A subset of high-titer anti–factor VIII A2 domain antibodies is responsive to treatment with factor VIII

Abstract

• Discrepancies exist between inhibitor titer and bleeding phenotype in anti-fVIII antibodies.• A subset of high-titer type 2 anti-A2 inhibitors can be treated with fVIII.The primary B-cell epitopes of factor VIII (fVIII) are in the A2 and C2 domains. Within the C2 domain, antibody epitope and kinetics are more important than inhibitor titer in predicting pathogenicity in a murine bleeding model. To investigate this within the A2 domain, the pathogenicity of a diverse panel of antihuman fVIII A2 domain monoclonal antibodies (MAbs) was tested in the murine model. MAbs were injected into hemophilia A mice, followed by injection of human B domain-deleted fVIII. Blood loss after a 4-mm tail snip was measured. The following anti-A2 MAbs were tested: high-titer type 1 inhibitors 4A4, 2-76, and 1D4; 2-54, a high-titer type 2 inhibitor; B94, a type 2 inhibitor; and noninhibitory MAbs GMA-012, 4C7, and B25. All high-titer type 1 MAbs produced blood loss that was significantly greater than control mice, whereas all non-inhibitory MAbs produced blood loss that was similar to control. The type 2 MAbs were not pathogenic despite 2-54 having an inhibitor titer of 34 000 BU/mg immunoglobulin G. In addition, a patient with a high-titer type 2 anti-A2 inhibitor who is responsive to fVIII is reported. The discrepancy between inhibitor titer and bleeding phenotype combined with similar findings in the C2 domain stress the importance of inhibitor properties not detected in the standard Bethesda assay in predicting response to fVIII therapy.

Anti‐C1 domain antibodies that accelerate factor VIII clearance contribute to antibody pathogenicity in a murine hemophilia A model

“…Anti-FVIII mAbs directed against non-C1 domain VWF epitopes increase FVIII clearance in FVIII À/À mice Previously we investigated the in vivo pathogenicity of antibodies against the A2 and C2 domains of FVIII [14,15]. A subset of anti-A2 and C2 mAbs produced bleeding in FVIII À/À mice at low and high doses of FVIII (180 units kg À1 and 360 units kg À1 , respectively) in the tail snip bleeding model.…”

Clot waveform analysis using CS‐2000i™ distinguishes between very low and absent levels of factor VIII activity in patients with severe haemophilia A

“…Moreover, assays of this nature can be time‐consuming and undertaken in laboratories outside a diagnostic centre, and inhibitor titres in patients with acquired HA may not predict prognosis . In addition, Eubanks et al . reported that a one‐stage clotting assay for residual FVIII:C appeared unlikely to reflect the inhibitory effects of some FVIII monoclonal antibodies on coagulation function.…”

A microengineered vascularized bleeding model that integrates the principal components of hemostasis

“…Antibodies were purchased from Fisher Scientific (CD41, NB100-2614, 1:400), Santa Cruz Biotechnology (P-selectin, sc-19996, 1 µg ml −1 ), Bio-Rad (vWF-FITC, AHP062F, 1:1000), BD (CD45, 555483, 1:400), and abcam (TF, ab35807, 1:200). Antibody AVW-3 and 59D8 (Alexa Fluor 488 conjugated) were provided by Dr. Shawn M. Jobe (Blood Center of Wisconsin, 10 µg ml −1 , 1:100) and mAb 2–76 by Dr. Shannon L. Meeks (Emory University, 10 µg ml −1 ) 37 , 38 .…”