Epitope mapping via selection of anti-FVIII antibody-specific phagepresented peptide ligands that mimic the antibody binding sites

Abstract

The most serious complication in today's treatment of congenital haemophilia A is the development of neutralising antibodies (inhibitors) against factor VIII (FVIII). Although FVIII inhibitors can be eliminated by immune tolerance induction (ITI) based on repeated administration of high doses of FVIII, 20-30% of patients fail to become tolerant. Persistence of FVIII inhibitors is associated with increased morbidity and mortality. Data from recent studies provide evidence for a potential association between ITI outcome and epitope specificity of FVIII inhibitors. Nevertheless the determination of epitopes and their clinical relevance has not yet been established. In this study a general strategy for the identification of anti-FVIII antibody epitopes in haemophilia A patient plasma was to be demonstrated. Phage-displayed peptide libraries were screened against anti-FVIII antibodies to isolate specific peptides. Peptide specificity was confirmed by FVIII-sensitive ELISA binding. Peptide residues essential for antibody binding were identified by mutational analysis and epitopes were predicted via FVIII homology search. The proposed mapping strategy was validated for the monoclonal murine antibody (mAb) 2-76. Binding studies with FVIII variants confirmed the location of the predicted epitope at the level of individual amino acids. In addition, anti-FVIII antibody-specific peptide ligands were selected for 10 haemophilia A patients with FVIII inhibitors. Detailed epitope mapping for three of them showed binding sites on the A2, A3 and C2 domains. Precise epitope mapping of anti-FVIII antibodies using antibody-specific peptide ligands can be a useful approach to identify antigenic sites on FVIII.

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.…”

Anti‐factor VIII antibodies in brothers with haemophilia A share similar characteristics

“…FVIII heavy chain (HC, A1‐A2‐B) and light chain (LC, A3‐C1‐C2) were purified from recombinant full‐length FVIII (rFVIII; Kogenate ™ FS, Bayer, Leverkusen or Helixate NexGen ® ; CSL Behring, Marburg, Germany) and individual human FVIII A2 and C2 domains were expressed in human embryonic kidney 293T cells as previously described . Recombinant porcine and single human domain (SHD) human/porcine hybrid FVIII (hpFVIII) proteins were purified from cell culture media of stable transfected baby hamster kidney cell lines as described before .…”

“…FVIII domain mapping by homologue‐scanning mutagenesis with SHD hpFVIII proteins was performed as previously described . A net absorbance (binding to pFVIII subtracted) within the linear binding range (Figure S1) above 0.15, corresponding to three standard deviations (SD) of IgG binding of 30 healthy individuals to pFVIII, was used for positive domain assignment.…”

Hemophilia A inhibitor treatment: the promise of engineered T-cell therapy

“…This scFv was isolated from a phage library and cloned into the human IgG1 heavy chain [56, 57], together with standard components for CAR design, including the CD28 transmembrane and intracellular domains and the intracellular domain of the CD3ζ chain (Figure 2B). Retroviral vectors encoding the ANS8 CAR were used to transduce human T effectors or human Treg cells [51].…”