Detection of Anti-drug Antibodies in Patients with Acquired Hemophilia A: Data from the GTH-AHA-EMI Study

Introduction: Acquired hemophilia A (AHA) is a rare bleeding disorder caused by the development of neutralizing antibodies against endogenous coagulation factor VIII (FVIII). Emicizumab, a humanized monoclonal bispecific antibody that mimics FVIII function, is expected to become a first-line therapy for AHA [1]. However, its administration has been associated with the development of anti-drug antibodies (ADA) in patients with congenital hemophilia A², raising concerns about potential immunogenicity in AHA as well. Moreover, there is currently no easy-to-access laboratory assay available to monitor ADA development in AHA patients receiving emicizumab treatment.

Objectives To develop an accessible enzyme-linked immunosorbent assay (ELISA) for the detection of binding ADA and to evaluate incidence of ADA in the GTH-AHA-EMI study cohort.

Method: The in-house ELISA was performed as follows: equal amounts of HRP- and biotin-labeled emicizumab (2 µg/ml each) were mixed and incubated overnight with 1:10 diluted plasma samples at room temperature (RT) to allow ADA-emicizumab complex formation. The mixture was then applied to a streptavidin-coated microplate (5 µg/ml) and incubated for 20 minutes at RT. After adding tetramethylbenzidine substrate, the absorbance was measured at 450 nm. To minimize inter-assay variability, a calibration curve prepared using goat anti-human antibodies (0–10 µg/ml) was utilized. The assay's lower limit of detection (LLOD) was determined using 21 plasma samples from patients with no history of coagulation disorders. Subsequently, 182 plasma samples collected from 47 patients enrolled in the GTH-AHA-EMI [3] study were tested. If the result exceeded the LLOD, the measurement was repeated, and a specificity assay was performed by adding an excess amount (160 µg/ml) of unlabeled emicizumab.

Results: A scalable and easy-to-use ELISA-based assay for binding ADA detection was developed. LLOD was determined to be 0.1 µg/ml based on the calibration curve. Of the 47 patients, 5 showed results above the LLOD at screening. The specificity assay confirmed that only one patient (2.1%) had binding ADA. Interestingly, these ADA were already detected at baseline, followed by a decrease, but remained detectable at week 13.

Conclusion: Using an in-house ELISA-based assay, we determined the incidence of binding ADA in the GTH-AHA-EMI study cohort to be 2.1%. This finding supports the previously reported low immunogenicity of emicizumab [2].

Publikationsverlauf

Artikel online veröffentlicht:
13. Februar 2025

• References

• 1 Iarossi M, Hermans C.. 2024; Emicizumab as first-line therapy in acquired hemophilia A. Res Pract Thromb Haemost 8 (04) 102438
• 2 Schmitt C. et al. 2021; Low immunogenicity of emicizumab in persons with haemophilia A. Haemophilia 27 (06) 984-992
• 3 Tiede A. et al. 2023; Emicizumab prophylaxis in patients with acquired haemophilia A (GTH-AHA-EMI): an open-label, single-arm, multicentre, phase 2 study. Lancet Haematol 10 (11) e913-e921