Identification of Novel Factor VIII Inhibitor Epitopes using Synthetic Peptide Arrays

Abstract

Objectives: Mapping the antibody-binding sites on the factor VIII (FVIII) protein opens the prospect of studying the development of FVIII inhibitors and the alteration of inhibitor specificities over time. This paper describes a novel approach to the mapping of FVIII antibody-binding sites. Methods: Immobilized synthetic peptide arrays covering 80% of the complete 2351 amino acid sequence of factor VIII (FVIII) were used to determine epitope specificity of 6 alloantibodies and 3 autoantibodies inhibitory to FVIII activity. This detailed assessment was carried out using a modified enzyme-linked immunosorbent assay with plasma from normal persons or hemophilia A patients without inhibitors as negative controls. Results: Antibody-combining sites could be differentiated in both a qualitative and quantitative manner and were patient-specific. Highly reactive peptides were restricted to specific sites in the A1-A3 and C1-C2 domains and were not proximal to known proteolytic cleavage sites. Free peptides incubated in vitro with the plasmas of 3 patients significantly reduced residual inhibitor titers in a dose-dependent manner. Conclusion: This technique permits the study of the development and specificity of FVIII inhibitors, can detect and differentiate between inhibitory and noninhibitory antibodies using immobilized or free peptides respectively, permits correlation of antibody-combining sites with inhibition of FVIII activity and provides a basis for the development of inhibitor adsorption or neutralization technology.

Impact of inhibitor epitope profile on the neutralizing effect against plasma‐derived and recombinant factor VIII concentrates in vitro

“…Alternatively, the conformation of the FVIII molecule could be influenced by the presence of the B‐domain and interacting antibodies rendering the molecule more resistant to activation by thrombin and FXa or susceptible to degradation. In this context, it is interesting to note that neutralizing antibodies against the B‐domain recognizing FVIII but not FVIIIa have also been described [18,19].…”

Epitope mapping of factor VIII inhibitor antibodies of Chinese origin

“…Moreover, we also found that the B‐domain epitopes were antigenic determinants and displayed neutralizing activity towards cognate inhibitors in some cases. Similarly, Palmer et al (1997) have also identified B domain‐specific inhibitors using synthetic peptide arrays. It remains unclear why the B‐domain epitopes can neutralize FVIII inhibitory activity, as the B domain is not required for function (Toole et al , 1986).…”

FVIII inhibitors: pathogenesis and avoidance

“…13 The neutralizing antibodies are mainly of the immunoglobulin (Ig) G1 and IgG4 subtypes and the epitopes recognized are located on both the light and heavy chains of FVIII with a preference for the A2 and C2 domains, 14 although several epitopes of both neutralizing and nonneutralizing types located outside these, some in the B domain, have also been described. 15,16 The main mechanism by which the antibodies neutralize the factor is by steric hindrance, but the formation of immune complexes and subsequent enhanced catabolism as well as hydrolysis have also been suggested. 17 Regarding nonneutralizing antibodies, it remains debated as to whether these antibodies, or at least any immune response they provoke, are of clinical significance and should be considered as well.…”