BACKGROUND: Coagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies. OBJECTIVES: To structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin. METHODS: The structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics. RESULTS: Here, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics. CONCLUSIONS: These improved structures yield a more confident model for next-generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels, and lower immunogenicity.
BACKGROUND: Coagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies. OBJECTIVES: To structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin. METHODS: The structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics. RESULTS: Here, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics. CONCLUSIONS: These improved structures yield a more confident model for next-generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels, and lower immunogenicity.
Authors: D Lillicrap; A Schiviz; C Apostol; P Wojciechowski; F Horling; C K Lai; C Piskernik; W Hoellriegl; P Lollar Journal: Haemophilia Date: 2015-08-17 Impact factor: 4.287
Authors: K Canis; J Anzengruber; E Garenaux; M Feichtinger; K Benamara; F Scheiflinger; L-A Savoy; B M Reipert; M Malisauskas Journal: J Thromb Haemost Date: 2018-06-11 Impact factor: 5.824
Authors: Po-Lin Chiu; George M Bou-Assaf; Ekta Seth Chhabra; Melissa G Chambers; Robert T Peters; John D Kulman; Thomas Walz Journal: Blood Date: 2015-06-11 Impact factor: 22.113
Authors: Philip M Zakas; Harrison C Brown; Kristopher Knight; Shannon L Meeks; H Trent Spencer; Eric A Gaucher; Christopher B Doering Journal: Nat Biotechnol Date: 2016-09-26 Impact factor: 54.908
Authors: Joseph S Gish; Lexi Jarvis; Kenneth C Childers; Shaun C Peters; Connor S Garrels; Ian W Smith; H Trent Spencer; Christopher B Doering; Pete Lollar; P Clint Spiegel Journal: Blood Date: 2021-05-27 Impact factor: 25.476
Authors: Kenneth C Childers; Shaun C Peters; Pete Lollar; Harold Trent Spencer; Christopher B Doering; Paul C Spiegel Journal: Blood Adv Date: 2022-06-14
Authors: Sriram Krishnaswamy; Walter Ageno; Yaseen Arabi; Tiziano Barbui; Suzanne Cannegieter; Marc Carrier; Audrey C Cleuren; Peter Collins; Laurence Panicot-Dubois; Jane E Freedman; Kathleen Freson; Philip Hogg; Andra H James; Colin A Kretz; Michelle Lavin; Frank W G Leebeek; Weikai Li; Coen Maas; Kellie Machlus; Michael Makris; Ida Martinelli; Leonid Medved; Marguerite Neerman-Arbez; James S O'Donnell; Jamie O'Sullivan; Madhvi Rajpurkar; Verena Schroeder; Paul Clinton Spiegel; Simon J Stanworth; Laura Green; Anetta Undas Journal: Res Pract Thromb Haemost Date: 2021-07-16
Authors: Estelle K Ronayne; Shaun C Peters; Joseph S Gish; Celena Wilson; H Trent Spencer; Christopher B Doering; Pete Lollar; P Clint Spiegel; Kenneth C Childers Journal: Front Immunol Date: 2021-06-10 Impact factor: 7.561