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

Fusion of the human IgG 1 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. 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. 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). 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 bymore » EM, and the affinities of the corresponding intact antibodies for BDD FVIII and rFVIIIFc were comparable by SPR analysis. Thus, 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.« less
Authors:
 [1] ;  [2] ;  [3] ;  [3] ;  [4] ;  [1] ;  [5] ;  [6] ;  [6] ;  [1] ;  [7] ;  [8]
  1. Biogen, Cambridge, MA (United States); Bioverativ Therapeutics, Waltham, MA (United States)
  2. Harvard Univ., Cambridge, MA (United States). Dept. of Cell Biology; Arizona State Univ., Mesa, AZ (United States). School of Molecular Sciences and Biodesign Inst.
  3. Biogen, Cambridge, MA (United States)
  4. Biogen, Cambridge. MA (United States); Compass Therpeutics, Cambridge, MA (United States)
  5. Harvard Univ., Cambridge, MA (United States). Dept. of Cell Biology
  6. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging
  7. Biogen, Cambridge, MA (United States); Rockefeller Univ., New York, NY (United States). Lab. of Molecular Electron Microscopy
  8. Biogen, Cambridge, MA (United States); Codiak BioSciences, Cambridge, MA (United States)
Publication Date:
Grant/Contract Number:
AC02-05CH11231; AC02‐06CH11357; R01GM105404
Type:
Accepted Manuscript
Journal Name:
Journal of Thrombosis and Haemostasis
Additional Journal Information:
Journal Volume: 15; Journal Issue: 6; Journal ID: ISSN 1538-7933
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); Eli Lilly and Co., Knoxville, TN (United States); National Institutes of Health (NIH)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; B-domain-deleted factor VIII; bleeding; factor VIII; hemophilia A; rFVIIIFc protein
OSTI Identifier:
1425419