Abstract
Introduction: Up to 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). Patients with congenital hemophilia who develop inhibitors usually have a polyclonal antibody response directed against the A2 and C2 domains of fVIII, but antibodies to all domains have been reported. We recently reported that anti-C1 domain antibodies were present in approximately 50% of plasmas from patients with hemophilia A and inhibitors. M6143 is a low titer (180 BU/mg IgG), tight-binding, anti-C1 monoclonal antibody that inhibits fVIII binding to von Willebrand Factor but not phospholipid. Surprisingly in a murine tail-snip bleeding model M6143 was pathogenic despite having a low inhibitory titer of 2.3 BU/ml (0.5 mg M6143/kg mouse body weight) and treatment with 180 U/kg of B-domain deleted (BDD) fVIII. The presence of M6143 resulted in increased clearance of fVIII/M6143 complexes. Given this data we hypothesized M6143 would remain pathogenic until fVIII was added in molar excess of M6143.
Methods: 8-12 week old E16 hemophilia A mice received 100 µl injections of M6143 at a concentration of 0.5 mg/kg (65 nM estimated peak plasma concentration), 0.25 mg/kg (~32.5 nM), 0.125 mg/kg (~16 nM), 0.0625 mg/kg (8 nM), or normal saline, followed by BDD fVIII 15 minutes later at a dose of 180 U/kg (~2.5 nM estimated peak plasma concentration), 360 U/kg (~5 nM), 720 U/kg (~10 nM), or 1440 U/kg (~20 nM). Tails were warmed then transected at 4 mm distally two hours post fVIII injection. Blood loss per mouse body weight (mg/g) following tail snips was measured using a pre-weighed 15 ml conical tube containing normal saline.
Results: Hemophilia A mice treated with 180 U/kg in the absence of antibody had minimal blood loss. M6143 at 0.5 mg/kg (2.3 BU/ml) produced significant bleeding at fVIII doses ranging from 180-1440 U/kg. Subsequently the concentration of M6143 was decreased while continuing extremely high fVIII dosing of 1440 U/kg (~20 nM). At all decreased concentrations of M6143, with high dose FVIII, bleeding was corrected (Figure 1).
Conclusion: M6143 is a minimally inhibitory antibody whose pathogenicity is only overcome in the presence of very high dose fVIII. The amount of fVIII needed relative to the inhibitor titer is paradoxical to the typical clinical strategy that low titer inhibitors can be overwhelmed with modest increases in the fVIII dosing. Inhibitors with similar characteristics to M6143 may be responsible for the subset of patients with low titer inhibitors who do not respond to treatment with fVIII.
Meeks:CSL Behring: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees.
