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

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Immune Tolerance and the Treatment of Hemophilacs with an Inhibitor 87 Table 1. Range of IP titers in patients without Bethesda titers. Group Type Patients Lowest IPU Highest IPU % of Range Patients Range No Immune Response 38 0-0.5 0-3.4 76 Immune Response ≥4.3 7 0 5.4-139 6 Table 2. Range of IP titers In inhibitor patients with Bethesda titers ≤2. Pt Lowest Highest Final Highest Lowest Final Tolerance IPU IPU IPU BU BU BU 1 0 122 3.6 1 0 0 + 2 0.4 4.4 0.7 0.6 0 0 + 3 0.6 20 3.4 0.7 0 0 + 4 0 169 0 10 0 0 + 5 0 10,644 1,310 634 0 41 - Methods and Results All hemophilic patient plasmas were tested for inhibitor activity by the Bethesda assay, 5 FVIII recovery, and FVIII half-life at the home institution. Immunoprecipitation assays (IP) were done in our laboratory as described elsewhere 8 by radio-labeling FVIII with Na 125 I and using it to bind serial dilutions of hemophilic plasmas. All antibodies were captured with protein G sepharose, and the unbound 125 I-FVIII was washed away. IP units/mL were calculated as ([bound/total 125 I – background without antibody/mL] x plasma dilution). This assay is approximately 10-fold more sensitive than the Bethesda assay. The first group of 11 hemophiliacs analyzed had Bethesda titers ranging from 1.1-255 at the start of therapy. They were subsequently treated with 100 units/kg/day FVIII. Seven of them had a negative Bethesda assay at the end of the treatment. Each sample was tested by IP assays with 125 I-FVIII to determine whether there were any remaining levels of either non-inhibitors and/or inhibitors at low levels. At the end of therapy, 4 patients had low but insignificant antibody levels in the IP assay, which confirmed the success of the tolerance procedure. Two had final IP titers of 5.6 and 228. Their respective FVIII half-life measurements at the same time were 9.1 and 5.2, which confirmed that only the latter had antibody levels high enough to clear FVIII more rapidly than usual. The 4 failures had remaining inhibitor titers of 0.7-31. A large study of previously untreated severe hemophilia A patients yielded additional interesting information. All plasmas collected from 50 evaluable patients of 72 enrolled were tested for inhibitor titers and by the IP assay for all anti- FVIII antibodies. A reliable immune response of 4.3 IP units/mL was defined as 3-fold above the average antibody levels determined in patients before FVIII therapy was begun. The patients in the study were evaluated by comparing the IP and Bethesda titers in multiple, serial plasmas drawn over the course of the study, and the data for those without inhibitor titers are summarized in Table 1. The patients’ data were divided into 2 groups based on the immune responses of the patients’ to rFVIII infusion. Immune responses that were either negative or below the cut-off defined above were obtained in 76% of the 50 evaluable patients, while 6 (12%) had immune responses ≥4.3 IP units/mL. Eight patients with Bethesda titers ≤3 became tolerant by the end of the study without any specific treatment for reduction of the inhibitor titer. Representative data are shown in Table 2. In addition, 2 of 3 patients with BU >3-10 also became spontaneously tolerant (Table 2, patient #4). Four patients with Bethesda titers of 11- 1,397 were given high dose rFVIII tolerance therapy, but success was achieved only in 1 who had pre-IT titers of 3 BU/mL compared to 38-1105 for the others. Two patients with 5 and 11 BU/mL given rFVIII prophylaxis treatment were also tolerized. The low titers and the use of prophylaxis may have enhanced the elimination of the antibodies, which should be verified in future studies. Data for 1 tolerance failure are shown in Table 2. Six non-inhibitor patients with immune responses ≥4.3 IP units/mL were IP negative at the end of the study period. The IP titers over time of anti-A2 or anti-C2 antibodies from 2 high titer inhibitor patients in tolerance therapy increased and decreased coordinately. Discussion Prior results from immune tolerance protocols in which 85% of individuals with 10 BU/mL were more easily tolerized by less FVIII than those with >10 BU/mL indicate that the lower titer antibodies can be more easily eradicted. 9 This was also the case in the patients described above who were receiving immune tolerance therapy. Three of 6 were tolerized. It was surprising, however, that 11 patients with inhibitor titers 0.6-≤10 lost their inhibitors without any specific treatment. Haematologica vol. 85(supplement to n. 10):October 2000
88 D. Scandella et al. Seven non-inhibitor patients had ≥1 antibody peaks detectable only in the immunoprecipitation assay at one or several times in their treatment history. In all of them the antibodies also disappeared spontaneously. The presence of such non-inhibitory antibodies (228 IP units/mL) in 1 patient led to a reduction of the FVIII half life to 5.2 hours compared to the normal value of 7.6 hours. Similar results were previously observed in ELISA experiments. 10 The non-inhibitory antibodies may therefore also lead to adverse effects on FVIII therapy if their titer is high enough. The paucity of half-life data did not allow a more precise determination of the minimum antibody concentration that affects FVIII half-life. In individuals without Bethesda titers that have increased, unexplained bleeding or shorter than normal half-life, the addition of the IP or similarly sensitive assay would be useful in determining whether anti-FVIII antibodies are the cause of these conditions. The immune response to FVIII in severe hemophiliacs, based on the Bethesda and the IP assays, is more heterogeneous than that found in earlier studies which used only the Bethesda assay. 1,2 In the large study described, 22 of the 72 total patients were positive by the Bethesda assay alone, and an additional 6 non-inhibitor patients were antibody positive (≥4.3 IP units/mL) by the IP assay. The percentage of individuals who responded immunologically to FVIII infusion was 34% of all study patients. Although 4 inhibitor patients were successfully tolerized by high dose FVIII therapy, 11 with a range of 0.6-10 BU/mL, and the 4 with low level antibodies spontaneously lost their anti-FVIII titer. By the end of the study, 68% of the inhibitor patients had become negative. REFERENCES 1. Schwartz RS, Abildgaard CF, Aledort LM, et al. The Recombinant Factor VIII Study Group. Human recombinant DNA-derived antihemophilic factor (factor VIII) in the treatment of hemophilia A. N Engl J Med 1990; 323:1800-5. 2. Bray GL, Gomperts ED, Courter S, et al. The Recombinate Study Group. A multicenter study of recombinant factor VIII (Recombinate ® ): safety, efficacy, and inhibitor risk in previously untreated patients with hemophilia A. Blood 1994; 83:2428-35. 3. Hoyer LW. The incidence of factor VIII inhibitors in patients with severe hemophilia A. Adv Exp Biol Med 1995; 386:35-45. 4. Schwaab R, Brackmann HH, Meyer C, et al. Haemophilia A: mutation type determines risk of inhibitor formation. Thromb Haemost 1995; 74: 1402-6. 5. Kasper CK, Aledort LM, Counts RB, et al. A more uniform measurement of factor VIII inhibitors. Thromb Diathes Haem1975; 34:869-72. 6. Mauser-Bunschoten EP, Niewenhuis HK, Roosendaal G, van den Berg HM. Low-dose tolerance induction in hemophilia A patients with inhibitors. Blood 1995; 86:983-8. 7. Brackmann HH. Induced immunotolerance in factor VIII inhibitor patients. Prog Clin Biol Res 1984; 150:181-95. 8. Thompson AR, Murphy MEP, Liu ML, et al. Loss of tolerance to exogenous and endogenous factor VIII in a mild hemophilia A patient with an Arg593 to Cys mutation. Blood 1997; 90:1902-10. 9. Mariani G, Ghirardini A, Bellocco R. Immune tolerance in hemophilia-principal results from the International Registry. Thromb Haemost 1994; 72:155-8. 10. Dazzi F, Tison T, Vianello F, et al. High incidence of anti-FVIII antibodies against non-coagulant epitopes in haemophilia A patients: a possible role for the halflife of transfused FVIII. Br J Haematol 1996; 93:688- 93. DISCUSSION 14 Characterization of antibodies to FVIII in hemophilia patients treated by immune tolerance therapy Scandella D. (Rockville, USA) KAZATCHKINE: I’m sure we have been underestimating these binding antibodies with no inhibiting activity. You may even be underestimating them yourself because of the arbitrary definition that you gave to the immune response. SCANDELLA: I wouldn’t exactly call it arbitrary. The titers I’m talking about, called “No Significant Immune Response”, are very low because they are very low in titers. KAZATCHKINE: Have you ever looked at a patient who has undergone tolerance induction and whether after absorbing factor VIII binding antibodies on factor VIII you found in the remaining IGg fraction antibodies that would inhibit your immuno-precipitation assay SCANDELLA: We haven’t done that. KAZATCHKINE: That may be another way of looking at those regulatory antibodies that we all wish we could find an easier assay for. SCANDELLA: One piece of data which I wasn’t able to show is that certainly in the no Bethesda titer immune responses they all disappeared without any particular therapy and concened inhibitors with less than ten BU, there was also a number where the inhibitor titer went away without any specific therapy. Haematologica vol. 85(supplement to n. 10):October 2000
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Acknowledgments We wish to thank th
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Haematologica 2000; 85(suppl. to n.
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18 J. Ingerslev sion monitoring dem
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20 J. Ingerslev REFERENCES 1. Hay C
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22 CRM Hay Side effects of Hyate:C
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24 CRM Hay tion by porcine factor V
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28 P. Lollar these epitopes by site
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30 P. Lollar titer and an anti-porc
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34 Immune Tolerance and the Treatme
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Page 71 and 72: 68 L. Nemes et al. trast to the ear
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