  He 
 
  14   is endeavoring, in his future studies, to try and define the 
 
  15   mechanism by which these SOCS protein inhibit 
 
  16   cytokine‑induced signalling.

  17             [Slide.] 
 
  18             In terms of the laboratory program of Dr. Johnson, 
 
  19   the primary interest here is in signalling through EGF 
 
  20   receptors and the role of the SHP‑2 phosphatase in this 
 
  21   signal transduction pathways.  So his studies mostly focus

  22   on mutational analyses of the erbB receptor, tyrosine 
 
  23   kinase‑‑ 
 
  24             [Slide.] 
 
  25             ‑‑as well as looking at the role of the SHP‑2 

                                                                 35 
 
   1   phosphatase by generating mutants and characterizing the

   2   activity of these mutants in vitro and in vivo. 
 
   3             [Slide.] 
 
   4             The laboratory program of Dr. Zoon is focused on 
 
   5   the structure/function relationships of interferon alpha.  
 
   6   It involves extensive characterization of newly generated

   7   hybrids in terms of what domains are essential for antiviral 
 
   8   activity, antiproliferative activity, competitive binding 
 
   9   activity and signal transduction pathways. 
 
  10             [Slide.] 
 
  11             The future goals include examining the interferon

  12   binding characteristics, characterizing the signal 
 
  13   transduction pathways and really delving into the 
 
  14   structure/function relationships as they apply to 
 
  15   antiproliferative activity. 
 
  16             [Slide.]

  17             Lastly, the laboratory program of Dr. Petricoin, 
 
  18   who I think may be a victim of the bad traffic this morning, 
 
  19   so I will just go over these a little more slowly, although, 
 
  20   in his former years, he was focused primarily on interferon 
 
  21   signalling, he has recently gone off in a very exciting new

  22   direction involving the use of proteomic technologies to 
 
  23   identify new proteins. 
 
  24             The power of this technique is enormous with 
 
  25   regard to many different areas.  So his goals are really to 

                                                                 36 
 
   1   use this technique in many settings.  One is to identify new

   2   proteins that track with normal and diseased cancer cells in 
 
   3   following human solid tumors, cancers of the prostate, 
 
   4   breast, ovary and esophagus. 
 
   5             He wants to use the proteomic technologies to 
 
   6   identify signal transduction pathways and to potentially

   7   identify new phosphoproteins in the setting of solid 
 
   8   malignancies, the erbB2 positive and negative breast 
 
   9   cancers, normal versus premalignant versus tumor breast 
 
  10   epithelium, normal versus premalignant versus invasive 
 
  11   prostate epithelium with low metastatic potential versus

  12   invasive, so, really, to try and get a correlate of what 
 
  13   proteins are involved, perhaps in malignant transformation 
 
  14   or in transformation to a more highly malignant form. 
 
  15             [Slide.] 
 
  16             He wants to use the proteomic technologies to

  17   identify biomarker profiles from body fluids for early 
 
  18   disease detection.  This would clearly be of enormous 
 
  19   benefit in a variety of malignancies, many of which are not 
 
  20   detected until late stages of disease.  So he wants to 
 
  21   examine the nipple‑fluid aspirates from volunteers in

  22   breast‑cancer patients to see if there is something that can 
 
  23   be picked up there as a precursor or malignancy. 
 
  24             He wants to examine serum from normal volunteers 
 
  25   and prostate‑cancer patients and also the ascitic and cystic 

                                                                 37 
 
   1   fluid from borderline non‑invasive and invasive

   2   ovarian‑cancer patients and he further wants to use these 
 
   3   proteomic technologies in a way that I think is very 
 
   4   exciting for the agency in general which is to identify 
 
   5   biomarker profiles for early toxicity screening using, 
 
   6   hopefully, serum, which is clearly one of the easiest fluids

   7   to get. 
 
   8             So, for instance, looking at whether one can 
 
   9   detect the early toxicity of adriamycin‑treated patients, 
 
  10   the cardiac toxicity; also to look at pre‑ and 
 
  11   post‑treatment of vasculitis and cardiotoxicity‑inducing

  12   agents in rat models. 
 
  13             That covers the presentation. 
 
  14             DR. SALOMON:  Thank you, Amy. 
 
  15             DR. BLUESTONE:  Amy, for the proteonic work, is 
 
  16   that being done using a mass‑spec approach, at 2D‑gel

  17   approach?  Is it in collaboration with a company or how is 
 
  18   that being‑‑ 
 
  19             DR. ROSENBERG:  He has gone to using the SELDI 
 
  20   which is a very high‑powered technique for identifying 
 
  21   proteins.  It is not terribly quantitative at this point but

  22   it is qualitative and very sensitive in terms of picking up 
 
  23   all protein forms. 
 
  24             There are a variety of chips that can be used with 
 
  25   this to really detect proteins of different molecular 

                                                                 38 
 
   1   weights.  If he were here, he could tell you better.

   2             DR. GOLDMAN:  If I may add, SELDI is, in fact, 
 
   3   laser desorption so it is like MALDI.  It is mass 
 
   4   spectrometry. 
 
   5             DR. CHAMPLIN:  Can I ask a question?  What are you 
 
   6   envisioning for this Office of Protein?

   7             MR. SIEGEL:  The Division of Therapeutic Proteins? 
 
   8             DR. CHAMPLIN:  Yes. 
 
   9             MR. SIEGEL:  Basically, we reorganized in October, 
 
  10   which was the start of this fiscal year.  The reorganization 
 
  11   can be described, I think, as having two major

  12   characteristics.  We went from laboratory‑based product 
 
  13   divisions to three.  That, among other things, largely 
 
  14   reflected the fact that our program has significantly 
 
  15   downsized, particularly its research aspects, over the last 
 
  16   four or five years.

  17             But also what was really the critical guiding 
 
  18   feature, and this is really to the point of your question of 
 
  19   this reorganization, was to reorganize the product‑review 
 
  20   divisions more along the lines of product classes which 
 
  21   shared common concerns regarding methods of production,

  22   methods of product testing and manufacturing control. 
 
  23             So, whereas in the past, we had cellular therapies 
 
  24   in all of our product divisions and protein therapies in all 
 
  25   of our product divisions, and antibody therapies in at least 

                                                                 39 
 
   1   two of the four product divisions, we kind of, by

   2   refocussing them, have created a situation where it is 
 
   3   easier to insure consistency in manufacturing and standards 
 
   4   as well as to develop policy and guidance in a concerted 
 
   5   way. 
 
   6             So that is how the products, some of which were in

   7   cytokine biology and some of which were in hematologic 
 
   8   products, kind of got merged into the Therapeutic Proteins 
 
   9   Division. 
 
  10             DR. CHAMPLIN:  I was struck by how 
 
  11   broad‑based‑‑everything from growth regulatory molecules to

  12   l‑asparaginase to biomarkers.  All of biology comes down to 
 
  13   proteins in the end and so it really is everything. 
 
  14             MR. SIEGEL:  That's true and that is true of all 
 
  15   of our divisions now.  The clinical diversity, of course, as 
 
  16   you all are probably aware, we have both a preclinical

  17   animal models group as well as a clinical group that is 
 
  18   together in Karen Weiss's division that is basically 
 
  19   organized by clinical specialty that works with all of these 
 
  20   product classes. 
 
  21             But it is true from the basic pharmacology and

  22   basic science that, just as in the Division of Monoclonal 
 
  23   Antibodies and in the Division of Cell and Gene Therapy, the 
 
  24   Division of Therapeutic Proteins has a diverse group of 
 
  25   products. 

                                                                 40 
 
   1             We have been aware that, in reorganizing, you can

   2   organize along clinical disciplines.  You can organize 
 
   3   around product class.  You can organize along scientific 
 
   4   disciplines or mechanisms of action.  Whatever you do has 
 
   5   advantages but requires that you pay careful attention to a 
 
   6   large number of cross‑cutting issues and interactions.  That

   7   is where we are. 
 
   8             DR. SALOMON:  I am getting a little concerned 
 
   9   about time here, so if we can keep on going.  Dr. Johnson? 
 
  10             DR. JOHNSON:  Thank you. 
 
  11             [Slide.]

  12             I thought, today, I would spend my five minutes 
 
  13   just bringing the committee up to date on some recent 
 
  14   progress that we have had since the site visit.  My 
 
  15   laboratory is interested in signalling by the erbB family of 
 
  16   receptors.  Ligands are comprised of four receptor tyrosine

  17   kinases which interact through process of a ligamerization 
 
  18   when the receptors engage a very large family of growth 
 
  19   factors which are structurally related to epidermal growth 
 
  20   factor. 
 
  21             [Slide.]

  22             ErbB1 is actually the epidermal growth‑factor 
 
  23   receptor, EGF receptor, and there is erbB2, erbB3 and erbB4.  
 
  24   This is an old slide, but actually, all of the erbs have 
 
  25   been shown to interact in a ligand‑dependent manner. 

                                                                 41 
 
   1             [Slide.]

   2             There are actually two projects which are ongoing 
 
   3   in the laboratory.  One is trying to understand the function 
 
   4   of the EGF receptor tyrosine kinase activity and signalling 
 
   5   by the EGF receptor. 
 
   6             [Slide.]

   7             As you might guess, the way we have addressed that 
 
   8   is by mutating the EGF receptor kinase activity into an EGF 
 
   9   receptor kinase‑inactive mutant form.  Just to kind of 
 
  10   describe some recent progress in this area and put it in a 
 
  11   nutshell, what we found is that, actually, the EGF receptor

  12   kinase activity is not essential to EGF‑induced signalling 
 
  13   in many cell types. 
 
  14             This appears to be due to the fact that there is 
 
  15   an erbB2‑EGF receptor heterodimer which signals in the 
 
  16   EGF‑dependent manner.  The signalling is not dependent upon

  17   the EGF receptor kinase activity.  This heterodimer can 
 
  18   actually activate two pathways which are essential to growth 
 
  19   and differentiation of cells‑‑that is, the mitogen‑activated 
 
  20   protein‑kinase pathway or MAPk and also the AkT kinase 
 
  21   pathway which is thought to be involved in cell survival.

  22             The heterodimer is incapable of activating signal 
 
  23   transducers and activators of STATs‑‑signal transducers and 
 
  24   activators of transcription, also known as STATs 1, 3 and 5.  
 
  25   We have actually set up two model systems to study the 

                                                                 42 
 
   1   signalling by the heterodimer.  One is in NR6 fibroblasts

   2   and the other is in 32D myeloid cells. 
 
   3             The biological response to EGF in these two cell 
 
   4   lines is actually different even though biochemically 
 
   5   signalling appears to be identical, in the fibroblast, EGF 
 
   6   can elicit proliferation.  In the 32D cells, all we see,

   7   really, is a weak survival and a delay in the onset of 
 
   8   apoptosis. 
 
   9             [Slide.] 
 
  10             A second project in the laboratory is trying to 
 
  11   understand the molecular basis for the role of the protein

  12   tyrosine phosphatase SHP‑2 in signalling by the erbB 
 
  13   receptors.  Several years ago, we were able to show that 
 
  14   SHP‑2 plays a positive and a central role in 
 
  15   mitogen‑activated protein kinase activation by the entire 
 
  16   erbB family of receptors.

  17             We are trying to identify how SHP‑2 functions in 
 
  18   that regard and what are the targets for SHP‑2. 
 
  19             [Slide.] 
 
  20             One of the ways that we have been addressing this 
 
  21   question is to generate a constituitively active form of

  22   SHP‑2, express it in cells and see what pathways and 
 
  23   transcription factors we can turn on in the absence of 
 
  24   receptor activation. 
 
  25             We have generated several constituitively active 

                                                                 43 
 
   1   forms of SHP‑2 that, in vitro, show at least a ten‑fold

   2   greater activity relative to the wild type and preliminary 
 
   3   experiments in intact cells show that we are able to turn on 
 
   4   a number of specific transcription factors in the absence of 
 
   5   receptor activation. 
 
   6             With that, I will end and answer any questions if

   7   there are any. 
 
   8             DR. SALOMON:  Is there a SHP‑2 knockout? 
 
   9             DR. JOHNSON:  It is lethal.  There actually is a 
 
  10   SHP‑2 knockout where a defective form of SHP‑2 still appears 
 
  11   to be made and so it is missing one of its SH‑2 domains, the

  12   protein that is made.  It has been useful even though there 
 
  13   is death in utero.  They have been able to establish some 
 
  14   cells and use them in some studies. 
 
  15             Whether that mutant form of SHP‑2 has any 
 
  16   signalling capacity is not exactly clear.  So there is not a

  17   knockout where there is no protein, that I am aware of. 
 
  18             DR. SALOMON:  I had a follow‑up question.  We are 
 
  19   interested in SHP‑2's association with the CXCR‑4 receptor.  
 
  20   Have you done anything with putting it into a T‑cell or any 
 
  21   other cell and looked at migration?

  22             DR. JOHNSON:  Putting in the constituitively 
 
  23   active forms? 
 
  24             DR. SALOMON:  Putting in the ten‑fold 
 
  25   constituitvely active‑‑ 

                                                                 44 
 
   1             DR. JOHNSON:  No; not in those cells.  We are

   2   mostly interested in fibroblasts and epithelial cells.  But 
 
   3   SHP‑2 appears to be doing a lot of different things in 
 
   4   different settings. 
 
   5             DR. SAUSVILLE:  So, in the model system that you 
 
   6   described of the EGFR independent activation, the kinase

   7   independent activation of the MAP kinase, does that require, 
 
   8   though, an active erbB2 tyrosine kinase domain or is it 
 
   9   actually independent of both? 
 
  10             DR. JOHNSON:  It appears to require erbB2 kinase 
 
  11   activity although, at times, we have seen a very weak signal

  12   generated by just heterodimerization where the EGFR is 
 
  13   kinase inactive and erbB2 is kinase inactive.  But is very 
 
  14   weak relative to the erbB2 kinase active heterodimer. 
 
  15             DR. SALOMON:  Thanks very much.  Very interesting 
 
  16   work.

  17             Next is Dr. Donnelly. 
 
  18             DR. DONNELLY:  Well, I am pleased to say that I 
 
  19   was not a victim of this morning's inclement weather, 
 
  20   although I was worried about this about thirty minutes ago 
 
  21   sitting out on the interstate.  I am pleased that I was able

  22   to make it here. 
 
  23             [Slide.] 
 
  24             As Dr. Rosenberg mentioned previously, the 
 
  25   research in my laboratory is primarily focused on exploring 

                                                                 45 
 
   1   the mechanisms by which certain cytokines and how cytokines

   2   cross‑regulate the actions of one another using principally 
 
   3   monocytes and, to some extent, murine macrophages as targets 
 
   4   for the actions of these cytokines. 
 
   5             In particular, we are focussing on how interferons 
 
   6   can inhibit IL4‑induced signalling, conversely how IL4 can

   7   inhibit the activation of monocytes and macrophages in 
 
   8   response to interferon gamma stimulation 
 
   9             Just very quickly, I would like to show you a 
 
  10   couple of slides that illustrate this second point‑‑that is, 
 
  11   now IL4 can inhibit interferon‑gamma‑induced responses. 

  12   There are a number of genes that are 
 
  13   interferon‑gamma‑inducible in both monocytes and 
 
  14   macrophages.  They include genes such as the high‑affinity 
 
  15   FC receptor of IgG, the B7 isoforms CD80 and CD86, ICAM1, 
 
  16   IP10 and iNOS.

  17             [Slide.] 
 
  18             This is simply to illustrate that when one looks 
 
  19   at the effects of interleukin 4 on induction of certain 
 
  20   genes that are interferon‑gamma‑inducible, in this case the 
 
  21   FCgammaR1 gene.  This is an RNA analysis of RNA from

  22   monocytes that were stimulated either with interferon gamma 
 
  23   alone, which gives a significant level of FCgammaR1 gene 
 
  24   expression. 
 
  25             However, when cells are preincubated with 

                                                                 46 
 
   1   interleukin for, there is virtually a complete inhibition of

   2   the ability of interferon gamma to induce expression of this 
 
   3   gene. 
 
   4             [Slide.] 
 
   5             This inhibitory effect of interleukin 4 on 
 
   6   interferon‑gamma‑inducible gene expression in human

   7   monocytes correlates with an inhibition of activation of the 
 
   8   transcription factor, Stat 1, which is the principle 
 
   9   interferon‑gamma‑inducible transcription factor.  The 
 
  10   inhibitory effect is not immediately apparent, so here this 
 
  11   suppression of activation of STAT 1 requires that the cells

  12   are preincubated for at least 60 minutes or so before this 
 
  13   effect becomes apparent and, thereafter, is fairly complete. 
 
  14             More importantly, we had found that this induction 
 
  15   of inhibition by IL4 of interferon gamma's ability to 
 
  16   activate STAT 1 and to activate expression of

  17   interferon‑gamma‑responsive genes correlates with the 
 
  18   ability of IL4 to induce expression of a novel gene known as 
 
  19   SOCS1, or suppressor of cytokine signalling 1, which is one 
 
  20   member of a family of genes which now numbers seven or 
 
  21   eight.

  22             IL4 activates expression of SOCS1 in human 
 
  23   monocytes.  The induction of SOCS1 mRNA which, it is quite 
 
  24   apparent, at 60 minutes correlates with the inhibition of 
 
  25   STAT 1 activation. 

                                                                 47 
 
   1             [Slide.]

   2             If you over‑express SOCS1 in a myeloid cell line, 
 
   3   in this case M1, this is, again, an RNA analysis of total 
 
   4   RNA, a panel of M1 cells, either the parental line or M1 
 
   5   transformance that over‑expressed SOCS1 or SOCS2.  When 
 
   6   these cells are stimulated with interferon gamma, the

   7   parental cells, you can see that there is strong induction 
 
   8   of FCgammaR1 and mRNA. 
 
   9             Forced expression of SOCS2 did not inhibit the 
 
  10   ability of interferon gamma to upregulate FCgammaR1 gene 
 
  11   expression.  However, forced expression of SOCS1 markedly

  12   inhibited the ability of interferon gamma to upregulate 
 
  13   FCgammaR1 gene expression. 
 
  14             [Slide.] 
 
  15             The SOCS proteins encoded by the SOCS genes act by 
 
  16   two principle mechanisms.  In future experiments, we are

  17   focussing in terms of how IL4 can inhibit 
 
  18   interferon‑gamma‑inducible gene expression specifically as 
 
  19   to the mechanism by which SOCS1 inhibits expression of 
 
  20   interferon‑gamma‑inducible genes. 
 
  21             I am not going to go through this in any detail in

  22   the interest of time, but, suffice it to say, when 
 
  23   expressed, the SOCS1 protein interacts specifically with the 
 
  24   receptor‑associated JAKs which thereby blocks the ability of 
 
  25   the kinase to phosphorylate the intercellular domain of the 

                                                                 48 
 
   1   cytokine receptor.

   2             For example, in our studies, we are interested in 
 
   3   the interferon gamma receptor complex.  Alternatively, other 
 
   4   members of the SOCS family, in this case Cis1, act by 
 
   5   binding directly to the phosphotyrosine motif on the 
 
   6   intercellular domain of the cytokine receptor and, again,

   7   block the ability of the latent STAT from docking and, in 
 
   8   turn, becoming activated by the receptor‑associated JAKs. 
 
   9             [Slide.] 
 
  10             On a more general level, the importance, I think, 
 
  11   of understanding how cytokines can cross‑regulate the

  12   actions of one another, and the role of the SOCS genes and 
 
  13   the SOCS proteins in mediating this inhibition is very 
 
  14   important.  In terms of understanding how the balance of 
 
  15   cytokines produced by either Th1‑ or Th2‑type T‑cells in 
 
  16   certain disease states predisposes to certain pathologies.

  17             For example, it has been generally considered 
 
  18   that, in many chronic autoimmune diseases such as rheumatoid 
 
  19   arthritis or multiple sclerosis, that there is an increased 
 
  20   frequency of Th1‑type T‑cells, increased production and 
 
  21   activity of interferon gamma which may, in fact, disrupt the

  22   normal balance of Th1 versus Th2. 
 
  23             Conversely, cytokines such IL4 and IL13 produced 
 
  24   by Th2‑type T‑cells normally control and prevent excessive 
 
  25   activation by interferon gamma. 

                                                                 49 
 
   1             It is also worth stating that IL4 is being tested

   2   as an antiinflammatory agent in certain autoimmune diseases 
 
   3   and the molecular basis for that may, in fact, involve a 
 
   4   role for the activation of the SOCS genes in this process. 
 
   5             Let me leave it at that and address any questions. 
 
   6             DR. SALOMON:  Thank you.  Very well done.

   7             DR. BLUESTONE:  I have a question.  I may have 
 
   8   missed this.  If you overexpress constitutive STAT, have you 
 
   9   actually been able to bypass the inhibition of IL4 in any 
 
  10   way by overexpressing any of these downstream targets? 
 
  11             DR. DONNELLY:  By overexpressing the STATs?

  12             DR. BLUESTONE:  Something that will actually 
 
  13   bypass‑‑as I understand it, the IL4 inhibits.  If the IL4 
 
  14   inhibits and you have got a number of readouts of that 
 
  15   inhibition, STAT inhibition as well as the SOCS inhibition, 
 
  16   I am just wondering if you can overcome the inhibition by

  17   bypassing that part of the pathway. 
 
  18             DR. DONNELLY:  We haven't actually designed 
 
  19   experiments to deliberately attempt that.  I think, in 
 
  20   theory, one could use a dominant negative SOCS to, perhaps, 
 
  21   overcome the inhibitory effect, something of that sort.

  22             DR. BLUESTONE:  Right. 
 
  23             DR. DONNELLY:  We haven't done those‑‑ 
 
  24             DR. BLUESTONE:  I am just trying to get a sense of 
 
  25   which of the effects you see are direct effects of the IL4 

                                                                 50 
 
   1   and which of them are downstream consequences of the IL4.

   2             DR. DONNELLY:  Very simply put, IL4 activates 
 
   3   STAT6.  STAT6, in turn, activates expression of SOCS1 which 
 
   4   then feedback‑inhibits‑‑well, certainly can feedback‑inhibit 
 
   5   IL4‑induced signalling; that is, the same cytokine that 
 
   6   elicits its expression but we have found that it can also

   7   cross‑regulate the activation of a cytokine. 
 
   8             It signals through an unrelated receptor, in this 
 
   9   case, the interferon‑gamma receptor.  We are specifically 
 
  10   looking at the mechanism of action by which SOCS1 inhibits, 
 
  11   for example, interferon‑gamma signalling.  We have now, in

  12   fact, been able to aminoprecipitate and show a correlation 
 
  13   in terms of a reduction of tyrosine phosphorylation of the 
 
  14   receptor, itself. 
 
  15             We are also hoping to, very soon, be able to show 
 
  16   a physical docking of SOCS1 on the receptor.

  17             DR. BLUESTONE:  On the interferon‑gamma receptor. 
 
  18             DR. DONNELLY:  That's correct. 
 
  19             DR. SALOMON:  Thank you. 
 
  20             I guess Dr. Petricoin is not here.  So, Kathy; 
 
  21   you're on.

  22             DR. ZOON:  It is a pleasure to be able to present 
 
  23   today.  I know Jeff was looking over.  He goes, "Now, where 
 
  24   do you belong in the organization?"  It is a bit confusion. 
 
  25             [Slide.] 

                                                                 51 
 
   1             I am in the Laboratory of Chemistry in the

   2   Division of Therapeutic Proteins in the Office of 
 
   3   Therapeutics.  So I am in the organization based on the 
 
   4   scope of the responsibilities of my lab's research program 
 
   5   as well as the regulatory responsibilities which is slightly 
 
   6   weird, but that is the way it is right now.

   7             My research in the laboratory involves the 
 
   8   interferon alpha's structure and function.  This is an area 
 
   9   that I have been actively engaged in for twenty‑five years.  
 
  10   One would say, "Gee, aren't you tired of studying interferon 
 
  11   alpha?"  I wish I could say I was.  But each time, I think,

  12   "Well, maybe I should do something else," something 
 
  13   interesting pops up and there is a lot yet to discover and 
 
  14   to really understand how interferon is working. 
 
  15             So this has still been a function of my 
 
  16   laboratory.  I have to, one, give credit to the members of

  17   the laboratory.  Mr. Joe Bekicz is here.  He is sitting in 
 
  18   the back.  Renchu Human, who is an research scientist who is 
 
  19   also in my laboratory.  And recently, as a result of some of 
 
  20   the recommendations of the site‑visit team, I have hired an 
 
  21   ORIS fellow, Hannah Schmietzer, to do some of the studies

  22   that were recommended by the site‑visit team. 
 
  23             I will discuss some further activities that we 
 
  24   have been doing as a result of the recommendations of the 
 
  25   site‑visit team. 

                                                                 52 
 
   1             [Slide.]

   2             What I am going to present in the first two slides 
 
   3   are what I believe are the scientific significance of the 
 
   4   work we have been doing on the interferon project.  We have 
 
   5   isolated twenty‑two components of natural human interferon 
 
   6   alpha.  In doing so, we have, then, in the purification

   7   process, determined their biological activities and their 
 
   8   physicochemical structural properties. 
 
   9             In doing so, we have identified a number of 
 
  10   important areas.  One is that there is a distinct spectrum 
 
  11   of biological activities associated with each of these

  12   components.  In saying that, we have looked at their 
 
  13   antiviral properties and their antiproliferative properties 
 
  14   and a number of immunomodulatory assays. 
 
  15             In summary, they each have a distinct combination 
 
  16   of antiviral, antiproliferative and immunomodulatory

  17   properties. 
 
  18             From a scientific and interesting point of view, 
 
  19   there are nine interferon‑‑at least nine genes and probably 
 
  20   in the order of fifteen to eighteen interferon alpha genes 
 
  21   that have been identified, located in human chromosome 9. 

  22   The question really that still is of great interest to the 
 
  23   field, why has nature made this redundancy in interferons 
 
  24   and what is their roles and responsibilities in eliciting 
 
  25   their biological activities under a variety of different 

                                                                 53 
 
   1   stimuli and in compartmentalization in terms of the types of

   2   tissues that they may be produced by or cells that they may 
 
   3   be produced by. 
 
   4             I think we are getting clues based on these 
 
   5   studies and some others that will contribute to this area.  
 
   6   We have also identified some very important cell‑binding

   7   properties.  In our examination of the interferon alphas, 
 
   8   the predominant interferon alpha, one that has been cloned 
 
   9   and has been used therapeutically, is interferon alpha 2. 
 
  10             In our system, we have identified two alpha‑2‑like 
 
  11   components.  In looking at competitive binding experiments

  12   using radiolabled interferon alpha 2, we have found a number 
 
  13   of interferons, in particular, that have interesting 
 
  14   properties, one being a component "o" which has a very high 
 
  15   antiviral activity, an extremely high antiproliferative 
 
  16   activity but competes poorly for the alpha 2B binding site.

  17             So this has led us to propose either that there is 
 
  18   a multicomponent receptor for which there may be a unique 
 
  19   component attached to the clearly defined interferon alpha 
 
  20   receptor 1 and alpha receptor 2 that have already been 
 
  21   defined or there may actually be a distinct receptor that we

  22   still have not determined. 
 
  23             So these studies are clearly important and 
 
  24   underway.  I will discuss some studies that we have recently 
 
  25   started as a result of some of the recommendations of the 

                                                                 54 
 
   1   site‑visit team.

   2             The third is in our structure‑function looking at 
 
   3   the different gylcoforms of interferon alpha.  We find 
 
   4   three.  Alpha‑2‑like interferons in natural cells are 
 
   5   glycosylated, which is an interesting aspect of these.  
 
   6   Those are o‑linked sugars.  There is alpha 14 which is one

   7   of our components which is also glycosylated at asparagine. 
 
   8             We are currently continuing these studies using 
 
   9   MALDI TOF mass spec to get a better handle on the particular 
 
  10   structure, those glycoforms. 
 
  11             [Slide.]

  12             We have also been involved in protein engineering 
 
  13   where we have taken two interferons, one the alpha 2 which 
 
  14   is the dominant form in lymphoblastoid interferon and, two, 
 
  15   we have cloned what was our component "o" that gave the 
 
  16   interesting biological characteristics of high

  17   antiproliferative activity, high antiviral activity but poor 
 
  18   alpha‑2‑B binding. 
 
  19             We have made a variety of forms of this.  What 
 
  20   this has allowed us to do is to identify critical regions in 
 
  21   the molecule with respect to binding and antiproliferative

  22   activity.  What we have found is that the amino terminal 
 
  23   portion of the molecule is very important in the binding 
 
  24   domain of the receptor. 
 
  25             The binding has a higher affinity with the 

                                                                 55 
 
   1   interferon alpha 2 domain at the amino terminus.  We have

   2   also found that the C terminal end of the molecule is very 
 
   3   important for the antiproliferative activity.  In fact, we 
 
   4   have assessed that one particular region from 75 to 95 is 
 
   5   also very important in the antiproliferative activity. 
 
   6             Taking our hybrids and making various versions, we

   7   have found that not only in the region 75 to 95 are there 
 
   8   critical amino acids but additional amino acids subsequent 
 
   9   from 95 to 166.  In doing some site‑directed mutagenesis, we 
 
  10   have found two tyrosines that are extremely important in the 
 
  11   antiproliferative activity, one at position 86 and one at

  12   position 90. 
 
  13             In addition, we have been doing some receptor 
 
  14   signal transduction experiments.  We have been doing these 
 
  15   in collaboration with Chip Petricoin from our division.  
 
  16   What we have found, in looking at a variety of systems, is

  17   that using our hybrids, the interferon pathway may not be 
 
  18   solely determined by the activation of STAT1 and STAT2.  We 
 
  19   have recently published. 
 
  20             Right now, this is an area that the lab will be 
 
  21   pursuing in greater depth.  I am, right now, looking and

  22   recruiting and talking to various individuals to pursue this 
 
  23   area in greater depth which was also a recommendation of the 
 
  24   site‑visit team. 
 
  25             [Slide.] 

                                                                 56 
 
   1             This summarizes the mission relevance of the work

   2   on interferon.  As Dr. Rosenberg outlined, interferon alpha 
 
   3   is a product that is regulated by the FDA.  It is a licensed 
 
   4   product.  It is also still under IND for a variety of 
 
   5   studies.  It is licensed for everything from hepatitis B and 
 
   6   C to AIDS‑related Kaposi's sarcoma.

   7             We are very interested in understanding the 
 
   8   structure/function for two reasons.  One is so that we can 
 
   9   get a better handle on the activities and effectiveness of 
 
  10   interferons using different clinical situations or 
 
  11   potentially used and, two, for safety profiles of these

  12   products because, by managing and understanding toxicities 
 
  13   of these interferons, one can eventually look at the 
 
  14   possibility of engineering an interferon specifically that 
 
  15   enhances its effectiveness and decreases it toxicities. 
 
  16             So those studies have as its basis for what is

  17   some of the underpinning of our work.  In addition, the work 
 
  18   we are doing‑‑the lab has been very important interesting 
 
  19   the development of methods and standards for interferon 
 
  20   alphas.  We have worked with the National Institutes of 
 
  21   Biological Standards and Control in the United Kingdom that

  22   makes standards available throughout the world. 
 
  23             Our methods and our interferons have often been 
 
  24   the lead in making determinations on some of these 
 
  25   standards, so that work has contributed much to the 

                                                                 57 
 
   1   interferon alpha standards world.

   2             Also, the uses of new technologies such as protein 
 
   3   engineering will enhance the expertise to review new 
 
   4   cytokines and interferons.  These techniques are currently 
 
   5   being used not only for interferons but other products as 
 
   6   well.

   7             [Slide.] 
 
   8             So where are we going in the future?  As part of 
 
   9   some of the recommendations of the team that came to visit 
 
  10   us in December, we were recommended to further study the 
 
  11   binding of alpha 21 and the hybrids to Daudi cells and

  12   characterize the receptor binding studies. 
 
  13             We have hired a post‑doc to do this.  Those 
 
  14   studies are underway and the data is in the process of being 
 
  15   collected.  We also plan to further study the binding 
 
  16   characteristics using soluble interferon receptors.  This

  17   work is being done in collaboration with the Weissman 
 
  18   Institute and Gideon Schreiber.  We have already got a fair 
 
  19   amount of data looking at the dissociation contents using 
 
  20   the soluble IFNR2 receptor. 
 
  21             These studies will be very important in looking at

  22   the interactions of our different interferons with not only 
 
  23   IFNR2 but IFNR1.  We have all the variants of the 
 
  24   interferons.  The Weissman has all the hybrids and mutants 
 
  25   of the IFNR1 and IFNR2.  So, by studying the characteristics 

                                                                 58 
 
   1   of these, we may get further insight into the structure and

   2   function and interaction of the interferons with their 
 
   3   receptors. 
 
   4             We are also, again, engaged in intracellular 
 
   5   signaling pathways.  Right now, this work is really being 
 
   6   generated in a number of areas.  We are hoping to narrow

   7   some of the important pathways by looking at our hybrids in 
 
   8   some of these microray chip technologies maybe to identify 
 
   9   more a subset of signalling pathways that might be more 
 
  10   advantageous to study. 
 
  11             We have a number of recommendations by the site

  12   visit that we are considering.  Right now, our first 
 
  13   priority is to look for an excellent candidate to help us 
 
  14   with these studies. 
 
  15             We are also looking at the role of the carboxy 
 
  16   terminus of interferon alpha maximizing the

  17   antiproliferative activity.  There are specific regions from 
 
  18   95 to 166 in the alphas, particularly in alpha 21, that we 
 
  19   will be doing some site‑specific mutagenesis in to really 
 
  20   further define what are the critical amino acids to elicit 
 
  21   the antiproliferative activity.

  22             I already mentioned our work on MALDI TOF looking 
 
  23   at the carbohydrate structures of those glycosylated 
 
  24   interferons.  We are also studying with a number of people 
 
  25   in the Center for Biologics and in the National Institutes 

                                                                 59 
 
   1   secondary and potentially tertiary structures of some of the

   2   interferon alphas so we can understand the three‑dimensional 
 
   3   interaction between these species ultimately with their 
 
   4   receptor. 
 
   5             The NMR studies are being done with Darren 
 
   6   Freeburg in the Office of Vaccines and the circular

   7   dichroism studies are being done in conjunction with Peter 
 
   8   McFee from the National Institutes of Health. 
 
   9             [Slide.] 
 
  10             Finally, in looking at some of the biological 
 
  11   functions in the next slide, we are looking with Dr.

  12   Kathleen Clouse's lab the effects of our hybrids on HIV 
 
  13   infection, of primary macrophages and T‑cells.  We have some 
 
  14   very exciting results with some of our site‑directed mutants 
 
  15   having extremely high antiviral activity against HIV that 
 
  16   looks very exciting.

  17             We are repeating those studies to make sure that 
 
  18   those data are reproducible.  We are working with Dr. Eda 
 
  19   Bloom and the Division of Cellular and Gene Therapy to 
 
  20   better understand the effects of interferon on natural 
 
  21   killer‑cell activity and we are also pursuing a number of

  22   other immunomodulatory activities to have a better sense of 
 
  23   how interferon alphas are affecting the immune system. 
 
  24             I would like to thank our site‑visit team.  They 
 
  25   gave a lot of excellent advice and, hopefully, we will be 

                                                                 60 
 
   1   able to follow those through and get the results that might

   2   be expected. 
 
   3             Thank you. 
 
   4             DR. SALOMON:  Thank you, Kathy. 
 
   5             Again, in the interest of time, I think we should 
 
   6   take the next half hour and go quickly into the closed

   7   session. 
 
   8             [Whereupon, at 9:40 a.m., the proceedings were 
 
   9   recessed, to be resumed at 10:15 a.m.] 

                                                                 61 
 
   1                       TOPIC I (Continued)         [10:15 a.m.]

   2         Islet Transplantation/Preclinical Animal Models 
 
   3             DR. SALOMON:  I would like to welcome everyone 
 
   4   back to the second day of the BRMAC's considerations of 
 
   5   islet transplantation.  Today, as promised, there are no 
 
   6   more limitations to the discussion on clinical issues as

   7   there were yesterday.  Apologies to everybody for that 
 
   8   artificiality. 
 
   9             So why don't we just get started.  We have to 
 
  10   finish this first session at five minutes to 12:00 which 
 
  11   sort of gives us an arbitrary finishing point, but there are

  12   some of us who need to check out, including me. 
 
  13             The other thing that I want to announce to 
 
  14   everybody is Rosanna Harvey who, if you don't know Rosanna, 
 
  15   please note if you can make travel arrangements with Rosanna 
 
  16   at least in any break that we have‑‑well, we won't have any

  17   break until five minutes before noon, but if you can make 
 
  18   any arrangements you can for travel with Rosanna. 
 
  19             Then, with that introduction, and I hope I am not 
 
  20   missing anything, I would like to introduce Karen Weiss from 
 
  21   the Division of Clinical Trial Design and Analysis to

  22   present an FDA introduction to this morning's events. 
 
  23                         FDA INTRODUCTION 
 
  24             DR. WEISS:  I will be very brief.  I just wanted 
 
  25   to welcome everybody back to the second day's discussion, to 

                                                                 62 
 
   1   thank the members of the BRM, the Endocrine and Metabolic

   2   Drugs Advisory Committee for joining us as well as all the 
 
   3   experts taking time from their busy schedule to come here 
 
   4   and discuss this extremely important topic with us. 
 
   5             As everybody now knows, today's discussion is 
 
   6   supposed to be focussing on the preclinical and the bridge

   7   from the preclinical to the clinical.  We have arranged the 
 
   8   afternoon for a series of questions based on topics that 
 
   9   have come before us at the FDA with these types of 
 
  10   therapies. 
 
  11             In almost every one of these, we have a number of

  12   questions with respect to issues in terms of clinical trials 
 
  13   as well as whether or not preclinical models can help sort 
 
  14   out and address some of these questions. 
 
  15             So we are very much looking forward to your advice 
 
  16   and discussions this afternoon.  I wanted to start the

  17   session, then, by introducing Dr. Lauren Black, a 
 
  18   pharmacologist from our division, who will present a brief 
 
  19   overview. 
 
  20                       Open Public Hearing 
 
  21             DR. SALOMON:  Just one minor thing.  I guess

  22   because, again, based on the format of the public hearing, 
 
  23   we have not had anybody officially request time at this 
 
  24   point but I am reminded that I should, again, ask if there 
 
  25   is anyone in the audience who would like a five‑minute 

                                                                 63 
 
   1   period to address the committee before we get started, you

   2   are more than welcome to step up now. 
 
   3             Lauren? 
 
   4         Animal Models of Islet Therapy: Utilization for 
 
   5           Clinical Trial Design and Safety Assessment 
 
   6             DR. BLACK:  I would like to thank the members of

   7   the advisory committee and the distinguished guests that are 
 
   8   here for participating in this session on animal models of 
 
   9   islet therapy. 
 
  10             [Slide.] 
 
  11             I will be presenting the FDA perspective on

  12   utilizing these models for clinical‑trial design.  Specific 
 
  13   comments on the model attributes and results generated in 
 
  14   these models will be addressed in the upcoming two 
 
  15   presentations. 
 
  16             Islet therapy models are viewed at FDA in the

  17   context of the larger field of solid‑organ transplantation.  
 
  18   Transplant therapy models have been very helpful in 
 
  19   prospectively designing clinical trials of investigational 
 
  20   immunosuppressant drugs and biologic therapies and, more 
 
  21   recently, of combined drug use and immunomodulatory

  22   strategies such as donor‑lymphocyte infusions. 
 
  23             [Slide.] 
 
  24             Preclinical‑model data are generated to advance 
 
  25   our scientific understanding and are utilized to support the 

                                                                 64 
 
   1   rationale for new clinical investigations.  In this context,

   2   animal models are used to assess the clinical utility of 
 
   3   procedures such as in identifying promising 
 
   4   immunosuppressive regimens, identifying effect islet doses 
 
   5   or administration methods or to ask if there are durable 
 
   6   effects of significant sequelae of effective treatments such

   7   as reductions in disease‑related morbidity. 
 
   8             In these aspects, well‑designed animal trials can 
 
   9   contribute on the benefit side to the evaluation of clinical 
 
  10   risk/benefit assessment as was mentioned by Dr. Zoon and 
 
  11   people yesterday morning.

  12             [Slide.] 
 
  13             Animal data also serve an important role in 
 
  14   supporting the safety of investigational data and are used 
 
  15   to determine safe doses and administration methods, for 
 
  16   instance, by examining surgery or infusion adverse

  17   reactions. 
 
  18             Additionally, the dynamics, nature and 
 
  19   dose‑response relationships of the toxicities are evaluated.  
 
  20   The nature of the toxicity may raise added concerns when 
 
  21   events are hard to monitor clinically, are irreversible or

  22   are sudden in onset.  Animal data are compared with the 
 
  23   proposed clinical protocol and utilized to guide choices 
 
  24   regarding clinical monitoring, endpoints and schedule, the 
 
  25   appropriate patient inclusion or exclusion criteria and are 

                                                                 65 
 
   1   utilized to suggest appropriate adjunctive therapies.

   2             Risks identified in animal studies often lead to 
 
   3   modifications in clinical‑trial design. 
 
   4             [Slide.] 
 
   5             To support clinical trials, animal models should 
 
   6   be chosen to be clinically relevant.  Islet allografting

   7   could be performed in an intact, healthy animal but the 
 
   8   islet metabolic function would likely go unchallenged in the 
 
   9   presence of a healthy pancreas. 
 
  10             It is an advantage for this field that 
 
  11   allotransplantation is feasible in outbred diabetic animals

  12   in a manner highly analogous to human allografting. 
 
  13             [Slide.] 
 
  14             The proposed clinical strategy and its perceived 
 
  15   risks and departures from knowns will influence the choice 
 
  16   in animal model and the degree of biologic comparability

  17   needed between the animal and the patient.  The more 
 
  18   relevant the animal model, the greater the degree of 
 
  19   confidence that an absence of safety problems in animals 
 
  20   provides strong safety support for patients. 
 
  21             In counterpoint, where models are viewed as poorly

  22   comparable, clinical trials such as those perhaps 
 
  23   tolerization therapies may be subject to more protocol 
 
  24   restrictions. 
 
  25             [Slide.] 

                                                                 66 
 
   1             In brief, animal models for diabetes range from

   2   inbred NOD mice to pancreatectomized non‑human primates and, 
 
   3   very rarely, natural models of adult‑onset diabetes.  No one 
 
   4   animal model could be expected to generate a perfect 
 
   5   predictor for human outcome.  The next speakers will 
 
   6   highlight the utilities and limitations of each model.

   7             [Slide.] 
 
   8             As in all thorough scientific investigations, 
 
   9   there are attributes of preclinical study design and conduct 
 
  10   that can provide the most convincing support for the safety 
 
  11   of an IND.  The study should provide a basis for

  12   comprehensive analysis of animal responses to all aspects of 
 
  13   treatment, overall health, drug regimen and islet‑induced 
 
  14   toxicity and disease or disease markers are all needed to be 
 
  15   monitored in detail. 
 
  16             Study design should also permit objective

  17   assessment of results, using prospectively designed 
 
  18   protocols, randomization and blinding wherever possible to 
 
  19   achieve this end.  From the perspectives of data integrity 
 
  20   and CBER review, the CFR outlines good laboratory‑practice 
 
  21   standards and requires that reporting of animal studies for

  22   INDs allows assessment of all aspects of animal safety. 
 
  23             In order to achieve this, animal‑study reports 
 
  24   need to be detailed and fully tabulated to include both 
 
  25   group and individual results. 

                                                                 67 
 
   1             [Slide.]

   2             There have been a number of shortcomings in 
 
   3   preclinical aspect of INDs submitted to date for islet 
 
   4   therapy and similar to those for other cell‑therapy fields.  
 
   5   These include incomplete datasets and designs that are 
 
   6   incompletely comparable to proposed clinical protocols.

   7             The concern is that, as proposed clinical 
 
   8   therapies move further from those with which we have current 
 
   9   clinical experience and preclinical experience, these 
 
  10   inadequacies in preclinical support could become limiting to 
 
  11   clinical development or miss an opportunity to predict

  12   clinical adverse reactions. 
 
  13             [Slide.] 
 
  14             In summary, clinical strategy and preclinical 
 
  15   study design should be closely integrated.  While 
 
  16   recognizing that some information can only come from

  17   clinical trials, some data useful in clinical‑trial design 
 
  18   can be gathered preclinically.  These data could include 
 
  19   regimens for islet‑only transplant. 
 
  20             In the past, animal models animal models have 
 
  21   demonstrated utility in predicting clinical drug toxicities

  22   and could generate data to aid in reducing islet toxicity or 
 
  23   rejection.  In contrast, many questions remain for 
 
  24   animal‑model us of immunomodulators such as for tolerization 
 
  25   approaches. 

                                                                 68 
 
   1             Current autoimmune animal models for these

   2   therapies have been open to criticism on account of clinical 
 
   3   comparability.  New research on the human pathophysiology of 
 
   4   diabetes may be needed to bridge current gaps in our 
 
   5   understanding of the models and human disease and the 
 
   6   utility of models for therapeutic testing.

   7             Lastly, to improve their utility in clinical‑trial 
 
   8   assessment, designs for preclinical studies of islet therapy 
 
   9   should involve a more detailed concurrently tracked approach 
 
  10   to both safety and activity monitoring.  Consideration of 
 
  11   the best model species, high comparability of animal and

  12   human trial designs, and more detailed documentation would 
 
  13   increase the scientific validity of the preclinical 
 
  14   evaluations and increase the impact of these investigations 
 
  15   on advanced and clinical therapy of diabetes. 
 
  16             Thank you.

  17             I would like to mention that this afternoon you 
 
  18   will be asked to address a number of questions that involve 
 
  19   both clinical and preclinical designs.  They will highlight 
 
  20   the role of preclinical models and the development of 
 
  21   clinical protocols and you will be asked to comment on areas

  22   in which there are unaddressed clinical questions. 
 
  23             Concomitantly, we will discuss how these might be 
 
  24   addressed in preclinical models.  Please consider where we 
 
  25   need to develop new animal models and how those models might 

                                                                 69 
 
   1   aid in identifying immunosuppressant regimens, appropriate

   2   measures of islet activity, evaluating the immunogenicity if 
 
   3   islets or the impact of multiple transplants on 
 
   4   sensitization, establishing the best methods for isolation, 
 
   5   defining quality for the best methods of delivery or 
 
   6   determining the impact of peri‑implant type glucose control

   7   or the effects of the animal models in determining islet 
 
   8   potency or the best determinants of dose. 
 
   9             At this time, Jack, would you like to come up?  I 
 
  10   would like to introduce the next two speakers who have been 
 
  11   invited as experts to provide detailed information on the

  12   models currently in use for islet therapy. 
 
  13             Jack O'Neil has developed and studied dog, pig and 
 
  14   rodent models to evaluate artificial pancreas, encapsulated 
 
  15   the allogeneic islets and allogeneic islet therapies in both 
 
  16   biotech industry and academic laboratories.  Jack will give

  17   a broad overview of the non‑human‑primate models.  He will 
 
  18   cover dog and pig models of islet therapy as well as rodent. 
 
  19             Immediately after Mr. O'Neil, Dr. Norman Kenyon 
 
  20   will address the non‑human‑primate models of islet therapy.  
 
  21   Dr. Kenyon is investigating immunointerventions in three

  22   different primate models of islet therapy and will discuss 
 
  23   the relevance of these models in developing clinical 
 
  24   strategies. 
 
  25              Animal Models of Islet Transplantation 

                                                                 70 
 
   1             MR. O'NEIL:  Good morning

   2             [Slide.] 
 
   3             I would like to thank the FDA for giving me the 
 
   4   opportunity to participate in this meeting as we embark on 
 
   5   one of the most exciting times and, certainly as we heard 
 
   6   yesterday from both the Juvenile Diabetes Foundation and the

   7   National Institutes of Health, one of the best finance times 
 
   8   of islet transplantation. 
 
   9             This presentation will cover some of the 
 
  10   preclinical animal models that are utilized in clinical 
 
  11   islet preclinical studies and development.

  12             [Slide.] 
 
  13             The major obstacles to successful clinical islet 
 
  14   transplantation faced today is to treat insulin‑dependent 
 
  15   diabetes in the type‑1 patient and the autoimmune 
 
  16   destruction of the islet graft.  Above and beyond that, you

  17   have the immuno‑attack that would accompany any allogeneic 
 
  18   tissue transplantation. 
 
  19             Currently islet transplantation is performed only 
 
  20   with a preexisting kidney islet transplant in most cases and 
 
  21   the conventional immunosuppression used to protect the

  22   kidney graft has been demonstrated to be cytotoxic to the 
 
  23   subsequent beta‑cell graft and, lastly, the insufficient 
 
  24   supply of allogeneic tissue has encouraged us to seek 
 
  25   alterative sources for beta‑cell replacement therapy. 

                                                                 71 
 
   1             [Slide.]

   2             The preclinical animal models used to investigate 
 
   3   clinical islet transplantation have a major limitation in 
 
   4   that transplantation incident animal models, there is not a 
 
   5   sufficient for the autoimmune destruction of the graft.  
 
   6   Currently, rodents are the only animal models available for

   7   this.  Most importantly, the NOD mouse, the BB rat and then 
 
   8   a model that I will talk about a little later is the 
 
   9   humanized autoimmunity transfer recipient. 
 
  10             [Slide.] 
 
  11             Basically, what I am going to do is go through

  12   each one of these obstacles and try to list the animal 
 
  13   models that would be appropriate in early development and 
 
  14   later end‑state development towards clinical studies. 
 
  15             The immuno‑attack of the islet graft can be looked 
 
  16   at in immunocompetent rodents, humanized immune‑transfer

  17   recipients and dogs and pigs in the non‑human primates. 
 
  18             [Slide.] 
 
  19             The conventional immunosuppressive regimen that 
 
  20   generally is used in kidney transplantation in order to 
 
  21   protect the islet graft is best studies in large animal

  22   models where the immune system is most closely similar to 
 
  23   the human with the dog, the pig and the non‑human‑primate 
 
  24   models. 
 
  25             [Slide.] 

                                                                 72 
 
   1             In the insufficient supply of allogeneic islets,

   2   although not directly under the scope of this particular 
 
   3   meeting, can be addressed with the same animal models 
 
   4   looking at immunocompromised rodents in early preclinical 
 
   5   studies and then moving up to the larger animal models as 
 
   6   you get closer to clinical development.

   7             [Slide.] 
 
   8             Basically, the methods that can be used for all of 
 
   9   these studies to evaluate both the safety and the efficacy 
 
  10   of preclinical transplantation are listed here.  Generally, 
 
  11   when preclinical studies are undertaken in the laboratory,

  12   efficacy is the primary concern of the investigator but I am 
 
  13   sure the FDA would certainly like us to implement safety 
 
  14   measures of these preclinical studies as well, starting with 
 
  15   the rodents and all the way up to the larger animal models. 
 
  16             For safety, by performing physical exams, a

  17   veterinary checks on the animals measuring body weight, 
 
  18   blood chemistry and hematology.  During the study, you can 
 
  19   get an idea on what the therapeutic effect to the recipient 
 
  20   is and then, in the post‑transplant period, to do a necropsy 
 
  21   and look at major organ systems, histopathologically, to see

  22   if there are any adverse effects to other organ systems in 
 
  23   the recipient animal. 
 
  24             For the efficacy, it is much like the clinical 
 
  25   islet transplant where you are looking at the blood glucose 

                                                                 73 
 
   1   which an exogenous insulin requirement of the patient,

   2   C‑peptide secretion, hemoglobin A1c levels, response to 
 
   3   secretogogue challenge looking at blood glucose, insulin and 
 
   4   C‑peptide secretion and then followed up, post‑transplant 
 
   5   period, with histopathology of the islet graft. 
 
   6             [Slide.]

   7             First, the animal model for transplantation, a 
 
   8   method to chemically induce diabetes in immunocompromised or 
 
   9   immunodeficient rodents with aloxan and streptozotocin, as 
 
  10   we heard yesterday.  It is a very useful model in looking at 
 
  11   an islet function in the absence of the immune system. 

  12   Alloxan and streptozotocin were demonstrated in the '40's 
 
  13   and the '60's to produce diabetes in rats and in larger 
 
  14   animal models with a cytotoxicity to pancreatic beta cells 
 
  15   resulting in insulin‑dependent diabetes characterized by 
 
  16   glucosuria and excessive weight loss and hyperglycemia.

  17             [Slide.] 
 
  18             The advantage to this model, as I said, is to 
 
  19   evaluate the islet function in the absence of the immune 
 
  20   response and the toxicity that is associated with 
 
  21   conventional immunosuppressive agents, as an accessible,

  22   cost‑effective animal model to be used in preclinical 
 
  23   development of therapeutic strategies. 
 
  24             The limitations of animal model is that there 
 
  25   occasionally is return to the spontaneous disease following 

                                                                 74 
 
   1   the induction of diabetes with diabetogenic agents.  The

   2   dose, the severity of the diabetes and the preclinical 
 
   3   outcome in these animal models have been shown to be 
 
   4   strain‑dependent. 
 
   5             [Slide.] 
 
   6             For each one of the animal models, I would like to

   7   go through what I call clinical comparability where we look 
 
   8   at some different aspects of the rodent or the animal model 
 
   9   to the clinic, the surgical methods and the islet dose.  As  
 
  10   far as organ procurement and islet isolation and then the 
 
  11   administration of islets is not comparable to the human

  12   situation for these immune‑compromised animals, 
 
  13   immunodeficient animals, there is no immunosuppression 
 
  14   necessary. 
 
  15             The clinical induction is not an autoimmune 
 
  16   disease and does not allow for the evaluation of therapy

  17   response to an autoimmune attack.  C‑peptide blood glucose 
 
  18   and IVGTT and body weight, all these can be measured, but 
 
  19   there is currently no method to correlate the results to the 
 
  20   engrafted cell mouse. 
 
  21             The histopathologic assessment of the graft can be

  22   performed much like it can be in the clinical setting. 
 
  23             [Slide.] 
 
  24             The spontaneous non‑obese diabetic mouse is a very 
 
  25   important animal model as it represents the best animal 

                                                                 75 
 
   1   model for the immune response in the islets in the type‑1

   2   diabetic setting.  The mouse was derived from an outbred 
 
   3   strain in the 1980s and has been extensively inbred since. 
 
   4             Insulitis occurs in all animals starting at about 
 
   5   four weeks of age and diabetes some time after twelve weeks 
 
   6   with a predominance of diabetes in about 80 percent of the

   7   females and only about 40 percent of the males.  The 
 
   8   diabetes is characterized by glucosuria, excessive weight 
 
   9   loss, hyperglycemia and ketoacidosis.  Without insulin 
 
  10   therapy, it is lethal. 
 
  11             [Slide.]

  12             The advantages of this animal model is that it is 
 
  13   an autoimmune model of insulin‑dependent diabetes.  It is a 
 
  14   cost‑effect animal model and it has extensively 
 
  15   characterized disease etiology.  There is also the 
 
  16   availability of immunological reagents were are not

  17   necessarily the case in the large animal models. 
 
  18             [Slide.] 
 
  19             The limitations; it has been criticized for 
 
  20   representing only one individual with type‑1 diabetes 
 
  21   because of its extensive inbreeding.  There are numerous

  22   interventions that are successful in influencing the onset 
 
  23   of the diabetes in this animal and, unfortunately, many 
 
  24   therapeutic strategies which prevail in rodents fail when 
 
  25   applied to larger animals and to humans. 

                                                                 76 
 
   1             [Slide.]

   2             The clinical comparability is much like that of 
 
   3   the immunocompromised rodents with the exception of the NOD 
 
   4   mouse, disease etiology share many clinical morphological 
 
   5   and immunological features with the human disease and, most 
 
   6   importantly, autoimmunity.

   7             Both immunosuppression and immune stimulation can 
 
   8   prevent the disease in the animal making therapeutic 
 
   9   strategies developed in the NOD mouse sometimes not relevant 
 
  10   to the clinical setting. 
 
  11             [Slide.]

  12             The humanized diabetic immunodeficient mouse is an 
 
  13   animal model that has been extensively studied over the last 
 
  14   decade or so and, basically, with the immunodeficient 
 
  15   environment of the animal, you can transfer human lymphoid 
 
  16   cells into the immunodeficient animal and generate the

  17   humanized mouse to evaluate immune responses to allografts. 
 
  18             Two examples of this type of animal model are the 
 
  19   SCID mouse or the RAG knockout mouse. 
 
  20             [Slide.] 
 
  21             The advantages are that you can evaluate the

  22   mechanisms of islet‑graft rejection.  There is the ability 
 
  23   to manipulate the cells that are transferred into this 
 
  24   recipient and, therefore, the immune system of the 
 
  25   recipient, and there is the potential to compare allo with a 

                                                                 77 
 
   1   normal patient and autoimmune responses with the transfer of

   2   lymphoid cells from a diabetic patient to the islet graft. 
 
   3             The limitations are the degree of the engraftment 
 
   4   and the susceptibility of the animal models to 
 
   5   graft‑versus‑host disease. 
 
   6             [Slide.]

   7             The BB rat was a spontaneous mutation of the 
 
   8   Wistar rat in the '70's and it has been extensively inbred 
 
   9   since.  Insulitis occurs at about four weeks of age and 
 
  10   diabetes after eight weeks.  Prevalence is equal both in 
 
  11   males and females and the insulin‑dependent diabetes is

  12   characterized by glucosuria and excessive weight loss, 
 
  13   hyperglycemia and ketoacidosis. 
 
  14             [Slide.] 
 
  15             The advantages, again, is that it is a spontaneous 
 
  16   autoimmune disease.  It is an accessible cost‑effective

  17   animal model.  It has, again, extensively characterized 
 
  18   disease etiology and the availability of the immunological 
 
  19   reagents. 
 
  20             Limitations are that the animal is T‑cell 
 
  21   deficient to start with.  It is prone to infection, has to

  22   be raised in SPF or VAF facilities and there is really no 
 
  23   real advantage compared to the insulin‑dependent diabetes 
 
  24   found in the NOD mouse. 
 
  25             [Slide.] 

                                                                 78 
 
   1             The clinical comparability would be shared with

   2   the other rodent models with the exception that the diabetes 
 
   3   does share many clinical, morphological and immunological 
 
   4   features with the human disease. 
 
   5             [Slide.] 
 
   6             As far as the dog used as a preclinical model for

   7   islet transplantation, there are basically three types of 
 
   8   diabetes in the dog.  There is the spontaneous diabetes.  
 
   9   You can also induce with the chemical induction with 
 
  10   streptozotocin and alloxan and perform a total 
 
  11   pancreatectomy to achieve insulin‑dependent diabetes.

  12             [Slide.] 
 
  13             Spontaneous diabetes in the dog is reported by the 
 
  14   veterinarians to have an incidence of about between 1:200 to 
 
  15   1:800 animals.  The most common cause of the diabetes is 
 
  16   reported as pancreatitis with diabetes secondary to a

  17   chronic pancreatitis. 
 
  18             There has been one dog breed, the keeshond dog, 
 
  19   that demonstrated a high incidence of diabetes at a young 
 
  20   age‑‑that was reported in the '80's and hasn't been heard 
 
  21   from since‑‑as well as the familial form of the diabetes in

  22   a colony of golden retrievers that has left the literature 
 
  23   since the '80's. 
 
  24             [Slide.] 
 
  25             Chemically induced diabetes with alloxan and/or 

                                                                 79 
 
   1   streptozotocin; dogs are more sensitive to the non‑beta‑cell

   2   toxicity of these agents and, generally, by combining the 
 
   3   two agents, you can miminize the toxic effect of each.  The 
 
   4   dogs following induction are suspectable to severe 
 
   5   hyperglycemia, have to be monitored very closely following 
 
   6   the chemical induction.

   7             [Slide.] 
 
   8             Pancreatectomy‑induced diabetes in a dog is a 
 
   9   fairly straightforward surgery and results in 
 
  10   insulin‑requiring diabetes.  It is essential to supplement 
 
  11   the dog's diet with pancreatic enzymes due to the exocrine

  12   deficiency caused by the pancreatectomy. 
 
  13             [Slide.] 
 
  14             The advantages of using a dog model is that it is 
 
  15   an accessible and well‑established animal model.  They are 
 
  16   easy to handle and train.  Most facilities in an academic

  17   setting, they do have housing available for dogs.  The diet 
 
  18   and metabolism resemble that of a human and it is a 
 
  19   cost‑effective large laboratory‑animal model. 
 
  20             [Slide.] 
 
  21             The limitations are that there is no consistent

  22   source of spontaneously diabetic dogs.  The chemical 
 
  23   induction is associated with a high rate of mortality.  
 
  24   Pancreatectomy results in brittle diabetes, digestive 
 
  25   deficiency and may compromise other organ systems. 

                                                                 80 
 
   1             As with all allograft settings, there is the

   2   inability to differentiate the contribution with the islet 
 
   3   graft to the native pancreatic function. 
 
   4             [Slide.] 
 
   5             For clinical comparability, the pancreas 
 
   6   procurement is generally optimized in the preclinical

   7   setting using a healthy donor in a single organ procurement.  
 
   8   Transplantation has been achieved in the liver by laparotomy 
 
   9   and the transhepatic method and has also been transplanted 
 
  10   in the renal and the splenic sites. 
 
  11             The dose studies support the clinical data in that

  12   the allograft survival and insulin independence are directly 
 
  13   related to the total islet‑cell mass transplanted. 
 
  14             [Slide.] 
 
  15             The immunosuppression requirements in a dog are 
 
  16   generally higher as compared to the human to achieve the

  17   same therapeutic effect.  Spontaneous diabetes in a dog is 
 
  18   usually related to the destruction of the islet secondary to 
 
  19   severe pancreatitis and is not an autoimmune response as 
 
  20   seen in type‑1 diabetes patients. 
 
  21             Some genetic disposition of diabetes was reported

  22   in the '80's but has not been reported since.  Diabetes 
 
  23   induced by chemical agents or pancreatectomy is certainly 
 
  24   different from the autoimmune disease experience in type‑1 
 
  25   patients. 

                                                                 81 
 
   1             [Slide.]

   2             Tissue typing can be done to look at matching and 
 
   3   mismatching.  It has been reported, but the data is limited 
 
   4   and is basically restricted to a few labs that have that 
 
   5   technology.  Again, C‑peptide, blood glucose, IVGTT, body 
 
   6   weight, can be measured but, again, there is no correlation

   7   to the engrafted islet mass.  Histopathologic assessment can 
 
   8   be performed much like it can be in the clinical setting. 
 
   9             [Slide.] 
 
  10             Diabetes in the pig; again, three different types 
 
  11   of diabetes in the pig.  There is spontaneous diabetes,

  12   chemical induction with alloxan and/or streptozotocin and 
 
  13   total pancreatectomy. 
 
  14             [Slide.] 
 
  15             Spontaneous diabetes, there is a line of Yucatan 
 
  16   minipigs that spontaneously developed diabetes.  It is a

  17   type‑1 diabetes with hyperinsulinemia and hyperglycemia.  
 
  18   There is an insulin resistance, especially during 
 
  19   gestational lactation.  They develop angiopathies and other 
 
  20   complications similar to the human disease.  However, there 
 
  21   has not been any extensive reports on that model since the

  22   1980s. 
 
  23             [Slide.] 
 
  24             The chemically induced diabetic pig with alloxan 
 
  25   and/or streptozotocin results in diabetes characterized, 

                                                                 82 
 
   1   again, by hyperglycemia, polyuria, glucosuria and weight

   2   loss.  A partial pancreatectomy can be supplemented with 
 
   3   these diabetogenic agents that result in insulin‑dependent 
 
   4   diabetes. 
 
   5             [Slide.] 
 
   6             A total pancreatectomy‑induced diabetes is a

   7   technically challenging surgery due to the close association 
 
   8   with the vasculature in the pig of the pancreas and the 
 
   9   vasculature.  Diabetes within the first week is 
 
  10   characterized by fatal hyperglycemia of not treated and 
 
  11   then, again, the removal of the exocrine function of the

  12   pancreas and it is essential to supplement the diet of the 
 
  13   animal with supplemental enzymes. 
 
  14             [Slide.] 
 
  15             The advantages are that the anatomy, physiology 
 
  16   and metabolism and diet are similar to human.  It is a

  17   relatively inexpensive animal model to purchase and it does 
 
  18   have the unique ability to evaluate the MCH disparities with 
 
  19   the NIH minipigs. 
 
  20             [Slide.] 
 
  21             The limitations of the animal model of the pig

  22   islet isolation is that it is probably the most technically 
 
  23   challenging procedure.  It is probably no coincidence that 
 
  24   there are very few labs that have had successful preclinical 
 
  25   studies in porcine islet transplantation. 

                                                                 83 
 
   1             They are difficult animals to handle and they are

   2   not easily trained.  Because of the difficulty of the close 
 
   3   association with the vasculature, this is a potential for 
 
   4   incomplete pancreatectomy.  Pigs are very susceptible to 
 
   5   post‑operative infection due to the nature of the beast, and  
 
   6   the inability, again, to differentiate the contribution of

   7   the drug graft versus the native pancreas. 
 
   8             [Slide.] 
 
   9             The clinical comparability for the pig is that, 
 
  10   again, the pancreas procurement is generally optimized with 
 
  11   a healthy donor and a single‑organ procurement. 

  12   Transplantation sites have included the spleen, the liver, 
 
  13   the kidney capsule by laparotomy. 
 
  14             Dose studies support clinical data again that the 
 
  15   graft survival and insulin independence are directly related 
 
  16   to the total islet mass transplanted.

  17             [Slide.] 
 
  18             Immunosuppression, again, in the animal model is 
 
  19   generally higher to achieve the same therapeutic effect.  
 
  20   Spontaneous diabetes and diabetes induced by the chemical 
 
  21   agents and/or pancreatectomy are certainly different than

  22   the autoimmune attack experienced in type‑1 diabetic 
 
  23   patients. 
 
  24             Minor and major histocompatibility matching can be 
 
  25   studied in the partially inbred NIH swine model 

                                                                 84 
 
   1             [Slide.]

   2             Again, the C‑peptide blood glucose, all the 
 
   3   clinically relevant parameters, can be followed but, there, 
 
   4   again, is no correlation to the engrafted mass and 
 
   5   histopathological assessment can be performed. 
 
   6             [Slide.]

   7             As far as future optimization of preclinical 
 
   8   animal models in islet transplantation, I think we need to 
 
   9   appreciate the limitations of each of the existing models 
 
  10   when trying to apply those strategies to the clinic; to 
 
  11   better characterize the etiology of spontaneous diabetes in

  12   large animal models; to closely mimic the clinical situation 
 
  13   for organ procurement, islet isolation and subsequent 
 
  14   transplant. 
 
  15             Certainly, one of the limitations is to develop 
 
  16   methods to quantitate engrafted islet mass and correlate

  17   with graft function post‑transplant and then to exploit any 
 
  18   large animal model of autoimmune diabetes to develop one 
 
  19   that more closely resembles the human disease. 
 
  20             [Slide.] 
 
  21             Progress in preclinical islet transplantation over

  22   the past has been basically cured "by the decade."  In the 
 
  23   1970's, rodents were cured.  In the 1980's, dogs were cured.  
 
  24   In the 1990's, pigs and non‑human primates were cured. 
 
  25             [Slide.] 

                                                                 85 
 
   1             Hopefully, 2000 is the decade of the human.

   2             Thank you very much. 
 
   3             DR. SALOMON:  Thank you.  Well done.  I think we 
 
   4   can take a few minutes for questions but I am going to try 
 
   5   and stay on time here so we can be done by noon. 
 
   6             DR. AUCHINCLOSS:  Jack, after listening to your

   7   talk, and thinking about it from my own point of view, would 
 
   8   you agree with this; I can think of using strep‑treated mice 
 
   9   for experiments, NOD mice for experiments, non‑human primate 
 
  10   for experiments. 
 
  11             I also think there are a very small number of

  12   indications for using pigs.  The two I have encountered are 
 
  13   demonstration that pig islets are working by using an 
 
  14   allograft transplant and I have seen at least one experiment 
 
  15   where taking advantage of the inbred NIH pigs was an 
 
  16   advantage.

  17             But, other than that, I can't think of any reason 
 
  18   to go to any other animal models.  Do you think that is 
 
  19   true? 
 
  20             MR. O'NEIL:  No, not necessarily.  I think the 
 
  21   field has learned a lot from using the dog as a preclinical

  22   animal model.  Certainly, the islet isolation and the 
 
  23   transplant and the maintenance of the animal is much easier 
 
  24   for most laboratory settings and I think we have learned a 
 
  25   lot from the dog model as far as applying that to the 

                                                                 86 
 
   1   clinical studies with different immunosuppression regimens

   2   that are necessary and different isolation techniques, and 
 
   3   so forth.  So I think that the dog does add value. 
 
   4             DR. AUCHINCLOSS:  Why would you do a dog instead 
 
   5   of a non‑human primate? 
 
   6             MR. O'NEIL:  The dog is less cost‑prohibitive, I

   7   think, if nothing else. 
 
   8             DR. AUCHINCLOSS:  Less costly, less relevant. 
 
   9             MR. O'NEIL:  Excuse me? 
 
  10             DR. AUCHINCLOSS:  But what you get in lower cost, 
 
  11   you lose in less relevant; no?

  12             MR. O'NEIL:  I don't know if you can clearly say 
 
  13   that one animal is more representative of the clinical 
 
  14   situation than the other.  They both have their limitations. 
 
  15             DR. KENYON:  Hugh, I would like to respond to 
 
  16   that, too.  I think I understand your point, but one of the

  17   advantages of the dog‑‑we actually use both models at the 
 
  18   DRI‑‑and, because it is less costly‑‑and they are easier to 
 
  19   handle.  It is not just the cost. 
 
  20             You could study some initial variables in the dog 
 
  21   and then, once you have narrowed them down, try them out in

  22   primates in order to move a little bit more quickly.  The 
 
  23   high‑dose donor bone‑marrow infusion studies that we have 
 
  24   done, we have been able to study dogs more quickly and do 
 
  25   more experiments with bone‑marrow infusion than with the 

                                                                 87 
 
   1   monkeys which is much more labor intensive and time

   2   consuming, in addition to the cost. 
 
   3             DR. HERING:  We are studying new 
 
   4   islet‑implantation sites, novel islet‑delivery systems 
 
   5   extensively in pigs.  I think this is a good model to study 
 
   6   this in and I am not sure whether the non‑human‑primate

   7   system is definitely more predictive than the pig system.  I 
 
   8   am not aware of any studies to demonstrate this. 
 
   9             So think there is reason to continue.  We also 
 
  10   think that studying novel immunosuppression regimens can 
 
  11   certainly be done in pigs, does not need to be done in

  12   primates, necessarily. 
 
  13             DR. BLUESTONE:  Jack, it seems to me that a lot of 
 
  14   what has come down here and what makes this different than 
 
  15   every other transplant setting we have talked about is this 
 
  16   autoimmune issue and how focussed should we as a community,

  17   should the FDA be as a regulatory agency, on the fact that, 
 
  18   with the exception of the NOD mouse and BB rat, which has 
 
  19   all its own problems, we really don't have, whether it be 
 
  20   dog or pig or monkey‑‑it doesn't matter‑‑we do not have a 
 
  21   model that, in a very fundamental way, mimics what is

  22   happening in the patients we are proposing. 
 
  23             How important is this issue in dominating all of 
 
  24   this discussion about, aside from where you put the islets, 
 
  25   but a discussion about immunemodulation and 

                                                                 88 
 
   1   immuneregulation.

   2             MR. O'NEIL:  I think it is critical.  As was 
 
   3   discussed yesterday, the attempts to try to develop 
 
   4   autoimmune animal models in the non‑human primate certainly 
 
   5   would be welcomed by the field.  In addition, I think it may 
 
   6   be important for us to get a message out to veterinarians

   7   and tell them that we are desperately seeking models of 
 
   8   autoimmune diabetes in large‑animal models, just to let them 
 
   9   know what to look for, and to try to develop strains from 
 
  10   that pool of animals. 
 
  11             DR. SALOMON:  Can we follow that up just a little

  12   bit in terms of discussion of the committee in that if we 
 
  13   think about the mechanisms of autoimmune diabetes, 
 
  14   obviously, there is this whole field of trying to come up 
 
  15   with ways of trying to understand better what the 
 
  16   immunologic events are and how you break tolerance in that

  17   compartment and then how you create injury. 
 
  18             At the same time, of course, we have got another 
 
  19   group of people working on islet transplantation.  Jeff has 
 
  20   asked an interesting question and that is, to what extent 
 
  21   are these two fields overlapping.  Can we talk just a little

  22   bit more specifically about in what ways would an islet 
 
  23   allograft‑‑I don't think we should go into xenografts right 
 
  24   now‑‑and islet allograft be affected in terms of its 
 
  25   survival and function in a target organ by anything that has 

                                                                 89 
 
   1   anything to do with autoimmunity.

   2             DR. BLUESTONE:  There is no doubt that there is 
 
   3   overlap.  All of that is true. 
 
   4             DR. SALOMON:  I wasn't tying to say there wasn't.  
 
   5   I just wanted to get it out into discussion, particularly as 
 
   6   there are members of the committee who are not experts in

   7   databases. 
 
   8             DR. BLUESTONE:  But if one is, even in a more 
 
   9   fundamental way, asked a question, and maybe here is where 
 
  10   the pigs might be an advantage, for instance, what is 
 
  11   fundamentally different about the autoimmune response than

  12   the allogeneic response. 
 
  13             One of the things that is potentially 
 
  14   fundamentally different is the stage in which the response 
 
  15   is being studied.  It is clear in an autoimmunity response, 
 
  16   you are studying a secondary response as a minimum in these

  17   patients, a long‑term memory response and a highly 
 
  18   established response, a response with a humoral component as 
 
  19   well as a cellular component. 
 
  20             Yet, very little of what we talk about in our 
 
  21   animal models are using presensitized or highly

  22   sensitized‑‑now, granted, we don't have an autoimmune model; 
 
  23   I understand that.  But we don't even use highly sensitized 
 
  24   animals for the most part for our animal models, something 
 
  25   that can be done, for instance, in the pig system where you 

                                                                 90 
 
   1   can have MHC sensitization; right‑‑a clear knowledge of what

   2   the MHC is going to be for sensitization which you can't do, 
 
   3   maybe, in the beagles or something.  I don't know. 
 
   4             You can use spleen or something to sensitize, a 
 
   5   skin graft or something like that.  All of those are 
 
   6   possible, but, to me, we have to be thinking a little bit

   7   more creatively about how we take‑‑granted, we should be 
 
   8   talking to every vet in the world and pull out our great 
 
   9   autoimmune type‑1 animal, but if that is not going to happen 
 
  10   quickly, are there ways that we can enhance the current 
 
  11   models that we have that might actually have some more

  12   similarities to the autoimmunes.  I don't know. 
 
  13             DR. CHAMPLIN:  The antigens involved in an 
 
  14   autoimmune response obviously are not alloantigens.  They 
 
  15   are not MHC or minor antigen, discrepant antigens, which are 
 
  16   likely very involved in graft rejection.  So there may well

  17   be some overlap ultimately in the patients, but I would 
 
  18   think that you have two problems; one is to try to develop a 
 
  19   system that performs pancreatic islet‑cell allografts in an 
 
  20   unperturbed recipient and then, secondarily, dealing with 
 
  21   the ongoing problem of diabetes and whatever ongoing immune

  22   response you would have against the transplanted tissue. 
 
  23             So those are two separate but equally important 
 
  24   questions in the ultimate solution. 
 
  25             DR. SALOMON:  If we look at the experience with 

                                                                 91 
 
   1   whole‑organ allotransplantation, pancreas

   2   allotransplantation, aside from I believe it is two reported 
 
   3   cases‑‑correct me if I am wrong‑‑but it is really a 
 
   4   minority.  Is there any evidence of recurrence of an 
 
   5   autoimmune diabetes leading to injury or destruction of 
 
   6   these whole‑organ allografts?

   7             So I go back to the question of what, if any, data 
 
   8   do we have that existing mechanisms in a diabetic patient 
 
   9   that induced the autoimmune diabetes at one point in their 
 
  10   course have anything to do with the survival and function of 
 
  11   an islet allograft‑‑

  12             DR. BLUESTONE:  But then you have to realize that, 
 
  13   also, as long as you keep a type‑1 diabetic on high doses of 
 
  14   cyclosporine, their diabetes doesn't get worse, either.  So, 
 
  15   to say, therefore, autoimmune diabetes is the same as 
 
  16   allograft rejection because cyclosporine inhibits both

  17   doesn't well‑‑because you are saying they are not getting 
 
  18   their recurrent allo while they are on cyclosporine. 
 
  19             They are not getting their autoimmunity while they 
 
  20   are on cyclosporine?  They are on drugs that are inhibiting 
 
  21   and might inhibit both but that doesn't mean that both are

  22   the same.  It just means they are both inhibitable by the 
 
  23   drugs that we are using in our patients. 
 
  24             So, if we are going to move forward, hopefully, 
 
  25   into therapies which are changing fundamentally the immune 

                                                                 92 
 
   1   system so that we are not working with long‑term

   2   immunosuppressive therapies, I think that that particular 
 
   3   piece of data may or may not be relevant to the long‑term 
 
   4   effects on the immune system. 
 
   5             DR. SALOMON:  Again, I have no agenda and I am not 
 
   6   pushing any scientific hypotheses yet.  I am just trying to

   7   get some of these issues on the table.  My point is that an 
 
   8   alternative hypothesis is that autoimmunity is 
 
   9   not‑‑mechanisms inducing islet autoimmunity in the patient 
 
  10   could potentially have absolutely nothing to do with the 
 
  11   survival and the immune reaction to the allograft because,

  12   again, the antigens are being presented in a different MCH 
 
  13   context and/or those mechanisms have been burnt out years 
 
  14   before you go ahead and do the allograft. 
 
  15             I am not saying I have any data, either, that that 
 
  16   is true but there seems to be‑‑I am not certain that I go

  17   along with this tacit assumption that studying islet 
 
  18   transplantation in non‑autoimmune diabetic models is a 
 
  19   limitation. 
 
  20             DR. BLUESTONE:  I will say one more thing and then 
 
  21   I will stop.  I think that is ridiculous.  There are lots of

  22   data.  Camillo has got data.  Everyone has got data.  But 
 
  23   when you try to suppress in an NOD mouse, an animal that is 
 
  24   already diabetic, the same drugs that work absolutely 
 
  25   perfectly in an NOD mouse where you have actually switched 

                                                                 93 
 
   1   the MHC so it is not diabetic work much, much better than

   2   they do in the autoimmune animal. 
 
   3             So I think that it is so unlikely that the 
 
   4   autoimmune response doesn't play any role in this‑‑the 
 
   5   reason I am being so strong about this is that I would hate 
 
   6   to come away from this discussion thinking that allo is all

   7   we should be caring about here and we shouldn't be caring 
 
   8   about the auto response. 
 
   9             I think the auto response is absolutely involved 
 
  10   in this thing and it doesn't burn out and be gone in these 
 
  11   autoimmune patients.  The antibodies are there.

  12             DR. SAUSVILLE:  I guess the question that comes 
 
  13   up, then, is would you then choose, if you were going to 
 
  14   recommend to the FDA which model they would potentially base 
 
  15   the design or advise potential sponsors in terms of using 
 
  16   immunosuppressive regimens, do you feel, therefore, that

  17   that should bias the selection of what animal model would be 
 
  18   most relevant then? 
 
  19             DR. SHERWIN:  There is no question that 
 
  20   ultimately‑‑you are going to have to approach it from both 
 
  21   sides initially and then work‑‑ultimately, there is no

  22   question.  I agree with Jeff, there is absolutely no 
 
  23   question that autoimmunity is a key player in the problems 
 
  24   of islet grafting. 
 
  25             It is so highly unlikely that it is not an issue 

                                                                 94 
 
   1   so I think, ultimately, we need to have, to be effective,

   2   autoimmune models, either human models which are, I guess, 
 
   3   the proof of the pudding or other‑‑we need to focus much 
 
   4   more‑‑ultimately, it is much easier to do allografts and it 
 
   5   is much harder to transplant in an autoimmune model 
 
   6   across‑‑doing allografts in autoimmune models.

   7             I think that ultimately that is where we have to 
 
   8   go.  I could just say that there are other potential ways of 
 
   9   developing autoimmune models that are more relevant to the 
 
  10   human situation‑‑I mean, just an example.  We have models 
 
  11   with human HLA transgenic animals, DQ8, DR4 animals, that

  12   get spontaneous autoimmune diabetes and it is due to 
 
  13   T‑cell‑‑you know, T‑cells.  It is an autoimmune model. 
 
  14             So it is conceivable that one can manipulate the 
 
  15   genetics of mice or even rats to develop humanized 
 
  16   autoimmune models that have some relevance, at least, to the

  17   human condition. 
 
  18             DR. AUCHINCLOSS:  I am going to make the 
 
  19   prediction here that there will not be any adequate 
 
  20   autoimmunity model, that we will never find a monkey or a 
 
  21   supply of monkeys in sufficient numbers with type‑1 diabetes

  22   to be useful. 
 
  23             I am going to suggest that I think all of the 
 
  24   SCID‑adoptive transfer models of human autoimmunity are 
 
  25   close to worthless and I am going to suggest that all of the 

                                                                 95 
 
   1   mouse autoimmunity models, even the humanized mice, have

   2   terrible limitations. 
 
   3             While we should use them, the fact of the matter 
 
   4   is we are never going to find the answer to this question 
 
   5   without testing it in diabetic patients. 
 
   6             DR. SHERWIN:  Nobody is arguing that.  But there

   7   are steps to take it to a human and I think that one can 
 
   8   learn a lot from autoimmune models even though some of the 
 
   9   answers may not be relevant to human. 
 
  10             I think it would be a big mistake to totally 
 
  11   ignore animal models and not strive to develop animal models

  12   that are more appropriate to the clinical situation. 
 
  13             DR. CHAMPLIN:  There are a lot of limitations, 
 
  14   particularly in trying to develop immunosuppressive 
 
  15   therapies in animal models and then translate, then, to man, 
 
  16   obviously major differences between species and in effects

  17   of steroids, major strain differences in one mouse strain 
 
  18   and another, at least as we see it in bone‑marrow 
 
  19   transplants.  Fludarabine had fundamentally different 
 
  20   metabolism in different species and so the effects in humans 
 
  21   are far different than they are in animals.

  22             So, although animals are certainly extremely 
 
  23   important in providing leads, ultimately, as you are trying 
 
  24   to develop an immunosuppressive regimen that works in human 
 
  25   patients, there is no substitute for testing it and 

                                                                 96 
 
   1   developing it in human patients.

   2             So I agree with Hugh's comment; even if you had a 
 
   3   perfect animal model, translating the therapy from that 
 
   4   animal to the human being isn't the direct translation, that 
 
   5   one has to do a lot of work within the human system. 
 
   6             DR. SHERWIN:  But you are dealing with, perhaps,

   7   the diseases that are different from diabetes in the sense 
 
   8   that we have other forms of therapy that are alternative and 
 
   9   relatively safe.  So, in the equation, even though I am very 
 
  10   strongly in favor of doing human islet transplantation, very 
 
  11   strongly in favor of it, one has to take into account the

  12   fact that there are alternative approaches that, in many 
 
  13   people, work very successfully. 
 
  14             They are improving continuously.  So one has to 
 
  15   take that into account when doing more invasive procedures. 
 
  16             DR. SALOMON:  That will be important this

  17   afternoon when we start to discuss what patients and what 
 
  18   kinds of clinical trials, specifically, should be done. 
 
  19             DR. BLUESTONE:  I think there is a middle ground 
 
  20   here.  I think, first of all, we haven't solved the allo 
 
  21   problem so it is not like we have solved half and we only

  22   have half to go.  We still have the allo problem and animal 
 
  23   models are probably very important in doing that. 
 
  24             That is number one.  Number two is, I think Hugh 
 
  25   is right, but I would state it differently.  I would say the 

                                                                 97 
 
   1   chances are we will probably solve it in humans before we

   2   get the models up that we want to get up but that doesn't 
 
   3   mean that the models, all along, haven't been helping 
 
   4   provide a road map for us. 
 
   5             So, although they may not give us the ultimate 
 
   6   dosing, and they may not even tell us‑‑but they are telling

   7   us where we are, where we are on target, which therapies are 
 
   8   on target and are moving us in the right direction, and 
 
   9   which therapies are not on target. 
 
  10             So where I see the question that I was asked 
 
  11   before is is there an animal model that should be used by

  12   the FDA as sort of the gold standard for saying this drug is 
 
  13   going to work in humans, I think the answer is absolutely 
 
  14   no.  But is there information that will be learned from the 
 
  15   animal models which will help inform us that the therapies 
 
  16   we are ultimately going to try in human beings have a better

  17   chance of working and, therefore, should be approved in an 
 
  18   IND.  I think the answer is absolutely yes. 
 
  19             So, to me, since there is no perfect model, then I 
 
  20   think we are best off keeping our options open, really not 
 
  21   sitting here and saying there are good models and bad models

  22   and no models, there is information that we need to learn.  
 
  23   The allo response is important and I wouldn't be surprised 
 
  24   if we don't learn something in a number of these other 
 
  25   models which at least sets us in a direction that we are not 

                                                                 98 
 
   1   currently going in.

   2             DR. RICARDI:  I would like to comment that, to me, 
 
   3   I agree with Dr. Champlin completely that the best model to 
 
   4   test these new therapeutic approaches is the clinical 
 
   5   setting and the model is the human and not the preclinical.  
 
   6   But I complete agree also with Jeff in the fact that you

   7   need this basic model and NOD for screening the development 
 
   8   of new tools like all the customary blockers and all the new 
 
   9   monoclonal antibodies may be tested. 
 
  10             But there are a series of situations where it is 
 
  11   either impossible or not practical, like anti‑CD3‑‑there are

  12   agents that do not cross‑react with non‑human primates.  
 
  13   There is the Edmonton protocol that has been developed‑‑ 
 
  14   these wonderful results have no animal model that prove the 
 
  15   concept of the potency of what turns out to be the most 
 
  16   effective way to prevent rejection of islets in an

  17   autoimmune background, like the trials in Pittsburgh with 
 
  18   FK506 would be that with the requirement of an animal model 
 
  19   because of the toxicity of the tacrilimus in dogs that 
 
  20   actually block development towards clinical application. 
 
  21             So I think we have to be very careful, meaning

  22   that we need an animal model.  We need to develop better 
 
  23   animal models for autoimmunity in large animals.  This is a 
 
  24   science kind of problem and concern, but I would not 
 
  25   necessarily require any preclinical model of proof of 

                                                                 99 
 
   1   principle or potency of what you want to demonstrate in a

   2   pilot clinical trial as a requisite for an IND for a 
 
   3   clinical islet transplant because you may have this 
 
   4   information from clinical trials in other diseases, just 
 
   5   from experimental models that may not provide direct 
 
   6   evidence, and the NOD mouse is a great model for basic

   7   research but is a fairly different disease than type‑1 
 
   8   diabetes in humans. 
 
   9             It is very much a violent onset and it happens in 
 
  10   a few weeks and it is completely different.  There are, 
 
  11   like, 127 ways right now to prevent diabetes in NOD mice and

  12   none in humans.  There is probably different relevance even 
 
  13   though I agree it is very important. 
 
  14             DR. SALOMON:  I also just wanted to stay on record 
 
  15   as, despite Dr. Bluestone's strong opinions, I am not at all 
 
  16   convinced that a mechanistic link between autoimmune and

  17   alloimmunity, between the mechanisms that destroy islets in 
 
  18   a diabetic patient and the mechanisms that challenge a 
 
  19   successful allotransplant are really very well connected. 
 
  20             I think that that is a very interesting area for 
 
  21   research.  There are a number of examples of autoimmune

  22   diseases that burn out.  I have transplanted many lupus 
 
  23   patients, very example, who have had the hell 
 
  24   immunosuppressed out of them and then actually completely 
 
  25   resolved their disease once they get kidney failure. 

                                                                100 
 
   1             I can give you several other examples.  There

   2   certainly is no data that I am aware of that you can just 
 
   3   cure patients with early diabetes using drugs like 
 
   4   cyclosporine, et cetera.  So the argument that this is all 
 
   5   an aberration of post‑transplant drug immunosuppression, I 
 
   6   am not certain I am convinced of that, either.

   7             But I don't know the answer.  I am just suggesting 
 
   8   that it is another area for research and uncertainty. 
 
   9             DR. SHERWIN:  When were those six patients studied 
 
  10   in Minnesota with minimal immunosuppression following 
 
  11   David's‑‑it wasn't two, as I remember.  It was two maybe

  12   with on immunosuppression, all of whom had recurrent 
 
  13   insulitis and T‑cell infiltration of islets after fifteen 
 
  14   years or more of post‑disease. 
 
  15             DR. HERING:  Yes; there are several lines of 
 
  16   evidence here.  One is the Minnesota pancreas transplants

  17   between identical twins.  The first patients received no 
 
  18   immunosuppression treatment and selective destroyed all beta 
 
  19   cells within four weeks. 
 
  20             The first patient had developed diabetes forty 
 
  21   years ago.  So this is very strong evidence.  Then, with

  22   immunosuppressive treatment, it was possible to prevent 
 
  23   recurrent autoimmune disease in this setting.  There is also 
 
  24   other evidence, bone‑marrow transplantation.  So you can 
 
  25   definitely transfer the disease. 

                                                                101 
 
   1             One bone‑marrow donor was, I guess, diagnosed with

   2   diabetes maybe ten years ago and then donated bone marrow, 
 
   3   and the recipient developed type‑1 diabetes soon after 
 
   4   bone‑marrow transplantation.  So that is another piece of 
 
   5   evidence to indicate the disease persists. 
 
   6             DR. SALOMON:  I mean, the identical‑twin data

   7   doesn't have anything to do with the argument I was making.  
 
   8   But the bone‑marrow data suggests that, in one patient, you 
 
   9   transferred disease. 
 
  10             DR. BLACK:  I just wanted to make one comment to 
 
  11   Dr. Ricordi's earlier about the utilization of preclinical

  12   data from FDA's perspective.  We treat the data on the 
 
  13   mechanism of action and clinical rationale lightly 
 
  14   understanding that the mechanisms that are present in the 
 
  15   animals' etiology of their disease‑‑for instance, the NOD 
 
  16   mice, are not necessarily equivalent to the human disease.

  17             We, therefore, feel it is necessary and are also 
 
  18   required by the CFR to really only utilize the animal data 
 
  19   for phase‑I trial design for the point of view of feasibly 
 
  20   ruling out what safety problems we can rule out. 
 
  21             Having addressed that issue, that is why I ruled

  22   out immunosuppressive toxicity or islet toxicity or 
 
  23   infusion‑related problems in my talk.  But I also wanted to 
 
  24   point out, Dr. Salomon, Dr. Kenyon is hoping for a half hour 
 
  25   for her presentation. 

                                                                102 
 
   1             DR. SALOMON:  We had cut that back.  I think we

   2   will try and stay on time. 
 
   3             DR. BLACK:  Okay.  Thank you. 
 
   4             DR. AUCHINCLOSS:  That was an extraordinary 
 
   5   statement.  In your view, what you are looking for, is 
 
   6   essentially safety data?  So you never want to see a mouse

   7   study in an IND. 
 
   8             MR. SIEGEL:  I think that, in many areas that we 
 
   9   regulate of biological therapies, there are immunological 
 
  10   issues and species‑specificity issues, receptor differences 
 
  11   and physiological differences that limit the function of

  12   animal models as models for efficacy. 
 
  13             We find that extensive safety data, while 
 
  14   sometimes is irrelevant, is sometimes relevant and, 
 
  15   therefore, almost always of some value if there is as model 
 
  16   in which you can‑‑in some cases, it is impossible to get any

  17   useful information from a model.  I don't this is the case. 
 
  18             That said, we do look importantly to rationale 
 
  19   data.  I am thinking back to not long ago when we had a very 
 
  20   relevant discussion largely between you and Dan as to how 
 
  21   much information one would need for a success of

  22   xenotransplantation of hearts or kidneys into non‑human 
 
  23   primates, say from pigs into non‑human primates, before 
 
  24   going into humans. 
 
  25             There, of course, the issues of both the 

                                                                103 
 
   1   availability of alternative therapies and the availabilities

   2   of rescue therapies should the transplant fail are very 
 
   3   different from here.  But the same issues of how relevant is 
 
   4   the model and how much rationale data do you need came up, 
 
   5   and, as you will recall from that context, the context of 
 
   6   committee and the context of the agency, as I indicated

   7   yesterday in talking about these issues, is that where have 
 
   8   an anticipated known or unknown risks to a patient such as 
 
   9   the risk of immunotherapy or the risks of removing their 
 
  10   heart, we will want to see some amount of rationale as 
 
  11   appropriate.

  12             As appropriate is put in there to take into 
 
  13   account the fact that if there are animal models of very 
 
  14   limited relevance, that may be very limited or it may come 
 
  15   from other sources, as Dr. Ricordi referred to.  There may 
 
  16   be therapies for other related diseases in humans and

  17   whatever that may provide the appropriate rationale 
 
  18   information. 
 
  19             But I would say, for many of our therapies, 
 
  20   including this one, if the animal models are not 
 
  21   particularly informative, we don't ask too much proof from

  22   them.  As you will come through our questions, we are going 
 
  23   to be asking you specifically how informative are they 
 
  24   regarding immunosuppression regimens, regarding dosing, 
 
  25   regarding other questions that we do need to face that will 

                                                                104 
 
   1   help us determine what we should expect sponsors to do with

   2   them beyond safety assessments. 
 
   3             DR. AUCHINCLOSS:  You have suggested that there is 
 
   4   a very big difference between this and xenotransplantation.  
 
   5   In this case, the vast majority of the immunosuppression 
 
   6   protocols will be ones that, in some variation or form, been

   7   tried in probably thousands of other patients.  Presumably, 
 
   8   your safety and efficacy data can be obtained from other 
 
   9   organ or tissue types of transplants in humans. 
 
  10             You also have 405 patients who have received islet 
 
  11   transplants and you know, I believe, a certain amount about

  12   the safety of islet transplantation simply as a technical 
 
  13   procedure.  It is becoming very unclear to me where any 
 
  14   issue data would become terribly useful to the FDA in those 
 
  15   circumstances. 
 
  16             MR. SIEGEL:  There are a lot of areas in which the

  17   safety data for things being tried including the safety of 
 
  18   some of the not just immunosuppression but the actual 
 
  19   products themselves, more data are needed. 
 
  20             I also do want to qualify that remark about 
 
  21   rationale which is to say we recognize the critical

  22   importance of these animal models to drug development, to 
 
  23   develop the hypotheses to figure out how to optimize a 
 
  24   therapy, which ones to take forward into humans. 
 
  25             We are not suggesting that they should not be done 

                                                                105 
 
   1   or that they are not useful, simply that, in terms of the

   2   amount of that work that will require, or the amount of 
 
   3   success in an animal model that will require before human 
 
   4   experimentation, that may be limited and will depend largely 
 
   5   on the relevance of the model and the risk to the patient 
 
   6   and the nature of the patient population because where there

   7   are more risks, there needs to be more rationale. 
 
   8             But where there are not relevant models, we don't 
 
   9   expect the impossible or irrelevant data. 
 
  10             DR. BLUESTONE:  It seems to me that one 
 
  11   intersection, though, is a drug that has been tried in

  12   another setting in islets as a target potentially for 
 
  13   toxicities associated with the drug so that one might think 
 
  14   that it might be appropriate to have some kind of safety 
 
  15   data, something like human islets transplanted into the SCID 
 
  16   mouse being treated with these drugs just to make sure that

  17   that drug doesn't have an adverse effect on the islet 
 
  18   function, itself, like if you gave steroids to a non‑SCID 
 
  19   who got human islets, would the steroids be wiping out those 
 
  20   islets. 
 
  21             So that may be a model.  It is not an efficacy

  22   model.  It is not even an immunological model.  But it is a 
 
  23   safety model for how your drugs might interact with the 
 
  24   islet transplant. 
 
  25             DR. AUCHINCLOSS:  I agree with that.  I think that 

                                                                106 
 
   1   makes perfectly good sense.  You can certainly imagine some

   2   very specific questions of safety that the FDA would be 
 
   3   interested in what the data is.  But they became much 
 
   4   smaller questions as I heard their real focus. 
 
   5             DR. CHAMPLIN:  I am not sure how reliable animal 
 
   6   models are for organ‑specific toxicity determination. 

   7   Often, one sees a totally different spectrum of toxicity in 
 
   8   various animal models than in humans although, obviously, 
 
   9   any information provides something to look for. 
 
  10             But if something was toxic to the islets in the 
 
  11   mouse, I am not sure that necessarily would‑‑

  12             DR. BLUESTONE:  No, no; the experiment that I am 
 
  13   suggesting is that if we were to say, based on yesterday's 
 
  14   experiments, have a mouse model in which you put human 
 
  15   islets, portal‑vein‑inject human islets into a SCID mouse so 
 
  16   it doesn't reject them and ask whether your drugs affect the

  17   human islets that you put into the mouse‑‑the mouse becomes 
 
  18   the vessel, not the islet target. 
 
  19             DR. CHAMPLIN:  The only difference, I guess, there 
 
  20   would be that you now have a mouse liver that is 
 
  21   metabolizing the drug and so it certainly would be an

  22   interesting system, again, for screening.  But one could 
 
  23   envision false results there, too. 
 
  24             DR. MILLER:  I just wanted to follow up on Dr. 
 
  25   Ricordi's comment that the advances in the Edmonton protocol 

                                                                107 
 
   1   would not have been predicted by any animal model.  Can you

   2   explain to us what animal models were tested to 
 
   3   see‑‑because, for those of us who are trying to sort of help 
 
   4   to be able to answer the afternoon questions about what 
 
   5   animal models do you require, I think if we have had an 
 
   6   advance in the field, it would be nice to know why the

   7   models did not predict it and whether you think that there 
 
   8   are any models that would have helped. 
 
   9             DR. SHAPIRO:  We carried out extensive experiments 
 
  10   over the previous five to six years in the dog, in 
 
  11   autografts and allografts, to try to test what were the

  12   optimal immunosuppressive regimes would could apply.  We 
 
  13   found that the sirolimus was a very effective agent.  We 
 
  14   knew that tacrolimus at standard dose was fairly toxic. 
 
  15             When we tried our regimen that we now use 
 
  16   clinically in that model in the autografts in the dog, it

  17   was very, very toxic.  We couldn't obtain the data but we 
 
  18   predicted it would be useful clinically.  We made a big step 
 
  19   and tried it clinically, and it worked. 
 
  20             DR. MILLER:  How about any of the autoimmune 
 
  21   models, the mouse models?  Did you look in any other‑‑

  22             DR. SHAPIRO:  We didn't.  Based on the comments 
 
  23   that standard dose or potent immunosuppression therapy is 
 
  24   also effective at controlling autoimmunity, we predicted 
 
  25   that that drug strategy would also control the autoimmune 

                                                                108 
 
   1   recurrence.

   2             DR. SAUSVILLE:  Although a point that I would make 
 
   3   and, again, we just saw the broad strokes of the regimen.  I 
 
   4   guess we are talking about a combination of individual 
 
   5   immunosuppressives that, in the setting of the human, now 
 
   6   has, apparently, been a major leap forward.

   7             It is notorious that combinations of agents, even 
 
   8   some of the chemotherapy agents we use, are not well 
 
   9   predicted, actually, in any animal model.  To that extent, I 
 
  10   actually would agree with Dr. Ricordi. 
 
  11             On the other hand, the activity of each of the

  12   individual components, at least getting to first base, that 
 
  13   this is a reasonable path to begin walking down, would 
 
  14   actually revealed in animal models at one level or another 
 
  15   at some point in the past. 
 
  16             So I think we have to recognize when the most

  17   appropriate time is to utilize the animal information as a 
 
  18   determinant of going forward.  There, I actually feel the 
 
  19   animals are highly valuable in setting these initial safety 
 
  20   issues for the first dose in humans. 
 
  21             But I would agree that, for subsequent uses, for

  22   combining, there you actually have to build on the more 
 
  23   clinical experience. 
 
  24             DR. SALOMON:  I think that is a good introduction 
 
  25   to Dr. Kenyon who is going to pick up the theme of animal 

                                                                109 
 
   1   models and bring us forward into non‑human primates.

   2               Non‑Human Primate Preclinical Models 
 
   3             DR. KENYON:  A lot of the issues I have on the 
 
   4   slides you all have already brought up. 
 
   5             [Slide.] 
 
   6             Really, I wanted to point out, too, that if you

   7   look in the literature for papers on non‑human‑primate 
 
   8   models of islet‑cell transplantation, you will find that 
 
   9   there really aren't very many.  There are several reasons 
 
  10   for this including the fact that, similar to clinical islet 
 
  11   isolation and transplantation, it has been difficult to

  12   isolate enough islets, viable islets, to get insulin 
 
  13   dependence post‑transplant. 
 
  14             The drugs that have worked routinely for 
 
  15   solid‑organ transplantation have not translated well to 
 
  16   islet until recently.  In addition, as we have already

  17   brought up, it is very labor intensive, time consuming and 
 
  18   costly. 
 
  19             [Slide.] 
 
  20             I thought Jack did a really nice job of talking 
 
  21   about safety parameters and efficacy parameters so I am not

  22   going to repeat that.  I just need to give you my 
 
  23   perspective on the fact that, with regards to the relevance 
 
  24   of non‑human‑primate models to islet‑cell transplantation, I 
 
  25   think we have to consider that when it comes to safety, 

                                                                110 
 
   1   clearly, data generated in monkeys, at least in my opinion,

   2   is going to be much more relevant to humans than data 
 
   3   generated in mice. 
 
   4             At the same time, we have to keep in mind that the 
 
   5   models we are using right now with induced diabetes are 
 
   6   generally healthy models, animals.  They don't have the

   7   underlying physiological changes associated with diabetes. 
 
   8             Then, with regards to efficacy, we have already 
 
   9   had intense discussion and I am sure we are going to have 
 
  10   some more, that there have been several techniques put 
 
  11   forward in mice that prevent rejection, can reverse

  12   autoimmunity, and none of those has translated consistently 
 
  13   or reproducibly to larger animal models including monkeys 
 
  14   and humans. 
 
  15             Another point to keep in mind is really what we 
 
  16   are looking at is efficacy for preventing rejection.  We are

  17   not looking at the autoimmune aspects. 
 
  18             [Slide.] 
 
  19             Then I just pointed this out, that until very 
 
  20   recently, protocols that work effectively in mice‑‑I think 
 
  21   the mice are very, very valuable.  They teach us pathways

  22   that we need to address, but in all my experience in dogs 
 
  23   and in non‑human primates, none of those approaches 
 
  24   singlehandedly has worked in a larger animal. 
 
  25             It has been already pointed out that there are 

                                                                111 
 
   1   differences in autoimmunity in rodents including, as Dr.

   2   Ricordi pointed out, that it is a very explosive onset of 
 
   3   autoimmunity.  I have been encouraging everyone that I have 
 
   4   talked to that works on the NOD mouse to let their mice get 
 
   5   diabetes and then put them on insulin and maintain them that 
 
   6   way for at least a few months or as long as they can, and

   7   then try and do an islet transplantation rather than doing 
 
   8   it at the time of onset when you have a superactivated 
 
   9   immune system. 
 
  10             [Slide.] 
 
  11             Just briefly, because I do think the non‑human

  12   primates have a lot of relevance to humans, there are a 
 
  13   couple of things, too, that you might not consider that are 
 
  14   not necessarily scientific. 
 
  15             The monkeys are more finicky.  A dog or a pig will 
 
  16   eat just about anything.  But monkeys, more like humans, can

  17   be finicky about what they eat.  It is harder to get them to 
 
  18   eat.  They are on two feet as opposed to on all fours.  In  
 
  19   different protocols, not so much for islet 
 
  20   allotransplantation, per se, that can have an effect. 
 
  21             Nevertheless, we use primarily a model of

  22   pancreatectomy‑induced diabetes.  It is possible to do a 
 
  23   total pancreatectomy in monkeys.  It is removed surgically.  
 
  24   The down side of that is that it requires enzyme 
 
  25   supplementation to replace the lost exocrine function and, 

                                                                112 
 
   1   because you don't have completely native exocrine function,

   2   it is possible‑‑you can't completely rule out‑‑that the 
 
   3   animals have malabsorption and, therefore, don't have the 
 
   4   same insulin requirements that a person would with a native 
 
   5   pancreas. 
 
   6             Again, this issue that we are addressing,

   7   rejection, I think is still a very critical point, though.  
 
   8   There is some exciting data recently that tolerance may be 
 
   9   possible but, until recently, it really has not been 
 
  10   possible to routinely prevent islet rejection. 
 
  11             We can't look at autoimmunity.  Again, I want to

  12   point out that we are looking at relatively healthy animals. 
 
  13             [Slide.] 
 
  14             With regard to chemical induction, and I know I am 
 
  15   expecting Hugh to ask me about this later, it is possible to 
 
  16   use streptozotocin.  There are a couple of key issues that I

  17   would like to bring out.  First of all, it is not a 
 
  18   non‑toxic thing.  It is definitely toxic to other organ 
 
  19   systems in addition to the beta cells. 
 
  20             When we have done streptozotocin induction, it has 
 
  21   been essential to monitor the animals very closely for at

  22   least twenty‑four hours and up to thirty‑six hours, until 
 
  23   they clearly stabilize with high blood sugar.  But you have 
 
  24   to keep them well hydrated to prevent kidney damage. 
 
  25             There is a point where they need bicarb.  When the 

                                                                113 
 
   1   beta cells start to die, we have to give them glucose for a

   2   period of eight hours.  So it is not an easy protocol, at 
 
   3   least the way that we have done it.  I know we will have 
 
   4   discussion on that necessarily to follow.  We have seen 
 
   5   evidence of kidney and liver damage that takes some time to 
 
   6   resolve.

   7             Also, with the chemical induction, it is possible 
 
   8   to have residual islet function.  I think that becomes more 
 
   9   important later.  I have another slide that I want to 
 
  10   discuss that.  As Jack mentioned, regeneration is possible, 
 
  11   although we did have a monkey that we induced diabetes and

  12   kept for over a year and I didn't see any evidence of this 
 
  13   at all. 
 
  14             Again, we are addressing issues of rejection as 
 
  15   compared to autoimmunity.  The animals are relatively 
 
  16   healthy in that they don't have the underlying disease

  17   changes of diabetes. 
 
  18             [Slide.] 
 
  19             With regards to spontaneous diabetes, and I know 
 
  20   we would all love to see a model of type‑1 diabetes in 
 
  21   monkeys, I want to be very clear here that when I say type

  22   1, I am referring to type‑1 autoimmune insulin‑dependent 
 
  23   diabetes.  That would obviously be the most desirable 
 
  24   non‑human‑primate model for transplantation because we could 
 
  25   address rejection, autoimmunity and diabetes‑related 

                                                                114 
 
   1   physiological changes.

   2             However, this is essentially nonexistent in 
 
   3   captivity.  I spoke with an individual a couple of years ago 
 
   4   who has been working with diabetes monkeys for twenty years.  
 
   5   He said in his career he had seen one monkey that he was 
 
   6   sure had type‑1 diabetes.

   7             [Slide.] 
 
   8             So the majority of monkeys with spontaneous 
 
   9   diabetes that are reported in the literature are actually 
 
  10   insulin‑requiring type‑2 diabetic monkeys.  So they are 
 
  11   frequently reported as type 1.  They are actually

  12   insulin‑requiring type 2. 
 
  13             They frequently have significant residual 
 
  14   islet‑cell function.  We actually had four cynomolgus 
 
  15   monkeys at the DRI with varying levels of function.  You 
 
  16   could get different degrees of metabolic control depending

  17   on just how much beta‑cell loss they had had. 
 
  18             So we are addressing issues of immunological 
 
  19   rejection but not autoimmunity.  However, if you had a 
 
  20   colony of animals with type‑2 diabetes, or had access to 
 
  21   them, this may be a setting where we can address the issue

  22   of safety in the setting of disease‑related changes. 
 
  23             [Slide.] 
 
  24             Other key differences in the design of preclinical 
 
  25   studies, and I have already alluded to this, is the duration 

                                                                115 
 
   1   of diabetes.  With our monkeys, we induced diabetes with

   2   streptozotocin or we pancreatectomized them, and they get a 
 
   3   transplant in a relatively short period of time whereas, and 
 
   4   here I am referring to clinical protocols that we have 
 
   5   approved at the DRI, our patients that are eligible for 
 
   6   transplant must have had diabetes for at least five years

   7             C‑peptide and all the clinical trials that I have 
 
   8   seen proposed to date, and I obviously have not seen all of 
 
   9   them, one of the criteria is that the patients are negative 
 
  10   for C‑peptide. 
 
  11             If you look in the literature, the monkeys

  12   reported are negative in some studies but clearly are 
 
  13   present in others.  So then, when you are looking at 
 
  14   efficacy, you have to try to determine the effect of the 
 
  15   islet transplant in a setting where the animal may not have 
 
  16   been completely diabetic.

  17             I don't think that necessarily makes it 
 
  18   irrelevant, but it is an important issue to consider. 
 
  19             [Slide.] 
 
  20             One of the great advantages of the 
 
  21   non‑human‑primate model is the ability to monitor them very

  22   similarly to what we do for humans.  We check blood glucose 
 
  23   with a glucometer and blood glucose strips, just as we would 
 
  24   do for people.  You can look at hemoglobin A1c and the 
 
  25   first‑phase insulin release, in an intravenous 

                                                                116 
 
   1   glucose‑tolerance test, can be correlated to functional

   2   islet mass. 
 
   3             With regards to insulin, a key difference to point 
 
   4   out is that humans, fully diabetic humans, need about a unit 
 
   5   of insulin per kilo per day whereas the monkeys require 3 to 
 
   6   6 units of insulin per kilo per day.

   7             So, for designing protocols where we are going to 
 
   8   look at reduction of exogenous insulin requirement as a 
 
   9   measure of graft function, we have to keep that in mind.  
 
  10   But one identical finding that we have had, that Dr. Ricordi 
 
  11   and Dr. Alejandro and I have discussed a lot, is that when

  12   you have a monkey with partial function, it clearly mimics 
 
  13   the clinical situation identically. 
 
  14             Depending on the degree of function you have with 
 
  15   minimal amounts of insulin, you can maintain relatively 
 
  16   normal metabolic control.

  17             [Slide.] 
 
  18             So what are some of the key differences?  Jack has 
 
  19   already alluded to this.  Humans, obviously, when it comes 
 
  20   to the organs, the donors and the procurement, in the 
 
  21   setting of clinical transplantation, we have variable health

  22   status preceding brain death, variable causes of death.  The 
 
  23   patients have been on life‑support for different periods of 
 
  24   time. 
 
  25             In the monkeys, they are generally healthy.  We do 

                                                                117 
 
   1   use, however, older non‑human primates as islet donors. 

   2   They are frequently animals that have been culled from the 
 
   3   colony for various problems such as a wasting syndrome in a 
 
   4   leg or diarrhea. 
 
   5             But, in general, they are relatively healthy.  
 
   6   Obviously, they are sacrificed for the purpose of organ

   7   donation and anesthetized at the time of donation.  So we 
 
   8   take the pancreas out in the OR and walk over to the lab.  
 
   9   The only thing that really holds up the isolation starting 
 
  10   is me on the telephone. 
 
  11             With regards to surgical technique, the removal is

  12   similar.  With the variable OPOs, you have different 
 
  13   surgeons removing organ.  I think someone said 
 
  14   yesterday‑‑was it you, Jonathan‑‑that that had been 
 
  15   correlated to the islet‑isolation outcome. 
 
  16             Obviously, usually, within a center, you have the

  17   same surgeon or surgeons removing the pancreas.  We don't 
 
  18   perfuse the organ because it is not necessary.  In the human 
 
  19   setting, you have to perfuse it with UW and you have longer 
 
  20   cold ischemia times.  Ours is generally less than an hour. 
 
  21             So those are key things to keep in mind as we

  22   design our trials. 
 
  23             [Slide.] 
 
  24             Then this issue of islet dose keeps coming up.  
 
  25   Here, also, we see a lot of similarities.  In the human 

                                                                118 
 
   1   pancreas, there are about a million islets.  I have not been

   2   able to find a report in the literature that details that in 
 
   3   a non‑human primate.  If anyone has seen that, I would like 
 
   4   to know about it. 
 
   5             It is really clear in the non‑human‑primate model, 
 
   6   just as it is for the clinic, that the number of functional

   7   viable islets you transplant is essential and critical to 
 
   8   the outcome of your transplant. 
 
   9             If you look at the data that Dr. Hering presented 
 
  10   from the International Islet Transplant Registry yesterday, 
 
  11   one of the factors that was found to be critical for a

  12   successful transplant was a minimum of 6,000 islet 
 
  13   equivalents per kilo.  Most of the data that you see 
 
  14   reported in the literature, insulin independence has been 
 
  15   achieved with the use of multiple donors. 
 
  16             Edmonton is now seeing that a minimum of

  17   10,000 islet equivalents per kilo appears to be essential 
 
  18   for insulin independence in humans.  This is exactly what we 
 
  19   have seen in both baboons, cynomolgus and rhesus monkeys at 
 
  20   the DRI.  I won't anymore do a transplant unless we have at 
 
  21   least 10,000 islet equivalents per kilo.

  22             In the literature, people either use multiple 
 
  23   donors to achieve enough or, in our case, we will take a 
 
  24   larger donor and transplant the islets that we get into a 
 
  25   small recipient. 

                                                                119 
 
   1             [Slide.]

   2             I was asked to comment, also, on the location of 
 
   3   the islets in the liver and the durability.  Similarly to 
 
   4   the human, islets lodge in the portal spaces with the larger 
 
   5   clusters in the portal triads.  Insulin‑positive islets have 
 
   6   been identified in human transplants at five years post

   7   transplant.  In the monkey, we now have data for 
 
   8   insulin‑positive islets within the liver at two years 
 
   9   post‑transplant. 
 
  10             [Slide.] 
 
  11             Also, the issue of matching has come up.  It is

  12   clear that we really don't know the answer to this question; 
 
  13   should we or should we not match?  Obviously, matching 
 
  14   favors engraftment and prevention of rejection.  However, 
 
  15   matching may also favor recurrent autoimmunity in the 
 
  16   setting of type‑1 diabetes.

  17             There are typing methods available for non‑human 
 
  18   primates.  The rhesus is the most well developed.  David 
 
  19   Watkins in Wisconsin, Judy Thomas in Alabama, have done a 
 
  20   lot of typing.  Dr. Gaur in Washington State is working on 
 
  21   the cynomolgus monkeys; others as well.

  22             So I think that, as time goes on, we will see more 
 
  23   and more reagents become available for that.  So it is 
 
  24   possible to type these animals and look back and see how 
 
  25   class I and class II mismatches played a role. 

                                                                120 
 
   1             But, again, our clinical trials do not require MHC

   2   matching and we have actually made it more difficult for 
 
   3   ourselves by purposely choosing the most mismatched animals 
 
   4   we can find to look at our tolerance‑induction protocols. 
 
   5             [Slide.] 
 
   6             The issue of immunosuppression has come up, as

   7   Jack mentioned, similar to the dog, the levels of FK506 and 
 
   8   cyclosporine that are needed to prevent rejection of a solid 
 
   9   organ in non‑human primates is clearly higher than that what 
 
  10   is needed in humans. 
 
  11             Now, with rapamycin, I am really not sure.  I

  12   think Dr. Bluestone and Dr. Hering have a little bit of 
 
  13   experience with this drug in the setting of islet 
 
  14   transplantation but that is going to be something that we 
 
  15   will all be looking at and shows a lot of promise, based on 
 
  16   the Edmonton data.

  17             But I think one clear advantage is that many‑‑not 
 
  18   all, and Dr. Ricordi specifically mentioned CD3 and the 
 
  19   CAMPAC CD52 antibody, but many, many of the humanized 
 
  20   monoclonal antibodies that are available for clinical 
 
  21   development do cross‑react with non‑human primates.  So if

  22   you can do your preclinical studies with these agents, you 
 
  23   can get, I think, some nice extrapolation to the clinical 
 
  24   setting. 
 
  25             Also, the anti‑thymus‑site globulins that are used 

                                                                121 
 
   1   clinically are clearly cross‑reactive in the non‑human

   2   primate. 
 
   3             [Slide.] 
 
   4             With regard to functional assessment of the islet 
 
   5   grafts, I have already touched on the fact that it is 
 
   6   possible to do the same types of studies in non‑human

   7   primates as in the clinic; blood‑glucose monitoring, 
 
   8   hemoglobin A1c, fasting‑plasma glucose insulin and 
 
   9   C‑peptide, and we can also use identical methods for 
 
  10   functional capacity, intravenous glucose tolerance testing, 
 
  11   arginine and glucagon stimulation

  12             It is also possible to do clamp studies in 
 
  13   non‑human primates although there are very few people, I 
 
  14   think, that can do that and I am certainly not one of them. 
 
  15             [Slide.] 
 
  16             So, really, I think, the issue that we were

  17   discussing in detail before I got up is when is it necessary 
 
  18   to generate preclinical data in support of clinical 
 
  19   protocols.  From my experience, and what we have had at the 
 
  20   institute, I would strongly argue that the most relevant 
 
  21   time for the non‑human‑primate studies is when we have novel

  22   immunotherapies, antibodies that cross‑react, approaches 
 
  23   that we can take to show some efficacy for prevention of 
 
  24   islet rejection, I think when it comes to using altered 
 
  25   doses or combinations of conventional and newer 

                                                                122 
 
   1   immunosuppressive drugs, that it is really going to be a

   2   case‑by‑case study because, in some situation, as has 
 
   3   already been brought up, the drugs may have been used 
 
   4   extensively in other settings and so there is some safety 
 
   5   and efficacy data. 
 
   6             [Slide.]

   7             So, obviously, and I feel like I am just 
 
   8   summarizing what you have already discussed, the critical 
 
   9   needs, if it was possible, would be to develop a model of 
 
  10   autoimmune diabetes in non‑human primates. 
 
  11             I, personally, also think that we will cure humans

  12   before we come up with that.  The only work that I am aware 
 
  13   of was published in abstract form from Gaur, Nepam and 
 
  14   Lernmark in Seattle.  They are working on a low‑dose 
 
  15   streptozotocin model of diabetes in non‑human primates. 
 
  16             I think another critical issue is the whole

  17   efficacy/safety thing.  We have prevention of rejection, but 
 
  18   then there is the issue of when you use this drug in an 
 
  19   animal with underlying disease, does that make a difference. 
 
  20             It would be possible to look at that, although 
 
  21   somewhat impractical, because you could induce diabetes and

  22   then maintain a animals for up to a year which is what it 
 
  23   takes to see some of the underlying kidney and vascular 
 
  24   changes, or we could work on transplanting animals with type 
 
  25   2; for example, treat them with streptozotocin to make them 

                                                                123 
 
   1   fully diabetic and then transplant them.

   2             Then something that hasn't really been discussed 
 
   3   much and isn't the focus of this session, but, obviously, 
 
   4   these models could be very critical for developing markers 
 
   5   for testing assays that could predict rejection and help us 
 
   6   more favorably protect our islet grafts.

   7             [Slide.] 
 
   8             Basically, what I would like to show you now‑‑we 
 
   9   have been talking about all these models and the theories 
 
  10   and the concepts.  So I just wanted to give you an example 
 
  11   of how we have used the model at the DRI.  So we have not

  12   used tissue typing to date.  We are starting to do that in 
 
  13   collaboration with other investigators, but we have used a 
 
  14   mixed leukocyte culture to identify strongly alloreactive 
 
  15   donor recipient pairs. 
 
  16             That consists of taking leukocytes from the

  17   recipient and stimulating them in vitro with irradiated 
 
  18   donor cells.  Then you can look at the proliferative 
 
  19   response of the recipient blood cells to the irradiated 
 
  20   donor.  It gives you an assessment of the degree of 
 
  21   alloreactivity.

  22             I am frequently asked, how can you know that this 
 
  23   really correlates.  In the work that I have done previously, 
 
  24   in the dog model, the MLC data clearly correlate with your 
 
  25   ability to prevent rejection.  If you use low‑dose 

                                                                124 
 
   1   cyclosporine in a dog, the animals will reject in seven to

   2   ten days if they are alloreactive. 
 
   3             If you take MLC non‑reaction pairs of dogs, they 
 
   4   will keep the graft for 30 days with low‑dose cyclosporine 
 
   5   as compared to seven in a highly alloreactive pair.  So it 
 
   6   is efficient for predicting rejection or alloreactivity.  In

   7   any case, we remove the pancreas from the donor and isolate 
 
   8   the islets on day ‑1. 
 
   9             In our studies so far, the islets have been 
 
  10   cultured overnight.  And then, in this particular study, we 
 
  11   were using anti CD145 from Biogen to test its ability to

  12   prevent rejection.  So, on day 0, the recipient's pancreas 
 
  13   was removed and it was given an intrahepatic islet‑cell 
 
  14   transplant. 
 
  15             Here is a key difference.  In humans, we are using 
 
  16   X‑ray and a catheter to do the percutaneous trans‑hepatic

  17   catheterization.  In our monkey model, animals are reopened 
 
  18   so we put a catheter in the portal vein leading to the liver 
 
  19   and drain the islets in. 
 
  20             Jack mentioned the different issues that have to 
 
  21   be addressed in islet transplant.  We included anti‑CD154 in

  22   the islet transplant because, in our thinking, it might 
 
  23   prevent some of the early nonspecific events that can lead 
 
  24   to early islet loss.  That clearly has been seen in the 
 
  25   monkey model. 

                                                                125 
 
   1             Maybe this is a good place to bring up something

   2   that Dr. Black mentioned the other day.  There is a learning 
 
   3   curve.  We were talking yesterday about clinical transplant 
 
   4   and how many isolations do you have to do before you can do 
 
   5   a clinical transplant. 
 
   6             We clearly have that same learning curve in the

   7   preclinical studies.  Our initial studies were in a baboon 
 
   8   and when we switched over the rhesus monkeys, we assumed 
 
   9   that it would be essentially the same.  But the islets were 
 
  10   much more fragile and our first several transplants actually 
 
  11   yielded primary nonfunction because we didn't have adequate

  12   viable islets. 
 
  13             Once we resolved that, we can consistently now, 
 
  14   and routinely get, insulin independence in our monkeys so 
 
  15   that the quality of the islets is obviously very important.  
 
  16   There is a learning curve.

  17             So, day 0, the recipient is pancreatectomized and 
 
  18   given an islet transplant.  Then we monitor the monkeys by 
 
  19   blood glucose.  We look at fasting two to three hour 
 
  20   post‑prandial and evening glucose. 
 
  21             [Slide.]

  22             We are trying to work very closely together in 
 
  23   whatever way we can to design our preclinical studies so 
 
  24   that they mirror exactly our clinical studies.  So one slide 
 
  25   that I don't have, we do the glucose‑stimulated insulin 

                                                                126 
 
   1   release and the viability on the islets.

   2             We have done over twenty monkeys this way, now.  
 
   3   In general, if the stimulation index is over 1‑‑usually, it 
 
   4   is higher than that, but I have had one animal that had a 
 
   5   stimulation index of 1.2 and the islets functioned very 
 
   6   effectively.

   7             The only case I have seen where the assay actually 
 
   8   may have really predicted that the islets would not work was 
 
   9   in a case where we used two donors.  Even though we had 
 
  10   gotten enough islets for transplant, it was not insulin 
 
  11   independent.  When we got the results back of the static

  12   incubation, one of the preps was less than 1.0 in the 
 
  13   stimulation index. 
 
  14             So I am not sure that the degree of the 
 
  15   stimulation index can help us, but it may be possible, 
 
  16   retrospectively, to say that it has some relevance.  But we

  17   do periodic physical exams.  The monkeys' weights are taken.  
 
  18   Every other week, we have a fasting plasma glucose C‑peptide 
 
  19   and insulin.  This is in addition to the daily monitoring; 
 
  20   periodic intravenous glucose‑tolerance testing; complete and 
 
  21   differential blood‑cell count and, because we are using

  22   immunomodulators and antibodies, immunophenotypic analysis 
 
  23   of the white blood‑cell subsets. 
 
  24             In our hands, we have done pre‑ and 
 
  25   post‑transplant mixed leukocyte culture to see if the animal 

                                                                127 
 
   1   becomes specifically nonreactive to the donor as compared to

   2   an unrelated third party. 
 
   3             We look for the development of antibodies to the 
 
   4   donor.  We do an extensive array.  We do P18s for the serum 
 
   5   chemistries and, also, in this case, using 5c8 from Biogen, 
 
   6   we were looking at 5c8 and anti‑5c8.

   7             [Slide.] 
 
   8             So this isn't as relevant to the model but just to 
 
   9   explain what we did.  We had induction therapy, 
 
  10   20 milligrams per kilogram with Hu5c8 on post‑operative days 
 
  11   ‑1, 0, 3, 10 and 18.  Then we initiated a maintenance

  12   therapy starting on post‑operative day 28 and the animals 
 
  13   were given a monthly injection of the antibody. 
 
  14             [Slide.] 
 
  15             So just to show you a little bit of what I have 
 
  16   been talking about.  These are the results that we get when

  17   we take a rhesus monkey, do a pancreatectomy and give an 
 
  18   islet transplant in the absence of any immunointervention.  
 
  19   Fasting blood glucose is the green line.  Post‑prandial is a 
 
  20   purple‑pink line.  This is milligrams per deciliter on this 
 
  21   axis, and this is the post‑operative day.

  22             This particular animal, and we have seen this now 
 
  23   on several occasions, the post‑prandial blood glucose became 
 
  24   elevated on post‑operative day 6 which, in our experience, 
 
  25   has been indicative of rejection. 

                                                                128 
 
   1             We initiated an insulin therapy on this day.  The

   2   fasting glucose started to rise later on, like around day 8 
 
   3   to 10 and what you can see is, even though we initiated 
 
   4   insulin therapy with three injections a day, when the 
 
   5   animals are fully diabetic, it is exactly like a human.  It 
 
   6   is very difficult to maintain a normal metabolic control.

   7             This animal was C‑peptide negative by 
 
   8   post‑operative day 10, so the islet were very rapidly 
 
   9   rejected. 
 
  10             [Slide.] 
 
  11             In striking contrast, using the anti‑CD154, these

  12   are the first three long‑term monkeys we had.  We had 
 
  13   long‑term graft survival.  Antibody was discontinued at 
 
  14   about one year post‑transplant in these monkeys.  All three 
 
  15   of them did eventually experience rejection, but you can see 
 
  16   there is excellent metabolic control.

  17             Yesterday, Dr. Ricordi showed the slide with a 
 
  18   child with type‑1 diabetes showing the glucoses all over the 
 
  19   place. 
 
  20             [Slide.] 
 
  21             This is just to show the post‑prandial glucose. 

  22   Since anti‑CD154 does not suppress islet function, we have 
 
  23   actually been able to determine that rejection may be 
 
  24   occurring by looking at post‑prandial glucose.  It elevates 
 
  25   before the fasting. 

                                                                129 
 
   1             [Slide.]

   2             So how can we use these metabolic assessments, and 
 
   3   I promise there are only a few more, to study the monkeys 
 
   4   like human?  The animals were given intravenous glucose and 
 
   5   then the glucose, insulin and C‑peptide response was 
 
   6   followed after the injection of the glucose which here is at

   7   time 0.  This axis, as Dr. Hering pointed out to me last 
 
   8   night, is incorrect. 
 
   9             But here is time 0 where the glucose is injected.  
 
  10   You can see the green line is pre‑transplant and then these 
 
  11   are the postoperative days.  At one year, is the blue line

  12   here.  What we have seen in our hands is that even in 
 
  13   animals with partial function, the glucose response is not 
 
  14   an adequate indicator that you have lost functional islet 
 
  15   mass. 
 
  16             It can be superimposable even in an animal with

  17   partial function but once it has fully rejected the graft, 
 
  18   we always see a clear difference. 
 
  19             [Slide.] 
 
  20             What we have found to be much more informative, it 
 
  21   has been shown by others that first‑phase insulin release in

  22   an intravenous glucose‑tolerance test is correlated to 
 
  23   functional islet mass.  We have seen that in our studies.  
 
  24             This is the same monkey that I showed in the 
 
  25   previous slide.  This is insulin on this axis and time after 

                                                                130 
 
   1   injection of glucose at 0 here.  The green line is the

   2   first‑phase insulin release before removal of the pancreas 
 
   3   and before the islet transplant. 
 
   4             Then, in the early post‑transplant period, the 
 
   5   light purple lines, you can see that the first‑phase insulin 
 
   6   release was blunted as compared to prior to pancreatectomy. 

   7   Now, the islets do have to revascularize and reenervate and 
 
   8   this could be a reflection of that. 
 
   9             However, the animal was insulin independent and 
 
  10   normal metabolic control.  Then, strikingly, at 155, 227, 
 
  11   296 days, which are represented in yellow here, we actually

  12   saw an improvement.  One year post transplant is represented 
 
  13   here by the blue line.  We have seen that in all of the 
 
  14   monkeys, that if we can prevent rejection, they actually 
 
  15   come up to prepancreatectomy levels at one year 
 
  16   post‑transplant.

  17             Then the antibody was discontinued at one year.  
 
  18   It experienced rejection at 498 days.  539 days, we did an 
 
  19   intravenous glucose‑tolerance test showing that it had fully 
 
  20   rejected the islet graft.  And then we did do a 
 
  21   retransplant.  You can see that we were able to fully

  22   restore insulin. 
 
  23             [Slide.] 
 
  24             This is just to be complete, the C‑peptide data 
 
  25   for the same monkey showing the pretransplant in the green, 

                                                                131 
 
   1   the early time post‑transplant and the midpoints here in the

   2   light purple and yellow and then, at rejection, you can see 
 
   3   there is clearly no function left.  And then the red line is 
 
   4   after the retransplant. 
 
   5             So I think that these models can very effectively 
 
   6   give us information on pathways that may lead to prevention

   7   of rejection and it can tell us as lot about the survival of 
 
   8   islets long‑term in the liver if we are able to prevent 
 
   9   rejection because that is still a question we frequently get 
 
  10   asked. 
 
  11             Thank you.

  12             DR. SALOMON:  Thank you, Dr. Kenyon, for a 
 
  13   excellent presentation. 
 
  14             I need a sense of the committee.  It always sort 
 
  15   of comes to the chair to try and stay on time but everybody 
 
  16   also wants to make sure that it all gets done.  We could

  17   have some discussion now of this which, certainly, there is 
 
  18   no question this is important area and hold Dr. Eggerman's 
 
  19   presentation to right after lunch, which would be my 
 
  20   preference.  Hugh; you are shaking your head. 
 
  21             Why don't we have some discussion now, invite some

  22   discussion, on all these issues including non‑human primate 
 
  23   and then, if we finish before 12:00, we will just break 
 
  24   before 12:00. 
 
  25             DR. SHERWIN:  Just a quick question.  Do non‑human 

                                                                132 
 
   1   primates express class II in beta cells?  In mice, class II

   2   is not expressed in islet cells whereas, in humans, it is 
 
   3   thought that it is and, perhaps, that is one of the 
 
   4   differences between the two that may have some impact. 
 
   5             Do you know anything about that? 
 
   6             DR. KENYON:  We haven't actually done studies with

   7   the non‑human primate islets.  It is a very good point.  I 
 
   8   have done a lot of studies, some years back, with human 
 
   9   islets.  I would guess that they are similar.  And we didn't 
 
  10   see class II in the human islets unless you treated them 
 
  11   with cytokines.  Then there was clearly dramatic

  12   upregulation primarily on the endothelial cells and that is 
 
  13   something we should look at. 
 
  14             Hugh, have you looked at that at all?  It is a 
 
  15   good point. 
 
  16             DR. BLUESTONE:  I think the answer is yes, that,

  17   at least cynomolgus upregulate class II and endothelial 
 
  18   cells in response to gamma interferon, at least.  I don't 
 
  19   know whether it is the same as human, but it is not exactly 
 
  20   like the mouse. 
 
  21             DR. SHERWIN:  The endothelial cells within the

  22   islet, they are coming from the patient or the recipient.  
 
  23   Do you know anything about perfusion?  I am just curious now 
 
  24   about perfusion of islets.  Normally, I assume that they are 
 
  25   coming from the recipient.  The islet gets revascularized? 

                                                                133 
 
   1             DR. KENYON:  Correct.

   2             DR. SHERWIN:  The perfusion of a normal islet goes 
 
   3   straight through the center and then percolates outward so 
 
   4   that there is sort of a unique kind of perfusion system for 
 
   5   the islet.  I wonder if you see the same thing in a grafted 
 
   6   islet, would the perfusion go out‑in?  Has anybody looked at

   7   that? 
 
   8             DR. KENYON:  That is a good question.  I have seen 
 
   9   papers on the microcirculation‑‑one paper on the 
 
  10   microcirculation in rhesus islets, but it was in the native 
 
  11   pancreas.  We haven't looked at that.

  12             DR. BLUESTONE:  The only potentially relevant 
 
  13   point is we tried to do some studies a couple of years ago 
 
  14   where we were using adenovirus with beta gal to try to get 
 
  15   stuff into islet.  We injected it into‑‑actually, this was 
 
  16   when they undid the kidney capsule and actually found most

  17   of the blue cells were around the outside. 
 
  18             DR. SHERWIN:  That is what I bet.  So there may be 
 
  19   some things about islet physiology that change during 
 
  20   transplantation that could influence results.  I mean, I 
 
  21   have no clue as to how.

  22             DR. SALOMON:  It is interesting to go back to 
 
  23   where we were yesterday where we had a fairly low opinion of 
 
  24   the necessity to look at glucagon‑ and 
 
  25   somatostatin‑producing cells yet today we are sort of taking 

                                                                134 
 
   1   the opposite tack that this may be important in terms of

   2   regulation of the‑‑I am just pointing out the‑‑ 
 
   3             DR. RICARDI:  Well, it may be important in terms 
 
   4   of research but I think one of the important findings with 
 
   5   these metabolic studies is that traditionally it was thought 
 
   6   that an islet transplant could not reproduce a pattern of

   7   first‑phase and second‑phase insulin release and has this 
 
   8   typical blunted first phase with delayed response in insulin 
 
   9   production. 
 
  10             What the study shows it that clearly in the 
 
  11   large‑animal preclinical model that actually this is an

  12   issue related mainly to islet mass transplant that survives 
 
  13   because if you have sufficient islets, you have a pattern 
 
  14   that mimics exactly what you would expect in a more 
 
  15   physiologic condition and actually demonstrates, also, the 
 
  16   ability of islets to improve in function over time in the

  17   absence of any rejection or autoimmune recurrence problem. 
 
  18             DR. BLUESTONE:  Norma, I have a question.  One of 
 
  19   the things that the large animal models offer that the 
 
  20   smaller ones don't is to look at where the islets go and 
 
  21   migrate.  You talked about the liver.  At least it has been

  22   our experience that we find islets all over the place.  We 
 
  23   find islets in the lung and stuff, and we probably don't 
 
  24   inject them as well as you do. 
 
  25             But I am just wondering, because the notion that 

                                                                135 
 
   1   the liver would be a good site for this because of immune

   2   privilege and things like that‑‑have you looked?  Is it 
 
   3   possible that some of the long‑term variability and success 
 
   4   depends on where the islets go as much as how good the 
 
   5   islets are? 
 
   6             DR. KENYON:  When you say "long‑term variability,"

   7   what are you referring to? 
 
   8             DR. BLUESTONE:  I am talking about long‑term 
 
   9   insulin independence of human beings after giving the 
 
  10   islets. 
 
  11             DR. KENYON:  That is a good point, Jeff.  We have

  12   not had a lot of tissues that we have started studying 
 
  13   extensively, but we have not looked for them in other 
 
  14   places.  And I will.  It is always a possibility. 
 
  15             Camillo, have you done any studies in the dog 
 
  16   looking at other tissues?

  17             DR. RICARDI:  There have been studies like 
 
  18   injection islet in the lungs as a site of transplantation.  
 
  19   But I wouldn't‑‑ 
 
  20             DR. BLUESTONE:  It was really very striking.  We 
 
  21   tried to do some biopsies in the liver, as you do.  As you

  22   know, it is depending on where you biopsy, you see them or 
 
  23   you don't.  We did two biopsies, couldn't find the islets.  
 
  24   We said, "What is going on here," because this monkey does 
 
  25   not need insulin. 

                                                                136 
 
   1             We were worried.  We did the pancreatectomy. 

   2   Still didn't need insulin.  So we just took other organs, 
 
   3   including the lung, and we found a lot of islets in the 
 
   4   lung. 
 
   5             DR. RICARDI:  And they were infusing the portal 
 
   6   vein.

   7             DR. BLUESTONE:  You bet.  Although, I can't vouch 
 
   8   for that.  I am not the surgeon. 
 
   9             DR. AUCHINCLOSS:  Let's start by putting this 
 
  10   strep issue to rest.  We work primarily with the cynos and I 
 
  11   know that is not your primary species, but I know you have

  12   also.  We find strep treatment to be very consistent, very 
 
  13   effective, no regeneration of islets in at least two dozen 
 
  14   examples that I can think of. 
 
  15             The cynos tolerate the strep treatment if you 
 
  16   hydrate them first.  There is some renal toxicity.  We get

  17   the C‑peptides well below one.  You clearly see big changes 
 
  18   after a successful islet transplant.  Blood sugars are 
 
  19   clearly abnormal and require insulin and then can be 
 
  20   normalized without insulin. 
 
  21             I think that the strep model is an equally

  22   acceptable model for islet transplantation in non‑human 
 
  23   primates; do you agree with that? 
 
  24             DR. SALOMON:  Can I also add our experience with 
 
  25   rhesus.  I would also acknowledge some help initially from 

                                                                137 
 
   1   Phil Padrid at the University of Chicago who is a good

   2   consultant.  We have done it now in about sixteen rhesus 
 
   3   macaque monkeys and had essentially the same results. 
 
   4             DR. KENYON:  My question, then, would be what are 
 
   5   the normal fasting C‑peptide levels in your monkeys and how 
 
   6   do they compare the monkeys after the streptozotocin

   7   because, in my experience now in baboons, rhesus and cyno, 
 
   8   not with strep, we have used strep in rhesus and baboon, the 
 
   9   fasting C‑peptide can be anywhere from 0.8 to 4.0 depending 
 
  10   on the monkey. 
 
  11             Even in the published literature, they will show

  12   the normal range of the C‑peptide and then the normal range 
 
  13   of the C‑peptide in the monkeys that are getting a 
 
  14   transplant, and some of them are in the normal range.  So do 
 
  15   you any IVGTT to prove that there is no C‑peptide release in 
 
  16   response to a stimulus.

  17             If I could see that, then I would be totally 
 
  18   satisfied and then it is just a matter of us, obviously, 
 
  19   working out the logistics because it has been very 
 
  20   labor‑intensive with the approach that we have been using. 
 
  21             But my main concern is does it really eliminate

  22   C‑peptide? 
 
  23             DR. BLUESTONE:  We routinely do IVGTT before we do 
 
  24   the transplants.  Our monkeys are usually two weeks out 
 
  25   post‑strep treatment.  I would say, in 80 percent of the 

                                                                138 
 
   1   monkeys, we see zero‑‑within the limits of the ELISA

   2   detection C‑peptide.  There are a subset, about 20 percent, 
 
   3   that we do see some, anywhere from 0.2 to 0.4, 0.6. 
 
   4             But in the majority of animals, we can wipe out 
 
   5   totally‑‑ 
 
   6             DR. KENYON:  I think if you show that, then it is

   7   actually a preferable model because you have the intact 
 
   8   exocrine function because they don't like the Viokase very 
 
   9   much. 
 
  10             DR. AUCHINCLOSS:  The second point I wanted to 
 
  11   bring out with respect to your presentation was the

  12   HLA‑matching issue where I think we ought to be clear that 
 
  13   we do not expect to accomplish HLA matching for islet 
 
  14   transplantation in the future.  That would essentially turn 
 
  15   it into the problem of trying to find a bone‑marrow 
 
  16   transplant for a 6‑antigen‑matched bone‑marrow transplant

  17   from a nonrelated individual, in which case, we might has 
 
  18   well forget islet transplantation as therapy. 
 
  19             So, to me, the only issue for HLA matching is to 
 
  20   make sure that your monkey model is not matched which is, of 
 
  21   course, what you were doing with your MLC cultures ahead of

  22   time, and the rest of the HLA matching, I would forget about 
 
  23   entirely as far as islet transplantation is concerned. 
 
  24             DR. KENYON:  I actually agree with you and I 
 
  25   should have explained it more clearly.  I didn't mean to 

                                                                139 
 
   1   match in the setting, if you would, in a bone‑marrow

   2   transplant.  I come from the solid‑organ perspective where 
 
   3   one DR, or something like that‑‑but even that, we are not 
 
   4   trying to achieve.  So that was my point, not to match 
 
   5   completely. 
 
   6             DR. SHERWIN:  I have two questions.  I am just

   7   curious about matching, just for my own education.  You are 
 
   8   talking about class I and class II?  Does it matter? 
 
   9             DR. AUCHINCLOSS:  You won't even know.  You won't 
 
  10   even look. 
 
  11             DR. SALOMON:  The only problem with an MLC is that

  12   it is more class II. 
 
  13             DR. AUCHINCLOSS:  It is class II, but you can 
 
  14   mismatch your monkeys for class I and II. 
 
  15             DR. HERING:  Let me ask you, 25 percent of the 
 
  16   donor population is haplo‑identical with type‑1 diabetic

  17   patients so it would not be completely inconceivable to find 
 
  18   a haplo‑identical donor for a type‑1 diabetic recipients.  
 
  19   Would you think that could have an impact? 
 
  20             DR. RICARDI:  Would that increase the possibility 
 
  21   of autoimmune recurrence?

  22             DR. AUCHINCLOSS:  I suspect the answer to 
 
  23   Camillo's question is yes.  I think the answer to your 
 
  24   question is probably yes, it would have an impact.  My 
 
  25   suspicion would be that the impact would be so small as to 

                                                                140 
 
   1   be absolutely insignificant compared to all else that we

   2   need to do. 
 
   3             DR. BLUESTONE:  I don't think the first part is 
 
   4   true.  So if I had to predict what happens is that 
 
   5   endothelial cells, which come from both‑‑probably something 
 
   6   from the donor, but also the recipient which are localized

   7   there‑‑will reprocess peptides and present them in the 
 
   8   context, whether there is a matching or a no matching, and 
 
   9   trigger release of cytokines locally which cause damage. 
 
  10             I don't know any reason to think that direct 
 
  11   recognition by class II cells is going to be the major

  12   pathway to destruction here. 
 
  13             DR. SHERWIN:  I would totally agree.  The question 
 
  14   I was really getting at was class I, which is a different 
 
  15   story.  I just think it is important to think about.  I 
 
  16   think class II matching is probably not as important as

  17   class I‑‑and to look at those issues; I think that is 
 
  18   important. 
 
  19             My other question really related to the liver, 
 
  20   itself.  Have you looked at what the liver looks like 
 
  21   metabolically?  When you put an islet into the portal vein,

  22   the levels of insulin around that islet are going to be 
 
  23   astronomical, as they are within the islet, many, many logs 
 
  24   higher.                                                       
 
  25             So, presumably, around the islet, there is a lot 

                                                                141 
 
   1   of glycogen.  That question is how have you changed the

   2   liver in any way in the area of the islet?  Have you looked 
 
   3   at that? 
 
   4             DR. KENYON:  We have done some initial 
 
   5   assessments.  When we sacrifice monkeys with partial 
 
   6   function, the intact islets actually don't appear to have

   7   any deposition around them.  But we haven't done the 
 
   8   staining yet.  We have really just started analyzing all the 
 
   9   tissues.  You see some lymphocytes if the animal is 
 
  10   undergoing rejection. 
 
  11             DR. SHERWIN:  I guess the only issue to think

  12   about down the road, and may probably not be an issue, is 
 
  13   those kinds of extremely high concentrations could be growth 
 
  14   factors.  So, it could theoretically lead to tumors or 
 
  15   things like that.  It is surely something to consider, even 
 
  16   though I am not saying that there is any evidence to support

  17   that view. 
 
  18             DR. KENYON:  Sure.  But, also, with regards to the 
 
  19   liver function, we do look at liver‑function tests every 
 
  20   other week.  In the immediate post‑transplant period, you 
 
  21   will see an elevation in some of the enzymes, but then they

  22   resolve within a week. 
 
  23             DR. AUCHINCLOSS:  I have another more general 
 
  24   question for the committee and for the FDA.  A lot of people 
 
  25   are talking about the animal studies but particularly, with 

                                                                142 
 
   1   the non‑human primates, we are talking about the efficacy of

   2   various drug combinations or antibodies, et cetera. 
 
   3             My question is, to what extent do you feel that 
 
   4   these tests have to be organ‑ or tissue‑specific.  I would 
 
   5   have suggested that 98 percent of what you learn from a 
 
   6   kidney or a heart transplant in a monkey with CD154 or

   7   reagent of choice is transferrable to islets, as well‑‑not 
 
   8   100 percent, but 98 percent.  Do you agree? 
 
   9             DR. KENYON:  No.  It hasn't been our experience.  
 
  10   Traditionally, things that have worked for solid‑organ 
 
  11   allografting in primates, including conventional

  12   immunosuppressive drugs, have not worked for islet.  
 
  13   Especially, in our hands, FK hasn't.  We haven't tried rapa 
 
  14   yet.  But, no; I don't think it is 98 percent. 
 
  15             Some of the newer things, the CD3 immunotoxin 
 
  16   being a prime example, is an exception.

  17             DR. CHAMPLIN:  If you think about vascularized 
 
  18   grafts and cellular grafts with kidneys on one end and a 
 
  19   bone‑marrow transplant on the other, certainly what works in 
 
  20   kidneys doesn't work for bone marrow and there is very 
 
  21   different immunosuppressive drug requirements for that type

  22   of transplant. 
 
  23             So we were chatting whether an islet is a tissue 
 
  24   or a cell transplant.  It is a small tissue, I guess‑‑lots 
 
  25   of them.  So it may very well have some unique 

                                                                143 
 
   1   characteristics and I wouldn't, necessarily, assume that

   2   things would cross over from solid organs. 
 
   3             DR. CARA:  I am sorry for this very sort of basic 
 
   4   question, but I need some education in terms of some of the 
 
   5   phenomena that you are talking about.  How is the 
 
   6   immunopathology, if you will, of rejection different from

   7   the immunopathology of type‑1 diabetes and is it important 
 
   8   to know the difference if you are going to be using 
 
   9   therapies designed to suppress rejection, perhaps even 
 
  10   autoimmunity, during islet‑cell transplantation? 
 
  11             DR. BLUESTONE:  Good question.  There has been

  12   very little‑‑if you want to get back to animal models like 
 
  13   the NOD mouse, we could talk a little bit.  But, certainly, 
 
  14   in human beings, we have very little information about 
 
  15   what‑‑there are questions, still.  People argue that 
 
  16   antibodies are not important.  I think there is no data in

  17   human being as to whether the antibodies are important or 
 
  18   not. 
 
  19             The issue about relative role of class‑I versus 
 
  20   class‑II‑specific cells, and stuff, I think it is an open 
 
  21   question.  There is enough controversy in the mouse model. 

  22   The human disease, I think, is really a totally open 
 
  23   question. 
 
  24             DR. SALOMON:  I think that is what was part of the 
 
  25   conflict earlier about even how relevant those two 

                                                                144 
 
   1   mechanisms are.

   2             DR. RICARDI:  In part, you may consider that in an 
 
   3   allo‑reaction, you would expect destruction of the entire 
 
   4   islets with an autoimmune kind of immune attack.  You would 
 
   5   have a selective beta‑cell damage.  But this is actually 
 
   6   more complex than that because if you have a failing islet

   7   autograft, you can find selective persistent alpha cells 
 
   8   alone and selective loss of beta cells. 
 
   9             So, because of the sensitivity of beta cells to 
 
  10   cytokine damage and other problems, you can find something 
 
  11   that mimics an autoimmune kind of islet destruction even in

  12   autotransplantation or in allotransplantation.  This is a 
 
  13   very difficult issue to be addressed. 
 
  14             Regarding the change in the liver, there are, 
 
  15   indeed, some early changes, even if you follow 
 
  16   liver‑function tests, there are normalized very soon after

  17   islet infusion‑‑there are some early changes that you can 
 
  18   find in animal models that is just the peri‑islet row of 
 
  19   hepatocytes in which you can see glycogen deposition. 
 
  20             As a matter of fact, the way to find islets at low 
 
  21   magnification, you just look for a glycogen around the liver

  22   and then you zoom in to get the islets in the rodent models.  
 
  23   But this seems to disappear with time and revascularization 
 
  24   and we have limited experience. 
 
  25             But, in the clinical setting, like long‑term, the 

                                                                145 
 
   1   five years or nine years islet functioning in human livers

   2   and in late biopsies, you will see pretty intact, what we 
 
   3   can say, hepatocytes around the islets and no late sign on 
 
   4   liver function. 
 
   5             But I agree that it is a field that could be 
 
   6   investigated more carefully.

   7             MR. SIEGEL:  You mentioned in the baboon, cyno and 
 
   8   rhesus, not much dissimilar to humans, you needed about 
 
   9   10,000 islet equivalents per kilogram.  Do you have data 
 
  10   regarding whether, for a given cell number, the viability of 
 
  11   the prep or in vitro functionality or the size distribution

  12   of the particles in the animals are predictive of success? 
 
  13             DR. KENYON:  Yes; Dr. Black asked me this 
 
  14   frequently.  The 10,000 number, basically, we came up with 
 
  15   based on experience.  That is the number of islet 
 
  16   equivalents that we can give and consistently and

  17   reproducibly get insulin independence for the first week 
 
  18   regardless of what they are treated with. 
 
  19             With regards to where they go and is that 
 
  20   predictive, those are things that we are trying to address 
 
  21   now with a lot of the tissues that we have.  I don't have

  22   the answer yet.  I think the functionality part, we have 
 
  23   looked at in vitro glucose‑stimulated insulin release in 
 
  24   twenty preps that got transplanted. 
 
  25             Unfortunately, when we had our initial learning 

                                                                146 
 
   1   curve, we were not doing those tests because I might be able

   2   to answer your question.  But now, the majority of the 
 
   3   transplants work.  The one that did not work, the animal had 
 
   4   islets from two donors.  So I was surprised, because it 
 
   5   clearly got enough islets. 
 
   6             But when we got the results of the in vitro

   7   studies, one of the preps had no stimulation at all and so 
 
   8   probably wasn't good.  So it does appear from the very 
 
   9   limited experience that we have that we might be able to 
 
  10   make a little bit of a correlation. 
 
  11             But the actual number of the stimulation index, if

  12   there is stimulation, I see a range of stimulation indices 
 
  13   in successfully transplanted animals.  So the only 
 
  14   correlation I can draw right now is that, in a prep where it 
 
  15   was actually the stimulation index, that animal didn't 
 
  16   become insulin dependent.

  17             But I don't think it is a high enough N to have an 
 
  18   impact on the clinical‑‑ 
 
  19             DR. RICARDI:  These were also done after culture 
 
  20   for one day overnight, so they are not fresh, they are not 
 
  21   immediately transplanted like in the Edmonton protocol.

  22             MR. SIEGEL:  Just one quick question on point of 
 
  23   fact; you mentioned that the intravenous glucose‑tolerance 
 
  24   test, the first‑phase insulin release, if I understood, was 
 
  25   correlated well with the functional islet cell mass. 

                                                                147 
 
   1             You said that earlier in your talk.  Later, you

   2   showed how that variable seemed to vary over time.  Were you 
 
   3   referring to the results that you obtained immediately post 
 
   4   operative, or the results a week later or a month or year 
 
   5   later, or what? 
 
   6             DR. KENYON:  That point has actually been shown by

   7   other investigators, primarily Paul Robertson, that there is 
 
   8   a correlation between the functional islet mass and 
 
   9   first‑phase insulin release. 
 
  10             But, interestingly, what we see in the first 
 
  11   couple of months post‑transplant, the height of that first

  12   phase is correlated to the number that you transplanted 
 
  13   whereas, later on, over time, as the islets revascularize 
 
  14   and settle in, at one year post‑transplant, they seem to 
 
  15   come together and achieve their pre‑pancreatectomy levels. 
 
  16             So, other than the fact that I see a correlation

  17   in the immediate post‑transplant period‑‑for example, one 
 
  18   monkey got 40,000 islet equivalents per kilo and it actually 
 
  19   had an insulin release post‑transplant that was much higher 
 
  20   than pre‑pancreatectomy.  So we, appropriately, named that 
 
  21   monkey Camillo.

  22             Our monkeys that get less than‑‑the one that I 
 
  23   showed you that had the blunted first phase at 42 days only 
 
  24   got about 11,000 islet equivalents per kilo.  So you can see 
 
  25   the first phase marching up.  But then, over time, they come 

                                                                148 
 
   1   together and it is not as indicative.

   2             DR. SALOMON:  I think, at this point, we are five 
 
   3   minutes after the afternoon and I would like to stop.  I 
 
   4   know some of us have to check out.  This is an excellent 
 
   5   discussion from some excellent presentations this morning.  
 
   6   I want to thank all the speakers.

   7             These are the issues we will be discussing the 
 
   8   rest of the afternoon, so I don't see any big issue to stop 
 
   9   here.  So I would like to have everyone back, if you don't 
 
  10   mind, at no later than 12:45 so we can get started on the 
 
  11   afternoon meeting.

  12             [Whereupon, at 12:05 p.m., the proceedings were 
 
  13   recessed to be resumed at 12:45 p.m.] 

                                                                149 
 
   1                A F T E R N O O N   S E S S I O N

   2                                                    [1:05 p.m.] 
 
   3             DR. SALOMON:  I would like to get started with the 
 
   4   meeting with Tom Eggerman from the CBER staff who is going 
 
   5   to sort of give us an introduction into some of the 
 
   6   questions we want to deal with this afternoon.

   7             I did want to tell everyone that we have had some 
 
   8   discussions just practically looking at when most of the 
 
   9   members are leaving to go to the airport, including me as 
 
  10   the chair.  I was going to delegate it, but everyone I 
 
  11   delegated to is also leaving at 4 o'clock.

  12             So, after discussion with the FDA, what we are 
 
  13   going to do is actually stick to finishing this meeting a 
 
  14   few minutes before 4 o'clock.  I hope that doesn't require 
 
  15   me to cut any important discussion off, but I think if we 
 
  16   can try and make clear, sharp comments and get all the

  17   discussion in, I think that will be better for everyone. 
 
  18             Okay, Tom.  You're on. 
 
  19                 FDA Perspective, Clinical Issues 
 
  20             DR. EGGERMAN:  Good afternoon. 
 
  21             [Slide.]

  22             My name is Tom Eggerman.  I am with the CBER 
 
  23   Division of Clinical Trials Design and Analysis as well as 
 
  24   the Division of Cell and Gene Therapy.  I would like to 
 
  25   discuss with you issues in early clinical‑trial development 

                                                                150 
 
   1   of allogeneic islet therapy from the FDA perspective.

   2             [Slide.] 
 
   3             As was excellently presented yesterday, islet 
 
   4   therapy has been developing for over fifteen years.  Over 
 
   5   this time, there have been a limited number of patients who 
 
   6   have been treated in a number of centers throughout the

   7   world and also, over this time, the technology for producing 
 
   8   islets has been refined, as have the clinical 
 
   9   immunosuppressive approaches. 
 
  10             Many potential sources for islets have been 
 
  11   evaluated including both fetal and non‑fetal allogeneic,

  12   autologous and multiple xenogeneic species.  A few islet 
 
  13   therapies have been associated with devices, both 
 
  14   encapsulated as well as macro‑device‑associated technologies 
 
  15   to help address the problem of immunologic rejection. 
 
  16             In today's discussion, we are focussing on

  17   allogeneic non‑fetal pancreas sources for islet therapy.  We 
 
  18   will be concentrating on the issues associated with early 
 
  19   clinical‑trial development, especially addressing the safety 
 
  20   and activity assessments.  Even though this therapy has been 
 
  21   used for over fifteen years, the limited successes have not

  22   allowed trials to really advance beyond phase I safety 
 
  23   studies. 
 
  24             Yesterday, some very encouraging data was alluded 
 
  25   to that will, hopefully, eventually translate into pivotal 

                                                                151 
 
   1   trials that will truly evaluate efficacy as well.

   2             [Slide.] 
 
   3             An important aspect of the evaluation of the 
 
   4   safety of proposed clinical studies submitted to the FDA is 
 
   5   evaluating the eligibility criteria to determine if there is 
 
   6   an acceptable risk/benefit for the patient population that

   7   is being studied. 
 
   8             In protocols submitted to the agency, the 
 
   9   eligibility criteria have included patients with type‑1 
 
  10   diabetes, with advanced disease.  The specific criteria have 
 
  11   included a negligible endogenous C‑peptide level, a history

  12   of diabetes for at least five to ten years, a history of 
 
  13   poor glycemic control including a number of documented 
 
  14   hypoglycemic episodes and an elevated hemoglobin A1c. 
 
  15             Most trials have enrolled patients who are already 
 
  16   under immunosuppression related to previous organ

  17   transplantation, usually kidney.  Some studies have 
 
  18   specified tissue matching such as ABO or HLA. 
 
  19             [Slide.] 
 
  20             In most studies of allogeneic islets submitted to 
 
  21   the FDA, patients are on concomitant immunosuppression which

  22   is associated with well‑known risks including infection, 
 
  23   nephrotoxicity and neoplasm.  For those patients already on 
 
  24   immunosuppression for other organ transplantation, there is 
 
  25   not the added risk of new immunosuppression but it is 

                                                                152 
 
   1   recognized that this may not be the optimal therapy for

   2   islet transplantation. 
 
   3             Some investigative studies are enrolling patients 
 
   4   for islet therapies that are using immunosuppressive 
 
   5   regimens specifically designed for optimized islet therapy.  
 
   6   Yesterday's presentation by Dr. Shapiro illustrated this

   7   approach. 
 
   8             In view of the risks associated with 
 
   9   immunosuppressive therapies, other techniques have been 
 
  10   developed to minimize or eliminate the need for 
 
  11   immunosuppression.  These have included devices to

  12   immunoisolate the islets, the development of tolerance 
 
  13   procedures and the use of epitope masking procedures. 
 
  14             [Slide.] 
 
  15             FDA evaluates general safety for the entire 
 
  16   therapy including the procedure used, the islet product as

  17   well as any concomitant therapies such as immunosuppression.  
 
  18   Routine evaluations include clinical lab monitoring such as 
 
  19   CBCs, chemistries and urinalysis as well as follow‑up clinic 
 
  20   visits. 
 
  21             When there is concomitant immunosuppressive

  22   therapies, clinical and laboratory assessments are preformed 
 
  23   appropriate for the specific regimen.  In addition, the 
 
  24   clinical protocol includes predetermined stopping rules 
 
  25   which require the cessation of patient enrollment for the 

                                                                153 
 
   1   development of severe or clinically significant toxicity.

   2             [Slide.] 
 
   3             Because of the specific disease aspects of 
 
   4   diabetes, the safety assessments also include 
 
   5   diabetes‑specific monitoring which include patient glucose 
 
   6   diaries, the number of hypoglycemic episodes, hemoglobin A1c

   7   and other gylcated proteins. 
 
   8             Some trials have also monitoring anti‑islet and/or 
 
   9   anti‑insulin antibody titers and, since islets produce other 
 
  10   proteins, there has been consideration of evaluating 
 
  11   antibodies to these other proteins as well.

  12             [Slide.] 
 
  13             Unlike many products, defining a dose in islet 
 
  14   therapy is not so straightforward as was discussed 
 
  15   yesterday.  There is a disagreement as to how a dose should 
 
  16   be defined.  The two methods most commonly used are related

  17   to the number of volume, so‑called islet equivalence, or 
 
  18   reflect an in vitro islet function prior to administration. 
 
  19             Hopefully, with more standardization in the field, 
 
  20   the best method will become clear and may reflect some 
 
  21   combination of these two elements.  In most studies, a

  22   single administration of islets has been evaluated.  Some 
 
  23   studies have used sequential administrations to reach a 
 
  24   predetermined dose. 
 
  25             Over time, islet function and/or number will 

                                                                154 
 
   1   likely diminish requiring a second administration of islets

   2   to maintain a certain level of islet‑related insulin 
 
   3   production. 
 
   4             The optimal timing for second and subsequent 
 
   5   administration and the potential success for second and 
 
   6   subsequent administration remains to be determined.  If

   7   allogeneic islet therapy becomes successful, source 
 
   8   limitations reflecting the limited number of potential 
 
   9   organs will greatly limit the use of second administrations 
 
  10   since the number of potential diabetes patients greatly 
 
  11   outnumbers the number of organs donated.

  12             It is hoped that advances in cell culturing, 
 
  13   genetic engineering and stem‑cell biology will eventually 
 
  14   allow either the expansion of islets of the establishment of 
 
  15   expandable pools that would allow the production of 
 
  16   unlimited numbers of islets so that all patients could be

  17   treated initially when appropriate and then retreated when 
 
  18   necessary. 
 
  19             Alternatively, sources such as xenogeneic islets 
 
  20   offer a relatively unlimited supply but raise other 
 
  21   potential infectious disease and immunologic issues.

  22             [Slide.] 
 
  23             A concern has been that immunosuppressive regimens 
 
  24   that have been developed for transplantation of organs such 
 
  25   as kidney may not be optimal for islets.  As was presented 

                                                                155 
 
   1   yesterday by Dr. Shapiro, one approach to optimize islet

   2   survival has been to develop islet‑specific 
 
   3   immunosuppression. 
 
   4             Many believe that elevated glucose levels, 
 
   5   particularly at the time of islet therapy, can be toxic to 
 
   6   islets and an approach for this potential problem has been

   7   to use tight glucose control immediately before, during and 
 
   8   for a period after islet therapy. 
 
   9             [Slide.] 
 
  10             The most commonly used route of administration has 
 
  11   been injection into the portal vein.  However, there have

  12   been serious adverse events associated with this approach 
 
  13   which is intended to reproduce the normal insulin secretion 
 
  14   which is transported through the portal vein from the 
 
  15   pancreas. 
 
  16             Other sites have been used which are usually

  17   associated with a device, primarily subcutaneous and 
 
  18   peritoneal sites.  The advantages of these sites include the 
 
  19   ease and decreased risk of administration and the ease of 
 
  20   product removal to better understand the survival of the 
 
  21   islets or to remove the product if there was an adverse

  22   event associated with its use. 
 
  23             [Slide.] 
 
  24             The informed‑consent documents include a 
 
  25   discussion about potential islet‑therapy procedure risks, 

                                                                156 
 
   1   potential infectious‑disease risks as well as the risks of

   2   any concomitant therapy. 
 
   3             The consent process also informs prospective study 
 
   4   participants of alternative therapies, of the potential 
 
   5   risks of alloimmunization including the potential negative 
 
   6   impact upon subsequent organ transplantation as well as

   7   repeat islet therapy. 
 
   8             [Slide.] 
 
   9             There have been multiple potential outcome 
 
  10   measures of activities that can be determined for islet 
 
  11   therapy.  These include glucose diaries, measures of glucose

  12   variability, fasting glucose levels, hemoglobin A1c or other 
 
  13   gylcated proteins, insulin usage and C‑peptide measurements, 
 
  14   either basal or stimulated. One of our questions to this 
 
  15   committee is whether levels of other islet proteins should 
 
  16   also be determined.

  17             [Slide.] 
 
  18             When islet therapy advances to the point of 
 
  19   pivotal trials, a major question will involve appropriate 
 
  20   efficacy endpoints.  If possible, insulin dependence would 
 
  21   be desirable.  However, other outcomes such as improved

  22   glucose control, may be a potential efficacy endpoint.  This 
 
  23   may be particularly important in brittle diabetics with 
 
  24   hypoglycemia unawareness and a history of life‑threatening 
 
  25   hypoglycemic episodes. 

                                                                157 
 
   1             Other clinical endpoints such as retinopathy,

   2   nephropathy or neuropathy that reflect improved glucose 
 
   3   control may also have the potential to demonstrate 
 
   4   meaningful benefit.  A question that will also need to be 
 
   5   answered is what durability of efficacy would be clinically 
 
   6   meaningful.

   7             These are examples of many important issues and 
 
   8   islet therapy.  We look forward to your insights and 
 
   9   perspectives this afternoon. 
 
  10             Thank you. 
 
  11             DR. SALOMON:  Thank you very much, Tom.

  12        Committee Discussion‑‑Preclinical/Clinical Issues 
 
  13             DR. SALOMON:  In preparing for the meeting, I had 
 
  14   several discussions with FDA staff.  I wanted to start off, 
 
  15   then, this last three hours or so of the meeting by trying 
 
  16   to do justice, very briefly, to what the FDA staff wanted to

  17   get out of this meeting. 
 
  18             Yesterday, we identified a series of issues that 
 
  19   relate to identifying and assuring the quality of the 
 
  20   product which is extremely important in terms of thinking 
 
  21   for a regulatory agency.  Again, I think the message the FDA

  22   is trying to get to the field is that, by doing this 
 
  23   proactively instead of reactively, would be to emphasize to 
 
  24   everyone that they want to be a partner in the development 
 
  25   of this moving forward and not create product criteria in a 

                                                                158 
 
   1   vacuum that would, in any way, impeded progress.

   2             From product to preclinical to clinical is kind of 
 
   3   where we are going now.  For the FDA, they were very 
 
   4   insistent that I get the message and stay on track in the 
 
   5   discussion, not to jump to clinical so far that we don't 
 
   6   deal with the implications of preclinical models, to the

   7   extent that we believe in preclinical models, because we 
 
   8   have already had some discussion of those issues and they 
 
   9   need to be on the table this afternoon. 
 
  10             So when we are talking about clinical‑trial 
 
  11   designs, the FDA wanted to always come back to what kinds of

  12   questions can be validly answered in what animal models 
 
  13   because, once again, when the question comes up to 
 
  14   initiating a particular kind of IND‑based proposal, how much 
 
  15   safety has to be demonstrated preclinically in an animal 
 
  16   model so that the FDA feels reassured and that the public,

  17   obviously, is reassured that we have done our diligence. 
 
  18             So I think these are sort of the key questions.  
 
  19   If we can kind of keep that in mind and remind ourselves to 
 
  20   comment on the preclinical models at each juncture, I think 
 
  21   we will be serving the FDA well.

  22             So I would like to begin this series of questions, 
 
  23   at least initially, in order.  The first question is that of 
 
  24   immunosuppression. 
 
  25             DR. AUCHINCLOSS:  Back to the issue of why is this 

                                                                159 
 
   1   conversation that is about to take place going to take place

   2   at all.  I have been rereading your proposed approach to 
 
   3   regulation of cellular and tissue‑based products from 1997.  
 
   4   If I understand it correctly, you feel that regulation of 
 
   5   islet transplantation is appropriate because there is a 
 
   6   metabolic component to the tissue but that if this were an

   7   autologous islet transplantation, regulation would not be 
 
   8   required except for the process of islet preparation. 
 
   9             Is that correct? 
 
  10             MR. SIEGEL:  If it were autologous and not more 
 
  11   than minimally manipulated, regulation would not be

  12   required, period.  Well, that is wrong‑‑as a product and, 
 
  13   therefore, much of the process of manipulation, many aspects 
 
  14   of it, would not be regulated. 
 
  15             However, your statement is more correct than I 
 
  16   initially indicated because it would still potentially be

  17   regulated under our authorities regarding transmission of 
 
  18   communicable disease which we use to regulate tissues 
 
  19   largely vis‑a‑vis issues of donor testing and screening but 
 
  20   also issues of insuring that the processing does not damage 
 
  21   the quality of the tissue.

  22             DR. AUCHINCLOSS:  So you are interested in the 
 
  23   safety of the tissue from infectious‑disease point of the 
 
  24   organ donor and you are interested in the process of the 
 
  25   preparation of the islets, but you would not regulate the 

                                                                160 
 
   1   trial use of autologous islets.

   2             And you feel that islet preparation, at least 
 
   3   according to this document, does not involve more than 
 
   4   minimum manipulation, at least as I read the document, which 
 
   5   says, "extraction or separation of the cells from structural 
 
   6   tissue," blah, blah, blah, "is not more than minimal

   7   manipulation." 
 
   8             So the only way in which I find that you are 
 
   9   interested in regulating allogeneic islet transplantation is 
 
  10   subsequently you say, "Well, metabolic function; if the cell 
 
  11   product has metabolic function, then we want to make sure

  12   that it has metabolic function and, at that point, we feel 
 
  13   an IND is necessary." 
 
  14             But then you go and you say, "Well, it is not 
 
  15   necessary for an autologous islet transplantation because 
 
  16   that is going back into the same recipient."  I don't

  17   understand the rationale for that. 
 
  18             MR. SIEGEL:  There is a rationale.  It was based 
 
  19   on months of back and forth to various regulatory committees 
 
  20   and discussion with various groups.  But, basically, there 
 
  21   is an attempt to draw a number of lines here between what

  22   should be regulated as a tissue and what should be regulated 
 
  23   as a biological product. 
 
  24             It was generally felt that, for example, tendons 
 
  25   or bone chips which are in, at least some sense, not alive 

                                                                161 
 
   1   have, in many cases, there is a higher a priori presumption

   2   of efficacy. 
 
   3             If you take a tendon and you use it to replace a 
 
   4   tendon in an individual, the regulatory concerns about 
 
   5   clinical efficacy and appropriate function are relatively 
 
   6   small.  However, there are a number of factors‑‑and,

   7   therefore, the regulatory focus of that guideline for that 
 
   8   class of products is on safety issues regarding making sure 
 
   9   that it is free of contamination and that it is stored in a 
 
  10   manner that wouldn't allow its intrinsic function to 
 
  11   deteriorate.

  12             Conversely, you have mentioned two of the three or 
 
  13   four types of issues that would cause such a product to 
 
  14   require marketing approval.  One is when a product is more 
 
  15   than minimally manipulated.  That involves what we would 
 
  16   generally consider manufacture.

  17             Examples might be expansion of cells, genetic 
 
  18   transduction of cells.  When one does that to a product, 
 
  19   while it is hard to draw hard‑and‑fast lines, it is 
 
  20   necessary to draw hard‑and‑fast lines in terms of telling 
 
  21   the world how you are going to regulate things.

  22             You can't just say, "I will know it when I see 
 
  23   it."  One of the lines that I think is a reasonable line is 
 
  24   that it is much more reasonable to presume that something is 
 
  25   functional when it is being used to do what it did and it is 

                                                                162 
 
   1   being used in its original form as opposed to when it is

   2   more than minimally manipulated. 
 
   3             Another factor that you mentioned is a combination 
 
   4   of allogeneic and metabolic or systemic activity in 
 
   5   allogeneic source.  The reason those are combined together 
 
   6   is because there is a reasonable presumption‑‑for example,

   7   if you take a blood vessel, a saphenous vein, say, or, say, 
 
   8   from a patient or you take an ovary out while you irradiate 
 
   9   their pelvis and restore that ovary, or you take a vein out 
 
  10   and put it into another area, there are reasonable 
 
  11   presumptions about clinical efficacy and safety that are not

  12   there when one uses allogeneic tissue, both because of 
 
  13   issues of rejection but also other issues of biological 
 
  14   compatibility. 
 
  15             Again, those are not lines that are certain.  You 
 
  16   can give counterexamples on either side of those lines where

  17   the concerns are higher in one group than in another group 
 
  18   but part of the goal here, and a critical part of the goal, 
 
  19   is to set out the rules in advance so somebody, when they 
 
  20   are deciding how to do their research, how to invest their 
 
  21   funds, what to develop, will understand what is the

  22   regulatory framework. Without that, significant damage can 
 
  23   be done to product development. 
 
  24             A third area is homologous usage.  So if you take 
 
  25   that tendon and you use it as a tendon, that is one thing.  

                                                                163 
 
   1   If you take that tendon and you use it as a ligature to

   2   prevent embolization in a vein, that is a different usage. 
 
   3   Of if you take that tendon and you make claims for it for 
 
   4   other uses such that we can implant this in your abdomen and 
 
   5   it will cure cancer or AIDS or something like that, we 
 
   6   consider that regulated.

   7             So that is the rationale why either more than 
 
   8   minimal manipulation, allogeneic and metabolic function, or 
 
   9   non‑homologous use are factors that cause products to be 
 
  10   regulated as products rather than as tissues. 
 
  11             DR. SALOMON:  Jay, I would also question if the

  12   fact is, to the extent that anyone around the table accepts 
 
  13   the premise that there are immunobiological features that 
 
  14   are unique to islet transplantation‑‑albeit there may be 
 
  15   some disagreement on the details, I think all of us accept 
 
  16   that overall premise‑‑then the use of different drug

  17   combinations‑‑in some clinical trials it is going to be new 
 
  18   drugs that haven't been tested before. 
 
  19             Certainly, the islet community is very excited 
 
  20   about the use of really new drugs that haven't been fully 
 
  21   tested.  And, certainly, biologics.  There is another

  22   rationale here, even with established drugs, that there 
 
  23   should be some oversight on the design and conduct of the 
 
  24   clinical trials to assure the fact that data obtained in the 
 
  25   experience of older or newer drugs in kidney or liver 

                                                                164 
 
   1   transplantation, let's say, is relevant to what is going to

   2   happen in our patients in islet transplantation. 
 
   3             MR. SIEGEL:  I think that is moot in the sense‑‑I 
 
   4   think that is right in the sense that I indicated.  In a 
 
   5   large majority of these cellular applications, it would be 
 
   6   under FDA purview.  We have also talked about encapsulation

   7   devices.  We have talked about concomitant experimental 
 
   8   immunosuppressive therapies. 
 
   9             Although, not at this meeting, we have every 
 
  10   anticipation that many of the technologies talked about here 
 
  11   will give use and be applied to cellular expansion and

  12   genetically modified cell technologies as well as in vivo 
 
  13   growth of cells with various regulated factors and products. 
 
  14             So, in most cases, that is the case.  But in some 
 
  15   cases, it does make a substantial difference whether the 
 
  16   cellular product, itself, is considered a regulated product.

  17             DR. AUCHINCLOSS:  Please, I do not suggest that I 
 
  18   don't want good trials of islet transplantation, that I 
 
  19   don't want oversight of those trials.  I just don't think 
 
  20   the FDA should be the source of that oversight. 
 
  21             Your rationale for regulating islets, when you

  22   read the whole thing‑‑you mentioned a variety of criteria.  
 
  23   It comes down to the fact that it is allogeneic and 
 
  24   metabolic.  You don't regulate all allogeneic, do you, 
 
  25   because organs evoke an immune response.  So it is not just 

                                                                165 
 
   1   allogeneic.

   2             It would make sense to me if you said, "I want to 
 
   3   prove, when you do an islet trial, that you produced an 
 
   4   islet that knows how to make insulin."  That would make 
 
   5   sense to me.  But then it would apply to an autologous 
 
   6   transplant just as much as an allogeneic transplant.

   7             So the FDA would be in a great position to help us 
 
   8   insure that the islets that are produced in facilities are 
 
   9   really islets and that they make insulin.  But there your 
 
  10   job can stop.  Once we show that the islet makes insulin, we 
 
  11   can design the clinical trials.

  12             MR. SIEGEL:  I should just say‑‑we could debate 
 
  13   this forever‑‑ 
 
  14             DR. SALOMON:  Let's not. 
 
  15             MR. SIEGEL:  And I am not sure it is particularly 
 
  16   useful.  I should say that there are different laws that

  17   apply to the area of solid vascularized organs.  So your 
 
  18   question, they are metabolic, yes.  But there are different 
 
  19   laws specifically that apply to how those are used in this 
 
  20   country. 
 
  21             DR. AUCHINCLOSS:  Yesterday, you suggested that if

  22   once we called it a product, it had to go the whole 
 
  23   distance.  But your document here does suggest that there 
 
  24   are kinds of products for which you want to insure safety of 
 
  25   the tissue and adequacy of the process but that do not 

                                                                166 
 
   1   require INDs and premarketing licensure.

   2             MR. SIEGEL:  The whole purpose of this structure 
 
   3   is to apply the level of regulation as appropriate to the 
 
   4   types and nature and extent of issues concerning safety and 
 
   5   efficacy raised by the product. 
 
   6             Those products that we do call tissues are

   7   regulated predominantly for infectious‑disease risk although 
 
   8   we believe we have some authority based on the 
 
   9   infectious‑disease risks to also make regulations pertinent 
 
  10   to product quality. 
 
  11             However, beyond that, we don't have options to

  12   regulate a product in the ways we are talking about, to just 
 
  13   regulate how it is manufactured and stop there and say it 
 
  14   has to be able to make insulin, but it is not a product.  
 
  15   That just doesn't fit into our regulatory‑‑ 
 
  16             DR. AUCHINCLOSS:  I don't understand this

  17   document, then. 
 
  18             DR. SALOMON:  I didn't want to interrupt until now 
 
  19   because I think Hugh's points should be a part of the 
 
  20   record.  If he has concerns about a discussion that now 
 
  21   follows, then I respect that from him.  I think we have got

  22   that in the record, now. 
 
  23             I don't think that the purpose here is for us to 
 
  24   debate what decisions the FDA has made on whether to 
 
  25   regulate or not, although I think Hugh's points, perhaps, 

                                                                167 
 
   1   should be considered in detail by the FDA.  So I would like

   2   to get back to the topics at hand. 
 
   3             Hugh, are you okay with that?  I don't want to 
 
   4   deprive you of a key point here. 
 
   5             So, the first thing would be immunosuppression.  I 
 
   6   think maybe sort of the overriding question on the

   7   immunosuppression is what do you guys think would be the 
 
   8   optimal immunosuppression to use in an islet transplant.  I 
 
   9   think we have got some data on the table on that already, 
 
  10   but let's put that as a specific question. 
 
  11             What data from current preclinical models justify,

  12   in your opinion, that decision.  We also touched on that a 
 
  13   little bit earlier but let's just make sure that we come to 
 
  14   some sort of conclusion on that. 
 
  15             DR. RICORDI:  Actually, I would like to make a 
 
  16   comment in support with Dr. Auchincloss' previous comment

  17   and that is that I don't think it is necessarily our 
 
  18   business to discuss the best immunosuppression for islets in 
 
  19   this site, but I completely agree on the fact that we should 
 
  20   address safety and product‑release criteria and what is the 
 
  21   best islet that we can put in patients.

  22             But we are here to develop a procedure over the 
 
  23   years that is extremely more safe than a pancreas organ 
 
  24   transplant, like maybe ten‑fold safer as to morbidity or 
 
  25   mortality, but in which, as things are going or developing, 

                                                                168 
 
   1   would be imposed on regulatory aspects that are ten‑fold

   2   more complex than what you have to do in organ 
 
   3   transplantation. 
 
   4             So I think the message is to go back to full 
 
   5   pancreas transplantation from the kind of discussion here.  
 
   6   I have to agree completely with Dr. Auchincloss that I am

   7   completely supportive of the idea of standardizing 
 
   8   product‑release criteria and safety concerns but it would be 
 
   9   severely damaging to the whole field of allotransplantation, 
 
  10   for example, to impose a unified protocol, that everybody 
 
  11   now does the same thing for the next two years in a stage

  12   where nobody knows which one is the best product development 
 
  13   of kind of tissue separation, et cetera. 
 
  14             DR. MILLER:  I actually don't think that is really 
 
  15   what they are asking.  I think they want some framework 
 
  16   around which experts feel are acceptable protocols for them

  17   to review, how much more information do the experts feel 
 
  18   that you need to collect before you can proceed with 
 
  19   clinical trials. 
 
  20             You may say that all the information is already 
 
  21   there, either from the clinical experience or the animal

  22   experience, and that is the question that they are wanting 
 
  23   to ask, is what I think we are here for. 
 
  24             DR. AUCHINCLOSS:  Carole, that is not true.  The 
 
  25   question says, "What is/are the most appropriate 

                                                                169 
 
   1   immunosuppression regimen(s) to use for islet‑only studies?" 

   2   That is an absurd question. 
 
   3             DR. CHAMPLIN:  I guess my fundamental issue or 
 
   4   question is what is the role of the FDA in defining these 
 
   5   sorts of things as opposed to individual institutions and 
 
   6   their IRB.  I would agree with the idea of trying to define

   7   the product and the safety issues and the product 
 
   8   description of things that really relate to the transplant 
 
   9   infusion. 
 
  10             But the issues of how best to treat patients, what 
 
  11   would be the eligibility criteria for people going on to

  12   clinical trials‑‑certainly the big area of immunosuppression 
 
  13   is something where one should not try to impose a 
 
  14   preconceived standard when there really is so little data of 
 
  15   efficacy. 
 
  16             MR. SIEGEL:  I think there is a lot of putting up

  17   of straw men here to shoot down.  There is no discussion of 
 
  18   imposing standards.  We have a requirement to insure that 
 
  19   these trials do not expose patients to unnecessary and 
 
  20   unreasonable risk. 
 
  21             In order to achieve that, we need, and we are

  22   looking to you, for advice, better understanding how to 
 
  23   assess those risks of when and whether additional animal 
 
  24   models are appropriate before going into humans, as to which 
 
  25   risks are more significant in which populations as a 

                                                                170 
 
   1   function of population, as a function of dosing, and, as

   2   products develop, we additionally need‑‑in addition to 
 
   3   insuring that there are not unnecessary and significant 
 
   4   risks, we need to insure that there are data of an adequate 
 
   5   nature and quality to be able to assess the safety and 
 
   6   efficacy of the product.

   7             Those are what our mandate is to do.  We do that 
 
   8   in many areas.  We don't, in those areas, tell people how to 
 
   9   do their clinical trials.  We don't tell people that there 
 
  10   is only one way to do a clinical trial.  We try to reflect 
 
  11   the best science and to add our expertise in clinical‑trial

  12   design, our expertise in what sorts of methodologies work, 
 
  13   what sorts of inferences can be made from what types of 
 
  14   study designs, add that to the expertise that we receive to 
 
  15   help people do trials that will be safe and will be 
 
  16   meaningful.

  17             DR. SALOMON:  So the question comes if you have a 
 
  18   clinical trial that you want to do‑‑let's back up a little 
 
  19   bit.  I don't want to tread in such sensitive ground 
 
  20   immediately.  But if there is a clinical trial that you want 
 
  21   to do, I assume you want to use immunosuppression or will

  22   want to use some form of immunosuppression. 
 
  23             How would you suggest that immunosuppression, at 
 
  24   the time you are going to initiate that trial, be justified?  
 
  25   What kind of data from a preclinical model would be 

                                                                171 
 
   1   reasonable to present?

   2             Jeff, do you have a comment you want to make on 
 
   3   that line? 
 
   4             DR. BLUESTONE:  No; obviously not, because I was 
 
   5   going to ask you what you said.  I was actually going try to 
 
   6   find a middle ground, here.

   7             DR. SALOMON:  I am not saying my middle ground is 
 
   8   any better than your middle ground, by the way. 
 
   9             DR. BLUESTONE:  So my middle ground would be to 
 
  10   ask your question somewhat differently which would be to ask 
 
  11   the question, is there anything that we can agree on that is

  12   a necessary component of the regimen that everyone should be 
 
  13   doing. 
 
  14             I think the answer we are going to say is not and, 
 
  15   therefore, it would be in Hugh's category.  For something 
 
  16   else, the answer may be absolutely yes, like 10,000 islet

  17   units which is maybe what we decided yesterday. 
 
  18             I actually don't see the distinction between 
 
  19   yesterday's discussion and today, only that we knew more 
 
  20   yesterday about what we liked and today we haven't had the 
 
  21   discussion yet.  I think your question is fine, but my

  22   answer to the question, if you ask it the way you did, is 
 
  23   no, I can't be prepared to sit here today and say that I 
 
  24   know the best immunosuppressive regimen or that there is 
 
  25   preclinical data to suggest what that is. 

                                                                172 
 
   1             DR. SALOMON:  I guess I was avoiding that.

   2             MR. SIEGEL:  Can I please‑‑let's try to look at 
 
   3   the questions before you jump to conclusions about what we 
 
   4   are asking and what advice we want.  There is not a question 
 
   5   here about is there a standard regimen, should there always 
 
   6   be one drug.

   7             The questions here are specifically focused on our 
 
   8   regulatory needs.  Each and every one of them is about 
 
   9   islet‑only therapy.  Why is each and every one of them about 
 
  10   islet‑only therapy?  Because islet‑only therapy is exposing 
 
  11   patients to immunosuppression who were not otherwise to be

  12   exposed to immunosuppression. 
 
  13             It is there where we have a significant burden to 
 
  14   determine whether this is a reasonable and unnecessary risk.  
 
  15   It is there where we must ask, do we yet know enough from 
 
  16   animal models to do this?  What information should we have

  17   from animal models?  What would be the most appropriate 
 
  18   patients from a potential benefit or rationale to risk to 
 
  19   make that determination? 
 
  20             We are not here to say, "This is the right 
 
  21   immunosuppressive regimen for islet transplants."  That is

  22   not even the question on the table. 
 
  23             DR. SALOMON:  Again, the question that I was 
 
  24   posing to try and follow, really, the spirit of what Jeff 
 
  25   said and what Jay said is based on the animal‑model 

                                                                173 
 
   1   experience that we discussed this morning and some

   2   yesterday, if you wanted to go forward into a clinical 
 
   3   trial, which animal models or model would provide the kind 
 
   4   of data to justify a given choice of immunosuppressive drug? 
 
   5             I am not trying to tell you to say that it is 
 
   6   rapamycin; just any approach, what approach?  If the answer

   7   is there is no approach, then we need to tell the FDA that 
 
   8   which means, to me, that no one is ready to go to a clinical 
 
   9   trial anytime soon, which is fine. 
 
  10             DR. AUCHINCLOSS:  I beg your pardon?  We are going 
 
  11   into a clinical trial with the Edmonton protocol which was

  12   developed in the human animal model. 
 
  13             DR. SALOMON:  The human animal model.  So the 
 
  14   idea, then, is that there is not preclinical animal‑‑if that 
 
  15   is what you want to say, that's fine.  Then what Hugh is 
 
  16   saying is that there is no preclinical data necessary.  You

  17   choose an immunosuppressive regimen based on what?  I am not 
 
  18   certain.  And then you start a human trial. 
 
  19             DR. RICORDI:  Maybe I can rephrase Hugh's comment.  
 
  20   I am saying there are many animal models valuable to develop 
 
  21   new strategies and a research base for immunosuppression and

  22   to screen drug combinations and everything. 
 
  23             There is not a single animal model, in my opinion, 
 
  24   that is a necessary prerequisite before moving to a pilot 
 
  25   clinical trial because of the lack of existence of a model 

                                                                174 
 
   1   similar to type‑1 diabetes and that can predict safety or

   2   efficacy in human patients. 
 
   3             DR. CHAMPLIN:  As I mentioned this morning, there 
 
   4   are big problems in trying to take drug trials from animals 
 
   5   directly into humans.  Certainly, we have the data that was 
 
   6   presented this morning.  We also have human data.  So I

   7   think that that is, perhaps, the most important data as one 
 
   8   considers going forward as to where are we now. 
 
   9             At least from the inklings of what we have heard 
 
  10   of the Edmonton protocol, it sounds to be a successful 
 
  11   starting point.

  12             DR. MILLER:  I agree with Dick.  The question we 
 
  13   are sort of struggling with here, I think, is how to 
 
  14   integrate what we already know about humans when you are now 
 
  15   asking us to go back to the animal models. 
 
  16             So a question that may help is do we feel that we

  17   can take a pilot trial without any further animal data and 
 
  18   generalize it and therefore leave the next steps to what 
 
  19   animal models you need to do before you then go back into a 
 
  20   different protocol for a human trial. 
 
  21             I am not exactly sure of the number of patients

  22   treated with islet‑only cells in the pilot trial, the 
 
  23   preliminary data, to then going on and building this 
 
  24   multicenter trial.  We don't know how strong the data is 
 
  25   even though we hear it. 

                                                                175 
 
   1             So my feeling is that there is enough clinical

   2   data already that can be reproduced or that could be 
 
   3   validated and looks good that shows that they followed so 
 
   4   many people out so long.  You are not going to get any 
 
   5   better than that for the study of this current regimen. 
 
   6             Therefore, if it is valid and reviewed, that

   7   probably is enough to go ahead and do the pilot trial.  If 
 
   8   that is our first answer, then the second question is, okay, 
 
   9   now we want to get away with no immunosuppression and what 
 
  10   kind of animal models do you need to do that.  That is a 
 
  11   separate, question.

  12             I think it is a separate question than whether or 
 
  13   not the pilot trial is adequately controlled by the human 
 
  14   experience we already have. 
 
  15             DR. BLACK:  Could I take one step back and say 
 
  16   just surely the team, Dr. Shapiro and Dr. Lakey, here, did

  17   not arrive at their FK506‑rapamycin combination without 
 
  18   preliminary work, perhaps dating back a number of years or 
 
  19   in several different models that gave them suggestions of 
 
  20   how to proceed in the clinic. 
 
  21             If you could clarify that a little bit?

  22             DR. SHAPIRO:  That is exactly right.  Our trial is 
 
  23   built on a synthesis of many years of preclinical 
 
  24   experimentation and also a substantial clinical knowledge of 
 
  25   use of these agents in different usage and combinations in 

                                                                176 
 
   1   kidney transplantation and other models.

   2             We haven't just come to the scene here with a 
 
   3   brand‑new therapy that hasn't been tested or applied in 
 
   4   other situations.  We have just used a cocktail of agents 
 
   5   that we believe are safe to use clinically in a different 
 
   6   way.

   7             DR. SALOMON:  What models in that development 
 
   8   process did you think gave you the best information or were 
 
   9   they all just pieces of a complicated puzzle? 
 
  10             DR. SHAPIRO:  I think it is like a jigsaw.  Our 
 
  11   preclinical in Edmonton had always been the adult

  12   islet‑transplantation model.  We knew that many of the drugs 
 
  13   we use are not compatible with adults and we knew that that 
 
  14   would only provide us information in terms of function of 
 
  15   islets and provide us a little bit of information in terms 
 
  16   of toxicity but not sufficient.

  17             And then you synthesize also what is available 
 
  18   clinically and what has occurred in that realm. 
 
  19             DR. SALOMON:  So, Dr. Kenyon, in that regard, 
 
  20   where does the non‑human primate studies that you are 
 
  21   doing‑‑they are very expensive.  They are very involving. 

  22   If this is just a complex jigsaw, maybe we could save some 
 
  23   serious money. 
 
  24             DR. KENYON:  It is a serious effort.  No; I agree 
 
  25   with everything that they have said so far, but, clearly. I 

                                                                177 
 
   1   would not be comfortable going into a human with an agent,

   2   for example, a new monoclonal or a new agent that had no 
 
   3   experience in the clinic. 
 
   4             So I think that is where the non‑human primate 
 
   5   models, the dog models, the animal models‑‑we get our 
 
   6   suggestions of efficacy from the rodents and then we have to

   7   move them up and see if they work in the larger animals. 
 
   8             I think that is very important there, but, 
 
   9   clearly, that doesn't give you the final answer either, so I 
 
  10   am not going to sit here and say that is the final answer.  
 
  11   It is clearly not true, but very important for new

  12   immunomodulatory agents. 
 
  13             DR. AUCHINCLOSS:  We need to be clear, again.  I 
 
  14   am not arguing that new immunoregulatory agents should not 
 
  15   be regulated by the FDA.  They are and they should be.  So 
 
  16   the FDA will certainly regulate a trial that had, for

  17   example, anti‑CD154.  That is not the question. 
 
  18             The question is whether they are regulating islet 
 
  19   transplantation, themselves. 
 
  20             MR. SIEGEL:  These are unapproved uses of all 
 
  21   these drugs.  Even if there was no cellular component, this

  22   trial would require FDA review.  The rapamycin, the FK506, 
 
  23   they are not approved for this use. 
 
  24             DR. KENYON:  Hugh, I think they are really asking 
 
  25   where do you draw the line?  Clearly, in the type of study 

                                                                178 
 
   1   they are doing in Edmonton, there is enough clinical data

   2   plus their experience to support it.  So I really thought 
 
   3   that what you were asking is where do you think it is 
 
   4   important to have some preclinical data. 
 
   5             I think, clearly, it is where you don't a lot of 
 
   6   clinical data.  But I, personally, would not want to do a

   7   human transplant with an agent that had really never been 
 
   8   used clinically without having some preclinical data.  I 
 
   9   think we do. 
 
  10             DR. BLUESTONE:  I think what we don't agree on and 
 
  11   what we haven't come to closure on‑‑and that is why I don't

  12   like the question.  What the question is posing, in a way, 
 
  13   is that there is a paradigm model out here, that there is 
 
  14   some set of three models, if you put them together you 
 
  15   should be able to‑‑so my answer to the question is that, in 
 
  16   the Edmonton case, then Hugh is right.  But in the CD154

  17   case, then Hugh wouldn't be right because there isn't a 
 
  18   human experience to rely on. 
 
  19             And he does not disagree with me.  So the answer, 
 
  20   from my perspective, goes back to what Jay says, you have to 
 
  21   make a rationale argument.  How do you make a rationale

  22   argument.  It has got to be a combination of preclinical 
 
  23   and/or clinical experience that demonstrates safety and some 
 
  24   degree of efficacy. 
 
  25             How you actually build that equation up is the 

                                                                179 
 
   1   same jigsaw puzzle that Jim has already done.  It is hard to

   2   sit here and say that an animal model, or two animal models, 
 
   3   are going to be the answer or not.  You have to base the 
 
   4   recommendations on a series of identifiable results that 
 
   5   make it a compelling rationale to go forward. 
 
   6             I don't think it is always going to be due to

   7   monkeys, and it is not always going to be a NOD mouse.  But 
 
   8   sometimes a NOD mouse might work because the antibody didn't 
 
   9   work in a monkey.  And sometimes the monkey will be workable 
 
  10   because it does work. 
 
  11             I think it would be a mistake to try to set a

  12   clear set of parameters of what those preclinical trials 
 
  13   should be other than the general principle that, in the 
 
  14   absence of clinical data, compelling clinical data, you need 
 
  15   preclinical data. 
 
  16             DR. SALOMON:  I don't disagree.  That is kind of

  17   what I was trying to get at when I used the analogy in 
 
  18   talking to Jim about the fact that it is a jigsaw.  So I 
 
  19   think, so far, the message that I am very comfortable in 
 
  20   giving to the FDA is that preclinical models are going to be 
 
  21   critical, but that there is no single preclinical model that

  22   will give you an answer or that you should require 
 
  23   information in. 
 
  24             It rather should be a presentation of a logical 
 
  25   series of preclinical experiments.  I think that also, and 

                                                                180 
 
   1   again I would put this out for discussion, it would be

   2   equally wrong that if someone came with a single preclinical 
 
   3   model and you wanted, based on a single preclinical model, 
 
   4   really without much evidence surrounding it, to go forward 
 
   5   and do a clinical trial, that probably would be equally 
 
   6   questionable, that there should be, probably, in any

   7   situation, a series of models, a series of lines of 
 
   8   investigation, hopefully independent, all of which 
 
   9   rationally support the decision made for the clinical 
 
  10   project. 
 
  11             Do you agree with that or not?

  12             DR. SHERWIN:  I do.  The only thing that I would 
 
  13   add is that, particularly with this disease being an 
 
  14   autoimmune disease, that one should consider the possibility 
 
  15   of using an autoimmune model of diabetes within that mix 
 
  16   because there is a certain level of complexity to the

  17   problem. 
 
  18             It is not a problem when you use big drugs.  The 
 
  19   drugs you used, it wouldn't matter at all.  But when you 
 
  20   start getting to refined‑‑because we are talking about 
 
  21   people without kidney transplant.  That is a different

  22   story.  Consequently, the kind of immunosuppression that you 
 
  23   use in that setting should be less toxic, I think, than the 
 
  24   kind of immunosuppression you would use with a kidney graft 
 
  25   as well. 

                                                                181 
 
   1             So when you get to the more selective agents where

   2   you are not going to be hitting it with a sledgehammer, I 
 
   3   think, at that point, an autoimmune model could give you 
 
   4   answers that are important and I wouldn't ignore that. 
 
   5             DR. BLUESTONE:  Would you say that the drug should 
 
   6   be less toxic than if it was a pancreas transplant?

   7             DR. SHERWIN:  I am not too in favor of pancreas 
 
   8   transplants in people who have no significant or serious 
 
   9   complications.  In other words, we use pancreas grafts, but, 
 
  10   usually, it is in people that are either having impending 
 
  11   renal failure or require a renal graft.  So it is not a

  12   routine thing, I think, to use pancreas transplantation in 
 
  13   people‑‑see; we disagree, obviously. 
 
  14             DR. HERING:  Let me ask you.  You are a 
 
  15   diabetologist. 
 
  16             DR. SHERWIN:  Yes.

  17             DR. HERING:  Would you admit that patients with 
 
  18   hypoglycemia unawareness and defective glucose‑count 
 
  19   irregulation, would you consider this a complication of 
 
  20   diabetes? 
 
  21             DR. SHERWIN:  Of course it is.  But with good

  22   care, generally speaking, that can be dealt with. 
 
  23             DR. HERING:  With good care in a clinical research 
 
  24   center; right? 
 
  25             DR. SHERWIN:  No, no, no, no.  You know, there are 

                                                                182 
 
   1   people that have recurrent hypoglycemia where it is a

   2   problem.  But, first of all, we have much better systems and 
 
   3   we are evolving much better systems for monitoring.  
 
   4   Consequently, in the next few years, those problems are 
 
   5   going to diminish. 
 
   6             So I rarely encounter somebody that I would

   7   think‑‑there are, like, one or two patients I have 
 
   8   encountered in the last ten years that I would think about 
 
   9   it.  There is a rare patient where you are correct.  But I 
 
  10   disagree.  I think with more effective insulin delivery 
 
  11   systems and good management, most people can be managed

  12   without enough of a problem to warrant immunosuppression. 
 
  13             I would rather have recurrent hypoglycemia 
 
  14   periodically that is manageable than immunosuppression. 
 
  15             DR. HERING:  I guess the DCCT was published in 
 
  16   '93, yet diabetes remains one of the leading causes of

  17   blindness and this and this and this, well, at least in the 
 
  18   type‑1 diabetic population.  Would you accept that 
 
  19   intensified insulin treatment is difficult to implement? 
 
  20             DR. SHERWIN:  Of course.  Of course it is 
 
  21   difficult to implement, but the improvements that one

  22   achieves with intensified therapy are sufficient, I think, 
 
  23   to warrant real caution doing a pancreas graft to try to 
 
  24   take it to the next level because the risks of imposing that 
 
  25   are greater than the risk of the disease, I think. 

                                                                183 
 
   1             We are getting into the wrong discussion, I know. 

   2   Maybe we should stop. 
 
   3             DR. CARA:  Actually, I think this is just the 
 
   4   right sort of discussion to have because I think we sort of 
 
   5   took the cart before the horse when we started talking about 
 
   6   different sorts of regimens in the sense that it seemed that

   7   we had all sort of accepted the notion that islet‑cell 
 
   8   transplantation in a patient without any evidence‑‑or on a 
 
   9   patient who is not already on immunosuppression therapy 
 
  10   should continue forward. 
 
  11             I am not sure that we all agree with that.  So I

  12   think the issue that we really need to sort of look at is 
 
  13   whether or not there is enough information, there is enough 
 
  14   data, to carry forward with islet‑cell transplantation in 
 
  15   patients that are not on any sort of immunosuppressive 
 
  16   therapy.

  17             MR. SIEGEL:  This is the question that is, I 
 
  18   think, the first point in the question‑‑ 
 
  19             DR. SALOMON:  We haven't forgotten that one, Jay. 
 
  20             MR. SIEGEL:  The first bullet is specifically 
 
  21   about which patients for the very reason of the nature of

  22   what the alternative treatments are that are that available 
 
  23   and what is the course of the disease‑‑this is an important 
 
  24   one in our construct in determining what are acceptable 
 
  25   risks or acceptable‑‑ 

                                                                184 
 
   1             DR. SALOMON:  I would point out that actually what

   2   Bernhard and Bob were doing was discussing this question. So 
 
   3   I don't want to derail that now because that is‑‑and I agree 
 
   4   with Dr. Cara that this is what we ought to ask now, and 
 
   5   that is what is the population of patients, if any, that you 
 
   6   believe today would be candidates for islet transplantation

   7   only, not in the setting of a kidney or a liver. 
 
   8             DR. SHAPIRO:  Patients essentially who have 
 
   9   documented evidence of failure of exogenous insulin therapy 
 
  10   for whatever reason. 
 
  11             DR. SALOMON:  Can you help us decide, what would

  12   that be?  Recurrent malignant hypoglycemia?  There has been 
 
  13   discussion in the literature on what that population should 
 
  14   be.  I think the FDA wants to hear us discuss what those 
 
  15   patients might be. 
 
  16             DR. SHAPIRO:  I think those opinions vary between

  17   expert diabetologists.  It is very difficult to define.  The 
 
  18   patient that we say has totally failed all efforts at 
 
  19   optimal glycemic control despite very intensive insulin 
 
  20   therapy, Dr. Sherwin would say he could easily treat that 
 
  21   patient.

  22             DR. SHERWIN:  I am not trying to say that.  I am 
 
  23   not glib.  I have as much trouble as anyone else.  I think 
 
  24   that the improvements that one can achieve with optimized 
 
  25   therapy‑‑there are now glucose sensors that are about to 

                                                                185 
 
   1   released that can give more continuous monitoring of glucose

   2   which is going to provide a lot of information. 
 
   3             DR. HERING:  I agree 100 percent.  Eventually, we 
 
   4   will have to perform a prospective clinical trial to address 
 
   5   this question.  Now we have to identify a patient population 
 
   6   to get islet transplantation to the level‑‑

   7             DR. SHERWIN:  I would say in people with incipient 
 
   8   creatinines, let's say, above two and a half or three who 
 
   9   still have function, to me, that would be not an 
 
  10   unreasonable group of people. 
 
  11             But I think you would have to be very, very

  12   careful about using hypoglycemia as a dominant reason to do 
 
  13   an islet graft.  It may be that there are selected 
 
  14   individuals who do that, but I would suggest that it would 
 
  15   require some sort of independent team of people to assess 
 
  16   what had been done clinically prior to subjecting someone,

  17   on that basis, for allotransplantation. 
 
  18             So I think you would need an independent team of, 
 
  19   perhaps, experts to evaluate the situation and be sure that 
 
  20   medical therapy had been exhausted.  I am not saying that 
 
  21   there aren't individuals like that, but there are not many.

  22             DR. HERING:  A fundamental point here is we are 
 
  23   not recommending islet transplantation for the treatment of 
 
  24   hypoglycemia unawareness.  But we are going to identify a 
 
  25   patient population and we want to see whether, in a 

                                                                186 
 
   1   controlled clinical trial, we can test whether islet

   2   transplantation can be performed safely and in an effective 
 
   3   manner. 
 
   4             So we are not recommending this as a treatment.  
 
   5   We are identifying a population that is considered suitable. 
 
   6             DR. SALOMON:  Can you be specific about what that

   7   patient population is, then, in the trial that you are 
 
   8   proposing? 
 
   9             DR. HERING:  Also, we can ask what are the 
 
  10   recommendations of the American Diabetes Association for 
 
  11   solitary pancreas transplantation.  ADA has concluded that

  12   it was "patients in whom all other measures have 
 
  13   consistently failed to ameliorate the situation;" this was 
 
  14   the definition. 
 
  15             DR. SHERWIN:  That's true.  It depends on how you 
 
  16   judge that.  So I am saying that it really would require, I

  17   think, some sort of assessment by an independent team of 
 
  18   diabetologists to say that it had been exhausted.  Then I 
 
  19   think it is acceptable. 
 
  20             DR. HERING:  I think that is a wonderful 
 
  21   agreement.  I guess nobody would argue that a diabetologist

  22   should be part of the team and should be approached and 
 
  23   should probably refer the patient. 
 
  24             DR. SHERWIN:  My only concern is that it really be 
 
  25   an independent sort of advisory committee or something like 

                                                                187 
 
   1   that because it is not a problem when you are dealing with

   2   people with renal disease who are going to require a kidney 
 
   3   graft.  If you don't want to have people on 
 
   4   immunosuppression, you could study people who one could 
 
   5   predict within a short period of time, they will require a 
 
   6   kidney graft even though they don't require

   7   immunosuppression‑‑ 
 
   8             DR. BLUESTONE:  Isn't that the job of the IRB? 
 
   9             DR. SHERWIN:  I would be careful about IRBs 
 
  10   because they don't treat people with diabetes.  They are not 
 
  11   sophisticated in the ways of patient selection.  I think

  12   IRBs can evaluate certain issues of importance but I don't 
 
  13   think, in really exposing people to initial trials where we 
 
  14   really don't know too much about outcomes yet, to me, if you 
 
  15   really want to avoid immunosuppression in a model that has 
 
  16   immunosuppression, you should focus on people who don't have

  17   it now, who are about to need it. 
 
  18             DR. MILLER:  But isn't it the role of clinical 
 
  19   trials and the informed‑consent process for the physician 
 
  20   taking care of the patient to discuss the risks and 
 
  21   benefits?  You have to have some minimum amount of disease

  22   in order to say, "This is a bad group."  But I don't think 
 
  23   you have to have every patient determined by a group to say 
 
  24   that, yes, this patient has failed everything else, because 
 
  25   that is part‑‑each person's risk/benefit ratio participating 

                                                                188 
 
   1   in the clinical trial is different.

   2             So I think you have to set standards saying what 
 
   3   is a minimum level of severe diabetes to make it, but I 
 
   4   think that as long as it is being done in the context of a 
 
   5   well‑designed clinical trial where the patients are informed 
 
   6   of the risks and benefits, and they have a disease which

   7   could potentially be benefitted by the intervention, that 
 
   8   that is what informed consent is all about. 
 
   9             DR. SALOMON:  Can you guys help me?  I guess that 
 
  10   question that I am thinking about is we have the possibility 
 
  11   of‑‑we are doing kidney transplants, obviously, in patients

  12   with diabetes.  Then the idea is that we have done a lot of 
 
  13   islet transplants and pancreas transplants in that group. 
 
  14             Now, and correct me if I am wrong, but you have 
 
  15   made a decision that you want to do the next group as islet 
 
  16   alone.  I am not objecting to that in any way.  Can you help

  17   us with how you‑‑ 
 
  18             DR. HERING:  I think the point to make is the 
 
  19   following.  Islet transplantation has been performed in 
 
  20   settings of simultaneous previous kidney or liver 
 
  21   transplantation.  This was probably okay to study some of

  22   the basic questions, but at the very same time, I guess 
 
  23   progress was slowed by the fact that you are doing 
 
  24   transplants only in patients that have received a kidney 
 
  25   transplant because now the kidney determines the protocol. 

                                                                189 
 
   1             This is not the way to proceed.  There are very

   2   well‑defined issues in islet transplantation such as whether 
 
   3   the treatment is diabetogenic, whether the treatment would 
 
   4   control autoimmunity, that are completely irrelevant, more 
 
   5   or less, in kidney transplantation. 
 
   6             So I think, for this specific reason, we think the

   7   field can only progress if you can address the questions 
 
   8   that matter without considering all the very important 
 
   9   issues in kidney transplantation. 
 
  10             DR. SALOMON:  I think that is well said, Bernhard.  
 
  11   So, can we begin to just analyze what is that population,

  12   then?  I have heard general statements like they should be 
 
  13   bad diabetics, I guess, is as close as I can get so far. 
 
  14             MR. SIEGEL:  Let me categorize what I have heard 
 
  15   because I am confused by this dialogue, and maybe I missed 
 
  16   something.  I thought there was general agreement among the

  17   speakers that that population is "failure of exogenous 
 
  18   insulin therapy." 
 
  19             What I heard Dr. Sherwin say, if I may 
 
  20   characterize, is that if one of the indicators of failure is 
 
  21   recurrent hypoglycemia or undetected unsymptomatic

  22   hypoglycemia, that one needs to be particularly cautious to 
 
  23   insure that that truly is failure of insulin therapy, that 
 
  24   that patient has been appropriately treated with the state 
 
  25   of the art before exposing him to these risks by appropriate 

                                                                190 
 
   1   independent experts.

   2             I don't think I heard disagreement.  I heard some 
 
   3   discussion of informed consent or IRB issues, but not 
 
   4   disagreement, that one should insure that that is the case 
 
   5   for those patients. 
 
   6             DR. LEVITSKY:  I actually heard the other day

   7   another category of patients which I commented on then and 
 
   8   would like to get back to again which was the patients who 
 
   9   were failures because they had ketoacidosis. 
 
  10             I am very concerned about including that group.  
 
  11   As a matter of fact, both groups are of concern to me

  12   because there is not only a biologic base for these 
 
  13   disorders but also a psychological base.  If you have a 
 
  14   group of patients who have not been very carefully screened 
 
  15   and not by a diabetologist who is fully committed to your 
 
  16   project and your patient and on the payroll, I am a little

  17   worried that you will actually be collecting a group of 
 
  18   patients who are going to be much less adherent to your 
 
  19   immunosuppressive regimen than if you went, for instance, to 
 
  20   the group which Bob Sherwin was discussing which is a group 
 
  21   which is about to go into renal failure but isn't yet and

  22   so, therefore, we can predict that decline in renal function 
 
  23   and when it is going to happen pretty well, even with ace 
 
  24   inhibitors and whatever. 
 
  25             So that population would tend to be a population 

                                                                191 
 
   1   which would offer you, I think, more stability from the

   2   psychological point of view, perhaps, than the group that I 
 
   3   heard selected out before. 
 
   4             DR. SHAPIRO:  As I have said yesterday, 
 
   5   transplanted patients who specifically have only metabolic 
 
   6   instability or only ketoacidosis‑‑these are patients who

   7   have real failure despite tremendous efforts on their part 
 
   8   independent evaluate to confirm that, independent 
 
   9   psychological evaluation in selected cases where 
 
  10   appropriate, to confirm that they really are failing on the 
 
  11   best management that we are aware of today.

  12             DR. LEVITSKY:  Failing with ketoacidosis as well 
 
  13   as hypoglycemia? 
 
  14             DR. SHAPIRO:  In certain cases, occasionally; yes.  
 
  15   We are talking about a very highly‑‑ 
 
  16             DR. LEVITSKY:  I would propose to you that any

  17   case that fails with ketoacidosis as well as hypoglycemia 
 
  18   doesn't have a biologic problem.  I cannot have that 
 
  19   biologic problem defined.  Maybe Dr. Sherwin can define 
 
  20   that, but I cannot. 
 
  21             DR. BLUESTONE:  I am a little confused.  There are

  22   two things here; right?  There is the islet transplant and 
 
  23   the immunosuppression.  It sounds to me like I have not 
 
  24   heard anybody think that that safety issues surrounding the 
 
  25   islets, themselves, is an issue to be concerned about, that 

                                                                192 
 
   1   the issues have been focused on immunosuppression.

   2             That is what I heard.  So, if that is true, you 
 
   3   would ask, what about if the patient doesn't get an islet 
 
   4   transplant.  The reality is that there have been and are 
 
   5   ongoing clinical trials in new‑onset diabetics who have none 
 
   6   of these very severe things with immunosuppressive drugs

   7   including cyclosporine which has been tested and things like 
 
   8   that in a patient population which I would imagine, on the 
 
   9   face of it, you would say is not a patient population you 
 
  10   would be submitting to these immunosuppressive therapies. 
 
  11             It has nothing to do with islet transplantation. 

  12   It has to do with whether or not we think it reasonable in 
 
  13   diabetics, given some of the morbidities and the outcomes, 
 
  14   of trying to test novel immunosuppressive therapies in those 
 
  15   patients, with or without islets. 
 
  16             So if the issue is about immunosuppression and not

  17   about islet transplantation, then I think it takes it into a 
 
  18   whole different realm of how do we treat our diabetics with 
 
  19   regards to immunosuppression. 
 
  20             DR. SALOMON:  The fact is that I was at the NIH at 
 
  21   a meeting with Joan.  It was in about 1998, where the

  22   discussion was with the experts, pediatric diabetologists, 
 
  23   transplant people.  I was there representing 
 
  24   immunosuppression. 
 
  25             The message we got pretty darned clearly was that 

                                                                193 
 
   1   we were not going to be allowed to do any sort of major

   2   trials of immunosuppression in these diabetics. 
 
   3             DR. BLUESTONE:  They didn't listen to you. 
 
   4             DR. SALOMON:  No, no; they did listen to us.  Most 
 
   5   of those trials were over. 
 
   6             DR. BLUESTONE:  Since then, there have been

   7   steroid trials.  There have been cyclosporine trials.  I was 
 
   8   at a meeting‑‑maybe it was the same meeting. 
 
   9             DR. SHERWIN:  They were short‑term trials. 
 
  10             DR. BLUESTONE:  Well, it was up to a year.  These 
 
  11   may be short‑term because if the islets don't take‑‑let's

  12   think about an outcome here. 
 
  13             DR. SHERWIN:  But this is a long‑term trial.  I 
 
  14   hope it is. 
 
  15             DR. BLUESTONE:  Right.  So the question is that if 
 
  16   you get normal glycemia after the islet transplant, and a

  17   year out, you are asking the question‑‑a year out, with a 
 
  18   normal glycemic patient with a functioning transplant, would 
 
  19   you be more worried about keeping them on immunosuppression 
 
  20   at that point or taking them off because of the long‑term 
 
  21   immunosuppression?

  22             You would keep them on immunosuppression because 
 
  23   what you have gained from making them normal glycemic 
 
  24   outweighs the risk of the long‑term immunosuppression or 
 
  25   that point, or would you take them off because you would 

                                                                194 
 
   1   rather have them lose the graft than have long‑term

   2   immunosuppression? 
 
   3             DR. SHERWIN:  I don't know. 
 
   4             DR. SALOMON:  I would just point out that I was 
 
   5   there in 1998 to convince them to do the immunosuppression, 
 
   6   Jeff, so it is not always clear what agendas we had at

   7   different times.  I am not against immunosuppression now.  I 
 
   8   think, though, we are getting away from what I wanted to do 
 
   9   to finish this first topic which was at least finish 
 
  10   discussing what would be the candidate for this first trial. 
 
  11             I think the comments of Bernhard and James have

  12   been on the point there.  One population that I would like 
 
  13   to ask you guys about would be relatively young patients 
 
  14   which, of course, means within five years of my current age, 
 
  15   who have microalbuminuria.  You get the idea; some sort of 
 
  16   microvascular disease that was easily objectifiable and yet

  17   were certainly far from the serious downstream complications 
 
  18   of diabetes. 
 
  19             What about that as a population that could be used 
 
  20   for these early studies? 
 
  21             DR. SHAPIRO:  Clearly, that is an important

  22   population group for studying the long‑term efficacy of 
 
  23   islet transplantation is the control of secondary 
 
  24   complications.  It is not the population that we are 
 
  25   targeting in our multicenter trial right now partly because 

                                                                195 
 
   1   we are a little concerned about the use of the low‑dose

   2   tacrolimus and its potential tacrolimus effect. 
 
   3             The last thing we want to do in that patient 
 
   4   population is accelerate the nephrotoxicity. 
 
   5             DR. RICORDI:  Actually, I think to evaluate the 
 
   6   patient population is very important to establish the

   7   baseline of what is the standard of treatment.  The most 
 
   8   worrisome thing that I heard today from the diabetologists 
 
   9   is that we are happy with what insulin can do today and that 
 
  10   hypoglycemia unawareness is not a real clinical problem. 
 
  11             DR. SHERWIN:  No; that is not what I said.

  12             DR. RICORDI:  No, no; I heard very well that you 
 
  13   said that it can be treated.  Incidentally, I want to put on 
 
  14   the record the reason next to my name there is Stacy Joy 
 
  15   Goodman Professor is that Stacy Joy, a sixteen‑year old, 
 
  16   died in a hypoglycemia crisis and this is for sure an

  17   element that can threaten the life of patients. 
 
  18             Maybe it can be managed at highly specialized 
 
  19   institutions or maybe the standard of care will improve the 
 
  20   life of patients with diabetes, but so far the gold standard 
 
  21   of insulin treatment through intensive‑care treatment and

  22   normalization of hemoglobin A1c through intensive insulin 
 
  23   treatment can be achieved in less than 5 percent of patients 
 
  24   with diabetes. 
 
  25             That is why I believe there is uniform agreement 

                                                                196 
 
   1   that insulin has been ineffective to prevent the

   2   complications of the disease that can develop and the reason 
 
   3   why we spend $120 billion a year on diabetes and it is one 
 
   4   of the leading causes of death, amputation, blindness and 
 
   5   kidney failure. 
 
   6             Otherwise, we would not be here.  I don't have the

   7   representatives from JDF or the parents of these children 
 
   8   who died or who have this complication, but I personally 
 
   9   consider it outrageous that since we can treat and control 
 
  10   everyone with insulin, why assume any risk. 
 
  11             These are calculated risks that have to be put

  12   forward to move a field of critical importance forward. 
 
  13             DR. SHERWIN:  I am not that naive.  Believe me.  I 
 
  14   think that the issue is to take a step‑wise approach in a 
 
  15   new form of therapy.  I want this to work and I really want 
 
  16   this to work.  It is not that I don't want it to work and it

  17   is not that I think that insulin therapy that we currently 
 
  18   offer is optimal. 
 
  19             But I do think that, in a situation where we don't 
 
  20   know outcomes yet, even though we think we may know from 
 
  21   short‑term experiments, we don't know long‑term results.  we

  22   don't have all the information and it is best to start in 
 
  23   small steps. 
 
  24             DR. RICORDI:  We started with six patients and we 
 
  25   are now going to 28 in a very controlled‑‑ 

                                                                197 
 
   1             DR. SHERWIN:  Well, six patients for how long? 

   2   See, I don't have the data.  But how long? 
 
   3             DR. RICORDI:  We are not talking about a 
 
   4   vaccination of all children. 
 
   5             DR. SHERWIN:  No.  What I am saying is somebody, 
 
   6   two years from now, may get cryptococcus or somebody else‑‑

   7             DR. SHAPIRO:  The precise risks of what we are 
 
   8   proposing to do I think will come out with a very carefully 
 
   9   conducted prospective clinical trial. 
 
  10             DR. SHERWIN:  All I am saying is, when you start a 
 
  11   trial, generally speaking, you would like to have very

  12   well‑defined criteria.  When you start to get into the 
 
  13   subjectivity of how much hypoglycemia is a problem or how 
 
  14   much ketoacidosis is a problem, that becomes a very 
 
  15   subjective assessment and is based upon lots of factors. 
 
  16             A certain level of creatinine or a certain level

  17   of proteinuria is a nice hard endpoint that allows you to 
 
  18   begin to approach a problem in a patient population that it 
 
  19   is a very high risk. 
 
  20             It seems to me that that is the population in 
 
  21   these early stages‑‑it is not that I don't want everybody to

  22   get islet transplants ultimately.  I think it is the way to 
 
  23   go over the long haul, but I do think, in the early stages, 
 
  24   it is a potential risk because our therapies are primitive, 
 
  25   still. 

                                                                198 
 
   1             We have so much to learn.  We don't know

   2   everything at this point.  I just think the highest‑risk 
 
   3   people that is not going to screw you up in terms of 
 
   4   assessing islets only would be the optimal patient to study.  
 
   5   That is not a subjective assessment. 
 
   6             DR. AUCHINCLOSS:  I think I disagree, essentially,

   7   with everybody here except for Carole because I think the 
 
   8   only criteria that is important here is informed consent.  
 
   9   In my view, if I had had diabetes for fifteen years and I 
 
  10   had zero complications of diabetes and I was under perfect 
 
  11   control, from the data that I have seen from Edmonton, I

  12   would take the choice to enter that trial because I would 
 
  13   see it as a no‑lose situation. 
 
  14             I either get an islet transplant working and I am 
 
  15   on, I think, a very non‑toxic program of immunosuppression 
 
  16   or it fails and I stop the immunosuppression.  I think it is

  17   an entirely justified trial for every patient with diabetes, 
 
  18   maybe with a minimum time period that they have had it as 
 
  19   long as you have informed consent. 
 
  20             DR. HARLAN:  My name is David Harlan.  I am from 
 
  21   the NIH.  I was going to make the point that Hugh made but

  22   also to add the point that if you wait until a person has 
 
  23   incipient renal failure or significant proteinuria, then you 
 
  24   run into a problem where we know that they are likely to 
 
  25   require a kidney transplant and that pancreas‑kidney 

                                                                199 
 
   1   transplant is known to be very curative, 90 percent

   2   curative. 
 
   3             Then you are depriving them of a known benefit in 
 
   4   that population where an islet transplant alone is not known 
 
   5   to be as effective.  It is 90 percent cure rate if you have 
 
   6   got a kidney‑transplant recipient.  We don't know that with

   7   islet. 
 
   8             DR. SHERWIN:  I thought the question was how do we 
 
   9   begin trials in people who are not getting kidney grafts?  
 
  10   That was the question.  What I am saying is that you are 
 
  11   absolutely right, that it is not going to be terribly

  12   effective in terms of preventing them from going into kidney 
 
  13   failure down the road.  I will accept that.  Is that what 
 
  14   you are saying? 
 
  15             DR. HARLAN:  My main point is the one that Hugh 
 
  16   Auchincloss made.

  17             DR. SHERWIN:  So you would take anybody who came 
 
  18   to you off the street‑‑ 
 
  19             DR. HARLAN:  With brittle diabetes‑‑ 
 
  20             DR. SHERWIN:  And has informed consent. 
 
  21             DR. HARLAN:  If it is truly informed consent.  We

  22   can argue about what is informed consent because that is a 
 
  23   difficult thing to truly achieve. 
 
  24             DR. SHERWIN:  Do you think we are that far along 
 
  25   at this point? 

                                                                200 
 
   1             DR. AUCHINCLOSS:  I would actually take a

   2   different point of view from Dr. Sherwin in the following 
 
   3   sense that you could make the argument, and I think it is a 
 
   4   reasonable argument, that the people with proteinuria or 
 
   5   elevated creatinines are exactly the wrong population to go 
 
   6   for, and the reason for that would be that I think that the

   7   major risk associated with islet transplantation alone is 
 
   8   sensitization for the subsequent transplant that they may 
 
   9   need. 
 
  10             So I would not target the population that you know 
 
  11   is heading there.  I would take the opposite population.

  12             DR. SALOMON:  I would point out that what I was 
 
  13   talking about was a patient with microalbuminuria and there 
 
  14   is some reasonable hope that, in that early population who 
 
  15   has got no elevation in creatinine or any change in 
 
  16   creatinine clearance, if anything, they might have actually

  17   an increase in creatinine clearance at that point, that a 
 
  18   successful islet transplant may prevent the kidney disease. 
 
  19             So I think if you choose that patient correctly, 
 
  20   the only argument I would have is that I think you could 
 
  21   reverse some of the disease.  Certainly, there is evidence

  22   of some reversal in kidney disease in pancreas‑alone 
 
  23   transplantations. 
 
  24             I think that would be a nice hope to the extent 
 
  25   that we all believe that there is some reversible component 

                                                                201 
 
   1   to the microvascular disease.

   2             DR. HERING:  I would like to argue in favor of 
 
   3   hypoglycemia unawareness.  I understand this is a 
 
   4   significant clinical problem in a small group of patients.  
 
   5   You are not talking about thousands of people here‑‑in a 
 
   6   small group of patients.

   7             The reason why I argue in favor of this subgroup 
 
   8   is they will benefit right away.  The problem of 
 
   9   hypoglycemia unawareness is eliminated right after 
 
  10   transplantation to the point that you have a clinical effect 
 
  11   obvious to everybody who wants to see it.

  12             If you argue here, now, kidney and maybe 
 
  13   neuropathy or whatever, this may take five or ten years 
 
  14   before you see a difference in a prospective clinical trial.  
 
  15   But, in hypoglycemia unawareness, the patient may benefit 
 
  16   immediately from the procedure whether you have complete

  17   insulin independence or whether you have some level of 
 
  18   islet‑graft function. 
 
  19             All the available clinical information clearly 
 
  20   supports this point. 
 
  21             DR. SHERWIN:  A lot of patients have diminished

  22   awareness from hypoglycemia and the reason is that the 
 
  23   threshold for releasing counter‑regulatory hormones is set 
 
  24   downward, but also their function is maintained better 
 
  25   during hypoglycemia because the brain metabolism changes as 

                                                                202 
 
   1   a result of that.

   2             But I do agree that that, in association with a 
 
   3   certain defined number of severe hypoglycemic events would 
 
   4   be a reasonable approach.  It is just that I think that 
 
   5   someone's hypoglycemia unawareness is someone else's not 
 
   6   hypoglycemia unawareness.  I think that it needs to have

   7   some sort of assessment of it from‑‑ 
 
   8             MR. SIEGEL:  I have a question about an underlying 
 
   9   assumption here which I assume is true because it is 
 
  10   unspoken but we are all assuming it.  Are we comfortable 
 
  11   enough that there will be normal homeostatic mechanisms in

  12   transplanted islets that, assuming once we have a therapy 
 
  13   where there is good tolerance or immunosuppression and good 
 
  14   survival of large numbers of islets that there is no concern  
 
  15   at all that those islets, themselves, would cause 
 
  16   hypoglycemia?

  17             DR. SALOMON:  Jay, can we hold that just for a 
 
  18   minute until we finish this one on candidates? 
 
  19             MR. SIEGEL:  It is quite relevant to whether the 
 
  20   hypoglycemic‑unaware patient is the best population because 
 
  21   if there is any risk that the treatment causes hypoglycemia,

  22   that wouldn't be a good population.  But it sounds like we 
 
  23   are all assuming that we know that that is not a concern. 
 
  24             I just wanted to make sure‑‑ 
 
  25             DR. AUCHINCLOSS:  The answer to that is yes, 

                                                                203 
 
   1   unequivocally yes.

   2             DR. SALOMON:  I think the animal data suggests 
 
   3   that if it works, it works really well and in the humans, 
 
   4   data that works‑‑ 
 
   5             DR. SHAPIRO:  The clinical data also clearly 
 
   6   indicates that that is the case.

   7             MR. SIEGEL:  I assume, from the discussion.  I 
 
   8   just wanted to get that out on the table. 
 
   9             DR. HARLAN:  I just wonder what is wrong with the 
 
  10   criteria that Hugh Auchincloss proposed.  There is a risk 
 
  11   associated with this procedure, no doubt, but diabetes is

  12   not a benign disease, now matter how well you care for it.  
 
  13   It is not a benign disease. 
 
  14             If the patients are aware of the risks of whatever 
 
  15   the protocol is that that study is going to do, it is 
 
  16   paternalistic for us to say you can't do it, if they

  17   understand those risks. 
 
  18             DR. EGGERMAN:  A fundamental question is do we 
 
  19   know what the risks are that we can tell the patient so they 
 
  20   can be truly informed? 
 
  21             DR. HARLAN:  They can be quantitated as much as is

  22   humanly possible.  There are some unknown risks.  Whenever 
 
  23   you enter into a protocol, you state that to a patient. 
 
  24             DR. EGGERMAN:  That needs to be clarified to the 
 
  25   patients, too, and it is not an assumption that we know all 

                                                                204 
 
   1   the potential risks in a field where there is investigative

   2   study. 
 
   3             DR. BLUESTONE:  On the immunosuppressive side, we 
 
   4   have tremendous amount of information and that seems to be 
 
   5   what we are focussing on here is the immunosuppression not 
 
   6   the transplant.

   7             DR. RICORDI:  So you could establish an 
 
   8   independent panel to discuss the informed consent with these 
 
   9   patients instead of to decide whether it was a treatment 
 
  10   failure of insulin. 
 
  11             MR. SIEGEL:  This is an experimental therapy.  I

  12   think this issue of it being paternalistic not to allow the 
 
  13   patient to accept the risk is not an inappropriate standard.  
 
  14   I think it is well establish, for example, if you can ask a 
 
  15   scientific question in population A or population B, and it 
 
  16   is safer to ask it in A than in B, you shouldn't ask patient

  17   B to answer that question for you, you should only ask 
 
  18   patient A. 
 
  19             If you have a healthy person who is willing to 
 
  20   volunteer to accept something very dangerous to advance 
 
  21   science, depending on the nature of that, it may well not be

  22   appropriate to ask him to do that.  So it is not so black 
 
  23   and white that informed consent solves all of the problems, 
 
  24   in part because of the proviso that you mentioned at first 
 
  25   which is there is no such thing as true and perfect informed 

                                                                205 
 
   1   consent.

   2             DR. HARLAN:  It is very difficult to achieve but 
 
   3   true Type‑1 diabetes with no demonstrable C‑peptide, 
 
   4   stimulatable C‑peptide, is not a benign disease. 
 
   5             DR. KENYON:  Can I just comment on all this?  I am 
 
   6   agreeing with Hugh and David that partly informed consent‑‑I

   7   think where I would draw the line right now is age.  I 
 
   8   certainly am not ready to go into a child with the protocols 
 
   9   that we have available until they are more proven in adults. 
 
  10             But I think that quality of life is really 
 
  11   important in the risk‑benefit ratio and David is right; it

  12   is not a benign disease.  It is a full‑time job.  The 
 
  13   hypoglycemia unawareness, when you have it, you have to 
 
  14   reset your target levels and then your hemoglobin A1c goes 
 
  15   up and your chances for complications go up. 
 
  16             We don't know.  Everybody differs so the true risk

  17   for the complications of diabetes are maybe just as unknown 
 
  18   as what the true long‑term risk of some of these drugs are.  
 
  19   So the only place that I would really draw the line right 
 
  20   now is not to go into children until we have a little more 
 
  21   data.

  22             But it is not a disease that is great to live with 
 
  23   and the quality of life should clearly be considered.  I 
 
  24   think each patient, as long as they have been carefully 
 
  25   evaluated, is best suited, with good informed consent, to 

                                                                206 
 
   1   make that decision.

   2             DR. SALOMON:  Can I ask a question?  If you take a 
 
   3   patient who has some objective viable end‑stage organ 
 
   4   injury, whether hypoglycemic unawarenss could be included in 
 
   5   that or recurrent keto‑‑any of things, microalbuminuria.  I 
 
   6   can follow that.

   7             If you go back and you say anyone who has had true 
 
   8   valid informed consent, who is an insulin‑dependent 
 
   9   diabetic, should go into the trial then the question I have 
 
  10   is if you have 100 insulin‑dependent diabetics today, and 
 
  11   you come back in thirty years, how much do we know

  12   about‑‑are 100 of the 100 that we started with going to be 
 
  13   blind and on dialysis at thirty years? 
 
  14             DR. SHERWIN:  No.  Clearly the kinds of results 
 
  15   that can be predicted now from intensified better therapies 
 
  16   that we offer today, up until twenty years ago, we didn't

  17   even have any way of assessing how things were going.  So it 
 
  18   was hopeless to manage diabetes.  We didn't try to manage it 
 
  19   because we couldn't. 
 
  20             But it is likely‑‑the data for renal failure in 
 
  21   Scandinavia now long‑term are about 9 percent.  It is

  22   predicted if people can achieve levels similar to the DCCT 
 
  23   that that would be‑‑over the course of one's life, renal 
 
  24   failure would be about a 9 percent risk as opposed to 30 to 
 
  25   35 percent previously. 

                                                                207 
 
   1             The chances of developing blindness is probably a

   2   similar percentage, about 8 percent. 
 
   3             DR. SALOMON:  I would just point out that if you 
 
   4   think about allotransplantation right now, the half‑life of 
 
   5   an allotransplant, and I am being vague‑‑kidney, heart, 
 
   6   liver‑‑maybe liver, I would put on the side for a

   7   minute‑‑but certainly kidney and heart‑‑would be fifteen 
 
   8   years?  Half‑life?  That is being pretty generous, actually.  
 
   9   There are people that could argue that it is less than that. 
 
  10             But, certainly, fifteen years, maybe twenty, if 
 
  11   you say these newer therapies are going to impact

  12   significantly.  So I don't think it is so unreasonable.  I 
 
  13   think we should be careful that if we take the attitude 
 
  14   that, of that 100, all of them should be offered this islet 
 
  15   transplant if they get true informed consent, based on the 
 
  16   fact that 50 of 100 will have lost their islet allograft by

  17   fifteen years, maybe twenty, and then what percentage is 
 
  18   left that would have been guaranteed to have complications 
 
  19   at thirty? 
 
  20             DR. BLUESTONE:  But then there are a couple of 
 
  21   points that you want to also make is that the JAMA article

  22   that says what happens if you don't get that kidney 
 
  23   transplant.  And, being on dialysis, you have a 
 
  24   eight‑year‑‑so there are morbidities associated with not. 
 
  25             DR. SALOMON:  If you said the hundred patients all 

                                                                208 
 
   1   had kidney failure at the time, then I am quiet.  I was

   2   talking about a hundred patients with no problems. 
 
   3             DR. BLUESTONE:  I understand.  But now you are 
 
   4   getting to a question‑‑so I am going to re‑ask my question 
 
   5   earlier because now I am hearing you and Bob say something 
 
   6   which I guess I would not have imagined.  But it is okay. 

   7   Bob has already said that short‑term immunosuppression, I 
 
   8   can live with that.  It is this long‑term thing that is a 
 
   9   real problem. 
 
  10             The islet transplant is okay.  There is not a big 
 
  11   safety issue there.  So I posed the question of if you

  12   transplanted a hundred people and, at one year, they were 
 
  13   all normoglycemic but all destined, now, to stay on 
 
  14   rapamycin in low dose, you guys would actually think that 
 
  15   that was a bad outcome because the immunosuppression 
 
  16   outweighs the benefit.

  17             If I am understanding what I am hearing correctly, 
 
  18   you think that, in the absence of both of those, the islet 
 
  19   transplant and the immunosuppression, that person can live a 
 
  20   better life because of better management of insulin and 
 
  21   stuff like that, because that is what it comes down to.  It

  22   comes down to a question of whether islet transplantation, 
 
  23   if successful under current regimens, is a good therapy. 
 
  24             DR. SHERWIN:  You think you are going to get 
 
  25   100 percent? 

                                                                209 
 
   1             DR. BLUESTONE:  Then we can have discussions about

   2   survival. 
 
   3             DR. SHERWIN:  Do you know anything about the 
 
   4   long‑term‑‑we haven't discussed the long‑term‑‑ 
 
   5             DR. SALOMON:  Let's not discuss the long‑term yet. 
 
   6             DR. RICORDI:  Excuse me.  You will have

   7   100 percent because if one fails, stop taking the 
 
   8   immunosuppression.  So those who will continue 
 
   9   immunosuppression are only the ones that are successful.  So 
 
  10   the risk will be calculated on 100 percent of the surviving 
 
  11   grafts.

  12             DR. BLUESTONE:  Right; from long‑term 
 
  13   immunosuppression.  You are only worrying about the guys 
 
  14   that are out a year, normoglycemic and on their drugs.  
 
  15   Would you want to take them off‑‑I asked you before and you 
 
  16   said, "I don't know if I wouldn't take them off."  That is

  17   what I think is the gating issue here because, to me, it 
 
  18   never occurred to me that a successful transplant at one 
 
  19   year in normal glycemia and normal well‑controlled normal 
 
  20   glycemia would be a possible bad outcome if it meant 
 
  21   immunosuppressive drugs for the long term.

  22             DR. SALOMON:  I want to make one thing clear.  My 
 
  23   last comments were simply to Dr. Harlan and Dr. Auchincloss' 
 
  24   concept that anybody with insulin‑dependent diabetic, if 
 
  25   they had informed consent, was a candidate for this. 

                                                                210 
 
   1             DR. RICORDI:  Maybe I can expand that criteria

   2   staying instead of anyone with diabetes, it should be anyone 
 
   3   where insulin treatment has been a failure, failure meaning 
 
   4   inability to achieve normal hemoglobin A1c levels in the 
 
   5   absence of a hypoglycemic episode.  That is treatment 
 
   6   failure in diabetes because it doesn't prevent the

   7   development of complications. 
 
   8             DR. SHERWIN:  I am not arguing that we would like 
 
   9   everybody to‑‑I would favor every type‑1 diabetic patient 
 
  10   getting an allograft.  I have no problem with that.  I hope 
 
  11   that happens.  I am just saying, in the very early stages of

  12   any trial where you don't really know what you are doing, it 
 
  13   seems to me to be very careful in that selection process in 
 
  14   the first line‑‑so you don't get caught up in some bad 
 
  15   incident like gene therapy. 
 
  16             DR. BLUESTONE:  The question was what do we mean

  17   by "we don't know what we are doing?"  The islet transplant 
 
  18   will either work or not work.  If it doesn't work, then they 
 
  19   go off their immunosuppression.  If it works, then we knew 
 
  20   what we were doing for that and then you are worried that we 
 
  21   don't know what we are doing vis‑a‑vis the

  22   immunosuppression. 
 
  23             I would argue that we already know what we are 
 
  24   doing.  It is not all good.  We know that.  It is not going 
 
  25   to worse for the islet transplants and all the other people 

                                                                211 
 
   1   we give the immunosuppression.  So what are you worried‑‑if

   2   it failed, then they are off the immunosuppression.  We 
 
   3   already agreed short‑term immunosuppression isn't the gating 
 
   4   issue here.  So what are we worried about? 
 
   5             DR. SHERWIN:  Somebody could die during the 
 
   6   procedure.  It could happen; right?

   7             DR. SALOMON:  As long as you start off with a 
 
   8   patient population that has a clear complication of 
 
   9   diabetes, not just diabetes on insulin, per se, then, after 
 
  10   that, I am okay with the idea. 
 
  11             MR. SIEGEL:  Maybe it is time to move on to other

  12   questions in the interest of time.  There is clearly not 
 
  13   consensus here.  I guess I did want to explore one 
 
  14   underlying assumption.  Is it also‑‑or it is not an 
 
  15   assumption.  I think Dr. Bluestone just stated it 
 
  16   explicitly.  Is it the general agreement here that the

  17   concerns of exposing somebody who is doing well to, say, 
 
  18   short‑term‑‑so he is only on immunosuppression for four or 
 
  19   five months and then he is off of it because the treatment 
 
  20   failed, that that is not a significant or worrisome risk? 
 
  21             DR. SALOMON:  We have to take the one sobering

  22   data that if you take patients who are on dialysis, get a 
 
  23   kidney transplant and the kidney transplant fails and they 
 
  24   go back to dialysis, they do very much more poorly than a 
 
  25   population that stayed on dialysis. 

                                                                212 
 
   1             So the idea here that even short‑term

   2   immunosuppression and a transplant procedure, per se, would 
 
   3   have no negative effect on the patient isn't necessarily 
 
   4   true. 
 
   5             MR. SIEGEL:  So you might affect the underlying 
 
   6   cause of the disease.

   7             DR. SALOMON:  The first thing, let's make sure‑‑I 
 
   8   am making an analogy to patients on dialysis with a kidney 
 
   9   transplant and immunosuppression and that may not be fair. 
 
  10             DR. BLUESTONE:  Again, that is apples and oranges.  
 
  11   Number one, how much of that poor outcome is because they

  12   are on immunosuppression versus the consequence of going 
 
  13   back on dialysis suddenly, in the metabolic aspect.  I think 
 
  14   there is no data to suggest that their worse outcome had 
 
  15   anything to do with the fact that they were on 
 
  16   immunosuppression for four months.

  17             The second thing is that there is no correlation, 
 
  18   in my mind, with the surgery that you went under to get the 
 
  19   transplant that failed four months later and the injury of 
 
  20   islets and the morbidity associated with major surgery and 
 
  21   the possible outcome of that.

  22             So, as you always say, I want it on the public 
 
  23   record that I would hate that the outcome of whether 
 
  24   immunosuppression on the spectrum is bad is based on 
 
  25   dialysis in kidney patients who reject it.  I think we can 

                                                                213 
 
   1   have a more objective view of this of a large cohort of

   2   patients, especially in the autoimmunity setting, who have 
 
   3   been given short‑term immunosuppression in phase I and some 
 
   4   phase II trials. 
 
   5             I don't know of any data that says that the 
 
   6   outcome of their disease has been negatively impacted by the

   7   fact that they have had immunosuppression for that short 
 
   8   period of time. 
 
   9             DR. SHERWIN:  Do we know anything about outcomes?  
 
  10   There have been a couple of hundred islet transplants.  Most 
 
  11   have failed up until now.  What are the results?  How are

  12   they done? 
 
  13             DR. RICORDI:  I think that the consideration of 
 
  14   risk has to be weighted on the proposed immunosuppressive 
 
  15   regimen and on the alternatives.  So here we are not 
 
  16   proposing, as has been for several years, to use massive

  17   T‑cell‑depletion agents.  We are not proposing to do total 
 
  18   infrared radiation or any major‑‑the induction is like with 
 
  19   xenapax with an anti‑IL2 receptor, there is low dose of 
 
  20   K506‑‑what I am afraid is happening is that we are 
 
  21   overregulating what we were thinking was the safe

  22   alternative to what is going to happen anyway that is the 
 
  23   whole‑organ pancreas graft with this same immunosuppressive 
 
  24   regimen because these patients will not just take the 
 
  25   opinion of whoever as an indication and say, "No; you have 

                                                                214 
 
   1   to stick with your 14,000 sticks every year to check glucose

   2   and get insulin," that they will go and try to get a 
 
   3   pancreas transplant which is a non‑regulated procedure, 
 
   4   easily accessible and performed increasingly at all major 
 
   5   institutions. 
 
   6             So I see the potential that we are overkilling

   7   what we consider the safe alternative to whole‑organ 
 
   8   transplantation when we will be using the exact same 
 
   9   immunosuppressive regimen.  But I agree that it should be 
 
  10   weighted, like what are we proposing?  Are we proposing 
 
  11   lethal radiation reconstitution with islets and life‑long

  12   important with methotrexate and cyclophosphamide? 
 
  13             No; we are proposing no steroids.  That was one of 
 
  14   the major concerns.  Low‑dose tacrolimus, rapamycin and an 
 
  15   induction with an anti‑IL2 receptor that has been so far the 
 
  16   safest induction treatment ever proposed in transplantation.

  17             DR. AUCHINCLOSS:  Before you leave the subject, 
 
  18   you just need to point out to the FDA that there might be 
 
  19   additional patient populations that should be considered; 
 
  20   namely, pancreatectomy patients and type‑2 diabetics with 
 
  21   minimal or no C‑peptides who have reasonable insulin

  22   requirements. 
 
  23             There are potentially patients who are not type‑1 
 
  24   diabetics who could be candidates. 
 
  25             DR. SALOMON:  Does anyone have any other comment 

                                                                215 
 
   1   directly on the question of what patient population because

   2   I would like to summarize and move on.  So it is just 
 
   3   patient‑population selection. 
 
   4             DR. EGGERMAN:  I just wanted to know, does anyone 
 
   5   have any idea of what risk islet therapy has on subsequent 
 
   6   organ transplantation or subsequent islet transplantation? 

   7   Is that just a theoretical risk or is there something real?  
 
   8   I know about the HLA sensitization which you mentioned but, 
 
   9   in terms of actual outcome of organ transplantation‑‑ 
 
  10             DR. AUCHINCLOSS:  If you sensitize some people to 
 
  11   HLA, then you are going to prevent some people from getting

  12   kidney transplants in the future.  It is not a theoretical 
 
  13   risk.  It is real. 
 
  14             DR. SALOMON:  Or at least they would go on these 
 
  15   lists looking for a perfect match or something like that, 
 
  16   which can happen, but is definitely detrimental in these

  17   days of organ shortage. 
 
  18             DR. EGGERMAN:  You have had experience with 
 
  19   several hundred patients who have been treated with islet 
 
  20   therapies.  Have any of them gone on to pancreatic 
 
  21   transplantation?

  22             DR. RICORDI:  Yes.  We actually have experience 
 
  23   both on the fact that in simultaneous islet and kidney 
 
  24   transplant where you have failure of the islet graft within 
 
  25   one year because of failure of the immunosuppressive 

                                                                216 
 
   1   strategies with that immune component, the kidney transplant

   2   maintains 100 percent graft survival and eventually, in the 
 
   3   four‑year follow up, are still without a sign of chronic 
 
   4   rejection. 
 
   5             We also have cases of patients who had an islet 
 
   6   transplant that failed who undergo a subsequent organ

   7   transplant with no apparent impact on the transplanted 
 
   8   organ.  But is this the final word?  I think these are very 
 
   9   anecdotal reports on very few patients. 
 
  10             But the early indication that we have from the 
 
  11   transplant surgeons‑‑Dr. Alejandro is here‑‑in pancreas

  12   transplantation or kidney transplantation, there has been no 
 
  13   reported negative effect. 
 
  14             DR. AUCHINCLOSS:  I think that you would have to 
 
  15   start with the assumption, however, that the first kidney 
 
  16   followed by a second kidney experience probably applies to

  17   islet transplantation as a rough approximation.  There, the 
 
  18   data would say that the outcome of the second transplant 
 
  19   statistically is not quite as good as the first and that the 
 
  20   waiting time tends to be longer because sensitization has 
 
  21   sometimes occurred.

  22             DR. BLUESTONE:  The other issue is that there are 
 
  23   a subset of patients who historically, when they get 
 
  24   monoclonal antibodies, have a reaction to that monoclonal 
 
  25   antibody that prevents retreatment with the monoclonal 

                                                                217 
 
   1   antibodies.  So, low as it may be, there is a possibility

   2   that that would happen. 
 
   3             DR. SALOMON:  I would also want to point out that 
 
   4   I haven't been convinced.  I think that if you transplant 
 
   5   somebody, immunosuppress them, they go through all the 
 
   6   metabolic gyrations of curing their diabetes for a short

   7   period of time, then losing their diabetic control possibly 
 
   8   getting some anti‑rejection therapy and then going back to 
 
   9   being diabetics is not a necessarily neutral event. 
 
  10             I don't think the experience with the problems we 
 
  11   have had with kidney‑transplant patients is absolutely

  12   irrelevant‑‑we seem to like analogies to kidney 
 
  13   transplantation in some settings.  I am just suggesting 
 
  14   there is something to be learned there as well. 
 
  15             Are there any other comments on this? 
 
  16             DR. HARLAN:  Two real quick comments on

  17   risk/benefit analysis.  Even if diabetes wasn't the leading 
 
  18   cause of blindness and kidney disease and a major risk 
 
  19   factor for blood‑vessel disease, you know where I am coming 
 
  20   from.  Even if it wasn't that, it is still a disease that 
 
  21   requires daily therapy, very expensive, very disruptive to a

  22   happy lifestyle.  That is on the risk side. 
 
  23             On the benefit side, we know from the UKPDS that 
 
  24   if you help someone control their blood sugar for one year, 
 
  25   it has sustained benefit for them down the road.  So even if 

                                                                218 
 
   1   this is beneficial for only a year or two, that has

   2   demonstrated long‑term health benefits if type‑2 diabetes is 
 
   3   a useful model.  And I think it is. 
 
   4             DR. SALOMON:  So to summarize this, and I think 
 
   5   that the discussion took a while, but I do think that there 
 
   6   were some very important points here.  I think, with respect

   7   to preclinical models of immunosuppression, we all agree 
 
   8   that it is a jigsaw.  It ought to be based on some 
 
   9   preclinical, some clinical, that there is no single model, 
 
  10   that probably more than one model ought to be used. 
 
  11             I don't think that is really committing anything

  12   that we haven't already discussed in detail. 
 
  13             With respect to the patient selection, per se, 
 
  14   that we spent the last half hour discussing, there I think 
 
  15   that there are some things that we probably will have to 
 
  16   agree to disagree on and the FDA can sort that out their own

  17   way. 
 
  18             Clearly, there are those who would say, "Give me a 
 
  19   diabetic on insulin and a good informed consent and leave me 
 
  20   alone."  That has been clearly heard from the committee.  
 
  21   There are others who feel that this is pretty reasonable but

  22   give us a patient who has clear objective evidence of 
 
  23   end‑organ, presumably microvascular or neurovascular, 
 
  24   disease, microalbuminuria or actually a reduction in renal 
 
  25   clearance, early changes in the eye, et cetera. 

                                                                219 
 
   1             Then there are those who say that, in addition to

   2   that, a very reasonable population to target would be those 
 
   3   with hypoglycemia unawareness or those with severe 
 
   4   complicating episodes of recurrent ketoacidosis.  However, 
 
   5   we heard from others on the committee that there it is 
 
   6   pretty gray and one has to be concerned about patient

   7   compliance, social and psychological factors in deciding 
 
   8   that that would be a population to do. 
 
   9             But I think there was general agreement that if 
 
  10   you had a process in place that was objective and verifiable 
 
  11   that, under those circumstances, it is possible that some of

  12   those patients might be candidates. 
 
  13             Am I missing anything?  That is kind of what I am 
 
  14   coming away with right now. 
 
  15             DR. MILLER:  Do you want to answer the first 
 
  16   bullet?  We still haven't answered the first bullet.

  17             DR. SALOMON:  I tried.  I really tried.  I don't 
 
  18   think these guys want to answer that question.  I think that 
 
  19   the decision here has been there are a number of people 
 
  20   sitting here at this table who are ready to go forward.  I 
 
  21   think the answer is obvious to me that they are ready to go

  22   forward. 
 
  23             DR. RICORDI:  I can try to summarize what I 
 
  24   understand is our consensus here that, in the absence of 
 
  25   data emerging from clinical trials in other settings or in 

                                                                220 
 
   1   other diseases that prove at least safety of the drugs.  If

   2   you have a completely new, like, monoclonal antibody 
 
   3   untested, that we all agree we would need some data emerging 
 
   4   from preclinical testing that shouldn't be limited to small 
 
   5   rodents but, potentially, include data from either dogs, 
 
   6   pigs or non‑human primate and that, in the non‑human

   7   primates, there is no set preference between total 
 
   8   pancreatectomy or streptozotocin‑induced diabetes because 
 
   9   they both have advantages and limitations. 
 
  10             In one setting, you have a reduced metabolic 
 
  11   absorption but a more complete beta‑cell exclusion.  You may

  12   have some residual beta‑cell function and you may require 
 
  13   additional testing to better define that your islets are 
 
  14   actually the ones responsible for the metabolic outcome. 
 
  15             But I think we all agree that some level of 
 
  16   preclinical testing is absolutely indicated for agents for

  17   which there is no testing available from the clinical 
 
  18   experience. 
 
  19             DR. SALOMON:  I think we should add in that Dr. 
 
  20   Sherwin also made another point and I left it out of my 
 
  21   summary.  That was, if possible, some data should be

  22   provided in an autoimmune model of diabetes because of this 
 
  23   outstanding issue of whether or not that is also an impact 
 
  24   on survival.  I'm sorry I left that one out. 
 
  25             DR. BLACK:  I would like to add one more point, 

                                                                221 
 
   1   just that, to add to Dr. Ricordi's comments, our criteria

   2   for trying the evaluate the risks of a procedure often ask 
 
   3   very carefully what is the adequacy of the database, whether 
 
   4   it is from in vitro studies, mechanistic studies, mouse 
 
   5   models of disease or the clinical field. 
 
   6             Sometimes, the nature of the clinical data makes

   7   it very difficult for us to assess a particular combination 
 
   8   of agents for whom the other part of the database suggests 
 
   9   that there may be concerns.  So I think it becomes complex 
 
  10   to try to pre‑caveat Dr. Ricordi's comments by saying that 
 
  11   we can only do preclinical models when we do not have

  12   clinical data. 
 
  13             So, sometimes, we will have to supplement the 
 
  14   clinical data. 
 
  15             DR. SALOMON:  Somewhere along the way, you will 
 
  16   have to explain to me the frightening concept that you could

  17   pre‑caveat something. 
 
  18             DR. PAPADOPOULOS:  I just have a question.  The 
 
  19   one thing that I am still not clear about is we have heard 
 
  20   that there is a proposed trial, multicenter trial, to do 
 
  21   twenty‑some‑odd of the these transplants.  I still have not

  22   heard what the exact eligibility criteria will be for that 
 
  23   specific trial. 
 
  24             I presume that that protocol must have been 
 
  25   written and there has to be a consensus amongst the experts 

                                                                222 
 
   1   as to who will be eligible.

   2             DR. SHAPIRO:  I actually presented that in slide 
 
   3   form in detail yesterday. 
 
   4             DR. PAPADOPOULOS:  Could you just review it? 
 
   5             DR. SHAPIRO:  The three patient categories; those 
 
   6   with hypoglycemia unawareness, those with metabolic

   7   instability and those with early progressive secondary 
 
   8   diabetic complications. 
 
   9             DR. PAPADOPOULOS:  What are you defining as these 
 
  10   early secondary complications?  Do you have creatinine 
 
  11   cutoffs?  What are they?

  12             DR. SHAPIRO:  They have got to have a creatinine 
 
  13   clearance greater than 60 mls per meter squared per minute. 
 
  14             DR. SALOMON:  Speaking as a nephrologist, a 
 
  15   creatinine clearance of 60 mls per minute in a 25‑year‑old 
 
  16   person is a terrible creatinine clearance.  I certainly

  17   would not want any of my children to have that creatinine 
 
  18   clearance. 
 
  19             MR. SIEGEL:  In diabetics, in particular, with 
 
  20   hyperfiltration; yes. 
 
  21             DR. SHERWIN:  But it is above; right?  There is no

  22   limit.  It is not below? 
 
  23             DR. BLUESTONE:  Correct.  They want it at a 
 
  24   certain goodness. 
 
  25             DR. SALOMON:  I understand.  I am just saying that 

                                                                223 
 
   1   the range means that you are going to‑‑that's fine.  I am

   2   just pointing out that it is a very broad range. 
 
   3             We are sort of where we could take a ten‑minute 
 
   4   break.  We are trying to get done by 4 o'clock.  What do you 
 
   5   guys think?  A ten‑minute break. 
 
   6             [Break.]

   7             DR. SALOMON:  One thing I would like to do is we 
 
   8   have got an hour and then this is over.  I discussed with 
 
   9   the FDA sort of what would be the key couple questions and 
 
  10   those we will try and get done now.  I would like to spend a 
 
  11   relatively short period of time, unless there is just

  12   overwhelming dispute is, on this question No. 2 which 
 
  13   actually, when it was initially presented to me, I was 
 
  14   willing to let go entirely because I agree with something 
 
  15   you had said earlier that insisting on HLA typing for 
 
  16   pancreatic islet transplantation was to really restrict its

  17   future. 
 
  18             But the FDA asked me to put it back on the table 
 
  19   and I think that the question I would like to start off with 
 
  20   was there was an interesting question from Camillo and that 
 
  21   is, given this autoimmunity and some of the data Bernhard

  22   came up with with HLA identicals; is there any concern on 
 
  23   the part of the group that‑‑not that you would have to HLA 
 
  24   match but that, actually, there might be a detriment to even 
 
  25   a haplotype match. 

                                                                224 
 
   1             DR. AUCHINCLOSS:  There is data and it is so

   2   controversial that it is not worth even thinking about.  The 
 
   3   answer is simply unknown, I think.  And that extends all the 
 
   4   way to the xenotransplant whether the xenotransplant is more 
 
   5   resistant or recurrent diabetes.  So there is no scientific 
 
   6   way to answer your question.

   7             I think the important thing in issue 2 is the 
 
   8   issue that is not included.  I would probably match for 
 
   9   blood group although there is no good evidence there either 
 
  10   that that is necessary, but it is just easy and it is 
 
  11   prudent.  I would not worry about HLA typing.

  12             The question is whether you should do a cross 
 
  13   match. 
 
  14             MR. SIEGEL:  Both of you have talked about HLA 
 
  15   typing.  I think what you are saying is you wouldn't worry 
 
  16   about HLA matching as an inclusion criteria, I would hope. 

  17   Or what part of what we are asking the committee on this is 
 
  18   that we believe that all of these studies should be 
 
  19   collecting HLA data. 
 
  20             DR. AUCHINCLOSS:  100 percent, for sure. 
 
  21             DR. SALOMON:  Nobody here would even have thought

  22   of that one. 
 
  23             DR. AUCHINCLOSS:  But the prospective question 
 
  24   that you have not addressed here is whether or not to do a 
 
  25   cross match.  If the recipient does have antibody against an 

                                                                225 
 
   1   HLA antigen of the donor, does that preclude the transplant?

   2             DR. RICORDI:  We always do a crossmatch and it is 
 
   3   excluded.  A positive crossmatch will exclude the islet 
 
   4   transplant. 
 
   5             DR. AUCHINCLOSS:  I think that would be prudent 
 
   6   way to approach it.  But maybe you or Bernhard or one of

   7   others can actually comment about how good the data is one 
 
   8   way or another. 
 
   9             DR. HERING:  There are no data, but I guess, in 
 
  10   the Edmonton protocol, it may be difficult to do a 
 
  11   crossmatch.  At the very same time, you can argue if a

  12   patient is PRA‑negative, has not received any blood 
 
  13   transfusion, no pregnancy, no previous transplant, it is 
 
  14   completely unlikely that this patient will have a positive 
 
  15   crossmatch. 
 
  16             I am not sure whether they actually did weight for

  17   the crossmatch.  I guess they transplanted right away. 
 
  18             DR. SALOMON:  I can't imagine that, by the time 
 
  19   they took the organ, by the way, they hadn't finished the 
 
  20   crossmatch.  But, certainly, by the time they processed the 
 
  21   islets‑‑do you guys disagree with that?

  22             DR. LAKEY:  It is all being done and it is all 
 
  23   done ahead of time before the transplant. 
 
  24             DR. AUCHINCLOSS:  So I would do it, but there is 
 
  25   no data to indicate that it is actually important, except, 

                                                                226 
 
   1   as Bernhard says, where they are PRA‑negative, I don't think

   2   I would bother to do it. 
 
   3             DR. MILLER:  This is a place where animal studies 
 
   4   may be of some help in that it won't help you with the 
 
   5   autoimmunity question but it will help about whether or not 
 
   6   there is any evidence that HLA matching in other species has

   7   any effect.  So, I don't know. 
 
   8             DR. SALOMON:  That is a really good point, 
 
   9   actually.  One of the things that we talked about was to 
 
  10   identify where preclinical models would help.  That is 
 
  11   possibly a good message to the FDA.

  12             DR. AUCHINCLOSS:  I have written that grant twice 
 
  13   and not gotten funded. 
 
  14             DR. MILLER:  Write it again. 
 
  15             DR. BLUESTONE:  I think there is a problem.  
 
  16   Number one is that in non‑human primates, that is not

  17   doable.  With rare exceptions, we don't have enough, yet, 
 
  18   markers to even do a lot of the matching there; right?  Dogs 
 
  19   is not doable. 
 
  20             DR. AUCHINCLOSS:  This isn't matching.  This is a 
 
  21   cross match.  Sensitize, get an antibody and then see what

  22   happens.  Does a positive crossmatch cause "hyperacute 
 
  23   rejection of islets?"  Nobody knows. 
 
  24             DR. SALOMON:  You could do A2, for example; isn't 
 
  25   that right‑‑in figuring? 

                                                                227 
 
   1             DR. AUCHINCLOSS:  Nobody knows whether blood group

   2   incompatibility is a problem. 
 
   3             DR. BLUESTONE:  There have actually been some 
 
   4   studies in mice, now, in which presensitization is done.  It 
 
   5   has been more difficult to suppress rejection.  I was always 
 
   6   thinking, use the antibody, but you don't get hyperacute. 

   7   You just can't suppress the regular rejection very well. 
 
   8             DR. SALOMON:  I still, though, think all the 
 
   9   animal data in tissue transplantation, whether it be skin 
 
  10   grafts and, certainly, any organ grafts, that if you are 
 
  11   presensitized against an MHC molecule, that you are going to

  12   have a detrimental immune reaction. 
 
  13             DR. AUCHINCLOSS:  It just is not the case.  We do 
 
  14   not have the data that HLA antibody causes an adverse 
 
  15   outcome for islet transplantation. 
 
  16             DR. SALOMON:  I would like to officially say that

  17   I was not on the study section nor a reviewer of any of your 
 
  18   grants.  Are we settled on that?  I don't think that is even 
 
  19   worth summarizing. 
 
  20             Then what I would like to do is turn the page to 
 
  21   page 2.  I am going to make the executive decision to skip

  22   organ quality, as we did discuss that yesterday.  I think 
 
  23   that is more getting back into product issues.  I would like 
 
  24   to, again, just setting up priority, look at this route and 
 
  25   site of islet product administration as a question. 

                                                                228 
 
   1             Is it desirable for islet preparations to be in

   2   direct contact with the portal circulation and what data 
 
   3   support this determination? 
 
   4             DR. HERING:  Insulin independence supports that 
 
   5   this is feasible.  It may not be the best site but, before 
 
   6   you move to a new site, I think here you should demonstrate

   7   preclinical data, that this is actual feasible. 
 
   8             DR. SALOMON:  The animal studies have all been 
 
   9   done by putting islets under‑‑excuse me; many animal studies 
 
  10   have been done putting islets under the kidney capsule.  
 
  11   There have been attempts in human patients to put islets

  12   under the kidney capsule not, I would say anyone would 
 
  13   agree, I'm sure, exhaustive studies. 
 
  14             But there have been some trials and they haven't 
 
  15   worked very well. 
 
  16             DR. HERING:  Every single transplant failed;

  17   right. 
 
  18             DR. RICORDI:  This is probably also because of 
 
  19   mechanical consideration that the kidney capsule in a rodent 
 
  20   or a small animal is like a very thin structure.  The human 
 
  21   kidney capsule is probably one of the worst places we can

  22   put islets.  But there are other sites like the spleen.  It 
 
  23   is an issue that is the object of continuous investigation. 
 
  24             The pancreas, itself, is a major target of future 
 
  25   potential new sites. 

                                                                229 
 
   1             DR. SALOMON:  We could say that a serious

   2   objective for preclinical studies would be taking the best 
 
   3   that we can do now with human islet verification and test at 
 
   4   alternative sites.  It probably really should be done with 
 
   5   human islets for at least some of these studies; right? 
 
   6             DR. RICORDI:  Or at least in a large animal where

   7   you have the same kind of mechanical consideration or 
 
   8   vascularization. 
 
   9             DR. SALOMON:  Good point. 
 
  10             DR. BLUESTONE:  I would just emphasize that one of 
 
  11   the interesting questions is that the thymus is a potential

  12   site as well, but we have to be prepared, at some point, 
 
  13   then, to think about younger recipients. 
 
  14             DR. SALOMON:  Right.  Again, there, though, it 
 
  15   would be nice particularly to see successful non‑human 
 
  16   primate models of that.

  17             DR. BLUESTONE:  You betcha. 
 
  18             DR. SALOMON:  We tried, with a grant from the 
 
  19   Juvenile Diabetes Foundation, and did eight animals with 
 
  20   human fetal islets‑‑so there is a difference; not adult 
 
  21   islets‑‑into the thymus of juvenile rhesus.  We tried it

  22   with immunosuppression and without immunosuppression and 
 
  23   were uniformly unsuccessful in even finding the tissue 
 
  24   afterwards so we would find the transplant site where it was 
 
  25   marked. 

                                                                230 
 
   1             Does that mean that it won't work?  No.  Does that

   2   mean we picked the perfect immunosuppression?  No.  I am 
 
   3   just saying that there has been experience.  I also know Eli 
 
   4   Naji has tried it as well and has not had much experience.  
 
   5   But I do think the animal data is compelling.  So I am not 
 
   6   saying it is not worth trying.  But that is definitely an

   7   interesting topic. 
 
   8             DR. SHERWIN:  Has the peritoneum been given up as 
 
   9   a potential site, the peritoneal cavity? 
 
  10             DR. RICORDI:  Not for encapsulated products but 
 
  11   the requirement for islet mass varies with the site that you

  12   consider so let's say if you need 1,000 islets in the liver, 
 
  13   you would need, like, 1,500 in the kidney capsule and maybe 
 
  14   2,500 intraperitonally to achieve the same metabolic 
 
  15   results. 
 
  16             So there are considerations also‑‑and the site may

  17   affect other variables.  That is why you may need data from 
 
  18   a large animal or a clinical model for new sites. 
 
  19             DR. BLACK:  What about splenic infusion? 
 
  20             DR. SALOMON:  I think Camillo mentioned that.  
 
  21   Camillo pointed out that that is another site.  There is

  22   animal data suggesting you can put it in the spleen or, as 
 
  23   you mentioned, the pancreas. 
 
  24             DR. RICORDI:  The only caution there is that the 
 
  25   human spleen is, in that case‑‑it has been used a lot in 

                                                                231 
 
   1   dogs, like intrasplenic infusion.  The spleen in a dog is

   2   probably one of the largest organs in the body.  It is a 
 
   3   huge spleen compared to humans.  So it has been used 
 
   4   traditionally when you have unpurified preparation or where 
 
   5   you are afraid of intraportal high pressure or portal 
 
   6   hypertension.

   7             Then it is rather safer to put an unpurified 
 
   8   preparation in the spleen.  But that is definitely another 
 
   9   potential site of islet implantation that may have the same 
 
  10   requirement of mass as the liver. 
 
  11             DR. HERING:  But the spleen is a site that needs a

  12   lot of attention.  Significant complications were noted in 
 
  13   clinical settings with intrasplenic transplantation, either 
 
  14   autotransplantation and I think, to some extent, with islet 
 
  15   allotransplantation because the anatomy is very complex.  In 
 
  16   a few instances, islets in majority of injected islets were

  17   found in the lung. 
 
  18             So it is something that needs attention.  The 
 
  19   spleen is not the site to go without careful consideration. 
 
  20             DR. RICORDI:  Or the patient with cirrhosis 
 
  21   because to get to the lung from the spleen, they have to

  22   have collateral‑‑ 
 
  23             DR. BLUESTONE:  So the question I would have, in 
 
  24   terms of site, is I think it is pretty safe to say that the 
 
  25   liver has been a site that is used a lot and successfully.  

                                                                232 
 
   1   So you don't want to necessarily throw out that baby.  What

   2   I think the critical issue is here is safety. 
 
   3             Of the things that one can identify in the 
 
   4   procedure that could have some morbidity associated with the 
 
   5   injection sites is going to be one.  What has been the 
 
   6   experience in terms of problems that have occurred with this

   7   kind of route of implant? 
 
   8             DR. RICORDI:  Bernard summarized them yesterday. 
 
   9             DR. HERING:  I think simply a lack of efficacy.  
 
  10   So why would you do a transplant with some sort of 
 
  11   immunotherapy or immunosuppression without any evidence‑‑

  12             DR. SALOMON:  Bernhard, we are not talking about 
 
  13   that.  We are talking about the idea of intraportal 
 
  14   hypertension, the possibility of infarcting a lobe of the 
 
  15   liver, the possibility of it not going to the liver but 
 
  16   infarcting something else or embolizing another organ

  17   system.  That is, I think, what Dr. Bluestone is asking. 
 
  18             DR. HERING:  There has been no single case 
 
  19   reported to the registry or communicated at any meeting or 
 
  20   published with any significant complications resulting from 
 
  21   intraportal infusion of purified and unpurified islets in

  22   the '90's, to the best of my knowledge. 
 
  23             DR. EGGERMAN:  Didn't you say yesterday that some 
 
  24   patients died that were associated‑‑ 
 
  25             DR. HERING:  This was in the early '80's and late 

                                                                233 
 
   1   '70's when significant volumes were infused into the portal

   2   circulation without heparinization, without maybe adequate 
 
   3   washing steps as they are now standard. 
 
   4             This is the experience that we have. 
 
   5             DR. SALOMON:  Have you had any instances in these 
 
   6   where patients will spike a fever?  Has there been any

   7   evidence for a disseminated intravascular coagulation?  I 
 
   8   know any kind of cytokine release syndrome, pulmonary leak, 
 
   9   pulmonary edema?  Hypoxia?  Anything? 
 
  10             DR. HERING:  The only complications that have been 
 
  11   noted are subcapsular hematoma and then there is one

  12   reported death resulting from injury to the hepatic artery 
 
  13   and one gall‑bladder injury requiring cholecystectomy.  That 
 
  14   is what has been reported. 
 
  15             DR. SALOMON:  Camillo, did you have a comment on 
 
  16   that?

  17             DR. RICORDI:  Actually, Dr. Alejandro has a paper 
 
  18   submitted‑‑I don't know if it is accepted, but reviewing the 
 
  19   whole‑‑of percutaneous intrahepatic catheterization of the 
 
  20   portal vein for islet infusion and complications that affect 
 
  21   hypotension.  I think there was that one perforated gall

  22   bladder was the worst. 
 
  23             DR. SALOMON:  Jeff actually asked a question about 
 
  24   thymus.  That opens up a door into questions of other 
 
  25   potentially immunoprivileged sites.  I know there has been 

                                                                234 
 
   1   some attention given for over a decade now to the testis,

   2   for example, as an immunoprivileged site. 
 
   3             I have always thought that was sexist, but there 
 
   4   have been some clinical trials proposed for that.  Do you 
 
   5   guys have any comments about any alternatives for 
 
   6   immunoprivileged sites?  Let me say, I don't want to get

   7   into fast gene‑‑I think that is for another day. 
 
   8             DR. RICORDI:  Then I would limit, definitely, the 
 
   9   volume that you infuse. 
 
  10             DR. SALOMON:  No argument there; right?  Well, I 
 
  11   am nonplussed, but let me summarize what came out of this

  12   again with the idea of making sure that we have some 
 
  13   consensus and that the FDA questions have been answered. 
 
  14             I think there is a consensus that the portal 
 
  15   circulation‑‑I'm sorry; let me back up before I summarize.  
 
  16   Do you have to put it in the portal circulation?  I realize

  17   we didn't answer that question specifically.  We talked 
 
  18   about how good it was to put it in the portal circulation 
 
  19   because that is way you have done it.  I buy that 
 
  20   100 percent.  I was getting ready to summarize that. 
 
  21             But then it dawned on me the question that we

  22   didn't talk about briefly was is there a metabolic argument 
 
  23   for the portal circulation?  We all know there is, but how 
 
  24   strong is that relative to, let's say, putting it in the 
 
  25   forearm? 

                                                                235 
 
   1             DR. SHERWIN:  It is more theoretical but I think

   2   it makes sense.  The question I have is whether injection 
 
   3   islets into the portal circulation and having them lodge in 
 
   4   the liver is the same as having insulin produced by the 
 
   5   pancreas and then diluted in the pancreatic circulation 
 
   6   because I would suspect that a lot of the insulin has got to

   7   leak‑‑much more insulin has got to leak out into the 
 
   8   circulation systemically than it would if it was in the 
 
   9   pancreas, for example. 
 
  10             DR. HERING:  There is more and more evidence now 
 
  11   from small animals studies that intraportal islet

  12   transplantation actually induces a state of insulin 
 
  13   resistance and is really not a physiological site. 
 
  14             But, at the very same time, it is the most 
 
  15   accessible site for clinical transplant studies.  There is a 
 
  16   long list of arguments in favor of other sites with true

  17   portal drainage.  Simply, we have to develop the sites in 
 
  18   preclinical models before we can proceed. 
 
  19             DR. SHERWIN:  That is really what I would 
 
  20   emphasize for the future is really sites that would allow 
 
  21   insulin to be released into the portal vein rather than

  22   directly in the liver.  I think that this is much closer to 
 
  23   being a peripheral site than people think. 
 
  24             DR. SALOMON:  Okay; excellent.  Let me start 
 
  25   again.  I think there is consensus here that, at the moment, 

                                                                236 
 
   1   the injection of the islets into the portal circulation

   2   makes a lot of sense from the basis of the preclinical 
 
   3   models, the clinical experience and I think everyone around 
 
   4   the table is comfortable with that. 
 
   5             We do acknowledge that the idea of other sites are 
 
   6   very critical to consider and that would be an excellent

   7   target for studies in the preclinical models; right? 
 
   8             I think that about summarizes it.  Amazing. 
 
   9             The next thing I would like to go to again, just 
 
  10   because I want to make sure that this gets covered before we 
 
  11   have to stop, is, on the next page, No. 8, and that is

  12   outcome measures.  I think that we have had some discussion 
 
  13   of these and I think Dr. Kenyon did a really nice job with 
 
  14   her last set of slides in giving us some sense of what 
 
  15   things look like in the non‑human primate model in terms of 
 
  16   what sorts of things correlated.

  17             Do you want to start, Dr. Kenyon, maybe in saying, 
 
  18   in a nutshell in your opinion and experience what should be 
 
  19   the best outcome variables? 
 
  20             DR. KENYON:  I think it is a series of things.  
 
  21   Clearly, blood‑glucose monitoring on a daily basis, at least

  22   in the first month post‑transplant and then you could 
 
  23   decrease later on; periodic metabolic assessment by either 
 
  24   intravenous glucose‑tolerance test or arginine or glucagon 
 
  25   stimulation which, I understand, the investigators in the 

                                                                237 
 
   1   network are discussing now; what is the best way to test

   2   that without stressing the islets; hemoglobin A1c, 
 
   3   periodically, clearly. 
 
   4             I think those are the primary ones‑‑reduction of 
 
   5   insulin requirement, insulin independence. 
 
   6             DR. SALOMON:  One point you made, I wanted you to

   7   comment specifically, was you did an IV glucose‑tolerance 
 
   8   test.  Two ways to interpret that‑‑well, actually, there are 
 
   9   three ways that you could look at it.  Glucose 
 
  10   disappearance, KG.  You could look at C‑peptide stimulation 
 
  11   and you could look at insulin levels.

  12             Do you want to comment?  Do you need to do all 
 
  13   three?  Is one superior to the other? 
 
  14             DR. KENYON:  I think, clearly, you need to look at 
 
  15   all three.  It has just been our experience in the monkeys 
 
  16   that looking at the glucose release alone, which if you look

  17   back at the literature historically, that is what has been 
 
  18   shown, that that, alone, really, does not give you a good 
 
  19   indication of the functional islet mass. 
 
  20             The glucose response curve can look fairly normal 
 
  21   in an animal with partial function whereas if you look at

  22   insulin, first‑phase insulin release, it correlates fairly 
 
  23   well with, in the short term, the number of islets we have 
 
  24   transplanted and, in the long term, with whether or not 
 
  25   there has been an episode of rejection. 

                                                                238 
 
   1             So you can see, in the absence of rejection, a

   2   maintenance of first‑phase insulin release.  Subsequent to a 
 
   3   rejection episode, you can see a decrease in that.  So I 
 
   4   think it gives you a nice measure of graft function.  And 
 
   5   then C‑peptide clearly is important to look at as well. 
 
   6             DR. SALOMON:  So, could we say that, again putting

   7   this out as something to discuss, that insulin release 
 
   8   associated with a glucose stimulation would be more 
 
   9   sensitive than looking at the glucose disappearance curve.  
 
  10   I guess the reason I bring that up is that, if you look at 
 
  11   the experience with islet autotransplants for chronic

  12   pancreatitis that I think has been very nicely documented 
 
  13   even just recently a follow‑up study from David Sutherland 
 
  14   and the Minnesota group, many of those patients are insulin 
 
  15   independent.  I think about 70 percent is the most recent 
 
  16   data.  Bernhard, you can correct me if I am wrong.  But 80

  17   percent of them or more have abnormal IV glucose‑tolerance 
 
  18   curves. 
 
  19             DR. HERING:  This is entirely acceptable. 
 
  20             DR. SALOMON:  I was just using it as a check 
 
  21   for‑‑if you have a normal IV glucose‑tolerance curve, and a

  22   normal disappearance, my point was that even without 
 
  23   insisting on normal insulin dynamics, that would be quite a 
 
  24   great result for an islet transplant. 
 
  25             DR. KENYON:  I agree.  The slide I showed showed 

                                                                239 
 
   1   you that at day 42, the first phase was significantly

   2   blunted as compared to pre‑pancreatectomy.  But that animal 
 
   3   was insulin independent and had normal metabolic control.  
 
   4   What I am strictly saying is as a measure of functional 
 
   5   islet mass, not as a measure of outcome as far as metabolic 
 
   6   control.

   7             So, in that case, hemoglobin A1c and the presence 
 
   8   of C‑peptide and insulin independence. 
 
   9             DR. AUCHINCLOSS:  I think there are two issues 
 
  10   her, Dan.  One is outcome measures and there are dozens of 
 
  11   them.  And they are relatively easy to list.  Norma has even

  12   mentioned‑‑and, obviously, you want to get into things like 
 
  13   long‑term complications, et cetera. 
 
  14             You might want to be thinking about imaging 
 
  15   techniques that might be applied to assess islet mass.  But 
 
  16   all of those are easy.  The hard question is which of them

  17   turn out to be easy to list. 
 
  18             The question is which outcome represents efficacy.  
 
  19   I think the way to frame that argument is to ask the $64,000 
 
  20   question, supposing you got an outcome of measurable 
 
  21   C‑peptide production but ongoing use of insulin.  How good

  22   an outcome is that?  I think you could make the argument‑‑I 
 
  23   would be curious to see what the committee says‑‑that that 
 
  24   is a surrogate marker for an acceptable outcome. 
 
  25             DR. HERING:  The question, Hugh, is is partial 

                                                                240 
 
   1   graft function‑‑let's say C‑peptide A1c is normal in the

   2   absence of hyperglycemia.  Would this justify 
 
   3   immunosuppressive treatment long‑term? 
 
   4             DR. SHERWIN:  Not an easy answer.  We don't know 
 
   5   the answer.  Obviously, it is much easier to manage diabetes 
 
   6   if there is some ongoing insulin production, even if it is

   7   not adequate to sustain normal glycemia. 
 
   8             So, clearly, there is benefit accrued if you can 
 
   9   produce some insulin.  On the other hand, we don't know 
 
  10   enough about the immunosuppressive‑‑the risk/benefit ratio 
 
  11   is really not clear.  So I wouldn't want to call it a

  12   success.  I would think that the outcome would be one for 
 
  13   investigation to try to determine whether it was successful 
 
  14   or not. 
 
  15             As far as the assessments are concerned, I would 
 
  16   propose that there are new methods you might think about. 

  17   One is now MiniMed has made a glucose sensor that allows 
 
  18   you, for three days, to continuously monitor glucose in an  
 
  19   ordinary day's circumstances with food. 
 
  20             So it seems to me that hooking these patients up 
 
  21   to a MiniMed sensor for three days periodically might give

  22   you a nice assessment of their everyday levels of glucose.  
 
  23   I think you might learn something from that. 
 
  24             The other thing I might suggest‑‑most of us in the 
 
  25   diabetes world are not too enthusiastic about IVGTTs as a 

                                                                241 
 
   1   measure of glucose disposal because a lot of glucose is lost

   2   in the urine when you give a big glucose load.  My sense is 
 
   3   if you want to look at glucose levels, you might do a 
 
   4   glucose‑tolerance test although an alternative approach, 
 
   5   which is reasonable, is a frequent sampled IVGTT to look at 
 
   6   insulin action and secretion simultaneously.

   7             There are models that have been set up by Bergman 
 
   8   that allow one to make an assessment of insulin secretory 
 
   9   rate as well as insulin action with using minimal‑model 
 
  10   techniques.  That might be something to do. 
 
  11             DR. HERING:  But those are research tools, I

  12   guess. 
 
  13             DR. SHERWIN:  But you are doing research.  I look 
 
  14   upon this as a research study, at this point.  I am not 
 
  15   saying it will be that way forever.  I think, because it is 
 
  16   a research study, you really want to get as much information

  17   as possible to satisfy people that have a metabolic 
 
  18   background, like myself. 
 
  19             DR. HERING:  We are doing studies like this but I 
 
  20   think one other question is is there any assay that can be 
 
  21   more or less done at all centers so that we can follow

  22   patients using the same assay.  I guess here the question is 
 
  23   whether this could be an arginine stimulation test which 
 
  24   just takes ten or fifteen minutes, can be done on an 
 
  25   outpatient basis regardless of blood‑glucose concentration. 

                                                                242 
 
   1             DR. SHERWIN:  Arginine, I wonder about.  One of

   2   the questions I didn't ask originally is as glucose 
 
   3   responsiveness fails, does amino‑acid responsiveness fail in 
 
   4   concert because, for example, with type‑2 diabetes, you lose 
 
   5   glucose response of beta‑cell function but not amino‑acid 
 
   6   response of beta‑cell function.

   7             So I don't know‑‑if you don't have the answer, 
 
   8   then I would not use arginine because it might be a less 
 
   9   sensitive measure because people with impaired beta‑cell 
 
  10   function of type‑2 diabetes can have a normal beta‑cell 
 
  11   response to something like arginine.

  12             DR. KENYON:  The reason that I had listed that in 
 
  13   the non‑human primate studies is there has been a lot of 
 
  14   discussion back and forth for a while now on what is the 
 
  15   optimal test to use clinically. 
 
  16             We haven't actually looked at that in the monkeys

  17   we have done in the past.  We are going to be looking at 
 
  18   that now, but, really, the use of the test came from 
 
  19   interaction with the clinical‑transplant people.  I think 
 
  20   one of them would have to answer, if you have seen a loss of 
 
  21   arginine responsiveness in patients who are losing a graft.

  22             DR. AUCHINCLOSS:  My impression was that the 
 
  23   arginine test became popular because people got so concerned 
 
  24   about high glucose loads being toxic to islets.  I have to 
 
  25   say I have got a lot of doubt about that particular feature 

                                                                243 
 
   1   which we will talk about in the moment.

   2             But I think that is really where the arginine test 
 
   3   crept into the transplant world in a big way. 
 
   4             DR. HERING:  But I think you are right.  Whatever 
 
   5   you do, you stress islets to release insulin.  Whether you 
 
   6   use arginine without hyperglycemia or whether you use

   7   glucose, I think it is pretty much the same thing you do. 
 
   8             DR. SHERWIN:  That is one of the nice things about 
 
   9   just getting glucose profiles.  You are not doing any 
 
  10   stressing to the system.  But insulin secretion is obviously 
 
  11   a critical measurement.  I don't know how useful it might be

  12   to do urinary C‑peptide over 24 hours as a way of trying to 
 
  13   assess integrated insulin secretory levels. 
 
  14             DR. BLUESTONE:  I am a little confused because it 
 
  15   is a great wish list, but if tomorrow there is, from the 
 
  16   FDA's perspective, an IND that goes in from a group of

  17   fourteen centers and they have to list the outcome studies 
 
  18   that they are going to all be able to do, whether that 
 
  19   center is in Edmonton, in the States, in Europe, that they 
 
  20   are all going to do the same minimal set that allows the IND 
 
  21   to be approved‑‑not all the good research, because that may

  22   not be done at all centers, or maybe it will have to be 
 
  23   centralized. 
 
  24             What is the subset of these that we agree on has 
 
  25   to be done at every center? 

                                                                244 
 
   1             DR. HERING:  It is a very short list.  I think you

   2   want to know hemoglobin A1c and you want to know whether 
 
   3   C‑peptide is present.  It basically comes down to very 
 
   4   simple measures because we don't want to study efficacy, we 
 
   5   don't want to compare whether this approach is now 
 
   6   preventing complications or is associated with improved

   7   quality of life; not in this trial. 
 
   8             So, basically, we want to identify a protocol that 
 
   9   can be utilized in a subsequent prospective study to address 
 
  10   this question.  But, at this point in time, this would be 
 
  11   too much and the Edmonton multicenter trial is not powered

  12   to address any of the efficacy questions that could be 
 
  13   addressed in subsequent studies. 
 
  14             MR. SIEGEL:  Let me suggest something about this 
 
  15   issue.  This question is, in fact, divided, as you see, into 
 
  16   activity measures and efficacy endpoints.  While I would not

  17   presume to suggest that I know what are the right activity 
 
  18   measures, I would urge both the funding bodies and the 
 
  19   investigators, in fact, to explore a broad variety of these 
 
  20   because to develop this therapy, you are going to need 
 
  21   something that is going to give you a faster feedback than

  22   whether the patient's kidneys fail. 
 
  23             You take a drug.  You may change the dose a little 
 
  24   bit but you don't change the molecule a little bit.  You do 
 
  25   that all the time with transplant procedures.  You not only 

                                                                245 
 
   1   change the drugs but you make minor changes in how you

   2   transplant them.  You need some rapid feedback as to some 
 
   3   indicator as to whether you are doing something that you 
 
   4   think might be better or might be worse. 
 
   5             Similarly, in this case, I think, you are 
 
   6   suggesting that you want early indicators of when you can

   7   stop exposing the patient to immunosuppression.  So there 
 
   8   are any of a number of reasons why you would want to have a 
 
   9   good measure so that, when you get to the efficacy‑‑so that, 
 
  10   A, you can have items that you can optimize to and B, then 
 
  11   you can see, in fact, which of them are predictors of

  12   efficacy when you are ultimately doing those larger efficacy 
 
  13   studies.  
 
  14             They don't have to be done at every center. 
 
  15             DR. SALOMON:  It seems to me that what I am 
 
  16   hearing now is that there ought to be at least three kinds

  17   of tests considered.  The first is kind of parameters like 
 
  18   how is the patient doing, what is their hemoglobin A1c, what 
 
  19   is their glucose doing over periods of time. 
 
  20             I thought the idea Dr. Sherwin had of doing close 
 
  21   monitoring for maybe three days in, let's say, a GCRC

  22   setting would be useful in some centers.  Then there should 
 
  23   be a second class of studies that ask the question, is there 
 
  24   functioning islet tissue there.  Those studies could be a 
 
  25   rather simple measurement of circulating C‑peptide, 

                                                                246 
 
   1   post‑prandial bump, maybe an oral glucose challenge.

   2             And then the third would be real functional tests 
 
   3   of an integrated nature such as a graded multi‑step IV 
 
   4   glucose tolerance measuring C‑peptide insulin and glucose 
 
   5   disappearance.  I think, Bernhard, we don't need to worry 
 
   6   about whether your trial decides to do all three levels of

   7   tests the first time through.  We don't have to be that 
 
   8   specific. 
 
   9             But, do you agree that that is sort of the general 
 
  10   idea? 
 
  11             DR. HERING:  I agree that detailed metabolic

  12   studies should be done in a selected group of recipients.  
 
  13   This is an independent study but cannot be done in the 
 
  14   majority of islet‑transplant recipients. 
 
  15             DR. MILLER:  Why not?  It is only 28 patients. 
 
  16             DR. KENYON:  These are simple.

  17             DR. HERING:  If you are talking about a stepped 
 
  18   hyperglycemia clamp assay, if you are talking about a 
 
  19   frequently sampled IVGTT, if you are talking about 
 
  20   euglycemic clamp studies and if you want to do it at 
 
  21   intervals, the people cannot leave the CRC in the first

  22   year, more or less. 
 
  23             DR. SHERWIN:  We are not talking about that at 
 
  24   all‑‑not at all.  In fact, if you want to assess insulin 
 
  25   secretion, you could do it in ten minutes with an 

                                                                247 
 
   1   intravenous bolus of glucose, if that is what you want to

   2   do.  But everything I said was outpatient.  Nothing is 
 
   3   inpatient.  I don't want people in the hospital.  They 
 
   4   should be out of the hospital in their ordinary environment. 
 
   5             Even the glucose‑monitoring system is an 
 
   6   outpatient procedure.  Even that.

   7             DR. AUCHINCLOSS:  Bob, tell me about the 
 
   8   twenty‑four hour C‑peptide in the urine because, to me, it 
 
   9   seems to me the surrogate maker ought to stay focussed on 
 
  10   C‑peptide.  Is the 24‑hour C‑peptide in the urine a pretty 
 
  11   good marker of sort of total production?

  12             DR. SHERWIN:  Renal function is reasonable; yes.  
 
  13   Not bad.  Fasting C‑peptide is fine, too.  But it is 
 
  14   something that should be considered.  It is not that hard to 
 
  15   do.  It is an easy measurement, basically. 
 
  16             DR. HERING:  I think you will learn so much by

  17   looking at insulin requirements before and after 
 
  18   transplantation, A1c levels, basic C‑peptide and stimulated 
 
  19   C‑peptide.  I agree, the MiniMed approach should probably be 
 
  20   added.  I guess the Edmonton protocol is proposing mean 
 
  21   amplitude of glycemic excursion which, basically, gives you

  22   the same kind of information. 
 
  23             I would limit it.  And then you have studies, 
 
  24   detailed metabolic studies, that can address an endless list 
 
  25   of other questions.  But that will not help us right now at 

                                                                248 
 
   1   this level.  Basically, we want to assess the proportion of

   2   patients with full or partial islet‑graft function. 
 
   3             This is what we need.  Once we have a protocol, 
 
   4   then we may want to proceed to the next level of complexity 
 
   5   and compare to intensified insulin treatment or pancreas 
 
   6   transplantation and then efficacy measures will be

   7   completely different. 
 
   8             DR. CARA:  I might be coming out of left field 
 
   9   asking this, but there is reason to believe, whether you 
 
  10   believe it or not is a different issue, but there is reason 
 
  11   to believe that insulin might "protect" the pancreas or at

  12   least the islet‑cell functions of the pancreas and may serve 
 
  13   some immunomodulatory role. 
 
  14             Do you know if concomitant insulin treatment 
 
  15   post‑transplant actually improves ultimate outcome?  Is it 
 
  16   something that should be considered?

  17             You indicated the need to evaluate the actual 
 
  18   function of the transplanted tissue but I am wondering 
 
  19   whether it would make sense, in the beginning, to at least 
 
  20   maintain some sort of insulin there or whether or not it 
 
  21   should.  I don't know.

  22             DR. HERING:  More or less every single 
 
  23   experimental study suggests that insulin administration in 
 
  24   the peri‑transplant period improves islet engraftment.  
 
  25   Insulin can put beta cells at rest.  That was the first 

                                                                249 
 
   1   hypothesis.  Insulin is a growth factor.  Insulin is

   2   antiapoptotic.  Insulin blocks macrophage NO production. 
 
   3             But the question is at what levels.  The Edmonton 
 
   4   protocol did not administer insulin in a routine manner 
 
   5   after transplantation, only if blood‑glucose levels, I 
 
   6   guess, exceeded 180 milligram per deciliter.

   7             But, at the very same time, the question was not 
 
   8   addressed whether peri‑transplant insulin administration 
 
   9   would have been helpful.  So I think you should prevent, 
 
  10   definitely, hyperglycemia in the peri‑transplant period and 
 
  11   the question is what is the threshold that you would like to

  12   see. 
 
  13             DR. SHAPIRO:  Some people believe that it may 
 
  14   actually stimulate the growth and function of the islets.  
 
  15   So it is not truly known. 
 
  16             DR. AUCHINCLOSS:  To put your question in context,

  17   for a period of time, it was sort of the lore that you 
 
  18   couldn't do islet transplantation unless you maintained 
 
  19   absolutely rigid tight glucose control for weeks after the 
 
  20   procedure.  But the fact of the matter is that the 
 
  21   successful islet transplants that have been done on people

  22   generally have been done without tight glucose control. 
 
  23             I don't know about Norma, but I suspect you 
 
  24   probably give no insulin after you do islet transplants in 
 
  25   monkeys.  We don't.  We don't bother at all.  None of the 

                                                                250 
 
   1   whole‑organ pancreas‑transplant programs worry about glucose

   2   control postoperative.  None of the small‑animal studies 
 
   3   maintain‑‑so the lore is a little bit hyped, I think. 
 
   4             DR. HERING:  But, Hugh, the point is what is the 
 
   5   number of islets required to restore insulin independence.  
 
   6   This number may depend on the degree of metabolic control

   7   post‑transplant.  I guess you would agree with this. 
 
   8             In some of the experimental studies where you 
 
   9   transplant a very good number of islets, yes; you may see 
 
  10   normoglycemia and insulin independence and you may not want 
 
  11   to overtreat the recipient.

  12             DR. AUCHINCLOSS:  I like James' approach.  Just 
 
  13   keep giving islets until they come off of insulin. 
 
  14             DR. RICORDI:  Actually, one of the debatable 
 
  15   issues like whether do you need systemic, like, insulin, 
 
  16   exogenous insulin injection, to have metabolic control in

  17   the post‑transplant period or whether a mild hyperglycemia 
 
  18   can stimulate more insulin secreted at the site of 
 
  19   implantation in the microenvironment where you really need 
 
  20   the growth factor and the antigenic factor of the 
 
  21   antiapoptotic.

  22             So I think there is still a little controversy 
 
  23   whether you should clamp very closely or whether a mild 
 
  24   hyperglycemia may be acceptable.  But I would like to echo 
 
  25   Dr. Siegel's point that we need to find out markers that can 

                                                                251 
 
   1   define good outcome in terms of what is good for the

   2   patients. 
 
   3             I think that these endpoints that we are defining 
 
   4   are exactly doing that because if you measure C‑peptide and 
 
   5   hemoglobin A1c, if you assume that you have two levels of 
 
   6   success in a cellular graft, it is not like an organ

   7   transplant where partial function virtually does not exist. 
 
   8             But, in islet transplantation, you can either 
 
   9   achieve complete failure, partial function or complete 
 
  10   insulin independence.  The measure to assess partial 
 
  11   function is C‑peptide and whether your normalize hemoglobin

  12   A1c levels, because those are outcome measurements that have 
 
  13   been, thanks to the studies of the DCCT and others that have 
 
  14   been correlated with positive endpoints for patients 
 
  15   because, even within the cohort of patients of the DCCT, it 
 
  16   has been shown that those patients with type‑1 diabetes that

  17   have residual C‑peptide secretion do better in terms of 
 
  18   complication development than other patients. 
 
  19             There have been urine studies in Europe on 
 
  20   C‑peptide treatment showing that even if you have a 
 
  21   C‑peptide secretion around 1 nanogram, it may be something

  22   beneficial for patients. 
 
  23             So I think C‑peptide hemoglobin A1c does not just 
 
  24   give us an immediate assessment of how much the islets are 
 
  25   working, whether they are working or not, but they also can 

                                                                252 
 
   1   be related to some kind of outcome long‑term predictions.

   2             DR. SALOMON:  Camillo, can I ask you a question, 
 
   3   following up on that‑‑well, to everyone, but to you‑‑what 
 
   4   criteria should be used to determine the loss of graft 
 
   5   function?  I guess my question is can you use any of these 
 
   6   criteria to diagnose rejection?

   7             DR. RICORDI:  It depends if you have partial 
 
   8   function or complete insulin independence.  But I would say 
 
   9   that the first‑‑if you have partial function, the first 
 
  10   index that you may have is hyperglycemia and higher insulin 
 
  11   requirement compared to what was your baseline when the

  12   transplant was functioning.  Absence of stimulated C‑peptide 
 
  13   production, basal and post‑prandial, are clear signs for 
 
  14   when you lose completely a graft. 
 
  15             You can do a glucagon test and have confirmation 
 
  16   of stimulated C‑peptide test and have confirmation that

  17   there is no more C‑peptide production. 
 
  18             DR. SHERWIN:   I would bet, although I am not 
 
  19   sure, that the best way of testing whether you are beginning 
 
  20   to lose function is insulin's response to intravenous 
 
  21   glucose over a ten‑minute period.  If you knew that and

  22   could follow it, if you began to lose islet mass, that 
 
  23   response would begin to fall off. 
 
  24             DR. RICORDI:  The reason I am saying depending on 
 
  25   the graft function, because we know from the preclinical 

                                                                253 
 
   1   study when we did these monkeys with normal, that if your

   2   baseline is insulin independence with an extremely 
 
   3   well‑regulated glucose level and basal in the 80's range, 
 
   4   even suspect rejection when basal or post‑prandial‑‑when you 
 
   5   have just a little blip in post‑prandial, it is already an 
 
   6   indication‑‑when you have a partial function, it is more

   7   complex because you may have more variable baseline or 
 
   8   post‑prandial glucoses. 
 
   9             Then unless you do very frequent IVGTT, it may be 
 
  10   very difficult to catch at an early time. 
 
  11             DR. HERING:  There is one important question in

  12   this context; should islet‑transplant rejection be treated. 
 
  13             DR. SALOMON:  You sort of anticipated my next 
 
  14   question.  What is the answer? 
 
  15             DR. HERING:  This depends on the availability of 
 
  16   early markers of rejection, I think.

  17             DR. AUCHINCLOSS:  That's right. 
 
  18             DR. KENYON:  I would say yes if we have an agent 
 
  19   that can effectively reverse it.  I agree with Camillo, what 
 
  20   he is saying.  It is so much cleaner if the animals are 
 
  21   clearly insulin independence.  Using the loss of first phase

  22   or a blunting in it as a predictor of rejection is difficult 
 
  23   because you would have to do it fairly often. 
 
  24             We have, retrospectively, seen animals that had a 
 
  25   rejection episode and, just serendipitously, we had done an 

                                                                254 
 
   1   IVGTT the week before and there was clearly a loss of first

   2   phase.  But, especially, depending on the initial mass 
 
   3   transplanted, they can be having some kind of an ongoing 
 
   4   rejection for a while, I think, without us detecting 
 
   5   hyperglycemia. 
 
   6             Just like when you are getting diabetes, you can

   7   lose a lot of cells before you actually detect 
 
   8   hyperglycemia.  With regards to treating them with insulin 
 
   9   post‑transplant, when we have done it, Hugh, is when the 
 
  10   animals have been hyperglycemic.  So usually that is 
 
  11   associated with the marginal islet mass and then we will

  12   treat them to keep them below 200 post‑prandial and around 
 
  13   100 fasting. 
 
  14             I have had monkeys that actually required 
 
  15   significant amounts of insulin that got a marginal mass and 
 
  16   actually came off insulin after the first 100 days.  But as

  17   far using that as a marker, it is difficult to say‑‑if the 
 
  18   animal has partial function, I think it is important to give 
 
  19   insulin because they may come off and it keeps their general 
 
  20   health status. 
 
  21             But, as far as protecting the islets,

  22   post‑transplant, we don't have any data to support it one 
 
  23   way or the other.  It is just a matter of metabolic control. 
 
  24             DR. AUCHINCLOSS:  I would say two things.  One is, 
 
  25   I think, I buy the point that probably glucose control 

                                                                255 
 
   1   matters somewhat but probably not to the degree of religion

   2   that we had about five years ago.  But I was actually 
 
   3   prompting you to point out that you believe you have treated 
 
   4   rejection episodes and reversed them an maintained islet 
 
   5   mass sufficient to maintain normal glycemia. 
 
   6             DR. KENYON:  We have; with anti‑CD154, multiple

   7   episodes of rejection in the monkeys. 
 
   8             DR. MILLER:  What is the registry data about 
 
   9   treating rejection?  Has that been captured in the registry, 
 
  10   because I think when we are looking at risk to patients, I 
 
  11   think that the protocol should very much standardize what

  12   gets done and how soon you stop, the less immunosuppression, 
 
  13   so that there are clear‑cut answers when the pilot is done 
 
  14   how you manage rejection. 
 
  15             DR. HERING:  There is only anecdotal data, and I 
 
  16   am not aware of any protocol that consistently reverses

  17   rejection.  So, hyperglycemia is a very late marker.  We 
 
  18   know this.  It is conceivable that 80 percent of the islet 
 
  19   mass is gone by the time you see hyperglycemia, or 
 
  20   50 percent, you can argue. 
 
  21             OKT3, ATG, steroids have been tested, but

  22   anecdotal data, at best, are available. 
 
  23             DR. SALOMON:  I have to say, it sounds like a 
 
  24   24‑hour urine for C‑peptide that was quantitative might 
 
  25   actually be‑‑ 

                                                                256 
 
   1             DR. HERING:  If you look at the normal range of

   2   C‑peptide in the urine, it is anything, I guess, from 5 to 
 
   3   200 microunits or whatever.  I don't know.  But it is a 
 
   4   wide, wide range.  There is one paper in the literature, The 
 
   5   Pitfalls of Urinary C‑Peptide Analysis.  This was published 
 
   6   by Ken Polonski.  If you really want to study C‑peptide,

   7   then you have to study the C‑peptide kinetics of a person.  
 
   8   So you have to use radioactive C‑peptide and have to go into 
 
   9   a lot of details if you really want to develop a sensitive 
 
  10   assay. 
 
  11             DR. SHERWIN:  That's correct.  The other thing I

  12   would emphasize again, and you are right about glucose being 
 
  13   a late marker, but one thing that I have been struck by with 
 
  14   the glucose monitoring is that, in people with normal A1c's 
 
  15   that we aggressively treat, let's say, with pumps, once you 
 
  16   put them on a continuous monitor, there are a lot of

  17   abnormalities because you are getting‑‑the immediate rise 
 
  18   and fall is much more abnormal than we suspected by getting 
 
  19   a continuous readout. 
 
  20             So I think you may detect subtle abnormalities in 
 
  21   post‑prandial glucose metabolism with that kind of an

  22   assessment.  I surely would try it as a trial in your study, 
 
  23   in not necessarily every patient, as a way of trying to pick 
 
  24   up early rejection because, obviously, that would be 
 
  25   critical. 

                                                                257 
 
   1             DR. HERING:  There is, now, I guess, some software

   2   some mathematical modeling software, so it could address the 
 
   3   question whether a given glycemic profile could be an early 
 
   4   marker of rejection; right. 
 
   5             DR. SALOMON:  We are getting a little bit toward 
 
   6   the end here.  I just want to make sure that we get the

   7   highlights.  One thing that we haven't‑‑we have sort of been 
 
   8   talking about 8a up until now, of course, on purpose.  Can 
 
   9   we just spend the last couple of minutes here on 8b, the 
 
  10   idea here being what kind of efficacy parameters would be 
 
  11   reasonable for really judging an outcome.

  12             What would you accept as a good outcome?  What is 
 
  13   the range of things?  Obviously, everyone understands that 
 
  14   perfect islet function is a good outcome, but what about the 
 
  15   intermediates there? 
 
  16             DR. HERING:  One outcome measure is healthcare

  17   dollars per quality‑adjusted life years saved. 
 
  18             DR. SALOMON:  Anything else? 
 
  19             DR. RICORDI:  I think C‑peptide secretion with 
 
  20   normalization of hemoglobin A1c levels in the absence of 
 
  21   severe hypoglycemia would be a gold standard of treatment

  22   now. 
 
  23             DR. SALOMON:  Even if the patient was still on 
 
  24   insulin. 
 
  25             DR. RICORDI:  Yes.  I am saying intermediate 

                                                                258 
 
   1   outcomes, not‑‑

   2             DR. SALOMON:  I understand. 
 
   3             DR. AUCHINCLOSS:  Are we sure we agree on that?  I 
 
   4   even saw you, Bob, nodding your head yes. 
 
   5             DR. SHERWIN:  Yes. 
 
   6             DR. AUCHINCLOSS:  I think that is a very

   7   fundamental point for this committee if it really agrees 
 
   8   that C‑peptide, normalization of hemoglobin and A1c is a 
 
   9   surrogate marker for a good outcome in a trial even if you 
 
  10   are on long‑term immunosuppression. 
 
  11             DR. SHERWIN:  I think‑‑would you say "good?"  It

  12   is an intermediate outcome that I would accept as acceptable 
 
  13   from an experimental perspective.  I don't know whether it 
 
  14   is a good outcome or not because I have no way of assessing 
 
  15   the long‑term effects of immunosuppression. 
 
  16             But I think it is something that is a reasonable

  17   one to look at.  It would be not an unacceptable outcome.  
 
  18   Let's put it that way. 
 
  19             DR. BLUESTONE:  I guess the question I have‑‑this 
 
  20   is my earlier question, now, an hour later.  If at one year, 
 
  21   somebody had a partial functional islet graft but was on

  22   severe immunosuppression with this protocol, would you leave 
 
  23   him on the immunosuppression? 
 
  24             DR. RICORDI:  You give him another informed 
 
  25   consent. 

                                                                259 
 
   1             DR. AUCHINCLOSS:  You would actually give him more

   2   islets. 
 
   3             DR. SHERWIN:  I would go on.  I think I would go 
 
   4   on.  I need to know more‑‑hopefully, I would have learned 
 
   5   some more as I went along.  I am not saying that it is 
 
   6   inappropriate therapy, really; no.  And I don't know the

   7   answer to it.  It is an experiment that, as long as it is 
 
   8   conformed consent and we are getting a good outcome from 
 
   9   A1c, I think it is an experiment worth seeing, if the 
 
  10   patient is informed. 
 
  11             MR. SIEGEL:  I guess more of what we are getting

  12   to with this question, though, would be what outcome would 
 
  13   be convincing that you have a favorable risk/benefit, either 
 
  14   a measure of benefit or reasonably likely to predict 
 
  15   clinical benefit, to the extent that you would feel this is 
 
  16   what you need to know to say yes, this is an effective

  17   treatment. 
 
  18             DR. SALOMON:  Give us another minute.  We are 
 
  19   getting there. 
 
  20             DR. AUCHINCLOSS:  Jay is asking the right question 
 
  21   because I was trying to phrase it that this was a surrogate

  22   marker for a good clinical outcome.  Bob answered the 
 
  23   question, I think, by saying, yes; you ought to go ahead and 
 
  24   study that. 
 
  25             DR. CHAMPLIN:  But the long‑term outcome is, 

                                                                260 
 
   1   obviously, reducing the end‑organ damage and that, at least

   2   right now, correlates with the hemoglobin A1c the best and, 
 
   3   at least for a surrogate, in the short term, that would be 
 
   4   the gold standard. 
 
   5             I guess the other aspect is is the patient 
 
   6   suffering from their immunosuppressive treatment.  If they

   7   are doing well without infection, without renal failure or 
 
   8   any toxicity from those drugs, then there is little harm in 
 
   9   continuing it.  So that has to be considered as well. 
 
  10             DR. CARA:  I have a couple of comments.  One is 
 
  11   that, regardless of whether or not the glycohemoglobin has

  12   been improved or normalized, I think one of the important 
 
  13   issues is to sort of continue the ongoing monitoring that we 
 
  14   routinely provide to individuals with diabetes to make sure 
 
  15   that we are, in fact, reducing the incidence of kidney 
 
  16   disease, eye disease, and so on and so forth.

  17             I think another important issue is the 
 
  18   quality‑of‑life issue.  We may not necessarily "cure" 
 
  19   individuals with diabetes, but if we can improve their 
 
  20   quality of life, either by reducing hypoglycemia or frequent 
 
  21   episodes of ketoacidosis or hospitalizations or whatever, I

  22   think that is of clear benefit. 
 
  23             MR. SIEGEL:  I would infer from that, though, that 
 
  24   when‑‑and I heard that the studies that are being planning 
 
  25   now are not at that stage of proving clinical benefit, when 

                                                                261 
 
   1   we are looking at that, then, those comments would also

   2   relate to what sorts of patients you would want to study. 
 
   3             If you want to show you are preventing 
 
   4   complications, you want to study a patient population in 
 
   5   which you know you have a reasonable incidence of what 
 
   6   complication to expect.

   7             Or if you want to improve quality of life, you 
 
   8   want to study patients whose quality of life needs 
 
   9   improvement. 
 
  10             DR. CARA:  But there are a couple of different 
 
  11   ways that you could, obviously, do that.  But I think the

  12   historical data that we have, thanks to the DCCT, provides 
 
  13   us with fairly substantial information as to the natural 
 
  14   history of diabetes. 
 
  15             If we can impact that in any positive way, I think 
 
  16   that is a very important issue.

  17             DR. SHERWIN:  There is one issue, actually, 
 
  18   revolving around that question.  Again, I don't know enough 
 
  19   about transplantation; most people with diabetes die of 
 
  20   heart disease and macrovascular complications.  My 
 
  21   impression is that people who get transplants have a high

  22   rate of macrovascular complications. 
 
  23             So the one issue that we have not focused on and I 
 
  24   am sorry I didn't really focus on it before is what do we 
 
  25   know about transplantation in general with respect to 

                                                                262 
 
   1   macrovascular disease and could we be accelerating

   2   macrovascular disease in this patient population. 
 
   3             That might be one of the things we monitor. 
 
   4             DR. LEVITSKY:   I thought most of that was due to 
 
   5   the glucocorticoid but maybe I am wrong about that, in 
 
   6   renal‑transplant people, people who are not too controlled. 

   7   My question, actually, related to, as we were discussing the 
 
   8   previous scenario with the patient who was sort of a 
 
   9   half‑way patient, it seemed to me that we were discussing an 
 
  10   N of 1, not an N of however many patients are going to go 
 
  11   into a study.

  12             I think I would like people to refocus the 
 
  13   question in terms of if the outcome of this study is that 
 
  14   everyone enrolled in it, or a certain percentage of people 
 
  15   enrolled in it, has that same scenario, that they are all 
 
  16   sort of halfway there but not completely off insulin, not if

  17   one single patient is in the study is there and you are 
 
  18   making an individual patient decision, what kind of outcome 
 
  19   do we want for the entire study. 
 
  20             Does 50 percent of the population have to be 
 
  21   completely off insulin?  If 50 percent of the population if

  22   on insulin but has a better hemoglobin A1c, is that an 
 
  23   effective outcome? 
 
  24             DR. SALOMON:  I think that is a good way of posing 
 
  25   the question.  I don't know that we need to come up with an 

                                                                263 
 
   1   answer to that, but I think that‑‑

   2             MR. SIEGEL:  Dr. Bluestone has argued, I think, 
 
   3   that the argument for posing it in terms of the individual 
 
   4   patient is that it is only the individual patient who is 
 
   5   experiencing that benefit who is also experiencing the risks 
 
   6   of prolonged immunosuppression.  So, if that is the main

   7   risk you are wanting to counterbalance, you can look at it 
 
   8   that way. 
 
   9             DR. CHAMPLIN:  I would argue that if you can be 
 
  10   successful in some patients, some meaningful fraction of the 
 
  11   patients, that would give you promise that improvements upon

  12   the procedure, giving more islets, optimizing 
 
  13   immunosuppression, doing things better, could get you up to 
 
  14   100 percent. 
 
  15             So the part‑way solution, I would view, is good 
 
  16   for a step that would justify carrying on.

  17             DR. SALOMON:  We are near the end here, so what I 
 
  18   would like to do is try summarizing this last question of 
 
  19   the two days. 
 
  20             DR. CARA:  Could I make one more point?  That is 
 
  21   the issue that‑‑I think we sort of agree on the things that

  22   we know we would need to be looking at from the point of 
 
  23   view of diabetes, but I am not so sure that we have a good 
 
  24   sense of what we routinely don't look for that we probably 
 
  25   should be looking for in a population of individuals that 

                                                                264 
 
   1   are undergoing islet‑cell transplantation.

   2             The sort of thoughts that occur are should we be 
 
   3   looking at issues like bone integrity as a result of 
 
   4   continued chemotherapy.  Should we be looking at a variety 
 
   5   of other issues related to either the treatment of the 
 
   6   concomitant therapy?

   7             DR. AUCHINCLOSS:  The answer is yes, we need the 
 
   8   long‑term data. 
 
   9             DR. CARA:  And, if so, what? 
 
  10             DR. AUCHINCLOSS:  The question is what is the 
 
  11   short‑term surrogate endpoint.  I just wanted to come back

  12   to that and say I don't personally agree with the statement 
 
  13   that I made about partial function. 
 
  14             I believe that an acceptable surrogate marker is 
 
  15   insulin independence.  I think if you had people insulin 
 
  16   independent, you could call that a success.  I would

  17   actually agree with Dr. Sherwin.  I would not feel 
 
  18   comfortable accepting, at this point, partial function as 
 
  19   demonstration of benefit. 
 
  20             I understand that, in both cases, you go ahead and 
 
  21   get long‑term data to verify the surrogate endpoint.  But I

  22   think, at this moment, I would have to say that insulin 
 
  23   independence is the surrogate marker I would take. 
 
  24             MR. SIEGEL:  Can I ask‑‑I am enjoying this 
 
  25   discussion, but I hope we won't leave Dr. Sherwin's question 

                                                                265 
 
   1   hanging because I would like to hear more before we go home

   2   as to whether are there specific concerns about making 
 
   3   macrovascular disease worse or should it be looked at‑‑can 
 
   4   you look at it as an endpoint for making it better and how 
 
   5   would you look at it. 
 
   6             DR. SALOMON:  I think that the point is, and I

   7   think that is sort of what Hugh is getting at, is that, 
 
   8   within a twelve‑month time frame, that is not so very easy 
 
   9   to do, frankly, in any objective way.  The fact that a risk 
 
  10   is there, however, which was alluded to by Dr. Levitsky, Dr. 
 
  11   Cara and Dr. Sherwin, is real.

  12             Steroids, alone, are not the only cause for 
 
  13   accelerated atherosclerotic vascular disease in transplants.  
 
  14   In terms of bone disease, it is known cyclosporine and FK506 
 
  15   do increase bone turnover and decrease bone deposition which 
 
  16   is something that has not often been made a big deal of, but

  17   it is actually a very real thing. 
 
  18             So I think that there are some potential deficits.  
 
  19   But, again, I don't think that you could do bone 
 
  20   densitometry in a twelve‑month time frame and significantly 
 
  21   see differences that would be relevant.

  22             DR. SHERWIN:  But you want to do it baseline, 
 
  23   then.  You are telling me you want to do it baseline because 
 
  24   you might continue this trial for five years.  It would be 
 
  25   good to have that baseline information. 

                                                                266 
 
   1             I also think you should do, then, carotid‑artery

   2   ultrasound.  Then the issue is, in other transplants that 
 
   3   people do, they do coronary angiography.  I am not trying to 
 
   4   say that is what I‑‑ 
 
   5             DR. SALOMON:  I think there is a lot of interest 
 
   6   in kidney and heart and liver transplantation with these

   7   cardiovascular risks.  Let's face it.  It is the most common 
 
   8   cause of death in vascularized organ allografting, more 
 
   9   common than dying because of the loss of your graft which is 
 
  10   sobering for all of us. 
 
  11             But we are very frustrated by the fact that there

  12   are very few and very poor measures of it.  You could spend 
 
  13   millions of dollars trying to do quantitative coronary 
 
  14   angiography or what they call IVUS, intravascular 
 
  15   ultrasound.  I don't think anybody wants to go there yet 
 
  16   with the first few islet transplants.

  17             DR. SHERWIN:  But doppler ultrasound of the 
 
  18   carotid artery seems to correlate reasonably well with 
 
  19   macrovascular disease.  It is surely being used by the DPP 
 
  20   as a surrogate marker.  It is not very difficult to do. 
 
  21             DR. SALOMON:  Again, this is not meant as an

  22   argument.  I do want to end at 4 o'clock because we are 
 
  23   going to lose people on the committee.  I thought, before we 
 
  24   walk away, that we should try and summarize this last part. 
 
  25             For 8a, my sense of the committee is that there 

                                                                267 
 
   1   are four kinds of tests that you go from simple measures

   2   such as their insulin dose, their daily glucose levels, to 
 
   3   more complicated tests such as the stimulated secretion of 
 
   4   C‑peptide, the glucose‑disappearance curve. 
 
   5             Then the third level would be more complicated 
 
   6   integrated tests which would include stimulated glucose,

   7   stimulated insulin, release, stimulated C‑peptide release 
 
   8   and then, finally, a fourth level that I think Dr. Sherwin 
 
   9   made me aware of that would even require a hospital 
 
  10   admission where you start getting into glucose clamping. 
 
  11             I know, Dr. Sherwin, you were not suggesting that

  12   we do any of those things, but there is a fourth level of 
 
  13   testing out there that could be done in a metabolic unit.  I 
 
  14   think Dr. Hering made the point that he thought you had to 
 
  15   keep that in mind, not to get completely carried away. 
 
  16             I think that the idea would that, at this point,

  17   it is probably too early to say what criteria would be used 
 
  18   to determine the loss of graft function short of someone 
 
  19   completely off of insulin who suddenly is back on their 
 
  20   maintenance dose of insulin, something really obvious with 
 
  21   no detectable C‑peptide.  You don't need to get hit over the

  22   head with a two‑by‑four to pick that one up, but more subtle 
 
  23   changes and, particularly in patients who might have partial 
 
  24   responses, this could be very difficult. 
 
  25             In terms of 8b, the idea of‑‑ 

                                                                268 
 
   1             MR. SIEGEL:  Before we leave that, am I not

   2   correct in having heard the committee say that, in fact, it 
 
   3   would be a very important goal to identify early measures of 
 
   4   dysfunction for any of a variety of reasons but, notably, to 
 
   5   treat rejection but potentially other reasons as well? 
 
   6             DR. SALOMON:  Yes; I think the committee agrees.

   7             MR. SIEGEL:  That there are potential markers out 
 
   8   there and that, for the most part, the only argument against 
 
   9   those potential markers was that, in some cases, they might 
 
  10   be inconvenient or costly to do. 
 
  11             DR. SALOMON:  I think that, overall, what we are

  12   trying‑‑I think what the committee is telling you is that, 
 
  13   right now, having not had much of an experience with 
 
  14   successful islet transplantations, certainly under this 
 
  15   particular circumstance and protocol, that it would be 
 
  16   premature to tell you what measures, that they should be

  17   measured and that that one of the major objectives of the 
 
  18   trial should be to answer that question with real data. 
 
  19             In terms of 8b, that, of course, is the idea of 
 
  20   what would be the endpoint benefits of a trial like this.  
 
  21   There, I think, there was a little bit of lack of clarity on

  22   the part of the committee.  I think concerns were raised 
 
  23   that it is easy, if everybody is off of insulin with great 
 
  24   insulin and glucose metabolism. 
 
  25             It is not quite so easy to decide on patients, 

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   1   let's say, who are still on insulin but have measures that

   2   clearly would suggest that they have a stability of their 
 
   3   diabetes. 
 
   4             However, I think that, for example, the Edmonton 
 
   5   group would say if these are patients who had really severe 
 
   6   hypoglycemic episodes or had severe ketoacidosis,

   7   complicated ketoacidosis, episodes that those patients 
 
   8   really would be getting a significant benefit from this 
 
   9   study and probably would justify that. 
 
  10             I think we are all clear about the fact that we 
 
  11   don't know what the long‑term consequences of this

  12   particular immunosuppressive regimen is in this particular 
 
  13   group of patients and that that makes this determination a 
 
  14   little bit more complicated. 
 
  15             DR. MILLER:  Could I just add something to what 
 
  16   you are saying.  It is not really clear that, if they are

  17   under better control‑‑i.e., no DKA or hypoglycemia‑‑that 
 
  18   that is an effect of the graft, itself.  I mean, these 
 
  19   patients will then be changed into a very intensive 
 
  20   monitoring roll and so it could be an epiphenomenon. 
 
  21             I disagree that that is an endpoint, especially in

  22   these early trials.  I really think that, in the earliest 
 
  23   trial, the goal is to get as much information as possible to 
 
  24   determine the safety and the preliminary efficacy. 
 
  25             This question about how to determine what is for a 

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   1   phase III trial, I think, is very premature at this time. 

   2   Let's get some data you can get these in before you start 
 
   3   planning a phase III trial. 
 
   4             MR. SIEGEL:  I would agree.  By the way, I think 
 
   5   that point is very well taken.  There is a lot of data out 
 
   6   there which suggests that, in almost all diseases, that

   7   patients who enter clinical trials do better on their trial 
 
   8   than they did in the period proceeding the trial. 
 
   9             Given what we have heard about the issues of poor 
 
  10   management and whether that represents optimization, one 
 
  11   might expect that here.  I will toss out something that I am

  12   sure nobody will think is a good idea, at least nobody is 
 
  13   actually doing the experiments, which is, at some point, and 
 
  14   I agree with you entirely, Carole, that this is not the 
 
  15   point, but at some point where one were studying this, an 
 
  16   interesting way to get at that question, although possibly

  17   not feasible, would be to take brittle diabetics and, 
 
  18   actually, to randomize them and have some of them in an 
 
  19   intensive management program. 
 
  20             Once you had a therapy that you knew was 
 
  21   reasonably effective, you could answer some important

  22   questions. 
 
  23             DR. SALOMON:  I think Carole's point is excellent.  
 
  24   The point I was making was simply that, if the Edmonton 
 
  25   group puts forward the idea that those are suitable patients 

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   1   for entry into such a trial, accepting the significant

   2   implications, risk, et cetera, to that patient, I didn't 
 
   3   think it was unreasonable to put it in the list of things 
 
   4   that would be reasonable outcome measures. 
 
   5             However, the potential that that might be a 
 
   6   clouded outcome measure is well taken.

   7             Then, lastly, but I don't at all unimportant, is 
 
   8   the point brought up at the end by Drs. Sherwin and Cara and 
 
   9   Levitsy that the potential of cardiovascular risk factors, 
 
  10   bone and other somatic complications of the therapy, really 
 
  11   are significant and they are clearly factors in allograft

  12   experience with these immunosuppressive drugs and will have 
 
  13   to be a parameter. 
 
  14             I think that kind of summarizes it. 
 
  15             DR. RICORDI:  May I make on brief comment.  I 
 
  16   completely agree that hemoglobin A1c alone could be a marker

  17   of better management of the patient.  That is why you 
 
  18   capolate with C‑peptide that cannot obviously emerge out 
 
  19   from nothing just because of management.  But it is very 
 
  20   important also that as soon as these patients enter in the 
 
  21   candidate list, they start‑‑they are treated with intensive

  22   insulin management. 
 
  23             We, indeed, are using this Teledox system that Dr. 
 
  24   Alejandro has been using with patients entering the 
 
  25   candidate list which manage very closely glucose levels that 

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   1   you can, indeed, improve even the insulin requirement before

   2   transplant.  You have to make sure that your baseline is 
 
   3   what you can achieve with close monitoring and ideal patient 
 
   4   treatment and then judge what is the additional component of 
 
   5   the transplant. 
 
   6             MR. SIEGEL:  If you enter somebody because of

   7   asymptomatic hypoglycemia or brittleness, you put them on 
 
   8   the candidate list, and you have this intensive monitoring, 
 
   9   do you then confirm that they remain brittle despite this 
 
  10   intensive therapy before you actually transplant them? 
 
  11             DR. RICORDI:  Yes, because you have to have tried

  12   to optimize treatment before. 
 
  13             DR. SALOMON:  I think, again, a couple of 
 
  14   different times, we have made the point that the baseline, 
 
  15   and establishing the baseline of these patients before 
 
  16   enrolling in the trial for any one of these parameters is

  17   going to be critical.  I think that is true.  It is true of 
 
  18   the cardiovascular and the bone diseases, et cetera. 
 
  19             I would like to end here.  I want to thank 
 
  20   everyone on the committee, all the speakers who joined us 
 
  21   today, the FDA, Gail Depolito, her staff at the FDA and

  22   everyone else for their participation today. 
 
  23             Thank you again. 
 
  24             MR. SIEGEL:  Thank you all; a very informative and 
 
  25   interesting discussion. 

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   1             [Whereupon, at 4:50 p.m., the meeting was

   2   adjourned.] 
 
   3                              ‑ ‑ ‑ 6