Protein C derivatives

Details Protein C derivatives Protein C derivatives

2002-07-22

2003-10-07

Prouty, Rebecca E. (Department: 1652)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

C435S320100, C435S252330, C435S226000, C536S023200

Reexamination Certificate

active

06630138

ABSTRACT:

This invention relates to novel polynucleotides, polypeptides encoded by them and to the use of such polynuoleotides and polypeptides. More specifically, the invention relates to human protein C derivatives with increased anti-coagulant activity, resistance to serpin inactivation, increased sensitivity to thrombin activation, or a combination thereof, when compared to wild-type activated protein C; to their production, and to pharmaceutical compositions comprising these human protein C derivatives.
Protein C is a serine protease and naturally occurring anti-coagulant that plays a role in the regulation of hemostasis by inactivating Factors V
a
and VIII
a
in the coagulation cascade. Human protein C is made in vivo as a single polypeptide of 461 amino acid a. This polypeptide undergoes multiple post-translational modifications including, 1) cleavage of a 42 amino acid signal sequence; 2) cleavage of lysine and arginine residues (positions 156 and 157) to make a 2-chain inactive precursor or zymogen (a 155 amino acid residue light chain attached via a disulfide bridge to a 262 amino acid residue heavy chain); 3) vitamin K-dependent carboxylation of nine glutamic acid residues located within the amino-terminal 45 residues (gla-domain): and, 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain). Finally, the 2-chain zymogen may be activated by removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C (aPC) possessing greater enzymatic activity than the 2-chain zymogen.
Blood coagulation is a highly complex process regulated by the balance between pro-coagulant and anti-coagulant mechanisms. This balance determines a condition of either normal hemostasis or abnormal pathological thrombus generation and the progression, for example, of coronary thrombosis leading to acute coronary syndromes (ACS; e.g. unstable angina, myocardial infarction). Two major factors control this balance; the generation of fibrin and the activation and subsequent aggregation of platelets. Both processes are controlled by the generation of the enzyme thrombin, which occurs following activation of the clotting cascade. Thrombin, in complex with thrombomodulin, also functions as a potent anti-coagulant since it activates protein C zymogen to aPC, which in turn inhibits the generation of thrombin. Thus, through the feedback regulation of thrombin generation via the inactivation of Factors Va and VIIIa, aPC functions as perhaps the most important down-regulator of blood coagulation resulting in protection against thrombosis. In addition, aPC has anti-inflammatory properties, and exerts profibrinolytic effects that facilitate clot lysis.
Various methods of obtaining protein C from plasma and producing protein C, aPC and protein C/aPC polypeptides through recombinant DNA technology are known in the art and have been described. See e.g., U.S. Pat. Nos. 4,775,624 and 5,358,932. Despite improvements in methods to produce aPC through recombinant DNA technology, aPC and derivatives thereof are difficult and costly to produce.
Unlike the zymogen protein C, activated protein C has an extremely short half-life. A major reason for the short half-life is that blood levels of aPC are regulated by molecules known as serpins (Serine Protease Inhibitors), which covalently bind to aPC forming an inactive serpin/aPC complex. The serpin/aPC complexes are formed when aPC binds and proteolytically cleaves a reactive site loop within the serpin; upon cleavage, the serpin undergoes a conformational change irreversibly inactivating aPC. The serpin/aPC complex is then eliminated from the bloodstream via hepatic receptors for the serpin/aPC complex. As a result, aPC has a relatively short half-life compared to the zymogen; approximately 20 minutes for aPC versus approximately 10 hours for human protein C zymogen (Okajima, et al.,
Thromb Haemost
63(1):48-53, 1990).
Therefore, an aPC derivative exhibiting resistance to serpin inactivation, while maintaining the desirable biological activities of aPC (e.g., anticoagulant, fibrinolytic, and anti-inflammatory activities), provides a compound that has an increased plasma half-life and is effectively more potent than the parent compound, requiring substantially reduced dosage levels for therapeutic applications. The potency advantages are especially important in disease states in which serpin levels are elevated.
Additionally, an aPC derivative exhibiting increased anti-coagulant activity, while maintaining the other biological activities of aPC (e.g., fibrinolytic, and anti-inflammatory activities), provides a compound that is effectively more potent than the parent compound, requiring substantially reduced dosage levels for therapeutic applications.
Enhancement of human protein C calcium and membrane binding activity by site-directed mutagenesis of the gla-domain has been reported by several investigator a, for example, Shen et al. (
J Biol. Chem.,
273(47) 31086-91, 1998.) and Shen et al. (
Biochemistry,
36(51) 16025-31, 1997). Through continued scientific experiments, analysis, and innovation, the present inventors identified specific sites and modified targeted amino acid residues in the gla-domain of the aPC molecule. Surprisingly, we found increased anti-coagulant activity of the aPC derivative when specific amino acid substitutions were performed. Therefore, an aPC derivative exhibiting increased anti-coagulant activity, while maintaining the other biological activities of aPC (e.g., fibrinolytic, and anti-inflammatory activities), provides a compound that is effectively more potent than the parent compound, requiring substantially reduced dosage levels for therapeutic applications.
Furthermore, human protein C derivatives with increased sensitivity to thrombin activation (hyper-activatable zymogens) are useful as site-activated anti-thrombotic agents, as described, for example, in U.S. Pat. No. 5,453,373 and in Richardson et al. (
Protein Science,
3:711-712, 1994). Such hyper-activatable zymogens can also be constructed to contain the gla-domain mutants and the serpin resistant derivatives described above. These derivatives have increased anti-coagulant activity, resistance to serpin inactivation, and increased sensitivity to thrombin activation when compared to wild-type human protein C.
Accordingly, the present invention describes novel human protein C derivatives. These human protein C derivatives retain the important biological activity when compared to wild-type protein C and have increased anti-coagulant activity, resistance to serpin inactivation, and increased sensitivity to thrombin activation when compared to wild-type human protein C. Other protein C derivatives of the present invention have increased sensitivity to thrombin activation and increased anti-coagulant activity or increased sensitivity to thrombin activation and resistance to serpin inactivation.
Therefore, these compounds provide various advantages, for example, site-activation, less frequent administration and/or smaller dosages and thus a reduction in the overall cost of production of the therapy. Thus, these compounds exhibit an advantage over current therapy in disease states of acute coronary syndromes such as unstable angina or myocardial infarction.
The present invention provides a human protein C derivative comprising SEQ ID NO: 1 wherein Asp at position 167 is substituted with Phe; Asp at position 172 is substituted with Lys and further comprising at least one amino acid substitution selected from the group consisting of:
His at position 10, Ser at position 11, or Ser at position 12 are independently substituted with any amino acid; Gln at position 32 is substituted with Glu; Asn at position 33 is substituted with Asp or Phe; and, amino acids at positions 194, 195, 228, 249, 254, 302, or 316 are substituted with an amino acid selected from Ser, Ala, Thr, His, Leu, Lys, Arg, Asn, Asp, Glu, Gly, and Gln.
The present invention also provides recombinant DNA molecules encoding the human protein C derivatives of the present in

Affiliated with

Also associated with

Rating

Protein C derivatives does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Protein C derivatives, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Protein C derivatives will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3167008