Generation and biochemical characterization of glycoPEGylated factor VIIa derivatives

Summary

Prophylaxis with 2–4 times weekly dosing of factor (F)VIII or FIX is established as an efficacious and safe treatment in haemophilia. Although prophylaxis is not readily available for the inhibitor patient,recent studies have demonstrated a reduction in bleeding episodes in inhibitor patients treated with daily infusions of FVIIa. In order to develop a treatment option comparable to prophylaxis with FVIII or FIX we looked to PEGylation which is an established method for prolonging the circulatory half-life of proteins. However, due to the numerous interactions of FVIIa with the cell surface,TF,FIX and FX there are limited options for unspecific chemical modification of FVIIa without loss of activity. Consequently, we explored the GlycoPEGylation™ technology for selective PEGylation of the two N-glycans in the FVIIa light chain and protease domain to generate seven specifically modified derivatives with PEG groups ranging from 2 to 40 kDa. These derivatives were evaluated in vitro for their ability to interact with small synthetic substrates as well as key molecules relevant to function in the coagulation pathway. The results demonstrate that modification of FVIIa using glycoPEGylation has only a very limited effect on the hydrolysis S-2288 and FX activation. However, the modification does to some extend alter the ability of FVIIa to interact with TF and more importantly, reduces the rate of ATIII inhibition by up to 50% which could allow for an extended active half-life in circulation.

Keywords

Long-acting - PEG - coagulation - serine protease - factor VIIa - prophylaxis

• References

• 1 Hoots WK, Ebbesen LS, Konkle BA. et al. Secondary prophylaxis with recombinant activated factor VII improves health-related quality of life of haemophilia patients with inhibitors. Haemophilia 2008; 14: 466-475.
• 2 Konkle BA, Ebbesen LS, Erhardtsen E. et al. Randomized, prospective clinical trial of recombinant factor VIIa for secondary prophylaxis in hemophilia patients with inhibitors. J Thromb Haemost 2007; 05: 1904-1913.
• 3 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30: 10363-10370.
• 4 Perona JJ, Craik CS. Structural basis of substrate specificity in the serine proteases. Protein Sci 1995; 04: 337-360.
• 5 Haya S, Moret A, Cid AR. et al. Inhibitors in haemophilia A: current management and open issues. Haemophilia 2007; 13 (Suppl. 05) 52-60.
• 6 Lindley CM, Sawyer WT, Macik BG. et al. Pharmacokinetics and pharmacodynamics of recombinant factor VIIa. Clin Pharmacol Ther 1994; 55: 638-648.
• 7 Fishburn CS. The pharmacology of PEGylation: Balancing PD with PK to generate novel therapeutics. J Pharm Sci. 2008 in print.
• 8 Harris JM, Chess RB. Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2003; 02: 214-221.
• 9 Harris JM, Martin NE, Modi M. Pegylation:a novel process for modifying pharmacokinetics. Clin Pharmacokinet 2001; 40: 539-551.
• 10 Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev 2002; 54: 459-476.
• 11 Beeby TL, Chasseaud LF, Taylor T. et al. Distribution of the recombinant coagulation factor 125I-rFVIIa in rats. Thromb Haemost 1993; 70: 465-468.
• 12 Thomsen MK, Wildgoose P, Nilsson P. et al. Accumulation of the recombinant factor VIIa in rat bone: importance of the Gla-domain and relevance to factor IX, another vitamin K-dependent clotting factor. Pharmacol Toxicol 1993; 73: 127-132.
• 13 Nelsestuen G. inventor University of Minnesota, assignee. Isolated vitamin K-dependent polypeptide linked to a polyethylene glycol (PEG) polymer, useful in anticoagulant therapy for treating clotting disorder e.g. hemophilia. USA. 2000
• 14 Hoffman M, Monroe 3rd DM. The action of highdose factor VIIa (FVIIa) in a cell-based model of hemostasis. Dis Mon 2003; 49: 14-21.
• 15 Sorensen BB, Karpf D, Groth A. et al. Effect of glycoPEGylation™ on the pharmacokinetic properties of FVIIa. J Thromb Haemost. 2007; 05 (Suppl. 02) P-T-012.
• 16 Ghosh S, Sen P, Ezban M, Pendurthi UR. et al. Activity and regulation of glycoPEGylated factor VIIa analogs. J Thromb Haemost. 2008 in press.
• 17 Freskgard PO, Olsen OH, Persson E. Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy. Protein Sci 1996; 05: 1531-1540.
• 18 Owenius R, Osterlund M, Lindgren M. et al. Properties of spin and fluorescent labels at a receptor-ligand interface. Biophys J 1999; 77: 2237-2250.
• 19 Bolt G, Kristensen C, Steenstrup TD. Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII. Glycobiology 2005; 15: 541-547.
• 20 Kruse S, Kleineidam RG, Roggentin P. et al. Expression and purification of a recombinant „small” sialidase from Clostridium perfringens A99. Protein Expr Purif 1996; 07: 415-422.
• 21 Defrees S, Zopf DA, Taudte S. et al. inventors; Neose Technologies, assignee. Making of Factor VII/ Factor VIIa peptide conjugate for enhancing clotting potency in mammal comprises contacting Factor VII/ Factor VIIa peptide comprising glycosyl moiety with polyethylene glycolsialic acid donor moiety and enzyme. 2007
• 22 DeFrees S, Wang ZG, Xing R. et al. GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli . Glycobiology 2006; 16: 833-843.
• 23 Larsen KS, Ostergaard H, Bjelke JR. et al. Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa. Biochem J 2007; 405: 429-438.
• 24 Payne MA, Neuenschwander PF, Johnson AE. et al. Effect of soluble tissue factor on the kinetic mechanism of factor VIIa: enhancement of p-guanidinobenzoate substrate hydrolysis. Biochemistry 1996; 35: 7100-7106.
• 25 Persson E, Kjalke M, Olsen OH. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity. Proc Natl Acad Sci USA 2001; 98: 13583-13588.
• 26 Persson E, Nielsen LS. Ca2+ in the first epidermal growth factor-like domain of activated factor VII. Blood Coagul Fibrinolysis 1998; 09 (Suppl. 01) S79-81.
• 27 Olson ST, Bjork I, Shore JD. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol 1993; 222: 525-559.
• 28 Sorensen BB, Persson E, Freskgard PO. et al. Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor. J Biol Chem 1997; 272: 11863-11868.
• 29 Jurlander B, Thim L, Klausen NK. et al. Recombinant activated factor VII (rFVIIa): characterization, manufacturing, and clinical development. Semin Thromb Hemost 2001; 27: 373-384.
• 30 Weber PL, Kornfelt T, Klausen NK. et al. Characterization of glycopeptides from recombinant coagulation factor VIIa by high-performance liquid chromatography and capillary zone electrophoresis using ultraviolet and pulsed electrochemical detection. Anal Biochem 1995; 225: 135-142.
• 31 Bjoern S, Foster DC, Thim L. et al. Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine. J Biol Chem 1991; 266: 11051-11057.
• 32 Jameson GW, Roberts DV, Adams RW. et al. Determination of the operational molarity of solutions of bovine alpha-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration. Biochem J 1973; 131: 107-117.
• 33 Schechter NM, Plotnick MI. Measurement of the kinetic parameters mediating proteaseserpin inhibition. Methods 2004; 32: 159-168.
• 34 Ahamed J, Versteeg HH, Kerver M. et al. Disulfide isomerization switches tissue factor from coagulation to cell signaling. Proc Natl Acad Sci USA 2006; 103: 13932-13937.
• 35 Petersen LC, Albrektsen T, Hjorto GM. et al. Factor VIIa/tissue factor-dependent gene regulation and procoagulant activity: effect of factor VIIa concentration. Thromb Haemost 2007; 98: 909-911.
• 36 Olsen OH, Rand KD, Ostergaard H. et al. A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulation. Protein Sci 2007; 16: 671-682.
• 37 Lormeau JC, Herault JP, Herbert JM. Antithrom binmediated inhibition of factor VIIa-tissue factor complex by the synthetic pentasaccharide representing the heparin binding site to antithrombin. Thromb Haemost 1996; 76: 5-8.
• 38 Hamamoto T, Kisiel W. The effect of cell surface glycosaminoglycans (GAGs) on the inactivation of factor VIIa--tissue factor activity by antithrombin III. Int J Hematol 1998; 68: 67-78.
• 39 Jesty J, Lorenz A, Rodriguez J. et al. Initiation of the tissue factor pathway of coagulation in the presence of heparin: control by antithrombin III and tissue factor pathway inhibitor. Blood 1996; 87: 2301-2307.
• 40 Banner DW, D’Arcy A, Chene C. et al. The crystal structure of the complex of blood coagulation factorVIIa with soluble tissue factor. Nature 1996; 380: 41-46.