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Background: Hemophilia A arises from dysfunctional or deficient coagulation factor (F)VIII and leads to inefficient fibrin clot formation and uncontrolled bleeding events. The development of antibody inhibitors is a clinical complication in hemophilia A patients receiving FVIII replacement therapy. LE2E9 is an anti-C1 domain inhibitor previously isolated from a mild/moderate hemophilia A patient and disrupts FVIII interactions with von Willebrand factor and FIXa, though the intermolecular contacts that underpin LE2E9-mediated FVIII neutralization are undefined., Objectives: To determine the structure of the complex between FVIII and LE2E9 and characterize its mechanism of inhibition., Methods: FVIII was bound to the antigen binding fragment (Fab) of NB2E9, a recombinant construct of LE2E9, and its structure was determined by cryogenic electron microscopy., Results: This report communicates the 3.46 Å structure of FVIII bound to NB2E9, with its epitope comprising FVIII residues S2040 to Y2043, K2065 to W2070, and R2150 to H2155. Structural analysis reveals that the LE2E9 epitope overlaps with portions of the epitope for 2A9, a murine-derived inhibitor, suggesting that these residues represent a shared antigenic region on the C1 domain between FVIII -/- mice and hemophilia A patients. Furthermore, the FVIII:NB2E9 structure elucidates the orientation of the LE2E9 glycan, illustrating how the glycan sterically blocks interactions between the FVIII C1 domain and the von Willebrand factor D' domain. A putative model of the FVIIIa:FIXa complex suggests potential clashing between the NB2E9 glycan and FIXa light chain., Conclusion: These results describe an antigenic "hotspot" on the FVIII C1 domain and provide a structural basis for engineering FVIII replacement therapeutics with reduced antigenicity., Competing Interests: Declaration of competing interests P.L. is listed as an inventor on a patent application describing ET3i and on patents owned by Emory University claiming compositions of matter that include modified FVIII proteins with reduced reactivity with anti-FVIII antibodies. C.B.D. and P.L. are cofounders of Expression Therapeutics and own equity in the company. Expression Therapeutics owns the intellectual property associated with ET3i. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conflict-of-interest policies. The remaining authors have no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. All rights reserved.)

The development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in ∼30% of patients with congenital hemophilia A receiving FVIII replacement therapy, as well as in all cases of acquired hemophilia A. KM33 is an anti-C1 domain antibody inhibitor previously isolated from a patient with severe hemophilia A. In addition to potently blocking FVIII binding to von Willebrand factor and phospholipid surfaces, KM33 disrupts FVIII binding to lipoprotein receptor-related protein 1 (LRP1), which drives FVIII hepatic clearance and antigen presentation in dendritic cells. Here, we report on the structure of FVIII bound to NB33, a recombinant derivative of KM33, via single-particle cryo-electron microscopy. Structural analysis revealed that the NB33 epitope localizes to the FVIII residues R2090-S2094 and I2158-R2159, which constitute membrane-binding loops in the C1 domain. Further analysis revealed that multiple FVIII lysine and arginine residues, previously shown to mediate binding to LRP1, dock onto an acidic cleft at the NB33 variable domain interface, thus blocking a putative LRP1 binding site. Together, these results demonstrate a novel mechanism of FVIII inhibition by a patient-derived antibody inhibitor and provide structural evidence for engineering FVIII with reduced LRP1-mediated clearance., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: The coagulation factors (F)V and VIII are homologous proteins that support hemostasis through their regulation of FX activity. Hemophilia A (HA) patients have reduced FVIII activity and a prolonged bleeding time that is corrected through the administration of exogenous FVIII. Around one-third of severe HA patients develop FVIII neutralizing antibodies, known as "inhibitors," which neutralize FVIII activity and preclude them from further FVIII therapy., Objectives: We hypothesized that, based on the degree of homology between FV and FVIII (~40%), FVIII-neutralizing antibodies could cross react with FV. To test this hypothesis, a panel of recombinant, patient-derived, FVIII-neutralizing antibodies were screened for cross-reactivity against FV., Methods: Factor V and FVIII activity was measured using one-stage clotting assays; structural analysis was carried out using a structural approach., Results: We detected FV neutralizing activity with the anti-FVIII A2 domain antibody NB11B2. Because this antibody was derived from an HA inhibitor patient, FV-neutralizing activity was then evaluated in a number of HA inhibitor patient plasma samples; nine alloimmune samples had FV-neutralizing activity whereas no FV neutralizing activity was found in the two autoimmune samples available. We next examined the degree of surface homology between FV and FVIII and found that structural similarity could explain the cross reactivity of the anti-A2 antibody and likely accounts for the cross reactivity we observed in patient samples., Conclusions: Although this novel observation is of interest, further work will be needed to determine whether FV neutralization in HA patient samples contributes to their bleeding diathesis., (© 2021 Crown copyright. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis. This article is published with the permission of the Controller of HMSO and the Queen‘s Printer for Scotland.)

The development of anti-drug antibodies (ADAs) is a serious outcome of treatment strategies involving biological medicines. Coagulation factor VIII (FVIII) is used to treat haemophilia A patients, but its immunogenicity precludes a third of severe haemophiliac patients from receiving this treatment. The availability of patient-derived anti-drug antibodies can help us better understand drug immunogenicity and identify ways to overcome it. Thus, there were two aims to this work: (i) to develop and characterise a panel of recombinant, patient-derived, monoclonal antibodies covering a range of FVIII epitopes with varying potencies, kinetics and mechanism of action, and (ii) to demonstrate their applicability to assay development, evaluation of FVIII molecules and basic research. For the first objective we used recombinant antibodies to develop a rapid, sensitive, flexible and reproducible ex vivo assay that recapitulates inhibitor patient blood using blood from healthy volunteers. We also demonstrate how the panel can provide important information about the efficacy of FVIII products and reagents without the need for patient or animal material. These materials can be used as experimental exemplars or controls, as well as tools for rational, hypothesis-driven research and assay development in relation to FVIII immunogenicity and FVIII-related products., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)