The structural basis for the functional comparability of factor VIII and the long-acting variant recombinant factor VIII Fc fusion protein

Abstract

Essentials Recombinant factor VIII (rFVIII) Fc fusion protein has a 1.5-fold longer half-life than rFVIII. Five orthogonal methods were used to characterize the structure of rFVIIIFc compared to rFVIII. The C-terminal Fc fusion does not perturb the structure of FVIII in rFVIIIFc. The FVIII and Fc components of rFVIIIFc are flexibly tethered and functionally independent.

Summary: Background Fusion of the human IgG₁ Fc domain to the C-terminal C2 domain of B-domain-deleted (BDD) factor VIII (FVIII) results in the recombinant FVIII Fc (rFVIIIFc) fusion protein, which has a 1.5-fold longer half-life in humans. Objective To assess the structural properties of rFVIIIFc by comparing its constituent FVIII and Fc elements with their respective isolated components, and evaluating their structural independence within rFVIIIFc. Methods rFVIIIFc and its isolated FVIII and Fc components were compared by the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS). The structure of rFVIIIFc was also evaluated by the use of X-ray crystallography, small-angle X-ray scattering (SAXS), and electron microscopy (EM). The degree of steric interference by the appended Fc domain was assessed by EM and surface plasmon resonance (SPR). Results HDX-MS analysis of rFVIIIFc revealed that fusion caused no structural perturbations in FVIII or Fc. The rFVIIIFc crystal structure showed that the FVIII component is indistinguishable from published BDD FVIII structures. The Fc domain was not observed, indicating high mobility. SAXS analysis was consistent with an ensemble of rigid-body models in which the Fc domain exists in a largely extended orientation relative to FVIII. Binding of Fab fragments of anti-C2 domain antibodies to BDD FVIII was visualized by EM, and the affinities of the corresponding intact antibodies for BDD FVIII and rFVIIIFc were comparable by SPR analysis. Conclusions The FVIII and Fc components of rFVIIIFc are structurally indistinguishable from their isolated constituents, and show a high degree of structural independence, consistent with the functional comparability of rFVIIIFc and unmodified FVIII.

Keywords: B-domain-deleted factor VIII; bleeding; factor VIII; hemophilia A; rFVIIIFc protein.

Figure 1. Structural comparison of rFVIIIFc and BDD rFVIII by HDX-MS

(A) Butterfly plot (top) and difference plot (bottom) comparing deuterium uptake of the FVIII component of rFVIIIFc with that of isolated BDD rFVIII. For the butterfly plot, the y-axis indicates deuterium uptake, expressed as the fraction of exchangeable amide protons displaced by deuterons, for a given peptide derived from rFVIIIFc (positive values) or from BDD rFVIII (negative values). For the difference plot, the y-axis indicates the average (N = 3) calculated mass difference (in Da) for peptides derived from rFVIIIFc and BDD rFVIII. The x-axis indicates the 416 FVIII-derived peptides ordered from N to C terminus based on their respective midpoints. The corresponding locations of FVIII domains (A1, A2, A3, C1, and C2) are indicated above with solid bars, and those of the acidic a1, a2, and a3 domains are denoted with dashed lines. (B) Selected deuterium uptake plots for FVIII-derived peptides. Peptide numbering (cyan) corresponds to the sequence positions indicated in Panel A. The domains in the ribbon representation of FVIII (PDB code: 2R7E) are colored as in Panel A, and calcium (yellow) and copper (purple) ions are depicted as spheres. (C) Corresponding butterfly plot (top) and difference plot (bottom) for peptides derived from the Fc domain of rFVIIIFc and isolated rFc. The x-axis indicates the 50 Fc-derived peptides ordered as in Panel A. The corresponding locations of the CH2 and CH3 sub-domains of the Fc domain are indicated with solid bars.

Figure 2. Crystal structure of rFVIIIFc

(A) Superposition of the FVIII element of rFVIIIFc (blue) with two previously solved structures of BDD FVIII (PDB code: 2R7E, green; PDB code: 3CDZ, orange) is shown in two views rotated by 180°. No electron density was observed for Fc, suggesting that it is not ordered in the crystal lattice. (B) The FVIII element of rFVIIIFc is shown in surface representation with its constituent domains shown in different colors (A1, red; A2, orange; A3, green; C1, blue; C2, purple). Symmetry mates of FVIII are also shown in surface representation and colored white, gray, and light blue, and reveal the presence of large solvent channels (left panel). One Fc conformation is modeled using the position of the Fc from one of the EM structures (right panel). The Fc is shown in surface representation while symmetry mates are shown in cartoon representation.

Figure 3. Solution structure of rFVIIIFc

(A) Comparison of the experimental (black) and calculated scattering profiles for the rFVIII atomistic model (green, χ = 1.1). (B) Comparison of the experimental (black) and calculated scattering profiles for the single best fit model (red, χ = 1.6) and the ensemble model (green, χ = 1.4) of rFVIIIFc. Residuals are shown below as green and red lines. (C) The atomistic models of rFVIII and rFVIIIFc (best fit) are shown on top with FVIII colored in blue and the Fc in green. The ensemble model of rFVIIIFc is shown below with FVIII colored in blue. The individual Fc conformers are shown in green, pink, and orange and their weights are indicated. The variable positions of the Fc in the ensemble model are also shown rotated by 90° around the y-axis.

Figure 4. Single-particle EM analysis of negatively stained rFVIIIFc

(A) Representative class averages obtained with the iterative stable alignment and classification procedure (see Supplementary Figure 3 for all averages). The side length of the individual panels is 27.3 nm. (B) 3D maps of six classes. (C) Superposition of models obtained by placing atomic models of FVIII and an Fc into EM density maps 1 and 4 (top) and 6 and 4 (bottom). The atomic model of FVIII (PDB code: 3CDZ) is shown in blue, and the atomic models of Fc (PDB code: 1HZH) placed into volumes 4, 1 and 6 are shown in green, red, and orange respectively. Scale bars in (B) and (C) represent 5 nm.

Figure 5. Binding of ESH8, GMA-8014 and GMA-8008 to rFVIII and rFVIIIFc

(A) The epitopes on FVIII of three different anti-C2 antibodies are highlighted in yellow (FVIII domains are colored as in Figure 2 with the C terminus in cyan). (B) Representative class averages of negatively stained BDD rFVIII in complex with the Fab of each antibody show that these antibodies bind to FVIII in a position similar to that occupied by the Fc in rFVIIIFc (Figure 4). See Supplementary Figure S9 for all class averages. The side length of the individual panels is 27.3 nm. (C) The affinity of each antibody for rFVIII and rFVIIIFc was determined by SPR in triplicate and indicates that the presence of the Fc domain in rFVIIIFc does not affect the binding of these antibodies to FVIII.