Haematologica 2000;85:supplement to no. 10 - Supplements ...

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Immune Tolerance and the Treatment of Hemophilacs with an Inhibitor 29 not be evident in a Bethesda assay and I think it’s really a question of how this epitope contributes overall to the average inhibitor plasma. It’s possible that in many plasmas some of the inhibitor reactivity isn’t seen in the Bethesda titer as clinically insignificant whereas there may be some patients in whom this epitope constitutes a major problem and the Bethesda titer isn’t reporting the problem. RYPERT: You’re talking about low antigenicity of your recombinant molecules meaning that you get a reduced binding of antibodies induced against human factor VIII. This, however doesn’t give us any reflection on the immunogenicity of the molecule that is the capacity to induce inhibitors which is the real problem of these products. LOLLAR: I also agree that it’s a more generally important issue but the antigenicity is also an important problem in those patients who have inhibitors. It’s important to distinguish the difference between antigenicity and immunogenicity. Antigenicity is defined as the ability of something to interact with the product of the immune response, usually an antibody, whereas immunogenicity is defined as an ability to elicit an immune response. One of the very interesting things about factor VIII epitopes which I think is unexplained at this point is that in patients with hemophilia A developing inhibitors and in patients who have acquired inhibitors we have very different immunological backgrounds including the fact that one has been treated with factor VIII exogenously and the other has not. If you look at patient’s epitopes in respect to the A2 polypeptide one sees these antibodies are binding to the same A2 molecule and to me this suggests something which is slightly against an immunological dogma and that is there are intrinsically important regions on the factor VIII molecule that drive the immune response whereas the immunological dogma is that the entire surface of a protein is potentially immunogenic. The fact is that the A2 epitope may be an immunodominant epitope; it could be that the intrinsic structures of that epitope are at least in part driving an immune response to the level that high affinity antibodies occur. This indicates that one could potentially modify those epitopes and create a less immunogenic molecule. One of the problems with immunogenicity is that we don’t have a nice in vitro test for it, whereas we have a fairly good test for antigenicity with the Bethesda assay. Therefore, it would be nice to have a test-tube or a cell-based assay in which one could predict the immunogenicity of factor VIII molecules, but we don’t have it yet. There is a hemophilia A model of immunogenicity which was developed by Dr.Hoyer’s group and I think that right now that would be the standard in which to try and test constructs on a bi- epitope basis evaluating their immunogenicity and we really intend to do that. LILLICRAP: Do you know whether any of the substitutions are making significant differences to the specific activity of the protein LOLLAR: We’ve made forty-one human porcine hybrids to date, and all of them have either equal activity or greater activity than human factor VIII. As you know porcine factor VIII has more activity than human factor VIII at least in a one stage clotting assay. It’s interesting and also a little surprising that some of the constructs did not have decreasing activity. What this means is that one can, not only with the A2 domain but also with the C2 and the A3 domains, make substitutions and create molecules which don’t bind to antibodies as they lose a micro-molecular interaction; yet they’re still able to interact with regions of the human clotting complex in an in vitro system and get a good specific activity. SAINT REMY: Have you any idea of the T-cell reactivity of your constructs LOLLAR: No, we haven’t. T-cell epitopes and B-cell epitopes are usually different. Just because the regions 484 to 508 on factor VIII are a problem area does not mean that’s what the T-cells recognize. T-cells recognize small peptides. There is a lot of research in this region: at the ISTH meeting, for example, Bianca Conti presented data on the T-cell epitopes which are present in human factor VIII with respect to infusion of human factor VIII into mice and also humans. She used a T-cell proliferation assay and identified several regions of the factor VIII molecule that indicate that at this point the T-cell epitopes are more frequent and much more complex than the B-cell epitopes and there are a lot more of them. HOYER: Have you looked at these hybrids with plasmas from patients who have developed inhibitors and then have been treated extensively with porcine factor VIII The reason I ask is to return to the issue of antigenicity versus immunogenicity. It is an imperfect tool but perhaps the only one we can use without animal studies. One wonders if the patients treated extensively with porcine factor VIII would develop antibodies that would react with your hybrids. On the other hand, if they don’t react with the hybrids, this would reinforce the notion that they may be used therapeutically for long periods of time. LOLLAR: This patient that I discussed who was an auto-antibody patient who had only received porcine factor VIII; he was the only patient we have had who had an anti-human Haematologica vol. 85(supplement to n. 10):October 2000
30 P. Lollar titer and an anti-porcine titer who we had a good clinical history for. The twenty-three patients that I showed you were patients for whom we did not have a clinical history and were just defined by their high Bethesda titers which ranged from ten to thousands. It would be interesting to accrue that data. I would like to have samples to do that. I think that a panel of porcine- human hybrids would be a potentially useful set of reagents to include in prospective studies to try to find out how the B-cell response varies with exposure to porcine factor VIII. Haematologica vol. 85(supplement to n. 10):October 2000
Page 1 and 2: Haematologica established in 1920 e
Page 3 and 4: Acknowledgments We wish to thank th
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II Immune Tolerance and the Treatme
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