Restricted Epitope Specificity of Anti-FVIII Antibodies that Appeared during a Recent Outbreak of Inhibitors

Summary

We recently described an outbreak of anti-factor VIII (FVIII) antibodies in a population of haemophilia A patients non-responsive to FVIII (1). To find out what part of the FVIII molecule had been altered, we purified specific anti-FVIII antibodies from the plasma of the five patients showing high titres of inhibitors. An average of 100 µg antibodies per ml of initial plasma was recovered by immunoadsorption on insolubilised FVIII. The antibodies followed the normal isotypic distribution, including the presence of specific IgG2 antibodies; the relative increase in IgG4 that is usually observed in patients with long-standing inhibitors, was not present. The regions of FVIII to which human antibodies bound were determined by a competition assay using a panel of murine monoclonal antibodies: two major regions were identified, one located in the A2 heavy chain domain, and the other made of determinants of both the A3 and C2 light chain domains. Affinity-purified antibodies inhibited the function of FVIII as determined in a chromo- genic assay. However, variations existed in the affinities with which antibodies bound to soluble FVIII. This study shows that the immu- nogenicity of two particular regions of FVIII has been altered. A screening for alterations located in these two regions should possibly be included in the preclinical evaluation of FVIII concentrates.

• References

• 1 Peerlinck K, Amout J, Gilles JG, Saint-Remy JM, Vermylen J. A higher than expected incidence of Factor VIII inhibitors in multitransfused haemophilia A patients treated with an intermediate purity pasteurized factor VIII concentrate. Thromb Haemost 1993; 69: 115-118
• 2 McMillan CW, Shapiro SS, Whitehurst D, Hoyer LW, Rao AV, Lazerson J. the Haemophilia Study Group. The natural history of factor VIII:c inhibitors in patients with hemophilia A: a national cooperative study. II. Observations on the initial development of factor VIII:c inhibitors. Blood 1988; 71: 344-348
• 3 Schwarzinger I, Pabinger I, Korninger C, Haschke F, Kundi M, Niessner H, Lechner K. Incidence of inhibitors in patients with severe and moderate hemophilia A treated with factor VIII concentrates. Am J Haematol 1987; 24: 241-245
• 4 Ehrenforth S, Kreuz W, Scharrerb I. et al. Incidence of FVIII and factor IX inhibitors in haemophiliacs. Lancet 1992; 339: 594-598
• 5 Horowitz M, Rook C, Horowitz B, Hugariner M. Virus safety of solvent/ detergent treated antihaemophilic factor concentrate. Lancet 1988; 19: 186-189
• 6 Schimpf K, Brackmann H, Kreuz W. Absence of hepatitis after treatment with pasteurized factor VIII concentrate in patients with hemophilia and no previous transfusions. New Engl J Med 1987; 316: 918-922
• 7 Morgenthaler J. Virus inactivation in plasma products. Current Study in Haematology. Blood Transfusion (Basel) 1989; 56: 55-58
• 8 Peerlinck K, Vermylen J. Epidemic of factor VIII inhibitors linked to factor VIII-P. Acta Clin Belg 1991; 46: 419-420
• 9 Rosendaal FR, Nieuwenhuis H, van de BergH, Heijboer H, MauserBunschoten EP, van der MeerJ, Smit C, Strengers PFW, Briët E. the Dutch Haemophilia Study Group. A sudden increase in factor VIII inhibitor development in multitransfused hemophilia A patients in the Netherlands. Blood 1993; 81: 2180-2186
• 10 Griffin B, Micklem L, McCann M, James K, Pepper D. The production and characterization of a panel of ten murine monoclonal antibodies to human procoagulant factor VIII. Thromb Haemost 1986; 55: 40-46
• 11 Burnouf T, Burnouf-Radosevich M, Huart J, Goudemand M. A highly purified factor VIII:c concentrate prepared from cryoprecipitate by ion-exchange chromatography. Vox Sang 1991; 60: 08-15
• 12 Hiemstra H, Nieuweboer C, Idoe M. et al. Evaluation of a wet pasteurization of a factor VIII concentrate produced by controlled-pore silica adsorption. Folia Haematology (Leipzig), 1990; 117: 557-563
• 13 Gilles JGG, Amout J, Vermylen J, Saint-Remy JMR. Anti-Factor VIII antibodies of haemophiliac patients are frequently directed towards non-functional determinants and do not exhibit isotypic restriction. Blood 1993; 82: 2452-2461
• 14 Steel HV, Sakariassen KS, Scholte BJ, Veerman ECI, van der KwastTH, de Groot PG, Sixma JJ, van Mourik JA. Characterization of 25 monoclonal antibodies to Factor VIII-von Willebrand factor: relationship between ristocetin-induced platelet aggregation and platelet adherence to subendothelium. Blood 1984; 63: 1408-1415
• 15 Leyte A, Mertens K, Distel B, Evers RF, De Keyzer-Nellen MJM, Groenenvan Dooren MMCL, De Bruin J, Pannekoek H, van Mourik JA, Verbeet MP. Inhibition of human coagulation factor VIII by monoclonal antibodies. BiochemJ 1989; 263: 187-194
• 16 Scandella D, Timmons L, Mattingly M, Trabold N, Hoyer LW. A soluble recombinant factor VIII fragment containing the A2 domain binds to some human anti-Factor VIII antibodies that are not detected by immunoblotting. Thromb Haemost 1992; 67: 665-671
• 17 Lenting PJ, van de Loo JWHP, Donath MJSH, van Mourik JA, Mertens K. The sequence Glu¹⁸¹¹-Lys¹⁸¹⁸ of human blood coagulation factor VIII comprises a binding site for activated factor IX. J Biol Chem 1996; 271: 1935-1940
• 18 Gilles JGG, Saint-Remy JMR. Healthy subjects produce both anti-factor VIII and specific anti-idiotypic antibodies. J Clin Invest 1994; 94: 1496-14505
• 19 Svendsen L, Brogli M, Lindeberg G, Stocker K. Differentiation of thrombin- and factor Xa-related amidolytic activity in plasma by means of synthetic thrombin inhibitor. Thromb Res 1984; 34: 457-462
• 20 Kasper CK, Aledort LM, Counts RB, Edson JR, Fratantone J, Green D, Hampton JW, Hilgartner MN, Lazarson J, Levin PH, Macmillan CN, Pool JC, Shapiro SS, Schulman NR, Van Eys J. A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh 1975; 34: 869-872
• 21 Barnett BC, Graham CM, Burt D, Skehel JJ, Thomas DB. The immune response of BALB/c mice to influenza hemagglutinin: commonality of the B cell and T cell repertoires and their relevance to antigenic drift. Eur J Immunol 1989; 19: 515-521
• 22 Aalberse R, van der Gaag R, van Leeuwen J. Serologic aspects of IgG4. I.Prolonged immunization results in an IgG4-restricted response. J Immunol 1983; 130: 722-726
• 23 Kemball-Cook G, Tubb J, Dawson N, Barrowcliffe T. The behaviour of different factor VIII concentrates in a chromogenic factor X-activating system. Br J Haematol 1993; 84: 274-278
• 24 Saenko EL, Shima M, Gilbert GE, Scandella D. Slowed release of thrombin-cleaved factor VIII from von Willebrand factor by a monoclonal and a human antibody is a novel mechanism for factor VIII inhibition. J Biol Chem 1996; 271: 27424-27431