Preclinical Testing of Human Recombinant von Willebrand Factor: ADAMTS13 Cleavage Capacity in Animals as Criterion for Species Suitability

ABSTRACT

Von Willebrand factor (VWF) is cleaved by the plasma metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin repeats, number 13) that regulates the hemostatic activity of VWF by limiting its multimeric size in the human system. In vitro and ex vivo studies have shown that human recombinant VWF (rVWF) is virtually resistant to the proteolytic activity of murine ADAMTS13. In contrast, rabbit and cynomolgus ADAMTS13 is able to cleave human rVWF. These findings were consistent with in vivo results showing distinct pharmacological behavior of human rVWF depending on the cleaving capacity of ADAMTS13 present in the species tested.

Studies were performed using three mouse strains (ADAMTS13 deficient, C57BL/6J [wild type], VWF deficient), rats, rabbits, and cynomolgus monkeys. All animals were infused once with different doses of human rVWF and, in addition, 14 daily doses were given to rats and cynomolgus monkeys. Exaggerated pharmacological effects were observed in mice, with the ADAMTS13 knockout mouse being the most sensitive strain. Similar findings with decreased incidence and severity were seen in normal C57BL/6J mice and also in VWF-deficient mice, where they were least pronounced. In rats, exaggerated pharmacological effects were observed only after 14 doses. Rabbits and cynomolgus monkeys showed no exaggerated pharmacological effects.

These differences between species and between mouse strains suggest that the efficiency of ADAMTS13 to cleave rVWF determines the severity of clinical, laboratory and pathohistological findings. These observations highlight the importance of evaluating species' suitability for the generation of meaningful preclinical data for determining the therapeutic safety margins for human patients. Only animals with a sufficient rVWF cleavage capacity by endogenous ADAMTS13 (rabbits and cynomolgus monkeys) are considered appropriate animal models for preclinical evaluation of the rVWF product.

KEYWORDS

Recombinant von Willebrand factor - rVWF - species suitability - ADAMTS13 cleavage

REFERENCES

• 1 Ruggeri Z M. Von Willebrand factor: looking back and looking forward.  Thromb Haemost. 2007;  98(1) 55-62
• 2 Sadler J E. von Willebrand factor: two sides of a coin.  J Thromb Haemost. 2005;  3(8) 1702-1709
• 3 Mannucci P M. Treatment of von Willebrand's disease.  N Engl J Med. 2004;  351(7) 683-694
• 4 Turecek P L, Mitterer A, Matthiessen H P et al.. Development of a plasma-and albumin-free recombinant von Willebrand factor.  Hamostaseologie. 2009;  29(Suppl 1) S32-S38
• 5 Turecek P L, Schrenk G, Rottensteiner H et al.. Structure and function of a recombinant von Willebrand factor (VWF) drug candidate.  Semin Thromb Hemost. 2010;  36(5) 522-528
• 6 Fischer B, Mitterer A, Schlokat U, DenBouwmeester R, Dorner F. Structural analysis of recombinant von Willebrand factor: identification of hetero- and homo-dimers [published correction appears in FEBS Lett 1994;353:337].  FEBS Lett. 1994;  351(3) 345-348
• 7 Fischer B E, Schlokat U, Mitterer A et al.. Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing recombinant furin.  FEBS Lett. 1995;  375(3) 259-262
• 8 Schlokat U, Fischer B E, Herlitschka S et al.. Production of highly homogeneous and structurally intact recombinant von Willebrand factor multimers by furin-mediated propeptide removal in vitro.  Biotechnol Appl Biochem. 1996;  24(Pt 3) 257-267
• 9 Turecek P L, Váradi K, Schlokat U, Pichler L, Dorner F, Schwarz H P. In vivo and in vitro processing of recombinant pro-von Willebrand factor.  Histochem Cell Biol. 2002;  117(2) 123-129
• 10 Fischer B E, Schlokat U, Reiter M, Mundt W, Dorner F. Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale.  Cell Mol Life Sci. 1997;  53(11–12) 943-950
• 11 Plaimauer B, Schlokat U, Turecek P L et al.. Recombinant von Willebrand factor: preclinical development.  Semin Thromb Hemost. 2001;  27(4) 395-403
• 12 Turecek P L, Gritsch H, Pichler L et al.. In vivo characterization of recombinant von Willebrand factor in dogs with von Willebrand disease.  Blood. 1997;  90(9) 3555-3567
• 13 Roussi J, Turecek P L, André P et al.. Effects of human recombinant, plasma-derived and porcine von Willebrand factor in pigs with severe von Willebrand disease.  Blood Coagul Fibrinolysis. 1998;  9(4) 361-372
• 14 Schwarz H P, Dorner F, Mitterer A et al.. Evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease.  Haemophilia. 1998;  4 (suppl 3) 53-62
• 15 Schwarz H P, Dorner F, Mitterer A et al.. Preclinical evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease.  Wien Klin Wochenschr. 1999;  111(5) 181-191
• 16 Turecek P L, Pichler L, Auer W et al.. Evidence for extracellular processing of pro-von Willebrand factor after infusion in animals with and without severe von Willebrand disease.  Blood. 1999;  94 1637-1647
• 17 Schwarz H P, Schlokat U, Mitterer A et al.. Recombinant von Willebrand factor—insight into structure and function through infusion studies in animals with severe von Willebrand disease.  Semin Thromb Hemost. 2002;  28(2) 215-226
• 18 Levy G G, Nichols W C, Lian E C et al.. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.  Nature. 2001;  413(6855) 488-494
• 19 Plaimauer B, Zimmermann K, Völkel D et al.. Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS13).  Blood. 2002;  100(10) 3626-3632
• 20 Tsai H M. Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion.  Blood. 1996;  87(10) 4235-4244
• 21 Moake J. Thrombotic thrombocytopenia purpura (TTP) and other thrombotic microangiopathies.  Best Pract Res Clin Haematol. 2009;  22(4) 567-576
• 22 Varadi K, Rottensteiner H, Vejda S et al.. Species-dependent variability of ADAMTS13-mediated proteolysis of human recombinant von Willebrand factor.  J Thromb Haemost. 2009;  7(7) 1134-1142
• 23 Laje P, Shang D, Cao W et al.. Correction of murine ADAMTS13 deficiency by hematopoietic progenitor cell-mediated gene therapy.  Blood. 2009;  113(10) 2172-2180
• 24 International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline: Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals, S6. 1997. http://Available at: www.fda.gov/downloads/Downloads/Drugs/GuidanceComplicaneRegulatoryInformation/Guidances/UCM074957.pdf Accessed June 17, 2010
• 25 Motto D G, Chauhan A K, Zhu G et al.. Shigatoxin triggers thrombotic thrombocytopenic purpura in genetically susceptible ADAMTS13-deficient mice.  J Clin Invest. 2005;  115(10) 2752-2761
• 26 Denis C, Methia N, Frenette P S et al.. A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis.  Proc Natl Acad Sci U S A. 1998;  95(16) 9524-9529