Characterization and Solution Structure of the Factor VIII C2 Domain in a Ternary Complex with Classical and Non-classical Inhibitor Antibodies

Abstract

Background:The development of antibodies against coagulation factor VIII (fVIII) is a serious complication of hemophilia A. Results: Small angle x-ray scattering reveals a molecular envelope solution structure of two inhibitor antibodies bound to the C2 domain of fVIII. Conclusion: Multiple inhibitor antibodies can bind to the fVIII C2 domain simultaneously, and modeling suggests the localization of key epitopes. Significance: Understanding fVIII-inhibitor interactions is crucial for developing more effective hemophilia A therapies.

Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes

“…The complete tertiary structure of the complex shows that each F AB recognizes opposing faces of the fVIII C2 domain, as previously suggested ( Figure 1A). 41 Interestingly, the overall morphology and geometry of the ternary complex agrees well with a recently described low-resolution SAXS model, with a measured angle between antibodies of ;135°( Figure 1B). 41 Buried surface areas for each antifVIII epitope were calculated to be approximately 690Å 2 and 810Å 2 between the fVIII C2 domain and the 3E6 and G99 interfaces, respectively.…”

“…52 Moreover, Lys2183 forms a direct contact with multiple 3E6 residues and is largely buried at the binding interface (Figure 2A), which is consistent with a Lys2183Ala mutant that decreases 3E6 binding by an order of magnitude. 41 By contrast, multiple residues at the C2 domain/G99 interface are suggested to be involved in binding both factors IXa and Xa. Specifically, Glu2228 and Trp2229 form direct contacts to G99 (Figure 3B), which are predicted to be involved in factor IXa binding by the fVIII C2 domain.…”

“…38,41 The 3E6 epitope partially consists of Lys2183, Asp2187, and Arg2215, each of which is expected to be involved in VWF binding (Figure 2). 52 Moreover, Lys2183 forms a direct contact with multiple 3E6 residues and is largely buried at the binding interface (Figure 2A), which is consistent with a Lys2183Ala mutant that decreases 3E6 binding by an order of magnitude.…”

“…[41][42][43] Monoclonal anti-human fVIII C2-specific antibodies (3E6 and G99) were produced from hybridomas following immunization of hemophilia A mice with intravenous, adjuvant-free injections of human fVIII, as described previously. 38,41 Large-scale antibody production was performed at the Antibody Production Facility at the Fred Hutchinson Cancer Research Center (FHCRC) (E. Wayner, Seattle, WA). Immunoglobulin (IgG) and F AB purifications were completed with the NAb Protein A Plus spin column and immobilized papain kits (Thermo Scientific, Rockford, IL) according to the manufacturer's protocols.…”

“…Moreover, our crystal structure confirms the suggested epitope regions previously characterized by small-angle x-ray scattering (SAXS) analysis. 41 This study, along with previous work, 38 establishes 3E6 and G99 as epitope probes, which have the potential to define further the role of specific regions of the fVIII C2 domain in procoagulant function and antibody recognition.…”