A Single Chain Variant of Factor VIII Fc Fusion Protein Retains Normal In Vivo Efficacy but Exhibits Altered In Vitro Activity

Buyue, Yang; Liu, Tongyao; Kulman, John D.; Toby, Garabet G.; Kamphaus, George D.; Patarroyo-White, Susannah; Lü, Qi; Reidy, Thomas J.; Mei, Baisong; Jiang, Haiyan; Pierce, Glenn F.; Sommer, Jürg M.; Peters, Robert

doi:10.1371/journal.pone.0113600

Cited by 12 publications

(16 citation statements)

References 28 publications

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“…This observation implies that the deleterious intracellular processing by furin is mediated by a nonproteolytic chaperone role, as has previously been ascribed to furin (49). Isolated SC species of the long-acting FVIII-BDD-Fc fusion protein actually demonstrate 2-stage and in vivo clotting activity comparable with that of the heterodimeric species (50). We speculate that the residual B-domain released after thrombin activation may have a biochemical role in coagulation, as has been observed in FV (51).…”

Section: Discussionsupporting

confidence: 68%

Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

et al. 2016

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Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645-1648) in the design of B-domain-deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector-based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy.

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Section: Discussionsupporting

confidence: 68%

Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

et al. 2016

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“…These studies also observed that the protein is fully activated by thrombin. Recently, novel single‐chain forms of FVIII that have increased affinity for VWF have been developed as a strategy for increasing the half‐life of the molecule; however, this results in only a modest half‐life extension . It is important to consider that each of these single‐chain forms is generated in a unique way (i.e.…”

Section: Discussionmentioning

confidence: 99%

Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A

Nguyen

George

Siner

et al. 2017

Journal of Thrombosis and Haemostasis

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Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a Paired basic Amino acid Cleaving Enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulate FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared to FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4 fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression thus lowering the vector dose, a critical factor for hemophilia A gene therapy.

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“…Recurrent bleeding was not detected in FXI or carboxypeptidase B 2 gene‐deleted animals. Consequently, the incidence of rebleeding, survival or delayed blood loss of hemophilic mice following tail vein transection has been used to assess the efficacy of coagulation factor replacement products in murine preclinical studies .…”

Section: Resultsmentioning

confidence: 99%

Suppressing protein Z‐dependent inhibition of factor Xa improves coagulation in hemophilia A

Girard

Lasky

Grunz

et al. 2019

Journal of Thrombosis and Haemostasis

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Background: Hemostasis requires a balance between pro- and anti-coagulant factors. Hemophiliacs bleed due to a procoagulant deficiency. The targeted reduction in the activity of endogenous anticoagulant pathways is currently being investigated as a means of improving hemostasis in hemophilia. Protein Z (PZ) is a co-factor that serves as a catalyst for PZ-dependent protease inhibitor (ZPI) inactivation of factor (F)Xa at phospholipid surfaces. Objectives: Evaluate the effects of 1) PZ or ZPI gene-deletion in hemophilia mice and 2) blocking PZ in human hemophilic plasma. Methods: 1) A Tail Vein Re-Bleeding assay (TVRB) was developed based on the serial disruption of clots forming over 15 minutes following a tail vein laceration in an anesthetized mouse. Wild type (WT)/FVIIIKO, PZKO/FVIIIKO and ZPIKO/FVIIIKO mice were evaluated in this model and their plasmas tested in thrombin generation assays. 2) A monoclonal antibody (Mab) against PZ was evaluated in human hemophilic plasma thrombin generation assays. Results: 1) Clot formations (mean ± SEM) in the TVRB were: 4.0 ± 0.9 for WT/FVIIIKO mice; 23.8 ± 1.1 for WT/FVIIIKO mice replaced with 100% FVIII; 15.2 ± 1.1 for PZKO/FVIIIKO mice; and 14.7 ± 1.2 for ZPIKO/FVIIIKO mice. Thrombin generation in PZKO/FVIIIKO and ZPIKO/FVIIIKO mouse plasmas was similar to FVIIIKO plasma replaced with ~15% rFVIII, 2) A Mab against PZ added to human hemophilia plasma enhanced thrombin generation to an extent similar to the addition of ~15% FVIII. Conclusions: Blockade of the PZ/ZPI system may be sufficient to ameliorate the phenotype of severe hemophilia.

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A Single Chain Variant of Factor VIII Fc Fusion Protein Retains Normal In Vivo Efficacy but Exhibits Altered In Vitro Activity

Cited by 12 publications

References 28 publications

Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A

Suppressing protein Z‐dependent inhibition of factor Xa improves coagulation in hemophilia A

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