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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6464—Protein C (3.4.21.69)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/745—Blood coagulation or fibrinolysis factors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21069—Protein C activated (3.4.21.69)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to novel polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides. More specifically, the invention relates to protein C derivatives resistant to serpin inactivation, to their production, and to pharmaceutical compositions comprising these protein C derivatives .
• Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of homeostasis by inactivating Factors V a and Villa in the coagulation cascade. Human protein C is made in vivo as a single polypeptide of 461 amino acids.
• 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-dependenr carboxylation of nine glutamic acid residues of the light chain, resulting in nine gamma-carboxyglutamic acid residues; and 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain) .
• 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.
• aPC functions as an anti-coagulant important in protecting against thrombosis, has anti-inflammatory effects through its inhibition of cytokine generation (e.g., TNF and IL-1), and exerts profibrinolytic properties that facilitate clot lysis.
• cytokine generation e.g., TNF and IL-1
• aPC provides a mechanism for anti-coagulation, anti-inflammation, and fibrinolysis .
• Plasma- derived and recombinantly produced aPC have been shown to be effective and safe antithrombotic agents in a variety of animal models of both venous and arterial thrombosis.
• Protein C levels have also been shown to be abnormally low in the following diseases and conditions : 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 (A DS) [Hasegawa, et al .
• DI disseminated intravascular coagulation
• HIT heparin-induced thrombocytopenia
• platelet inhibition is efficacious in both prevention and treatment of thrombotic disease.
• antiplatelet agents such as aspirin
• aPC and antiplatelet agents results in a synergy that allows the reduction of the dosages of both aPC and the antiplatelet agent (s) .
• the reduction of the dosages of the agents in combination therapy in turn results in reduced side effects such as increased bleeding often observed in combination anti- coagulant/anti-platelet therapy.
• a 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.
• serpins Serine Protease Inhibitors
• 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.
• 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(l):48-53, 1990). It has been shown that changes to serine protease amino acid sequences at residues which interact directly with the substrate (generally within or near the active site) can alter the specificity of the serine protease, potentially providing increased specific activity towards appropriate coagulation factors, as well as increased resistance to serpins (Rezaie, J Biol Che 271 (39) : 23807-23814 , 1996; Rezaie and Esmon, Eur. J.
• an aPC polypeptide exhibiting increased resistance to serpin inactivation, while maintaining the desirable biological activities of aPC e.g., anticoagulant, fibrinolytic, and anti-inflammatory activities
• aPC e.g., anticoagulant, fibrinolytic, and anti-inflammatory activities
• PCI protein C inhibitor
• ⁇ -antitrypsin ⁇ -AT
• Both PCI and ⁇ -AT have been demonstrated to be the primary physiological inactivators of aPC in disease states such as disseminated intravascular coagulation (Scully, et al . , Thromb Haemost 69 (5) : 448-53 , 1993)
• elevated levels of ⁇ -AT have been observed in a number of disease states involving an inflammatory response (Somayajulu, et al .
• the present invention describes novel protein C derivatives. These protein C derivatives retain the important biological activity of the wild-type protein C (SEQ ID NO: 7) and have substantially longer half-lives in human blood. Therefore, these compounds provide various advantages, eg. less frequent administration and/or smaller dosages and thus a reduction in the overall cost of production of the therapy. Additionally, these compounds exhibit an advantage in disease states with significantly elevated ⁇ -AT levels such as sepsis. Importantly, the increases in protein C derivative plasma half-lives may be achieved via single amino acid substitutions, which are less likely to be immunogenic in comparison to molecules which contain multiple amino acid substitutions (U.S. Patent No. 5,358,932; Holly, et al . , Biochemistry 33:1876-1880, 1994).
• the present invention provides a protein C derivative comprising SEQ ID NO: 1 and the corresponding amino acids in SEQ ID NO: 2, wherein one or more of amino acids 194, 195,
• the invention further provides the activated form of the above-identified protein C derivatives .
• the present invention also provides recombinant DNA molecules encoding the protein C derivatives in the preceding paragraph, in particular those comprising SEQ ID NOS: 8, 9, and 10.
• Another aspect of the present invention provides protein sequences of these same protein C derivatives, particularly those comprising SEQ ID NOS: 3, 4, and 5 and the activated forms of these protein C derivatives.
• the present invention comprises methods of treating vascular occlusive disorders and hypercoagulable states 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, and hemolytic uremic syndrome.
• the invention further provides treating these same diseases and conditions employing the activated form of the above- identified protein C derivatives.
• Another embodiment of the present invention is a method of treating sepsis comprising the administration to a patient in need thereof a pharmaceutically effective amount of a protein C derivative of this invention in combination with bacterial permeability increasing protein.
• the present invention comprises methods of treating acute coronary syndromes such as myocardial infarction and unstable angina.
• the present invention further comprises methods of treating thrombotic disorders.
• thrombotic disorders include, but are not limited to, stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery.
• the present invention also provides a pharmaceutical composition comprising a protein C derivative of this invention.
• Human protein C derivatives for the above-mentioned indications and pharmaceutical compositions are preferably selected from L194S, L194S:T254S, and L194A:T254S.
• an aspect of the invention comprises treating the diseases and conditions caused or resulting from protein C deficiency as defined herein, by inhibiting binding to inhibitor recognition sequences S2 , S3', and S4 ' of the serpins, PCI and ⁇ -AT.
• This final aspect of the invention contemplates any and all modifications to any aPC molecule resulting in inhibition of the binding to said inhibitor recognition sequences of the serpins PCI and ⁇ _-AT.
• the inhibition of binding to the specific inhibitor recognition sequences of the serpins (S2, S3', and S4 ' ) being an important contribution to this aspect of the invention.
• Figure 1 Inactivation of human aPC polypeptides during incubation with normal human plasma.
• Activated protein C levels were determined using immunocapture assay, and compared to a standard curve generated from dilutions of the purified protein in rabbit plasma; the standard curve ranged from 1 to 250 ng/mL, with the calculated values within 10% of the standard samples. Data are shown for the wild-type protein (WT, circles) , T254S (squares) , L194S (triangles) , and L194S/T254S (diamonds) . The values plotted are the mean and standard error for the three animals. For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below. Antiplatelet agent - one or more agents alone or in combination which reduces the ability of platelets to aggregate.
• Agents understood and appreciated in the art include those cited in, for example, Remington, The Science and Practice of Pharmacy, Nineteenth Edition, Vol II, pages 924-25, Mack Publishing Co., herein incorporated by reference. Such agents include but are not limited to aspirin (ASA) , clopidogrel, ReoPro® (abciximab) , dipyridamole, ticlopidine and Ilb/lIIa antagonists.
• ASA aspirin
• clopidogrel ReoPro®
• abciximab dipyridamole
• ticlopidine ticlopidine
• Ilb/lIIa antagonists e.g., aPC or activated protein C refers to recombinant aPC .
• aPC includes and is preferably recombinant human aPC although aPC may also include other species having protein C proteolytic, amidolytic, esterolytic, and biological (anti- coagulant, anti-inflammatory, or pro-fibrinolytic) activities .
• Protein C derivative (s) refers to the recombinantly produced polypeptides of this invention that differ from wild-type human protein C but when activated retain the essential properties i.e., proteolytic, amidolytic, esterolytic, and biological (anti-coagulant, anti- inflammatory, pro-fibrinolytic activities) .
• the definition of protein C derivatives as used herein also includes the activated form of the above identified protein C derivatives . Treating - describes the management and care of a patient for the purpose of combating a disease, condition, or disorder whether to eliminate the disease, condition, or disorder, or prophylactically to prevent the onset of the symptoms or complications of the disease, condition, or disorder.
• Bolus injection the injection of a drug in a defined quantity (called a bolus) over a period of time up to about
• Unit dosage form - refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient .
• Hypercoagulable states - excessive coagulability associated with disseminated intravascular coagulation, pre- thrombotic conditions, activation of coagulation, or congenital or acquired deficiency of clotting factors such as aPC.
• Zymogen - protein C zymogen refers to secreted, inactive forms, whether one chain or two chains, of protein C .
• compositions comprising: a pharmaceutically effective amount - a therapeutically efficacious amount of a pharmaceutical compound.
• the particular dose of the compound administered according to this invention will, of course, be determined by the attending physician evaluating the particular circumstances surrounding the case, including the compound administered, the particular condition being treated, the patient characteristics and similar considerations.
• Acute coronary syndromes clinical manifestations of coronary atherosclerosis complicated by coronary plaque rupture, superimposed coronary thrombosis, and jeopardized coronary blood flow resulting in coronary ischemia and/or myocardial infarction.
• the spectrum of acute coronary syndromes includes unstable angina, non-Q-wave (i.e., non- ST-segment elevation) myocardial infarction, and Q-wave (i.e., ST-segment elevation) myocardial infarction.
• Thrombotic disorders a disorder relating to, or affected with the formation or presence of a blood clot within a blood vessel. Such disorders include, but are not limited to, stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery.
• Serpin any of a group of structurally related proteins that typically are serine protease inhibitors whose inhibiting activity is conferred by an active site in a highly variable and mobile peptide loop and that include but are not limited to protein C inhibitor (PCI) and ⁇ - antitrypsin ( ⁇ ]_-AT) .
• PCI protein C inhibitor
• ⁇ ]_-AT ⁇ - antitrypsin
• Inhibitor recognition sequence S2 the 2 nd residue N-terminal to the cleavage site of PCI or ⁇ -AT.
• Inhibitor recognition sequence S3' the 3 rd residue C-terminal to the cleavage site of PCI or ⁇ -AT.
• Inhibitor recognition sequence S4 ' the 4 th residue C-terminal to the cleavage site of PCI or ⁇ -AT.
• Wild-type protein C the type of protein C that predominates in a natural population of humans in contrast to that of natural or laboratory mutant or polypeptide forms of protein C.
• Bactericidal permeability increasing protein includes naturally and recombinantly produced bactericidal permeability increasing (BPI) protein; natural, synthetic, and recombinant biologically active polypeptide fragments of BPI protein; biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins and dimers; biologically active variant analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs; and BPI-derived peptides.
• BPI bactericidal permeability increasing
• the complete amino acid sequence of human BPI, as well as the nucleotide sequence of DNA encoding BPI have been elucidated by Gray, et al . , 1989, J. Biol. Chem 264:9505.
• Recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI are disclosed in U.S. Patent No. 5,198,541, herein incorporated by reference .
• the activated form of aPC or isolated human aPC polypeptides may be produced by activating recombinant human protein C zymogen or recombinant protein C derivative zymogen in vitro or by direct secretion of the activated form of protein C.
• the means by which the activation occurs is not critical and the process aspects of this invention include any and all means of activation.
• Protein C derivatives may be produced in 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.
• the present invention provides protein C derivatives, including activated forms thereof, which have increased resistance to serpins, and consequently result in extended plasma half-lives. Specific protein C derivatives include L194S, L194S:T254S, and L194A:T254S and activated forms thereof .
• Protein C derivative L194S preferably contains a serine residue at position 194 rather than a leucine residue normally found at this position.
• amino acid substitutions at residue 194 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, and Gin.
• the activated form of protein C derivative L194S demonstrates prolonged half-life in plasma ( Figure 1) and increased resistance to serpins, for example, ⁇ _-antitrypsin ( ⁇ -AT) , Figure 4.
• Protein C derivative L194S:T254S preferably contains a serine residue at position 194 rather than a leucine residue normally found at this position and a serine residue at position 254 rather than a threonine residue normally found at this position. It is apparent to one with skill in the art that other amino acid substitutions at residues 194 and 254 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gin, and Gly, provided that amino acid 254 is not substituted with Thr.
• the activated form of human protein C derivative L194S:T254S demonstrates a prolonged half-life in normal human plasma compared to wild- type protein C, Figure 2.
• Protein C derivative L194A:T254S preferably contains an alanine residue at position 194 rather than a leucine residue normally found at this position and a serine residue at position 254 rather than a threonine residue normally found at this position. It is apparent to one with skill in the art that other amino acid substitutions at residues 194 and 254 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gin, and Gly, provided that amino acid 254 is not substituted with Thr.
• the activated form of human protein C derivative L194A:T254S demonstrates a prolonged half-life in normal human plasma compared to wild-type protein C, Figure 2.
• Further embodiments of the present invention include protein C derivatives: L194T, L194A, A195G, L228Q, T254S, F316N, Y249E, and Y302Q, and activated forms thereof which have increased resistance to serpins.
• Protein C derivatives L194T or L194A preferably contain a threonine residue or an alanine residue at position 194 rather than a leucine residue normally found at this position.
• amino acid substitutions at residue 194 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, His, Lys, Arg, Asn, Asp, Glu, and Gin.
• Protein C derivative A195G preferably contains a glycine residue at position 195 rather than an alanine residue normally found at this position.
• amino acid substitutions at residue 195 in addition to Gly may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, and Gin.
• the activated form of protein C derivative A195G demonstrates prolonged half-life in plasma ( Figure 1) and increased resistance to serpins, for example, a ] _-antitrypsin ( ⁇ -AT) , Figure 4.
• Protein C derivative L228Q preferably contains a glutamine residue at position 228 rather than a leucine residue normally found at this position.
• amino acid substitutions at residue 228 in addition to Gin may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, and Gly.
• Protein C derivative T254S preferably contains a serine residue at position 254 rather than a threonine residue normally found at this position. It is apparent to one with skill in the art that other amino acid substitutions at residue 254 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gin, and Gly, provided that amino acid 254 is not substituted with Thr.
• the activated form of protein C derivative T254S demonstrates prolonged half-life in plasma (Figure 1) and increased resistance to serpins, for example, ⁇ -antitrypsin ( ⁇ -AT) , Figure 4.
• Protein C derivative F316N preferably contains an asparagine residue at position 316 rather than a p enylalanine residue normally found at this position.
• amino acid substitutions at residue 316 in addition to Asn may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asp, Glu, Gin, and Gly.
• Protein C derivative Y249E preferably contains a glutamic acid residue at position 249 rather than a tyrosine residue normally found at this position.
• An additional polypeptide contains an Asp at position 249 rather than a tyrosine residue.
• amino acid substitutions at residue 249 in addition to Glu and Asp may impart increased resistance to serpins in the resulting polypeptide molecule.
• amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asn, Gin, and Gly.
• Protein C derivative Y302Q preferably contains a glutamine residue at position 302 rather than a tyrosine residue normally found at this position.
• An additional polypeptide contains a Glu at position 302 rather than a tyrosine residue.
• amino acid substitutions at residue 302 in addition to Glu or Gin may impart increased resistance to serpins in the resulting polypeptide molecule.
• amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, and Gly.
• protein C derivatives may include proteins that represent functionally equivalent gene products.
• Such an equivalent protein C derivative may contain deletions, additions, or substitutions of amino acid residues within the amino acid sequence encoded by the protein C polypeptide gene sequences described above, but which result in a silent change, thus producing a functionally equivalent protein C derivative gene product.
• Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
• polypeptides of the present invention include polypeptides having an amino acid sequence at least identical to that of SEQ ID NOS: 3, 4, or 5, or fragments thereof with at least 90% identity to the corresponding fragment of SEQ ID NOS: 3, 4, or 5.
• all of these polypeptides retain the biological activity of human aPC.
• Preferred polypeptides are those that vary from SEQ ID NOS: 3, 4, or 5 by conservative substitutions i.e., those that substitute a residue with another of like characteristics. Typical substitutions are among Ala, Val , Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues
• the invention also provides DNA compounds for use in making the protein C derivatives. These DNA compounds comprise the coding sequence for the light chain of human protein C zymogen or protein C derivative zymogen positioned immediately adjacent to, downstream of, and in translational reading frame with the prepropeptide sequence of human protein C zymogen or protein C derivative zymogen.
• 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 protein C derivative .
• DNA compounds of the present invention were prepared by the use of site-directed mutagenesis to change particular positions within human protein C zymogen. The methods used for the identification of residues which form critical contacts in these particular positions are described in Example 1.
• the protein C derivatives 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-MK 2 , CHO-K1, 293, or AV12 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 (Invitrogen) ; and, pIRES/Hygro, and pIRES/neo (Clonetech) .
• the preferred vector of the present invention is pIG3 as described in Example 2.
• the protein C derivatives made by any of these methods must undergo post-translational modifications such as the addition of nine 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) .
• post-translational modifications such as the addition of nine 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) .
• post-translational modifications such as the addition of nine gamma-carboxy-glutamates, the addition of one erythro-beta-hydroxy-Asp (beta- hydroxylation) , the addition
• Human protein C may be activated by thrombin alone, by a thrombin/thrombomodulin complex, by RW-X, a protease from Russell's Viper venom, by pancreatic trypsin or by other proteolytic enzymes.
• the recombinant protein C derivatives of the present invention are useful for the treatment of vascular occlusive disorders or hypercoagulable states associated with sepsis, disseminated intravascular coagulation, 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.
• the recombinant protein C derivatives of the present invention are useful for the treatment of sepsis in combination with bacterial permeability increasing protein.
• the activated protein C derivatives of the present invention are combined with an antiplatelet agent (s) to treat or prevent various disorders, such as, thrombotic disease .
• the present invention further provides for the treatment of acute coronary syndromes comprising myocardial infarction, and unstable angina with human protein C derivatives with resistance to serpin inactivation as compared to wild-type aPC.
• the recombinant human protein C derivatives of the present invention are also useful for the treatment of thrombotic disorders such as stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery.
• the protein C derivatives can be formulated according to known methods to prepare a pharmaceutical composition comprising as the active agent an aPC polypeptide and a pharmaceutically acceptable solid or carrier.
• a desired formulation would be one that is a stable lyophilized product of high purity comprising a bulking agent such as sucrose, a salt such as sodium chloride, a buffer such as sodium citrate and an activated protein C derivative.
• a preferred stable lyophilized formulation comprises: 2.5 mg/ml activated protein C polypeptide, 15 mg/ml sucrose, 20 mg/ml NaCl and a citrate buffer, said formulation having a pH of 6.0.
• An additional stable lyophilized formulation comprises: 5.0 mg/ml activated protein C polypeptide, 30 mg/ml sucrose, 38 mg/ml NaCl and a citrate buffer, said formulation having a pH of 6.0.
• the human aPC polypeptides will be administered parenterally to ensure delivery into the bloodstream in an effective form by injecting the appropriate dose as a continuous infusion for 1 to 240 hours. More preferably, the human aPC polypeptides will be administered as a continuous infusion for 1 to 192 hours. Even more preferably, the human aPC polypeptides will be administered as a continuous infusion for 1 to 144 hours. Yet even more preferably, the aPC polypeptides will be administered as a continuous infusion for 1 to 96 hours.
• the amount of human aPC polypeptide administered will be from about 0.01 ⁇ g/kg/hr to about 50 ⁇ g/kg/hr. More preferably, the amount of human aPC polypeptide administered will be about 0.1 ⁇ g/kg/hr to about 25 ⁇ g/kg/hr. Even more preferably the amount of human aPC polypeptide administered will be about 1 ⁇ g/kg/hr to about 15 ⁇ g/kg/hr. The most preferable amounts of human aPC polypeptide administered will be about 5 ⁇ g/kg/hr or about 10 ⁇ g/kg/hr.
• the human aPC polypeptide will be 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 .
• the human aPC derivatives will be administered by injecting a dose of 0.01 mg/kg/day to about 1.0 mg/kg/day, B.I.D. (2 times a day), for one to ten days. More preferably, the human aPC derivatives will be administered B.I.D. for three days.
• the human aPC polypeptides will be administered subcutaneously to ensure a slower release into the bloodstream.
• Formulation for subcutaneous preparations will be done using known methods to prepare such pharmaceutical compositions.
• An additional aspect of the invention comprises treating the diseases and conditions caused or resulting from protein C deficiency as defined herein, by inhibiting binding to inhibitor recognition sequences S2 , S3', and S4 ' of the serpins, PCI and ⁇ _-AT, as described in Example 1.
• This final aspect of the invention contemplates any and all modifications to any aPC molecule resulting in inhibition of the binding to said inhibitor recognition sequences of the serpins PCI and ⁇ -AT.
• the human aPC polypeptides described in this invention have essentially the same type of biological activity as the wild-type human aPC, with substantially longer half-lives in human blood. Therefore, these compounds will require either less frequent administration and/or smaller dosage.
• Table I depicts the sequences recognized by aPC.
• the cleavage site occurs between the two residues shown in italics. Residues occupying the specific subsites, S2, S3', and S4 ' , are underlined.
• the recognized sites in factor Va are different from the sites in either factor Villa or the inhibitors, therefore, it is possible to engineer the active site of aPC to preferentially cleave the more critical coagulant factor Va, while at the same time decrease aPC's likelihood of being inhibited by serpins.
• S2 the 2 nd residue N-terminal to the cleavage site
• S3' site the S3' site
• S4' the S2 site
• the S2 site is primarily occupied by polar residues in the factor Va sequences; unlike PCI and ⁇ -AT, which have hydrophobic residues at this position.
• the S3' site occupied by polar side chains in all of the substrate sequences, but notably, a hydrophobic side chain in the ⁇ - AT sequence.
• the S4' site is occupied by charged residues in all three factor Va sequences, but is occupied by hydrophobic residues in the factor Villa and inhibitor sequences .
• Protein C Polypeptide Construction and Production Protein C derivatives were constructed using the polymerase chain reaction (PCR) following standard methods.
• the source of the wild-type coding sequence was plasmid pLPC (Bio/Technology 5:1189-1192, 1987).
• the universal PCR primers used include: PCOOlb; 5'-
• GCGATGTCTAGAccaccATGTGGCAGCTCACAAGCCTCCTGC -3' which encodes for an Xbal restriction site (underlined) used for subcloning, a Kozak consensus sequence (lowercase) (Kozak, J Cell Biol 108 (2) .229-41, 1989), and the 5' end of the coding region for protein C: PC002E; 5'-
• CAGGGATGATCACTAAGGTGCCCAGCTCTTCTGG-3 ' which encodes for the 3' end of the coding region for human protein C, and includes a Bell restriction site (underlined) for subcloning.
• Mutagenic PCR primers include: PC194SF, 5'- CTCAAAGAAGAAGTCCGCCTGCGGGGCAGTGC-3 ' and PCI94SR, 5 ' - GCACTGCCCCGCAGGCGGACTTCTTCTTTGAG-3' which encode for a Leu (CTG) to Ser (TCC) mutation at position 194 (boldfaced type) ; PCA195GF , 5 ' -GAAGAAGCTGGGGTGCGGGGCAGTGC-3 ' , and PCA195GR, 5' -GCACTGCCCCGCACCCCAGCTTCTTC-3 ' , which encode for a Ala(GCC) to Gly(GGG) mutation; PCT254SF, 5'- GC
• the first round of PCR was used to amplify two fragments of the protein C gene; the 5' fragment was generated using PCOOlb and the antisense mutagenic primer, and the 3' fragment was generated using PC002e and the sense mutagenic primer.
• the resulting amplified products were purified by standard procedures. These fragments were combined and then used as a template for a second round of PCR using primers PCOOlb and PC002e.
• the final PCR product was digested with Xbal and Bell and subcloned into similarly digested expression vector pIG3.
• a wild-type construct was similarly generated by PCR using the two universal primers and the plasmid pLPC as the template, followed by subcloning into pIG3.
• the mutations were confirmed by DNA sequencing of both the coding and non-coding strands.
• the completed expression plasmids were designated pIG3-HPC (wild-type protein C) , pGH41 (T254S) , pGH51 (A195G) , and pGH94 (L194S) .
• the pIG3 vector was generated by the insertion of an "internal ribosome entry site" (IRES) (Jackson, et al . , Trends Biochem Sci 15 (12) : 447-83 , 1990) and green fluorescent protein (GFP) (Cormack, et al . , Gene 173:33-38, 1996) gene into the mammalian expression vector pGTD (Gerlitz,et al . , Biochem J 295(Pt l):131-40, 1993).
• IRS internal ribosome entry site
• GFP green fluorescent protein
• the GBMT promoter (Berg, Nucleic Acids Res 20(20) : 5485-6, 1992) drives expression of a bicistronic mRNA (5'- cDNA - IRES - GFP - 3'). Efficient translation of the first cistron is initiated by classical assembly of ribosome subunits on the 5' -methylated cap structure of the mRNA; while the normally inefficient translation of a second cistron is overcome by the IRES sequence which allows for internal ribosome assembly on the mRNA.
• the coupling of the cDNA and reporter on a single mRNA, translated as separate proteins, allows one to screen for the highest-producing clones on the basis of fluorescence intensity.
• the expression vector also contains an ampicillin resistance cassette for maintenance of the plasmid in E. coli , and a murine DHFR gene with appropriate expression sequences for selection and amplification purposes in mammalian tissue expression.
• the adenovirus-transformed Syrian hamster AV12-664 cell line was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 ⁇ g/mL gentamicin, 200 ⁇ g /mL Geneticin (G418) , and 10 ⁇ g /mL vitamin Kl .
• Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 ⁇ g/mL gentamicin, 200 ⁇ g /mL Geneticin (G418) , and 10 ⁇ g /mL vitamin Kl .
• G418 ⁇ g /mL
• Fspl- linearized plasmids were transfected using either the calcium phosphate method (ProFection, Gibco BRL-Life Technologies) or FuGene-6 (Boehringer Mannheim) , following the manufacturer's instructions.
• Example 3 Activation of Recombinant Protein C Complete activation of the zymogen forms of protein C and polypeptides was accomplished by incubation with thrombin-sepharose . Thrombin-sepharose was washed extensively with Buffer A. 200 ⁇ L of packed thrombin- sepharose was mixed with 250 ⁇ g of protein C in 1 mL of the same buffer and incubated at 37 °C for 4 hours with gentle shaking on a rotating platform.
• the degree of protein C activation was monitored by briefly pelleting the thrombin-sepharose, and assaying a small aliquot of the supernatant for aPC activity using the chromogenic substrate S-2366 (DiaPharma) . Following complete activation, the thrombin-sepharose was pelleted, and the supernatant collected. aPC concentration was verified by Pierce BCA assay, and the aPC was either assayed directly, or frozen in aliquots at -80°C.
• Assays were performed at 25 °C, in Buffer A containing 1 mg mL "1 BSA, 3 mM CaCl 2 , and 0.5 nM aPC . Reactions (200 ⁇ L/well) were performed in a 96 -well microtiter plate, and amidolytic activity was measured as the change in absorbance units/min at 405 nm as monitored in a ThermoMax kinetic micrometer plate reader. Kinetic constants were derived by fitting velocity data at varying substrate concentrations (16 ⁇ M to 2 mM) to the Michaelis-Menten equation.
• Example 5 Inactivation of aPC Polypeptides
• the rates of inactivation of aPC polypeptides were determined by incubating normal human plasma (Helena Labs) with 20 nM aPC (or either polypeptide) at 37°C ( Figure 1) .
• Plasma concentration was 90% (v/v) in the final reaction buffer containing 150 mM NaCl, 20 mM Tris, pH 7.4, and 1 mg mL "1 BSA. Aliquots were removed at selected times, and activity was measured as amidolytic activity using S-2366 at a final concentration of ImM. The measured half-lives are summarized in Table IV.
• heparin (10 U mL " 1 ) , which is known to cause about 100- fold stimulation in the inactivation of aPC by PCI (Heeb, et al . , J Biol Chem 263(24) .11613-11616, 1988; Espana, et al . , Thromb Res 55 (3) :369-84, 1989; Aznar, et al . , Thromb Haemost 76 (6) :983-988, 1996), was added to a similar reaction ( Figure 3) .
• ⁇ -antitrypsin ⁇ -AT
• ⁇ -AT ⁇ -antitrypsin
• reaction buffer consisting of 3 mM CaCl 2 , 150 mM NaCl, 20 mM Tris, pH 7.4, and 1 mg mL "1 BSA. Aliquots were removed at selected times, and activity was measured as amidolytic activity using S-2366 at a final concentration of ImM.

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