Data Coming out of the Human Inhibitor PUP Study (HIPS) Reveal 4 Subgroups of Patients with Distinct Antibody Signatures

Background and objectives FVIII inhibitors remain the major complication of replacement therapy in hemophilia A. Why some patients develop inhibitors whereas others do not is poorly understood. The Hemophilia Inhibitor PUP Study (HIPS, clinicaltrials.gov NCT01652027), a prospective multicenter observational study, aim to identify early immune biomarkers which predict inhibitor development in previously untreated patients (PUPs) with severe hemophilia A. Such biomarkers would facilitate early risk identification and the initiation of potential immune intervention strategies. Previously we reported time-resolved antibody data for the first 15 patients who completed antibody analysis (Gangadharan 2017). Here we present data from all 25 patients enrolled in HIPS, identifying four distinct patient subgroups based on their unique antibody signatures. We also summarize data for F8 mutations and longitudinal data of circulating immune cells such as FoxP3+ T cells (Tregs), Th17 cells, and granulysin+ cells. Methods FVIII inhibitor testing (Nijmegen method, performed in the central laboratory Medical University of Vienna) and antibody analytics were assessed prior to first exposure, 7-9 days after exposure day (ED) 1 and 5-7 days after EDs 5, 10, 20, 30, 40, and 50. FVIII-specific advanced antibody analytics were performed using methodology described by Whelan 2013 and Hofbauer 2015. FVIII mutational analysis was performed at Bloodworks Northwest, Seattle. Longitudinal epigenetic cell counting in whole blood using quantitative PCR-based methylation assays (www.epiontis.com) was done to monitor changes in peripheral FoxP3+ T cells (Tregs), pro-inflammatory Th17 cells, and granulysin+ cells. Results Our data reveal 4 different subgroups of patients associated with distinct signatures of FVIII-specific antibodies. The first subgroup includes 7 subjects who developed FVIII inhibitors by study criteria (B.U. > 0.6 B.U. x 2 measurements from consecutive study days in central laboratory). All 7 subjects had high-risk F8 gene mutations including large deletions (2), intron 22 inversions (4) and duplications (1). Inhibitors in these patients were associated with the development of high-affinity class-switched FVIII-specific antibodies which were detected prior to the first detection of inhibitors. High-affinity IgG1 was generally observed first, followed by high affinity IgG3 and subsequently high-affinity IgG4. Other isotypes and IgG subclasses of anti-FVIII antibodies were