Sabrina Grailly

Inhibition of factor VIII with a partially inhibitory human recombinant monoclonal antibody prevents thrombotic events in a transgenic model of type II HBS antithrombin deficiency in mice

Summary.  Venous thromboembolic disease is a major cause of morbidity and mortality, necessitating antithrombotic therapy. A human monoclonal anti‐factor (F)VIII antibody, LCL‐mAb‐LE2E9, produced by a lymphoblastoid cell line derived from a hemophilia A patient with inhibitor to wild‐type but not mutant self FVIII, was previously reported to achieve efficient inhibition of thrombosis in an experimental vena cava thrombosis model in mice. Here, the antithrombotic efficacy of a recombinant DNA‐derived version of this anti‐FVIII antibody (rec‐mAb‐LE2E9) was tested in mice which carry a type II heparin binding site antithrombin deficiency mutation and display spontaneous chronic thrombosis in several sites including the penile vein of sexually active males. The recombinant anti‐FVIII antibody (100 µg, repeated after 3 days) prevented thrombotic priapism in all treated males, whereas all control animals treated with saline (group of four animals) developed priapism within 6 days after mating (P < 0.05 for treated vs. saline). The rec‐mAb‐LE2E9 and the original LCL‐mAb‐LE2E9 were equally effective (five and seven males/group, respectively). These results confirm that FVIII inhibition represents a potent antithrombotic strategy, and show that both LCL‐mAb‐LE2E9 and rec‐mAb‐LE2E9 efficiently prevent thrombosis in a physiological model representative of thrombosis in patients with a severe prothrombotic risk.

Efficient factor VIII affinity purification using a small synthetic ligand.

Summary.  Background: Hemophilia A is currently treated by infusions of the coagulation factor (F) VIII, of which production and purification remain a challenging task. Current purification procedures using immunoaffinity chromatography are cumbersome, expensive, and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified using octapeptide ligands, but their use is limited due to the low resistance to proteases. Objective: Our goal was to develop and evaluate a novel ligand for FVIII purification, overcoming the drawbacks of current procedures. Methods: Peptide ligands were screened for binding of 125I‐plasma‐derived‐FVIII (pdFVIII) in a microbead assay. A selected ligand‐coated Toyopearl resin was then used for pdFVIII purification from cell‐conditioned Delbucco’s modified Eagle’s medium (DMEM) containing fetal bovine serum. The proteolytic stability of ligand was measured by incubating with human serum and proteinase K, and its cytotoxicity towards human OV‐MZ‐6 cells was assayed. Results: A high‐affinity octapeptidic FVIII ligand was modified into the small, highly stable and non‐toxic peptidomimetic ligand L4 by rational and combinatorial design without affecting its affinity for FVIII. Using ligand L4‐coated Toyopearl resin, pdFVIII was isolated from cell‐conditioned medium with high purity and 89% column retention after elution with a mild buffer containing 0.6 m NaCl at pH 6.8. Conclusions: Ligand L4 offers a valuable alternative to antibody‐based procedures for laboratory and industrial production. Its synthesis by established solid‐phase procedures is straightforward and considerably cheaper than the biotechnological production of antibodies, and safety concerns associated with the use of biological material are overcome.