The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/ Phe2200 phospholipid binding ␤-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 ␤-sandwich.

Recombinant human factor VIII expression levels, in vitro and in vivo, are significantly lower than levels obtained for other recombinant coagulation proteins. Here we describe the generation, high level expression and characterization of a recombinant B-domain-deleted porcine factor VIII molecule. Recombinant B-domaindeleted porcine factor VIII expression levels are 10-to 14-fold greater than recombinant B-domain-deleted human factor VIII levels by transient and stable expression in multiple cell lines. Peak expression of 140 units⅐10 6 cells ؊1 ⅐24 h ؊1 was observed from a baby hamster kidneyderived cell line stably expressing recombinant porcine factor VIII. Factor VIII expression was performed in serum-free culture medium and in the absence of exogenous von Willebrand factor, thus greatly simplifying protein purification. Real time reverse transcription-PCR analysis demonstrated that the differences in protein production were not caused by differences in steady-state factor VIII mRNA levels. The identification of sequence(s) in porcine factor VIII responsible for high level expression may lead to a better understanding of the mechanisms that limit factor VIII expression.Factor VIII (fVIII) 1 is a large (ϳ 300 kDa) glycoprotein that functions as an integral component of the intrinsic pathway of blood coagulation. Mutations in the fVIII gene that result in decreased or defective fVIII protein give rise to the genetic disease, hemophilia A, which is phenotypically characterized by recurrent bleeding episodes. Treatment of hemophilia A entails intravenous infusion of either human plasma-derived or recombinant fVIII material. Approximately 25% of all hemophilia A patients treated with fVIII products develop antibodies that inhibit fVIII activity and limit treatment efficacy (1). Patients with fVIII-inhibitory antibodies can be treated using porcine plasma-derived fVIII products, which generally display low cross-reactivity with the human fVIII antibodies (2, 3). Currently there is not a recombinant porcine fVIII product available for clinical use.Since the introduction of recombinant fVIII for the treatment of hemophilia A, commercial suppliers have struggled to keep up with high patient demand (4). The shortage of recombinant fVIII material has precluded prophylactic treatment of severely affected patients, limited the implementation of immune-tolerance regimens, and kept treatment costs high. Unfortunately, fVIII is expressed and recovered at low levels in the heterologous mammalian cell culture systems used for commercial manufacture. The importance of this problem has fueled significant research efforts to overcome the low fVIII expression barrier, and several basic mechanisms have been identified that limit fVIII expression (for review, see Kaufman et al. (5)) Despite these findings, fVIII expression levels remain low, and a product shortage persists.The porcine fVIII cDNA sequence has been reported and shown to encode the homology-defined internal protein domain structure, A1-A2-B-ap-A3-C1-C2 (6, 7). Porcin...

Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII. Novel hybrid human/porcine factor VIII molecules were constructed to identify porcine factor VIII domains that confer high level expression. Hybrid human/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human factor VIII. Additionally, hybrid murine/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency. Factor VIII (fVIII)1 is a plasma protein that functions in proteolytically activated form as a cofactor within the intrinsic pathway of blood coagulation to increase the rate of proteolytic activation of factor X by activated factor IX. fVIII contains a domain structure designated A1-A2-B-ap-A3-C1-C2 that is defined based on internal sequence homology (1, 2). The fVIII A domains share homology with the copper-binding protein ceruloplasmin (3, 4), which has an A1-A2-A3 domain structure in which the three A domains are arranged along a pseudo-3-fold axis of symmetry (5). Before cell secretion, fVIII is cleaved at the B/ap-A3 domain junction into A1-A2-B (heavy chain) and ap-A3-C1-C2 (light chain) subunits. fVIII circulates in the plasma as an inactive heavy chain/light chain heterodimeric procofactor that is non-covalently bound to von Willebrand factor. Proteolytic activation of fVIII by thrombin results from cleavages at Arg-372 between the A1 and A2 domains, Arg-740 between the A2 and B domains, and Arg-1689 between the ap and A3 domains. During this process, the covalent linkage between the A1 and A2 domains is lost, and the B domain and 41-residue ap are released, producing a heterotrimeric,

SummaryHemophilia A is the inherited bleeding disorder that results from mutation of blood coagulation factor VIII (fVIII). Described here is the generation of a regulated expression system producing recombinant murine fVIII. Murine B-domainless fVIII was expressed at a peak level of 4 units/106 cells/24 h in serum-free media. Subsequently, a two-step purification procedure resulted in 5,300-fold enrichment and a 70% yield. Highly purified recombinant murine fVIII had a specific coagulant activity of 660 units per nanomole. It underwent proteolytic processing by thrombin to yield an activated heterotrimer that demonstrated significantly greater stability than activated human fVIII. Recombinant murine fVIII was utilized to generate an anti-fVIII polyclonal antibody. Intravenous injection of recombinant murine fVIII into hemophilia A mice failed to induce a significant anti-fVIII immune response using a schedule that yielded high titer inhibitory antibodies to human fVIII. This may provide an important model for the study of immune tolerance to fVIII.