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

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.

doi:10.1111/jth.13700

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

Journal Article · Wed May 03 00:00:00 EDT 2017 · Journal of Thrombosis and Haemostasis

DOI:https://doi.org/10.1111/jth.13700· OSTI ID:1425419

Leksa, N. C. ^[1]; Chiu, P. -L. ^[2]; Bou-Assaf, G. M. ^[3]; Quan, C. ^[3]; Liu, Z. ^[4]; Goodman, A. B. ^[1]; Chambers, M. G. ^[5]; Tsutakawa, S. E. ^[6]; Hammel, M. ^[6]; Peters, R. T. ^[1]; Walz, T. ^[7]; Kulman, J. D. ^[8]

Biogen, Cambridge, MA (United States); Bioverativ Therapeutics, Waltham, MA (United States)
Harvard Univ., Cambridge, MA (United States). Dept. of Cell Biology; Arizona State Univ., Mesa, AZ (United States). School of Molecular Sciences and Biodesign Inst.
Biogen, Cambridge, MA (United States)
Biogen, Cambridge. MA (United States); Compass Therpeutics, Cambridge, MA (United States)
Harvard Univ., Cambridge, MA (United States). Dept. of Cell Biology
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging
Biogen, Cambridge, MA (United States); Rockefeller Univ., New York, NY (United States). Lab. of Molecular Electron Microscopy
Biogen, Cambridge, MA (United States); Codiak BioSciences, Cambridge, MA (United States)

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. 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 by 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.

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Research Organization:: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); Eli Lilly and Co., Knoxville, TN (United States); National Institutes of Health (NIH)

Grant/Contract Number:: AC02-05CH11231; AC02‐06CH11357; R01GM105404

OSTI ID:: 1425419

Journal Information:: Journal of Thrombosis and Haemostasis, Vol. 15, Issue 6; ISSN 1538-7933

Country of Publication:: United States

Language:: English

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Cited by: 7 works

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References (52)

Visualization of an N-terminal fragment of von Willebrand factor in complex with factor VIII Yee, Andrew; Oleskie, Austin N.; Dosey, Anne M. Blood, Vol. 126, Issue 8 https://doi.org/10.1182/blood-2015-04-641696	journal	August 2015
A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models Stennicke, Henning R.; Kjalke, Marianne; Karpf, Ditte M. Blood, Vol. 121, Issue 11 https://doi.org/10.1182/blood-2012-01-407494	journal	March 2013
Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation Meeks, Shannon L.; Healey, John F.; Parker, Ernest T. Blood, Vol. 110, Issue 13 https://doi.org/10.1182/blood-2007-06-096842	journal	December 2007
Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy Böttcher, B.; Wynne, S. A.; Crowther, R. A. Nature, Vol. 386, Issue 6620 https://doi.org/10.1038/386088a0	journal	March 1997
Overview of the CCP 4 suite and current developments Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D. Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444910045749	journal	March 2011
Effect of pegylation on pharmaceuticals Harris, J. Milton; Chess, Robert B. Nature Reviews Drug Discovery, Vol. 2, Issue 3 https://doi.org/10.1038/nrd1033	journal	March 2003
Exploring “one-shot” kinetics and small molecule analysis using the ProteOn XPR36 array biosensor Bravman, Tsafrir; Bronner, Vered; Lavie, Kobi Analytical Biochemistry, Vol. 358, Issue 2 https://doi.org/10.1016/j.ab.2006.08.005	journal	November 2006
Biochemical and functional characterization of a recombinant monomeric factor VIII-Fc fusion protein Peters, R. T.; Toby, G.; Lu, Q. Journal of Thrombosis and Haemostasis, Vol. 11, Issue 1 https://doi.org/10.1111/jth.12076	journal	January 2013
Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein McCue, Justin; Kshirsagar, Rashmi; Selvitelli, Keith Biologicals, Vol. 43, Issue 4 https://doi.org/10.1016/j.biologicals.2015.05.012	journal	July 2015
Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs Dumont, Jennifer A.; Liu, Tongyao; Low, Susan C. Blood, Vol. 119, Issue 13 https://doi.org/10.1182/blood-2011-08-367813	journal	March 2012
Recombinant factor VIII Fc fusion protein for the prevention and treatment of bleeding in children with severe hemophilia A Young, G.; Mahlangu, J.; Kulkarni, R. Journal of Thrombosis and Haemostasis, Vol. 13, Issue 6 https://doi.org/10.1111/jth.12911	journal	April 2015
The ribosome at improved resolution: New techniques for merging and orientation refinement in 3D cryo-electron microscopy of biological particles Penczek, Pawel A.; Grassucci, Robert A.; Frank, Joachim Ultramicroscopy, Vol. 53, Issue 3 https://doi.org/10.1016/0304-3991(94)90038-8	journal	March 1994
The tertiary structure and domain organization of coagulation factor VIII Shen, Betty W.; Spiegel, Paul Clint; Chang, Chong-Hwan Blood, Vol. 111, Issue 3 https://doi.org/10.1182/blood-2007-08-109918	journal	February 2008
Accurate SAXS Profile Computation and its Assessment by Contrast Variation Experiments Schneidman-Duhovny, Dina; Hammel, Michal; Tainer, John A. Biophysical Journal, Vol. 105, Issue 4 https://doi.org/10.1016/j.bpj.2013.07.020	journal	August 2013
Long-term safety and efficacy of recombinant factor VIII Fc fusion protein (rFVIIIFc) in subjects with haemophilia A Nolan, B.; Mahlangu, J.; Perry, D. Haemophilia, Vol. 22, Issue 1 https://doi.org/10.1111/hae.12766	journal	July 2015
Negative staining and image classification — powerful tools in modern electron microscopy Ohi, Melanie; Li, Ying; Cheng, Yifan Biological Procedures Online, Vol. 6, Issue 1 https://doi.org/10.1251/bpo70	journal	January 2004
Genetic diagnosis of haemophilia and other inherited bleeding disorders Peyvandi, F.; Jayandharan, G.; Chandy, M. Haemophilia, Vol. 12, Issue s3 https://doi.org/10.1111/j.1365-2516.2006.01263.x	journal	July 2006
Rational design of a fully active, long-acting PEGylated factor VIII for hemophilia A treatment Mei, Baisong; Pan, Clark; Jiang, Haiyan Blood, Vol. 116, Issue 2 https://doi.org/10.1182/blood-2009-11-254755	journal	July 2010
Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds McCue, Justin T.; Selvitelli, Keith; Walker, Joshua Journal of Chromatography A, Vol. 1216, Issue 45 https://doi.org/10.1016/j.chroma.2009.09.045	journal	November 2009
UCSF Chimera?A visualization system for exploratory research and analysis Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C. Journal of Computational Chemistry, Vol. 25, Issue 13 https://doi.org/10.1002/jcc.20084	journal	January 2004
3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography Zhang, Xing; Zhang, Lei; Tong, Huimin Scientific Reports, Vol. 5, Issue 1 https://doi.org/10.1038/srep09803	journal	May 2015
Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli Radermacher, M.; Wagenknecht, T.; Verschoor, A. Journal of Microscopy, Vol. 146, Issue 2 https://doi.org/10.1111/j.1365-2818.1987.tb01333.x	journal	May 1987
Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS) Hura, Greg L.; Menon, Angeli L.; Hammel, Michal Nature Methods, Vol. 6, Issue 8 https://doi.org/10.1038/nmeth.1353	journal	July 2009
Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS) Hammel, Michal European Biophysics Journal, Vol. 41, Issue 10 https://doi.org/10.1007/s00249-012-0820-x	journal	May 2012
Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa-Factor VIIIa Complex Ngo, Jacky Chi Ki; Huang, Mingdong; Roth, David A. Structure, Vol. 16, Issue 4 https://doi.org/10.1016/j.str.2008.03.001	journal	April 2008
SPARX, a new environment for Cryo-EM image processing Hohn, Michael; Tang, Grant; Goodyear, Grant Journal of Structural Biology, Vol. 157, Issue 1 https://doi.org/10.1016/j.jsb.2006.07.003	journal	January 2007
Low-resolution refinement tools in REFMAC 5 Nicholls, Robert A.; Long, Fei; Murshudov, Garib N. Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4 https://doi.org/10.1107/S090744491105606X	journal	March 2012
Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source Classen, Scott; Hura, Greg L.; Holton, James M. Journal of Applied Crystallography, Vol. 46, Issue 1 https://doi.org/10.1107/S0021889812048698	journal	January 2013
A randomized comparison of two prophylaxis regimens and a paired comparison of on-demand and prophylaxis treatments in hemophilia A management: Comparisons of treatment in hemophilia A management Valentino, L. A.; Mamonov, V.; Hellmann, A. Journal of Thrombosis and Haemostasis, Vol. 10, Issue 3 https://doi.org/10.1111/j.1538-7836.2011.04611.x	journal	February 2012
Iterative Stable Alignment and Clustering of 2D Transmission Electron Microscope Images Yang, Zhengfan; Fang, Jia; Chittuluru, Johnathan Structure, Vol. 20, Issue 2 https://doi.org/10.1016/j.str.2011.12.007	journal	February 2012
Use of TLS parameters to model anisotropic displacements in macromolecular refinement Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Acta Crystallographica Section D Biological Crystallography, Vol. 57, Issue 1 https://doi.org/10.1107/S0907444900014736	journal	January 2001
Phase 3 study of recombinant factor VIII Fc fusion protein in severe hemophilia A Mahlangu, Johnny; Powell, Jerry S.; Ragni, Margaret V. Blood, Vol. 123, Issue 3 https://doi.org/10.1182/blood-2013-10-529974	journal	January 2014
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Pegylated, full-length, recombinant factor VIII for prophylactic and on-demand treatment of severe hemophilia A Konkle, Barbara A.; Stasyshyn, Oleksandra; Chowdary, Pratima Blood, Vol. 126, Issue 9 https://doi.org/10.1182/blood-2015-03-630897	journal	August 2015
Evaluation of the toxicology and pharmacokinetics of recombinant factor VIII Fc fusion protein in animals Dumont, Jennifer A.; Loveday, Kenneth S.; Light, David R. Thrombosis Research, Vol. 136, Issue 6 https://doi.org/10.1016/j.thromres.2015.07.019	journal	December 2015
REFMAC 5 for the refinement of macromolecular crystal structures Murshudov, Garib N.; Skubák, Pavol; Lebedev, Andrey A. Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444911001314	journal	March 2011
The Utility of Hydrogen/Deuterium Exchange Mass Spectrometry in Biopharmaceutical Comparability Studies Houde, Damian; Berkowitz, Steven A.; Engen, John R. Journal of Pharmaceutical Sciences, Vol. 100, Issue 6 https://doi.org/10.1002/jps.22432	journal	June 2011
Population pharmacokinetics of recombinant factor VIII Fc fusion protein: Clinical Pharmacology in Drug Development Nestorov, Ivan; Neelakantan, Srividya; Ludden, Thomas M. Clinical Pharmacology in Drug Development, Vol. 4, Issue 3 https://doi.org/10.1002/cpdd.167	journal	October 2014
High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance Nguyen, Phuong-Cac T.; Lewis, Kenneth B.; Ettinger, Ruth A. Blood, Vol. 123, Issue 17 https://doi.org/10.1182/blood-2013-09-527275	journal	April 2014
Improvements in factor concentrates Lillicrap, David Current Opinion in Hematology, Vol. 17, Issue 5 https://doi.org/10.1097/MOH.0b013e32833c06c6	journal	January 2010
The Acidic Region of the Factor VIII Light Chain and the C2 Domain Together Form the High Affinity Binding Site for von Willebrand Factor Saenko, Evgueni L.; Scandella, Dorothea Journal of Biological Chemistry, Vol. 272, Issue 29 https://doi.org/10.1074/jbc.272.29.18007	journal	July 1997
Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry Chiu, Po-Lin; Bou-Assaf, George M.; Chhabra, Ekta Seth Blood, Vol. 126, Issue 8 https://doi.org/10.1182/blood-2015-04-641688	journal	August 2015
New and Emerging Agents for the Treatment of Hemophilia: Focus on Extended Half-Life Recombinant Clotting Proteins Ragni, Margaret V. Drugs, Vol. 75, Issue 14 https://doi.org/10.1007/s40265-015-0451-5	journal	August 2015
Structure and flexibility within proteins as identified through small angle X-ray scattering Pelikan, M.; Hura, G.; Hammel, M. General Physiology and Biophysics, Vol. 28, Issue 2 https://doi.org/10.4149/gpb_2009_02_174	journal	January 2009
Recombinant factor VIII Fc (rFVIIIFc) fusion protein reduces immunogenicity and induces tolerance in hemophilia A mice Krishnamoorthy, Sriram; Liu, Tongyao; Drager, Douglas Cellular Immunology, Vol. 301 https://doi.org/10.1016/j.cellimm.2015.12.008	journal	March 2016
Crystal Structure of a Neutralizing Human IgG Against HIV-1: A Template for Vaccine Design Saphire, E. O. Science, Vol. 293, Issue 5532 https://doi.org/10.1126/science.1061692	journal	August 2001
Recombinant factor VIII Fc fusion protein: extended-interval dosing maintains low bleeding rates and correlates with von Willebrand factor levels Shapiro, A. D.; Ragni, M. V.; Kulkarni, R. Journal of Thrombosis and Haemostasis, Vol. 12, Issue 11 https://doi.org/10.1111/jth.12723	journal	October 2014
SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs Shaikh, Tanvir R.; Gao, Haixiao; Baxter, William T. Nature Protocols, Vol. 3, Issue 12 https://doi.org/10.1038/nprot.2008.156	journal	October 2008
Phaser crystallographic software McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Journal of Applied Crystallography, Vol. 40, Issue 4 https://doi.org/10.1107/S0021889807021206	journal	July 2007
EMAN: Semiautomated Software for High-Resolution Single-Particle Reconstructions Ludtke, Steven J.; Baldwin, Philip R.; Chiu, Wah Journal of Structural Biology, Vol. 128, Issue 1 https://doi.org/10.1006/jsbi.1999.4174	journal	December 1999
A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine Shima, Midori; Scandella, Dorothea; Yoshioka, Akira Thrombosis and Haemostasis, Vol. 69, Issue 03 https://doi.org/10.1055/s-0038-1651588	journal	March 1993
Immunodominant T-cell epitopes in the factor VIII C2 domain are located within an inhibitory antibody binding site Pratt, Kathleen; Qian, Jiahua; Ellaban, Ekram Thrombosis and Haemostasis, Vol. 92, Issue 09 https://doi.org/10.1160/TH03-12-0755	journal	January 2004

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59 BASIC BIOLOGICAL SCIENCES
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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

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