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

Abstract

The pathogenicity of anti-human factor (F) VIII monoclonal antibodies (MAbs) was tested in a murine bleeding model. 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 approximately 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. 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. 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.