High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance

Abstract

• Amino acid residues comprising B-cell epitopes recognized by neutralizing antifactor VIII antibodies (inhibitors) have been identified. • Amino acids contributing significant antigen-antibody binding avidity are candidates for mutagenesis in the design of less antigenic proteins.Neutralizing anti-factor VIII (FVIII) antibodies that develop in patients with hemophilia A and in murine hemophilia A models, clinically termed "inhibitors," bind to several distinct surfaces on the FVIII-C2 domain. To map these epitopes at high resolution, 60 recombinant FVIII-C2 proteins were generated, each having a single surface-exposed residue mutated to alanine or a conservative substitution. The binding kinetics of these muteins to 11 monoclonal, inhibitory anti-FVIII-C2 antibodies were evaluated by surface plasmon resonance and the results compared with those obtained for wild-type FVIII-C2. Clusters of residues with significantly altered binding kinetics identified "functional" B-cell epitopes, defined as those residues contributing appreciable antigen-antibody avidity. These antibodies were previously shown to neutralize FVIII activity by interfering with proteolytic activation of FVIII by thrombin or factor Xa, or with its binding to phospholipid surfaces, von Willebrand factor, or other components of the intrinsic tenase complex. Fine mapping of epitopes by surface plasmon resonance also indicated surfaces through which FVIII interacts with proteins and phospholipids as it participates in coagulation. Mutations that significantly altered the dissociation times/half-lives identified functionally important interactions within antigenantibody interfaces and suggested specific sequence modifications to generate novel, less antigenic FVIII proteins with possible therapeutic potential for treatment of inhibitor patients. (Blood. 2014;123(17):2732-2739 IntroductionThe development of neutralizing anti-factor VIII (FVIII) antibodies is a serious complication that may be encountered when FVIII replacement therapy is administered to patients with hemophilia A (HA). It affects 25% to 30% of the treated HA population, with a peak occurrence after ;14 FVIII infusions.1-3 Autoimmune responses to FVIII can also occur, 4 and although this happens only rarely, the resulting bleeding phenotype can be severe. Inhibitors can be difficult and extremely expensive to manage clinically. Interestingly, porcine FVIII has been used effectively in the clinic as a "bypass" therapy; that is, a therapeutic protein that can evade neutralization by anti-FVIII antibodies in many allo-and autoimmune inhibitor patients. [5][6][7] However, some patients have or could develop antibodies that neutralize porcine FVIII as well, 8 because of antigenic cross-reactivity 9 or because regions in which the porcine sequence differs from the human FVIII sequence stimulate effector T cells, leading to antibody production. Identification of the binding sites (B-cell epitopes) on FVIII that are recognized by inhibitors would allow rational design of novel therapeutic FVIII...

T cells from hemophilia A subjects recognize the same HLA-restricted FVIII epitope with a narrow TCR repertoire

“…Thus FVIII-A2 peptides would definitely be foreign and possibly immunogenic. Furthermore, FVIII antigen was not detected when his plasma was tested by ELISA using monoclonal antibody ESH-8, which recognizes a C2 domain epitope, 55,56 suggesting that most or all of the *Cells were sorted from this polyclonal line based on their binding avidity for the DRB1*01:01-FVIII 2194-2213 tetramer into high-, medium-, and low-avidity populations (P2, P3, and P4 gates, respectively, in supplemental Figure 4); 200 000 cells were collected from each population except for the smaller high-avidity population, which consisted of 112 000 cells. Genomic DNA was isolated from cells, and deep TCRB-CDR3 sequencing was performed by Adaptive Biotechnologies.…”

Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A

“…Patients who develop inhibitors typically have antibodies that bind to the FVIII-A2 and FVIII-C2 domains, 62 and there has been substantial progress in characterizing FVIII B-cell epitopes. [63][64][65][66][67] Risk factors for development of inhibitors in HA include genetic factors such as the type of F8 gene mutation, the severity of hemophilia, HLA haplotype, and polymorphisms in genes involved in immunoregulation 68 as well as environmental factors such as the intensity of FVIII exposure and concomitant immunologic "danger signals." 69 Identification of risk factors related to rFVIII vs plasma-derived FVIII has been more controversial.…”

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

“…[8][9][10] Healey et al 11 demonstrated a predominance of anti-fVIII antibodies to the A2 and C2 domains of fVIII in a murine hemophilia A model, but antibodies to the other domains were also detected. Characterization of the structural and functional properties of anti-A2 and anti-C2 domain monoclonal antibodies (mAbs) has advanced our understanding of the epitope spectrum of anti-fVIII antibodies [12][13][14][15] and their mechanisms of inhibition. [16][17][18][19] However, there is increasing evidence of the C1 domain's contribution to fVIII function and immunogenicity.…”