Detection of Non Inhibitory Binding Antibodies to Von Willebrand Factor Affecting the Clearance of VWF:Ag in Von Willebrand Disease

Abstract ²²⁷⁵

Von Willebrand Disease (VWD) is an inherited rare bleeding disorder caused by a deficiency of von Willebrand factor (VWF). VWF, the largest multimeric plasma glycoprotein, facilitates platelet aggregation and stabilizes FVIII in the circulation. Inhibitory antibodies to VWF have been reported in 10% – 15% of type 3 VWD patients repetitively treated with plasma-derived VWF/FVIII concentrates. The clinical impact of non-inhibitory antibodies to VWF has not been previously reported. To investigate the immunogenicity of a novel recombinant human VWF (rhVWF), both total binding anti-VWF antibodies and inhibitory anti-VWF antibodies (VWF:Ristocetin Cofactor Activity [RCo], VWF:Collagen Binding Activity [CB], VWF:FVIII Binding [FVIIIB]) were assessed in a Phase 1 multicenter clinical study. Total binding anti-VWF antibodies were determined by an enzyme-linked immunosorbent assay (ELISA) employing polyclonal anti-human IgG antibodies. Plasma samples were analyzed in two steps employing a screening assay determining the titer of the binding antibodies and in a second step, confirming the specificity of the antibody in a competition assay. Samples were considered positive, when a sample was at least two titer steps lower than the antibody titer detected in the screening assay necessitating a screening titer of at least 1:80. Neutralizing antibodies to the key functional activities of VWF, such as VWF:RCo, VWF:CB and VWF:FVIIIB, were measured by assays based on the Bethesda assay established for quantitative analysis of FVIII inhibitors (Nijmegen modification of the Bethesda assay). To exclude false positive results, the detection limit for anti-VWF inhibitors was set to 1 BU/mL for all 3 assays. Three of 39 subjects screened had a pre-existing high titer non-neutralizing binding antibody (all 1/1280) to VWF. One of these 3 subjects was excluded from the study due to the presence of an inhibitory antibody to VWF:CB (1.3 BU/mL) at screening. Two of the 3 subjects were enrolled and treated with rVWF and/or pdVWF concentrate as part of the safety, tolerability and PK assessments. The high titer binding anti-VWF antibodies were associated with a significant decreased VWF:Ag activity post infusion of either pdVWF or rVWF and consequential decreased activity of VWF:RCo, VWF:CB and FVIII:C. For example, one of the subject had a reduced VWF:Ag incremental recovery of 1.1 IU/dL/(U VWF:RCo/kg) (mean of cohort 1.6 IU/dL/[U VWF:RCo/kg]) and a reduced VWF:Ag t_1/2 2.4 hours (mean of cohort t_1/2 25.3 hours) post infusion of rhVWF dosed at 50 IU VWF:RCo/kg. Relevant data of all PK assessments and antibody titers will be presented. None of the study subjects developed an inhibitory antibody to either VWF or FVIII and there was no impact on the subsequent treatment of bleeding episodes with commercial pdFVIII/VWF concentrates noted by the treating physician. The clinical relevance of non-neutralizing antibodies to VWF requires additional investigation.