High-Resolution Mapping of B-Cell Epitopes on the Factor VIII C2 Domain Using Surface Plasmon Resonance

Abstract 203

Neutralizing anti-factor VIII (FVIII) antibodies that develop in hemophilia A mice following exposure to human FVIII bind to several distinct regions (B-cell epitopes) on the FVIII C2 domain. This 19-kDa region of FVIII binds to thrombin and factor Xa during proteolytic activation of FVIII, and it also binds to phospholipids, von Willebrand factor and factor IXa; all of these interactions are essential for the cofactor effect of FVIII in the intrinsic tenase reaction, which is a critical control point in blood coagulation. The binding of antibodies to any of these surfaces on the C2 domain can block FVIII activity, causing serious bleeding that is difficult to control.

In order to fine-map B-cell epitopes on FVIII, 60 recombinant N-terminal His-tagged FVIII-C2 proteins were generated in an E. coli expression system and purified to homogeneity. Each had a surface-exposed amino acid residue mutated to alanine. The binding kinetics of these FVIII-C2 muteins to 10 inhibitory anti-FVIII-C2 monoclonal antibodies (mAbs) was then evaluated by surface plasmon resonance (SPR). The muteins were injected over biosensors on which the mAbs were captured using a high affinity goat anti-mouse IgG that was covalently attached to the Biacore CM5 biosensor surface by amine coupling. The anti-FVIII-C2 mAbs were: ESH8, ESH4, 2–77, 2–117, 3D12, 3E6, I109, 1B5, 3G6 and I54. Single-cycle kinetic experiments were carried out for each FVIII-C2 mutein/mAb pair, and variants having a dissociation rate >4X that measured for wild-type FVIII-C2 were marked as candidates for the corresponding B-cell epitope. The dissociation rate, rather than the binding affinity, was found to be the best diagnostic measurement to identify side-chain substitutions that altered the antigen-antibody binding interaction. Initial SPR kinetics experiments were run in a “blinded” fashion, meaning the FVIII-C2 crystal structure (Pratt et al., Nature 402, 439–42, 1999) was not consulted either before or during analysis of the binding kinetics. Subsequent visualization of the crystal structure using a molecular graphics program allowed us to locate the sites of amino acid substitutions that perturbed the binding of particular mAbs to the FVIII-C2 surface. Amino acid substitutions that affected binding to multiple mAbs, or that occurred at sites that were non-contiguous with clusters of surface-exposed residues identified in this manner, were not considered part of a given epitope as they clearly affected the protein folding/stability. FVIII-C2 muteins used for this analysis bound to most of the mAbs with affinities similar to that of wild-type FVIII-C2, indicating that they were properly folded. For these proteins, observation of altered kinetics in binding to a particular mAb indicated a change had occurred at the antigen-antibody binding interface. Averaged kon and koff values were obtained from 10 SPR runs per mAb, measuring the binding kinetics to WT-FVIII-C2. These experiments yielded accurate kinetics and affinities of each mAb for the FVIII C2 domain.

Clusters of surface-exposed residues with significantly altered binding kinetics identified specific sites comprising “functional” B-cell epitopes, defined as those residues contributing appreciable antigen-antibody binding affinity. All of these mAbs were previously shown, by competition ELISAs, to correspond to partially overlapping epitopes, designated A, AB, B, BC and C; binding of antibodies to these epitopes interfered with: (1) proteolytic activation of FVIII by thrombin or Factor Xa; (2) binding of FVIII to phospholipid surfaces and/or von Willebrand factor; (3) a chromogenic factor X activation assay (Meeks et al., Blood 110:4234–4242, 2007). Fine-mapping of these epitopes by SPR indicated that the mAbs assigned to classes A, B and C indeed bound to distinct regions of the FVIII-C2 surface. Identification of these epitopes also pinpoints functionally important regions on the FVIII C2 domain, and these results contribute further insight into the binding interactions leading to FVIII activation to FVIIIa and its participation in the intrinsic tenase complex at negatively charged phospholipid surfaces.