The humoral response to human factor VIII in hemophilia A mice

Abstract

Summary. Background: Inhibitory antibodies (Abs) to factor VIII (FVIII inhibitors) constitute the most significant complication in the management of hemophilia A. The analysis of FVIII inhibitors is confounded by polyclonality and the size of FVIII. Objectives: The goal of this study was to dissect the polyclonal response to human FVIII in hemophilia A mice undergoing a dosage schedule that mimics human use. Methods: Splenic B-cell hybridomas were obtained following serial i.v. injections of submicrogram doses of FVIII. Results of a novel, anti-FVIII domain-specific enzyme-linked immunosorbent assay were compared to Ab isotype and anti-FVIII inhibitory activity. Results: The robust immune response resulted in the production of 300 hybridomas per spleen. We characterized Abs from 506 hybridomas, representing the most comprehensive analysis of a protein antigen to date. Similar to the human response to FVIII, anti-A2 and anti-C2 Abs constituted the majority of inhibitors. A novel epitope was identified in the A2 domain by competition ELISA. Anti-A2 and anti-C2 Abs were significantly associated with IgG 1 and IgG 2a isotypes, respectively. Because the IgG 2a isotype is associated with enhanced Fc receptor-mediated effector mechanisms, this result suggests that anti-C2 Abs and inflammation may be linked. Additionally, we identified a novel class of Abs with dual specificity for the A1 and A3 domains. Forty per cent of the Abs had no detectable inhibitory activity, indicating that they are prominent and potentially pathologically significant. Conclusion: The expanded delineation of the humoral response to FVIII may lead to improved management of hemophilia A through mutagenesis of FVIII B-cell epitopes.

Comparison of the immunogenicity of different therapeutic preparations of human factor VIII in the murine model of hemophilia A

“…However, a recent study has reported an unrestricted repertoire of anti-FVIII IgG in H-2D b FVIII-deficient mice. 8 In addition, while anti-VWF IgG have never been reported in patients treated with pdFVIII, we observed the development of VWF-specific IgG. This might be related to the sequence differences between the endogenous murine VWF and the exogenously administered human VWF (83% homology in amino acid sequences).…”

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

“…11,29 The supernatants of hybridomas producing anti-C1 domain antibodies were collected and antibodies purified using SP Sepharose Fast Flow or Protein G agarose chromatography columns. The immunoglobulin G (IgG) isotype and subclass were determined by enzyme-linked immunosorbent assay (ELISA) using alkaline-phosphatase isotype and subclass-specific antibodies.…”

“…7 Most inhibitor patients with congenital hemophilia A develop antibodies against the A2 and C2 domains. [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.…”

Auditing Protein Therapeutics Management by Professional APCs: Toward Prevention of Immune Responses against Therapeutic Proteins

“…To study the immunogenicity of proteins used in replacement therapy, animals genetically stripped of the analogous protein are better suited because they mimic the actual patient situation. These cases have been well documented in the hemophilia model (62,63).…”