Workshop on Factor VIII Inhibitors Lister Hill Auditorium, Building 38A National Institutes of Health, Bethesda, MD November 21, 2003 Dear Factor VIII Inhibitor Workshop Stakeholders Letter PROCEEDINGS DR. LOZIER: I would like to welcome you to the FDA International Association of Biologicals Workshop on Factor VIII inhibitors. My name is Jay Lozier. I am the Chairman of the Organizing Committee for this event. Before we start the program, I need to go through some housekeeping announcements. First of all, you would all have a registration package of written materials with speaker slides and handouts. Be sure you get the extra handouts that were made available at the last minute which should have been given to you at the front desk. The registration handouts include an evaluation form that we would like for you to turn in at the end of the day to help us with the design of future workshops. We will have a transcript of the proceeding available about fifteen working days after the meeting and you will find sheet in your handouts with the web page and the address for obtaining that. There will also be a videotape available from FTC Reports for a price yet to be determined, which I am given to believe is on the order of $500 to $700. So that will be available later than the transcript, obviously. The rest rooms are in the lobby of the Lister Hill Center. When you go in and out of the auditorium, please use the back or side exits. Please turn pagers and cell phones to silent ring or vibrate mode as a courtesy to the rest of us. There is a message center at 301 496-4062 that you can use to relay messages. Lunch, we presume, will be at noon if we keep on schedule in the cafeteria that is down the stairs in the lobby. There is some renovation there and it may advisable--some people may want to go to the Natcher Building which has a larger cafeteria which is just essentially across the parking lot. I think if you follow the NIH regulars, you will be able to find that. We have breaks scheduled in the middle of the morning session, in the middle of the afternoon session. Please come back from those promptly to keep our program on time. Finally, I would like to thank our speakers for their hard work in preparing their talks and getting the material to us. Some of them come quite a long way, as you will see, and I think you will appreciate their hard work. At this time, I would like to introduce Dr. Jay Epstein, the Director of the Office of Blood Research and Review in the FDA Center for Biologics. He will make introductory remarks to start the conference. Introductory Remarks DR. EPSTEIN: Thank you very much, Jay. It is my pleasure to welcome everyone to this workshop Factor VIII inhibitors. Before we start, though, I would like to acknowledge the hard work of the Planning Committee and, in particular, Jay Lozier who was the Chair. I think that their effective planning is reflected in the excellent turnout that we have and I am very pleased to see the luminaries of the field here among us. I just want to acknowledge Mark Weinstein, Andrew Chang, Nisha Jain, Anthony Miraslus who put the program together and Joe Wilczek who has provided administrative support. Let me also note that this is a co-sponsored workshop with the IABS and that we are very pleased to host this workshop as part of a continuing effort to assure the safety, efficacy and quality of the products that we regulate. Today, with HIV and other viral contaminants under control, inhibitor formation presents itself as the chief adverse event associated with the use of antihemophilic factor. Therefore, the central question at this time, with respect to safety, is how can we ensure that new Factor VIII products or products that have undergone significant manufacturing changes won't induce inhibitor formation in previously treated patients. Therefore, to reduce the occurrence of inhibitor formation, we need to understand to what extent immunogenicity is a property of the products rather than a phenomenon intrinsic to the patients. The answer to this scientific question will lie in conducting appropriate preclinical studies, clinical studies, and postmarket surveillance. Today, at this workshop, we will devote most of our time to defining what are appropriate means in regard to preclinical trials inhibitor assays and surveys in patient populations. Our specific objectives, then, will be to improve clinical-trial design, to review the available data on the prevalence and the incidence of inhibitor formation, to examine the limitations and the potential of assays for Factor VIII inhibitors, to attempt to foster international harmonization in this area and to explore the future directions that are feasible and collaborations that could emerge. I would like to just quickly review the structure of the program for you. The scientific program will begin with an overview of Factor VIII inhibitors and the historical context, particularly noting the Dutch and the Belgian experience with inhibitor formation to marketed products. Next, we will be examining environmental and genetic factors that may influence antibody formation. Can we separate inhibitor formation that is caused by neoantigens in the products from inhibitor formation that is due to the genetic makeup or environmental circumstances of the patient? Following this presentation, we will hear about the potential of preclinical studies to predict inhibitor formation. What role, if any, can animals play in predicting inhibitor formation? One of the major dilemmas that we face in evaluating clinical trials is deciding what should be counted as a positive inhibitor response. This depends, in part, on the sensitivity, specificity and reproducibility of the assay used to detect the inhibitor. Two of our four speakers will address these topics as well as the results from a surveillance study and the development of new assays at increased sensitivity. I think most of you are aware that the International Society on Thrombosis and Hemostasis has recommended that previously treated patients be studied first when conducting clinical trials of inhibitor formation. We will be hearing about the rationale for this recommendation as well as ISTH recommendations about what should constitute a positive and what should constitute a high-titer result. The next two speakers will describe surveys of Factor VIII inhibitor formation in Canada and in the United States. The results of these studies should give us insight into the prevalence in inhibitor formation and provide a baseline for expected inhibitor formation when switching patients from one product to another product. We then get a lunch break. After lunch, we will focus more on the regulatory aspects of inhibitor formation. Colleagues from the U.S. Food and Drug Administration and the European EMEA will discuss their agencies' current thinking about preclinical testing, clinical trials and postmarket surveillance for inhibitor formation. These discussions will help us to better understand the rationale behind our respective policies and thereby help us to move toward greater international harmonization. We will further examine the FDA rationale for clinical-trial design with a presentation on statistical considerations that influence the number of patients required for a given trial. Then, after a report on the role of the Data Safety Monitoring Board in clinical trials, we will hear in-depth presentations from industry representatives about specific clinical trials that they have conducted. These reports should allow us to compare the various trials with respect to patient numbers, patient exclusion criteria, the assays used and threshold levels considered as positive and the results of postmarket surveillance. The last part of the meeting will focus on future directions. We will discuss preliminary ideas on prospective international collaborative studies on product-related Factor VIII inhibitor formation. This presentation will be followed by an open panel discussion where FDA staff and audience members will be able to pose questions to the speakers. I should note that a brief question period will also be available following most of the talks. So, before we begin our scientific program, it is my great pleasure to introduce Mrs. Laurie Shumway who is the mother of a son with hemophilia and a Factor VIII inhibitor. Mrs. Shumway has graciously volunteered to give us the personal perspective on coping with an inhibitor in addition to hemophilia. It is my pleasure to invite up Mrs. Shumway. Thank you. (Applause.) Consumer Perspective MRS. SHUMWAY: Thanks, Jay. Good morning. My son, Scott, is a 14-year-old high-school freshman with severe Factor VIII deficiency and he has an inversion. When he was two-years old, we discovered that he had a high titer inhibitor. We had no family history of hemophilia so we had very limited knowledge of it and no practical experience at all. However, through our hematologist, our local chapter and the treatment center here in D.C., we had a huge body of knowledge and experience that we could draw on and we learned a lot in those first months and years. Hemophilia really did seem manageable. But once we discovered the inhibitor, it suddenly didn't seem so manageable anymore. We didn't know whether we would be able to treat Scott effectively. Scott was diagnosed with a bleeding disorder on a Saturday in January, 1989, when he was eight-days old. You know, it is always on a weekend. The hospital we were at was able to diagnose that he had bleeding disorder but they couldn't, until Monday came, tell us what type of bleeding disorder he had. So they treated him with fresh-frozen plasma and that did stop the bleeding. In the next few weeks, we discussed possible treatment options and their pluses and minuses. Since recombinant factor was sort of on the horizon at that time, we decided that we were going to treat our son with donor-directed cryoprecipitate. Cryo was an effective treatment for Scott in the first two years of his life. During that time, we really didn't perceive any changes in how he responded to treatment. So the discovery of an inhibitor in 1991 was a real surprise. His inhibitor was 280 Bethesda units. So the first serious issue we faced was how were we going to treat Scott in the presence of such a high-titer inhibitor. It is really startling when you realize that your son may not be able to be treated effectively. Porcine Factor VIII might work but what happens if the bleeding doesn't stop or he needs treatment again? For the day-to-day bleeds that he might have experienced, our treatment choices were basically Factor IX products that really, at that time, were not considered as safe as the monoclonal Factor VIII products that we had rejected two years earlier. So that was a really difficult thing for us to sort of come to terms with. From 1991 until 1995, we were forced to treat Scott with Factor IX products. They didn't work particularly effectively. When Scott would have a bleed, we would often have to treat him several days in a row, immobilize the joint, and we used a lot of ice. During that period, our refrigerator quit and we bought a brand-new refrigerator with an ice crusher. I still say it is the best investment we ever made. We also became pretty adept at managing pain. Scott spent many nights sleeping in a bean-bag chair because it was really the only way we could support a bleeding knee so that he would be comfortable enough to sleep. It was really frustrating not to be able to stop a bleed. We knew that immune tolerance was out there and that it offered a way to deal with the inhibitor. But, back in the early '90's, there really wasn't a lot of information on it or experience with the various protocols. Because Scott had a high-titer inhibitor, we were advised, at that time, that we should wait until it was lower before we started immune tolerance. We might not get that same advice today. Also, we knew that we would need to place a central line for venous access before we started such a program. There was a precipitating event that really pushed us into immune tolerance and to try to improve venous access. I shut his arm in the van door. So we treated him but his forearm continued to swell. You know, he had tingly fingers or couldn't quite feel them. And then we were--after two or three days, we really could not get access to his veins. So off we went to the hospital where he was treated more aggressively with higher doses and more frequently than we had at home. But we were really worried about getting clotting where we didn't want it and knew that there was a danger of that. But we also knew there was a danger of really damaging his arm without treating. So this event made us realize that we couldn't wait any longer. If Scott had a more serious bleed, we could have lost him, is the bottom line. In fact, during the time when Scott was young, there was a child in our chapter who had inhibitors who did die. But the decision to begin immune-tolerance program was program was primarily left up to us. There were no definite answers as to when to start or which protocols had higher probabilities of success. In fact, as I said earlier, we were advised to wait until the inhibitor titer had come down. Waiting had significant impacts for us. We couldn't treat Scott effectively for a four- or five-year period. Once we started immune tolerance, there was a higher cost associated with it. Scott was larger. He weighed more and required more factor. We started the immune tolerance in 1993. His inhibitor had come down to 28 Bethesda units. We had a central line placed using porcine factor. It was a Broviac catheter which Scott had for nine years. Scott hit his peak inhibitor titer two to three months after starting immune tolerance. His peak titer was about 3500 Bethesda units and he had an antiporcine inhibitor titer of about 1,000. So we had lost our ability to use porcine factor to treat any serious bleeds for a while. What we did in immune tolerance was we treated Scott twice a day with fairly large doses of Factor VIII for about two years and then we treated him daily for another two-and-a-half years. During that time, we were getting ready to give up because we didn't seem to be making any progress. While Scott's inhibitor titers had been unmeasurable or low for several years, his progress towards normal Factor VIII levels had sort of stagnated. Like, his one-hour post-infusion levels were, I think, around 30 percent. So we were thinking this is it, maybe; this is as good as we are going to do. But a lucky conversation with some Scandinavian hematologists after a session on inhibitors at an annual NHF meeting caused us to continue. They had had a patient and they had kept going for six to seven years and had achieved near normal Factor VIII levels. So we decided, okay, let's stay the course. So we continued treating for another year and a half to treat him daily with factor. We did gradually see improvements in his Factor VIII recovery. Since 1999, Scott has received factor every other day. We haven't been able to able to go to a three-day prophylaxis regimen. We tried it once, but, in the first months, on the second day without factor, every single time he had a bleed. So we went back to the every other day. That prevents virtually all bleeding. Maybe two or three times a year, we have something we need to treat but it is usually because he has had an injury or something. You can imagine the cost that is associated with an immune-tolerance program. Scott's factor costs were over $1 million in several years. Both my husband and I work for the federal government so we have access to insurance that does not have a lifetime cap. We consider ourselves very lucky. However, every year, the first time we get factor in January, we reach the catastrophic limit in our insurance and we owe the maximum out-of-pocket expense for the year, which is $4,000 or $5,000, depending on where they set that in a particular year. Then, after that, our insurance pays 100 percent of factors costs. However, we still face some challenges from time to time in dealing with insurance. For example, we periodically take a look at the price we are paying for factor to try and keep the cost of Scott's treatment to a minimum. We are obtaining factor from a preferred provider with our insurance company but found another provider that had the same factor, the same product, for a significantly lower cost. We switched providers and that meant we would save our insurance company tens of thousands of dollars. However, the new provider was not a preferred provider so, under our insurance company, there was a higher out-of-pocket expense that we needed to pay. So the insurance company wanted us to pay another $1,000 towards factor in that year even though we had talked to them about changing providers before we did it. So, after several phone calls and letters and stuff, they did, ultimately, waive that $1,000. But, in closing, let me leave you with a few questions that we and Scott face. For instance, this past June, when we went in for his annual clinic visit, Scott's inhibitor titer was 0.5 Bethesda units but his recoveries were not quite as good as they had been. When things change, we are always concerned about what does this mean. In this case, we haven't seen any change in his bleeding patterns or in his response to factor but, in the past year, he has grown five or six inches and put on twenty or thirty pounds and probably what happened is we just didn't increase his dosage sufficiently during the year. While Scott is aware that he has hemophilia, its effects on him on minimal. He plays all kinds of sports and participates in other activities. How are we going to help him understand the importance of continuing regular infusions to keep that inhibitor at bay? What role will insurance issues and possible discrimination in employment play in determining what he does and where he lives? How could changing products affect his inhibitor levels and recoveries? How will his inhibitors affect his ability to take advantage of a possible cure? Inhibitors really added more uncertainty to our lives. But, for us, after immune tolerance, hemophilia, once again, seems manageable. That's it. Thanks. (Applause.) DR. LOZIER: Thank you very much for bringing us that perspective. I think it is an important one. Overview of Factor VIII Inhibitors DR. LOZIER: At this time, I would like to provide a succinct, I hope, overview of something that took two-and-a-half days, I guess, to present in its entirety in Chapel Hill, recently, and that is an overview of Factor VIII inhibitors. First, I will talk about the definition of inhibitors and their characteristics and briefly mention their frequency of occurrence and discuss the problem of inhibitors from the regulatory standpoint, then discuss the issue of how we assess the inhibitor risk in clinical trials and then just briefly explain the workshop agenda. During the course of prophylaxis or treatment of hemophilia with Factor VIII concentrates, antibodies to Factor VIII can develop in patients with hemophilia A. These inhibitor antibodies will manifest themselves by neutralizing Factor VIII activity and/or accelerating the clearance of Factor VIII from the circulation. The inhibitor-neutralizing ability is measured in vitro by assessing the Factor VIII activity after incubation of patient plasma with normal source of Factor VIII, usually plasma, the so-called Bethesda assay and all its variants. The in vivo assessment of Factor VIII inhibitors is the fall-off study where the elimination of infused Factor VIII from the circulation of a patient is measured over time. Factor VIII inhibitors are interesting for many reasons but one of the interesting points is complement fixation and immune-complex disease and anaphylaxis are relatively rare in contrast to the Factor IX inhibitors. The Factor VIII inhibitors are typically IgG4 antibodies with specificities for Factor VIII epitopes that may, in fact, interfere with binding of von Willebrand factor, phospholipids, Factor IX or Factor X. Recent evidence also shows that some of these inhibitor antibodies may catalyze proteolytic cleavage of Factor VIII. The inhibitor epitopes are typically clustered at the A2, A3, and C2 domains as well as the acidic animo acids that fall between the A1, A2, domains and the B domain and the A3 domain. The antibody response to Factor VIII is characterized by the titer at any point in time of antibody as measured in vitro and also as measured dynamically in the patient by the nature of the anamnestic response. There are inhibitors that are high titer versus low titer and the anamnestic response, meaning the increase in the titer upon reexposure can be high or low. Factor VIII inhibitor incidence depends on a number of patient factors, environmental factors and sometimes the Factor VIII product, itself. We have all read and written, in many cases, about the overall rate of Factor VIII inhibitor development being on the order of 20 percent. There is, though, quite a bit of variability in this data depending on the severity of the patient population that you are assessing, the frequency of the assessment for inhibitors will change data and the threshold for a positive inhibitor will certainly factor into the calculation of the incidence. The greatest inhibitor incidence, as far as new inhibitor development, is in those patients who have no prior exposure to Factor VIII, the previously untreated patients or so-called PUPs. The lowest inhibitor incidence is in those patients who have previously been treated and not yet shown an inhibitor, the so-called PTPs. As you can see from this summary slide from Earnest Briet's metaanalysis of seven prospective inhibitor studies in patients with severe hemophilia A and high-titer Factor VIII inhibitors, there is, when you normalize to a common starting point--and the follow-up here is in years as opposed to exposure days. But the point is, over the course of time, we will asymptotically approach the 20 percent inhibitor frequency in these patients. Of course, the greatest rate of new development of inhibitors is in the early time frame, typically in the first year or two which would correspond typically, on a weekly-treatment basis, to about 75 exposure days. After five years, the new inhibitor development rate is much decreased, but there is still, even out late in the course of hemophilia, a small but finite incidence of new onset of inhibitors. The patient factors that play into this are the severity of the hemophilia, the nature of the mutation--namely the inversions, deletions and nonsense mutations--have a greater incidence of inhibitors than missense mutations and small deletions. Presumably, this difference is a reflection of the issue of how much cross-reactive material that may tolerize the patient over time or the so-called CRM-positive status versus those who have no cross-reacting material. Other genetic factors that may play into this are possibly HLA haplotypes, race. African-Americans may have a higher incidence of Factor VIII inhibitors than Caucasians. There may be cytokine and immune-response modifier genes that factor into this and I will be presenting some animal data suggesting that may be the case, at least in a mouse model, that I have developed. I will talking about that at the ASH meeting in San Diego in December. Comorbid disease states including infection, perhaps autoimmune conditions, pregnancy or malignancy, which are really more relevant more acquired inhibitors, not specifically our topic today, but these are factors that can influence the incidence of inhibitors in hemophilia. Concomitant surgery or trauma, perhaps acting as an adjuvant, so to speak, for the immune system. There has been discussion of whether the infusion method or treatment intensity is a factor for influencing the rate inhibitor development. The Factor VIII concentrates that we have available are those derived from plasma which we have had from the 1960s beginning with the low specific activity, cryoprecipitate, preparations which have usually more protein than units of Factor VIII. In the '60s, chromatographic and precipitation maneuvers were developed that gave intermediate purity, Factor VIII products that had on the order of 10 to 20 units per milligram of protein. Then, finally, the monoclonal-antibody purification process yielded the high-purity concentrates that typically have 2,000 or 3,000 units of Factor VIII per milligram of protein. In the 1980s, recombinant product came along which were derived from fermentation of Factor VIII transduced cells and purified by monoclonal antibody or other affinity chromatography preparations or methods and also have the very high specific activity of 2,000 or 3,000 units per milligram protein. The manufacturing process, which we are interested in, of course, can influence immunogenicity of Factor VIII and seemingly minor changes in virus inactivation procedures are associated with an outbreak of inhibitors in heavily treated patients using one particular product. This is the Dutch-Belgian inhibitor epidemic alluded to earlier which 8 of 140 previously treated patients with extensive Factor VIII exposure with severe hemophilia A developed inhibitors in short order after use of a new plasma-derived Factor VIII concentrate that was solvent-detergent treated and heated at 63 degrees for ten hours. The patients demonstrated both low- and high-titer Factor VIII inhibitors that had complex inhibition kinetics and appeared to have specificity for the Factor VIII light chain. When this product was discontinued in these patients, the inhibitors gradually declined. The problem we have at FDA and the other regulatory agencies that we communicate with is to evaluate the new Factor VIII products for safety, efficacy and potency and inhibitor antibodies now are the chief adverse event that we are concerned with since the virtual elimination of HIV and hepatitis risk. The inhibitor risk assessment depends on the definition of what an inhibitor is, what is positive and what is negative, where is your cutoff. What is the significance of a transient inhibitor and how do we define and decide what the cutoff is between a high- and low-titer inhibitor? We are interested in who should participate in trials and we are going to hear from Dr. White on the ISTH recommendations on studying PTPs preferentially in initial trials of new products. We want to know how clinical trials should be designed. With regard to clinical trials, we are interested in what size trial, how many arms we should be asking sponsors to bring to a licensure proceeding. In addition to how many arms, what are the appropriate comparators? Should we use historic controls? Should we compare with current products, with plasma products, or comparable recombinants? We will also hear from Dr. Aledort about the role of the Data Safety Monitoring Board this afternoon. We also need to know how, once we have this data, do we evaluate the clinical trials and to assess the inhibitor risk for new Factor VIII products and can this regulatory approach be harmonized between the FDA and the other worldwide authorities. We will certainly be hearing from Dr. Rainer Seitz on that topic this afternoon, to some extent. We will be interested to know opinions of those in the audience about the role and the importance of postmarketing surveillance in the regulatory decision-making process. The morning sessions will address definitions and laboratory issues and measurements of inhibitors and will discuss some of the clinical epidemiology from the United States and Canada. Our afternoon sessions will address the design of clinical trials including FDA and industry perspectives. Donna DiMichele will be a moderator of a panel discussion at the end of this conference and will be presenting, immediately before that, her thoughts on the possible role for postmarket surveillance. So I would like to thank you and try to keep on time here. Immediately, I would like to introduce Dr. Joan Cox Gill of the Blood Center of Southeast Wisconsin. She has been a hemophilia care provider for many years and has done a lot of work on inhibitor and hemophilia research. She will be discussing the environmental and genetic factors that may influence inhibitor antibody formation. (Applause.) Joan? Environmental and Genetic Factors That May Influence Antibody Formation DR. GILL: Thank you very much. I want to thank the sponsors for the privilege of presenting at this meeting this morning. I am going to talk to you about environmental and genetic factors that may influence inhibitor antibody formation. We all know that inhibitors impact the outcome of replacement therapy and also impact the assessment of efficacy and safety of new therapeutic replacement products and is likely to impact the outcome of gene therapy. Therefore, it would be highly desirable to predict risk for inhibitor development and to identify factors that predispose to inhibitor development. What is the evidence for genetic factors playing a role in inhibitor development? First of all, just by natural history, we know that inhibitors develop early within a median of nine to 11 exposure days to replacement therapy. This suggests that there is a predisposition to inhibitors on the part of the patient's genetic makeup. There has also been shown to be an increased inhibitor risk in African Americans in several studies that have been published. Finally, there are animal studies that show that the introduction of an out-bred female into a hemophilic dog colony in Canada resulted in progeny with inhibitors whereas other lines in that colony, the dogs did not develop inhibitors. Also, there is a differential development of inhibitors in hemophilic mouse strains. We decided to try to address this issue by doing a survey of sib pairs across the United States and Canada. This slide just summarizes the number of families that were surveyed and the inhibitor incidence. So, as one would expect, 30 percent of the severe hemophilia-A patients, families, there was at least one family member with an inhibitor whereas in moderate hemophilia-A patients, it was lower. Again, as has been shown in many studies, the incidence, or prevalence, of inhibitors in hemophilia-B is much lower. In this slide, I would like you to concentrate on these purple numbers. If one calculates the expected number of families in which there would be two patients affected with an inhibitor and the incidence of inhibitors is 15 percent, one would expect, by chance alone, five families. If the inhibitor incidence is 20 percent, one would expect nine families. We observed a significantly higher number of families in which there was concordance of inhibitor development in 28 of the families and this was highly statistically significant. This study was a confirmed study by Astermark in an international survey of sibling pairs of 460 families. Again, African Americans or blacks had a higher incidence of inhibitors than Caucasians. Concordant inhibitor families were higher than expected by chance alone. There was a much higher risk of an inhibitor if you have a positive family history of inhibitors, so about 48 percent would be expected to develop an inhibitor if their family members had an inhibitor. What are some of the factors that might be influencing this increased risk of inhibitors in families? Well, first of all, the factor-mutation type has been shown to be in influence so the more severe mutations result in higher prevalence and incidence of inhibitors than the less severe mutations. However, if one looks at intron-22 inversion mutation which accounts for about 40 percent of severe hemophilia only 20 percent of the patients affected by that mutation do develop an inhibitor. So this suggests that there must be other factors involved in inhibitor formation. Indeed, if one looks at all of the other mutations causing severe hemophilia A, one has a similar incidence of inhibitor development. We then asked the question that, since each hemophilic member of a single family has the same Factor VIII mutation, if additional important genetic factors play a role, the risk of inhibitor development should be greater in the hemophilic siblings of an inhibitor patient than in the extended hemophilic relatives; that is, grandfathers, cousins, nephews, et cetera. We looked at data from two studies. One was the inhibitor survey undertaken by the Hemophilia Research Society where we identified 113 inhibitor patients with severe hemophilia A and found that 41 percent of those families had one or more family members affected. There was a 52 percent risk of inhibitor development in your sibling had an inhibitor whereas only 11 percent inhibitor risk, if only your extended family members had an inhibitor and not your brother. This was born out also in the sibling study I just mentioned where, again, we saw only 9 percent in extended family members. So, again, this suggests that there are other genetic factors important in inhibitor development other than the specific Factor VIII mutation in the family. So what could some of these other genetic risk factors be? First of all, we know that T-cells are important in inhibitor formation. There is concomitant disappearance of inhibitor with loss of CD4 helper T-cells in patients who are infected with HIV. We know that the IgG isotype is predominantly IgG4 and this is evidence for the TH2-like nature of the response; that is, the response requires T-cell help for B-cell differentiation and immunoglobulin isotype switching. In addition, tolerance to Factor VIII can be induced in inhibitor patients, again suggesting a role of T-cells and in animal studies tolerance induction has been shown by blocking accessory molecule interaction in some of the mouse models. We know that the initiation of the immune response occurs when antigen-presenting cells present peptides in the context of MHC that are recognized by the T-cell receptor and, in order to have a proliferative response, one needs to have interaction of accessory cells that provide a second stimulus for proliferation and expansion of the immune response. Then, depending upon the cytokine environment in which that response occurs, one can have antibody synthesis or we now know we can have suppression of antibody production and, if there is lack of secondary responses, then tolerance occurs. One of the genetic variability factors that has been studied quite extensively is that of HLA. This is a fairly logical step to look at. We found that, in several studies, there was no significant difference in HLA type in patients who developed inhibitors or who didn't develop inhibitors and only weak associations were found when only patients with intron-22 inversions were studied. So, in looking at the overall immune system, these are some candidate genes that may be important in inhibitor development. MHC, of course, as I have mentioned already, immunoglobulin genes, T-cell receptor genes, cytokine and cytokine receptor genes that have defined cell subsets and then accessory molecules. I think that there is a lot of suggestive evidence but, to date, we don't have any firm polymorphism or mutations that have been found that would substantiate this hypothesis. So we have a lot of work to do in this area. So what are some of the factors that may predispose to inhibitor formation that are non-genetic or environmental? There has been a lot of work done to examine the type and purity of Factor VIII concentrate effect on inhibitor development. In a nice, systematic overview of studies over time by White published in Hemophilia, a number of these studies were examined. This slide shows the weighted mean percent cumulative risk of all inhibitors and the weighted mean percent cumulative risk of high responder inhibitors in studies of patients who receive multiple low and intermediate purity plasma-derived concentrates, single plasma-derived concentrates and recombinant concentrates. What we can see from an examination of these studies is that the overall cumulative risk is about the same, if one looks at plasma-derived concentrates, multiple plasma-derived concentrates versus recombinant concentrates. But, interestingly, a few studies, there was a much lower risk in people who had received single plasma-derived concentrates. Now, these studies were very small and there were some older patients entered into these studies. So I think this is an intriguing observation that will probably not be studied further because we now are using primarily recombinant concentrates, or at least desire to use primarily recombinant concentrates because of their improved safety. Because some of the older studies did not measure inhibitor titers as frequently as the newer studies and, thus, patients who have transient inhibitors can be missed by those studies. The incidence of high responders was also looked at. What was very interesting here is that cumulative risk for a high-responder inhibitor is actually lower in patients who receive recombinant clotting-factor concentrates versus those that were treated with multiple, low and intermediate purity plasma-derived concentrates. I think this answers, or at least partially answers, an important question about use of recombinant clotting factors which we were concerned for a long time about an increased risk of inhibitor development using recombinant factor but, in fact, the high-responder inhibitors, which are the most clinically significant, actually had a lower risk. What about differences in recombinant clotting-factor concentrates? Kogenate and Recombinate, as you know, are both full-length recombinant Factor VIII concentrates where as ReFacto is B-domain deleted. You can see from this summary slide that the cumulative risk is almost virtually identical. The median age of the patients treated in those previously untreated studies or PUP studies was virtually identical and the median exposure days to the time of inhibitor development was also almost identical, so suggesting that, at least in these three studies, again small studies, that deletion of the B domain does not have an effect on cumulative risk for inhibitor development. What are some of the other factors that might predispose to inhibitor formation? The age at initial therapeutic exposure has, in two very small studies by the Swedes and I think it was Spanish groups, suggested that if the patient was exposed earlier, the patient would have an increased risk for inhibitor development. This is counterintuitive where most of us, I think, were brought up to believe that if one received a foreign antigen early, it would be more likely that tolerance would develop. Well, those two small studies suggest that that is not true although I must say that they are both very small and especially the older patient groups in whom the inhibitor risk was lower, there were only four or five patients in that group. So I think we need to do more studies to further define that possibility. In all of the PUP studies that have been done, the dose and frequency of initial therapeutic exposures has not seemed to play a role. There are some intriguing possibilities that, if a patient were exposed in utero via a maternal-fetal transfusion to maternal Factor VIII, perhaps that may be one of the factors that could explain the difference in inhibitor development among patients who have the same Factor VIII mutation. However, there is, to date, no evidence to suggest that that is true. There has been one study by, I think this was the Swedish group also, who examined the possibility that breast feeding might be protective in inhibitor development. There are homologous proteins in breast milk that have a significant amount of homology to Factor V and Factor VIII and therefore might be expected to provide some measure of protectiveness against inhibitor development if oral tolerance is a true phenomenon that might prevent inhibitor development. However, in that study, there was no evidence that breast-fed infants had a lower incidence of inhibitor development. We also know that there are many concomitant illness--for example, HIV infection--that might predispose to a patient developing an inhibitor perhaps even later on in life. There have been also several anecdotal reports of patients who had serious infections who were treated with high-doses of a new product at the time of surgery who then developed an inhibitor even though they had been previously exposed to many, many doses of Factor VIII concentrate. I think those kinds of studies to have a better definition of the risk of inhibitor development later on in life after many exposures to Factor VIII, those studies really need to be done and we need to have a better definition of that aspect of inhibitor development because, if we are going to attempt to determine whether or not there are neoantigens being formed with new products that are being developed, we need to know what the baseline natural history of inhibitor development is in these patients in whom we are now adding a new product. So, if one looks at some of the PUP studies--I was asked to address the question as to what we can learn from PUP studies to apply to our design of studies for previously treated patients. As Dr. Lozier has already shown you, if one looks at the cumulative risk of inhibitor development in PUPs, the inhibitors develop early and then the inhibitor incidence levels off. However, there are some patients, as we mentioned, who do develop inhibitors later on in life. So I think that, in addressing this question, first of all, we need more information about the long-term natural history of inhibitor incidence, either or just-appearing inhibitors, in patients who are treated with single products versus multiple products. We need careful follow up of patients during a switch to a new product and we need to evaluate the effect of illnesses and medications on inhibitor development during product changes so that we need to understand the pattern of inhibitor development in order to then evaluate whether or not a new product actually has neoantigens that provoke a new inhibitor response. So, important variables that we need to look at to evaluate both PUP and PTP studies are hemophilia and mutational analyses, the ethnic background of patients, family history of inhibitors, a previous history of inhibitors, any concomitant immunologic disorders and medications and whether or not an anti-inflammatory disorder might occur at the time of exposure. I think that there are several organizations in this country and in Europe that should be able to develop new studies that will help us better define the long-term natural history of inhibitor development in heavily treated patients as well as in previously untreated patients. I hope that this workshop will stimulate the development of some of those studies. Thank you very much. (Applause.) DR. LOZIER: Joe, why don't you go ahead and load Dr. Saint-Remy's talk. We have time for a few questions. I forgot to mention if you wish to ask a question from the bench here in the front, you can actually activate your microphone by pushing a button at the base of the microphone. Those in the back who may have a question can go to either of the freestanding microphones in the back. We have time here for a question or two for Dr. Gill. Dr. Golding? DR. GOLDING: I don't have a microphone so I am going to shout. One of the issues with reducing an antibody response to an antigen, as you pointed out, requires T-cell help. Now, there have been several papers that have shown that there is such a thing as bystander help. In other words, if someone has an intercurrent infection or some other immune stimulus, you can get help to the B-cells without the actual, in this case, Factor VIII providing the help. When you look at the patients and the history, you see that, in very young people, they are getting inhibitors. So what I am wondering is how carefully people have looked at intercurrent infections, for example, in young children and the association that that may have development of inhibitors and whether very aggressive treatment of infections or avoidance, if possible, of treatment during times when there is acute infection could be helpful in this situation. DR. GILL: I think that is a very important point. Unfortunately, we have not, to date, looked carefully at that question. I think, in all of the previous studies that have been published, that possibility has not been looked at and I think that that is something that we need to do. We could possibly get some preliminary information from some of the PUP studies in which all adverse events were recorded during the study. But, as far as I know, no one has done that to date. But it is something that we certainly should do in the future. DR. LOZIER: Dr. Chang of FDA has a question. DR. CHANG: Joan, I enjoyed your talk very much. DR. GILL: Thank you. DR. CHANG: One of your slides, you had a very good comparison on the product type and also the accumulated inhibitor formation versus high-titer patients. I assume that is on the PUPs patients. I was just wondering whether or not there is a systematic analysis on the PTP with a similar layout of the analysis. DR. GILL: In that same paper, published by White in Hemophilia, there was a discussion of PTP patients as well but the conclusion was that there really wasn't enough definitive data to answer the question, which I think is why we need to do additional cooperative studies. We are going to be hearing about some cooperative studies, I think, later on this morning with the CDC studies and I know that the Hemophilia and Thrombosis Research Society is interested in initiating some natural-history studies, too, so that we can get enough patients to begin to answer those questions. DR. LOZIER:Thanks very much, Joan. We will go to our next speaker, Dr. Jean-Marie Saint-Remy of the Center for Molecular and Vascular Biology at the University of Leuven, Belgium. He has been a leading researcher in the field of Factor VIII inhibitors and was one of those who showed that certain Factor VIII inhibitors have proteolytic activity against the Factor VIII molecule. He will provide his talk entitled What Can Preclinical Testing of Factor VIII concentrates tell us; a Cautionary Tale. What Can Preclinical Testing of Factor VIII Concentrates Tell us; a Cautionary Tale DR. SAINT-REMY: Good morning. Just to comply with the European habit of starting a talk by making apologies, I have two apologies to offer you. The first one that you should have the handout with old slides and I apologize. I just probably overlooked the mail asking me to send the slides. But you will get them later on. The second apology is that, as you probably already realize, I have a kind of flu-like illness. This is the true influenza coming from Australia. The funny thing about this is that I got it in Brussels from one of my patients living in Scotland. So the world is small. I like the title I have given because this is really open and probably very much in the spirit of what we do in our lab. I would like to start by making just a couple of statements. The first one is that we might not speak the same language but I am trying to combine the scientific approach and the clinical approach because I have been educated as a M.D. On the clinical perspective, preclinical testing of Factor VIII means that you wish to know in advance which of those Factor VIII concentrates are going to produce inhibitors. But, of course, inhibitors, in terms of immunological setup, comprehension, understanding, does not mean anything, just a case in point. The point is to evaluate whether a factor VIII concentrate increased risk of immunogenicity in general not only the 25 percent of those antibodies which might interfere with Factor VIII activity. When I am saying evaluate the risk of increased immunogenicity, that is really what I mean. Factor VIII is a foreign protein for every hemophilia-A patient. It is going to be fully foreign if you no Factor VIII at all. It is going to be partially foreign if you have parts of your Factor VIII functional and not functional. If you are in good health, well, you should consider Factor VIII as a foreign body and then make an immune response against Factor VIII. I would be very anxious not to see any kind of immune response against Factor VIII in healthy individuals with or without Factor VIII, as a matter of fact. Everything has to be considered not in terms of whether or not you see antibodies, you see an immune response against Factor VIII, but whether or not the equilibrium in between immunity and tolerance is established or reestablished as soon as you come with Factor VIII. You see that this is a very complicated slide. But basically the message is simple. You have a constant exposure to an antigen, Factor VIII in this case. This is going to trigger an immunity which is compensated by tolerance in the periphery. Nowadays, it is almost impossible to open a journal on immunology not to see a paper on a new mechanism of tolerance induction in the periphery. The new one is certainly to a version of the B-cell receptor which is a very interesting finding which I think has whole new consequences on the way we should look at anti-Factor-VIII antibodies. We all, including hemophilia-A patients, have specific B and T-cells in the periphery with the capacity to make a full-blown immune response against Factor VIII with about 20 percent of those antibodies having inhibitory capacity. The B-cells are produced, as you, I guess, know, continuously during your lifetime and you make each day 100 millions of new B-cells which have the capacity to respond to Factor VIII. On the other hand, the T-cell repertoire, at least in men, is almost entirely fixed at birth so we have a huge capacity there. As soon as you have a good pair of B and T specific for the same antigen and the right conditions, which might be triggered by inflammation, for instance, you will stop making a full functional immune response against Factor VIII. The other part I would like to stress which was already alluded to by one of the questions after the previous speaker is the fact that once you have been exposed to an antigen, again, if you are a healthy individual including, of course, hemophilia-A patients, you have got to memorize your immune response. It means that you have memory T-cells, memory B-cells. It is a kind of surveillance. They are there just to react whenever necessary. If you expose, and this is certainly the case, Factor VIII on a regular basis to the same antigen, what the antigen is going to do is first, of course, to trigger the memory response and, in this case, the B-cell memory response against Factor VIII and trigger a new set of somatic mutation from the memory B-cell there from this new set of somatic permutations, you will get a new population of memory B-cells and then your population of plasmacytes which are going to be clone to the bone marrow and sit there probably for a few weeks to produce the high-affinity antibodies. You have to understand that each time you lose the Factor VIII in such a setup, you will, again, embark on this circle and create new memory B-cells and new plasmacytes. The point is to decide what happens exactly in Factor VIII. Basically, we don't know. It would be dishonest just to say that we know everything about the immune response against Factor VIII for many reasons. Perhaps, as someone said at a previous meeting, there are not many immunologists interested in this Factor VIII immune response. It might not be very reason. The second reason by be that Factor VIII is a huge molecule. Whenever you work with mice, you like to use what we call circus antigen, antigen which are as small as possible you can define a single epitope. That is much, much easier. Now, the knowledge we have acquired over the last let's say ten years about the way those B-cells, when they are activated by interaction with T-cells, what their fate is, is really tremendous. You have basically two pathways. The first one is not a classical one but which is terminal-center-independent which is going to generate B-cells with a short life but producing antibodies with the IgG4 isotype if it is programmed with no somatic mutation but with the capacity of cloning to expand. On the other hand, you have a size 8, a new run of memory B-cells and long-lived plasma cells which are going to sit in the bone marrow. That is about it for the homeostasis for the Factor VIII immune response except that, on the other side, you have regulatory T-cells and anti-idiotypic B-cells. I am not going to say a word about anti-idiotypic B-cells but I would like just to show one slide about regulatory T-cells. This is certainly an emerging field and especially in the field of Factor VIII. If I have to make kind of a guess for the forthcoming five years, we will learn a lot about the role of those regulatory T-cells in the immune response, the control of the immune of response against Factor VIII. Basically, we distinguish now a not suppressive but regulatory T-cell which is characterized by the presence of CD25 receptor. This is the IL2 receptor. These cells were trained to the CD4 lineage and, more recently, the transcription-repressor factor, FoxP3, is now considered as the main characteristics of those T-cells. We know for a while, for almost ten years now, that the thymus is actively selecting some of those T-cells. They are hosted in the periphery and act there as a natural counterpart for immune response. But, on the other hand, and this is somewhat new and there is as paper last month in the Journal of Clinical Investigation showing interesting data in man that, indeed, even those CD4 T-cells without the two characteristic markers, whenever they are in the periphery exposed under specific conditions, they can just change themselves and acquire to those markers and become part of what we call the adaptive regulatory pathways which, of course, means, the message is there, that this is probably amenable to manipulation. I would say that, perhaps, we speak much about the role of immunogenicity of Factor VIII as a molecule, per se. We should program another symposium on regulatory T-cells and the way the immune system could program down the immune response against Factor VIII. Now, what about the animal model? I said and I promised to make a kind of cautionary tale, so I will try to keep on my promises. Optimal criteria for an animal model; well, you have to act with an animal where the genetic background is identical. Otherwise, you end up with consideration about extra MHG factors or whatever which could have, indeed, an influence on the capacity to mount an immune response against Factor VIII. Ideally, the model should be deficient in the antigen, and I mean fully deficient, which is probably not the case right now with hemophilia in mice. You should, and this is maybe the main point, be in a possibility for evaluating the immune response at the clonal level, be it, at least in vitro but, if possible, also in vivo. Of course, no or limited adjuvants because, as soon as you use adjuvants, like CFA or IFA, or even milder adjuvant, you distort the immune response one way or another. What are the models available? Well, why not start with the kind of them which is wild type mouse. You have the hemophilia-A mouse, immunodeficient mice, the combination of the Factor VIII deficient, immunodeficient, and more recently the transgenic mouse strains. Now, why should I spend two slides on normal mice? Of course, in normal mice, the mice have Factor VIII so whatever you do with your human Factor VIII, you will skew the immune response against the determinants which make the difference between human and mouse Factor VIII. In this case, the repertoire of those mice should be purged of all the T-cells recognizing not all the T-cell epitopes present on Factor VIII but all the immunodominant T-cell epitope on Factor VIII. This analysis is of very much importance. The usefulness, I think, and has been over the years is to establish just a library of monoclonal antibodies. What for, you would say. Well, we learn a lot about the mechanism by which Factor VIII is inactivated. We learn a lot about how the structure and the function of Factor VIII are related and I think it is still a good tool to compare the antigenicity of factors. I will give you an example of this. This is an old study, at least five years old, I think, just to illustrate the point. This is the heat-denatured Factor VIII. It is a plasma-derived Factor VIII which was heated as we normally do for factorization, 63 degrees centigrade. We use a mouse monoclonal antibody recognized in acidic A3 domain. This is the type of activity in optical density on the vertical axis and, of course, increased amount of Factor VIII on the horizontal axis. You see that, as soon as you heat for one minute, at this temperature, you will lose quite a lot of the reactivity. The message is that an antibody against the acidic A3 domain, which is, of course, a crucial part of Factor VIII not only for the binding of von Willebrand factor, you will lose a lot of reactivity with some of the monoclonal antibodies. This one is a conformation-dependent antibody against the acidic A3 domain. So what about the knockouts? Everyone is picking the knockout mice. The knockout mice should be fully immunocompetent in terms of the immune response against Factor VIII. So the repertoire should not be skewed towards the non-specific for human Factor VIII. I think it is interesting but with limitation first of all because it is not quite sure that the Factor VIII hemophilia-A mice which is now available, and there are basically two strains, are totally devoid of Factor VIII. To the contrary, the XLM17 knockout mice have been shown to have a part of the heavy chain of Factor VIII floating around which might have an influence on the way tolerance is induced against Factor VIII. We don't know for sure, but it might be the case. So ideally speaking, in an ideal world, we still are expecting to have a fully Factor VIII mouse strain available. But, with this type of model, it is possible, I think, to directly compare the immunogenicity of different batches of Factor VIII and possibly also to evaluate the impact of added factors, like, of course, von Willebrand factor. You probably remember this slide coming from a paper we published with a student from Frankfort working at that time in biotests. AUDIENCE: What are the yellow and white on the graph. DR. SAINT-REMY: Yes. I am going to explain it. This is Bethesda units. This is the influence of von Willebrand factor on the immunogenicity of Factor VIII concentrate. What we did is to take a population of hemophilia-A mice. They were injected with plasma-derived Factor VIII or recombinant Factor VIII or von Willebrand alone. In some cases, plasma-derived Factor VIII No. 2 and the two recombinant Factor VIIIs--these were, of course, then deprived of any von Willebrand factor in the final formulation. This one is containing a kind of degraded form of von Willebrand factor. So, what you see in the yellow bar is just the Bethesda units obtained in groups of those mice injected with product as it is commercially available. So, what we did is to add, in some cases, some von Willebrand factor just to see that, in this plasma-derived Factor VIII, additional fully functional von Willebrand factor would decrease by 50 percent at least the level of inhibitors against Factor VIII. The same for this recombinant Factor VIII here. So the point is not to make kind of a lot of fuss about this but we think that we that we should look much more closely on the way von Willebrand factor is probably modulating the immunogenicity of Factor VIII. What I am saying is von Willebrand factor. It may be that other factors do play a role, too. Immunodeficient strains; we have been playing a lot with SCID mice. SCID stands for severe combined immunodeficiency. It means that those mice have no functional B and T-cells. They are unable to mount an immune response against any protein, any cells and, of course, not against Factor VIII. The downside of this is that, because of having no function B and T-cells, they have no lymphoid organs so it is impossible to mount a primary immune response in those mice which is basically a limitation. So, what you have to do is to reconstitute those mice with cells and they will accept cells because they are unable to reject them, and you can reconstitute groups of those mice with cells pertaining to different patients that are, of course, already primed cells, B and T-cells. I think this is useful because you can still compare different Factor VIII preparations or different individuals together injected with the same Factor VIII preparation in a setup which is a mouse environment. I will just show you the way we do it. This is the donor. We have to take quite a lot of those cells, but this is not unlimited because, if you go too far, of course, the graft is going to reject the host. So the SCID mice are injected with IP, with a number of those PBMC. Then you inject either nothing or either Factor VIII. Just to illustrate the point--this is not published--again, you have the inhibition of Factor VIII activity here. You see that when you take the plasma IgG from the patient from which the cells were taken to reconstitute the SCID mice, you have a good capacity to inhibit Factor VIII as a function of total IgG concentration. This is the result of finding a number of six of those SCID mice reconstituted with the cells of those patients just to make the point that it is possible, as you see, to reconstitute an inhibitory response, secondary response, in those mice. You see that the variation from one mouse to another is certainly very high and this is not the ideal model. So we switch to another type of model which is available which is combining no Factor VIII and no immune system. For the time being, this has been extremely useful to evaluate the immunogenicity of different vectors used for gene therapy and especially in collaboration with Ivan Dandridge in our lab. Now, the caveats, because I promised to be cautious, the number of FH genes in the mouse is about 100-fold higher than what you see in man. So the capacity to start with the diversification of the B-cell compartment in the mouse is about 100-fold higher in man so it is difficult to extrapolate at the molecular level what you see in the mouse to man. Of course, the MHG-S2 determinants are distinct in the mouse as they are in humans. We know that if you take human Factor VIII, inject human recombinant Factor VIII, in hemophilia-A mice, you will get an immune response. If you do the same with mouse recombinant Factor VIII, you will get not only another type qualitatively different immune response but also the titer of inhibitor is going to be much lower. So there is some difference between mouse and human Factor VIII although the two molecules are highly homologous. The effect of inflammation is, to me, something we should absolutely look at. There are many, many reasons why the induction of an acute inflammation or joint bleeding or so could, indeed, trigger the production of inhibitor antibodies and I think we should set up protocols to understand this more. Of course, the caveat which is probably the first one is that hemophilia, being heterogenous, I don't know if many of the human population where the inbreeding is complete, where everyone has, of course, the same deletion in the Factor VIII gene, which have treated at the same time and the same place and the same conditions. Now, the way we have chosen to try to circumvent at least some of those difficulties is to say, well, it is almost impossible to take a mouse model and extrapolate everything up to the human situation. So why don't we try to go back to the mouse and only to the mouse? Why don't we try to use an hemophilia-A mouse, inject it the normal way with mouse urine, with murine recombinant Factor VIII, just to avoid differences between the two molecules which we do not understand, to be honest. As I said, the best thing about animal models is the capacity to follow the immune response not only in vitro but in vivo too at the clonal level. So we have opted to work on the transgenic mouse model. But, of course, if you now bombard the genome of a mouse with a few of those receptors coding for the BCR or TCR against Factor VIII, and insert them at random into the genome, you will end up with something which might not be very interesting. So, what we do is a target replacement, what we call a knock-in system, where you integrate the receptor you are interested in at the right place in the genome. The idea is that, by doing so, it should be amenable to all the physiological modulations later on. So the usefulness of such a model, which is ongoing now, is to allow a clonal analysis of the anti-Factor VIII immune response not only in vitro and in vivo but also to take the cells, separate the cells in vitro, and reconstitute all the mice with this, just to put those transgenic cells into a more normal physiological environment. It should be useful for evaluating treatment strategies. It should be useful, too, to evaluate more so-called physiological conditions like inflammation, sub-QT injections and so on. Now, I have to give some kind of conclusion about what I think we should do. I think, with the tools we have now, and this is probably provocative, we should take the pain of comparing the immunogenicity of Factor VIII preparation in the Factor VIII knockout mice we have now. We should evaluate the level of specific antibodies, level in inhibitors, but also run a kind of epitope mapping. You know, I am pretty convinced that, if we look carefully, it might be possible to have a kind of a footprint, each product having a specific pattern of antibodies recognizing different regions. The B-cell epitope mapping is now something which is routinely used. We use, on a routine basis, about 55 different fragments of Factor VIII and we can, using this technique, identify almost any possible B-cell repertoire on the Factor VIII molecule. The technique is simple. From the DNA of Factor VIII, we made a vector. This is transcripted in the system containing ribosomes from rabbit reticulocyte lysate. So everything is there in terms of amino acid to make small bits of peptides except one marker which is methionine which is radiolabeled. Now, if you have your antibody, you can mix it with a solid phase in which you have protein A or protein G. the mixture is then incubated with the polypeptide of interest, one out of the 50 we are now currently using. You have a kind of complex bond which can be precipitated and you just can do radioactivity on it or you can disassociate the peptide and look for the real signs of the peptide you have been precipitating. That is very handy. It takes less than one day to run the assay and, with the well-skilled technician, you can run about 50 assays a week. I should skip on this because the previous speaker already said that. Just if we wish to make a prospective trial in PTPs, as I was asked to speak about this, I would certainly not restrict myself to assess the level of inhibitor antibody. I would also look for not only the titer, all the antibodies against Factor VIII. I would also try to make the picture more clear about the capacity of those antibodies to interfere with physiological partners like von Willebrand factor and phospholipids and I would run an epitope mapping in a systematic way. I can tell you that we have now data on the intron-22 inversion where it seems that, indeed, the mapping--this is coming from human beings--the mapping of epitopes made by--antibodies made by those patients, seem to emerge as a clear picture. Last, but certainly not least, what we should do at the T-lymphocyte level. I have no time to speak about this but this is going to be crucial in the coming five years. Thank you so much. (Applause.) DR. LOZIER: Again, we have time for questions, perhaps one or two. Go ahead and activate your microphone. AUDIENCE: I was a bit intrigued about the mice experiments that you did where you add von Willebrand's factor and you saw a reduced, I guess, immunogenicity of the Factor VIII products. I was just wondering how that was done. Was the von Willebrand-containing product mixed with the Factor VIII prior to being administered to these mice, or was it a sequential thing where-- DR. SAINT-REMY: We did both. AUDIENCE: You did both. And the results were the same. I guess one of the questions is, as a result, can we get any information from that in terms of how much endogenous von Willebrand's factor in a factor and is there a correlation between that and inhibitor risk, and what about giving DDAVP to these mice who presumably do have von Willebrand's factor in them and you can certainly almost duplicate what you did just by increasing their endogenous von Willebrand's factor. DR. SAINT-REMY: To answer the first part of your question, the answer is no. We have absolutely no clue through such experiments to give you an answer to this. About the DDAVP, this is another story. We are working quite hard on DDAVP and we have reached surprising findings; basically, that is to say that the prediction of von Willebrand's factor and Factor VIII might be completely dissociated under certain circumstances and maybe Factor VIII is not only produced from the hepatocyte as many people do think. We have a system--I cannot speak about it too long--is about the fact that we just perfuse human lungs with a system containing or not DDAVP and it is possible to produce Factor VIII in this system. DR. LOZIER: Could you identify yourself and give your affiliation for the transcript. AUDIENCE: Oh; I'm sorry. Manuel Carcao from the Hospital for Sick Children in Toronto. DR. LOZIER: I think we can take Dr. Aledort's question. DR. ALEDORT: On the same vein, do you think it may have something--the von Willebrand's factor presence may have something to do with your assay of inhibitor function because of the inhibition of inhibition of catalytic activity that you have also shown in the presence of more von Willebrand's factor, that it is less immunogenic but that the ability to measure it may be altered? DR. SAINT-REMY: That is a pretty good point. I think we already discussed this. In this system, we check for the presence of catalytic antibodies but it was not possible to detect it, of course, at the polyclonal level. DR. LOZIER: There is just one question here, please. DR. BERGEDRIVER: Bergedriver, Baxter. I think we have discussed the limitations of these results with von Willebrand's factor in mice for quite some detail in the past. My question is what do you think you see effects of von Willebrand's factor with one recombinant Factor VIII product but not with the other recombinant Factor VIII product? Have you got any explanation for that. If that would be a more sort of general feature of von Willebrand's factor, you should see a reduction in both recombinant Factor VIII products that you used for your studies. DR. SAINT-REMY: You are perfectly right. But, I mean, these are experimental data. That is all I can say. We were really surprised to see those data and we thought it might be interesting enough just to try to publish them. But I have no explanation. Of course, you and me, we have about fifteen different hypotheses to explain this, but-- DR. LOZIER: I think we need to move on to our next speaker, in the interest of staying on schedule. Dr. Sanj Raut of the Division of Hematology at the National Institute for Biological Standards and Controls at Hertfordshire in the U.K. will give us a talk on the regulatory aspects of the Factor VIII inhibitor assay. Thank you. Regulatory Aspects of Factor VIII Inhibitor Assay DR. RAUT: I would just like to thank Dr. Weinstein, Dr. Lozier and colleagues for inviting me to give my talk today. In my talk, I will be concentrating on issues related to the standardization of Factor VIII inhibitor assays and, in particular, I will be showing you some a number of collaborative studies that have addressed the issues of the difficulties in measurement of Factor VIII inhibitor. One of the early studies that actually specifically addressed these difficulties was a study carried about by Austen and colleague who published their data in Thrombosis Hemostasis 1982. In this study, they essentially compared two inhibitor assays, the standard Bethesda and the New Oxford methods. In the study, they had eight plasma from inhibited patients. Seven of it were from hemophiliacs and one was from a patient who developed spontaneous antibody to Factor VIII. These samples were distributed to eleven laboratories and these participants were asked to carry out both the New Oxford and the Bethesda assays on these samples. So here is a table just showing you, perhaps alarmingly, the large interlab variability we see when using both methods. You can see that, for the Bethesda, we see CVs between 38 and 78 percent compared to a range of 47 to 128 percent for the New Oxford method. Incidentally, the spontaneous antibody sample 4, again we see a relatively large CV there and for sample 7, which was, in fact a sample with a low inhibitor titer--and I haven't got the slide here, but, essentially the Bethesda assay, the majority of the labs showed some sort of detection when it came to sensitivity whereas the New Oxford method couldn't find any antibody at all. The majority of the labs couldn't find any antibodies. So this study also looked at interlab variability. We can see that, again, although it is relatively large, 37 and 65 for the two methods, they were, in fact, much lower than the previous interlab variabilities. Another study here which looked at the difficulties measuring the Factor VIII inhibitors. It was a study carried out in 2001, in fact, by Eric Preston and Tim Woods. This was a study, it is a UK-NEQAS study, which looked at essentially the Bethesda assays across a number of laboratories. In fact, 60 U.K. labs were involved and 18 international labs. The samples that they included in the study, first of all, two samples, was the 110 sample which is essentially a plasma from a hemophilic patient with inhibitor which also, incidently, cross-reacted with porcine Factor VIII. Now, this sample was HCV-positive and was essentially only sent out to the U.K. laboratories. The second sample, the 110A, was a plasma from a patient with an acquired inhibitor and this was sent out to both the international and the U.K. laboratories. Participants were asked to carry out a single assay on each sample, primarily by the Bethesda assay. They were also asked to declare the sort of local limit of detection of these assays and they obtained quite a varied response. But, in general, the majority of the labs showed that the lowest limit of detection was around 0.5 Bethesda units. So, looking at the actual human Factor VIII:C inhibitor assay, let's concentrate first primarily on the CVs. Again, we see a relatively large interlab variability. We see these around 47. The same sample was assayed in a porcine inhibitor assay. They obtained a slightly large CV of around 60, 60.5, percent. On the second sample, 110A was assessed. This was the acquired inhibitor. Once again, we see large, relatively large, CVs for both the international and the U.K. labs varying from 68 to 86 percent. Now I am going to talk about a study that was carried out at NRBSC which is a NIBS wet workshop. This was a controlled study to compare the Bethesda assay where a number of participants, 16 U.K. labs, in fact, where invited. This is an unpublished study in which a number of hemophilic plasma samples were included which had inhibitors and also plasma samples which didn't have inhibitors. Now, these participants actually came with their own reagents and materials sufficient enough to carry out their own normal assay. The only thing that was provided was coagulation machines, coagulometers. Each participants were asked to carry out replica assays repeatedly over a three-day period in eight different sessions with different conditions. The findings of the study are shown here. Essentially, the plasma samples without inhibitor were assessed. We obtained between 15 to 26 percent CVs which is the kind of range we would expect. However, when an inhibitor was present, the CVs jumped to between 53 to 80 percent. Now, in the red, we have here, is a figure, 20 to 30 percent, which represents the same assay when repeated relatively to a reference standard. Moving on, when the inhibitor samples were assessed, when an incubation state was standardized--i.e., for all the labs--one, group, actually, assessed the incubation state--the CVs actually dropped down to 33 to 43 percent. Once again, the presence of a standard, this dropped even further, between 13 to 34 percent. When the same assays were repeated with both the incubation and Factor VIII assay, stage standardized--i.e., essentially the one-stage assay--the CVs dropped further, between 14 to 20 percent, this relative to the standard, came down even further to between 6 to 29 percent. We also looked at the intraoperator or interlab CV compared to the interlab CV and this, once again, appeared to be much smaller than the interlab CV. After these studies, it was decided by the ISTHSSC Factor VIII/Factor IX Subcommittee that we should carry out a collaborative study in the hope to possibly standardize Factor VIII inhibitor assay and, in particular, to develop a reference standard which may be useful in these assays. This study was carried out in collaboration with Dr. Steve Kitchen at Royal Halamshire Hospital. In this study, we had two samples containing human anti-Factor VIII monoclonals, both Type 1 and Type 2, and one sample containing a rabbit antipolyclonal These were assayed in a multicenter study, 15 centers involved. 17 sets of data were collected using the local Bethesda method, some 5 percent of which were the Nijmegen modification, and variability of inhibitor assays were assessed. We asked the question, could any of these reference materials assist in the standardization of this drug. Looking at the patient samples, once again, we obtained a relatively large interlab CV varying from 33 to 52 percent. For the individual reference standards, again, we obtained relatively large CVs when measuring the Bethesdas varying from 26 to 30 percent. Interesting, we also looked at the difference between one stage in chromogenic assays. Here we see Bethesda titers right across the board with the patient samples on the reference preparations and we see that, generally, the chromogenic assays gave much lower Bethesda titers compared to the one-stage assays. This was significant for the patient 1, 3 and the rabbit polyclonal antibody. Two points here, really. First of all, the chromogenic assay, you could speculate that it may have something to do with the larger dilution that has been used. Also, the one-stage assay tends to have, or can have, a longer lag phase in the dilution perhaps with a shorter incubation time and this could, then, perhaps, create a large Bethesda titer. Secondly, across here, we see patient 1, 2, 3 and their other polyclonal antibodies are intact, polyclonal in nature, and it may not be surprising to see that they behave quite similarly. This is just illustrating using stacking history. I am illustrating the effect of a particular standard. Here we have, first of all, in the top slide, we see the variability of inhibitor 1 without any standards in the presence of, in this case, the polyclonal antibody reference standard. We do see a better agreement. This can be seen in a sort of correlation graph. Here, once again, we are looking at inhibitor 1 patient relative to the polyclonal antibody and we see you get a very good correlation in all the different types of assays used. So the relationship in terms of correlation coefficients; really, we obtained a significant correlation primarily with the polyclonal antibody. We did get some correlation for patient 1 using one of the human monoclonals but, primarily, it was the polyclonal. That seemed to have good correlation with these inhibitor samples. So, now looking at a table showing, first of all, the effects on CVs. Without a standard, we see that the CVs are generally large for the three patient samples and, when we look at the CVs relative to the standards, we do see a drop in CVs. But the best drop is primarily with the rabbit polyclonal antibody and dropping down to between 26 and 33 percent. The conclusion really here is the high interlab variability that we see is expected compared across the previous studies. The high interlab variabilities are actually much greater than normal Factor VIII assays. We do see some improvement in CVs between centers using standards reference particularly for the rabbit polyclonal. So the next step really is, perhaps, to look at a large-scale production of one of these materials as a possible standard with a full multi-assay, multicenter-study assay. If we were to carry out, it would be nice to find what sort of level we should put into these standards which may be useful. Possible advantages of a reference preparation would be, obviously, to reduce interlab CVs. It may also be useful to have QC material for labs for clinical studies and also as a common sample in evaluating a number of new methodologies that are currently being developed. So the proposal is to seek out feedback from participants and the hemophiliac community in general before embarking on this study. Before I finish, I would just like to raise a number of points which I think we should consider. We know that there is a high interlab variability and this is directly due to the presence of inhibitor. We know that the intralab CVs are better than interlab CVs by a physician within a lab but it is difficult to get an agreement between lab and better CVs are obtained with standard; hence, the need for a standard. Other points to consider. There is a mistake here. The chromogenic--Bethesda titers obtained using chromogenic assays are relatively better, smaller, compared to one stage. And this may be due to a plasma-matrix dilution effect due to the chromogenic assay, itself. We may need to look at modifying the actual assessment of the residual Factor VIII activity. This could be, for example, looking at high dilution stage in a one-stage assay, for example, and, also, perhaps, we could look at the reduced or standardizing the actual incubation time of these assays. Same points time for a different Factor VIII assay to complete and to vary and this will affect inhibitor time to neutralize Factor VIII. Finally, should we consider standardizing the different stages of the individual assays; for example, the incubation stage such as the antibody dilution step which is critical and, perhaps, we should look at Factor VIII assay stages, itself. These points are difficult for labs to agree on but they may serve as useful guidelines. I would just to finish. I would like to thank Drs. Jorgen Ingerslev and Marc Jacquemin for providing antibodies and the various participants, the various labs, that took part in these studies. Thank you. (Applause.) DR. LOZIER: We have time for a few questions. Once again, activate your microphone and please identify yourself and give your affiliation. DR. KEY: Nigel Key from the University of Minnesota. These antibodies, do they have Type 1 or Type 2 kinetics and, if so, are they also standard--have you looked for the acquired antibody patients or mild inhibitor patients with antibodies? DR. RAUT: The two monoclonals that we used as the reference standard, one was Type 1. The other was Type 2. But we haven't specifically looked at the antibodies, the spontaneous or the quiet inhibitor examples using this specifically. DR. LOZIER: Any other questions? DR. LAWLER: Pete Lawler, Emory University. The Bethesda assay is defined as the dilution of manamide that produces 50 percent inhibition. In practice, when you look at the original paper that described the assay, there is a line that is drawn between 25 and 75 percent inhibition for which there is no experimental basis for the slope of that line. In practice, when you talk to technicians about how they define that 50 percent level, you get a variety of different answers because it is frequently just left up to the technician and there are no standardized protocols by which that 50 percent inhibition is found. In our experience, we experimentally tried to find that point by doing a series of dilutions and then doing a regression to find that value and find that there is a fair amount of scatter associated with it, much higher than the precision of the assay. In your studies, how do you actually--or have you proposed to try to more rigorously define the 50 percent inhibition points to try to decrease interlab variation? DR. RAUT: I think these are the points we are going to address in the next study which I forgot to mention. But essentially, you are right in what you are saying. Different labs effectively do different things when it comes down to the actual inhibitor assay. The stage you are talking about, the level between the 25 and 75 percent, they do tend to vary considerably. These assays are carried out as one-point assays because primarily they are clinical labs and they are going through a huge number of assays. In a research lab, you always do varying degrees of dilution steps. to find your exact 50 percent level. So, yes; we would like to address this point in the next study without being too prescriptive. This kind of thing we could discuss with the Factor VIII-Factor IX Subcommittee when it comes down to actual putting together the protocol. DR. LAWLER: I would also like to make a comment. I think, in principle, using a rabbit polyclonal standard is not a good idea because you are going to create an apples and oranges comparison because of the fact that there is a lot of heterogeneity among human samples in terms of what epitopes are being recognized and it is not really possible to define a reference standard for an inhibitory antibody, in my opinion, because of the polyclonal and heterogeneous functional properties of the different antibodies that we are looking at clinically. DR. RAUT: You are probably right there. I think we are limited as to what we could use as a standard. It would be very, practically, impossible to get human plasmas as a reference standard at this level. So, realistically, do you propose monoclonals? We have access to both polyclonals and monoclonals. This, again, is something we could discuss before proceeding with the protocol. DR. LOZIER: Do we have one more question? Dr. Kessler? DR. KESSLER: From a practical standpoint, on all of the assays that you performed on the specimens, was there any difficulty in determining the difference between a high-titer inhibitor and a low-titer inhibitor or are you just talking about laboratory precision on your assay? DR. RAUT: In our particular study, the last study, we were looking at the interlab variability and the precision within the lab should have been addressed but wasn't, which we would hope to do that for the next study. But, really, we are trying to compare the precision with the agreements between labs for these assays. DR. LOZIER: Thanks very much. DR. RAUT: Okay. DR. LOZIER: We will move on to our next speaker, Dr. Bert Verbruggen of Nijmegen, the Netherlands, who has worked for many years on modifications of the Bethesda assay that people used to assay the Factor VIII inhibitors. His modifications to the Bethesda assay have been known commonly as the Nijmegen modifications and we are pleased to have him present his talk on innovations in the Factor VIII inhibitor assay. Innovations in the Factor VIII Inhibitor Assay DR. VERBRUGGEN: Thank you, Jay. First of all, I want to thank the organizers for inviting me to give this lecture here. I am going to talk about the specificity, accuracy, precision and interlab variation and sensitivity. I am going to limit myself to the Bethesda-based methods. Concerning specificity, we have done some research on the normal pool plasma and the control sample. It is imidazole buffer in the classical Bethesda assay. Concerning the sensitivity, we have done some work on the patient for treatment of patient plasma and a mix of patient plasma and normal pool plasma. Concerning the accuracy, I show you some results on the study that we have done on Factor VIII activity assays, especially on the use Factor VIII deficient plasma and also the use of Factor VIII deficient plasma and the Nijmegen modification. Specificity is the ability to obtain normal results in a normal situation. We have modified the classical Bethesda assay in two ways. We have changed the control sample and we have proposed to use Factor VIII deficient plasma instead of imidazole buffer to be sure that the protein content and the control mixture and the test mixture are equivalent and we have buffered the normal pool plasma at pH 7 to obtain a stable pH and a stable Factor VIII activity. The modification has a better specificity. Allen Giles has done some research on it. I think in this afternoon's session, there will be more about this research. He found that in 887 samples of the same number of patients an increase in data of zero Nijmegen Bethesda units compared to the classical Bethesda units, an increase of 32 patients with an inhibitor activity of zero. Of course, he found a decrease in the number of samples with an inhibitor activity in the grey zone between zero and 0.5 Bethesda units and in the number of patients with higher Bethesda units. So these data are not, per se, an indication for a better specificity because they should be correlated to a golden standard. But, at least, it is an indication that the Nijmegen Bethesda assay is more specific. Currently, in our laboratory, we are trying to correlate both the classical Bethesda assay and the Nijmegen assay to a gold standard like the kinetic measurements. Concerning the specificity, these are our data of the last year. We analyzed 79 patient samples. Some of the samples were from the same patient but, in the last year, we analyzed 79 patient samples for Factor VIII inhibitor activity and we found 74, less than 0.2 Bethesda units and Nijmegen--but, of course, we analyzed it with the Nijmegen Bethesda assay--we found 74 samples with less than 0.2 Nijmegen Bethesda units and we found five samples with an increased inhibitor activity. Two samples were from a patient with a lupus anticoagulant. One sample was from a patient with a history of inhibitor at the end of his eventual onset of therapy. And there were two samples of one patient with an inhibitor titer of about 0.4 Nijmegen-Bethesda units. This patient did not have--these were recent data. This patient does not have clinical signs of the presence of an inhibitor and currently we are doing some kinetic experiments. So we are not sure whether this patient has, indeed, a Factor VIII inhibitor. So our conclusions with respect to the specificity is that we figured false-positive inhibitor results have been eliminated in the Nijmegen-Bethesda assay by buffering the normal pool plasma and by replacing the imidazole buffer by Factor VIII deficient plasma. Accuracy; accuracy is a measure of agreement between the estimates of a value and a true value. I think there at least two important theoretical items; that is, that the Factor VIII activity in normal pool plasma that is used as a Factor VIII source in the incubation mixture may have some influence on the accuracy; that means when you have normal pool plasma with varying Factor VIII activity, this also causes variations in the accuracy and also, of course, the variations in the Factor VIII:C activity assays will have an influence on the accuracy. But Dr. Raut has also showed you a lot of data on it. We have done some research on the influence of the Factor VIII deficient plasmas that are used in the Factor VIII activity drug is used as substrate plasma in the Factor VIII deficient plasma that are used as a control sample. These are the results. We measured some four or five different samples. These are the results of one typical sample. We analyzed this sample with all types of--with the four types of Factor VIII deficient plasma, a substrate plasma and a Factor VIII activity assay and with the same samples of the same Factor VIII deficient plasma as control samples in the mixture. We saw that the highest inhibitor titers were in samples with control when we used chemical depleted plasma as a control sample and other deficient plasma as substrate plasma in the activity assay. The reason for these high titers was the presence of activated Factor V in the chemical depleted plasma. The lowest data we received with the immuno-depleted plasma and especially with one type of the immuno-depleted plasma with a very low, or the absence, of von Willebrand's factor in this Factor VIII deficient plasma. In fact, intermediate data we received with an almost homogenous system with the chemical depleted plasma so can be depleted plasma used in the Factor VIII activity assay and used as control in the mixing studies and also, but a little bit higher, with congenital deficient plasma, in the homogeneous system with congenital deficient plasma. I think that the conclusion is that the accuracy of the Nijmegen-Bethesda assay is influenced by the type of Factor VIII deficient plasma. Of course, it is important before you can define the accuracy, you need a standard. Until now, there is no standard but Dr. Raut also has talked about that in the last talk so I won't go further on it. Interlaboratory variation; I shall spend only a few words on it because Dr. Raut has shown these results already. But our conclusion is that there was, in the survey of Dr. Raut and Steven Kitchen, no difference in the interlaboratory CV between the Nijmegen assay and the Bethesda assay. So the Nijmegen modification does not influence the interlaboratory variation. Sensitivity; the sensitivity is the ability to detect an abnormal situation. So when you use a golden standard which says whether an inhibitor is present or not, and you use a test to assay this inhibitor with a certain cutoff value, the sensitivity is defined as the number of true positives divided by the number of--sorry; the number of positives with the golden standard and inhibitor test divided by the total number of true positives. But do we have a golden standard? Secondly, can we define a clear cutoff point in order to decide which test is positive and which is negative. I think the answer to both questions is no. I think the answer is no because Factor VIII kinetics after replacement and bleeding status are golden standards. But I think they are not 18 carat. The cutoff value for the Nijmegen-Bethesda assay in the literature is 0.4 units per ml. However, this is not evidence based and it is only slightly better than in the classical Bethesda assay. The sensitivity of an inhibitor assay depends on the ability to detect small changes in Factor VIII:C activity in the test mixture. But normally the coagulation assays only have limited precision to detect small differences in Factor VIII activity. So, we tried to improve the sensitivity of the inhibitor assays and we looked at the patient plasma and at this mix of patient plasma and normal plasma. We developed a test which we called the Nijmegen low titer inhibitor assay. In short, in the test, the plasma of the patient and the control are concentrated by selective protein filtration by a centrifuge technique and the concentrate rate is about four times. It depends on the filter you use. The inhibitor type is measured in the mixture with normal pool plasma that is stabilized at a pH of 7.4 in a 1 to 3 ratio, so three parts concentrate patient plasma and one part normal pool plasma. Any residual Factor VIII in the patient plasma which is disturbing this test is removed, destroyed, by incubation with EDTA. We have got these results. When we spiked a normal hemophilic plasma with an antibody to inhibit to Factor VIII to a concentration of about 0.2 Bethesda units per ml, and we measured with the normal Nijmegen modification the inhibitor activity. Then we get a correlation between the spike concentration and the measured concentration that is not significant. So this method is not able to detect these low inhibitor activities. When we analyzed the same samples with the low titer assay, and here are the inhibitor activities. We express it as a low titer inhibitor unit. Then we see a highly significant correlation between the spike concentrations and the measured concentration. Our conclusion is that, with the low-titer inhibitor assay, the detection limit is increased about 10 to 15 times so it is 10 to 15 times lower than in the normal Nijmegen-Bethesda assay. But should low titers be of concern, the important question. We are only a small hemophilia-treating center and we have about 35 severe hemophiliacs in our center. At this moment, we only have two patients with an inhibitor, a low inhibitor. I show you some results with these two patients. Patient 1 is a man born in '72 with severe hemophilia. Von Willebrand's factor is 40 percent or 0.4 per units per ml and genotype is an inversion intron 1. Because of his Factor VIII inhibitor--he is a long-standing Factor VIII inhibitor that started already in 1983. But, after all these years, the inhibitor titer became zero about three years ago and remained zero over the last three years. But there were some clinical problems with this patient. The patient needed more Factor VIII concentrate than could be expected and the patient has still, unless inhibitor activity was zero, still had a severe bleeding tendency so we performed a kinetic of Factor VIII concentrate and of Factor VIII von Willebrand's factor concentrate and we found that the half-life of Factor VIII in this patient was extremely low, 2.9 hours where the normal range was between 8 and 12 hours. The same was, in a lesser extent, true for the Factor VIII von Willebrand's concentrate. Case 2 is a man born in 1942 also with severe hemophilia, with a normal von Willebrand's factor and the genotype inversion intron 22. This was a fast-growing inhibitor. It was a low response with the highest activity of about 2.1 and also this patient returned to activity of zero about two years ago and also remained zero until now, with the normal Nijmegen-Bethesda assay. But also this patient has much higher need for Factor VIII than could be expected in a patient with a chance of an inhibitor. Also, these patients suffered from abnormal bleeding tendency. The inhibitor titer was zero. We also performed a half-life, a kinetic measurement, and we found the half-life of 4.1 hours. It is also very much decreased. The clinical presentation in both patients suggest the presence of inhibitor because of the unusual bleeding tendency and because of the fast disappearance of the Factor VIII. But the Nijmegen-Bethesda assay was negative. So we performed the low titer assay on a number of samples which we had in stock of these patients. At the time that we performed the kinetic studies when the inhibitor titer with the Nijmegen-Bethesda was zero, we found an increased inhibitor titer with the low titer assay. This sample we had in stock and we found a very high inhibitor titer. These are not Nijmegen-Bethesda units but these are some other units. We have to define it further. At this moment, these are the latest data which I got from the laboratory the day before yesterday. Still, this patient has an undetectable inhibitor with the Nijmegen-Bethesda but still these patients have an increased inhibitor activity with the low titer assay and, moreover, this patient still has a big need for Factor VIII and still suffers from abnormal bleedings. Case 2, this patient reached the maximum level of 3.2 Nijmegen-Bethesda units and then the inhibitor rapidly decreased to zero. At this point, we performed the kinetic of Factor VIII and, at this point, we also analyzed the low titer inhibitor assay and found 1.2 low titer units. At this moment, the patient has both with the Nijmegen-Bethesda assay and with the low titer assay, zero activity of inhibitor. At this moment, this patient doesn't have any abnormal bleeding and we are performing at this moment again the Factor VIII kinetics. But this patient does not need any more than normal Factor VIII. So, again, the question should low titers be of concern. I think maybe yes. Low titers of Factor VIII:C inhibitors may be of clinical relevance but we only have two patients. So I think we have to cooperate with other big centers to get more insight in this problem and I think more data have to be gathered to get more evidence about this problem. These are my coworkers. Thank you very much. (Applause.) DR. LOZIER: We have some time for some questions. Please activate your microphone or go to the stand-up mike and identify yourself. I see Dr. Aledort. DR. ALEDORT: Dr. Aledort, New York. Just a question. When the low titer was 7.0, what was the normal Bethesda unit without the Nijmegen? DR. VERBRUGGEN: So what you are asking for is the normal-- DR. ALEDORT: The regular Bethesda unit without the Nimegen addition. When you were at 7.2 at the time you had a low Nimegen. DR. VERBRUGGEN: Yes. At that time, I think the Nijmegen-Bethesda assay was 0.4. And so we think our experience is that the low titer assay is about 15 times more sensitive than the Nijmegen-Bethesda. So there is a good correlation between the Nijmegen-Bethesda and the low titer assay at that moment. DR. ALEDORT: But what about the normal Bethesda unit without the Nimegen part. DR. VERBRUGGEN: Oh; okay. I'm sorry; we did not perform that. DR. LOZIER: Dr. Lusher? DR. LUSHER: Jeanne Lusher, Detroit. I have two questions. One is when would you do the original Nimegen assay in assaying patients? I mean, at what level of Bethesda unit would you do that? Second, with this low titer Nimegen assay which your results look very fascinating, currently all of the trials, clinical trials with new products, use a cutoff as part of the inclusion/exclusion criterion, a cutoff of 0.6 standard Bethesda units. May we, by using that 0.6 standard Bethesda unit be missing people who really do have a tendency to form inhibitors because we are not doing this Low Titer Nimegen assay as an inclusion/exclusion criterion? DR. VERBRUGGEN: Yes. We have only little experience. We only had two patients. I think it is too early to draw conclusions. But I think it should be possible that we are missing--we are also missing these two patients with our normal Nijmegen-Bethesda assay. DR. LUSHER: But, just in the general population when you find--the hemophilia population when you are assaying for an inhibitor, when should we use the Nimegen versus a standard? I mean, just the ones below 5 Bethesda units or everybody? What would you suggest? DR. VERBRUGGEN: I should say always. But I think that the classical Bethesda assay has some limitations regarding the standardization. The Nijmegen-Bethesda assay is more standardized. So I think, if you have the chance to choose between a good standardized method or a less standardized method, I think you should use the good standardized method. I think we are able, we are more able, to define a good cutoff point with the Nijmegen-Bethesda assay compared to the classical Bethesda assay. But still there is no good definition. Also, not in the Nijmegen-Bethesda, say, of the cutoff. DR. LOZIER: In the interest of time, let's try one more question. Keith Hoots has a question back there. DR. HOOTS:n Keith Hoots, Houston. There was unusual PK on Case 2. I mean, you gave them 25 per kilo and you jumped all the way up to 80 percent. So the area under the curve was kind of unusual to begin with. How do you account for that? DR. VERBRUGGEN: Excuse me? I did not understand your question. DR. HOOTS: In Case 2, your in vivo recovery was 80 percent, at least according to the graph, after 25 per kilo, which is extraordinarily high in vivo recovery. Even though the half-life was short, the recovery was high, suggesting that this individual has unusual pharmacokinetics to begin with. Any explanation for that? DR. VERBRUGGEN: No. I don't know. I don't have an explanation. I think, with 25 units per kilogram, it is normal to get a top activity of about 80 percent and, in this patient, there was a rapid decrease of Factor VIII activity because of the presence of an inhibitor. DR. LOZIER: Before we take a break, I want to point out that we have some speaker handouts for this afternoon at the front and, in addition, we have a couple of this morning's speakers' handouts that are new and improved. So let's take a 15-minute break and return for business just a little bit before 10:45. (Break.) DR. LOZIER: In the interest of time, I would like to reconvene the workshop here after the break. We will be resuming with Dr. Gilbert White who is Professor of Medicine at the University of North Carolina at Chapel Hill. He has been, as you all know, a very prominent hemophilia healthcare provider for many, many years and a researcher in bleeding disorders and very, very active in the International Society for Thrombosis and Hemostasis. He will now present a discussion of the ISTH rationale of recommendations for use of previously treated patients in clinical trials. ISTH Rationale of Recommendations for Use in Previously Treated Patients in Clinical Trials DR. WHITE: I was sitting in the back before and having trouble hearing so can everybody hear me? I will try and stand close to the mike here. I think everybody knows that the stimulus for the current products that we use in the treatment of hemophilia, the stimulus for their development dates back into the mid-1980s when various bloodborne infections got into the blood supply and into the products that patients with hemophilia were treated. The first plasma concentrates were developed in the late 1960s. Hemophilia was recognized in the early 1970s but it was really the development of AIDS in patients with hemophilia that really was the stimulus to the development of new Factor VIII and Factor IX concentrates. Within two or three years, Factor VIII was cloned. Monoclonal Factor VIII first was used clinically a few years after that. Recombinant Factor VIII was introduced in 1987. Gene therapy followed and now we have second and third-generation products. It was, I guess, hoped and probably expected that monoclonal-antibody purified plasma and recombinant concentrates of Factor VIII would be free of those bloodborne infectious agents but, of course, in the initial clinical trials, the challenge was to demonstrate that. So I think the first trials, the clinical evaluation, was carried out in two phases, a first phase to demonstrate that the new products made by new technologies were at least therapeutically equivalent to the old products, and then a second phase. That first phase was generally carried out in previously treated patients as previous had been done, but then there was second phase to demonstrate the viral safety of the new products and those generally had to be carried out in uninfected patients. So, often, there was a phase I study in previously treated patients and then the a phase II study that was carried out in previously uninfected patients. Those studies, I think, we are all familiar with. One of the things that came out of those initial studies is that there appeared to be, at least in the studies in uninfected patients, a fairly high prevalence of inhibitors, much higher than we were used to seeing, the usual 15 percent that we were all used to seeing as clinicians. So, in the three initial recombinant Factor VIII studies, there were prevalences that were up in the 30 percent range, nearly twice what we were used to seeing. And so that raised questions in a lot of people's minds about these products and whether or not they had been immunologically altered, perhaps because they were made in animal cells, perhaps because they were purer than previous forms of Factor VIII. So there was considerable concern in the community about immunogenicity and the emphasis of those clinical trials really switched from there being questions about viral safety to there being questions about immunogenicity. I think we now recognize, although there is still some debate about this--I think we now recognize that this high prevalence of antibodies, one, did not reflect true high response inhibitors, that it was both low response, transient and high response inhibitors, and that the prevalence of high response inhibitors in these studies really was not greatly different from what we had been used to seeing. So the current thinking is that, at least in part, this increased occurrence of inhibitors is due to closer scrutiny of patients during these clinical trials. We certainly were looking for inhibitors much more closely than we had ever done before. I think, as a result of that, we began to see, really for the first time, what the natural history of inhibitors were. We began to see that inhibitors formed transiently and then go away, that some people do develop low-titer inhibitors that stay low-titer inhibitors and then the traditional high response inhibitors or high titer inhibitors that we had seen back in the 50s and 60s and 70s. That notion that, perhaps, these products were not as immunologically altered as the PUP studies made us first wonder was the study in previously treated patients where the occurrence of inhibitors was very low, 2 percent, 3 percent, even 1 percent, often one or two patients in the clinical trials. So, with that in mind, the idea developed that previously treated patients who had had a high number of exposure days to Factor VIII or Factor IX had really shown themselves to be tolerant to exogenous Factor VIII and Factor IX and, therefore, were considered to be at low risk to develop inhibitors. A corollary of that, if you will, was that, if one then used previously treated patients in these studies to assess immunogenicity that any increase in inhibitor formation in previously treated patients would be consistent with the product being neoantigenic. Examples of that that we will hear throughout this meeting are the Belgian and Dutch examples of Factor VIII concentrates that were somehow altered during their manufacture and became neoantigenic. One of the critical aspects of this is the number of exposure days. This is the rate, or the occurrence, of inhibitors versus exposure days that was published by McMillan from the Cooperative Inhibitor Study a number of years ago. You can see that, although, as was previously mentioned, the rate of inhibitor formation is highest down here in young individuals, that inhibitor formation continues with exposure days so that, by 50 exposure days, maybe 75 to 80 percent of patients who are going to develop an inhibitor will have done so but 15 to 20 percent of patients who are going to develop an inhibitor will not have done so. You really don't reach an asymptote with 100 percent until you get out here to close to 250 days. So there are differences, I think, between studies that are carried out with 150 exposure days as an eligibility criteria versus 250 exposure days. Certainly, when one gets down here to 50 exposure days as an eligibility criteria, the number of inhibitors that one might expect to see, even in a previously treated patient cohort, is not an insignificant number. So based on these, in 1999, the Factor VIII and Factor IX Subcommittee of the SSC of the ISTH deliberated about this and came up with a recommendation that previously deliberated about this and came up with a recommendation that previously treated patients with greater than 250 exposure days should be used to assess the immunogenicity of new products and previously untreated patients, or newly--somebody remind me what NIPs are--noninfected; that is what it is, not newly--and noninfected patient should not be used to assess immunogenicity because, in all likelihood, their number of exposure days will be so small that the risk of inhibitor development would be increased. I think that that has made some sense. I think the FDA and European agencies have recognized that and that has become a standard. That is certainly the rationale behind why we currently use PTPs to assess immunogenicity. The only real point that I would make is that this number here is not a trivial number. It is a number that has some experimental backing and is a number that needs to be looked at with great care when one is designing clinical trials. The Factor VIII and Factor IX Subcommittee later, in 2001, also addressed, because of concerns about differences between the definition of inhibitors and concerns that that might muddy comparisons between studies, also decided to make an official recommendation about what was a high response and what was a low response inhibitor, and those definitions are shown here. A high response is greater than or equal to 5 Bethesda units and a low response inhibitor is less than 5 Bethesda units. So my charge was to go over those recommendations. I think that they are still valid today. I think that previously treated patients are the right population to look at neoantigenicity of new product and, as we go to new generations of Factor VIII and Factor IX that are structurally altered in order to make better molecules, I think that this is going to continue to be an issue that we need to be cognizant of and work on in our clinical trials. So I will stop there, Jay, and if there is time for questions, I would be glad to answer questions. (Applause.) DR. LOZIER: We have a few minutes for questions. Again, state your affiliation and activate your microphone. Dr. Aledort. DR. ALEDORT: Aledort, New York. Do you have any assessment from the literature, which I think is really very difficult to do, if you really looked at a population of greater than 150 versus a population of greater than 250, what incremental or likelihood of the number of inhibitors you might find given 100 patients. The other question is, unfortunately, all those data do not, in any way, talk about the genetic predisposition to inhibitor in those previously treated patients in terms of how far out you go. I think you would have to know a little bit more about that to feel sanguine about the population that you are actually studying. DR. WHITE: I think your latter point is a good point, Lou. Let me answer your question first and then I will comment. The data was from McMillan. You know the study. It was not a study--it was a prospective study, certainly a valid trial at the time, but it was a mixture of patients, PUPs and PTPs and so on. So I think that data is a little bit tainted based on what we know today. If you actually look at the curve, and there are not many studies that have looked prospectively at inhibitor development as long as that study did. It was about seven to ten-years study, as I recall. It certainly was not a 25-year study, but it was a fairly long study. If you look at the curve, the difference between 150 and 250 exposure days in 100 patient would probably be one or two patients, so very small. I think my real point is that 50 is a number that has been used in some studies and I think that is a significant difference from 250 and 150. As far as the genetic data, you know what I believe about that. You know that I think that there are a lot of answers in the genetics that Joan talked about but I don't think we are at the point where we can predict individuals yet. I think maybe we are not too far from it, but we are not there yet. I do think genetics will play a role in when a person develops an inhibitor. I don't know that it will be as simple that nonsense mutations are going to develop in inhibitors if they are going to develop them in 50 days and deletion mutations are going to develop them in five days. I don't think it will wash out to be that simple. But I think molecular defect and other genetic factors are going to play a role in when inhibitors develop upon exposure to Factor VIII, but I couldn't predict as to how it would affect that. DR. LOZIER: I think one other point about the McMillan study, as I recall, the criterion was 2 percent as opposed to 1 percent for severe. So that is just another factor. Could we have one more question? Okay. Then, at this point, we had expected to have three speakers talk about epidemiology of Factor VIII inhibitors, particularly from the U.K., Canada and the U.S. Unfortunately, Dr. Charlie Hay from England was unable to make due to a last-minute emergency so we will be proceeding on here, when we have our slides, with Dr. Carcao who will talk about the Canadian experience and Dr. Evatt with the U.S. experience. Canadian Experience with Factor VIII Inhibitors During Conversion to Recombinant Products DR. CARCAO: I would like to thank the sponsors for inviting me to present some of our Canadian experience with inhibitors. I also want to thank Dr. David Lillycrap who is here in the audience. You should also know that Dr. Lillycrap was actually invited to do this talk but he graciously asked me to do it on his behalf. Part of the reason why I have been asked to do this is that I am the Chair of the Inhibitor Subcommittee of the Association of Hemophilia Clinic Directors. The other members of the Association are shown there and, as you can see, alphabetically, I am the first on the list and I think that is probably why they asked me to be the Chair. A little bit about hemophilia in Canada. Canada is a country of about 30 million people so you can put that into context in terms of your own countries. In Canada, there are 2,561 hemophiliacs. This is as of July of this year. How accurate are those numbers? We believe that they are probably close to 98 or 99 percent accurate but we certainly can't claim 100 percent accuracy. Of those numbers, 2063 of the patients are hemophilia-A patients and that represents 81 percent of all hemophilia in Canada. 498, or 19 percent, are hemophilia B patients. Virtually all of these patients are followed and, hence, registered in 25 hemophilia treatment centers that are scattered throughout Canada. If you work out the numbers, given that the population is about 30 million people, you will see that the prevalence of hemophilia is about 1 in 5,740 males. In Canada, presently more than 90 percent of the patients receive recombinant factor concentrates. Specifically, I was asked to address hemophilia A and so, from now forward, I will be speaking about hemophilia A. In Canada, we have 2,063 total hemophilia-A patients. Of these, the severes constitute 30 percent or 614 patients. Moderate represent 12 percent of the total and mild patients represent 58 percent of the total. If you look at inhibitors versus no inhibitors, and this is prevalence data not incidence data, currently there are, of the 614 severes, 72 patients currently having inhibitors representing a prevalence of 12 percent. For moderate, the number is 3 percent, the current prevalence. For mild, it is 0.3 percent. If you want to translate this into incidence--we certainly can't--but you can probably estimate that maybe the incidence might be three times the level of the prevalence. So, in total, inhibitors in Canada, we have presently 83. This i