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. 2024 Sep;22(9):2449-2459.
doi: 10.1016/j.jtha.2024.05.024. Epub 2024 Jun 5.

Structural basis for inhibition of coagulation factor VIII reveals a shared antigenic hotspot on the C1 domain

Kenneth C Childers  1 , Ben Cowper  2 , Jordan D Vaughan  1 , Juliet R McGill  1 , Omar Davulcu  3 , Pete Lollar  4 , Christopher B Doering  5 , Carmen H Coxon  2 , Paul C Spiegel Jr  6
Affiliations

Structural basis for inhibition of coagulation factor VIII reveals a shared antigenic hotspot on the C1 domain

Kenneth C Childers et al. J Thromb Haemost. 2024 Sep.

Abstract

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.

Keywords: antibody inhibitor; blood coagulation; cryoelectron microscopy; factor VIII; hemophilia.

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Conflict of interest statement

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.

Figures

Figure 1.
Figure 1.. ET3i:NB2E9 cryo-EM map and structure.
(A,B) Cryo-EM map (A) and model (B) of the ET3i:NB2E9 complex. A1 domain, dark blue; A2 domain, light blue; A3 domain, cyan; C1 domain, orange; C2 domain, yellow; NB2E9 light chain, light green; NB2E9 heavy chain, dark green. Glycans are colored white in (A) and represented as sticks in (B).
Figure 2.
Figure 2.. The ET3i:NB2E9 interface.
Intermolecular contacts between ET3i C1 domain (orange) and (A) NB2E9 light chain (light green) and (B,C) heavy chain (dark green).
Figure 3.
Figure 3.. Structures of ET3i:NB2E9 glycans.
Cryo-EM map of the ET3i:NB2E9 complex. Insets depict glycans on ET3i residues N280 and N1810 and NB2E9 residue N27. A1 domain, dark blue; A2 domain, light blue; A3 domain, cyan; C1 domain, orange; C2 domain, yellow; NB2E9 light chain, light green; NB2E9 heavy chain, dark green. Glycans are colored white and represented as sticks.
Figure 4.
Figure 4.. Structural comparison between LE2E9/2A9 epitopes and vWF binding site.
Surface representation of the A3–C1 domains from ET3i (PDB ID: 6MF0) (grey). (A) Blue and red colored surfaces highlight FVIII amino acids which exclusively bind inhibitors 2A9 and LE2E9, respectively. Purple colored surface illustrates FVIII amino acids which overlap between 2A9 and LE2E9 epitopes. (B) Orange and light purple surfaces highlight FVIII amino acids which exclusively bind vWF and LE2E9, respectively. Green colored surface illustrates FVIII amino acids which overlap between vWF and LE2E9 binding sites.
Figure 5.
Figure 5.. Superposition of ET3i:NB2E9 and BIVV001 structures.
Structures of BIVV001 (PDB ID: 7KWO) and ET3i:NB2E9 were aligned, demonstrating significant steric clashing between the NB2E9 glycan (white sticks) and VWF D’ domain (pink). C1 domain, orange; NB2E9 light chain, light green; NB2E9 heavy chain, dark green.
Figure 6.
Figure 6.. Superposition of ET3i:NB2E9 structure and model of the intrinsic tenase complex.
(A) Model of the human intrinsic tenase complex was generated by the ROBETTA structure prediction server using the structure of the prothrombinase complex as a template (PDB ID: 7TPP). (B) The C1:NB2E9 structure was superimposed with the model of the intrinsic tenase complex, showing significant steric clashing between the NB2E9 glycan and FIXa light chain. A1 domain, dark blue; A2 domain, light blue; A3 domain, cyan; C1 domain, orange; C2 domain, yellow; FIXa heavy chain, light red; FIXa light chain, maroon; NB2E9 light chain, light green; NB2E9 heavy chain, dark green. Glycans are colored white and represented as sticks.
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