title: A single chain variant of factor VIII Fc fusion protein retains normal in vivo efficacy but exhibits altered in vitro activity.
abstract: Recombinant factor VIII Fc (rFVIIIFc) is a fusion protein consisting of a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC), we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF), with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc.
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Yang
authors.initials: Y
authors.lastname: Buyue
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Tongyao
authors.initials: T
authors.lastname: Liu
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: John D
authors.initials: JD
authors.lastname: Kulman
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Garabet G
authors.initials: GG
authors.lastname: Toby
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: George D
authors.initials: GD
authors.lastname: Kamphaus
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Susannah
authors.initials: S
authors.lastname: Patarroyo-White
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Qi
authors.initials: Q
authors.lastname: Lu
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Reidy
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Baisong
authors.initials: B
authors.lastname: Mei
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Glenn F
authors.initials: GF
authors.lastname: Pierce
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Jurg M
authors.initials: JM
authors.lastname: Sommer
authors.affiliation: Hematology Research, Biogen Idec, Cambridge, Massachusetts, United States of America.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Peters
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D004195:Disease Models, Animal; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics* / Q000493:pharmacokinetics* / Q000627:therapeutic use; D006207:Half-Life; D006467:Hemophilia A / Q000097:blood / Q000188:drug therapy*; D006470:Hemorrhage; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000493:pharmacokinetics* / Q000627:therapeutic use; D013917:Thrombin / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D005169:Factor VIII; D013917:Thrombin