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. 2023 May 9;7(9):1831-1848.
doi: 10.1182/bloodadvances.2022007267.

Prospective Hemophilia Inhibitor PUP Study reveals distinct antibody signatures during FVIII inhibitor eradication

Helmut Paul  1 , Verena Berg  1 , Bagirath Gangadharan  2 , Joel Bowen  3 , Petra LeBeau  4 , Jan Blatný  5 , Christoph Male  6 , Vlad C Radulescu  7 , Rosa Diaz  8 , Maria Elisa Mancuso  9   10 , Deborah L Brown  11 , Birgit M Reipert  1   2
Affiliations

Prospective Hemophilia Inhibitor PUP Study reveals distinct antibody signatures during FVIII inhibitor eradication

Helmut Paul et al. Blood Adv. .

Abstract

Factor VIII (FVIII) inhibitor formation is a major clinical concern during replacement therapy in patients with hemophilia A. Immune tolerance induction (ITI) is the only therapeutic approach to attempt inhibitor eradication and establishment of long-term immune tolerance to FVIII. Hemophilia Inhibitor Previously Untreated Patient (PUP) Study (HIPS) was a prospective clinical trial to investigate changes in the immune system of PUPs with severe hemophilia A. Five patients who developed persistent FVIII inhibitors during HIPS entered an ITI extension arm (HIPS-ITI). During HIPS-ITI, inhibitor patients received ITI with the same FVIII product (a single source of recombinant, human full-length FVIII) used in HIPS until successful tolerance, declared failure, or a maximum of 2 years after HIPS-ITI enrollment, whichever came first. Blood samples and clinical data were collected monthly. Longitudinal FVIII-binding antibody signatures, associated binding specificities, and apparent affinities were determined for each patient at each sampling time point. ITI was successful or partially successful in 2 patients and failed in 3. Both groups presented with distinct FVIII-specific antibody signatures. ITI success required the disappearance of FVIII inhibitors, which was associated with the eradication or sustained titer minimization of high-affinity FVIII-specific antibodies, particularly of the immunoglobulin G1 (IgG1) and IgG4 subclasses. In contrast, ITI failure, as reflected by FVIII inhibitor persistence, was associated with persistent high-affinity FVIII-specific antibodies. Interestingly, 1 patient with partial ITI success and 1 patient with ITI failure developed apparent oligoreactive FVIII-binding antibodies during ITI. The explanation of the true nature of these antibodies requires more comprehensive follow-ups in future studies. This trial was registered at www.clinicaltrials.gov as #NCT01652027.

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Conflict of interest statement

Conflict-of-interest disclosure: H.P., V.B., and D.L.B. received research funding for institution from Baxalta Innovations GmbH, a Takeda company. B.G. is an employee of Baxalta Innovations GmbH, a Takeda company, and holds stock options of Takeda. J. Bowen received research funding from the study sponsor, University of Texas Health Science Center. P.L. received research funding from the study sponsor, University of Texas Health Science Center, and was an employee of Rho Inc. at the time of the study. J. Blatný received consultation/speakers fee from Takeda, Novo Nordisk, Sobi, LFB, Roche, Pfizer, CSL Behring, and Octapharma. C.M. received fees to institution for study participation from Bayer, Baxalta/Shire/Takeda, Biotest, CSL Behring, Novo Nordisk, and Sobi; unrestricted grant to institution from Biotest and CSL Behring; and personal honoraria (consultancy, speaker, chair) from Bayer, Biotest, CSL Behring, Novo Nordisk, Roche, and Pfizer. V.C.R. received research funding to institution and fees to institution for study participation from Takeda, Pfizer, and Grifols. R.D. was an employee of Baylor College of Medicine at the time of the study. M.E.M. received consultation, advisory, and speaker fees from Bayer, BioMarin, Catalyst Bioscience, CSL Behring, Grifols, Kedrion, LFB, Novo Nordisk, Octapharma, Pfizer, Roche, Sanofi, Sobi, Spark Therapeutics, Takeda, and uniQure. B.M.R. was an employee of Baxalta Innovations GmbH, a Takeda company, at the time of the study.

Figures

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Graphical abstract
Figure 1.
Figure 1.
Longitudinal monitoring of FVIII-specific antibodies and FVIII inhibitors in HIPS-ITI patient 1 during HIPS and HIPS-ITI. (A) FVIII-specific antibody titers (IgG1, IgG2, IgG3, IgG4, IgA, IgM as indicated) and FVIII inhibitors (BU/mL) for HIPS-ITI patient 1. The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The yellow star marks the initiation of ITI treatment at ED11. The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI. (B) Mean KA for FVIII-specific antibodies differentiated for individual IgG subclasses and FVIII inhibitors (BU/mL) for HIPS-ITI patient 1. Data for apparent KAs include the 95% confidence intervals (CIs) for up to 2 affinity clusters for each IgG subclass (dark colors: cluster 1; light colors: cluster 2). The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The asterisks mark antibodies with apparent affinities that were too high (KAs > 1011 M−1) to be assessed. Therefore, their apparent KAs were reported as 1011 M−1. The yellow star marks the initiation of ITI treatment at ED11. The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI. ∗IgG1 cluster 1, ∗∗IgG4 cluster 1. B, baseline; ND, not detectable (below the detection limit of 1:20 for FVIII-binding antibodies).
Figure 2.
Figure 2.
Longitudinal monitoring of specific and apparent nonspecific FVIII-binding antibodies and FVIII inhibitors in HIPS-ITI patient 2 during HIPS and HIPS-ITI. (A) FVIII-binding (specific) antibody titers (IgG1, IgG2, IgG3, IgG4, IgA, IgM; open circles, open squares, open triangles as indicated), FVIII-binding (apparent nonspecific) antibody titers (IgG1, IgG2, IgG3, IgG4, IgA; filled circles, filled squares as indicated), and FVIII inhibitors (BU/mL) for HIPS-ITI patient 2. The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The yellow star marks the initiation of ITI treatment at ED16. The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI. (B) Mean KA for FVIII-binding (specific) antibodies differentiated for individual IgG subclasses and FVIII inhibitors (BU/mL) for HIPS-ITI patient 2. Data for apparent KAs include the 95% CIs for up to 2 affinity clusters for each IgG subclass (dark colors: cluster 1; light colors: cluster 2). The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The yellow star marks the initiation of ITI treatment at ED16. The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI.
Figure 3.
Figure 3.
Longitudinal monitoring of FVIII-specific antibodies and FVIII inhibitors in HIPS-ITI patients 3 and 4 during HIPS and HIPS-ITI. (A,C) FVIII-specific antibody titers (IgG1, IgG2, IgG3, IgG4, IgA, IgM as indicated) and FVIII inhibitors (BU/mL) for HIPS-ITI patients 3 (A) and 4 (C). The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The yellow star marks the initiation of ITI treatment at ED12 (HIPS-ITI patient 3) and ED13 (HIPS-ITI patient 4). The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI. (B,D) Mean KA for FVIII-specific antibodies differentiated for individual IgG subclasses/Ig isotypes and FVIII inhibitors (BU/mL) for HIPS-ITI patients 3 (B) and 4 (D). Data for apparent KAs include the 95% CIs for up to 2 affinity clusters for each IgG subclass and for IgA (dark colors: cluster 1; light colors: cluster 2). The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The asterisks mark antibodies with apparent affinities that were too high (KAs > 1011 M−1) to be assessed. Therefore, their apparent KAs were reported as 1011 M−1. The yellow star marks the initiation of ITI treatment at ED12 (HIPS-ITI patient 3) and ED13 (HIPS-ITI patient 4). The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI. ∗IgG1 cluster 1.
Figure 3.
Figure 3.
Longitudinal monitoring of FVIII-specific antibodies and FVIII inhibitors in HIPS-ITI patients 3 and 4 during HIPS and HIPS-ITI. (A,C) FVIII-specific antibody titers (IgG1, IgG2, IgG3, IgG4, IgA, IgM as indicated) and FVIII inhibitors (BU/mL) for HIPS-ITI patients 3 (A) and 4 (C). The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The yellow star marks the initiation of ITI treatment at ED12 (HIPS-ITI patient 3) and ED13 (HIPS-ITI patient 4). The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI. (B,D) Mean KA for FVIII-specific antibodies differentiated for individual IgG subclasses/Ig isotypes and FVIII inhibitors (BU/mL) for HIPS-ITI patients 3 (B) and 4 (D). Data for apparent KAs include the 95% CIs for up to 2 affinity clusters for each IgG subclass and for IgA (dark colors: cluster 1; light colors: cluster 2). The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The asterisks mark antibodies with apparent affinities that were too high (KAs > 1011 M−1) to be assessed. Therefore, their apparent KAs were reported as 1011 M−1. The yellow star marks the initiation of ITI treatment at ED12 (HIPS-ITI patient 3) and ED13 (HIPS-ITI patient 4). The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI. ∗IgG1 cluster 1.
Figure 4.
Figure 4.
Longitudinal monitoring of specific and apparent nonspecific FVIII-binding antibodies and FVIII inhibitors in HIPS-ITI patient 5 during HIPS and HIPS-ITI. (A) FVIII-binding (specific) antibody titers (IgG1, IgG2, IgG3, IgG4, IgA, IgM; open circles, open squares, open triangles as indicated), FVIII-binding (apparent nonspecific) antibody titers (IgG1, IgG2, IgG3, IgG4, IgA; filled circles, filled squares as indicated), and FVIII inhibitors (BU/mL) for HIPS-ITI patient 5. The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The yellow star marks the initiation of ITI treatment at ED13. The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI. (B) Mean KA for FVIII-binding (specific) antibodies differentiated for individual IgG subclasses and FVIII inhibitors (BU/mL) for HIPS-ITI patient 5. Data for apparent KAs include the 95% CIs for up to 2 affinity clusters for each IgG subclass (dark colors: cluster 1; light colors: cluster 2). The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The yellow star marks the initiation of ITI treatment at ED13. The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI.
Figure 5.
Figure 5.
Apparent oligoreactive FVIII- and FIX-binding antibodies in HIPS-ITI patient 2 during HIPS and HIPS-ITI and timeline of clinical events. (A) Timeline of clinical events documented for HIPS-ITI patient 2 throughout HIPS and HIPS-ITI including (i) adverse events with short descriptions, (ii) infections and administered treatment, (iii) hospitalizations, duration of hospitalizations and administered treatment, (iv) immunizations and administered vaccinations, and (v) additionally administered medications and supplements. The yellow star marks the initiation of ITI treatment at ED16. (B) Apparent oligoreactive FVIII- (points, filled squares) and FIX-binding (asterisks) antibody titers as well as FVIII-specific (circles, open squares, open triangles) antibody titers (total Ig, IgG1, IgG2, IgG3, IgG4, IgA, IgM as indicated) and FVIII inhibitors (BU/mL) for HIPS-ITI patient 2. The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The yellow star marks the initiation of ITI treatment at ED16. The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI. DTaP-IPV-Hib, diphtheria, tetanus, pertussis, inactivated polio vaccine, Haemophilus influenzae type b; IIV4, quadrivalent-inactivated influenza vaccine; HAV, hepatitis A virus; PCV, pneumococcal-conjugated vaccine; PO, by mouth.
Figure 6.
Figure 6.
Apparent oligoreactive FVIII- and FIX-binding antibodies in HIPS-ITI patient 5 during HIPS and HIPS-ITI and timeline of clinical events. (A) Timeline of clinical events documented for HIPS-ITI patient 5 throughout HIPS and HIPS-ITI including (i) adverse events with short descriptions, (ii) infections and administered treatment, (iii) hospitalizations, duration of hospitalizations and administered treatment, (iv) immunizations and administered vaccinations, and (v) additionally administered medications and supplements. The yellow star marks the initiation of ITI treatment at ED13. (B) Apparent oligoreactive FVIII- (points, filled squares) and FIX-binding (asterisks) antibody titers as well as FVIII-specific (circles, open squares, open triangles) antibody titers (total Ig, IgG1, IgG2, IgG3, IgG4, IgA, IgM as indicated) and FVIII inhibitors (BU/mL) for HIPS-ITI patient 5. The red dotted line represents the limit for positive evaluation of FVIII inhibitors (0.6 BU/mL). The yellow star marks the initiation of ITI treatment at ED13. The continuous vertical line indicates the end of HIPS and the start of HIPS-ITI. AV, arteriovenous; DTaPolio, diphtheria, tetanus, polio.
Figure 7.
Figure 7.
Correlation between titers of apparent oligoreactive FVIII- and FIX-binding antibodies in HIPS-ITI patient 2 during HIPS-ITI. (A-B) Correlation analyses between apparent oligoreactive FVIII-binding IgG1 respectively IgG2 titers and FIX-binding total Ig (A) as well as apparent oligoreactive FVIII-binding IgA and FIX-binding total Ig (B) during HIPS-ITI (ED70 to ED388) of HIPS-ITI patient 2. Spearman correlation coefficients (r) with 95% CIs (given in brackets) are presented in the figure legends. Statistically significant correlations are highlighted, ∗P ≤ .05 and ∗∗P ≤ .01.
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