Affinity of FVIII-specific antibodies reveals major differences between neutralizing and nonneutralizing antibodies in humans

Abstract

Key Points• A new technology is presented to assess apparent affinities of FVIII-specific antibodies, differentiated for isotypes and IgG subclasses.• Affinities of FVIII-specific antibodies in patients with FVIII inhibitors are up to 100-fold higher than in patients without inhibitors.Recently, we reported that distinct immunoglobulin (Ig) isotypes and IgG subclasses of factor VIII (FVIII)-specific antibodies are found in different cohorts of patients with hemophilia A and in healthy individuals. Prompted by these findings, we further investigated the distinguishing properties among the different populations of FVIII-specific antibodies. We hypothesized that the affinity of antibodies would discriminate between the neutralizing and nonneutralizing antibodies found in different study cohorts. To test this idea, we established a competition-based enzyme-linked immunosorbent assay technology to assess the apparent affinities for each isotype and IgG subclass of FVIIIspecific antibodies without the need for antibody purification. We present a unique data set of apparent affinities of FVIII-specific antibodies found in healthy individuals, patients with congenital hemophilia A with and without FVIII inhibitors, and patients with acquired hemophilia A. Our data indicate that FVIII-specific antibodies found in patients with FVIII inhibitors have an up to 100-fold higher apparent affinity than that of antibodies found in patients without inhibitors and in healthy individuals. High-affinity FVIII-specific antibodies could be retrospectively detected in longitudinal samples of an individual patient with FVIII inhibitors 543 days before the first positive Bethesda assay. This finding suggests that these antibodies might serve as potential biomarkers for evolving FVIII inhibitor responses. (Blood. 2015;125(7):1180-1188

FVIII-binding IgG modulates FVIII half-life in patients with severe and moderate hemophilia A without inhibitors

“…Antibodies were analyzed by using fully validated semiquantitative, directbinding enzyme-linked immunosorbent assays (ELISAs) as described in Whelan et al 6 Details of the validation procedure as well as details of the cutoff determination and the sensitivity of the ELISAs are provided in Whelan et al 6 and Hofbauer et al 7 Initially, all samples were screened in a 1:20 dilution for FVIII-binding IgG antibodies. Samples that tested positive were characterized for titers of FVIIIbinding IgG subclasses and apparent affinities.…”

Nonneutralizing antibodies against factor VIII and risk of inhibitor development in severe hemophilia A

“…In previous reports, NNA prevalence ranged from 2% to 3% in healthy individuals 16,18,21,24 and from 12.2% to 53.8% in patients with hemophilia A. [13][14][15][16][17][18][19][20][21][22]24,25,29,30 Several factors, particularly that all previous studies predominantly included patients with multiple transfusions, explain this large variation and the gaps in knowledge about their potential clinical role.…”

“…[13][14][15][16][17][18][19][20][21][22]24,25,29,30 Several factors, particularly that all previous studies predominantly included patients with multiple transfusions, explain this large variation and the gaps in knowledge about their potential clinical role. For instance, the intron 22 inversion of FVIII mutation was associated with an increased frequency of NNAs in patients who had had multiple transfusions in 1 study 29 that was not confirmed in another study.…”

“…Their prevalence is approximately 2% to 3% in healthy individuals, 16,18,21,24 but estimates in patients who have hemophilia with different degrees of severity vary widely from 12% to 54%. [13][14][15][16][17][18][19][20][21][22]24,25,[29][30][31] Although NNAs and inhibitors cannot be distinguished on the basis of their isotypes, clonality, and epitopes, 32 recent data published by Hofbauer et al 16 indicate that anti-FVIII immunoglobulin G (IgG) with inhibitory activity has an up to 100-fold higher affinity for FVIII than IgG without inhibitory activity. On the basis of cross-reactivity studies, it has also been suggested that FVIII inhibitors in hemophilia A patients originate from the expansion of a natural anti-FVIII clone of B lymphocytes that exists before any treatment with FVIII and secretes anti-FVIII antibodies similar to the natural antibodies found in healthy individuals.…”

Inhibitory antibodies against factor VIII C1 domain

“…8 This raises the intriguing possibility that dangerous antibodies might be detected before bleeding occurs. Detecting the risk of bleeding before it occurs will require assay (s) that better measure the risk of bleeding.…”