Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization

Abstract

Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light- chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.

Epitope mapping of factor VIII inhibitor antibodies of Chinese origin

“…There was no data to differentiate the exact fraction of their findings that recognized a3 from those that recognized A3-C1. Human antibodies directed to the a1 region are rare, occurring only at an incidence of 1´8±7% (Fulcher et al, 1987;Lubahn et al, 1989;Scandella et al, 1989;Prescott et al, 1997). We found 14´3% (4 out of 28) for a1, which is the highest number reported; however, explanations for this high incidence are not clear.…”

Human factor VIII can be packaged and functionally expressed in an adeno‐associated virus background: applicability to haemophilia A gene therapy

“…), and MAb 413 was as previously described (Saenko et al, 1994). Epitopes of MAbs ESH8 and 413 are localized to C2 domain residues 2248-2285 and A2 domain residues 373-606, respectively (Scandella et al, 1989), whereas the ESH4 epitope is localized to the C2 domain of the factor VIII light chain (Saenko & Scandella, 1995). Biotinylation of MAb ESH8 was performed as previously described (Scandella et al, 1992).…”

Interferon‐γ secretion defects in haemophilia A patients receiving highly purified plasma‐derived or recombinant factor VIII

“…Humoral immunity to FVIII develops in approximately 25% of severe haemophilia A patients (reviewed by Ehrenforth et al, 1992). In these patients, FVIII-specific antibodies (FVIII inhibitors) bind to functionally important regions of the FVIII molecule, inhibiting its procoagulant function (Lollar et al, 1994;Scandella et al, 1989Scandella et al, , 1993Shima et al, 1993;Arai et al, 1989). Significant morbidity and mortality results from complications associated with treatment of bleeding episodes in haemophilia A patients with FVIII inhibitors.…”