Longitudinal Antibody Signatures Following FVIII Replacement Therapy in Previously Untreated Patients with Severe Hemophilia Α- New Insights from the Hemophilia Inhibitor PUP Study (HIPS)
Authors: Gangadharan Bagirath , Reipert Birgit , Scheiflinger Fritz , Bowen Joel , Donnachie Elizabeth , Fijn van Draat Karin , Gruppo Ralph A. , Klintman Jenny , Male Christoph , McGuinn Catherine E. , Meeks Shannon L. , Recht Michael , Ragni Margaret V. , Yaish Hassan M. , Shapiro Amy D. , Yee Donald L , Radulescu Vlad Calin , Santagostino Elena , Brown Deborah 
Published: April 2021

Abstract
Background and objectives
The Hemophilia Inhibitor PUP Study (HIPS, clinicaltrials.gov NCT01652027) is a prospective multicenter observational study of PUPs with severe hemophilia A during their first 50 exposure days (EDs) to recombinant Factor VIII (FVIII, Advate TM). The objective is to elucidate immune system changes and identify potential early biomarkers of inhibitor development in PUPs. Previously we reported that the appearance of high-affinity anti-FVIII antibodies preceded clinical diagnosis of FVIII inhibitors in patients (Reipert 2016). Here we present longitudinal antibody data including titers of FVIII-specific IgM, IgG1-4, IgA, apparent affinities of antibodies and titers of FVIII inhibitors for the first 15 patients who completed the antibody analysis of the HIPS study. Moreover, we present longitudinal monitoring data for key circulating immune cells such as FoxP3+ T cells (Tregs), pro-inflammatory Th17 cells, total T cells and granulysin+ NK cells.
Methods
FVIII inhibitor testing (Nijmegen methodology, performed in the central laboratory Medical College of Vienna) and antibody analytics were done prior to first exposure, 7-9 days after ED 1 and 5-7 days after EDs 5, 10, 20, 30, 40, and 50. FVIII-specific antibody analytics, including specifications of isotypes, IgG subclasses and apparent affinities were done using methodology described by Whelan 2013 and Hofbauer 2015. Circulating immune cells including FoxP3+ T cells (Tregs), pro-inflammatory Th17 cells, total T cells and granulysin+ NK cells were monitored by epigenetic cell counting in whole blood using quantitative PCR-based methylation assays (www.epiontis.com)
Results
25 subjects have been enrolled among 15 study sites. Data for 15 subjects who had completed 50 exposure days were available for analysis. Among these 15 subjects, 4 developed FVIII inhibitors by study criteria (&gt; 0.6 B.U. x 2 measurements in central laboratory) at exposure days 6, 10 and 20. All inhibitors were associated with the development of high affinity FVIII-specific antibodies. High-affinity IgG1 antibodies were detected first and subsequently switched to IgG3 and IgG4, switching to IgG2 was observed only in one of the 4 patients. Two of the 4 inhibitor patients initially developed low affinity IgG1 antibodies which were subsequently accompanied by high affinity IgG1 before switching to high-affinity IgG3 and IgG4. One of the 4 inhibitor patients expressed non-neutralizing FVIII-specific IgG1 antibodies prior to first FVIII infusion already and developed a high-titer inhibitor as early as ED6
Six of the 15 patients who had completed 50 exposure days developed non-neutralizing antibodies (detected on at least 2 time points). Five of them expressed low affinity IgG1 which was never accompanied by high affinity IgG1 or by any other IgG subclass. One of the 6 patients initially expressed low affinity IgG1 which was later accompanied by non-neutralizing high affinity IgG1. Switching to other IgG subclasses was not observed.
Three patients had no antibodies detected and 2 had non-neutralizing antibodies detected at a single time point only.
Levels of circulating FoxP3+ Tregs in the infant patients were in the same range as in healthy adults with some variation between individuals. In contrast, circulating TH17 cells were barely detectable. The levels of granulysin+ NK cells showed substantial variations between patients as well as during longitudinal monitoring in the same patient.
Conclusion
Antibody data obtained from the first 15 patients who completed 50 exposure days in the HIPS study confirms that FVIII inhibitor development is preceded by the detection of high-affinity FVIII-specific antibodies. High affinity IgG1 antibodies appeared first and subsequently switched to IgG3 and IgG4 which indicates a T-cell dependent pathway in FVIII inhibitor development. Switching to IgG2 was only seen in 1 of the 4 inhibitor patients. In contrast, patients with non-neutralizing antibodies only expressed IgG1 antibodies and never switched to another IgG subclass. 5 of the 6 patients with non-neutralizing antibodies only expressed low affinity IgG1 which might reflect a T-cell independent pathway of antibody development.
In summary, our data indicate that comprehensive analysis of FVIII-specific antibodies might provide early biomarkers which indicate evolving FVIII inhibitors.

Disclosures
Gangadharan: Shire: Employment. Reipert: Shire: Employment. Scheiflinger: Shire: Employment. Bowen: Shire: Research Funding. Fijn van Draat: ZonMW,CSL Behring: Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees; Pfizer, Novo Nordisk: Other: Lectures. Gruppo: Shire: Other: Honorarium for participation on physician advisory board. Male: Bayer, Baxalta, Biotest, CSL Behring, Novo Nordisk, Pfizer: Other: Speaker Honoraria and/Travel support. McGuinn: Shire/Baxalta: Consultancy; Shire / Baxalta, Spark, Biogen, Roche/Genetehc: Research Funding. Recht: Baxalta, Novo Nordisk, Biogen, Pfizer: Research Funding; Biogen , Kediron: Consultancy. Ragni: A Anylam,Biomarin,Tecere, Benitec: Honoraria; Alnylam, CSL Behring, Dimensions, Genetech/Roche,Pfizer, Shire, SPARK: Research Funding. Yaish: Baxalta, Bayer and Octapharma, CSL behring: Consultancy, Membership on an entity's Board of Directors or advisory committees; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees. Santagostino: : Bayer, Shire, Pfizer, Novo Nordisk, Kedrion, Roche, Sobi, Bioverativ, CSL Behring, Grifols, Octapharma: Speakers Bureau; Bayer, Shire, Pfizer, Novo Nordisk, Kedrion, Roche, Sobi, Bioverativ: Other: Advisory board. Brown: Shire: Research Funding.