Leksa N C, Chiu P-L, Bou-Assaf G M, Quan C, Liu Z, Goodman A B, Chambers M G, Tsutakawa S E, Hammel M, Peters R T, Walz T, Kulman J D
Biogen, Cambridge, MA, USA.
Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
J Thromb Haemost. 2017 Jun;15(6):1167-1179. doi: 10.1111/jth.13700. Epub 2017 May 3.
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.
Background Fusion of the human IgG 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.
要点 重组因子VIII（rFVIII）Fc融合蛋白的半衰期比rFVIII长1.5倍。使用五种正交方法来表征rFVIIIFc与rFVIII相比的结构。C末端Fc融合不会干扰rFVIIIFc中FVIII的结构。rFVIIIFc的FVIII和Fc成分通过灵活连接且功能独立。
背景 将人IgG Fc结构域融合到缺失B结构域（BDD）的因子VIII（FVIII）的C末端C2结构域上，产生重组FVIII Fc（rFVIIIFc）融合蛋白，其在人体内的半衰期长1.5倍。目的 通过将rFVIIIFc的组成成分FVIII和Fc与其各自分离的成分进行比较，并评估它们在rFVIIIFc中的结构独立性，来评估rFVIIIFc的结构特性。方法 使用氢-氘交换质谱（HDX-MS）比较rFVIIIFc及其分离的FVIII和Fc成分。还通过X射线晶体学、小角X射线散射（SAXS）和电子显微镜（EM）评估rFVIIIFc的结构。通过EM和表面等离子体共振（SPR）评估附加的Fc结构域的空间干扰程度。结果 rFVIIIFc的HDX-MS分析表明，融合不会对FVIII或Fc造成结构扰动。rFVIIIFc晶体结构表明，FVIII成分与已发表的BDD FVIII结构无法区分。未观察到Fc结构域，表明其具有高流动性。SAXS分析与一组刚体模型一致，其中Fc结构域相对于FVIII以大致伸展的方向存在。通过EM观察到抗C2结构域抗体的Fab片段与BDD FVIII的结合，并且通过SPR分析相应完整抗体对BDD FVIII和rFVIIIFc的亲和力相当。结论 rFVIIIFc的FVIII和Fc成分在结构上与其分离的成分无法区分，并且显示出高度的结构独立性，这与rFVIIIFc和未修饰FVIII的功能可比性一致。
