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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

Key Points This study is the first to assess the prognostic value of FVIII-specific antibody data in patients with AHA. Anti-FVIII IgA, but not immunoglobulin G, autoantibodies at baseline are potential predictors of recurrence and poor outcome of AHA.

The substantial variability in pharmacokinetic parameters in hemophilia patients A poses a challenge for optimal treatment with factor VIII (FVIII) products. We investigated the effect of FVIII-specific immunoglobulin G (IgG) on FVIII half-life in a cohort of 42 adult patients with severe and moderate hemophilia A without inhibitors. Fifteen (35.7%) of 42 patients tested positive for FVIII-binding IgG with titers ‡1:20 in the initial antibody screen, 9 of these 15 patients had FVIII-specific antibodies with titers ‡1:40, mostly low-tomoderate-affinity IgG1 and IgG3, and 1 had high-affinity IgG4 and later developed low-titer FVIII inhibitors. His brother with low-to-moderate-affinity IgG1 and IgG3 also later developed low-titer FVIII inhibitors. The presence of FVIII-specific IgG subclass titer ‡1:40 antibodies was significantly associated with shorter FVIII half-life (median, 7.8 hours [interquartile range, 6.6-9.2 hours]) vs 10.4 hours [interquartile range, 8.9-13.8 hours]); the regression coefficient adjusted for log age and log von Willebrand factor (VWF) antigen was 20.32 (P 5 .004), accounting for 16.9% of the observed variability of FVIII half-life in our cohort. Our data indicate a significant contribution of non-neutralizing FVIII-specific IgG to FVIII half-life reduction in hemophilia A patients. Thus, screening for FVIII-specific IgG could be beneficial in tailoring FVIII prophylactic regimens. (Blood. 2016;128(2):293-296) IntroductionProphylactic treatment of hemophilia A patients with factor VIII (FVIII) products is presently state of the art.1 FVIII pharmacokinetics differ significantly, which poses a challenge for optimal treatment design. It is generally accepted that the von Willebrand factor (VWF) level significantly influences pharmacokinetic (PK) parameters such as FVIII half-life.2-5 Different VWF levels only partially explain the variability in FVIII half-life, which leaves the question of which other parameters are accountable. This study was conducted to evaluate the effect of non-neutralizing, FVIII-specific immunoglobulin G (IgG) antibodies on FVIII half-life. Study designPlasma samples from a cohort of 42 patients with hemophilia A (recently described by Kepa et al 2 ) were screened for FVIII-binding IgG antibodies. 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. The specificity of FVIII-binding antibodies was tested in all samples with antibody titers $1:40 by using an affinity ELISA.7 Antibodies without conclusive apparent affinity were considered unspecific. Antibodies with titers ,1:40 (eg, 1:20)...

Preventing factor VIII (FVIII) inhibitors following replacement therapies with FVIII products in patients with hemophilia A remains an unmet medical need. Better understanding of the early events of evolving FVIII inhibitors is essential for risk identification and the design of novel strategies to prevent inhibitor development. The Hemophilia Inhibitor Previously Untreated Patients (PUPs) Study (HIPS; www.clinicaltrials.gov #NCT01652027) is the first prospective cohort study to evaluate comprehensive changes in the immune system during the first 50 exposure days (EDs) to FVIII in patients with severe hemophilia A. HIPS participants were enrolled prior to their first exposure to FVIII or blood products (“true PUPs”) and were evaluated for different immunological and clinical parameters at specified time points during their first 50 EDs to a single source of recombinant FVIII. Longitudinal antibody data resulting from this study indicate that there are 4 subgroups of patients expressing distinct signatures of FVIII-binding antibodies. Subgroup 1 did not develop any detectable FVIII-binding immunoglobulin G (IgG) antibodies. Subgroup 2 developed nonneutralizing, FVIII-binding IgG1 antibodies, but other FVIII-binding IgG subclasses were not observed. Subgroup 3 developed transient FVIII inhibitors associated with FVIII-binding IgG1 antibodies, similar to subgroup 2. Subgroup 4 developed persistent FVIII inhibitors associated with an initial development of high-affinity, FVIII-binding IgG1 antibodies, followed by IgG3 and IgG4 antibodies. Appearance of FVIII-binding IgG3 was always associated with persistent FVIII inhibitors and the subsequent development of FVIII-binding IgG4. Some of the antibody signatures identified in HIPS could serve as candidates for early biomarkers of FVIII inhibitor development.