WO2006044294A2

WO2006044294A2 - Human protein c analogs

Info

Publication number: WO2006044294A2
Application number: PCT/US2005/036310
Authority: WO
Inventors: Barbara Anne Swanson
Original assignee: Eli Lilly And Company
Priority date: 2004-10-14
Filing date: 2005-10-12
Publication date: 2006-04-27
Also published as: WO2006044294A3

Abstract

This present invention provides human protein C analogs having increased activity as compared to wild-type human activated protein C.

Description

HUMAN PROTEIN C ANALOGS

FIELD OF THE INVENTION

This invention is in the field of medicine. More particularly, this invention is directed to human protein C analogs.

BACKGROUND OF THE INVENTION

Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of homeostasis by deactivating Factors Va and VIII_a in the coagulation cascade. Human protein C is made in vivo primarily in the liver as a single polypeptide of 461 amino acids. This precursor molecule undergoes multiple post- translational modifications including 1) cleavage of a 42 amino acid signal sequence to yield a 419 amino acid, one chain zymogen; 2) proteolytic removal from the one chain zymogen of the lysine residue at position 156 and the arginine residue at position 157 to make the 2-chain form of the molecule, (i.e., a light chain of 155 amino acid residues attached through a disulfide bridge to the serine protease-containing heavy chain of 262 amino acid residues); 3) vitamin K-dependent carboxylation of nine glutamic acid residues clustered in the first 42 amino acids of the light chain, resulting in 9 gamma- carboxyglutamic acid residues; and 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain). Finally, the circulating 2-chain zymogen is activated in vivo by thrombin in complex with thrombomodulin at a phospholipid surface in the presence of calcium ion. Activation results from removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C (aPC) possessing enzymatic activity.

Blood coagulation is a highly complex process regulated by the balance between pro-coagulant and anticoagulant mechanisms. This balance determines a condition of either normal hemostasis or abnormal pathological thrombus generation. Two major factors control this balance, the generation of fibrin and the activation and subsequent aggregation of platelets, both processes controlled by the generation of the enzyme thrombin, which occurs following activation of the clotting cascade. Thrombin, when bound to thrombomodulin, also functions as a potent anticoagulant 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 inhibition of Factors Va and Villa, aPC functions as perhaps the most important down-regulator of blood coagulation resulting in protection against thrombosis. In addition to anticoagulation, aPC has anti-inflammatory effects and exerts profibrinolytic properties that facilitate clot lysis.

Various diseases and conditions have been linked to protein C. For example, protein C levels have been shown to be abnormally low for persons having disseminated intravascular coagulation (DIC; Fourrier, et al., Chest 101:816-823, (1992)), sepsis (Gerson, et al., Pediatrics 91:418-422, (1993)), major trauma/major surgery (Thomas, et al., Am J Surg. 158:491-494, (1989)), burns (Lo, et al., Burns 20:186-187 (1994)), adult respiratory distress syndrome (ARDS; Hasegawa, et al., Chest 105(l):268-277, (1994)), and transplantations (Gordon, et al., Bone Marrow Trans. 11 :61-65 (1993)). In addition, there are numerous diseases with thrombotic abnormalities or complications that aPC may be useful in treating, such as: heparin-induced thrombocytopenia (HIT; Phillips, et al., Annals of Pharmacotherapy 28: 43-45, (1994)), sickle cell disease or thalassemia

(Karayalcin, et al., The American Journal of Pediatric Hematology/Oncologv 11(3):320- 323, (1989)), viral hemorrhagic fever (Lacy, et al., Advances in Pediatric Infectious Diseases 12:21-53, (1997)), thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS; Moake, Seminars in Hematology 34(2):83-89, (1997)). Furthermore, a study published in The New England Journal of Medicine (344: 699-709

(2001)) demonstrated that recombinant human aPC significantly reduces mortality in patients with severe sepsis. Thus, aPC is a therapeutic agent having both proven and potential use for a broad array of indications.

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. Patent Nos. 4,775,624 and 5,358,932. Despite improvements in recombinant DNA technology, these proteins and analogs thereof remain difficult and costly to produce. Additionally, higher aPC doses can expose patients to safety risks. See e.g., U.S. Patent No. 6,008,199. One remedy to these issues involves providing a human protein C analog that maintains the desirable biological activities of wild-type activated protein C (e.g., anticoagulant, fibrinolytic, and anti- inflammatory activities) while also being more active than wild-type activated protein C, thereby requiring substantially reduced dosage levels for therapeutic applications.

SUMMARY OF THE INVENTION

The human protein C analogs of the present invention have increased activity as compared to wild-type activated protein C due to specific amino acid substitutions in the analogs. The human protein C analogs of the present invention require less frequent administration and/or lower dosages of the analog for therapeutic applications, thereby reducing manufacturing costs. Reducing the amount of compound administered to patients via the use of human protein C analogs of the present invention may also minimize potential side effects. One embodiment of the present invention is a human protein C analog comprising

SEQ ID NO:1 having at least one amino acid substitution and as many as five amino acid substitutions selected from the group consisting of:

Ser at position 3 is substituted with Tyr, Phe, or Arg; Leu at position 5 is substituted with Phe; Leu at position 8 is substituted with Trp;

GIn at position 32 is substituted with Met; VaI at position 34 is substituted with Leu; Ser at position 94 is substituted with Tyr; His at position 202 is substituted with Leu; GIu at position 242 is substituted with GIn or Asn;

VaI at position 243 is substituted with Ala; Phe at position 244 is substituted with Tyr; Asn at position 248 is substituted with Ala; Thr at position 254 is substituted with He or VaI; Leu at position 261 is substituted with He;

Ala at position 264 is substituted with He, VaI, Thr, or Pro; GIn at position 265 is substituted with Met;

Thr at position 272 is substituted with VaI;

GIn at position 293 is substituted with Lys;

His at position 303 is substituted with Arg; GIu at position 309 is substituted with Ser, Leu, or GIy;

Ala at position 310 is substituted with Leu;

Asn at position 313 is substituted with Lys or Arg;

Pro at position 324 is substituted with GIn; lie at position 348 is substituted with Arg; GIn at position 353 is substituted with Arg;

Ser at position 367 is substituted with Ala; and

VaI at position 375 is substituted with He.

Another embodiment of the present invention is a human protein C analog comprising SEQ ID NO:1 having at least one amino acid substitution and as many as five amino acid substitutions selected from the group consisting of:

Ser at position 3 is substituted with Tyr, Phe, or Arg;

Leu at position 5 is substituted with Phe;

Leu at position 8 is substituted with Tip;

His at position 202 is substituted with Leu; GIn at position 293 is substituted with Lys;

GIu at position 309 is substituted with Leu or GIy;

Ala at position 310 is substituted with Leu;

Asn at position 313 is substituted with Lys or Arg;

Pro at position 324 is substituted with GIn; and GIn at position 353 is substituted with Arg.

Yet another embodiment of the present invention is a human protein C analog comprising SEQ ID NO:1, wherein Ser at position 3 is substituted with Tyr.

Another embodiment of the present invention is a human protein C analog comprising SEQ ID NO:1, wherein Ser at position 3 is substituted with Phe. Yet another embodiment of the present invention is a human protein C analog comprising SEQ ID NO:1, wherein Leu at position 8 is substituted with Tip. Another embodiment of the present invention is a human protein C analog comprising SEQ ID NO:1, wherein GIn at position 32 is substituted with Met.

Yet another embodiment of the present invention is a human protein C analog comprising SEQ ID NO:1, wherein Ala at position 264 is substituted with He. Another embodiment of the present invention is a human protein C analog comprising SEQ ID NO:1, wherein Asn at position 313 is substituted with Lys.

Yet another embodiment of the present invention is a human protein C analog comprising SEQ ID NO:1, wherein GIn at position 353 is substituted with Arg.

Another embodiment of the present invention is a human protein C analog comprising SEQ ID NO: 1 , wherein VaI at position 375 is substituted with He.

A further embodiment of the present invention is a human protein C analog for use as a medicament. Another embodiment of the present invention is a human protein C analog for the manufacture of a medicament for use in the treatment of human thrombotic disease. Yet another embodiment of the present invention is a human protein C analog for the manufacture of a medicament for use in the treatment of vascular occlusive disorder, arterial thromboembolic disorder, thrombotic disorder, and disease states predisposing to thrombosis. An additional embodiment of the present invention is a human protein C analog for the manufacture of a medicament for use in the treatment of acute coronary syndromes. An additional embodiment of the present invention is a method of treatment using a human protein C analog. Another embodiment of the present invention is a method of treating human thrombotic disease using a human protein C analog. Yet another embodiment of the present invention is method of treating vascular occlusive disorder, arterial thromboembolic disorder, thrombotic disorder, and disease states predisposing to thrombosis using a human protein C analog. An additional embodiment of the present invention is a method of treating acute coronary syndromes using a human protein C analog.

Another embodiment of the present invention is a pharmaceutical composition comprising the human protein C analog. Another embodiment of the present invention is a pharmaceutical composition comprising the human protein C analog and further comprising at least one pharmaceutically acceptable excipient. Another embodiment of the present invention is a pharmaceutical container comprising the pharmaceutical composition. The present invention also provides a liquid pharmaceutical preparation comprising the pharmaceutical composition. Another embodiment of the present invention is an intravenous infusion apparatus comprising the liquid pharmaceutical preparation. In all of the embodiments of the present invention, the human protein C analog can be in the two-chain zymogen form or the activated form.

Other embodiments of the present invention are a recombinant DNA molecule comprising a nucleotide sequence encoding the human protein C analog, a vector comprising the recombinant DNA molecule, and a host cell comprising the vector. In a preferred embodiment, the vector is an expression vector. In another preferred embodiment, the host cell is a eukaryotic cell.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below. "Wild-type protein C" is the type of protein C that predominates in a natural population of humans, in contrast to variant or analog polypeptide forms of protein C from natural or laboratory sources, respectively.

"Wild-type protein C zymogen" as used herein includes the 419 amino acid, non- activated, single-chain polypeptide (SEQ ID NO:1) and the two-chain non-activated zymogen. Wild-type protein C zymogen is preferably human.

"Wild-type activated protein C" or "wild-type aPC" refers to the activated form of wild-type protein C. Wild-type activated protein C is preferably human.

"Activated protein C" or "aPC" refers to recombinantly-produced activated protein C. Activated protein C includes and is preferably wild-type human activated protein C. Activated protein C may be produced by activating protein C zymogen in vitro or by direct secretion of the activated form of protein C. Activation results from removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C possessing enzymatic activity. Protein C may be produced in cells, eukaryotic cells, transgenic animals, or transgenic plants, including, for example, secretion from human kidney 293 cells as a zymogen then purified and activated by techniques known to the skilled artisan. "Human protein C analog" of "analog" refers to the analogs of the invention that differ from the amino acid sequence of wild-type protein C while retaining essential properties i.e., proteolytic, amidolytic, esterolytic, and biological (anticoagulant, anti¬ inflammatory, pro-fibrinolytic activities). Human protein C analog includes protein C analog zymogen and activated protein C analog. Furthermore, human protein C analog is a protein that differs from single chain zymogen (SEQ ID NO:1), two-chain zymogen, or activated protein C by at least one amino acid substitution.

"Protein C analog zymogen" as used herein includes the 419 amino acid, non- activated, single-chain polypeptide having at least one amino acid substitution and the two-chain, non-activated zymogen having at least one amino acid substitution. Protein C analog zymogen is preferably a human analog.

"Activated protein C analog" or "aPC analog" refers to the activated form of a human protein C analog.

"Activated form" of a human protein C analog refers to an analog wherein activation is achieved by activating protein C analog zymogen in vitro or by direct secretion of the activated form of the analog. Activation results from removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C analog possessing enzymatic activity.

"Zymogen" or "zymogen form" refers to the wild-type protein C zymogen or protein C analog zymogen. Zymogen refers to the non-activated form of wild-type protein C or protein C analog, whether as a single chain (e.g. "single chain zymogen form") or as a two-chain form (e.g. "two-chain zymogen form"). The zymogen exists in the single chain form unless cleavage of lysine and arginine residues (positions 156 and 157) occurs, resulting in a two-chain (light chain and heavy chain) non-activated zymogen.

"Amino acid substitution" refers to replacing the amino acid, which includes all twenty naturally occurring human amino acids (e.g. alanine, arginine, etc.), at a particular position in the given amino acid sequence with a different amino acid using site-directed mutagenesis. The numbering of protein C amino acid positions used herein refers to the 419 amino acid, one chain protein C zymogen. As detailed herein, post-translational modification of the single chain zymogen includes removal of two amino acids to generate a two-chain zymogen having a light and heavy chain, and subsequent removal of a dodecapeptide from the N-terminus of the heavy chain to yield aPC.

All of the human protein C analogs of the present invention can be prepared by the use of site-directed mutagenesis to change particular positions within the wild-type protein C zymogen. These analogs are activated by activating protein C analog zymogen in vitro or by direct secretion of the activated form of the analog. Activation results from removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C analog possessing enzymatic activity. The technique for modifying nucleotide sequences by site-directed mutagenesis is well known to those skilled in the art. See e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning : A Laboratory Manual, second Edition (1989). Abbreviations used herein for the twenty naturally occurring human amino acids are those known in the art (e.g. Ala or A for alanine, Arg or R for arginine, etc.). A substitution at a given position is indicated by the position number having the native amino acid shown to the left of the position number and the substituted amino acid shown to the right of the position number. For example, S3 Y means that the native amino acid at position 3 in wild-type protein C zymogen (or the corresponding position in wild-type aPC) is serine and the substituted amino acid at position 3 is tyrosine.

"Increased activity" refers to the ability of a human protein C analog to inhibit thrombin generation compared to the ability of wild-type aPC to inhibit thrombin generation. Increased activity can be assessed by methods known in the art, including using a IIase assay. The IIase assay, as used herein, directly measures thrombin generation by measuring thrombin activity towards a chromogenic substrate. Results from the IIase assay are generally predictive of activity in coagulation based assays because coagulation times are directly related to the rate and amount of thrombin generation. As such, IIase assay results reflect targets for activated partial thromboplastin time (APTT) or for an animal model of thrombosis. Human protein C analogs of the present invention have increased activity as determined by the IIase assay (see Example

1).

"Pharmaceutically acceptable excipient" as used herein refers to a substance used in preparing a human protein C analog into a viable medium of administration for a human. Pharmaceutically acceptable excipients refer to various substances including but not limited to bulking agents, pH buffering agents, salts, chelating agents, and diluents. Human protein C analogs can be prepared in a variety of ways. For example, human protein C analogs can be freeze dried so to provide a lyophilized material or formulated so to provide a liquid pharmaceutical preparation, defined hereinafter.

"Diluent" or "pharmaceutically acceptable diluent" refers to a reconstitution diluent or an intravenous infusion solution. A reconstitution diluent is a solution used to restore a lyophilized material containing analogs of the present invention to the liquid state. An intravenous infusion solution is a solution used as a vehicle for the administration of pharmaceutical compositions or formulations containing analogs of the present invention to a patient. A lyophilized formulation is reconstituted prior to its addition to the intravenous infusion solution. Some examples of diluents, as either reconstitution solutions or intravenous infusion solutions, include 0.9% Sodium Chloride, Sodium Chloride with Potassium Chloride, Glucose and Sodium Chloride, 5% Dextrose, Lactated Ringers, 3% Sodium Chloride, Sterile Water for Injection, and Ringers Injection.

"Pharmaceutical composition" as used herein is a pharmaceutical therapeutic agent comprising a human protein C analog, where the analog can be in the zymogen or the activated form. These pharmaceutical compositions can further comprise pharmaceutically acceptable excipients. Additionally, these compositions can be lyophilized or comprise a diluent.

"Liquid pharmaceutical preparation" as used herein is a formulation or solution comprising a human protein C analog that is appropriate to be given as a therapeutic agent.

"Pharmaceutically effective amount" represents an amount of an analog of the invention that is capable of treating particular disease states or disorders in mammals, preferably humans. The particular dose of the analog administered according to this invention will, of course, be determined by the particular circumstances surrounding the case, including the analog administered, the particular condition being treated, and similar considerations.

"Pharmaceutical container" means a receptacle such as a vial or cartridge that is used to receive the designated material, i.e., a human protein C analog. Pharmaceutical container, as used herein, refers to a sterile receptacle that can hold a medicament for later administration to a human. "Intravenous infusion apparatus" means equipment used to administer a human protein C analog to a human including, but not limited to, an infusion bag, an infusion tubing, and an infusion pump chamber.

"Unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material, herein a human protein C analog of the present invention, calculated to produce the desired therapeutic effect. In a preferred embodiment, the unit dose also contains a suitable pharmaceutical excipient.

"Effective amount" is a therapeutically efficacious amount of a pharmaceutical compound or compounds, particularly human protein C analogs of the present invention.

"Thrombotic disorder" is a disorder relating to, or affected with the formation or presence of a blood clot within a blood vessel. Thrombotic disorders include, but are not limited to, stroke, myocardial infarction, unstable angina, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery. "Hypercoagulable states" refer to excessive coagulability associated with disseminated intravascular coagulation, pre-thrombotic conditions, activation of coagulation, or congenital or acquired deficiency of clotting factors. Furthermore, "vascular occlusive disorders" and "hypercoagulable states" are disorders including but not limited to sepsis, disseminated intravascular coagulation, purpura fulminans, major trauma, major surgery, burns, adult respiratory distress syndrome, transplantations, deep vein thrombosis, heparin-induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome.

"Treating" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a human protein C analog of the present invention either therapeutically or prophylactically to prevent the onset of the symptoms or complications of the disease, condition, or disorder. The present invention provides human protein C analogs that retain the important biological activity (e.g., anticoagulant, fibrinolytic, and anti-inflammatory activities) of wild-type activated protein C and have greater activity than wild-type activated protein C.

These analogs provide various advantages, e.g. less frequent administration and/or smaller dosages and thus a reduction in the overall manufacturing costs as well as more potent compounds for treating disease states while minimizing potential side effects. Human protein C analogs having a specific amino acid substitution at one, two, three, four or five positions among positions 3, 5, 8, 32, 34, 94, 202, 242, 243, 244, 248, 254, 261, 264, 265, 272, 293, 303, 309, 310, 313, 324, 348, 353, 367, or 375 of SEQ ID NO: 1 have increased activity when compared to wild-type activated protein C. Preferably, a human protein C analog having increased activity when compared to wild-type activated protein C is an activated protein C analog.

A preferred human protein C analog of the present invention comprises SEQ ID NO:1 having one, two, three, four, or five amino acid substitution(s) selected from the group consisting of:

Ser at position 3 is substituted with Tyr, Phe, or Arg; Leu at position 5 is substituted with Phe; Leu at position 8 is substituted with Trp; GIn at position 32 is substituted with Met; VaI at position 34 is substituted with Leu;

Ser at position 94 is substituted with Tyr; His at position 202 is substituted with Leu; GIu at position 242 is substituted with GIn or Asn; VaI at position 243 is substituted with Ala; Phe at position 244 is substituted with Tyr;

Asn at position 248 is substituted with Ala; Thr at position 254 is substituted with He or VaI; Leu at position 261 is substituted with lie; Ala at position 264 is substituted with He, VaI, Thr, or Pro; GIn at position 265 is substituted with Met;

Thr at position 272 is substituted with VaI; GIn at position 293 is substituted with Lys; His at position 303 is substituted with Arg; GIu at position 309 is substituted with Ser, Leu, or GIy; Ala at position 310 is substituted with Leu;

Asn at position 313 is substituted with Lys or Arg; Pro at position 324 is substituted with GIn; He at position 348 is substituted with Arg; GIn at position 353 is substituted with Arg; Ser at position 367 is substituted with Ala; and VaI at position 375 is substituted with He. More preferably, such a human protein C analog of the present invention is in the two- chain zymogen form or activated form.

Even more preferably, a human protein C analog of the present invention comprises SEQ ID NO:1 having one, two, three, four, or five amino acid substitution(s) selected from the group consisting of: Ser at position 3 is substituted with Tyr or Phe;

Leu at position 8 is substituted with Trp; GIn at position 32 is substituted with Met; Ala at position 264 is substituted with He; Asn at position 313 is substituted with Lys; GIn at position 353 is substituted with Arg; and

VaI at position 375 is substituted with lie.

Still more preferably, such a human protein C analog of the present invention is in the two-chain zymogen form or activated form.

Another preferred human protein C analog of the present invention comprises SEQ ID NO:1 having one, two, three, four, or five amino acid substitution(s) selected from the group consisting of:

Ser at position 3 is substituted with Tyr, Phe, or Arg; Leu at position 5 is substituted with Phe; Leu at position 8 is substituted with Trp; His at position 202 is substituted with Leu;

GIn at position 293 is substituted with Lys;

GIu at position 309 is substituted with Leu or GIy;

Ala at position 310 is substituted with Leu;

Asn at position 313 is substituted with Lys or Arg; Pro at position 324 is substituted with GIn; and

GIn at position 353 is substituted with Arg. More preferably, such a human protein C analog of the present invention is in the two- chain zymogen form or activated form.

Even more preferably, a human protein C analog of the present invention comprises SEQ ID NO:1 having one or two amino acid substitution(s) selected from the group consisting of:

Ser at position 3 is substituted with Tyr or Phe; Leu at position 8 is substituted with Trp; Asn at position 313 is substituted with Lys; and GIn at position 353 is substituted with Arg. Still more preferably, such a human protein C analog of the present invention is in the two-chain zymogen form or activated form.

Yet more preferably, a human protein C analog of the present invention comprises SEQ ID NO:1, wherein Ser at position 3 is substituted with Tyr, Ser at position 3 is substituted with Phe, Leu at position 8 is substituted with Trp, GIn at position 32 is substituted with Met, Ala at position 264 is substituted with He, Asn at position 313 is substituted with Lys, GIn at position 353 is substituted with Arg, or VaI at position 375 is substituted with He. Still more preferably, such a human protein C analog of the present invention is in the two-chain zymogen form or activated form.

In a preferred embodiment, the activated form of the analog of the present invention has increased activity, relative to the activated form of the wild-type activated protein C, in the IIase assay disclosed herein.

The invention also provides DNA molecules that can be used in making the human protein C analogs. These DNA molecules comprise the coding sequence for the light chain of human wild-type protein C or human protein C analog positioned immediately adjacent to, downstream of, and in translational reading frame with the pre- propeptide sequence of human wild-type protein C or human protein C analog. The DNA sequences preferably encode the Lys-Arg dipeptide, which is processed during maturation of the protein C molecule, the activation peptide and the heavy chain of the human wild- type protein C or human protein C analog. Thus, the human protein C analogs of the present invention are modified polypeptides having at least one amino acid substitution, thereby differentiating the analog from the wild-type human protein C zymogen, single chain sequence identified as SEQ ID NO: 1, the wild-type human protein C zymogen, two-chain sequence, and the wild-type human activated protein C sequence.

Those skilled in the art will recognize that, due to the degeneracy of the genetic code, a variety of DNA molecules can encode the analogs described herein. U.S. Patent No. 4,775,624, the entire teaching of which is herein incorporated by reference, discloses the wild-type form of the human protein C molecule. The skilled artisan could readily determine which changes in the DNA sequences which could encode the exact analogs as disclosed herein. The invention is not limited to the specific DNA sequences disclosed. Consequently, the construction described below and the preferred DNA molecules are merely illustrative and do not limit the scope of the invention.

All of the DNA molecules of the present invention can be prepared by the use of site-directed mutagenesis to change particular positions within the human wild-type protein C zymogen. The technique for modifying nucleotide sequences by site-directed mutagenesis is well known to those skilled in the art. See e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning : A Laboratory Manual, second Edition (1989).

The human protein C analogs can be made by techniques well known in the art utilizing eukaryotic cell lines, transgenic animals, or transgenic plants. Skilled artisans will readily understand that appropriate host eukaryotic cell lines include but are not limited to HepG2, LLC-MK2, CHO-Kl, HEK293, or AV 12 cells, examples of which are described in U.S. Patent No. 5,681 ,932, herein incorporated by reference. Furthermore, examples of transgenic production of recombinant proteins are described in U.S. Patent Nos. 5,589,604 and 5,650,503, herein incorporated by reference.

Skilled artisans recognize that a variety of vectors are useful in the expression of a DNA sequence of interest in a eukaryotic host cell. Vectors that are suitable for expression in mammalian cells include, but are not limited to: pGT-h, pGT-d; pCDNA 3.0, pCDNA 3.1, pCDNA 3.1+Zeo, and pCDNA 3.1+Hygro (mvitrogen), pMH (Roche); and, pIRES/Hygro, and pIRES/neo (Clonetech). The preferred vectors of the present invention are pIG3 and pMH.

Other sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.

In some cases it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e. to maintain the proper reading frame. The human protein C analogs made by any of these methods must undergo post- translational modifications such as the addition of nine or ten gamma-carboxy-glutamates, the addition of one erythro-beta-hydroxy-Asp (beta-hydroxylation), the addition of four Asn-linked oligosaccharides (glycosylation) and, the removal of the leader sequence (42 amino acid residues). Such post-translational modifications are necessary for efficient production and secretion of the human protein C analogs from mammalian cells.

It is known in the art that post-translational modifications of recombinant proteins such as the human protein C analogs of the present invention may vary depending on which host cell line is utilized for the expression of the recombinant protein. For example, the post-translational modification of gamma-carboxylation, which is essential for the anticoagulant activity of the human protein C analogs of the present invention, may be higher, slightly lower, or much lower than plasma derived wild-type protein C gamma-carboxylation, depending on the host cell line used (Yan et al., Bio/Technolo gy 8(7):655-661, (1990)).

The human protein C analogs of the present invention may be administered as a zymogen or in the activated form. Methods for the activation of zymogen forms of human wild-type protein C and human protein C analogs to human wild-type activated protein C and human activated protein C analogs are old and well known in the art. Activation may be achieved by thrombin alone, by a thrombin/thrombomodulin complex, by RVV-X, a protease from Russell's Viper venom, by pancreatic trypsin or by other proteolytic enzymes.

Human protein C analogs of the present invention can be prepared in a similar fashion as hereinafter described for human wild-type protein C. Human wild-type protein C is produced recombinantly in Human Kidney 293 cells by techniques well known to the skilled artisan such as those set forth in Yan, U.S. Patent No. 4,981,952, the entire teaching of which is herein incorporated by reference. The gene encoding human wild- type protein C is disclosed and claimed in Bang, et al., U.S. Patent No. 4,775,624, the entire teaching of which is incorporated herein by reference. The plasmid used to express human wild-type protein C in 293 cells is plasmid pLPC which is disclosed in Bang, et al., U.S. Patent No. 4,992,373, the entire teaching of which is incorporated herein by reference. The construction of plasmid pLPC is also described in European Patent Publication No. 0 445 939, and in Grinnell, et al., 1987, Bio/Technology 5:1189-1192, the teachings of which are also incorporated herein by reference. Briefly, the plasmid is transfected into 293 cells, then stable transformants are identified, subcultured and grown in serum-free media. After fermentation, cell-free medium is obtained by microfiltration. Alternatively, the vector used is pMH (Roche). The plasmid is transiently transfected into 293 cells and grown in serum-free media. After the appropriate time, the cell-free medium is removed without filtration.

The human wild-type protein C is separated from the culture fluid by an adaptation of the techniques of Yan, U.S. Patent No. 4,981,952. The clarified medium is made 4 mM in EDTA before it is absorbed to an anion exchange resin (Fast-Flow Q, Pharmacia). After washing with 4 column volumes of 20 mM Tris, 200 mM NaCl, pH 7.4 and 2 column volumes of 20 mM Tris, 150 mM NaCl, pH 7.4, the bound recombinant human wild-type protein C is eluted with 20 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4. The eluted protein is greater than 95% pure after elution as judged by SDS- polyacrylamide gel electrophoresis. Further purification of the protein is accomplished by making the protein 3 M in NaCl followed by adsorption to a hydrophobic interaction resin (Toyopearl Phenyl 650 M, TosoHaas) equilibrated in 20 mM Tris, 3 M NaCl, 10 mM

CaCl2, pH 7.4. After washing with 2 column volumes of equilibration buffer without CaCl2, the recombinant wild-type human protein C is eluted with 20 mM Tris, pH 7.4.

The eluted protein is prepared for activation by removal of residual calcium. The recombinant human wild-type protein C is passed over a metal affinity column (Chelex- 100, Bio-Rad) to remove calcium and again bound to an anion exchanger (Fast Flow Q,

Pharmacia). Both of these columns are arranged in series and equilibrated in 20 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 6.5. Following loading of the protein, the Chelex-100 column is washed with one column volume of the same buffer before disconnecting it from the series. The anion exchange column is washed with 3 column volumes of equilibration buffer before eluting the protein with 0.4 M NaCl₃ 20 mM Tris-acetate, pH 6.5. Protein concentrations of recombinant human wild-type protein C and recombinant wild-type activated protein C solutions are measured by UV 280 nm extinction EO.1%=I .85 _or i.95_; respectively.

Human protein C analogs of the present invention can be activated in a similar fashion as hereinafter described for human wild-type protein C. To activate the human wild-type protein C zymogen, bovine thrombin is coupled to Activated CH-Sepharose 4B (Pharmacia) in the presence of 50 mM HEPES, pH 7.5 at 4°C. The coupling reaction is done on resin already packed into a column using approximately 5000 units thrombin/mL resin. The thrombin solution is circulated through the column for approximately 3 hours before adding MEA to a concentration of 0.6 mL/L of circulating solution. The MEA- containing solution is circulated for an additional 10 to 12 hours to assure complete blockage of the unreacted amines on the resin. Following blocking, the thrombin-coupled resin is washed with 10 column volumes of 1 M NaCl, 20 mM Tris, pH 6.5 to remove all non-specifically bound protein, and is used in activation reactions after equilibrating in activation buffer.

Purified human wild-type protein C is made 5mM in EDTA (to chelate any residual calcium) and is diluted to a concentration of 2 mg/mL with 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5. This material is passed through a thrombin column equilibrated at 37 °C with 50 mM NaCl and either 20 mM Tris pH 7.4 or 20 mM Tris-acetate pH 6.5. The flow rate is adjusted to allow for approximately 20 minutes of contact time between the human wild-type protein C and thrombin resin. The effluent is collected and immediately assayed for amidolytic activity. If the material does not have a specific activity

(amidolytic) comparable to an established standard of human wild-type activated protein C, it is recycled over the thrombin column to activate the human wild-type protein C to completion. Alternatively, the thrombin resin can be added directly to the given protein C solution. This solution is shaken at 37 ⁰C, often for 16 hours, followed by filtration of the sample to recover the activated protein C from the resin. This is followed by 1 : 1 dilution of the material with 20 mM buffer as above, with a pH of anywhere between 7.4 or 6.0 (lower pH being preferable to prevent autodegradation) to keep the human wild-type activated protein C at lower concentrations while it awaits the next processing step. Removal of leached thrombin from the human wild-type activated protein C material is accomplished by binding the human wild-type activated protein C to an anion exchange resin (Fast Flow Q, Pharmacia) equilibrated in activation buffer (either 20 mM Tris, pH 7.4 or preferably 20 mM Tris-acetate, pH 6.5) with 150 mM NaCl. Thrombin passes through the column and elutes during a 2-6 column volume wash with 20 mM equilibration buffer. Bound human wild-type activated protein C is eluted with a step gradient using 0.4 M NaCl in either 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4. Higher volume washes of the column facilitate more complete removal of the dodecapeptide. The material eluted from this column is stored either in a frozen solution (- 20°C) or as a lyophilized powder.

The human protein C analogs of the present invention can be formulated according to known methods to prepare a pharmaceutical composition. More preferably, this pharmaceutical composition comprises the two-chain zymogen form or the activated form of the analog. Even more preferably, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient. Preferable pharmaceutically acceptable excipients include a bulking agent, a salt, a pH buffering agent, a chelating agent, and a diluent. Still more preferably, the pharmaceutical composition comprising a human protein C analog is lyophilized. For example, a desired formulation is a stable lyophilized product of high purity comprising a bulking agent such as sucrose, trehalose or raffmose; a salt such as sodium chloride or potassium chloride; a pH buffering agent such as sodium citrate, Tris acetate, or sodium phosphate, at a pH of about 5.5 to about 6.5; and a human protein C analog. Additionally, this formulation also comprises a chelating agent such as EDTA disodium salt. Reconstitution of this formulation preferably involves a reconstitution diluent or an intravenous infusion solution, thereby providing a liquid pharmaceutical preparation.

Pharmaceutical compositions as described herein are stored in a pharmaceutical container. Preferably, the pharmaceutical container is sterile. More preferably, the pharmaceutical container is a vial or a cartridge. Additionally, a liquid pharmaceutical preparation as described herein is placed into an intravenous infusion apparatus. Preferably, the intravenous infusion apparatus is an infusion bag, an infusion tubing, or an infusion pump chamber.

A human protein C analog of the present invention is useful as a medicament. More specifically, a human protein C analog of the present invention will be effective in the treatment of human thrombotic disease including replacement therapy in the treatment of protein C deficiency, vascular occlusive disorder, and hypercoagulable state including sepsis, disseminated intravascular coagulation, purpura fulminans, major trauma, major surgery, burns, adult respiratory distress syndrome, transplantations, deep vein thrombosis, heparin-induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, meningococcemia, complications during pregnancy, and venous thromboembolic complications associated with bone marrow transplantation, other organ transplantation, surgery, or trauma. The a human protein C analog of the present invention will also be effective in the treatment of vascular occlusive disorder, arterial thromboembolic disorder, thrombotic disorder, and disease state predisposing to thrombosis, such as, myocardial infarction, stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery. Additionally, a human protein C analog of the present invention will be effective hi the treatment of acute coronary syndromes including unstable angina. Preferably, such a human protein C analog of the present invention is in the two-chain zymogen form or activated form.

The invention further provides treating the above-mentioned diseases, conditions, and disorders by administering to a patient a pharmaceutically effective amount of a human protein C analog. Preferably, such a human protein C analog of the present invention is in the two-chain zymogen form or activated form. A human protein C analog of the present invention can be administered at an appropriate dose level understood and appreciated in the art and determined by the attending physician evaluating the particular circumstances surrounding the case. Preferably, the amount of human protein C analog administered is from about 0.01 μg/kg/hr to about 50 μg/kg/hr. More preferably, the amount of human protein C analog administered is about 0.1 μg/kg/hr to about 25 μg/kg/hr. Yet more preferably the amount of human protein C analog administered is about 0.1 μg/kg/hr to about 15 μg/kg/hr. Even more preferably the amount of human protein C analog administered is about 1 μg/kg/hr to about 15 μg/kg/hr. A still more preferable amount of human protein C analog administered is about 5 μg/kg/hr or about 10 μg/kg/hr. A further preference provides that such a human protein C analog of the present invention is in the two-chain zymogen form or activated form. Preferably, the human protein C analog is administered parenterally to ensure delivery into the bloodstream in an effective form by injecting a dose of 0.01 mg/kg/day to about 1.0 mg/kg/day, one to six times a day, for one to ten days. More preferably, the human protein C analog is administered B.I.D. (2 times a day) for three days.

Alternatively, the human protein C analog is administered at a dose of about 0.01 μg/kg/hr to about 50 μg/kg/hr, by continuous infusion for 1 to 240 hours.

The preferred plasma ranges obtained from the amount of human protein C analog administered is 0.02 ng/niL to less than 100 ng/mL.

In another embodiment, the human protein C analog is administered by injecting a portion (1/3 to 1/2) of the appropriate dose per hour as a bolus injection over a time from about 5 minutes to about 120 minutes, followed by continuous infusion of the appropriate dose for up to 240 hours. hi another embodiment, the human protein C analog is administered by local delivery through an intracoronary catheter as an adjunct to high-risk angioplasty (with and without stenting). The amount of human protein C analog administered is from about 0.01 mg/kg/day to about 1.0 mg/kg/day by continuous infusion, bolus injection, or a combination thereof.

In another embodiment, the human protein C analog is administered subcutaneously at a dose of 0.01 mg/kg/day to about 10.0 mg/kg/day, to ensure a slower release into the bloodstream. Formulation for subcutaneous preparations will be done using known methods to prepare such pharmaceutical compositions.

AU patents and articles referred to herein are incorporated by reference. The following examples are intended to illustrate but not limit the invention.

Example 1 ELISA Analysis

ELISA analysis is used to quantitate the protein C analog zymogen and the wild- type protein C zymogen. Greiner ELISA plates are coated overnight with 50 μL of 2.5 μg/mL goat polyclonal antibody (Enzyme Research Laboratories) in carbonate buffer (50 mM carbonate, pH 8.3) at 4 ⁰C. The next day, after washing with PBST (which is used in all subsequent washes after each step), the wells are blocked with 3% BSA for 1 hour at room temperature. After washing, samples diluted in fresh media are added. The wild- type protein C zymogen standard curve is typically prepared at 0.025, 0.05, 0.07, 0.09, 0.12, 0.17 and 0.3 μg/mL, while samples are diluted to be within that range. Samples are incubated for 1 hour at room temperature, then are washed. The primary antibody (HPC- 4 (Roche), 0.15 μg/mL in 20 mM Tris, pH 7.4, with 1 mM CaCl₂) is added to the samples for 1 hour at room temperature. Following washing, secondary antibody (anti-mouse IgG- peroxidase from sheep (Sigma), diluted 10,000x in PBS) is added for 1 hour at room temperature. After a final wash, the samples are developed with TMB substrate and are stopped with an equal volume of 2.5M H₂SO₄. Absorbances are measured at 450 nm. Concentrations are calculated using the wild-type protein C zymogen standard curve using a SOFTmax program (see below). An ELISA of wild-type activated protein C and activated protein C analog is also conducted by coating Greiner ELISA plates with 50 μL of a murine monoclonal anti- protein C antibody that does not interfere with the activity or activation of protein C (e.g. HPC-3; 2.5 μg/mL (Geiger, et al, Thrombosis and Haemostasis 61(l):86-92 (1989))) in PBS and are stored overnight at 4°C. The following day, after washing with PBS-T, the plate is blocked with 3% BSA for 1 hour at room temperature. (Note that all washes are performed with PBS-T buffer and are done after each step.) Solutions of wild-type activated protein C or activated protein C analog diluted in media are added for 1 hour at room temperature. The wild-type activated protein C standard curve is typically prepared with 0.025, 0.05, 0.07, 0.09, 0.12, 0.17, 0.3 μg/mL wild-type activated protein C. The samples are diluted to be within that range. The primary antibody (e.g. biotinylated HPC-

1, 0.15 μg/mL in PBS (ClA, ClB: Heeb, et al., Thrombosis Research 52:33-43 (1988))) is then added for 1 hour at room temperature. NeutrAvidin- Alkaline Phosphatase conjugate (Pierce) is added for 30 to 60 minutes at room temperature. The ELISA is developed with PPNP and is stopped with NaOH. Absorbance is read at 405 nm. Concentrations of samples are calculated using the generated standard curve. IIase Assay

For the IIase assay, reagent preparation includes making Tris buffer (30 mM Tris/HCl, 150 mM NaCl, pH 7.4); 2x Tris buffer (60 mM Tris/HCl, 300 mM NaCl, pH 7.4); Tris/HSA buffer (1 mg/mL HSA in Tris buffer); EDTA buffer (47 mM EDTA in Tris buffer); and calcium buffer (50 mM CaCl₂ in 2x Tris buffer). Vesicles are 200 μM phosphatidyl serine / phosphatidyl choline (PS:PC) sonicated vesicles (1:25 of 5 mM PS:PC stock).

An aliquot of Factor Va is diluted every week to 25 nM (8.4 μg/mL) in 1 :1 glycerol:2x Tris and is stored at -20 ⁰C. Factor Xa is diluted to 160 nM in Tris/HSA and is stored at -20 ⁰C.

All assays are performed in 96-well round-bottom microtiter plates. For each microtiter plate, the reagents are prepared as follows. Reagent A is prepared immediately before use by mixing 5 mM vesicles (80 μL), 50 mM CaCl₂ buffer (400 μL), 2x Tris buffer (1408 μL) and the appropriate amount of fVa (typically around 0.3 nM final concentration) for a final volume of 2 mL.

The factor Xa solution (2.3 nM) is prepared immediately before use in Tris/HSA buffer. The prothrombin solution (5.6 μM) is prepared immediately before use in 2x Tris buffer. The colorimetric substrate, S-2238, is prepared by dissolving 25 mg in 100 mL water and adding 50 mL 2x Tris. The wild-type activated protein C standard curve is generated by preparing 0.1,

0.2, 0.4, 0.6, 1, 1.75 and 2.5 μg/mL wild-type activated protein C diluted into media (1:1 DMEM:Ham's F12) containing 10 mg/mL BSA. The wild-type activated protein C standards and activated protein C analog samples are diluted 4x into Tris/HSA, with 20 μL placed per microtiter plate well. Reagent A and Factor Xa solution are mixed 1:1, with 4 mL prepared per microtiter plate. After sitting at room temperature for 15 minutes,

40 μL of this solution is added to each well containing sample and are quickly mixed. After incubating for 3.5 minutes at room temperature, 20 μL of prothrombin solution is added. The reaction is incubated at room temperature for 15 minutes. The reaction is stopped by the addition of 120 μL of EDTA buffer. The production of thrombin is assayed by adding an aliquot (20 μL) of the stopped reaction to 100 μL of S-2238. The rate of cleavage is monitored at 405 nm over 5 minutes. The rate is calculated for each well by fitting the linear portion of each curve using the Vmax kinetic program in SOFTmax. A wild-type activated protein C standard curve is generated for each plate and is used to calculate the relative activities of all of the activated protein C analog samples on that plate (see below).

The amount of fVa added in the assay is critical. Each week, the fVa for the reaction is calibrated by doing test curves in duplicate with varying amounts of fVa (usually between 0.2 and 0.5 nM final concentration). The conditions giving the best duplicates and spread between the high and low wild-type activated protein C concentrations are used for that week's assays.

The absorbance reader used during this study is a SPECTRAmaxPLUS384 Microplate Spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA). The instrument control and data collections software used is SOFTmax PRO V4.0.

Raw assay data for calculating zymogen concentration (ELISA) and aPC activity (Ilase assay) is collected by reading at 450 nm and 405 nm respectively in the 96-well format. SOFTmax PRO is used for plate specific curve fitting and data extrapolation. Analog sample specific data is extrapolated using the standard curve generated for each plate. The standard curve is fitted using the 4-parameter logistic curve fitting equation. The fit is based on the equation:

Y = ((A-D)/(l+(x/C)^ΛB))+D

where FJt and A are the Y- values corresponding the asymptote at the high and low values of the X-axis respectively. The coefficient C is the X- value corresponding the midpoint between A and D. The coefficient B describes the transition rate between the asymptotes from the center of the curve. Further discussion of this equation is also found in Softmax PRO for Life Sciences and Drug Discovery User 's Manual.

Relative Activity (RA) is determined by applying the following equation:

RA = (AA * Ilase dilution factor) / (AC * ELISA dilution factor)

where AA (Analog Activity) is the extrapolated activity value from the Ilase assay and

AC (Analog Concentration) is the extrapolated value from the quantitative ELISA assay. Analogs with RA values greater than 20% of wild-type activated protein C are selected for further analysis. This group is then processed to remove analogs that had ELISA values lower than 0.025 μg/mL or IIase values corresponding to the activity produced by 0.2 μg/mL of wild-type activated protein C or less. This is done to eliminate false positives generated by analogs that express poorly. The remaining analogs are then evaluated. Analogs with a specific activity greater than wild-type activated protein C are considered primary hits. The activity results in Table 1 are relative to wild-type activated protein C (Activity = 1) and Sl lG/Q32E/N33D-activated protein C (GED-aPC; Activity = 10). H10Q/S1 lG/S12K-activated protein C (QGK-aPC; Activity = 3) produces an activity result that exceeds the variability of the assay. Hence, an analog having an activity result of 3 or more is an analog having increased activity over wild-type activated protein C.

Table 1

The second result listed is from purified material. ²ND = Not Determined

Example 2 Activated Partial Thromboblastin Time (APTT) Assay

The assay is performed using a CoAScreener (American Labor Corporation, Durham, NC) to measure coagulation times. Wild-type activated protein C or activated protein C analog dilutions are prepared in Tris / BSA buffer (20 mM Tris, pH 7.4, containing 150 mM NaCl and 1 mg/mL BSA), and are added to reconstituted human plasma (Citrol, Dade Behring) and Actin APTT Reagent (Dade Behring). Coagulation is initiated with the addition of calcium chloride solution. The clotting time is determined using the CoAScreener.

Table 2

. Example 3 Protein S assay

The assay to determine whether protein S stimulated aPC cleavage of fVa is performed as a modified IIase assay. Wild-type activated protein C and activated protein C analogs are diluted in a series of concentrations ranging between 0.5 and 0.025 μg/mL. Human protein S (Enzyme Research Laboratories) is diluted to 82 μg/mL in Tris/HSA. AU samples are diluted 4x with Tris/HSA in one column and 4x with Tris/HSA/Protein S in another column. Final volume is 20 μL per well. Prothrombin and Reagent A are prepared as in the IIase assay (see Example 1; fVa calibrated for this set of conditions), while fXa is diluted to 2.8 μM. All subsequent steps are done as in the IIase assay (see Example 1), except prothrombin is added to the reaction mix 1.5 to 2 minutes after Reagent A/fXa addition. Each well is assayed by addition of 20 μL to the S-2238 reaction solution (DiaPharma). The results are then plotted.

For each protein (wild-type activated protein C or activated protein C analog), two curves are generated, one without protein S and one with protein S. Rates without protein S are compared to the wild-type activated protein C curve, and likewise rates with protein S are compared to the wild-type activated protein C plus protein S curve. From this, the amount each activated protein C analog is stimulated by protein S compared to wild-type activated protein C is determined. Table 3

N^χτDτ» — = Not Determined

Example 4 Half-life of Activated Protein C in Pooled Plasma

A deep 96-well polypropylene plate is set in a 37 °C water bath for 5 to 10 minutes and is covered with a lid or adhesive in order to prevent evaporation. Pooled human plasma (150 μL) is added and warmed for 5 minutes. Then 30 μL of the activated protein C analog sample (2 μg/mL) is added to the plasma and mixed together, with 25 μL immediately removed to a Costar 96 well microtiter plate for the zero time point. To this mixture is added 175 μL of S-2366 (DiaPharma) in Tris buffer containing 1 mg/mL BSA. The rate is measured at 405 nm over 5 minutes. Aliquots are removed over the course of 60 minutes and are assayed as above (see Example 3). For each set of samples (either wild-type activated protein C or activated protein C analogs), the rates (reflective of remaining activity) are plotted versus time to determine half-life.

Table 4

ND = Not Determined ²Half-life is likely similar to wild-type activated protein C.

Example 5 Endothelial Protein C Receptor (EPCR) Assay

Greiner U-bottom plates are coated with 2 μg/mL soluble EPCR in PBS and are stored overnight at 4 ⁰C. The next day, plates are washed with PBST followed by PBS. The wells are blocked with 3% BSA/Tris buffer (30 mM Tris/HCl, 150 mM NaCl₅ pH 7.4) for 1 hour at room temperature. Plates are then washed with Tris buffer before addition of the activated protein C analog samples. Samples are diluted to seven concentrations, usually ranging from 6 to 0.5 μg/mL, in Tris buffer containing 1% BSA and 3 mM calcium (Tris/B S A/Calcium). Typically 50 μL are added per well, and are incubated either 1 to 2 hours at room temperature or overnight at 4 ⁰C. After washing with Tris buffer containing 3 mM calcium, primary antibody (goat anti-protein C, 10,000x dilution in Tris/B S A/Calcium) is added for 1 hour at room temperature. After washing with Tris buffer containing 3 mM calcium, secondary antibody (anti-goat, 12,000x dilution in Tris/B S A/Calcium) is added for 1 hour at room temperature. The final washes are with PBST. Then, TMB substrate is added until sufficiently developed. The absorbances at 450 nm are plotted to determine a K_D for each set of samples.

Table 5

SEQ ID NO:1 : Wild-Type Human Protein C Zymogen, Single Chain

1 ANSFLEELRH SSLERECIEE ICDFEEAKEI FQNVDDTLAF WSKHVDGDQC

51 LVLPLEHPCA SLCCGHGTCI DGIGSFSCDC RSGWEGRFCQ REVSFLNCSL

101 DNGGCTHYCL EEVGWRRCSC APGYKLGDDL LQCHPAVKFP CGRPWKRMEK 151 KRSHLKRDTE DQEDQVDPRL IDGKMTRRGD SPWQWLLDS KKKLACGAVL

201 IHPSWVLTAA HCMDESKKLL VRLGEYDLRR WEKWELDLDI KEVFVHPNYS

251 KSTTDNDIAL LHLAQPATLS QTIVPICLPD SGLAERELNQ AGQETLVTGW

301 GYHSSREKEA KRNRTFVLNF IKIPWPHNE CSEVMSNMVS ENMLCAGILG 351 DRQDACEGDS GGPMVASFHG TWFLVGLVSW GEGCGLLHNY GVYTKVSRYL 401 DWIHGHIRDK EAPQKSWAP

Claims

We claim:

1. A human protein C analog comprising SEQ ID NO: 1 having one, two, three, four, or five amino acid substitution(s) selected from the group consisting of: Ser at position 3 is substituted with Tyr, Phe, or Arg;

Leu at position 5 is substituted with Phe;

Leu at position 8 is substituted with Trp;

GIn at position 32 is substituted with Met;

VaI at position 34 is substituted with Leu; Ser at position 94 is substituted with Tyr;

His at position 202 is substituted with Leu;

GIu at position 242 is substituted with GIn or Asn;

VaI at position 243 is substituted with Ala;

Phe at position 244 is substituted with Tyr; Asn at position 248 is substituted with Ala;

Thr at position 254 is substituted with He or VaI;

Leu at position 261 is substituted with He;

Ala at position 264 is substituted with He, VaI, Thr, or Pro;

GIn at position 265 is substituted with Met; Thr at position 272 is substituted with VaI;

GIn at position 293 is substituted with Lys;

His at position 303 is substituted with Arg;

GIu at position 309 is substituted with Ser, Leu, or GIy;

Ala at position 310 is substituted with Leu; Asn at position 313 is substituted with Lys or Arg;

Pro at position 324 is substituted with GIn; He at position 348 is substituted with Arg; GIn at position 353 is substituted with Arg; Ser at position 367 is substituted with Ala; and VaI at position 375 is substituted with He.

2. The human protein C analog of Claim 1 wherein the amino acid substitution is selected from the group consisting of:

Ser at position 3 is substituted with Tyr or Phe; Leu at position 8 is substituted with Trp; GIn at position 32 is substituted with Met;

Ala at position 264 is substituted with He; Asn at position 313 is substituted with Lys; GIn at position 353 is substituted with Arg; and VaI at position 375 is substituted with He.

3. A human protein C analog comprising SEQ ID NO: 1 having one, two, three, four, or five amino acid substitution(s) selected from the group consisting of:

Ser at position 3 is substituted with Tyr, Phe, or Arg;

Leu at position 5 is substituted with Phe; Leu at position 8 is substituted with Trp;

His at position 202 is substituted with Leu;

GIn at position 293 is substituted with Lys;

GIu at position 309 is substituted with Leu or GIy;

Pro at position 324 is substituted with GIn; and

GIn at position 353 is substituted with Arg.

4. The human protein C analog of Claim 3, having one, two, three, or four amino acid substitutions) selected from the group consisting of:

Ser at position 3 is substituted with Tyr or Phe; Leu at position 8 is substituted with Trp; Asn at position 313 is substituted with Lys; and GIn at position 353 is substituted with Arg.

5. The human protein C analog of Claim 1 , having one amino acid substitution, wherein Ser at position 3 is substituted with Tyr.

6. The human protein C analog of Claim 1 , having one amino acid substitution, wherein Ser at position 3 is substituted with Phe.

7. The human protein C analog of Claim 1 , having one amino acid substitution, wherein Leu at position 8 is substituted with Trp.

8. The human protein C analog of Claim 1 , having one amino acid substitution, wherein GIn at position 32 is substituted with Met.

9. The human protein C analog of Claim 1 , having one amino acid substitution, wherein Ala at position 264 is substituted with He.

10. The human protein C analog of Claim 1 , having one amino acid substitution, wherein Asn at position 313 is substituted with Lys.

11. The human protein C analog of Claim 1 , having one amino acid substitution, wherein GIn at position 353 is substituted with Arg.

12. The human protein C analog of Claim 1 , having one amino acid substitution, wherein VaI at position 375 is substituted with He.

13. Use of the human protein C analog of any one of Claims 1 to 12 as a medicament.

14. Use of the human protein C analog of any one of Claims 1 to 12 in the manufacture of a medicament for use in the treatment of human thrombotic disease.

15. The human protein C analog of Claim 14 wherein the human thrombotic disease is selected from the group consisting of replacement therapy in the treatment of protein C deficiency, vascular occlusive disorder, and hypercoagulable state.

16. The human protein C analog of Claim 15 wherein the human thrombotic disease is selected from the group consisting of sepsis, disseminated intravascular coagulation, purpura fulminans, major trauma, major surgery, burns, adult respiratory distress syndrome, transplantations, deep vein thrombosis, heparin-induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, meningococcemia, complications during pregnancy, and venous thromboembolic complications associated with bone marrow transplantation, other organ transplantation, surgery, and trauma.

17. Use of the human protein C analog of any one of Claims 1 to 12 in the manufacture of a medicament for use in the treatment of vascular occlusive disorder, arterial thromboembolic disorder, thrombotic disorder, and disease state predisposing to thrombosis.

18. Use of the human protein C analog of any one of Claims 1 to 12 in the manufacture of a medicament for use in the treatment of myocardial infarction, stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery.

19. Use of the human protein C analog of any one of Claims 1 to 12 in the manufacture of a medicament for use in the treatment of acute coronary syndromes.

20. Use of the human protein C analog of any one of Claims 1 to 12 in the manufacture of a medicament for use in the treatment of unstable angina.

21. An activated form of the human protein C analog of any one of Claims 1 to 12.

22. A recombinant DNA molecule comprising a nucleotide sequence encoding the human protein C analog of any one of Claims 1 to 12.

23. A vector comprising the recombinant DNA molecule of Claim 22.

24. A host cell comprising the vector of Claim 23.

25. A pharmaceutical composition comprising the human protein C analog of any one of Claims 1 to 12.

26. A pharmaceutical composition comprising an activated form of the human protein C analog of Claim 25.

27. The pharmaceutical composition of any of Claims 25 or 26 further comprising at least one pharmaceutically acceptable excipient.

28. The pharmaceutical composition of Claim 27 wherein the pharmaceutically acceptable excipient is selected from the group consisting of a bulking agent, a pH buffering agent, a salt, a chelating agent, and a diluent.

29. The pharmaceutical composition of Claim 28 wherein the pharmaceutical composition is lyophilized.

30. A liquid pharmaceutical preparation comprising the preparation obtained by admixing the pharmaceutical composition of any one of Claims 25 to 29 in a pharmaceutically acceptable diluent.