Rachel T. Barrow

Analysis of the Human Factor VIII A2 Inhibitor Epitope by Alanine Scanning Mutagenesis

Antibodies directed to the A2 domain of factor VIII (fVIII) are usually an important component of the polyclonal response in patients who have clinically significant inhibitory antibodies to fVIII. A major determinant of the A2 epitope has been located by homolog scanning mutagenesis using recombinant hybrid human/porcine fVIII molecules to a sequence bounded by Arg484–Ile508 (Healey, J. F., Lubin, I. M., Nakai, H., Saenko, E. L., Hoyer, L. W., Scandella, D., and Lollar, P. (1995) J. Biol. Chem.270, 14505–14509). Within this region, human residues Arg484, Pro485, Tyr487, Ser488, Arg489, Pro492, Val495, Phe501, and Ile508 differ from porcine fVIII. We stably expressed in mammalian cells nine active B-domainless human fVIII molecules containing single alanine substitutions at these sites. Their inhibition by a murine anti-A2 monoclonal antibody, monoclonal antibody (mAb) 413, and by three A2-specific alloimmune and two A2-specific autoimmune human inhibitor plasmas was measured by the Bethesda assay. The inhibition of Arg484 → Ala, Tyr487 → Ala, Arg489 → Ala, and Arg492 → Ala by mAb413 was reduced by greater than 90% compared with wild-type, B-domainless human fVIII. mAb413 inhibited the most severely affected mutant, Arg489 → Ala, 0.01% as well as wild-type fVIII. For all five patient plasmas, the Tyr487 → Ala mutant displayed the greatest reduction in inhibition. The inhibition of the Tyr487 → Ala mutant by these antibodies ranged from 10% to 20% that of wild-type fVIII. The inhibition of the Ser488 → Ala, Arg489 → Ala, Pro492 → Ala, Val495 → Ala, Phe501 → Ala, and Ile508 → Ala mutants by most of the plasmas also was significantly reduced. In contrast, the Arg484 → Ala and Pro485 → Ala mutants were relatively unaffected. Thus, although mAb413 binds to the same region as human A2 inhibitors, it recognizes a different set of amino acid side chains. The side chains recognized by human A2 inhibitors appear to be similar, despite the differing immune settings that give rise to fVIII alloantibodies and autoantibodies.

Nonclassical anti-C2 domain antibodies are present in patients with factor VIII inhibitors

The antihuman factor VIII (fVIII) C2 domain immune response in hemophilia A mice consists of antibodies that can be divided into 5 groups of structural epitopes and 2 groups of functional epitopes. Groups A, AB, and B consist of classical C2 antibodies that inhibit the binding of fVIII to phospholipid and von Willebrand factor. Groups BC and C contain nonclassical C2 antibodies that block the activation of fVIII by thrombin or factor Xa. Group BC antibodies are the most common and display high specific inhibitory activity and type II kinetics. The C2 epitope groups recognized by 26 polyclonal human anti-fVIII inhibitor plasmas were identified by a novel competition enzyme-linked immunosorbent assay using group-specific murine monoclonal antibodies. Most of the anti-C2 inhibitor plasmas inhibited the binding of both classical and nonclassical antibodies. These results suggest that nonclassical anti-C2 antibodies contribute significantly to the pathogenicity of fVIII inhibitors.

The humoral response to human factor VIII in hemophilia A mice

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 IgG1 and IgG2a isotypes, respectively. Because the IgG2a 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.

Comparative immunogenicity of recombinant B domain‐deleted porcine factor VIII and Hyate:C in hemophilia A mice presensitized to human factor VIII

Summary.  Hyate is a commercial plasma‐derived porcine factor (F)VIII concentrate that is used in the treatment of patients with inhibitory antibodies to FVIII. OBI‐1 is a recombinant B domain‐deleted form of porcine FVIII that is in clinical development for the same indication. Hemophilia A mice were presensitized with human FVIII to simulate clinical inhibitory antibody formation and then were randomized to receive OBI‐1 or Hyate:C in a comparative immunogenicity trial. OBI‐1 or Hyate:C were given in a series of four intravenous injections at weekly intervals at doses of 1, 10, or 100 U kg−1. Inhibitory antibodies to porcine FVIII were not detected by Bethesda assay in most of the mice given OBI‐1 or Hyate:C at doses of 1 or 10 U kg−1, but were identified in 81% and 94% of mice given 100 U kg−1 of OBI‐1 or Hyate:C, respectively. There was no significant difference between OBI‐1 and Hyate:C in inhibitory antibody formation at any dose, although there was a trend toward a lower Bethesda titer in OBI‐1‐treated mice at 10 U kg−1 (P = 0.09). Total anti‐FVIII antibodies to Hyate:C and OBI‐1 were also measured by ELISA using immobilized purified plasma‐derived porcine FVIII and OBI‐1, respectively, as antigens. At the 10 and 100 U kg−1 doses, the mean anti‐FVIII response was higher in Hyate:C‐treated‐mice than in OBI‐1‐treated mice (P = 0.02 and P = 0.004, respectively). The results using this model suggest that OBI‐1 may be less immunogenic and safer than Hyate:C in FVIII inhibitor patients.

Non-classical anti-factor VIII C2 domain antibodies are pathogenic in a murine in vivo bleeding model.

Summary.  Objective: The pathogenicity of anti‐human factor (F) VIII monoclonal antibodies (MAbs) was tested in a murine bleeding model. Methods: MAbs were injected into the tail veins of hemophilia A mice to a peak plasma concentration of 60 nm, followed by injection of human B domain‐deleted FVIII at 180 U kg−1, producing peak plasma concentrations of ∼2 nm. At 2 h, blood loss following a 4‐mm tail snip was measured. The following MAbs were tested: (i) 4A4, a type I anti‐A2 FVIII inhibitor, (ii) I54 and 1B5, classical type I anti‐C2 inhibitors, (iii) 2–77 and B45, non‐classical type II anti‐C2 inhibitors, and (iv) 2–117, a non‐classical anti‐C2 MAb with inhibitory activity less than 0.4 Bethesda Units per mg IgG. Results: All MAbs except 2–117 produced similar amounts of blood loss that were significantly greater than control mice injected with FVIII alone. Increasing the dose of FVIII to 360 U kg−1 overcame the bleeding diathesis produced by the type II MAbs 2–77 and B45, but not the type I antibodies, 4A4, I54, and 1B5. These results were consistent with the in vitro Bethesda assay in which 4A4 completely inhibited both 1 U mL−1 and 3 U mL−1 FVIII, while there was 40% residual activity at saturating concentrations of 2–77 at either concentration of FVIII. Conclusions: For patients with an inhibitor response dominated by non‐classical anti‐C2 antibodies both the in vivo and in vitro results suggest that treatment with high‐dose FVIII rather than bypassing agents may be warranted.

Factor VIII inhibitors.

The differential diagnosis in the bleeding patient includes inhibitory antibodies to blood coagulation proteins. Factor VIII (fVIII) is the most commonly targeted coagulation protein by the immune system. FVIII inhibitors arise as alloantibodies in transfused hemophiliacs and as autoantibodies in nonhemophiliac populations (1, 2, 3, 4). They develop in response to fVIII infusions in approximately 25% of patients with hemophilia A (5). In nonhemophiliacs, fVIII autoantibodies develop in a variety of clinical settings, including the postpartum period, systemic erythematosus, and chronic lymphocytic leukemia. Interestingly, autoantibody patients tend to present with more severe bleeding than hemophilia A patients with a de novo inhibitor. In both settings, fVIII inhibitors are polyclonal IgG populations directed against multiple epitopes.

The comparative immunogenicity of human and porcine factor VIII in haemophilia A mice

Inhibitory antibodies to factor VIII (FVIII inhibitors) are the most significant complication in the management of haemophilia A. The immunogenicity of FVIII may be driven in part by structural determinants within the FVIII molecule itself. Regions of nonidentity between human and porcine FVIII possibly could drive differential immune responses. The goal of this study was to compare the overall antibody response and levels of antibodies to the individual FVIII domains in naïve haemophilia A mice immunised with human or porcine FVIII. Haemophilia A mice were immunised with human or porcine FVIII using a protocol that mimics human clinical use. Inhibitor and total anti-FVIII antibody titers were measured and the domain-specificity of antibodies from 1,759 anti-FVIII hybridomas was determined. The overall immunogenicity of human and porcine FVIII was similar but significant differences in domain recognition were discovered. Anti-A2 and anti-C2 antibodies constituted the majority of inhibitors in both the human and porcine FVIII groups, similar to inhibitors that develop in humans. The proportions of anti-A2 or anti-C2 antibodies were not significantly different between the two groups. However, the specific inhibitory activity of anti-A2 antibodies was higher in the human FVIII group. Additionally, proportion of anti-C1 antibodies was significantly higher in the human FVIII group. In contrast, anti-A3 antibodies were more common in the porcine FVIII group. The differential immune response to human and porcine FVIII suggests that it may be possible to reduce the immunogenicity of FVIII by mutagenesis of the A2, A3 and C1 domains.