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Observational Study
. 2020 Nov 24;4(22):5785-5796.
doi: 10.1182/bloodadvances.2020002731.

The prospective Hemophilia Inhibitor PUP Study reveals distinct antibody signatures prior to FVIII inhibitor development

B M Reipert  1 , B Gangadharan  1 , C J Hofbauer  1 , V Berg  2 , H Schweiger  2 , J Bowen  3 , J Blatny  4 , K Fijnvandraat  5 , E S Mullins  6 , J Klintman  7 , C Male  8 , C McGuinn  9 , S L Meeks  10 , V C Radulescu  11 , M V Ragni  12 , M Recht  13 , A D Shapiro  3 , J M Staber  14 , H M Yaish  15 , E Santagostino  16 , D L Brown  17
Affiliations
Observational Study

The prospective Hemophilia Inhibitor PUP Study reveals distinct antibody signatures prior to FVIII inhibitor development

B M Reipert et al. Blood Adv. .

Abstract

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.

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Conflict of interest statement

Conflict-of-interest disclosure: B.M.R. was an employee of Baxalta Innovations at the time of the study but left after study completion. B.G. is an employee of Baxalta Innovations and holds Takeda stock options. C.J.H. was an employee of Baxalta Innovations at the time of the study but is now an employee of Takeda Pharmaceutical (Cambridge, MA) and holds Takeda stock options. V.B. and H.S. received institutional research funding from Baxalta Innovations. J. Bowen received research funding from the study sponsor, University of Texas Health Science Center (Houston, TX). J. Blatny has received consultation/speakers fees from Takeda, Novo Nordisk, Sobi, LFB, Roche, Pfizer, CSL Behring, and Octapharma. K.F. has received unrestricted institutional research grants from CSL Behring and Novo Nordisk and institutional consultancy fees from Grifols, Takeda, Novo Nordisk, and Roche. E.S.M. has received advisory board fees from Bayer and Takeda. C. Male has received institutional fees for study participation from Bayer, Baxalta/Shire/Takeda, Biotest, CSL Behring, Novo Nordisk, and Sobi; an unrestricted institutional grant from Biotest and CSL Behring; and personal honoraria (consultancy, speaker, chair) from Bayer, CSL Behring, Novo Nordisk, and Roche. C. McGuinn received consultancy/advisory board fees from Roche, and Bioverat, Kendrion, Octapharma, BPL. and Biomarin and institutional fees for study participation from Baxalta/Shire/Takeda, Pfizer/Spark, Novo Nordisk, Roche, and Bioverat/Roche, and Bioverativ/Sanofi. S.L.M. received consultancy/advisory board fees from Bayer, Baxalta/Shire/Takeda, Bioverativ/Sanofi, CSL Behring, Roche, and Bioverat, Novo Nordisk, Octapharma, Pfizer, Sangamo, Spark/Roche, and Sobi. V.C.R. received institutional research funding and fees for study participation from Takeda, Pfizer, and Grifols. M.V.R. received research funding and advisory board fees from Alnylam, Biomarin, Bioverativ, Sangamo, and Spark. M.R. received institutional research funding from Bioverativ/Sanofi, Biomarin, Roche, and Bioverat, Novo Nordisk, Shire/Takeda, Spark/Roche, uniQure and consultancy/advisory board fees from Bioverativ/Sanofi, CSL Behring, Roche, and Bioverat, Kedrion, Novo Nordisk, Pfizer, Shire/Takeda, and uniQure. A.D.S. received institutional research funding from Agios, Biomarin, Bioverativ, Daiichi Sankyo, Roche, and Bioverat, Glover Blood Therapeutics, Kendrion, Novartis, Novo Nordisk, OPKO, Octapharma, Pfizer, Prometic, Sangamo, and Takeda and consultant/advisory board fees from Bioverativ, Roche, and Bioverat, Novo Nordisk, Prometic, Sangamo, and Shire/Takeda; no funding was personally accepted (all was paid to the Indiana Hemophilia & Thrombosis Center). J.M.S. received consultancy/advisory boards from Spark, Roche, and Bioverat, Novo Nordisk, Bayer, Takeda, and Sanofi. H.M.Y. received institutional research funding from Novo Nordisk, Takeda, Bayer, Agio, Genentics, and CSL and advisory/consultancy board fees from Novo Nordisk, Takeda, Bayer, Genentics, and Octapharma. E.S. received advisory boards and/or speaker bureaus fees from Bayer, Pfizer, CSL Behring, Novo Nordisk, Shire/Takeda, Sobi, Bioverativ, Grifols, Kedrion, Octapharma, Roche, Spark, and uniQure. D.L.B. received research funding from Baxalta/Takeda. J.K. declares no competing financial interests.

Figures

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Graphical abstract
Figure 1.
Figure 1.
Longitudinal monitoring of FVIII-binding antibodies and FVIII inhibitors in 2 representative examples of patients in subgroup 1. (A-B) Results of the analysis of FVIII-binding antibodies (IgG1, IgG2, IgG3, IgG4, IgA, IgM as indicated) and FVIII inhibitors (BU/mL) for 2 representative examples of patients in subgroup 1, patient 3 (A) and patient 6 (B), who did not develop FVIII inhibitors throughout the study period. The red dotted lines represent the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The data for the remaining 5 patients of subgroup 1 are shown in supplemental Figure 2. B, baseline; ND, not detectable (below the detection limit of 1:20 for FVIII-binding antibodies).
Figure 2.
Figure 2.
Longitudinal monitoring of FVIII-binding antibodies and FVIII inhibitors in 2 representative examples of patients in subgroup 2. (A-B) Results of the analysis of FVIII-binding antibodies (IgG1, IgG2, IgG3, IgG4, IgA, and IgM, as indicated) and FVIII inhibitors (BU/mL) for 2 representative examples of patients in subgroup 2, patient 9 (A) and patient 14 (B), who developed FVIII-binding IgG1 antibodies but did not develop FVIII inhibitors throughout the study period. The red dotted lines represent the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). (C-D) Apparent affinity constants of FVIII-binding IgG1 antibodies (mean KA) and FVIII inhibitors (BU/mL) in patient 9 (C) and patient 14 (D). Data for apparent affinity constants include the 95% CIs for up to 2 IgG1 affinity clusters (open blue bars, IgG1 population 1; closed blue bars, IgG1 population 2). The red dotted lines represent the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). KA, apparent affinity constant. The data for the remaining 5 patients of subgroup 2 are shown in supplemental Figure 3.
Figure 3.
Figure 3.
Longitudinal monitoring of FVIII-binding antibodies and FVIII inhibitors in the 2 patients in subgroup 3. (A-B) Results of the analysis of FVIII-binding antibodies (IgG1, IgG2, IgG3, IgG4, IgA, and IgM, as indicated) and FVIII inhibitors (BU/mL) for the 2 patients in subgroup 3, patient 15 (A) and patient 16 (B), who developed FVIII-binding IgG1 antibodies and transient low-titer FVIII inhibitors. The red dotted lines represent the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). (C-D) Apparent affinity constants of FVIII-binding IgG1 antibodies (mean KA) and FVIII inhibitors (BU/mL) in patient 15 (C) and patient 16 (D). Data for apparent affinity constants include the 95% CIs for up to 2 IgG1 affinity clusters (open blue bars, IgG1 population 1; closed blue bars, IgG1 population 2). The red dotted lines represent the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL).
Figure 4.
Figure 4.
Longitudinal monitoring of FVIII-binding antibodies and FVIII inhibitors in 2 representative examples of patients in subgroup 4. (A-B) Results of the analysis of FVIII-binding antibodies (IgG1, IgG2, IgG3, IgG4, IgA, and IgM, as indicated) and FVIII inhibitors (BU/mL) for 2 representative examples of patients in subgroup 4, patient 17 (A) and patient 21 (B), who developed FVIII-binding IgG subclass-switched antibodies and persistent FVIII inhibitors. The red dotted lines represent the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). (C-D) Apparent affinity constants of FVIII-binding antibodies (mean KA), differentiated for individual IgG subclasses and FVIII inhibitors (BU/mL), in patient 17 (C) and patient 21 (D). Data for apparent affinity constants include the 95% CIs for ≤2 affinity clusters for each IgG subclass (open bars, population 1; closed bars, population 2). The red dotted lines represent the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The asterisk indicates antibodies with apparent affinities that were too high to be assessed. Therefore, they were set to 10e11. The data for the remaining 5 patients of subgroup 4 are shown in supplemental Figure 4.
Figure 5.
Figure 5.
Summary of FVIII-binding antibodies for all patients in subgroups 2 and 4. (A-B) Medians and IQRs for titers and apparent affinity constants of FVIII-binding antibodies, differentiated for IgG subclasses 1-4, IgM and IgA, as detected in patients in subgroup 2, who did not develop FVIII inhibitors (A), and patients in subgroup 4, who developed persistent FVIII inhibitors (B). The calculation of medians and IQRs for titers and apparent affinity constants included all antibody data for each patient in the respective subgroup at each time point analyzed.
Figure 6.
Figure 6.
Comparison of titers and apparent affinities for FVIII-binding IgG1 antibodies in patients in subgroups (SG) 2 and 4. Medians and IQRs for titers and apparent affinity constants of FVIII-binding IgG1 antibodies, as detected in patients of subgroup 2, who did not develop FVIII inhibitors, and patients in subgroup 4, who developed persistent FVIII inhibitors. Median IQRs for titers and apparent affinity constants include all antibody data for each patient in the respective subgroup at each time point analyzed. There is a significant difference in both titers and apparent affinity constants between patients of subgroups 2 and 4. ****P < .0001.
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