WO2003106666A2 - Protein c variants with altered properties - Google Patents

Protein c variants with altered properties Download PDF

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Publication number
WO2003106666A2
WO2003106666A2 PCT/DK2003/000392 DK0300392W WO03106666A2 WO 2003106666 A2 WO2003106666 A2 WO 2003106666A2 DK 0300392 W DK0300392 W DK 0300392W WO 03106666 A2 WO03106666 A2 WO 03106666A2
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use according
amino acid
variant
substitutions
protein
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PCT/DK2003/000392
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French (fr)
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WO2003106666A3 (en
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Bobby Soni
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Maxygen Aps
Maxygen Holdings Ltd.
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Priority to AU2003232176A priority Critical patent/AU2003232176A1/en
Publication of WO2003106666A2 publication Critical patent/WO2003106666A2/en
Publication of WO2003106666A3 publication Critical patent/WO2003106666A3/en

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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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4866—Protein C (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P31/04—Antibacterial agents
    • 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)

Definitions

  • the present invention relates to protein C variants which comprise at least one modification in the loop region composed of positions 306-314 for the treatment of hypercoagulable states or acquired protein C deficiency, such as sepsis.
  • Such variants have a reduced anticoagulant activity and a similar anti-inflammatory effect as compared to the corresponding human activated protein C (hAPC) polypeptide.
  • Blood coagulation is a process involving a complex interaction of various blood components, or factors, which eventually give rise to a fibrin clot.
  • blood components participating in the coagulation "cascade” are proenzymes or zymogens, i.e. enzymatically inactive proteins that are converted into an active form by action of an activator.
  • Regulation of blood coagulation is accomplished enzymatically by proteolytic inactivation of the procoagulation factors Va and Villa achieved by activated protein C (Esmon, JBiol Chem 1989; 264; 4743-4746).
  • Protein C is a serine protease that circulates in the plasma as a zymogen with a half-life of approximately 7 hours, and plasma levels are typically in the range of 3-5 ⁇ g/1. It is produced in vivo in the liver as a single chain precursor polypeptide of 461 amino acids.
  • the protein C precursor comprises a 42 amino acid residue signal and propeptide sequence that includes a conserved 18 amino acid propeptide sequence found in all vitamin Independent proteins (Stanley et al., Biochemistry (1999) 38:15681-7).
  • This precursor polypeptide undergoes multiple post-translational modifications, including a) cleavage of the signal sequence and the propeptide sequence; b) cleavage of lysine and arginine residues (positions 156 and 157) to make a two-chain inactive zymogen (a 155 amino acid light chain attached via a disulfide bridge to a 262 amino acid heavy chain); c) vitamin Independent carboxylation of nine glutamic acid residues of the light chain resulting in nine gamma-carboxyglutamic acid residues in the N-terminal region of the light chain; and d) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain).
  • the two-chain zymogen may be activated by removal of a dodecapeptide (the activation peptide) at the N-terminus of the heavy chain (positions 158-169), producing the activated protein C (APC).
  • the activation peptide is activated by limited proteolysis by thrombin in complex with throm- bomodulin on the lumenal surface of the endothelial cell. As explained above, activation liberates a 12 amino acid activation peptide from the N-terminal of the heavy chain.
  • the APC has a half-life of approximately 15 minutes in plasma.
  • C4b-binding protein In the presence of its cofactor, protein S, APC proteolytically inactivates factors Va and Villa, thereby reducing thrombin generation (Esmon, Thromb Haemost 1993; 70; 29- 35). Protein S circulates reversibly bound to another plasma protein, C4b-binding protein. Only free protein S serves as a cofactor for APC. Since C4b-binding protein is an acute phase reactant, the plasma levels of this protein vary greatly in many diseases and thus in- fluence the anticoagulant activity of the protein C system.
  • the gene encoding human protein C maps to chromosome 2ql3-ql4 (Patracchini et al., Hum Genet 1989; 81; 191-192), spans over 11 kb, and comprises a coding region (exons II to IX) and a 5' untranslatable region encompassing exon I.
  • the protein domains encoded by exons II to IX show considerable homology with other vitamin K-dependent coagulation proteins such as factor IX and X.
  • Exon II codes for a signal peptide
  • exon III codes for a propeptide and a 38 amino acid sequence containing 9 Glu residues.
  • the propeptide contains a binding site for the carboxylase that transforms the Glu residues into dicarboxylic acid (Gla) able to bind calcium ions, a step required for phospholipid binding (Cheung et al., Arch Biochem Biophys 1989; 274; 574-581).
  • Exons IV, V and VI encode a short connection sequence and two EGF-like domains, respectively.
  • Exon VII encodes both a domain encompassing the 12 amino acid activation peptide and the dipeptide 156-157 which, when cleaved off, yields the mature two-chain form of the protein.
  • Exons VIII and IX encode the serine protease domain.
  • APC is inhibited in plasma by the protein C inhibitor as well as by alpha- 1- antitrypsin and alpha-2-macroglobulin.
  • the experimental three-dimensional structure of human APC (in a Gla-domainless form) has been determined to 2.8 A resolution and reported by Mather et al., EMBO J 1996; 15; 6822-6831.
  • the structure includes a covalently bound inhibitor (D-Phe-Pro-Arg chloromethylketone, PPACK).
  • APC is used for the treatment of genetic and acquired protein C deficiency and has been suggested for use as an anticoagulant in patients with some forms of lupus, following stroke or myocardial infarction, after venous thrombosis, disseminated intravascular coagulation (DIC), septic shock, emboli such as pulmonary emboli, transplantation, such as bone marrow transplantation, burns, pregnancy, major surgery/trauma and adult respiratory stress syndrome (ARDS).
  • DIC disseminated intravascular coagulation
  • emboli such as pulmonary emboli
  • transplantation such as bone marrow transplantation
  • Recombinant APC is produced by Eli Lilly and Co. and is marketed under the name Xigris®.
  • PEGylated wild-type APC is described in JP 8-92294.
  • WO 91/09960 discloses a hybrid protein comprising modifications in the heavy chain part of protein C.
  • WO 00/66754 reported that substitution of the residues naturally occurring in the positions 194, 195, 228, 249, 254, 302 or 316 lead to an increased half-life of APC in human blood as compared to the wild-type APC.
  • WO 99/20767 and WO 00/66753 disclose vitamin K-dependent polypeptide variants containing modifications in the Gla domain.
  • WO 98/44000 broadly describes protein C variants with an increased amidolytic activity.
  • US 5,453,373 discloses human protein C derivatives which have altered glycosyla- tion patterns and altered activation regions, such as N313Q and N329Q.
  • US 5,460,953 discloses DNA sequences encoding zymogen forms of protein C which have been engineered so that one or more of the naturally occurring glycosylation sites have been removed. More specifically, US 5,460,953 discloses the variants N97Q, N248Q, N313Q and N329Q. Conjugated protein C variants, e.g. with one or more introduced glycosylation sites, are disclosed in WO 02/32461.
  • Xigris® One major problem associated with use of hAPC (Xigris®) in the treatment of sepsis is the severe bleeding risks associated therewith. Accordingly, Xigris® has been approved by the United States Food and Drug Administration (FDA) only for "adult patients with severe sepsis who have a high risk of death (at least one dysfunctional major organ, APACHE II score above 25) with low risk of bleeding complications". As a result, a large group of sepsis patients with a high bleeding risk are excluded from treatment with Xigris®.
  • FDA United States Food and Drug Administration
  • a modified product which is suitable for treatment of sepsis, but which is safe to use, i.e. which has a reduced anticoagulant activity and thus is available to sepsis patients with a significant bleeding risk but which at the same time retains its anti- inflammatory properties.
  • a direct role of activated protein C in the prevention of apoptosis has recently been reported (Cheng et al., Nature Medicine 9(3):338-42, 2003). Cheng et al. found that APC directly prevents apoptosis in hypoxic human brain endothelium, and that this is not merely a secondary effect to its anti-coagulant and anti-inflammatory effects but rather is independent of its anti-coagulant effect. Further, they found that the neuroprotective effect of APC is
  • EPCR endothelial protein C receptor
  • PAR-1 pro- tease-activated receptor-1
  • the present invention is based on modifications in the autolysis loop of protein C
  • variants open up the possibility for treatment of diseases and conditions in which an anti-inflammatory activity is required, such as in the treatment of acquired hypercoagulable states or acquired protein C deficiency, in particular sepsis, since the important anti-inflammatory properties are substantially retained, and the product is safe to use due to the significantly reduced anticoagulant properties of the variants.
  • the anti- apoptotic effect of APC is not believed to be related to its anti-coagulant activity, as suggested by Cheng et al. (2003), it is contemplated that the variants of the invention will sub- stantially maintain the anti-apoptotic properties of activated protein C despite having reduced anticoagulant properties.
  • an object of the present invention is to provide a safer protein C polypeptide, i.e. a protein C polypeptide with a significantly reduced anticoagulant activity but which substantially retains the anti-mflammatory and/or anti-apoptotic properties of hAPC, and which is suitable for treatment of various diseases such as sepsis.
  • the present invention relates to the use of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2 for the manufacture of a medicament for the treatment of a hypercoagulable state or acquired protein C deficiency, in particular when the hypercoagulable state or protein C deficiency is associated with sepsis.
  • the present invention relates to a method of treating a patient with a hypercoagulable state or acquired protein C deficiency, in particular where the hyperco- agulable state or protein C deficiency is associated with sepsis, which comprises administering to said patient an effective amount of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2.
  • the present invention relates to a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2, with the proviso that the variant is not human protein C or hAPC comprising the following modifications: R306A, E307A, K308A, E309A, K311A, R312A, R314A, R306A+R312A, R306A+K311A+R312A+R314A, H303*+S304*+S305*+K308*+E307D+A310T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, K308N+A310S, K308N+A310T, E309N+K311
  • R312N+R314T R306C, E307C, K308C, E309C, A310C, and R312C, and that the variant does not contain a mutation in position 313.
  • the present invention relates to pharmaceutical compositions comprising the variants of the invention; to variants of the invention or pharmaceutical composi- tions of the invention for use as a medicament; to nucleotide sequences encoding the variants of the invention; to expression vectors comprising the the nucleotide sequences of the invention; to host cells comprising the nucleotide sequences of the invention or the expression vectors of the invention; and to methods for preparing the variants of the invention.
  • precursor protein C or "precursor human protein C” refers to the DNA-encoded form of protein C, i.e. it includes the signal peptide and propeptide (residues -42 to -1), the light chain (residues 1-155), the Lys- Arg dipeptide (residues 156-157) and the heavy chain (residues 158-419), including the activation peptide (residues 158-169), shown in SEQ ID NO:2.
  • one-chain zymogen protein C or “one-chain zymogen human protein C refers to the one-chain inactive form of protein C, which includes the light chain (residues 1-155), the heavy chain (residues 158-419) including the activation peptide (residues 158-169), and the Lys-Arg dipeptide (residue 156-157) shown in SEQ ID NO:2.
  • two-chain zymogen protein C or “two-chain zymogen human protein C” refers to the two-chain inactive form of protein C, which includes the light chain (residues 1 -155) and the heavy chain (residues 158-419) including the activation peptide (residues 158-169), shown in SEQ ID NO:2.
  • zymogen protein C or “zymogen human protein C” refers to both the one-chain form and the two-chain form of the zymogen protein C.
  • activated protein C or “activated human protein C” refers to both the one-chain form and the two-chain form of the zymogen protein C.
  • activated protein C or “activated human protein C” refers to both the one-chain form and the two-chain form of the zymogen protein C.
  • activated protein C activated human protein C
  • APC activated human protein C
  • hAPC hAPC
  • protein C or "human protein C” is intended to encompass all of the above-mentioned forms of protein C, i.e. the "precursor protein C” form, the "zymogen protein C” form (the one-chain form as well as the two-chain form) and the "activated protein C form".
  • the “autolysis loop”, as used herein, refers to amino acid residues 306-314 of SEQ ID NO:2.
  • the "Gla domain”, as used herein, refers to amino acid residues 1-45 of SEQ ID NO:2.
  • the “EGF domains”, as used herein, refers to amino acid residues 55-134 of SEQ ID NO:2.
  • the "active site region” is defined as including those amino acid residues that are described as belonging to the active site in WO 02/32461, namely: L170, 1171, D172, G173, Q184, V185, V186, L187, L188, D189, S190, K191, K192, K193, L194, A195, C196, G197, A198, T208, A209, A210, H211, C212, M213, D214, E215, S216, K217, K218, L219, L220, L228, 1240, V243, V245, N248, Y249, S250, K251, S252, T253, T254, D255, N256, D257, 1258, A259, L261, T295, L296, V297, T298, G299, W300, G301, Y302,
  • variant is intended to cover a polypeptide which differs in one or more amino acid residues from human protein C, in particular from hAPC.
  • a variant differs from human protein C or hAPC (SEQ ID NO:2) in 1-15 amino acid residues, such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, e.g. in 1-10 amino acid residues, 1-7 amino acid residues or 1-5 amino acid residues.
  • the variants disclosed herein must contain at least one amino acid modification in the loop region constituted by amino acid residues 306-314. However, other modifications outside this region may also be present.
  • the term "differs" or “differs from” when used in connection with specific modifi- cations is intended to allow for additional differences being present apart from the specified amino acid difference.
  • the variant in addition to the modifications of amino acid residues located in the autolysis loop, the variant can comprise other substitutions, insertions or deletions which are not related to the modification of amino acid residues present in the autolysis loop. Such other alterations may e.g. be modifications performed in the active site region aimed at increasing the functional half-life of the variant.
  • Other examples include truncation of the N- and/or C-terminus by one or more amino acid residues, or addition of one or more extra residues at the N- and/or C-terminus, e.g.
  • conservative substitutions include amino acids within the respective groups listed in the table below.
  • substitution and “substitution” are used interchangeably herein.
  • modified or “modification” includes a substitution, an insertion or a deletion.
  • introduction is primarily intended to mean substitution of an existing amino acid residue, but may also mean insertion of an additional amino acid residue.
  • conjugated is primarily intended to mean substitution of the amino acid residue to be removed by another amino acid residue, but may also mean deletion (without substitution) of the amino acid residue to be removed.
  • conjugated polypeptide or conjugated variant
  • conjugated variant is intended to indicate a heterogenous (in the sense of composite or chimeric) molecule formed by the covalent attachment of a variant to one or more non-polypeptide moieties such as polymer molecules, lipophilic compounds, sugar moieties or organic deri- vatizing agents.
  • the conjugated variant is soluble at relevant concentrations and conditions, i.e. soluble in physiological fluids such as blood. Examples of conjugated variants include glycosylated variant polypeptides and PEGylated variant polypeptides.
  • covalent attachment means that the variant polypeptide and the non-polypeptide moiety are either directly covalently joined to one another or are indirectly covalently joined to one another through an intervening moiety or moieties such as a bridge, spacer or linker moiety or moieties.
  • non-polypeptide moiety refers to a non-polypeptide molecule that is capable of conjugating to an attachment group of the polypeptide. Preferred examples of such molecules include polymer molecules and sugar moieties.
  • polymer molecule or “polymer moiety” is a molecule formed by cova- lent linkage of two or more monomers, wherein none of the monomers is an amino acid residue, except where the polymer is human albumin or another abundant plasma protein.
  • polymer can be used interchangeably with the term “polymer”.
  • sugar moiety is intended to indicate a carbohydrate-containing molecule comprising one or more monosaccharide residues, capable of being attached to the polypeptide by way of in vivo or in vitro glycosylation.
  • in vivo glycosylation is intended to mean any attachment of a sugar moiety occurring in vivo, i.e. during posttransla- tional processing in a glycosylating cell used for expression of the polypeptide, e.g. by way of N-linked or O-linked glycosylation. The exact oligosaccharide structure depends, to a large extent, on the glycosylating organism in question.
  • in vitro glycosylation is intended to refer to a synthetic glycosylation produced in vitro, normally involving covalently linking a sugar moiety to an attachment group of a polypeptide, optionally using a cross-linking agent. In vivo and in vitro glycosylation are discussed in more detail below.
  • N-glycosylation site has the sequence N-X-S/T/C", wherein X is any amino acid residue except proline, N is asparagine and S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine.
  • S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine.
  • An "O-glycosylation site” is the OH-group of a serine or threonine residue.
  • attachment group is intended to indicate a functional group of the polypeptide, in particular of an amino acid residue thereof or a carbohydrate moiety, capable of attaching a non-polypeptide moiety such as a polymer molecule, a sugar moiety, a lipophilic molecule or an organic derivatizing agent.
  • a non-polypeptide moiety such as a polymer molecule, a sugar moiety, a lipophilic molecule or an organic derivatizing agent.
  • attachment group is used in an unconventional way to indicate the amino acid residues constituting an N-glycosylation site.
  • asparagine residue of the N-glycosylation site is the one to which the sugar moiety is attached during glycosylation, such attachment cannot be achieved unless the other amino acid residues of the N-glycosylation site are present.
  • amino acid residue comprising an attachment group for the non-polypeptide moiety as used in connection with alterations of the amino acid sequence of the polypeptide of interest is to be understood as meaning that one or more amino acid residues constituting an N-glycosylation site are to be altered in such a manner that either a functional N-glycosylation site is introduced into the amino acid sequence or removed from the sequence.
  • Amino acid names and atom names are used as defined by the Protein DataBank (PDB) (www.pdb.org-), which is based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atom names, etc.), Eur. J. Biochem., 138, 9-37 (1984) together with their corrections in Eur. J. Biochem., 152, 1 (1985)).
  • PDB Protein DataBank
  • amino acid residue is intended to include any natural or synthetic amino acid residue, and is primarily intended to indicate an amino acid residue contained in the group consisting of the 20 naturally occurring amino acids, i.e. selected from the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y) residues
  • A39 in a given amino acid sequence indicates that position number 39 is occupied by an alanine residue in the amino acid sequence shown in SEQ ID NO:2.
  • A39S indicates that the alanine residue of position 39 is substituted with a serine residue.
  • Alterna- tive substitutions are indicated with a "/", e.g., A39S/T means that the alanine residue of position 39 is substituted with either a serine residue or a threonine residue, i.e. A39S/T should be construed as covering two individual substitutions, namely A39S and A39T.
  • A39S+K251N Multiple substitutions are indicated with a "+", e.g., A39S+K251N means that the alanine residue of position 39 is substituted with a serine residue and that the lysine residue in position 251 is substituted with an asparagine residue.
  • the insertion of an additional amino acid residue is indicated in the following way: Insertion of a serine residue after A39 is indicated by A39AS.
  • a deletion of an amino acid residue is indicated by an asterix.
  • deletion of the alanine residue of position 39 is indicated by A39*. Unless otherwise indicated, the numbering of amino acid residues made herein is made relative to the amino acid sequence of SEQ ID NO:2.
  • nucleotide sequence is intended to indicate a consecutive stretch of two or more nucleotide molecules.
  • the nucleotide sequence may be of genomic, cDNA, RNA, semi-synthetic or synthetic origin, or any combination thereof.
  • vector refers to a plasmid or another nucleotide sequence that is capable of replicating within a host cell or being integrated into the host cell genome.
  • Cell "host cell”, “cell line” and “cell culture” are used interchangeably herein and all such terms should be understood to include progeny resulting from growth or cultur- ing of a cell.
  • Transformation and “transfection” are used interchangeably to refer to the process of introducing DNA into a cell.
  • operably linked refers to the covalent joining of two or more nucleotide sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences can be performed.
  • operably linked means that the nucleotide sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleo- tide adaptors or linkers are used, in conjunction with standard recombinant DNA methods.
  • the term "functional in vivo half-life” is used in its normal meaning, i.e. the time at which the activity of the variant is 50% of the initial value. The functional in vivo half-life may be determined by any suitable method known in the art.
  • the term "increased" as used about the functional in vivo half-life is used to indicate that the half-life of the variant is statistically significantly increased relative to that of a reference molecule, e.g. hAPC, determined under comparable conditions. Normally, functional in vivo half-life is increased when inhibition of the polypeptide is decreased. Thus, preferred variants are those which, in their activated form, have an increased functional in vivo half-life as compared to hAPC, such as Xigris®.
  • Particularly preferred variants are those where the ratio between the functional in vivo half-life of the variant, in its activated form, and the functional in vivo half-life of hAPC is at least 1.25, more preferably at least 1.50, such as at least 1.75, e.g. at least 2, even more preferably at least 3, such as at least 4, e.g. at least 5, most preferably at least 6, such as at least 7, e.g. at least 8, at least 9 or at least 10.
  • Clearance mechanisms of relevance for a variant polypeptide may include one or more of the reticuloendothelial systems (RES), kidney, spleen or liver, receptor-mediated degradation, or specific or non-specific proteolysis.
  • RES reticuloendothelial systems
  • renal clearance is used in its normal meaning to indicate any clearance taking place by the kidneys, e.g. by glomerular filtration, tubular excretion or tubular elimination. Normally, renal clearance depends on physical characteristics of the variant polypeptide, including molecular weight, size (relative to the cutoff for glomerular filtration), symmetry, shape/rigidity and charge. A molecular weight of about 67 kDa is normally considered to be a cut-off- value for renal clearance. Renal clearance may be measured by any suitable assay, e.g. an established in vivo assay. For instance, renal clearance may be determined by administering a labelled (e.g. radio- labelled or fluorescence labelled) variant polypeptide or conjugated variant to a patient and measuring the label activity in urine collected from the patient. Reduced renal clearance is determined relative to the reference molecule, such as human APC.
  • a labelled e.g. radio- labelled or fluorescence labelled
  • the terms “activity”, “APC activity” and “activated protein C activity” refer to the amidolytic activity.
  • a suitable in vitro APC activity assay (entitled “APC Amidolytic Assay") is described in Example 7 herein. More particularly, a variant is classified as having "APC activity” if the variant, in its activated form, has an activity of at least 10% of the hAPC activity when tested in the "APC Amidolytic Assay” described in Example 7 herein.
  • the variant has an activity of at least 20% of the hAPC activity, such as an activity of at least 30% of the hAPC activity, more preferably the variant has an activity of at least 40% of the hAPC activity, such as an activity of at least 50% of the hAPC activity, even more preferably the variant has an activity of at least 60% of the hAPC activity, e.g.
  • the variant in its activated form, has an activity when tested in the "APC Amidolytic Assay" described in Example 7 herein which is essentially the same as or higher than the activity of hAPC.
  • anti-inflammatory effect refers to the fact that the variants, in their acti- vated form, prevent induction of various pro-inflammatory cytokines and adhesion molecules, such as tumor necrosis factor alpha, interleukin-1, interleukin-8 and E-selectin, either in vitro or in vivo.
  • anti-inflammatory effect of a variant of the invention as compared to hAPC may easily be assessed by the skilled person using the "APC Anti-inflammatory Assay I” or “APC Anti -inflammatory Assay II” disclosed in Examples 12 and 13 herein or other similar assays.
  • the terms “increased affinity”, “higher affinity” or “improved affinity” when used in connection with the protein C-EPCR (endothelial protein C receptor) interaction refer to a higher affinity binding to the EPCR, due to increased binding energy, than for human protein C. Whether a variant has an increased affinity to the EPCR as compared to human protein C may easily be assessed by the skilled person using the "EPCR Binding Assay I” or the “EPCR Binding Assay II” disclosed in Examples 14 and 15 herein or other similar assays.
  • the ratio between the dissociation constant for human protein C (Kd ;Wt ) and the dissociation constant for the variant (Kd >va rian t ) is at least 1.5, when determined in accordance with one of the EPCR binding assays described herein.
  • this ratio is at least 2, more preferably at least 3, such as at least 5, e.g. at least 10, even more preferably at least 25, such as at least 40, at least 50, at least 75, at least 90, or at least 100, e.g. at least 250, at least 500 or at least 1000.
  • the terms “increased resistance towards inactivation by alpha- 1-antitrypsin” and “increased resistance towards inactivation by human plasma”, respectively, are intended to refer to a variant which, in its activated form, is inhibited by alpha- 1-antitrypsin or human plasma, respectively, to a lesser degree than hAPC.
  • the present inventor has developed suitable preliminary tests which can easily be carried out by the skilled person in order to initially assess the performance of the variant in question.
  • the "Alpha- 1-Antitrypsin Inactivation Assay” (described in Example 9 herein), the “Human Plasma Inactivation Assay I” (described in Example 10 herein) and the “Human Plasma Inactivation Assay II” (described in Example 11 herein) may be employed to initially assess the potential of a selected variant. Using either the first, the second, the third or all of these tests, the suitability of a selected variant to resist inactivation by either alpha- 1-antitrypsin and/or human plasma can be assessed.
  • the variant which, in its activated form, has a residual activity of at least 20% when tested in the "Alpha- 1-Antitrypsin Inactivation Assay" described in Example 9 herein using an inhibitor concentration of 16.6 ⁇ M.
  • the variant has a residual activity of at least 25% or at least 30%, such as a residual activity of at least 40%, more preferably the variant has a residual activity of at least 50%, such as a residual activity of at least 60%, even more preferably the variant has a residual activity of at least 70%, such as a residual activity at least 75%, most preferably a residual activity of at least 80%, such as at least 85%.
  • the suitability of a selected variant may be tested in the "Human Plasma Inactivation Assay I".
  • a preferred variant is one which, in its activated form, has a residual activity of at least 20% when tested in the "Human Plasma Inactivation Assay I" described in Example 10 herein.
  • the variant has a residual activity of at least 25% or at least 30%, such as a residual activity of at least 40%, more preferably a residual activity of at least 50%, such as a residual activity of at least 60%, even more preferably a residual activity of at least 70%, such as a residual activity at least 75%.
  • the suitability of a se- lected variant may be tested in the "Human Plasma Inactivation Assay II".
  • a variant which, in its activated form, has a functional in vitro half-life that is increased compared to that of hAPC.
  • the term "functional in vitro half-life” is used in its normal meaning, i.e. the time at which the activity of the variant is 50% of its initial value.
  • the functional in vitro half-life may suitably be determined by the "Human Plasma Inactivation Assay II" described in Example 11 herein.
  • the term “increased” as about the functional in vitro half-life is used to indicate that the half-life of the variant is significantly increased relative to that of a reference molecule, e.g. hAPC, determined under comparable conditions.
  • the ratio between the functional in vitro half-life of the variant, in its activated form, and the functional in vitro half-life of hAPC is at least 1.25 when tested in the "Human Plasma Inactivation Assay II" described in Example 11 herein.
  • the ratio is at least 1.5, such as at least 2, more preferably at least 3, such as at least 4, even more preferably at least 5, such as at least 6, at least 7, at least 8, at least 9 or at least 10.
  • an interest-ing variant is one which, in its activated form, has an anticoagulant activity of 0-75% of the anticoagulant activity of hAPC when tested in this assay.
  • the variant in its activated form has an anticoagulant activity of 0-70%, such as 0-65%, e.g. 0-60%, more preferably 0-55%), such as 0-50%, e.g.
  • the variant has essentially no anticoagulant activity when tested in the "APC Clotting Assay" described in Example 8.
  • reduced immunogenicity is intended to indicate that the variant gives rise to a measurably lower immune response than a reference molecule, e.g. hAPC, as determined under comparable conditions.
  • the immune response may be a cell or antibody mediated response (see, e.g., Roitt: Essential Immunology (8th Edition, Blackwell) for further definition of immunogenicity).
  • reduced antibody reactivity is an indication of reduced immunogenicity.
  • Reduced immunogenicity may be determined by use of any suitable method known in the art, e.g. in vivo or in vitro.
  • the variants of the invention comprise at least one modfica- tion in position 306-314 of SEQ ID NO:2, i.e at least one modification in a position selected from the group consisting of 306, 307, 308, 309, 310, 311, 312, 313 and 314.
  • protein C contains an N-glycosylation site at position N313.
  • this particular N-glycosylation site is not modified, i.e. the vari- ant does not comprise any amino acid modification in position N313 or T315.
  • the variant comprises at least one modification in a position selected from the group consisting of 306, 307, 308, 309, 310, 311, 312 and 314, in particular position 308, 314 and 308+314.
  • the modification may be an amino acid deletion, an amino acid insertion or an amino acid substitution.
  • the modification is an amino acid substitution.
  • the variant contains 1-6, such as 1-5, 1-4 or 1-3 modifications, such as 1 or 2 substitutions, in position 306-314.
  • the autolysis loop contains a number of charged residues, namely R306, E307, K308, E309, K311, R312 and R314, and in a preferred embodiment one or more of these charged residues have been substituted with an uncharged residue.
  • At least one charged amino acid amino acid residue has been substituted with an uncharged or, in the case of positively charged residues in the native protein, optionally a negatively charged amino acid residue, in particular with an un- charged amino acid residue.
  • At least one negatively charged amino acid residue has been substituted with an uncharged amino acid residue.
  • substitutions which may be performed in position 306 include substitutions selected from the group consisting of R306A, R306V, R306L, R306I, R306F, R306W, R306P, R306G, R306S, R306T, R306Y, R306N and R306Q, preferably selected from the group consisting of R306A, R306V, R306L and R306I, such as R306A.
  • substitutions which may be performed in position 307 include substitutions selected from the group consisting of E307A, E307V, E307L, E307I, E307F, E307W, E307P, E307G, E307S, E307T, E307Y, E307N and E307Q, preferably selected from the group consisting of E307A, E307V, E307L and R307I, such as E307A.
  • substitutions which may be performed in position 308 include substitutions selected from the group consisting of K308A, K308V, K308L, K308I, K308F, K308W, K308P, K308G, K308S, K308T, K308Y, K308N and K308Q, preferably selected from the group consisting of K308A, K308V, K308L and K308I, such as K308A.
  • substitutions which may be performed in position 309 include substitutions selected from the group consisting of E309A, E309V, E309L, E309I, E309F, E309W, E309P, E309G, E309S, E309T, E309Y, E309N and E309Q, preferably selected from the group consisting of E309A, E309V, E309L and R309I, such as E309A.
  • substitutions which may be performed in position 311 include substitutions selected from the group consisting of K311 A, K311 V, K31 1 L, K3111, K311 F, K311W, K311P, K311G, K311 S, K31 1T, K311Y, K31 1N and K31 1Q, preferably selected from the group consisting of K311A, K31 IV, K31 IL and K31 II, such as K311A.
  • substitutions which may be performed in position 312 include substitutions selected from the group consisting of R312A, R312V, R312L, R312I, R312F, R312W, R312P, R312G, R312S, R312T, R312Y, R312N and R312Q, preferably selected from the group consisting of R312A, R312V, R312L and R312I, such as R312A.
  • substitutions which may be performed in position 314 include substitutions selected from the group consisting of R314A, R314V, R314L, R314I, R314F, R314W, R314P, R314G, R314S, R314T, R314Y, R314N and R314Q, preferably selected from the group consisting of R314A, R314V, R314L and R314I, such as R314A.
  • the variant comprises two or more, e.g. two, substitutions in position 306-314, in particular two or more substitutions in positions selected from the group consisting of 306, 307, 308, 309, 310, 311, 312 and 314.
  • the variant comprises substitutions in positions K308 and R314, specific examples of which include K308A+R314A, K308A+R314V, K308A+R314L, K308A+R314I, K308V+R314A, K308V+R314V, K308V+R314L, K308V+R314I, K308L+R314A, K308L+R314V, K308L+R314L, K308L+R314I, K308I+R314A, K308I+R314V, K308I+R314L and K308I+R314I, in particular K308A+R314A.
  • the variant comprises three or more, e.g. three, substitutions in position 306-314, in particular three or more substitutions in positions selected from the group consisting of 306, 307, 308, 309, 310, 311, 312 and 314.
  • the variant comprises substitutions in positions K308,
  • substitutions which may be performed in positions 306, 308 and 314 include substitutions selected from the group consisting of R306A/V/L/I+K308A/V/L/I+ R314A/V/L/I, such as R306A+K308A+R314A.
  • substitutions which may be performed in positions 308, 311 and 314 include substitutions selected from the group consisting of K308A/L/N/I+K311 A L/N/I+ R314A/L/N/I, such as K308A+K311 A+R314A.
  • substitutions which may be performed in positions 308, 312 and 314 include substitutions selected from the group consisting of
  • K308A/L/N/I+R312A/L/V/I+ R314A/L/N/I such as K308A+R312A+R314A.
  • the variant comprises four or more, e.g. four, substitutions in position 306-314, in particular four or more substitutions in positions selected from the group consisting of 306, 307, 308, 309, 310, 311, 312 and 314.
  • the variant comprises substitutions in positions K308,
  • 311, 312 and 314 include substitutions selected from the group consisting of R306A V/L/I+K308A/V/L/I+K311 A/V/L/I+R314A/V/L/I, R306A/V/L/I+K308A/V/L/I+R312A/V/L/I+R314A/V/L/I and
  • the variant comprises five or more, e.g. five, substitutions in position 306-314, in particular five or more substitutions in positions selected from the group consisting of 306, 307, 308, 309, 310, 311, 312 and 314.
  • the variant comprises substitutions in positions K308,
  • R314 and at least three further residues selected from the group consisting of R306, E307, E309, K311 and R312, preferably R306, K311 and R312.
  • substitutions which may be performed in positions 306, 308, 311, 312 and 314 include substitutions selected from the group consisting of R306A/V/L/I+K308A/V/L/I+K311A/V/L/I+R312A/V/L/I+R314A/V/L/I, such as the following substitutions: R306A+K308A+K311A+R312A+R314A.
  • the variant comprises one or more of the above-mentioned substitutions in combination with one or more modifications in the active site region (see the section entitled "Modifications in the active site region' ' ' below).
  • Recombinant hAPC is produced by Eli Lilly and Co. and the product (Xigris®) has been approved by the FDA for certain sepsis patients.
  • Xigris® the product
  • relatively high doses and frequent administration are necessary to reach and sustain the desired therapeutic or prophylactic effects of hAPC due to its short half-life.
  • adequate dose regulation is difficult to obtain and the need for frequent intravenous administrations of high levels of hAPC makes treatment expensive.
  • a molecule with a longer functional half-life would decrease the number of necessary administrations and potentially provide more optimal therapeutic hAPC levels with concomitant enhanced therapeutic effect.
  • the functional half-life of hAPC may be increased, e.g. as a consequence of re- prised inhibition, for example by conjugation of hAPC to a non-polypeptide moiety, e.g. PEG (polyethylene glycol) or carbohydrates. Furthermore, this may also be achieved by mutating the protein C molecule in such a way that it remains active but blocks the binding of inhibitors to the protein.
  • a non-polypeptide moiety e.g. PEG (polyethylene glycol) or carbohydrates.
  • One strategy includes introducing an amino acid residue comprising an attachment group for a non-polypeptide moiety, which makes it possible to specifically adapt the polypeptide so as to make the molecule more susceptible to conjugation to the non-polypeptide moiety of choice, thereby optimizing the conjugation pattern, e.g. to ensure an optimal distribution and number of non-polypeptide moieties on the surface of the active site region and to ensure that only the attachment groups intended to be conjugated is present in the molecule, and thereby obtain a new conjugated molecule which has APC activity and in addition one or more improved properties.
  • a non-polypeptide moiety it is possible to design the attachment of a non-polypeptide moiety to an attachment group present in the active site region so that inactivation by human plasma or certain inhibitors, such as alpha- 1-antitrypsin, is significantly reduced.
  • the amino acid residue comprising an attachment group for a non-polypeptide moiety is selected on the basis of the nature of the non-polypeptide moiety of choice and, in most instances, on the basis of the method in which conjugation between the variant polypeptide and the non-polypeptide moiety is to be achieved.
  • the non- polypeptide moiety is a polymer molecule such as a polyethylene glycol amino acid residues comprising an attachment group may be selected from the group consisting of lysine, cys- teine, aspartic acid, glutamic acid, histidine, and tyrosine, preferably cysteine or lysine.
  • the attachment group is e.g. an in vivo glycosylation site, preferably an N-glycosylation site.
  • variants having an increased resistance towards inactivation by alpha- 1- antitrypsin or an increased resistance towards inactivation by human plasma comprise at least one modification in the active site region (as defined above), the rationale being that modifications in this particular region of the protein C molecule will impair binding of inhibitors (such as alpha- 1-antitrypin) to the protein C variant while still retaining a substan- tial APC activity and a substantial anti-inflammatory and/or anti-apoptotic effect.
  • inhibitors such as alpha- 1-antitrypin
  • the modification in the active site region comprises at least one amino acid substitution at a position which is occupied by an amino acid residue having at least 25% of its side chain exposed to the surface, such as at least 50% of its side chain exposed to the surface.
  • the substitution comprises introduction of an amino acid residue comprising an attachment group for a non- polypeptide moiety, such as a sugar moiety or a polymer molecule.
  • residues of the hAPC molecule are defined as having zero side chain accessibility: G67, C89, C98, G103, C105, HI 07, C109, Y124, G142, G173, V186, L187, A198, V199, 1201, V206, L207, T208, A210, C212, V221, E235, 1258, A259, L260, L261, L263, A267, V274, 1276, L283, V297, C331, M335, A346, G361, M364, T371, F373, L374, G376, L377, V392 and 1403.
  • H211 active site histidine
  • cysteine residues listed above are normally not candidates for modification in the present context either; in particular, C384, which is located in the active site, is not a candidate for modification.
  • the variant comprises 1-5 modifications in the active site region, such as 1-4 or 1-3 modifications in the active site region, for example 1 , 2 or 3 modifications in the active site region.
  • the introduced amino acid resi- due comprising an attachment group for a non-polypeptide residue creates a glycosylation site, in particular an in vivo N-glycosylation site.
  • substitutions where the introduced amino acid residue creates an in vivo N-glycosylation site include substitutions selected from the group consisting of D172N+K174S, D172N+K174T, D189N+K191 S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, K192N+L194S, K192N+L194T, K193N+A195S, K193N+A195T, D214N, D214N+S216T, E215N+K217S, E215N+K217T, S216N+K218S, S216N+K218T, K217N+
  • the introduced in vivo N-glycosylation site is selected from the group consisting of D189N+K191 S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, D214N, D214N+S216T, K217N+L219S, K217N+L219T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, T253N+D255S, T253N+D255T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385S, G
  • the introduced in vivo N-glycosylation site is selected from the group consisting of D189N+K191 S, D189N+K191T, K191N+K193T, D214N, D214N+S216T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S and L386N+H388T.
  • the introduced in vivo N-glycosylation site is selected from the group consisting of D189N+K191T, K191N+K193T, D214N, K251N, S252N, T253N+D255T, Y302N, S336N+M338T, V339T, M338N, G383N+G385T and L386N+H388T, in particular D189N+K191T, D214N, K251N and L386N+H388T.
  • substitutions include D189N+K191T, D214N and L386N+H388T as well as K251N, S252N and Y302N.
  • the variant may contain 1-5 in vivo glycosylation sites, such as a single in vivo glycosylation site (in addition to the already present glycosylation sites at positions 97, 248, 313 and 329) in the active site region.
  • 1-5 in vivo glycosylation sites such as a single in vivo glycosylation site (in addition to the already present glycosylation sites at positions 97, 248, 313 and 329) in the active site region.
  • the variant comprises more than one in vivo glycosylation site in the active site region, in particular 2-5 (additional) in vivo glycosylation sites, such as 2, 3, 4 or 5 (additional) in vivo glycosylation sites in the active site region, preferably introduced by one or more of the substitutions described in any of the above lists.
  • the variant in order to prepare a variant according to this aspect of the invention, the variant must be expressed in a glycosylating host cell capable of attaching sugar moieties at the glycosylation sites or alternatively subjected to in vitro glycosylation.
  • glycosylating host cells are given in the section below entitled "Coupling to a sugar moiety”.
  • variants which contain one or more non-utilized glycoylation sites (i.e. no sugar moiety is actually attached to the introduced glycosylation site) still have interesting properties, in particular with respect to increased resistance towards inhibition by alpha- 1-antitrypsin and increased resistance towards inactivation by human plasma.
  • Such variants comprise at least one substitution in the active site region (as defined above), in particular they comprise a substitution of an amino acid residue which is located in the active site region and which has at least 25% of its side chain exposed to the surface of the molecule (as defined above).
  • preferred variants according to this aspect of the invention comprise a substitution in a position selected from the group consisting of D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, V334, S336, N337, M338, 1348, L349, D351, R352, E357, E382, G383, L386, L387 and H388, in particular a substitution selected from the group consisting of K251N, S252N, Y302N and S190+K192T, especially K251N and S252N.
  • the introduced amino acid residue comprising an attachment group for a non-polypeptide residue is a cysteine residue.
  • the cysteine residue is preferably introduced in a position which is within the active site region (as defined herein) and which is occupied by an amino acid residue having at least 25% of its side chain exposed to the surface of the molecule (as defined herein), i.e.
  • the cysteine residue is preferably introduced in a position selected from the group consisting of D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, V334, S336, V339, M338, 1348, L349, D351, R352, E357, G383, E385, L386, L387 and H388.
  • the cysteine residue is introduced in a positions selected from the group consisting of D189, S190, K191, D214, K217, K251, S252, T253, Y302, S336, N337, M338, G383 and L386. Even more preferably, the cysteine residue is introduced in a position selected form the group consisting of D189, K191, K251, S252, T253, Y302, S336, N337, M338, G383 and L386. Most preferably, the cysteine residue is introduced in a position selected from the group consisting of D189, D214, K251 and L386.
  • the conjugated variant according to this embodiment typically comprises 1-5 introduced cysteine residues in the active site region, in particular 2-5 or 1-3 introduced cysteine residues, e.g. 1, 2 or 3 introduced cysteine residues.
  • any introduced cysteine residue are preferably conjugated (i.e. covalently attached) to a non-polypeptide moiety, e.g. a polymer molecule, such as PEG or more preferably mPEG.
  • a polymer molecule such as PEG or more preferably mPEG.
  • the conjugation between the cysteine-containing polypeptide variant and the polymer molecule may be achieved in any suitable manner, e.g. as described in the section entitled "Conjugation to a polymer molecule", e.g. using a one step method or using the stepwise manner referred to in said section.
  • the preferred method for PEGylating the variant is to covalently attach PEG to cysteine residues using cysteine-reactive PEGs.
  • a number of highly specific, cysteine-reactive PEGs with different groups e.g. orthopyridyl- disulfide, maleimide and vinylsulfone
  • different size PEGs 2-20 kDa, such as 5 kDa, 10 kDa, 12 kDa or 15 kDa
  • the conjugated variant according to this embodiment may comprise at least one second non-polypeptide moiety, such as 1-5 or 1-3 such moieties.
  • the second non- polypeptide moiety is preferably a sugar moiety, in particular an in vivo attached sugar moi- ety.
  • the sugar moiety may be present at one or more of the naturally-occurring glycosylation sites present in the parent polypeptide, or at an introduced glycosylation site. Suitable introduced glycosylation sites, in particular N-glycosylation sites, are described above
  • amino acid substitutions which are particularly interesting according to this aspect of the present invention are those wherein a charged amino acid residue located in the active site region and also having at least 25% of its side chain exposed to the surface is substituted with an amino acid residue having no charge, in particular an amino acid residue having no charge but a polar side chain (Gly, Ser, Thr, Cys, Tyr, Asn or Gin), as well as amino acid substitutions wherein a charged amino acid residue located in the active site region and also having at least 25% of its side chain exposed to the surface is substituted with an amino acid resi- due having an opposite charge.
  • amino acid substitutions wherein the charge of the amino acid residue in question is changed to an opposite charge include D172K, D172R, D189K, D189R, K191D, K191E, K192D, K192E, K193D, K193E, D214K, D214R, E215K, E215R, K217D, K217E, K218D, K218E, K251D, K251E, D255K, D255R, D351K, D351R, R352D, R352E, E357K, E357R, E382K and E382R, such as D214K, D214R, E215K, E215R, K251D, K251E, E357K and E357R, e.g. D214K, D214R, K251D and K251E, in particular K25 ID.
  • amino acid substitutions wherein the charged amino acid residue in question is substituted with an amino acid side chain having a polar side chain include D172G/S/T/C/Y/N/Q, D189G/S/T/C/Y/N/Q, K191G/S/T/C ⁇ 7N/Q, K192G/S/T/C/Y/N/Q, K193G/S/T/C ⁇ 7N/Q, D214G/S/T/C/Y/N/Q, E215G/S/T/C/Y/N/Q, K217G/S/T/C/Y/N/Q, K218G/S/T/C/Y/N/Q, K251G/S/T/C/Y/N/Q, D255G/S/T/C/Y/N/Q, D351G/S/T/C/Y/N/Q, R352G/S/T/C/Y/N/Q, E357G/S/S/
  • K251G/S/T/C/Y/N/Q and E357G/S/T/C/Y/N/Q e.g. D214Q, E215Q, K251Q and E357Q, in particular K251Q.
  • Another interesting substitution may be K251N+T253A.
  • the increased resistance towards inactivation by alpha- 1- antitrypsin and/or human plasma may be determined and assessed by the "Alpha- 1- Antritrypsin Inactivation Assay", the “Human Plasma Inactivation Assay I” or the “Human Plasma Inactivation Assay II” disclosed herein. These assays enable the skilled person to assess, at an early stage, whether the constructed variant can be expected to have an increased functional half-life.
  • modifications discussed in connection with the active site region may be combined with modifications in the autolysis loop in order to obtain a protein C variant having an increased functional half-life, a reduced anticoagulant activity, and a substantially retained anti-inflammatory and/or anti-apoptotic effect.
  • the variant comprises i) at least one amino acid modification in position 306-314; and ii) at least one amino acid modification in the active site region. More particularly, a preferred variant is one wherein i) at least one amino acid residue selected from the group consisting of R306, E307, K308, E309, K311, R312 and R314 has been substituted with an uncharged amino acid residue; and ii) the at least one modification in the active site region is selected from the group consisting of D172N+K174S, D172N+K174T, D189N+K191S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, K192N+L194S, K192N+L194T,
  • the variant is one wherein i) at least one amino acid residue selected from the group consisting of R306, K308, K311, R312 and R314 has been substituted with an uncharged amino acid residue; and ii) the at least one modification in the active site region (as defined herein) is selected from the group consisting of D189N+K191S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, D214N, D214N+S216T, K217N+L219S, K217N+L219T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, S336N+M338S, S336N+M338T, S336
  • the variant is one wherein i) at least one amino acid residue selected from the group consisting of K308 and R314 has been substituted with an uncharged amino acid residue; and ii) the at least one modification in the active site region (as defined herein) is selected from the group consisting of D189N+K191S, D189N+K191T, K191N+K193T, D214N, D214N+S216T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S and
  • the variant is one wherein i) at least one amino acid residue selected from the group consisting of K308 and R314 has been substituted with an uncharged amino acid residue selected from the group consisting of A, V, L and I; and ii) the at least one modification in the active site region (as defined herein) is selected from the group consisting of D189N+K191T, K191N+K193T, D214N, K251N, S252N, T253N+D255T, Y302N, S336N+M338T, V339T, M338N, G383N+G385T and L386N+H388T.
  • An example of a highly preferred variant is one wherein i) at least one amino acid residue selected from the group consisting of K308 and R314 has been substituted with an uncharged amino acid residue selected from the group consisting of A, V, L and I; and ii) the at least one modification in the active site region (as defined herein) is selected from the group consisting of D189N+K191T, D214N, D251N and L386N+H388T.
  • Another example of a highly preferred variant is one wherein i) at least one amino acid residue selected from the group consisting of K308 and R314 has been substituted with an uncharged amino acid residue selected from the group consisting of A, V, L and I; and ii) the at least one modification in the active site region (as defined herein) is selected from the group consisting of K251D, S252N and Y302N.
  • the non-polypeptide moiety Amino acid residues comprising other attachment groups may be introduced by substitution into the parent polypeptide using the same approach as that illustrated above with in vivo N-glycosylation sites and cysteine residues. For instance, one or more amino acid residues comprising an acid group (glutamic acid or aspartic acid), tyrosine or lysine may be introduced into the positions discussed above.
  • the non-polypeptide moiety of the conjugated variant is preferably selected from the group consisting of polymer molecules and sugar moieties (by way of in vivo glycosylation). These agents may confer desirable properties to the variant polypeptide, in particular increased functional in vivo half-life.
  • the variant polypeptide is normally conjugated to only one type of non-polypeptide moiety, but may also be conju- gated to two or more different types of non-polypeptide moieties, e.g. to a polymer molecule and a sugar moiety.
  • a conjugated variant may be produced by culturing an appropriate host cell under conditions conducive for the expression of the variant polypeptide, and recovering the variant polypeptide, wherein a) the variant polypeptide comprises at least one N- or O- glycosylation site and the host cell is an eukaryotic host cell capable of in vivo glycosyla- tion, and/orb) the variant polypeptide is subjected to conjugation to a non-polypeptide moiety in vitro.
  • the polymer molecule to be coupled to the variant polypeptide may be any suitable polymer molecule, such as a natural or synthetic homo-polymer or hetero-polymer, typically with a molecular weight in the range of about 300-100,000 Da, such as about 500-20,000 Da, more preferably in the range of about 500-15,000 Da, even more preferably in the range of about 2-12 kDa, such as in the range of about 3-10 kDa.
  • a suitable polymer molecule such as a natural or synthetic homo-polymer or hetero-polymer, typically with a molecular weight in the range of about 300-100,000 Da, such as about 500-20,000 Da, more preferably in the range of about 500-15,000 Da, even more preferably in the range of about 2-12 kDa, such as in the range of about 3-10 kDa.
  • homo-polymers examples include a polyol (i.e. poly-OH), a polyamine (i.e. poly-NH 2 ) and a polycarboxylic acid (i.e. poly-COOH).
  • a hetero-polymer is a polymer comprising different coupling groups, such as a hydroxyl group and an amine group.
  • suitable polymer molecules include polymer molecules selected from the group consisting of polyalkylene oxide (PAO), including polyalkylene glycol (PAG), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, poly- vinyl alcohol (PVA), poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid anhydride, dextran, including carboxymethyl- dextran, or any other biopolymer suitable for reducing immunogenicity and/or increasing functional in vi o half-life.
  • PEO polyalkylene oxide
  • PAG polyalkylene glycol
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • PVA poly- vinyl alcohol
  • PVA poly-carboxylate
  • poly-(vinylpyrolidone) polyethylene-co-maleic acid anhydride
  • polystyrene-co-maleic acid anhydride de
  • PEG is the preferred polymer molecule, since it has only few reactive groups capable of cross-linking compared to, e.g., polysaccharides such as dextran.
  • mono- functional PEG e.g. methoxypolyethylene glycol (mPEG)
  • mPEG methoxypolyethylene glycol
  • the hydroxyl end groups of the polymer molecule must be provided in activated form, i.e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succina- mide (SSA), succinimidyl propionate (SPA), succinimidyl butyrate (SBA), succinimidy carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS),
  • reactive functional groups examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succina- mide (SSA), succinimidyl propionate (SPA), succinimidyl butyrate (SBA), succinimidy carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NH
  • Suitable activated polymer molecules are commercially available, e.g. from Nektar Therapeutics, Huntsville, AL, USA, or from PolyMASC Pharmaceuticals pic, UK.
  • the polymer molecules can be activated by conventional methods known in the art, e.g. as disclosed in WO 90/13540. Specific examples of activated linear or l o branched polymer molecules for use in the present invention are described in the Shearwater Corp. 2001 Catalog (Polyethylene Glycol and Derivatives for Biomedical Applications, incorporated herein by reference).
  • activated PEG polymers include the following linear PEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG,
  • NHS-PEG e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG
  • activated PEG polymers particularly preferred for coupling to cysteine residues include the following linear PEGs: vinylsulfone-PEG (VS-PEG),
  • PEG 20 preferably vinylsulfone-mPEG (VS-mPEG); maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) and orthopyridyl-disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG).
  • MAL-PEG maleimide-PEG
  • OPSS-PEG orthopyridyl-disulfide-PEG
  • OPSS-mPEG orthopyridyl-disulfide-mPEG
  • the expression host cell may be selected from fungal (filamentous fungal or yeast), insect or animal cells or from transgenic plant cells.
  • the host cell is a mammalian cell, such as a CHO cell, BHK or HEK, e.g. HEK 293, cell, or an insect cell, such as an SF9 cell, or a yeast cell, e.g. S. cerevisiae or Pichia pas tons, or any of the host cells mentioned hereinafter.
  • sugar moieties such as dextran to amino acid residues of the variant polypeptide
  • covalent in vitro coupling of sugar moieties such as dextran to amino acid residues of the variant polypeptide may also be used, e.g. as described in WO 02/32461 (incorporated by reference) and the references cited therein.
  • the polypeptide variant may be produced by any suitable method known in the art. Such methods include constructing a nucleotide sequence encoding the variant polypeptide and expressing the sequence in a suitable transformed or transfected host.
  • the host cell is a garnma-carboxylating host cell such as a mammalian cell.
  • variant polypeptides may be produced, albeit less efficiently, by chemical synthesis or a combination of chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.
  • a nucleotide sequence encoding a polypeptide variant may be constructed by iso- lating or synthesizing a nucleotide sequence encoding the parent protein C, such as protein C with the amino acid sequence shown in SEQ ID NO:2, and then changing the nucleotide sequence so as to effect introduction (i.e. insertion or substitution) or removal (i.e. deletion or substitution) of the relevant amino acid residue(s).
  • nucleotide sequence is conveniently modified by site-directed mutagenesis in accordance with conventional methods.
  • the nucleotide sequence may be prepared by chemical synthesis, e.g. by using an oligonucleotide synthesizer, wherein oligonu- cleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced.
  • oligonucleotides coding for por- tions of the desired polypeptide may be synthesized and assembled by PCR (polymerase chain reaction), ligation or ligation chain reaction (LCR) (Barany, PNAS 88:189-193, 1991).
  • the individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • the recombinant vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector in which the nucleotide sequence encoding the polypeptide variant is operably linked to additional segments required for tran- scription of the nucleotide sequence.
  • the vector is typically derived from plasmid or viral DNA.
  • suitable expression vectors for expression in the host cells mentioned herein are commercially available or described in the literature.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalo virus.
  • yeast cells include the 2 ⁇ plasmid and derivatives thereof, the POT1 vector (US 4,931,373), the pJSO37 vector described in Okkels, Ann. New York Acad. Sci. 782, 202-207, 1996, and pPICZ A, B or C (Invifrogen).
  • Useful vectors for insect cells include pVL941, pBG311 (Cate et al., Cell 45: 685-98 (1986)), pBluebac 4.5 and pMelbac (both available from Invifrogen).
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E.
  • coli including pBR322, pET3a and ⁇ ET12a (both from Novagen Inc., WI, USA), wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages.
  • phage DNAs e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages.
  • Other vectors for use in this invention include those that allow the nucleotide sequence encoding the variant polypeptide to be amplified in copy number.
  • amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g.
  • the recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question.
  • An example of such a sequence is the SV40 origin of replication.
  • suitable sequences enabling the vector to replicate are the yeast plasmid 2 ⁇ replication genes REP 1-3 and origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespom.be TPI gene (described by Russell, Gene 40:125- 30, 1985), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • selectable markers include ura3 and leu2.
  • selectable markers include amdS, pyrG, arcB, niaD and sC.
  • control sequences is defined herein to include all components which are necessary or advantageous for the expression of the variant polypeptide.
  • Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide.
  • control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activating sequence, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter.
  • expression control sequences may be used in the present invention.
  • useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors as well as any sequence known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • control sequences for directing transcription in mammalian cells include the early and late promoters of S V40 and adenovirus, e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovi- rus immediate-early gene promoter (CMV), the human elongation factor l ⁇ (EF-l ⁇ ) promoter, the Drosophila minimal heat shock protein 70 promoter, the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC) promoter, the human growth hormone terminator, SV40 or adenovirus Elb region polyadenylation signals and the Kozak consensus sequence (Kozak, JMol Biol 1987, 196(4):947-50).
  • adenovirus 2 major late promoter e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovi- rus
  • a synthetic intron may be inserted in the 5' untranslated region of the nucleotide sequence encoding the polypeptide.
  • An example of a synthetic intron is the synthetic intron from the plasmid pCI-Neo (available from Promega Corporation, WI, USA).
  • control sequences for directing transcription in insect cells include the polyhedrin promoter, the PI 0 promoter, the Autographa califomica polyhedro- sis virus basic protein promoter, the baculovirus immediate early gene 1 promoter and the baculovirus 39K delayed-early gene promoter, and the SV40 polyadenylation sequence.
  • suitable control sequences for use in yeast host cells include the promoters of the yeast ⁇ -mating system, the yeast triose phosphate isomerase (TPI) promoter, promoters from yeast glycolytic genes or alcohol dehydrogenase genes, the ADH2-4c promoter, and the inducible GAL promoter.
  • TPI yeast triose phosphate isomerase
  • suitable control sequences for use in filamentous fungal host cells include the ADH3 promoter and terminator, a promoter derived from the genes encoding Aspergillus oryzae TAKA amylase triose phosphate isomerase or alkaline protease, an A. niger ⁇ -amylase, A. niger or A. nidulans glucoamylase, A. nidulans acetami- dase, Rhizomucor miehei aspartic proteinase or lipase, the TPI1 terminator and the ADH3 terminator.
  • suitable control sequences for use in bacterial host cells include promoters of the lac system, the trp system, the TAC or TRC system, and the major pro- moter regions of phage lambda.
  • the nucleotide sequence encoding a protein C polypeptide variant may optionally include a nucleotide sequence that encode a signal peptide.
  • the signal peptide is present when the polypeptide is to be secreted from the cells in which it is expressed. Such signal peptide, if present, should be one recognized by the cell chosen for expression of the polypeptide.
  • the signal peptide may be homologous (e.g. be that normally associated with human protein C) or heterologous (i.e. originating from another source than human protein C) to the polypeptide or may be homologous or heterologous to the host cell, i.e.
  • the signal peptide may be prokaryotic, e.g. derived from a bacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian, or insect or yeast cell.
  • Any suitable eukaryotic or prokaryotic host may be used to produce the variant polypeptide, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants, although eu- karyotic hosts are preferred.
  • bacterial host cells include gram-positive bacteria such as strains of Bacillus, e.g. B. brevis orB. subtilis, Pseudomonas or Streptomyces, or gram-negative bacteria, such as strains of E. coli.
  • the introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang et al., 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., Young et al., 1961, Journal of Bacteriology 81 : 823-829, or Dubnau et al., 1971, JMolBiol 56: 209-221), electroporation (see, e.g., Shigekawa et al., 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler et al., 1987, Journal of Bacteriology 169: 5771-5278).
  • protoplast transformation see, e.g., Chang et al., 1979, Molecular General Genetics 168: 111-115
  • competent cells see, e.g., Young et al., 1961, Journal of Bacteriology
  • suitable filamentous fungal host cells include strains o ⁇ Aspergillus, e.g. A. oryzae, A. niger, or A. nidulans, Fusarium or Trichoderma. Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and US 5,679,543. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Examples of suitable yeast host cells include strains of Saccharomyces, e.g. S.
  • Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
  • suitable insect host cells include a Lepidoptora cell line, such as S. frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (High Five) (US 5,077,214).
  • Transformation of insect cells and production of heterologous polypeptides therein may be performed as described by Invifrogen.
  • suitable mammalian host cells include Chinese hamster ovary (CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL- 1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL-1573)), as well as plant cells in tissue culture.
  • COS Green Monkey cell lines
  • BHK Baby Hamster Kidney
  • HEK 293 ATCC CRL-1573
  • mammalian cell such as a CHO cell
  • sialyltransferase e.g. 1,6-sialyltransferase, e.g. as described in US 5,047,335, in order to provide improved glycosylation of the protein C polypeptide.
  • an endoprotease in particular a PACE (Paired basic amino acid converting enzyme) (e.g. as described in US 5,986,079), such as a Kex2 endoprotease (e.g. as described in WO 00/28065).
  • PACE Paned basic amino acid converting enzyme
  • Kex2 endoprotease e.g. as described in WO 00/28065
  • Methods for introducing exogeneous DNA into mammalian host cells include calcium phosphate-mediated transfection, electroporation, DEAE-dextran mediated transfec- tion, lipo some-mediated transfection, viral vectors and the transfection method described by Life Technologies Ltd, Paisley, UK using Lipofectamin 2000. These methods are well known in the art and e.g. described by Ausbel et al. (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. The cultivation of mammalian cells are conducted according to established methods, e.g. as disclosed in (Animal Cell Biotechnology, Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc, Totowa, New Jersey, USA and Harrison MA and Rae IF, General Techniques of Cell Culture, Cambridge University Press 1997).
  • the cells are cultivated in a nutrient medium suitable for production of the variant polypeptide using methods known in the art.
  • the cell may be cultivated by shake flask cultivation, small-scale or large- scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
  • the resulting variant polypeptide may be recovered by methods known in the art.
  • the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, ultra-filtration, extraction or precipitation.
  • the variant polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelec- tric focusing), differential solubility (e.g., ammonium sulfate precipitation) or extraction (see, e.g., Protein Purification (2nd Edition), Janson and Ryden, editors, Wiley, New York, 1998).
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelec- tric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • extraction see, e.g., Protein Purification (2nd Edition), Janson and Ryden, editors, Wiley, New York, 1998).
  • the variant polypeptide may be activated by any suitable method known in the art, e.g. by thrombin as described in EP 1 087 011 or by the venom protein C activator, ACC-C (Nakagaki et al., Thrombosis Research 58:593-602, 1990).
  • variants of the invention also provides novel human protein C polypeptides.
  • a further aspect of the present invention relates to a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2, with the proviso that the variant is not human protein C or hAPC comprising the following modifica- tions: R306A, E307A, K308A, E309A, K311 A, R312A, R314A, R306A+R312A, R306A+K311A+R312A+R314A, H303*+S304*+S305*+K308*+E307D+A310T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, E30
  • the variant is as described in the above sections entitled “Modifications in the autolysis loop", “Modifications in the active site region” and “Preferred combination variants”.
  • the present invention relates to the use of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2 for the manufacture of a medicament for reducing inflammation.
  • the invention relates to the use of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2 for the manufacture of a medicament for the prevention or treatment of a hypercoagulable state or acquired protein C deficiency, e.g. when the hypercoagulable state or protein C deficiency is associated with sepsis; transplantations, such as bone marrow transplantation; bums; pregnancy; major surgery; trauma; or adult respiratory distress syndrome (ARDS), in particular sepsis.
  • ARDS adult respiratory distress syndrome
  • the present invention relates to the use of a human protein C variant comprising at least one amino acid modification in position 306- 314 relative to SEQ ID NO:2 for the manufacture of a medicament for the treatment of sepsis.
  • the present invention relates to a method of reducing inflammation in a patient, which comprises administering to the patient an effective amount of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2.
  • the invention also relates to a method of treating a patient with a hypercoagulable state or acquired protein C deficiency, e.g.
  • hypercoagulable state or protein C deficiency is associated with sepsis; transplantations, such as bone marrow transplantation; burns; pregnancy; major surgery; trauma; or adult respiratory distress syndrome (ARDS), in particular sepsis, which comprises administering to the patient an effective amount of a hu- man protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2.
  • this method is for the treatment of sepsis
  • hypocoagulable state(s) refers to excessive coagulability associated with disseminated intravascular coagulation, prethrombotic conditions, activation of coagulation, or congenital or acquired deficiency of clotting factors, such as APC.
  • Sepsis is defined as a systemic inflammatory response to infection, associated with and mediated by the activation of a number of host defense mechanisms including the cyto- kine network, leukocytes, and the complement and coagulation/fibrinolysis systems (Mest- ers et al., Blood 88:881-886, 1996).
  • Disseminated intravascular coagulation (DIC) with widespread deposition of fibrin in the microvasculature of various organs, is an early manifestation of sepsis/septic shock. DIC is an important mediator in the development of the multiple organ failure syndrome and contributes to the poor prognosis of patients with septic shock ( founder et al., Chest 101 :816-823, 1921).
  • LPS lipopolysaccharide
  • results obtained in these animals are not necessarily predictive to the treatment of humans, and the efficacious dose level of human activated protein C is extremely variable depending upon the animal model selected.
  • the serum half-life of human activated protein C in humans is 30 to 40 minutes, compared to a half-life of 8 to 10 minutes in baboons and 90 minutes in rabbits.
  • agents that block inflammatory mediators associated with the pathophysiology of this disease.
  • clinical studies with a variety of agents that block inflammatory mediators have been unsuccessful (reviewed in Natanson et al., Ann. Intern.
  • phase III trials for the treatment of sepsis (Bernard et al., NEnglJ Med, 344, 699-709, 2001) were completed. Patients suffering from severe sepsis were given doses of 24 ⁇ g/kg/h for a total duration of 96 hours as infusion. A total of 1520 persons were involved in the trial and it was found that the 28 days mortality rate was reduced from 31% to 25%.
  • a "patient” for the purposes of the present invention includes both humans and other mammals. Thus the methods are applicable to both human therapy and veterinary ap- plications.
  • the variant is administered to patients in an effective dose.
  • effective dose herein is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered.
  • the exact dose will depend on the disorder to be treated, and will be ascertainable by one skilled in the art using known techniques.
  • 24 ⁇ g/kg/h of hAPC is administered for 96 hours as continous infusion, which conesponds to a total amount of protein of about 230 mg for a patient having a body weight of about 100 kg.
  • Those variants disclosed herein that have an increased functional half-life are contemplated to be more efficient, in particular in the treatment of sepsis, than the corresponding hAPC.
  • the increased efficacy means that the effective dose needed to obtain the desired effect for a particular disorder can be smaller (less protein need to be administered) than the effective dose of hAPC or, alternatively, that by using the same dose as presently being used for hAPC, the treatment of patients with the variants disclosed herein will be more efficient in that a decrease in the mortality rate, as compared to a similar treatment with hAPC, will be observed.
  • such variants permit less frequent administration, such as bolus injections, of the variants.
  • the variants may be administered by a either a bolus or infusion or as a combination thereof with doses which range from 1 ⁇ g/kg body weight as a bolus every 2 nd hour for several days (e.g.
  • a dose as possible is administered as infrequently as possible, e.g. 1-500 ⁇ g/kg body weight, preferably 1-250 ⁇ g/kg body weight, such as 1-100 ⁇ g/kg body weight, more preferably 1-50 ⁇ g/kg body weight is administered as a bolus every 4-96 hours, e.g. every 8-96 hours, such as every 16-96 hours, every 24-96 hours, every 40-96 hours, every 48-96 hours, every 56-96 hours, or every 72-96 hours.
  • the ratio between the AUC (area under the curve) of the variant, in its activated form, and the AUC of hAPC is at least 1.25 when tested in the "Human Plasma Inactivation Assay II" described in Example 11 herein.
  • the ratio is at least 1.5, such as at least 2, e.g. at least 3, more preferably the ratio is at least 4, such as at least 5, e.g. at least 6, even more preferably the ratio is at least 7, such as at least 8, e.g. at least 9, most preferably the ratio is at least 10.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2, with the proviso that the variant is not human protein C or hAPC comprising the following modifications: R306A, E307A, K308A, E309A, K311A, R312A, R314A, R306A+R312A, R306A+K311A+R312A+R314A, H303*+S304*+S305*+K308*+E307D+A310T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, K308N+A310S, K308N+A310T,
  • pharmaceutically acceptable means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the patients to which they are administered.
  • Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceuti- cal Sciences, 19th edition, A. R.
  • the variant can be used "as is” and/or in a salt form thereof.
  • Suitable salts include, but are not limited to, salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, as well as e.g. zinc salts. These salts or complexes may by present as a crystalline and/or amorphous structure.
  • the pharmaceutical composition of the invention may be administered alone or in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the variant, either concurrently or in accordance with another treatment schedule. In addition, the variant or pharmaceutical composition may be used as an adjuvant to other therapies.
  • composition of the invention may be formulated in a variety of forms, e.g. as a liquid, gel, lyophilized, or as a compressed solid.
  • the preferred form will depend upon the particular indication being treated and will be readily able to be determined by one skilled in the art.
  • the administration of the formulations of the present invention can be performed in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, in- tracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other acceptable manner.
  • the formulations can be administered continuously by infusion, although bolus injection is acceptable, using techniques well known in the art, such as pumps or implantation. In some instances the formulations may be directly applied as a solution or spray.
  • compositions designed for parenteral administration.
  • parenteral formulations may also be provided in frozen or in lyophilized form.
  • the composition must be thawed prior to use.
  • the latter form is often used to enhance the stability of the active compound contained in the composition under a wider variety of storage conditions, as it is recognized by those skilled in the art that lyophilized preparations are generally more stable than their liquid counterparts.
  • Such lyophilized preparations are reconstituted prior to use by the addition of one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.
  • parenterals In case of parenterals, they are prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the polypeptide having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers typi- cally employed in the art (all of which are termed "excipients"), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives.
  • excipients for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives.
  • Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are typically present at a concentration ranging from about 2 mM to about 50 mM Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate- disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid- monosodium fumarate mixture, fumaric acid-disodium
  • Preservatives are added to retard microbial growth, and are typically added in amounts of e.g. about 0.1%-2% (w/v).
  • Suitable preservatives for use with the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octade- cyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g. benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.
  • Isotonicifiers are added to ensure isotonicity of liquid compositions and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Polyhydric alcohols can be present in an amount between 0.1% and 25% by weight, typically 1% to 5%, taking into account the relative amounts of the other ingredients.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall.
  • Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, omithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as
  • proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins
  • hydrophilic polymers such as polyvinylpynolidone
  • monosaccharides such as xylose, mannose, fructose and glucose
  • disaccharides such as lactose, maltose and sucrose
  • trisaccharides such as raffi- nose, and polysaccharides such as dextran.
  • Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on the active protein weight.
  • Non-ionic surfactants or detergents may be present to help solubilize the therapeutic agent as well as to protect the therapeutic polypeptide against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the polypeptide.
  • Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers (Tween®-20, Tween®-80, etc.).
  • Additional miscellaneous excipients include bulking agents or fillers (e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) and cosolvents.
  • bulking agents or fillers e.g. starch
  • chelating agents e.g. EDTA
  • antioxidants e.g., ascorbic acid, methionine, vitamin E
  • cosolvents e.g., ascorbic acid, methionine, vitamin E
  • the active ingredient may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example hydroxy- methylcellulose, gelatin or poly-(methylmethacylate) microcapsules, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Parenteral formulations to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.
  • sustained-release preparations include semi-permeable matri- ces of solid hydrophobic polymers containing the variant, the matrices having a suitable form such as a film or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethyl- ene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the ProLease® technology or Lupron Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • polyesters for example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)
  • polymers such as ethyl ene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for long periods such as up to or over 100 days
  • certain hydrogels release proteins for shorter time periods.
  • encapsulated polypeptides When encapsulated polypeptides remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved.
  • stabilization may be achieved by modifying sulfhydryl residues, ly- ophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • a gene encoding the human protein C precursor is constructed by assembly of synthetic oligonucleotides by PCR using methods similar to the ones described in Stemmer et al. (1995) Gene 164, pp. 49-53.
  • the native protein C signal sequence is maintained in order to allow secretion of the gene product.
  • the synthetic gene is designed with a Nhel site at the 5 '-end and a Xbal site at the 3 '-end and subcloned behind the CMV promoter in pcDNA3.1/Hygro (Invifrogen) using these sites.
  • the sequence encoding the protein C pre- cursor in the resulting plasmid, termed pCR4 is given in SEQ ID NO: 1.
  • the synthetic gene is cloned into the Kpnl-Xbal sites of pcDNA3.1 Hygro containing an intron (from pCI-Neo (Promega)) in the 5' untranslated region of the gene.
  • Mutants of protein C are constructed using Quick-Change (Stratagene). Primers are e.g. purchased from TAG Technology (Copenhagen, Denmark) containing the appropriate mutations. PCR reactions are performed according to the manufacturer's manual and the plasmids are transformed into TGI competent cells. Plasmid preparations are made on sin- gle clones and the sequences are verified using a DNA sequencer 3100 genetic Analyser (ABI)
  • CHO Kl cells for expression of protein C and variants thereof
  • CHO Kl cells are grown to near confluence in T-25 tissue culture flasks.
  • the medium on the cells is changed with 5 ml of fresh medium (e.g. MEM ⁇ (Invifrogen Gibco #32571-028), 10% FCS, 100 U/ml of penicillin, 100 ⁇ g/ml streptomycin and 5 ⁇ g/ml Vitamin K).
  • fresh medium e.g. MEM ⁇ (Invifrogen Gibco #32571-028)
  • FCS fetal calf serum
  • the cells are transfected using the Lipofec- tamin2000 (Invifrogen #116687-019) transfection agent according to the manufactures instructions. After 24 hours, 360 ⁇ g/ml Hygromycin B selection is applied to the cells. During the next few days the selective medium is changed daily until cell shedding stops. At confluence in T-25 tissue culture flask the cells are passed to a T-175 tissue culture flask and allowed to propagate under selection until confluence. The cells in the resulting pool of stable transfectants are dilution cloned and cryo-preserved for future use.
  • Dilution cloning Cells are trypsinised and counted in a hemacytometer. A dilution with approximately 10 cells/ml is made and 100 ⁇ l aliquots are dispensed into the wells of a 96 well tissue culture plate. After a week wells with single clones are identified by microscopy and marked. At confluence productive clones are screened by ELISA analysis of 24 hours cul- ture supematants. The expression level of productive clones are quantified by ELISA analysis of supematants from confluent T-25 tissue culture flasks. The best clones are then transferred to T-175 tissue culture flasks, propagated to confluence and cryo-preserved.
  • the clone with the highest expression level is chosen for production purposes. After thawing, the clone is propagated to confluence in MEM ⁇ (Invifrogen Gibco #32571-028), 10% FCS, 100 U/ml of penicillin, 100 ⁇ g/ml streptomycin and 5 ⁇ g/ml Vitamin K in a T-175 tissue culture flask and used as inoculum for one tissue culture roller bottle. The cells are propagated in roller bottles until confluent in MEM ⁇ (Invifrogen Gibco #32571 -028), 10% FCS, 100 U/ml of penicillin, 100 ⁇ g/ml streptomycin and 5 ⁇ g/ml Vitamin K.
  • the medium is changed to UltraCHO (BioWhittaker #12-724Q), 0,1% ExCyte (Serologicals Proteins Inc. # 81-129-1), 100 U/ml of penicillin, 100 ⁇ g/ml streptomycin and 5 ⁇ g/ml Vitamin K for four days with a medium shift after 2 days.
  • the medium is changed to the production medium, DMEM F-12 (Invifrogen # 11039-021), 1 : 100 ITS- A, 0,1 % ExCyte (Serologicals Proteins Inc. # 81-129-1), 100 U/ml of penicillin, 100 ⁇ g/ml sfreptomycin and 5 ⁇ g/ml Vitamin K and the medium harvested daily for purification purposes.
  • the supematants are concentrated and diafiltrated into 20 mM tris-HCl, pH 7.4, 150 mM NaCl (equilibration buffer) using a TFF-system (Benchscale, Millipore) equipped with pellicon Biomax lOkDa mwco membranes.
  • the diafiltrated supernatant is then applied onto 20 ml of equilibrated Q- sepharose FF matrix packed in a XK26 column at 20 ml/min.
  • the column is washed with the equilibration buffer until a stable baseline (measured by the absorbance at 280 nm) is obtained.
  • Elution of protein C is performed by using 20 mM tris-HCl, pH 7.4, 120 mM NaCl and 15mM CaCl 2 at a flow rate of lOml/min.
  • Fractions are analysed by SDS-page, and protein C-containing fractions are pooled.
  • the conductivity is adjusted by the addition of NaCl either as a solid or as a stock solution for optimal binding onto a phenyl sepharose 6 FF (Pharmacia).
  • the sample is then applied onto 10 ml matrix packed in a XK16/10 column equilibrated with 20 mM tris-HCl, pH 7.4, 1 M NaCl, lOmM CaCl 2 .
  • the column is washed until stable base line with the equilibration buffer.
  • protein C is specifically eluted from the column. Protein C-containing fractions are pooled and prepared for activation.
  • Protein C polypeptides are activated using the venom protein C activator, ACC-C (Nakagaki et al., Thrombosis Research 58:593-602, 1990).
  • the zymogen forms are incubated at 37°C for about 60 min in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, using a final concentration of 1 ng/ml of ACC-C.
  • the activation process is checked using the APC amidolytic activity assay and polyacrylamide gel electrophoresis analysis.
  • EPCR endothelial protein C receptor
  • Example 7 Determination of amidolytic activity APC Amidolytic Assay Amidolytic activity is determined using the peptide substrate SPECTROZYME PCa with the formula H-D-Lys( ⁇ -Cbo)-Pro-Arg-pNA.2AcOH (American Diagnostica Inc., product # 336) at a final concentration of 0.5 mM. Assays are performed at 23°C in 50 mM Tris-HCl (pH 8.3), 100 mM NaCl, 5 mM CaCl 2 . The rate of hydrolysis is recorded for 3 min at 405 nm as the change in absorbance units/min in a plate reader.
  • Anticoagulant activity is assessed by monitoring the prolongation of clotting time in the activated partial thromboplastin time (APTT) assay using Nycoplastin (Nycomed, product no. 1002448) together with Normal Hemostasis Reference Plasma (American Diagnostica Inc., catalogue no. 258N). Coagulation is started by mixing the APTT reagent containing hAPC with the normal hemostasis reference plasma at 37°C and measuring the clotting time by manual mixing.
  • APTT activated partial thromboplastin time
  • Example 9 Inactivation by alpha- 1-antitrypsin Alpha- 1-Antitrypsin Inactivation Assay hAPC is incubated with 16.6 or 42.3 ⁇ M human alpha- 1-antitrypsin (Sigma) in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM CaCl 2 containing 0.1% BSA at 37°C.
  • Example 11 In vitro half-life in human plasma Human Plasma Inactivation Assay II hAPC is incubated in 90% normal human plasma (Sigma Diagnostics, AccuclotTM reference plasma) containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM CaCl 2 at 37°C. Aliquots are removed at various time-points and assayed for APC amidolytic activity as described in the "APC Amidolytic Assay ". The residual APC activity at the various time- points is expressed in percentage of the APC activity measured at the start of the experiment. The functional in vitro half-life (expressed in minutes) is calculated as the time at which 50% of the APC activity is still present.
  • TNF ⁇ tumor necrosis factor ⁇
  • HUVEC human umbilical vein endothelial cells
  • concentrations (0 - 200 nM) of hAPC and the variants are then incubated for up to 20 hours.
  • the cells are removed by trypsination and analysed using flow cytometry measuring the surface expression of ICAM-1, VCAM-1 and/or E-selectin.
  • a FITC-conjugated anti-human E-selectin monoclonal antibody (catalog number: BBA21, R&D Systems, Minneapolis, USA) is used.
  • the anti- inflammatory effect of hAPC and the variants is determined by calculating the APC concentration needed to suppress the TNF ⁇ stimulation of E-selectin to 50% compared to the effect of TNF ⁇ obtained without hAPC.
  • This hAPC concentration is used to indicate the half maximum inhibitory concentration (IC 50 ), and these values are determined for hAPC and each individual variant.
  • Example 13 Determination of anti-inflammatory effect APC Anti-inflammatory Assay II
  • monocytic THP-1 cells (10 5 ) (ATCC, Rockville MD) are pre-incubated with various concentrations (0-1600 nM) of the variants for 16 hours. Cells are then stimulated using 0.5ug/ml LPS (Cat# 314, List for 16 hours. Cells are then stimulated using 0.5ug/ml LPS (Cat# 314, List Biological la- barotories) for 4 hours.
  • TNF alpha production in the cell culture supernatant is subsequently measured using a TNF alpha ELISA (Cat# DY210, R and D systems).
  • the anti- inflammatory effect of hAPC and the variants is determined by calculating the hAPC con- 5 centration needed to obtain 50% reduction in LPS induced TNF alfa production as compared to the effect of LPS alone. This hAPC concentration is used to indicate the half maximum inhibitory concentration (IC 50 ), and these values are determined for human hAPC and each individual polypeptide.
  • THP-1 cells are pre-incubated with the polypeptide for 30 min.
  • the off-rate constant (k off ) and the on-rate constant (k o n) of wild-type human protein C and the protein C variants are measured using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the measurements are performed on a Biacore 2000 (Biacore AB, Uppsala, Sweden).
  • the extracellular part of EPCR is immobilized on a sensor chip SA (Biacore AB) and the dissocia-
  • Ka k of i/ on
  • the obtained K d values are used to evaluate the affinity of each obtained protein C variant.
  • the assay may be conducted using either the zymogen form of wild-type human protein C (and the corresponding zymogen forms of the variants) or the activated form of wild-type human protein C (and the conesponding acti-
  • Stably transfected EPCR human 293 cells are used to measure the binding of wild- 30 type human protein C and the variants of protein C to cellular membrane bound EPCR. Increasing concentrations of 125 I-labeled human protein C or protein C variants are incubated with the human 293 cells in suspension and washed to remove excess of radio labeled APC. The K d value for each variant is determined by Scatchard analysis. A similar analysis can be performed using competition binding between the protein C variants and 125 I-labeled human protein C.
  • the assay may be conducted using either the zymogen form of wild-type human protein C (and the conesponding zymogen forms of the variants) or the activated form of wild-type human protein C (and the conesponding activated forms of the variants).
  • Protein C libraries may be displayed on the surface of COS cells using the protoplast fusion technique (Tan et al. (1998) PNAS 95:4247-4252).
  • Protoplast fusion is a technique that enables the expression of bacterial DNA in a eukaryotic system.
  • the protoplast fusion protocol has two distinct manipulations: the formation of bacterial protoplasts and the fusion of bacterial and recipient cells.
  • the cell wall of the bacteria must first be sufficiently degraded to enable fusions.
  • the formation of these bacterial protoplasts is accomplished by exposure to lysozyme, followed by short periods of incubation. Fusions are facilitated by exposure to polyethylene glycol.
  • An advantage of protoplast fusions, compared to SuperFect® (Qiagen) transfections, is that fusions may be carried out immediately following transformation; fransfected DNA does not have to be isolated and prepared. More important, the fusions are nearly clonal, allowing for the expression of single plasmid constructs.
  • the following protocol has been optimized for a specific plasmid, and rates of transfection will vary with changes in the protocol.
  • the fransfected E. coli cells containing the appropriate plasmids with the protein C library are grown at 37°C in Luria Broth containing appropriate selection antibiotic (i.e. 100 ⁇ g/ml ampicillin or 40 ⁇ g/ml kanomycin) to an absorbance of 0.6-0.7 measured at 666 nm. Chloramphenicol is added to 200 ⁇ g/ml and the culture is incubated at 37°C for 12-16 hours to amplify plasmid copy numbers.
  • appropriate selection antibiotic i.e. 100 ⁇ g/ml ampicillin or 40 ⁇ g/ml kanomycin
  • Bacteria from 25 ml of culture are pelleted by centrifugation at 3500 rpm for 15 minutes at room temperature. The culture supernatant is removed by aspiration.
  • the bacterial pellet is resuspended in 1.25 ml of chilled 20% sucrose/0.05 M Tris-HCl, pH 8.
  • T 4 Lysozyme is prepared immediately before as a 5 mg/ml in 0.25 M Tris-HCl, pH 8 stock. 0.25 ml of lysozyme is added to the bacterial suspension and incubated on ice for six minutes.
  • Plasmid expression may be analyzed and sorted using FACS at 24, 48, or 72 hours.
  • cells can be isolated based on the expression of protein C on their cell surface, using the V5-tag, as well as the affinity of the displayed protein C variants for soluble EPCR.
  • Soluble EPCR is FITC-labeled in order to identify high affinity binders.
  • the EPCR binding affinity of each surface-displayed protein C variant can be determined from equilibrium binding tifration curves.
  • Cells displaying the protein C variants are incubated in varying concentrations of FITC-labeled soluble EPCR.
  • a flow cytometer e.g. a FACSCaliburTM is used to measure the mean fluorescence of the cell populations.
  • the equilibrium dissociation constant, K d can be fitted using a suitable model (e.g. non-linear least-squares curve fit). Variants with high affinity are subjected to DNA sequencing to determine the amino acid sequence in each particular protein C variant that binds with high affinity to EPCR.
  • a suitable model e.g. non-linear least-squares curve fit
  • Cleavage of PAR-1 by APC was determined by incubating the endothelial cell line EaHy926 with serially diluted APC and then examining the residual expression of intact PAR-1 by flow cytometry. In order to compare the PAR-1 cleavage activity of the tested variants and hAPC, equal amounts - based on amidolytic activity - of the variants and hAPC were used in the assay.

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Abstract

The invention relates to the use of a human protein C variant comprising at least one amino acid modification in position 306-314, e.g. substitution of a charged residue with an uncharged residue, for the manufacture of a medicament for the treatment of a hypercoagulable state or acquired protein C deficiency, e.g. for the treatment of sepsis. The variants have a reduced anticoagulant activity compared to non-modified human activated protein C.

Description

PROTEIN C VARIANTS WITH ALTERED PROPERTIES
FIELD OF THE INVENTION
The present invention relates to protein C variants which comprise at least one modification in the loop region composed of positions 306-314 for the treatment of hypercoagulable states or acquired protein C deficiency, such as sepsis. Such variants have a reduced anticoagulant activity and a similar anti-inflammatory effect as compared to the corresponding human activated protein C (hAPC) polypeptide.
BACKGROUND OF THE INVENTION
Blood coagulation is a process involving a complex interaction of various blood components, or factors, which eventually give rise to a fibrin clot. Generally, blood components participating in the coagulation "cascade" are proenzymes or zymogens, i.e. enzymatically inactive proteins that are converted into an active form by action of an activator. Regulation of blood coagulation is accomplished enzymatically by proteolytic inactivation of the procoagulation factors Va and Villa achieved by activated protein C (Esmon, JBiol Chem 1989; 264; 4743-4746).
Protein C is a serine protease that circulates in the plasma as a zymogen with a half-life of approximately 7 hours, and plasma levels are typically in the range of 3-5 μg/1. It is produced in vivo in the liver as a single chain precursor polypeptide of 461 amino acids. The protein C precursor comprises a 42 amino acid residue signal and propeptide sequence that includes a conserved 18 amino acid propeptide sequence found in all vitamin Independent proteins (Stanley et al., Biochemistry (1999) 38:15681-7). This precursor polypeptide undergoes multiple post-translational modifications, including a) cleavage of the signal sequence and the propeptide sequence; b) cleavage of lysine and arginine residues (positions 156 and 157) to make a two-chain inactive zymogen (a 155 amino acid light chain attached via a disulfide bridge to a 262 amino acid heavy chain); c) vitamin Independent carboxylation of nine glutamic acid residues of the light chain resulting in nine gamma-carboxyglutamic acid residues in the N-terminal region of the light chain; and d) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain). Finally, the two-chain zymogen may be activated by removal of a dodecapeptide (the activation peptide) at the N-terminus of the heavy chain (positions 158-169), producing the activated protein C (APC). Protein C is activated by limited proteolysis by thrombin in complex with throm- bomodulin on the lumenal surface of the endothelial cell. As explained above, activation liberates a 12 amino acid activation peptide from the N-terminal of the heavy chain. The APC has a half-life of approximately 15 minutes in plasma. In the presence of its cofactor, protein S, APC proteolytically inactivates factors Va and Villa, thereby reducing thrombin generation (Esmon, Thromb Haemost 1993; 70; 29- 35). Protein S circulates reversibly bound to another plasma protein, C4b-binding protein. Only free protein S serves as a cofactor for APC. Since C4b-binding protein is an acute phase reactant, the plasma levels of this protein vary greatly in many diseases and thus in- fluence the anticoagulant activity of the protein C system.
The gene encoding human protein C maps to chromosome 2ql3-ql4 (Patracchini et al., Hum Genet 1989; 81; 191-192), spans over 11 kb, and comprises a coding region (exons II to IX) and a 5' untranslatable region encompassing exon I. The protein domains encoded by exons II to IX show considerable homology with other vitamin K-dependent coagulation proteins such as factor IX and X. Exon II codes for a signal peptide, while exon III codes for a propeptide and a 38 amino acid sequence containing 9 Glu residues. The propeptide contains a binding site for the carboxylase that transforms the Glu residues into dicarboxylic acid (Gla) able to bind calcium ions, a step required for phospholipid binding (Cheung et al., Arch Biochem Biophys 1989; 274; 574-581). Exons IV, V and VI encode a short connection sequence and two EGF-like domains, respectively. Exon VII encodes both a domain encompassing the 12 amino acid activation peptide and the dipeptide 156-157 which, when cleaved off, yields the mature two-chain form of the protein. Exons VIII and IX encode the serine protease domain.
The complete amino acid sequence of human protein C has been reported by Foster et al., PNAS USA 1986; 82; 4673-4677 and includes a signal peptide, a propeptide, a light chain, a heavy chain and an activation peptide. The sequence is available from the Swiss- Prot protein sequence database under entry name PRTC_HUMAN and primary accession number P04070.
APC is inhibited in plasma by the protein C inhibitor as well as by alpha- 1- antitrypsin and alpha-2-macroglobulin.
The experimental three-dimensional structure of human APC (in a Gla-domainless form) has been determined to 2.8 A resolution and reported by Mather et al., EMBO J 1996; 15; 6822-6831. The structure includes a covalently bound inhibitor (D-Phe-Pro-Arg chloromethylketone, PPACK). APC is used for the treatment of genetic and acquired protein C deficiency and has been suggested for use as an anticoagulant in patients with some forms of lupus, following stroke or myocardial infarction, after venous thrombosis, disseminated intravascular coagulation (DIC), septic shock, emboli such as pulmonary emboli, transplantation, such as bone marrow transplantation, burns, pregnancy, major surgery/trauma and adult respiratory stress syndrome (ARDS).
Recombinant APC is produced by Eli Lilly and Co. and is marketed under the name Xigris®.
PEGylated wild-type APC is described in JP 8-92294. WO 91/09960 discloses a hybrid protein comprising modifications in the heavy chain part of protein C.
WO 00/66754 reported that substitution of the residues naturally occurring in the positions 194, 195, 228, 249, 254, 302 or 316 lead to an increased half-life of APC in human blood as compared to the wild-type APC. WO 99/20767 and WO 00/66753 disclose vitamin K-dependent polypeptide variants containing modifications in the Gla domain.
WO 98/44000 broadly describes protein C variants with an increased amidolytic activity.
US 5,453,373 discloses human protein C derivatives which have altered glycosyla- tion patterns and altered activation regions, such as N313Q and N329Q.
US 5,460,953 discloses DNA sequences encoding zymogen forms of protein C which have been engineered so that one or more of the naturally occurring glycosylation sites have been removed. More specifically, US 5,460,953 discloses the variants N97Q, N248Q, N313Q and N329Q. Conjugated protein C variants, e.g. with one or more introduced glycosylation sites, are disclosed in WO 02/32461.
One major problem associated with use of hAPC (Xigris®) in the treatment of sepsis is the severe bleeding risks associated therewith. Accordingly, Xigris® has been approved by the United States Food and Drug Administration (FDA) only for "adult patients with severe sepsis who have a high risk of death (at least one dysfunctional major organ, APACHE II score above 25) with low risk of bleeding complications". As a result, a large group of sepsis patients with a high bleeding risk are excluded from treatment with Xigris®. Clearly, there is a need for a modified product which is suitable for treatment of sepsis, but which is safe to use, i.e. which has a reduced anticoagulant activity and thus is available to sepsis patients with a significant bleeding risk but which at the same time retains its anti- inflammatory properties.
Several investigators have focused on the importance of the so-called autolysis
5 loop in protein C. WO 98/4400 and Shen et al. (Thromb. Haemost. 82, 1078-1087, 1999) describe that substituting the entire autolysis loop of hAPC with the corresponding, and shorter, autolysis loop from bovine protein C leads to a variant which has an increased ami- dolytic activity (about 4-fold) as well as an increased anticoagulant activity (2-3 fold). Furthermore, it has been reported that this molecule was inhibited by alpha- 1-antitrypsin in a l o fashion similar to that of hAPC (Shen et al., Biochemistry 39, 2853-2860, 2000).
Gale et al., Blood, 96, 585-593, 2000, studied the hypothesis that the autolysis loop of protein C binds factor Va by preparing alanine mutants of charged residues in the autolysis loop. In general, it was found that by substituting these residues with alanine the resulting variants exhibited a significantly reduced anticoagulant activity, whereas the amidolytic
15 activity was reduced to a lesser extent.
It is now well established that an excessive inflammatory response accompanies the initial stages of sepsis and appears to contribute to associated organ system failure and death (Arndt et al. Intensive Care Med 27, S104-S115, 2001). Thus, the anti-inflammatory properties of an anti-sepsis drug are crucial.
20 A direct role of activated protein C in the prevention of apoptosis has recently been reported (Cheng et al., Nature Medicine 9(3):338-42, 2003). Cheng et al. found that APC directly prevents apoptosis in hypoxic human brain endothelium, and that this is not merely a secondary effect to its anti-coagulant and anti-inflammatory effects but rather is independent of its anti-coagulant effect. Further, they found that the neuroprotective effect of APC is
25 dependent upon binding to the endothelial protein C receptor (EPCR) and activation of pro- tease-activated receptor-1 (PAR-1). This EPCR-dependent activation of PAR-1 as a requirement for the anti-apoptotic effect of APC was subsequently confirmed by Mosnier et al. (Biochem J, online publication ahead of print, 8 April 2003).
The present invention is based on modifications in the autolysis loop of protein C
30 to result in variants having a reduced anti-coagulant activity and a substantially retained anti-inflammatory effect and/or anti-apoptotic effect as compared to hAPC.
Such variants open up the possibility for treatment of diseases and conditions in which an anti-inflammatory activity is required, such as in the treatment of acquired hypercoagulable states or acquired protein C deficiency, in particular sepsis, since the important anti-inflammatory properties are substantially retained, and the product is safe to use due to the significantly reduced anticoagulant properties of the variants. In addition, since the anti- apoptotic effect of APC is not believed to be related to its anti-coagulant activity, as suggested by Cheng et al. (2003), it is contemplated that the variants of the invention will sub- stantially maintain the anti-apoptotic properties of activated protein C despite having reduced anticoagulant properties.
Thus, an object of the present invention is to provide a safer protein C polypeptide, i.e. a protein C polypeptide with a significantly reduced anticoagulant activity but which substantially retains the anti-mflammatory and/or anti-apoptotic properties of hAPC, and which is suitable for treatment of various diseases such as sepsis.
BRIEF DISCLOSURE OF THE INVENTION
Thus, in a first aspect the present invention relates to the use of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2 for the manufacture of a medicament for the treatment of a hypercoagulable state or acquired protein C deficiency, in particular when the hypercoagulable state or protein C deficiency is associated with sepsis.
In a second aspect the present invention relates to a method of treating a patient with a hypercoagulable state or acquired protein C deficiency, in particular where the hyperco- agulable state or protein C deficiency is associated with sepsis, which comprises administering to said patient an effective amount of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2.
In a third aspect the present invention relates to a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2, with the proviso that the variant is not human protein C or hAPC comprising the following modifications: R306A, E307A, K308A, E309A, K311A, R312A, R314A, R306A+R312A, R306A+K311A+R312A+R314A, H303*+S304*+S305*+K308*+E307D+A310T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, K308N+A310S, K308N+A310T, E309N+K311S, E309N+K311T, A310N+R312S, A310N+R312T, R312N+R314S,
R312N+R314T, R306C, E307C, K308C, E309C, A310C, and R312C, and that the variant does not contain a mutation in position 313.
In further aspects the present invention relates to pharmaceutical compositions comprising the variants of the invention; to variants of the invention or pharmaceutical composi- tions of the invention for use as a medicament; to nucleotide sequences encoding the variants of the invention; to expression vectors comprising the the nucleotide sequences of the invention; to host cells comprising the nucleotide sequences of the invention or the expression vectors of the invention; and to methods for preparing the variants of the invention.
DETAILED DISCLOSURE OF THE INVENTION Definitions
In the context of the present application and invention the following definitions apply: When used in the present context the term "precursor protein C" or "precursor human protein C" refers to the DNA-encoded form of protein C, i.e. it includes the signal peptide and propeptide (residues -42 to -1), the light chain (residues 1-155), the Lys- Arg dipeptide (residues 156-157) and the heavy chain (residues 158-419), including the activation peptide (residues 158-169), shown in SEQ ID NO:2. The term "one-chain zymogen protein C" or "one-chain zymogen human protein C refers to the one-chain inactive form of protein C, which includes the light chain (residues 1-155), the heavy chain (residues 158-419) including the activation peptide (residues 158-169), and the Lys-Arg dipeptide (residue 156-157) shown in SEQ ID NO:2.
The term "two-chain zymogen protein C" or "two-chain zymogen human protein C" refers to the two-chain inactive form of protein C, which includes the light chain (residues 1 -155) and the heavy chain (residues 158-419) including the activation peptide (residues 158-169), shown in SEQ ID NO:2.
The term "zymogen protein C" or "zymogen human protein C" refers to both the one-chain form and the two-chain form of the zymogen protein C. The terms "activated protein C", "activated human protein C", "APC" or "hAPC" are used about the activated zymogen and include the light chain (residues 1-155) and the heavy chain without the activation peptide (i.e. residues 170-419) of SEQ ID NO:2.
Unless stated otherwise, the term "protein C" or "human protein C" is intended to encompass all of the above-mentioned forms of protein C, i.e. the "precursor protein C" form, the "zymogen protein C" form (the one-chain form as well as the two-chain form) and the "activated protein C form".
The "autolysis loop", as used herein, refers to amino acid residues 306-314 of SEQ ID NO:2. The "Gla domain", as used herein, refers to amino acid residues 1-45 of SEQ ID NO:2.
The "EGF domains", as used herein, refers to amino acid residues 55-134 of SEQ ID NO:2. The "active site region" is defined as including those amino acid residues that are described as belonging to the active site in WO 02/32461, namely: L170, 1171, D172, G173, Q184, V185, V186, L187, L188, D189, S190, K191, K192, K193, L194, A195, C196, G197, A198, T208, A209, A210, H211, C212, M213, D214, E215, S216, K217, K218, L219, L220, L228, 1240, V243, V245, N248, Y249, S250, K251, S252, T253, T254, D255, N256, D257, 1258, A259, L261, T295, L296, V297, T298, G299, W300, G301, Y302,
H303, S304, S305, R306, E307, K308, E309, A310, K311, R312, N313, R314, T315, F316, 1321, 1323, P324, V326, C331, V334, M335, S336, N337, M338, V339, M343, L344, C345, A346, G347, 1348, L349, D351, R352, Q353, D354, A355, C356, E357, G358, D359, S360, G361, G362, P363, M364, G376, L377, V378, S379, W380, G381, E382, G383, C384, G385, L386, L387, H388, N389, Y390, G391, V392, Y393 and T394.
The term "variant" is intended to cover a polypeptide which differs in one or more amino acid residues from human protein C, in particular from hAPC. Normally, a variant differs from human protein C or hAPC (SEQ ID NO:2) in 1-15 amino acid residues, such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, e.g. in 1-10 amino acid residues, 1-7 amino acid residues or 1-5 amino acid residues. As will be understood, the variants disclosed herein must contain at least one amino acid modification in the loop region constituted by amino acid residues 306-314. However, other modifications outside this region may also be present.
The term "differs" or "differs from" when used in connection with specific modifi- cations is intended to allow for additional differences being present apart from the specified amino acid difference. For instance, in addition to the modifications of amino acid residues located in the autolysis loop, the variant can comprise other substitutions, insertions or deletions which are not related to the modification of amino acid residues present in the autolysis loop. Such other alterations may e.g. be modifications performed in the active site region aimed at increasing the functional half-life of the variant. Other examples include truncation of the N- and/or C-terminus by one or more amino acid residues, or addition of one or more extra residues at the N- and/or C-terminus, e.g. addition of a methionine residue at the N- terminus as well as "conservative amino acid substitutions", i.e. substitutions performed within groups of amino acids with similar characteristics, e.g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids.
Examples of conservative substitutions include amino acids within the respective groups listed in the table below.
1 Alanine (A) Glycine (G) Serine (S) Threonine (T)
2 Aspartic acid (D) Glutamic acid (E)
3 Asparagine (N) Glutamine (Q)
4 Arginine (R) Histidine (H) Lysine (K)
5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V)
6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)
The terms "mutation" and "substitution" are used interchangeably herein. The term "modified" or "modification" includes a substitution, an insertion or a deletion. The term "introduce" is primarily intended to mean substitution of an existing amino acid residue, but may also mean insertion of an additional amino acid residue.
The term "remove" is primarily intended to mean substitution of the amino acid residue to be removed by another amino acid residue, but may also mean deletion (without substitution) of the amino acid residue to be removed. The term "conjugate" (or interchangeably "conjugated polypeptide" or "conjugated variant") is intended to indicate a heterogenous (in the sense of composite or chimeric) molecule formed by the covalent attachment of a variant to one or more non-polypeptide moieties such as polymer molecules, lipophilic compounds, sugar moieties or organic deri- vatizing agents. Preferably, the conjugated variant is soluble at relevant concentrations and conditions, i.e. soluble in physiological fluids such as blood. Examples of conjugated variants include glycosylated variant polypeptides and PEGylated variant polypeptides.
The term "covalent attachment" or "covalently attached" means that the variant polypeptide and the non-polypeptide moiety are either directly covalently joined to one another or are indirectly covalently joined to one another through an intervening moiety or moieties such as a bridge, spacer or linker moiety or moieties. The term "non-polypeptide moiety" refers to a non-polypeptide molecule that is capable of conjugating to an attachment group of the polypeptide. Preferred examples of such molecules include polymer molecules and sugar moieties.
The term "polymer molecule" or "polymer moiety" is a molecule formed by cova- lent linkage of two or more monomers, wherein none of the monomers is an amino acid residue, except where the polymer is human albumin or another abundant plasma protein. The term "polymer" can be used interchangeably with the term "polymer".
The term "sugar moiety" is intended to indicate a carbohydrate-containing molecule comprising one or more monosaccharide residues, capable of being attached to the polypeptide by way of in vivo or in vitro glycosylation. The term "in vivo glycosylation" is intended to mean any attachment of a sugar moiety occurring in vivo, i.e. during posttransla- tional processing in a glycosylating cell used for expression of the polypeptide, e.g. by way of N-linked or O-linked glycosylation. The exact oligosaccharide structure depends, to a large extent, on the glycosylating organism in question. The term "in vitro glycosylation" is intended to refer to a synthetic glycosylation produced in vitro, normally involving covalently linking a sugar moiety to an attachment group of a polypeptide, optionally using a cross-linking agent. In vivo and in vitro glycosylation are discussed in more detail below.
An "N-glycosylation site" has the sequence N-X-S/T/C", wherein X is any amino acid residue except proline, N is asparagine and S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine. An "O-glycosylation site" is the OH-group of a serine or threonine residue.
The term "attachment group" is intended to indicate a functional group of the polypeptide, in particular of an amino acid residue thereof or a carbohydrate moiety, capable of attaching a non-polypeptide moiety such as a polymer molecule, a sugar moiety, a lipophilic molecule or an organic derivatizing agent. Useful attachment groups and their matching non-polypeptide moieties are apparent from the table below.
Figure imgf000010_0001
Figure imgf000011_0001
For in vivo N-glycosylation, the term "attachment group" is used in an unconventional way to indicate the amino acid residues constituting an N-glycosylation site. Although the asparagine residue of the N-glycosylation site is the one to which the sugar moiety is attached during glycosylation, such attachment cannot be achieved unless the other amino acid residues of the N-glycosylation site are present. Accordingly, when the non-polypeptide moiety is a sugar moiety and the conjugation is to be achieved by N-glycosylation, the term "amino acid residue comprising an attachment group for the non-polypeptide moiety" as used in connection with alterations of the amino acid sequence of the polypeptide of interest is to be understood as meaning that one or more amino acid residues constituting an N-glycosylation site are to be altered in such a manner that either a functional N-glycosylation site is introduced into the amino acid sequence or removed from the sequence.
Amino acid names and atom names (e.g. CA, CB, CD, CG, SG, NZ, N, O, C, etc.) are used as defined by the Protein DataBank (PDB) (www.pdb.org-), which is based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atom names, etc.), Eur. J. Biochem., 138, 9-37 (1984) together with their corrections in Eur. J. Biochem., 152, 1 (1985)).
The term "amino acid residue" is intended to include any natural or synthetic amino acid residue, and is primarily intended to indicate an amino acid residue contained in the group consisting of the 20 naturally occurring amino acids, i.e. selected from the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y) residues.
The terminology used for identifying amino acid positions/substitutions is illustrated as follows: A39 in a given amino acid sequence indicates that position number 39 is occupied by an alanine residue in the amino acid sequence shown in SEQ ID NO:2. A39S indicates that the alanine residue of position 39 is substituted with a serine residue. Alterna- tive substitutions are indicated with a "/", e.g., A39S/T means that the alanine residue of position 39 is substituted with either a serine residue or a threonine residue, i.e. A39S/T should be construed as covering two individual substitutions, namely A39S and A39T. Multiple substitutions are indicated with a "+", e.g., A39S+K251N means that the alanine residue of position 39 is substituted with a serine residue and that the lysine residue in position 251 is substituted with an asparagine residue. The insertion of an additional amino acid residue is indicated in the following way: Insertion of a serine residue after A39 is indicated by A39AS. A deletion of an amino acid residue is indicated by an asterix. For example, deletion of the alanine residue of position 39 is indicated by A39*. Unless otherwise indicated, the numbering of amino acid residues made herein is made relative to the amino acid sequence of SEQ ID NO:2.
The term "nucleotide sequence" is intended to indicate a consecutive stretch of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semi-synthetic or synthetic origin, or any combination thereof.
The term "vector" refers to a plasmid or another nucleotide sequence that is capable of replicating within a host cell or being integrated into the host cell genome.
"Cell", "host cell", "cell line" and "cell culture" are used interchangeably herein and all such terms should be understood to include progeny resulting from growth or cultur- ing of a cell.
"Transformation" and "transfection" are used interchangeably to refer to the process of introducing DNA into a cell.
"Operably linked" refers to the covalent joining of two or more nucleotide sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences can be performed. Generally, "operably linked" means that the nucleotide sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleo- tide adaptors or linkers are used, in conjunction with standard recombinant DNA methods. The term "functional in vivo half-life" is used in its normal meaning, i.e. the time at which the activity of the variant is 50% of the initial value. The functional in vivo half-life may be determined by any suitable method known in the art.
The term "increased" as used about the functional in vivo half-life is used to indicate that the half-life of the variant is statistically significantly increased relative to that of a reference molecule, e.g. hAPC, determined under comparable conditions. Normally, functional in vivo half-life is increased when inhibition of the polypeptide is decreased. Thus, preferred variants are those which, in their activated form, have an increased functional in vivo half-life as compared to hAPC, such as Xigris®. Particularly preferred variants are those where the ratio between the functional in vivo half-life of the variant, in its activated form, and the functional in vivo half-life of hAPC is at least 1.25, more preferably at least 1.50, such as at least 1.75, e.g. at least 2, even more preferably at least 3, such as at least 4, e.g. at least 5, most preferably at least 6, such as at least 7, e.g. at least 8, at least 9 or at least 10. Clearance mechanisms of relevance for a variant polypeptide may include one or more of the reticuloendothelial systems (RES), kidney, spleen or liver, receptor-mediated degradation, or specific or non-specific proteolysis. The term "renal clearance" is used in its normal meaning to indicate any clearance taking place by the kidneys, e.g. by glomerular filtration, tubular excretion or tubular elimination. Normally, renal clearance depends on physical characteristics of the variant polypeptide, including molecular weight, size (relative to the cutoff for glomerular filtration), symmetry, shape/rigidity and charge. A molecular weight of about 67 kDa is normally considered to be a cut-off- value for renal clearance. Renal clearance may be measured by any suitable assay, e.g. an established in vivo assay. For instance, renal clearance may be determined by administering a labelled (e.g. radio- labelled or fluorescence labelled) variant polypeptide or conjugated variant to a patient and measuring the label activity in urine collected from the patient. Reduced renal clearance is determined relative to the reference molecule, such as human APC.
Unless stated otherwise, the terms "activity", "APC activity" and "activated protein C activity" refer to the amidolytic activity.
A suitable in vitro APC activity assay (entitled "APC Amidolytic Assay") is described in Example 7 herein. More particularly, a variant is classified as having "APC activity" if the variant, in its activated form, has an activity of at least 10% of the hAPC activity when tested in the "APC Amidolytic Assay" described in Example 7 herein. Preferably, the variant has an activity of at least 20% of the hAPC activity, such as an activity of at least 30% of the hAPC activity, more preferably the variant has an activity of at least 40% of the hAPC activity, such as an activity of at least 50% of the hAPC activity, even more preferably the variant has an activity of at least 60% of the hAPC activity, e.g. an activity of at least 70% of the hAPC activity, most preferably the variant has an activity of at least 80% of the hAPC activity, such as an activity of at least 90% of the hAPC activity. In a very interesting embodiment, the variant, in its activated form, has an activity when tested in the "APC Amidolytic Assay" described in Example 7 herein which is essentially the same as or higher than the activity of hAPC.
The term "anti-inflammatory effect" refers to the fact that the variants, in their acti- vated form, prevent induction of various pro-inflammatory cytokines and adhesion molecules, such as tumor necrosis factor alpha, interleukin-1, interleukin-8 and E-selectin, either in vitro or in vivo.
The anti-inflammatory effect of a variant of the invention as compared to hAPC may easily be assessed by the skilled person using the "APC Anti-inflammatory Assay I" or "APC Anti -inflammatory Assay II" disclosed in Examples 12 and 13 herein or other similar assays.
The terms "increased affinity", "higher affinity" or "improved affinity" when used in connection with the protein C-EPCR (endothelial protein C receptor) interaction refer to a higher affinity binding to the EPCR, due to increased binding energy, than for human protein C. Whether a variant has an increased affinity to the EPCR as compared to human protein C may easily be assessed by the skilled person using the "EPCR Binding Assay I" or the "EPCR Binding Assay II" disclosed in Examples 14 and 15 herein or other similar assays. Of particular interest are variants wherein the ratio between the dissociation constant for human protein C (Kd;Wt) and the dissociation constant for the variant (Kd>variant) is at least 1.5, when determined in accordance with one of the EPCR binding assays described herein. In a preferred embodiment, this ratio is at least 2, more preferably at least 3, such as at least 5, e.g. at least 10, even more preferably at least 25, such as at least 40, at least 50, at least 75, at least 90, or at least 100, e.g. at least 250, at least 500 or at least 1000. When used herein, the term "dissociation constant" is defined as the ratio between the off-rate constant (koff) and the on-rate constant (kon) for the protein C-EPCR complex, i.e. ICj = koff kon- A high affinity for the EPCR receptor corresponds to a lower ICj value.
The terms "increased resistance towards inactivation by alpha- 1-antitrypsin" and "increased resistance towards inactivation by human plasma", respectively, are intended to refer to a variant which, in its activated form, is inhibited by alpha- 1-antitrypsin or human plasma, respectively, to a lesser degree than hAPC. In order to enable the skilled person to select effective and preferred variants at an early stage, the present inventor has developed suitable preliminary tests which can easily be carried out by the skilled person in order to initially assess the performance of the variant in question. Thus, the "Alpha- 1-Antitrypsin Inactivation Assay" (described in Example 9 herein), the "Human Plasma Inactivation Assay I" (described in Example 10 herein) and the "Human Plasma Inactivation Assay II" (described in Example 11 herein) may be employed to initially assess the potential of a selected variant. Using either the first, the second, the third or all of these tests, the suitability of a selected variant to resist inactivation by either alpha- 1-antitrypsin and/or human plasma can be assessed.
Of particular interest is thus a variant which, in its activated form, has a residual activity of at least 20% when tested in the "Alpha- 1-Antitrypsin Inactivation Assay" described in Example 9 herein using an inhibitor concentration of 16.6 μM. Preferably, the variant has a residual activity of at least 25% or at least 30%, such as a residual activity of at least 40%, more preferably the variant has a residual activity of at least 50%, such as a residual activity of at least 60%, even more preferably the variant has a residual activity of at least 70%, such as a residual activity at least 75%, most preferably a residual activity of at least 80%, such as at least 85%.
Alternatively, or in addition to the above-mentioned test, the suitability of a selected variant may be tested in the "Human Plasma Inactivation Assay I". A preferred variant is one which, in its activated form, has a residual activity of at least 20% when tested in the "Human Plasma Inactivation Assay I" described in Example 10 herein. Preferably, the variant has a residual activity of at least 25% or at least 30%, such as a residual activity of at least 40%, more preferably a residual activity of at least 50%, such as a residual activity of at least 60%, even more preferably a residual activity of at least 70%, such as a residual activity at least 75%.
Alternatively, or in addition to the above-mentioned test, the suitability of a se- lected variant may be tested in the "Human Plasma Inactivation Assay II". Of interest is thus a variant which, in its activated form, has a functional in vitro half-life that is increased compared to that of hAPC.
The term "functional in vitro half-life" is used in its normal meaning, i.e. the time at which the activity of the variant is 50% of its initial value. The functional in vitro half-life may suitably be determined by the "Human Plasma Inactivation Assay II" described in Example 11 herein. The term "increased" as about the functional in vitro half-life is used to indicate that the half-life of the variant is significantly increased relative to that of a reference molecule, e.g. hAPC, determined under comparable conditions.
More particularly, of interest is a variant where the ratio between the functional in vitro half-life of the variant, in its activated form, and the functional in vitro half-life of hAPC is at least 1.25 when tested in the "Human Plasma Inactivation Assay II" described in Example 11 herein. Preferably, the ratio is at least 1.5, such as at least 2, more preferably at least 3, such as at least 4, even more preferably at least 5, such as at least 6, at least 7, at least 8, at least 9 or at least 10. The terms "decreased anticoagulant activity", "reduced anticoagulant activity" and
"lowered anticoagulant activity" are intended to refer to a variant whose capability to function as an anticoagulant has been reduced compared to a reference molecule, in particular compared to hAPC. Whether a variant has a decreased anticoagulant activity may be assessed by using the "APC Clotting Assay" described in Example 8 herein. Thus, an interest- ing variant is one which, in its activated form, has an anticoagulant activity of 0-75% of the anticoagulant activity of hAPC when tested in this assay. Preferably, the variant in its activated form has an anticoagulant activity of 0-70%, such as 0-65%, e.g. 0-60%, more preferably 0-55%), such as 0-50%, e.g. 0-45%, more preferably 0-40%, such as 0-35%, e.g. 0- 30%, most preferably 0-25%, such as 0-20%, 0-20%, 0- 15%, 0- 10% or 0-5% of the anticoagulant activity of hAPC when tested in the "APC Clotting Assay".
In one interesting embodiment of the invention, the variant has essentially no anticoagulant activity when tested in the "APC Clotting Assay" described in Example 8.
The term "reduced immunogenicity" is intended to indicate that the variant gives rise to a measurably lower immune response than a reference molecule, e.g. hAPC, as determined under comparable conditions. The immune response may be a cell or antibody mediated response (see, e.g., Roitt: Essential Immunology (8th Edition, Blackwell) for further definition of immunogenicity). Normally, reduced antibody reactivity is an indication of reduced immunogenicity. Reduced immunogenicity may be determined by use of any suitable method known in the art, e.g. in vivo or in vitro.
The terms "at least 25% of its side chain exposed to the surface of the molecule" and "at least 50% of its side chain exposed to the surface of the molecule" are defined with reference to WO 02/32461, which describes determination of the degree of side chain exposure of human protein C. It should be noted that when these terms are used in connection with introduction of an in vivo N-glycosylation site, they refer to the surface accessibility of the amino acid side chain in the position where the sugar moiety is actually attached. In many cases it will be necessary to introduce a serine or a threonine residue in position +2 relative to the asparagine residue to which the sugar moiety is actually attached (unless, of course, this position is already occupied by a serine or a threonine residue), and the posi- tions where the serine or threonine residues are introduced are allowed to be buried, i.e. to have less than 25% or 50% of their side chains exposed to the surface of the molecule.
Variants
Modifications in the autolysis loop In its broadest aspect, the variants of the invention comprise at least one modfica- tion in position 306-314 of SEQ ID NO:2, i.e at least one modification in a position selected from the group consisting of 306, 307, 308, 309, 310, 311, 312, 313 and 314.
It should be noted that protein C contains an N-glycosylation site at position N313. In a preferred embodiment, this particular N-glycosylation site is not modified, i.e. the vari- ant does not comprise any amino acid modification in position N313 or T315. Thus, in a preferred embodiment the variant comprises at least one modification in a position selected from the group consisting of 306, 307, 308, 309, 310, 311, 312 and 314, in particular position 308, 314 and 308+314. The modification may be an amino acid deletion, an amino acid insertion or an amino acid substitution. Preferably, the modification is an amino acid substitution. Normally, the variant contains 1-6, such as 1-5, 1-4 or 1-3 modifications, such as 1 or 2 substitutions, in position 306-314.
The autolysis loop contains a number of charged residues, namely R306, E307, K308, E309, K311, R312 and R314, and in a preferred embodiment one or more of these charged residues have been substituted with an uncharged residue.
Thus, in one embodiment at least one charged amino acid amino acid residue has been substituted with an uncharged or, in the case of positively charged residues in the native protein, optionally a negatively charged amino acid residue, in particular with an un- charged amino acid residue.
In another ambodiment at least one negatively charged amino acid residue has been substituted with an uncharged amino acid residue.
Examples of specific substitutions which may be performed in position 306 include substitutions selected from the group consisting of R306A, R306V, R306L, R306I, R306F, R306W, R306P, R306G, R306S, R306T, R306Y, R306N and R306Q, preferably selected from the group consisting of R306A, R306V, R306L and R306I, such as R306A.
Examples of specific substitutions which may be performed in position 307 include substitutions selected from the group consisting of E307A, E307V, E307L, E307I, E307F, E307W, E307P, E307G, E307S, E307T, E307Y, E307N and E307Q, preferably selected from the group consisting of E307A, E307V, E307L and R307I, such as E307A.
Examples of specific substitutions which may be performed in position 308 include substitutions selected from the group consisting of K308A, K308V, K308L, K308I, K308F, K308W, K308P, K308G, K308S, K308T, K308Y, K308N and K308Q, preferably selected from the group consisting of K308A, K308V, K308L and K308I, such as K308A. Examples of specific substitutions which may be performed in position 309 include substitutions selected from the group consisting of E309A, E309V, E309L, E309I, E309F, E309W, E309P, E309G, E309S, E309T, E309Y, E309N and E309Q, preferably selected from the group consisting of E309A, E309V, E309L and R309I, such as E309A. Examples of specific substitutions which may be performed in position 311 include substitutions selected from the group consisting of K311 A, K311 V, K31 1 L, K3111, K311 F, K311W, K311P, K311G, K311 S, K31 1T, K311Y, K31 1N and K31 1Q, preferably selected from the group consisting of K311A, K31 IV, K31 IL and K31 II, such as K311A. Examples of specific substitutions which may be performed in position 312 include substitutions selected from the group consisting of R312A, R312V, R312L, R312I, R312F, R312W, R312P, R312G, R312S, R312T, R312Y, R312N and R312Q, preferably selected from the group consisting of R312A, R312V, R312L and R312I, such as R312A.
Examples of specific substitutions which may be performed in position 314 include substitutions selected from the group consisting of R314A, R314V, R314L, R314I, R314F, R314W, R314P, R314G, R314S, R314T, R314Y, R314N and R314Q, preferably selected from the group consisting of R314A, R314V, R314L and R314I, such as R314A.
In a further embodiment the variant comprises two or more, e.g. two, substitutions in position 306-314, in particular two or more substitutions in positions selected from the group consisting of 306, 307, 308, 309, 310, 311, 312 and 314.
In a preferred embodiment the variant comprises substitutions in positions K308 and R314, specific examples of which include K308A+R314A, K308A+R314V, K308A+R314L, K308A+R314I, K308V+R314A, K308V+R314V, K308V+R314L, K308V+R314I, K308L+R314A, K308L+R314V, K308L+R314L, K308L+R314I, K308I+R314A, K308I+R314V, K308I+R314L and K308I+R314I, in particular K308A+R314A.
In another embodiment the variant comprises three or more, e.g. three, substitutions in position 306-314, in particular three or more substitutions in positions selected from the group consisting of 306, 307, 308, 309, 310, 311, 312 and 314. In a preferred embodiment the variant comprises substitutions in positions K308,
R314 and at least one further residue selected from the group consisting of R306, E307, E309, K311 and R312, preferably R306, K311 and R312, such as substitutions in positions selected from the group consisting of R306+K308+R314, K308+K311+R312 and K308+R312+R314. Specific examples of substitutions which may be performed in positions 306, 308 and 314 include substitutions selected from the group consisting of R306A/V/L/I+K308A/V/L/I+ R314A/V/L/I, such as R306A+K308A+R314A. Specific examples of substitutions which may be performed in positions 308, 311 and 314 include substitutions selected from the group consisting of K308A/L/N/I+K311 A L/N/I+ R314A/L/N/I, such as K308A+K311 A+R314A.
Specific examples of substitutions which may be performed in positions 308, 312 and 314 include substitutions selected from the group consisting of
K308A/L/N/I+R312A/L/V/I+ R314A/L/N/I, such as K308A+R312A+R314A.
In a still further embodiment the variant comprises four or more, e.g. four, substitutions in position 306-314, in particular four or more substitutions in positions selected from the group consisting of 306, 307, 308, 309, 310, 311, 312 and 314. In a preferred embodiment the variant comprises substitutions in positions K308,
R314 and at least two further residues selected from the group consisting of R306, E307, E309, K311 and R312, preferably R306, K311 and R312, such as substitutions in positions selected from the group consisting of R306+K308+K311+R314, R306A+K308+K312+R312 and K308+K311+R312+R314. Specific examples of substitutions which may be performed in positions 306, 308,
311, 312 and 314 include substitutions selected from the group consisting of R306A V/L/I+K308A/V/L/I+K311 A/V/L/I+R314A/V/L/I, R306A/V/L/I+K308A/V/L/I+R312A/V/L/I+R314A/V/L/I and
K308AΛ L/I+K311A/V/L/I+R312A/V/L/I+R314A/V/L/I, such as substitutions selected from the group consisting of R306A+K308 A+K311 A+R314 A,
R306A+K308A+R312A+R314A and K308A+K311A+R312A+R314A.
In a still further embodiment the variant comprises five or more, e.g. five, substitutions in position 306-314, in particular five or more substitutions in positions selected from the group consisting of 306, 307, 308, 309, 310, 311, 312 and 314. In a preferred embodiment the variant comprises substitutions in positions K308,
R314 and at least three further residues selected from the group consisting of R306, E307, E309, K311 and R312, preferably R306, K311 and R312.
Specific examples of substitutions which may be performed in positions 306, 308, 311, 312 and 314 include substitutions selected from the group consisting of R306A/V/L/I+K308A/V/L/I+K311A/V/L/I+R312A/V/L/I+R314A/V/L/I, such as the following substitutions: R306A+K308A+K311A+R312A+R314A.
In further interesting embodiments of the invention, the variant comprises one or more of the above-mentioned substitutions in combination with one or more modifications in the active site region (see the section entitled "Modifications in the active site region''' below).
Modifications in the active site region Recombinant hAPC is produced by Eli Lilly and Co. and the product (Xigris®) has been approved by the FDA for certain sepsis patients. However, relatively high doses and frequent administration are necessary to reach and sustain the desired therapeutic or prophylactic effects of hAPC due to its short half-life. As a consequence, adequate dose regulation is difficult to obtain and the need for frequent intravenous administrations of high levels of hAPC makes treatment expensive.
A molecule with a longer functional half-life would decrease the number of necessary administrations and potentially provide more optimal therapeutic hAPC levels with concomitant enhanced therapeutic effect.
The functional half-life of hAPC may be increased, e.g. as a consequence of re- duced inhibition, for example by conjugation of hAPC to a non-polypeptide moiety, e.g. PEG (polyethylene glycol) or carbohydrates. Furthermore, this may also be achieved by mutating the protein C molecule in such a way that it remains active but blocks the binding of inhibitors to the protein.
One strategy includes introducing an amino acid residue comprising an attachment group for a non-polypeptide moiety, which makes it possible to specifically adapt the polypeptide so as to make the molecule more susceptible to conjugation to the non-polypeptide moiety of choice, thereby optimizing the conjugation pattern, e.g. to ensure an optimal distribution and number of non-polypeptide moieties on the surface of the active site region and to ensure that only the attachment groups intended to be conjugated is present in the molecule, and thereby obtain a new conjugated molecule which has APC activity and in addition one or more improved properties. For instance, it is possible to design the attachment of a non-polypeptide moiety to an attachment group present in the active site region so that inactivation by human plasma or certain inhibitors, such as alpha- 1-antitrypsin, is significantly reduced. The amino acid residue comprising an attachment group for a non-polypeptide moiety is selected on the basis of the nature of the non-polypeptide moiety of choice and, in most instances, on the basis of the method in which conjugation between the variant polypeptide and the non-polypeptide moiety is to be achieved. For instance, when the non- polypeptide moiety is a polymer molecule such as a polyethylene glycol amino acid residues comprising an attachment group may be selected from the group consisting of lysine, cys- teine, aspartic acid, glutamic acid, histidine, and tyrosine, preferably cysteine or lysine. When the non-polypeptide moiety is a sugar moiety, the attachment group is e.g. an in vivo glycosylation site, preferably an N-glycosylation site. Thus, variants having an increased resistance towards inactivation by alpha- 1- antitrypsin or an increased resistance towards inactivation by human plasma comprise at least one modification in the active site region (as defined above), the rationale being that modifications in this particular region of the protein C molecule will impair binding of inhibitors (such as alpha- 1-antitrypin) to the protein C variant while still retaining a substan- tial APC activity and a substantial anti-inflammatory and/or anti-apoptotic effect. This, in turn, has the consequence that such variants will exhibit a significantly prolonged functional half-life compared to human hAPC since elimination of the inhibitor/ APC complex via hepatic receptors is avoided or at least reduced.
In a particularly preferred embodiment of the invention, the modification in the active site region comprises at least one amino acid substitution at a position which is occupied by an amino acid residue having at least 25% of its side chain exposed to the surface, such as at least 50% of its side chain exposed to the surface. In particular, the substitution comprises introduction of an amino acid residue comprising an attachment group for a non- polypeptide moiety, such as a sugar moiety or a polymer molecule. As described in WO 02/32461, the following residues of the hAPC molecule are defined as having zero side chain accessibility: G67, C89, C98, G103, C105, HI 07, C109, Y124, G142, G173, V186, L187, A198, V199, 1201, V206, L207, T208, A210, C212, V221, E235, 1258, A259, L260, L261, L263, A267, V274, 1276, L283, V297, C331, M335, A346, G361, M364, T371, F373, L374, G376, L377, V392 and 1403. The following residues are defined as having more than 25% of their side chain exposed to the surface: Q49, L51, V52, P54, L55, E56, H57, P58, C59, A60, S61, G65, H66, T68, 170, D71, G72, 173, G74, S75, F76, S77, D79, R81, S82, G83, W84, E85, R87, F88, Q90, R91, E92, F95, L96, N97, S99, L100, D101, LI 10, El 11, El 12, VI 13, Gl 14, W115, R117, S119, P122, G123, K125, G127, D128, D129, L130, L131 , Q132, H134, P135, A136, V137, K138, R143, W145, K146, D172, K174, M175, R177, R178, D180, D189, S190, K191, K192, K193, H202, P203, H211, D214, E215, S216, K217, K218, L220, R229, R230, W231, K233, W234, L236, D237, D239, K241, E242, V243, F244, V245, P247, N248, S250, K251, S252, T253, T254, D255, A264, Q265, P266, T268, S270, Q271, D280, S281, G282, E285, R286, E287, Q290, A291, G292, Q293, E294, L296, Y302, H303, S304, S305, R306, E307, K308, E309, A310, K311, R312, N313, R314, T315, F316, F320, K322, P327, H328, N329, E330, S332, E333, V334, S336, N337, M338, S340, E341, 1348, L349, G350, D351, R352, E357, S367, H369, G370, E382, G383, C384, L386, L387, H388, R398, D401, H404, G405, H406, R408 and D409. Although the active site histidine (H211) is surface exposed, H211 is not a candidate for modification according to the present invention. Furthermore, the cysteine residues listed above are normally not candidates for modification in the present context either; in particular, C384, which is located in the active site, is not a candidate for modification. The following residues are defined as having more than 50% of their side chain exposed to the surface: Q49, L51, V52, P54, L55, E56, A60, S61, G65, 170, D71, G72, 173, G74, S75, S77, D79, R81, S82, R87, R91, E92, F95, L96, N97, S99, El 11, VI 13, Gl 14, Wl 15, RI 17, P122, K125, D128, D129, L130, Q132, H134, V137, K138, K146, D172, K174, R177, S190, K191, K192, K193, D214, E215, K217, K218, R229, R230, W231, K233, D239, K241, E242, P247, N248, K251, S252, Q265, P266, T268, Q271, S281, E285, Q290, G292, Y302, S305, R306, E307, K308, E309, A310, R312, N313, T315, K322,
N329, E330, E333, S336, N337, M338, E341, 1348, G350, R352, E357, G370, G383, H388, R398, D401, H404, G405, R408 and D409.
The residues Al, N2, S3, F4, L5, E6, E7, L8, R9, H10, Sl l, S12, L13, E14, R15, E16, C17, 118, E19, E20, 121, C22, D23, F24, E25, E26, A27, K28, E29, 130, F31, Q32, N33, V34, D35, D36, T37, L38, A39, F40, W41, S42, K43, H44, V45, D46, G47, D48, R147, M148, E149, K150, K151, R152, S153, H154, L155, K410, E411, A412, P413, Q414, K415, S416, W417, A418, P419 were not included in the structure analysed in WO 02/32461 and are, in the context of the present application, regarded as being 100% exposed to the surface. Combining the list of amino acids having at more than 25% of their side chain exposed to the surface with the list of amino acids defined as in the active site region (see above), the following amino acid residues are determined to be within the active site region and to have at least 25% of their side chains exposed to the surface:
D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, H211, L220, V243, V245, N248, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, T315, F316, V334, S336, N337, M338, 1348, L349, D351, R352, E357, E382, G383, C384, L386, L387 and H388. Typically, the variant comprises 1-5 modifications in the active site region, such as 1-4 or 1-3 modifications in the active site region, for example 1 , 2 or 3 modifications in the active site region.
In a highly preferred embodiment of the invention the introduced amino acid resi- due comprising an attachment group for a non-polypeptide residue creates a glycosylation site, in particular an in vivo N-glycosylation site. Specific examples of substitutions where the introduced amino acid residue creates an in vivo N-glycosylation site include substitutions selected from the group consisting of D172N+K174S, D172N+K174T, D189N+K191 S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, K192N+L194S, K192N+L194T, K193N+A195S, K193N+A195T, D214N, D214N+S216T, E215N+K217S, E215N+K217T, S216N+K218S, S216N+K218T, K217N+L219S, K217N+L219T, K218N+L220S, K218N+L220T, L220N+R222S, L220N+R222T, V243N+V245S, V243N+V245T, V245N+P247S, V245N+P247T, S250N, S250N+S252T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, T254N+N256S, T254N+N256T, D255N+D257S, D255N+D257T, L296N, L296N+T298S, Y302N, Y302N+S304T, H303N, H303N+S305T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, V334N, V334N+S336T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, I348N+G350S, I348N+G350T, L349N+D351 S, L349N+D351T, D351N+Q353S, D351N+Q353T, R352N+D354S, R352N+D354T, E357N+D359S, E357N+D359T, G383N+G385S, G383N+G385T, L386N+H388S, L386N+H388T, L387N+N389S, L387N+N389T, H388N+Y390S and H388N+Y390T.
More preferably, the introduced in vivo N-glycosylation site is selected from the group consisting of D189N+K191 S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, D214N, D214N+S216T, K217N+L219S, K217N+L219T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, T253N+D255S, T253N+D255T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S and L386N+H388T. Even more preferably, the introduced in vivo N-glycosylation site is selected from the group consisting of D189N+K191 S, D189N+K191T, K191N+K193T, D214N, D214N+S216T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S and L386N+H388T.
Most preferably, the introduced in vivo N-glycosylation site is selected from the group consisting of D189N+K191T, K191N+K193T, D214N, K251N, S252N, T253N+D255T, Y302N, S336N+M338T, V339T, M338N, G383N+G385T and L386N+H388T, in particular D189N+K191T, D214N, K251N and L386N+H388T.
Specific examples of highly preferred substitutions include D189N+K191T, D214N and L386N+H388T as well as K251N, S252N and Y302N.
The variant may contain 1-5 in vivo glycosylation sites, such as a single in vivo glycosylation site (in addition to the already present glycosylation sites at positions 97, 248, 313 and 329) in the active site region. However, in order to obtain efficient shielding of protease cleavage sites on the surface of the parent polypeptide and/or to efficiently impair inhibitor binding, it may in some instances be desirable that the variant comprises more than one in vivo glycosylation site in the active site region, in particular 2-5 (additional) in vivo glycosylation sites, such as 2, 3, 4 or 5 (additional) in vivo glycosylation sites in the active site region, preferably introduced by one or more of the substitutions described in any of the above lists.
It will be understood that in order to prepare a variant according to this aspect of the invention, the variant must be expressed in a glycosylating host cell capable of attaching sugar moieties at the glycosylation sites or alternatively subjected to in vitro glycosylation. Examples of glycosylating host cells are given in the section below entitled "Coupling to a sugar moiety".
It is well known to the person skilled in the art that even though in vivo glycosylation sites such as N-glycosylation sites are introduced, they are not necessarily utilized. It is contemplated that such variants which contain one or more non-utilized glycoylation sites (i.e. no sugar moiety is actually attached to the introduced glycosylation site) still have interesting properties, in particular with respect to increased resistance towards inhibition by alpha- 1-antitrypsin and increased resistance towards inactivation by human plasma. Such variants comprise at least one substitution in the active site region (as defined above), in particular they comprise a substitution of an amino acid residue which is located in the active site region and which has at least 25% of its side chain exposed to the surface of the molecule (as defined above). Thus, preferred variants according to this aspect of the invention comprise a substitution in a position selected from the group consisting of D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, V334, S336, N337, M338, 1348, L349, D351, R352, E357, E382, G383, L386, L387 and H388, in particular a substitution selected from the group consisting of K251N, S252N, Y302N and S190+K192T, especially K251N and S252N. Most preferably the substitution is K251N. In another preferred embodiment of the invention the introduced amino acid residue comprising an attachment group for a non-polypeptide residue is a cysteine residue. In a similar way as described above the cysteine residue is preferably introduced in a position which is within the active site region (as defined herein) and which is occupied by an amino acid residue having at least 25% of its side chain exposed to the surface of the molecule (as defined herein), i.e. the cysteine residue is preferably introduced in a position selected from the group consisting of D172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, D255, L296, Y302, H303, S304, S305, V334, S336, V339, M338, 1348, L349, D351, R352, E357, G383, E385, L386, L387 and H388. More preferably, the cysteine residue is introduced in a positions selected from the group consisting of D189, S190, K191, D214, K217, K251, S252, T253, Y302, S336, N337, M338, G383 and L386. Even more preferably, the cysteine residue is introduced in a position selected form the group consisting of D189, K191, K251, S252, T253, Y302, S336, N337, M338, G383 and L386. Most preferably, the cysteine residue is introduced in a position selected from the group consisting of D189, D214, K251 and L386. The conjugated variant according to this embodiment typically comprises 1-5 introduced cysteine residues in the active site region, in particular 2-5 or 1-3 introduced cysteine residues, e.g. 1, 2 or 3 introduced cysteine residues.
As will be understood any introduced cysteine residue are preferably conjugated (i.e. covalently attached) to a non-polypeptide moiety, e.g. a polymer molecule, such as PEG or more preferably mPEG. The conjugation between the cysteine-containing polypeptide variant and the polymer molecule may be achieved in any suitable manner, e.g. as described in the section entitled "Conjugation to a polymer molecule", e.g. using a one step method or using the stepwise manner referred to in said section. The preferred method for PEGylating the variant is to covalently attach PEG to cysteine residues using cysteine-reactive PEGs. A number of highly specific, cysteine-reactive PEGs with different groups (e.g. orthopyridyl- disulfide, maleimide and vinylsulfone) and different size PEGs (2-20 kDa, such as 5 kDa, 10 kDa, 12 kDa or 15 kDa) are commercially available, e.g. from Nektar Therapeutics (formerly Shearwater Corp.), Huntsville, AL, USA. The conjugated variant according to this embodiment may comprise at least one second non-polypeptide moiety, such as 1-5 or 1-3 such moieties. When the first non- polypeptide moiety is a polyalkylene oxide or PEG derived polymer, the second non- polypeptide moiety is preferably a sugar moiety, in particular an in vivo attached sugar moi- ety. The sugar moiety may be present at one or more of the naturally-occurring glycosylation sites present in the parent polypeptide, or at an introduced glycosylation site. Suitable introduced glycosylation sites, in particular N-glycosylation sites, are described above
As is evident from the above list of residues that are located in the active site region and that also have at least 25% side chain exposure, a substantial number of the these positions are occupied by charged amino acid residues. Analysing the three-dimensional structure of protein C, in particular the above-identified region, it can be observed that at least some of the charged residues interact with each other. For example, K251 is believed to form a salt bridge to D214. Moreover, it can be seen that a cluster of negatively charged amino acid residues (D214, E215 and E357) is present. Without being bound by any par- ticular theory it is contemplated that the charged amino acid residues within the above- identified region, or at least some of the charged amino acid residues within this particular region, are important for capturing and/or binding the substrate/inhibitor. Therefore, amino acid substitutions which are particularly interesting according to this aspect of the present invention are those wherein a charged amino acid residue located in the active site region and also having at least 25% of its side chain exposed to the surface is substituted with an amino acid residue having no charge, in particular an amino acid residue having no charge but a polar side chain (Gly, Ser, Thr, Cys, Tyr, Asn or Gin), as well as amino acid substitutions wherein a charged amino acid residue located in the active site region and also having at least 25% of its side chain exposed to the surface is substituted with an amino acid resi- due having an opposite charge.
Specific examples of amino acid substitutions wherein the charge of the amino acid residue in question is changed to an opposite charge include D172K, D172R, D189K, D189R, K191D, K191E, K192D, K192E, K193D, K193E, D214K, D214R, E215K, E215R, K217D, K217E, K218D, K218E, K251D, K251E, D255K, D255R, D351K, D351R, R352D, R352E, E357K, E357R, E382K and E382R, such as D214K, D214R, E215K, E215R, K251D, K251E, E357K and E357R, e.g. D214K, D214R, K251D and K251E, in particular K25 ID.
Other specific examples of amino acid substitutions wherein the charged amino acid residue in question is substituted with an amino acid side chain having a polar side chain include D172G/S/T/C/Y/N/Q, D189G/S/T/C/Y/N/Q, K191G/S/T/CΛ7N/Q, K192G/S/T/C/Y/N/Q, K193G/S/T/CΛ7N/Q, D214G/S/T/C/Y/N/Q, E215G/S/T/C/Y/N/Q, K217G/S/T/C/Y/N/Q, K218G/S/T/C/Y/N/Q, K251G/S/T/C/Y/N/Q, D255G/S/T/C/Y/N/Q, D351G/S/T/C/Y/N/Q, R352G/S/T/C/Y/N/Q, E357G/S/T/C/Y/N/Q and E382G/S/T/CΛ7N/Q, such as D214G/S/T/C/Y/N/Q, E215G/S/T/C/Y/N/Q,
K251G/S/T/C/Y/N/Q and E357G/S/T/C/Y/N/Q, e.g. D214Q, E215Q, K251Q and E357Q, in particular K251Q. Another interesting substitution may be K251N+T253A.
As explained above, the increased resistance towards inactivation by alpha- 1- antitrypsin and/or human plasma may be determined and assessed by the "Alpha- 1- Antritrypsin Inactivation Assay", the "Human Plasma Inactivation Assay I" or the "Human Plasma Inactivation Assay II" disclosed herein. These assays enable the skilled person to assess, at an early stage, whether the constructed variant can be expected to have an increased functional half-life.
Preferred combination variants
As will be understood from the previous sections, the modifications discussed in connection with the active site region may be combined with modifications in the autolysis loop in order to obtain a protein C variant having an increased functional half-life, a reduced anticoagulant activity, and a substantially retained anti-inflammatory and/or anti-apoptotic effect.
Thus, in one embodiment the variant comprises i) at least one amino acid modification in position 306-314; and ii) at least one amino acid modification in the active site region. More particularly, a preferred variant is one wherein i) at least one amino acid residue selected from the group consisting of R306, E307, K308, E309, K311, R312 and R314 has been substituted with an uncharged amino acid residue; and ii) the at least one modification in the active site region is selected from the group consisting of D172N+K174S, D172N+K174T, D189N+K191S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, K192N+L194S, K192N+L194T,
K193N+A195S, K193N+A195T, D214N, D214N+S216T, E215N+K217S, E215N+K217T, S216N+K218S, S216N+K218T, K217N+L219S, K217N+L219T, K218N+L220S, K218N+L220T, L220N+R222S, L220N+R222T, V243N+V245S, V243N+V245T, V245N+P247S, V245N+P247T, S250N, S250N+S252T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, T254N+N256S, T254N+N256T, D255N+D257S, D255N+D257T, L296N, L296N+T298S, Y302N, Y302N+S304T, H303N, H303N+S305T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, V334N, V334N+S336T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, I348N+G350S, I348N+G350T, L349N+D351S, L349N+D351T, D351N+Q353S, D351N+Q353T, R352N+D354S, R352N+D354T, E357N+D359S, E357N+D359T, G383N+G385S, G383N+G385T, L386N+H388S, L386N+H388T, L387N+N389S, L387N+N389T, H388N+Y390S and H388N+Y390T. More preferably, the variant is one wherein i) at least one amino acid residue selected from the group consisting of R306, K308, K311, R312 and R314 has been substituted with an uncharged amino acid residue; and ii) the at least one modification in the active site region (as defined herein) is selected from the group consisting of D189N+K191S, D189N+K191T, S190N+K192S, S190N+K192T, K191N+K193S, K191N+K193T, D214N, D214N+S216T, K217N+L219S, K217N+L219T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S and L386N+H388T.
Even more preferably, the variant is one wherein i) at least one amino acid residue selected from the group consisting of K308 and R314 has been substituted with an uncharged amino acid residue; and ii) the at least one modification in the active site region (as defined herein) is selected from the group consisting of D189N+K191S, D189N+K191T, K191N+K193T, D214N, D214N+S216T, K251N, K251N+T253S, S252N, S252N+T254S, T253N+D255S, T253N+D255T, Y302N, Y302N+S304T, S336N+M338S, S336N+M338T, V339S, V339T, M338N, M338N+S340T, G383N+G385S, G383N+G385T, L386N+H388S and L386N+H388T.
Most preferably, the variant is one wherein i) at least one amino acid residue selected from the group consisting of K308 and R314 has been substituted with an uncharged amino acid residue selected from the group consisting of A, V, L and I; and ii) the at least one modification in the active site region (as defined herein) is selected from the group consisting of D189N+K191T, K191N+K193T, D214N, K251N, S252N, T253N+D255T, Y302N, S336N+M338T, V339T, M338N, G383N+G385T and L386N+H388T. An example of a highly preferred variant is one wherein i) at least one amino acid residue selected from the group consisting of K308 and R314 has been substituted with an uncharged amino acid residue selected from the group consisting of A, V, L and I; and ii) the at least one modification in the active site region (as defined herein) is selected from the group consisting of D189N+K191T, D214N, D251N and L386N+H388T.
Another example of a highly preferred variant is one wherein i) at least one amino acid residue selected from the group consisting of K308 and R314 has been substituted with an uncharged amino acid residue selected from the group consisting of A, V, L and I; and ii) the at least one modification in the active site region (as defined herein) is selected from the group consisting of K251D, S252N and Y302N.
The non-polypeptide moiety Amino acid residues comprising other attachment groups may be introduced by substitution into the parent polypeptide using the same approach as that illustrated above with in vivo N-glycosylation sites and cysteine residues. For instance, one or more amino acid residues comprising an acid group (glutamic acid or aspartic acid), tyrosine or lysine may be introduced into the positions discussed above. As indicated further above the non-polypeptide moiety of the conjugated variant is preferably selected from the group consisting of polymer molecules and sugar moieties (by way of in vivo glycosylation). These agents may confer desirable properties to the variant polypeptide, in particular increased functional in vivo half-life. The variant polypeptide is normally conjugated to only one type of non-polypeptide moiety, but may also be conju- gated to two or more different types of non-polypeptide moieties, e.g. to a polymer molecule and a sugar moiety.
Methods of preparing a conjugated variant
In the following sections "Conjugation to a polymer molecule " and "Conjugation to a sugar moiety", conjugation to specific types of non-polypeptide moieties is described. In general, a conjugated variant may be produced by culturing an appropriate host cell under conditions conducive for the expression of the variant polypeptide, and recovering the variant polypeptide, wherein a) the variant polypeptide comprises at least one N- or O- glycosylation site and the host cell is an eukaryotic host cell capable of in vivo glycosyla- tion, and/orb) the variant polypeptide is subjected to conjugation to a non-polypeptide moiety in vitro.
Conjugation to a polymer molecule The polymer molecule to be coupled to the variant polypeptide may be any suitable polymer molecule, such as a natural or synthetic homo-polymer or hetero-polymer, typically with a molecular weight in the range of about 300-100,000 Da, such as about 500-20,000 Da, more preferably in the range of about 500-15,000 Da, even more preferably in the range of about 2-12 kDa, such as in the range of about 3-10 kDa. When the term "about" is used herein in connection with a certain molecular weight, the word "about" indicates an approximate average molecular weight and reflects the fact that there will normally be a certain molecular weight distribution in a given polymer preparation.
Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine (i.e. poly-NH2) and a polycarboxylic acid (i.e. poly-COOH). A hetero-polymer is a polymer comprising different coupling groups, such as a hydroxyl group and an amine group.
Examples of suitable polymer molecules include polymer molecules selected from the group consisting of polyalkylene oxide (PAO), including polyalkylene glycol (PAG), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, poly- vinyl alcohol (PVA), poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid anhydride, dextran, including carboxymethyl- dextran, or any other biopolymer suitable for reducing immunogenicity and/or increasing functional in vi o half-life. Another example of a polymer molecule is human albumin or another abundant plasma protein. Generally, polyalkylene glycol-derived polymers are biocompatible, non-toxic, non-antigenic, non-immunogenic, have various water solubility properties, and are easily excreted from living organisms.
PEG is the preferred polymer molecule, since it has only few reactive groups capable of cross-linking compared to, e.g., polysaccharides such as dextran. In particular, mono- functional PEG, e.g. methoxypolyethylene glycol (mPEG), is of interest since its coupling chemistry is relatively simple (only one reactive group is available for conjugating with attachment groups on the polypeptide). Consequently, as the risk of cross-linking is eliminated, the resulting conjugated variants are more homogeneous and the reaction of the polymer molecules with the variant polypeptide is easier to control.
To effect covalent attachment of the polymer molecule(s) to the variant polypeptide, the hydroxyl end groups of the polymer molecule must be provided in activated form, i.e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succina- mide (SSA), succinimidyl propionate (SPA), succinimidyl butyrate (SBA), succinimidy carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS),
5 aldehyde, nifrophenylcarbonate (NPC), and tresylate (TRES)). Suitable activated polymer molecules are commercially available, e.g. from Nektar Therapeutics, Huntsville, AL, USA, or from PolyMASC Pharmaceuticals pic, UK.
Alternatively, the polymer molecules can be activated by conventional methods known in the art, e.g. as disclosed in WO 90/13540. Specific examples of activated linear or l o branched polymer molecules for use in the present invention are described in the Shearwater Corp. 2001 Catalog (Polyethylene Glycol and Derivatives for Biomedical Applications, incorporated herein by reference).
Specific examples of activated PEG polymers include the following linear PEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG,
15 and SCM-PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-NHS and those disclosed in US 5,932,462 and US 5,643,575.
Specific examples of activated PEG polymers particularly preferred for coupling to cysteine residues include the following linear PEGs: vinylsulfone-PEG (VS-PEG),
20 preferably vinylsulfone-mPEG (VS-mPEG); maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) and orthopyridyl-disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG). Typically, such PEG or mPEG polymers will have a size of about 5 kDa, about 10 kD, about 12 kDa or about 20 kDa.
Further information on PEGylation methods and technologies may be found in WO
25 02/32461 (incorporated by reference) and the references cited therein.
Coupling to a sugar moiety
In order to achieve in vivo glycosylation of a protein C molecule comprising one or more glycosylation sites the nucleotide sequence encoding the variant polypeptide must be 30 inserted in a glycosylating, eukaryotic expression host. The expression host cell may be selected from fungal (filamentous fungal or yeast), insect or animal cells or from transgenic plant cells. In a preferred embodiment the host cell is a mammalian cell, such as a CHO cell, BHK or HEK, e.g. HEK 293, cell, or an insect cell, such as an SF9 cell, or a yeast cell, e.g. S. cerevisiae or Pichia pas tons, or any of the host cells mentioned hereinafter. Although in v vo glycosylation is preferred, covalent in vitro coupling of sugar moieties such as dextran to amino acid residues of the variant polypeptide may also be used, e.g. as described in WO 02/32461 (incorporated by reference) and the references cited therein.
Methods of preparing a protein C variant
The polypeptide variant, optionally in glycosylated form, may be produced by any suitable method known in the art. Such methods include constructing a nucleotide sequence encoding the variant polypeptide and expressing the sequence in a suitable transformed or transfected host. Preferably, the host cell is a garnma-carboxylating host cell such as a mammalian cell. However, variant polypeptides may be produced, albeit less efficiently, by chemical synthesis or a combination of chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.
A nucleotide sequence encoding a polypeptide variant may be constructed by iso- lating or synthesizing a nucleotide sequence encoding the parent protein C, such as protein C with the amino acid sequence shown in SEQ ID NO:2, and then changing the nucleotide sequence so as to effect introduction (i.e. insertion or substitution) or removal (i.e. deletion or substitution) of the relevant amino acid residue(s).
The nucleotide sequence is conveniently modified by site-directed mutagenesis in accordance with conventional methods. Alternatively, the nucleotide sequence may be prepared by chemical synthesis, e.g. by using an oligonucleotide synthesizer, wherein oligonu- cleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced. For example, several small oligonucleotides coding for por- tions of the desired polypeptide may be synthesized and assembled by PCR (polymerase chain reaction), ligation or ligation chain reaction (LCR) (Barany, PNAS 88:189-193, 1991). The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
Persons skilled in the art will be capable of selecting suitable vectors, expression control sequences, hosts, markers, etc. for expressing the polypeptide, e.g. as described in WO 02/32461.
The recombinant vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
The vector is preferably an expression vector in which the nucleotide sequence encoding the polypeptide variant is operably linked to additional segments required for tran- scription of the nucleotide sequence. The vector is typically derived from plasmid or viral DNA. A number of suitable expression vectors for expression in the host cells mentioned herein are commercially available or described in the literature. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalo virus. Specific vectors are, e.g., pCDNA3.1(+)\Hyg (Invifrogen, Carlsbad, CA, USA) and pCI-neo (Stratagene, La Jolla, CA, USA). Useful expression vectors for yeast cells include the 2μ plasmid and derivatives thereof, the POT1 vector (US 4,931,373), the pJSO37 vector described in Okkels, Ann. New York Acad. Sci. 782, 202-207, 1996, and pPICZ A, B or C (Invifrogen). Useful vectors for insect cells include pVL941, pBG311 (Cate et al., Cell 45: 685-98 (1986)), pBluebac 4.5 and pMelbac (both available from Invifrogen). Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pBR322, pET3a and ρET12a (both from Novagen Inc., WI, USA), wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Other vectors for use in this invention include those that allow the nucleotide sequence encoding the variant polypeptide to be amplified in copy number. Such amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g. US 4,470,461 ; and Kaufman et al., Mol. Cell. Biol. 2:1304- 19, 1982) and glutamine synthetase ("GS") amplification (see, e.g., US 5,122,464 and EP 338841).
The recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 origin of replication. When the host cell is a yeast cell, suitable sequences enabling the vector to replicate are the yeast plasmid 2μ replication genes REP 1-3 and origin of replication.
The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespom.be TPI gene (described by Russell, Gene 40:125- 30, 1985), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. For S. cerevisiae, selectable markers include ura3 and leu2. For filamentous fungi, selectable markers include amdS, pyrG, arcB, niaD and sC. The term "control sequences" is defined herein to include all components which are necessary or advantageous for the expression of the variant polypeptide. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activating sequence, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter.
A wide variety of expression control sequences may be used in the present invention. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors as well as any sequence known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
Examples of suitable control sequences for directing transcription in mammalian cells include the early and late promoters of S V40 and adenovirus, e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovi- rus immediate-early gene promoter (CMV), the human elongation factor lα (EF-lα) promoter, the Drosophila minimal heat shock protein 70 promoter, the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC) promoter, the human growth hormone terminator, SV40 or adenovirus Elb region polyadenylation signals and the Kozak consensus sequence (Kozak, JMol Biol 1987, 196(4):947-50). In order to improve expression in mammalian cells a synthetic intron may be inserted in the 5' untranslated region of the nucleotide sequence encoding the polypeptide. An example of a synthetic intron is the synthetic intron from the plasmid pCI-Neo (available from Promega Corporation, WI, USA).
Examples of suitable control sequences for directing transcription in insect cells include the polyhedrin promoter, the PI 0 promoter, the Autographa califomica polyhedro- sis virus basic protein promoter, the baculovirus immediate early gene 1 promoter and the baculovirus 39K delayed-early gene promoter, and the SV40 polyadenylation sequence. Examples of suitable control sequences for use in yeast host cells include the promoters of the yeast α-mating system, the yeast triose phosphate isomerase (TPI) promoter, promoters from yeast glycolytic genes or alcohol dehydrogenase genes, the ADH2-4c promoter, and the inducible GAL promoter. Examples of suitable control sequences for use in filamentous fungal host cells include the ADH3 promoter and terminator, a promoter derived from the genes encoding Aspergillus oryzae TAKA amylase triose phosphate isomerase or alkaline protease, an A. niger α-amylase, A. niger or A. nidulans glucoamylase, A. nidulans acetami- dase, Rhizomucor miehei aspartic proteinase or lipase, the TPI1 terminator and the ADH3 terminator. Examples of suitable control sequences for use in bacterial host cells include promoters of the lac system, the trp system, the TAC or TRC system, and the major pro- moter regions of phage lambda.
The nucleotide sequence encoding a protein C polypeptide variant, whether prepared by site-directed mutagehesis, synthesis, PCR or other methods, may optionally include a nucleotide sequence that encode a signal peptide. The signal peptide is present when the polypeptide is to be secreted from the cells in which it is expressed. Such signal peptide, if present, should be one recognized by the cell chosen for expression of the polypeptide. The signal peptide may be homologous (e.g. be that normally associated with human protein C) or heterologous (i.e. originating from another source than human protein C) to the polypeptide or may be homologous or heterologous to the host cell, i.e. be a signal peptide normally expressed from the host cell or one which is not normally expressed from the host cell. Accordingly, the signal peptide may be prokaryotic, e.g. derived from a bacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian, or insect or yeast cell.
Any suitable eukaryotic or prokaryotic host may be used to produce the variant polypeptide, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants, although eu- karyotic hosts are preferred. Examples of bacterial host cells include gram-positive bacteria such as strains of Bacillus, e.g. B. brevis orB. subtilis, Pseudomonas or Streptomyces, or gram-negative bacteria, such as strains of E. coli. The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang et al., 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., Young et al., 1961, Journal of Bacteriology 81 : 823-829, or Dubnau et al., 1971, JMolBiol 56: 209-221), electroporation (see, e.g., Shigekawa et al., 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler et al., 1987, Journal of Bacteriology 169: 5771-5278). Examples of suitable filamentous fungal host cells include strains oϊ Aspergillus, e.g. A. oryzae, A. niger, or A. nidulans, Fusarium or Trichoderma. Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and US 5,679,543. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Examples of suitable yeast host cells include strains of Saccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Klyveromyces, Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H. polymorpha or Yarrowia. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; Ηinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920: and as disclosed by Clontech Laboratories, Inc, Palo Alto, CA, USA (in the product protocol for the Yeastmaker™ Yeast Transformation System Kit). Examples of suitable insect host cells include a Lepidoptora cell line, such as S. frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (High Five) (US 5,077,214). Transformation of insect cells and production of heterologous polypeptides therein may be performed as described by Invifrogen. Examples of suitable mammalian host cells include Chinese hamster ovary (CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL- 1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL-1573)), as well as plant cells in tissue culture. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. Also, the mammalian cell, such as a CHO cell, may be modified to express sialyltransferase, e.g. 1,6-sialyltransferase, e.g. as described in US 5,047,335, in order to provide improved glycosylation of the protein C polypeptide.
In order to increase secretion it may be of particular interest to produce the variant polypeptide together with an endoprotease, in particular a PACE (Paired basic amino acid converting enzyme) (e.g. as described in US 5,986,079), such as a Kex2 endoprotease (e.g. as described in WO 00/28065).
Methods for introducing exogeneous DNA into mammalian host cells include calcium phosphate-mediated transfection, electroporation, DEAE-dextran mediated transfec- tion, lipo some-mediated transfection, viral vectors and the transfection method described by Life Technologies Ltd, Paisley, UK using Lipofectamin 2000. These methods are well known in the art and e.g. described by Ausbel et al. (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. The cultivation of mammalian cells are conducted according to established methods, e.g. as disclosed in (Animal Cell Biotechnology, Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc, Totowa, New Jersey, USA and Harrison MA and Rae IF, General Techniques of Cell Culture, Cambridge University Press 1997).
In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the variant polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large- scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
The resulting variant polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, ultra-filtration, extraction or precipitation.
The variant polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelec- tric focusing), differential solubility (e.g., ammonium sulfate precipitation) or extraction (see, e.g., Protein Purification (2nd Edition), Janson and Ryden, editors, Wiley, New York, 1998).
The variant polypeptide may be activated by any suitable method known in the art, e.g. by thrombin as described in EP 1 087 011 or by the venom protein C activator, ACC-C (Nakagaki et al., Thrombosis Research 58:593-602, 1990).
Variants of the invention The present invention also provides novel human protein C polypeptides. Thus, a further aspect of the present invention relates to a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2, with the proviso that the variant is not human protein C or hAPC comprising the following modifica- tions: R306A, E307A, K308A, E309A, K311 A, R312A, R314A, R306A+R312A, R306A+K311A+R312A+R314A, H303*+S304*+S305*+K308*+E307D+A310T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, K308N+A310S, K308N+A310T, E309N+K311S, E309N+K311T, A310N+R312S, A310N+R312T, R312N+R314S, R312N+R314T, R306C, E307C, K308C, E309C, A310C and R312C, and that the variant does not contain a mutation in position 313. The invention further includes the use of such protein C polypeptides as a medicament, in particular for the treatment of sepsis and other conditions treatable with protein C as described below.
In preferred embodiments of the invention the variant is as described in the above sections entitled "Modifications in the autolysis loop", "Modifications in the active site region" and "Preferred combination variants".
It will be understood that details and particulars concerning the variants mentioned above in connection with the use or method aspect of the invention will also apply to the variant aspect of the invention, whenever appropriate.
Pharmaceutical compositions and use
In one embodiment, the present invention relates to the use of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2 for the manufacture of a medicament for reducing inflammation. In another embodiment, the invention relates to the use of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2 for the manufacture of a medicament for the prevention or treatment of a hypercoagulable state or acquired protein C deficiency, e.g. when the hypercoagulable state or protein C deficiency is associated with sepsis; transplantations, such as bone marrow transplantation; bums; pregnancy; major surgery; trauma; or adult respiratory distress syndrome (ARDS), in particular sepsis. Thus, in a particular embodiment the present invention relates to the use of a human protein C variant comprising at least one amino acid modification in position 306- 314 relative to SEQ ID NO:2 for the manufacture of a medicament for the treatment of sepsis. Further, the present invention relates to a method of reducing inflammation in a patient, which comprises administering to the patient an effective amount of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2. The invention also relates to a method of treating a patient with a hypercoagulable state or acquired protein C deficiency, e.g. where the hypercoagulable state or protein C deficiency is associated with sepsis; transplantations, such as bone marrow transplantation; burns; pregnancy; major surgery; trauma; or adult respiratory distress syndrome (ARDS), in particular sepsis, which comprises administering to the patient an effective amount of a hu- man protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2. In a particular preferred embodiment, this method is for the treatment of sepsis
In the present context the term "hypercoagulable state(s)" refers to excessive coagulability associated with disseminated intravascular coagulation, prethrombotic conditions, activation of coagulation, or congenital or acquired deficiency of clotting factors, such as APC.
Sepsis is defined as a systemic inflammatory response to infection, associated with and mediated by the activation of a number of host defense mechanisms including the cyto- kine network, leukocytes, and the complement and coagulation/fibrinolysis systems (Mest- ers et al., Blood 88:881-886, 1996). Disseminated intravascular coagulation (DIC), with widespread deposition of fibrin in the microvasculature of various organs, is an early manifestation of sepsis/septic shock. DIC is an important mediator in the development of the multiple organ failure syndrome and contributes to the poor prognosis of patients with septic shock (Founder et al., Chest 101 :816-823, 1921). Several encouraging pre-clinical studies using protein C in various animal models of sepsis have been reported. A study in a baboon sepsis model by Taylor et al. (J. Clin. Invest. 79:918-25, 1987) used plasma-derived human activated protein C. The animals were treated prophylactically (i.e., the APC was given at the start of the two hour infusion of the LD100 E. coli). Five out of five animals survived 7 days and were considered permanent survivors to the experimental protocol. In control animals receiving an identical infusion of E. coli, five out of five animals died in 24 to 32 hours. The efficacious dose was 7 to 8 mg/kg.
In a lipopolysaccharide (LPS; E. coli) sepsis model in rats (Murakami et al., Blood 87:642-647, 1996), the pulmonary vascular injury induced by LPS was inhibited by human plasma derived activated protein C at a dose of 100 μg/kg. Furthermore, in a ligation and puncture sepsis model in rabbits, Okamoto et al. (Gastroenterology 106:A747, 1994), demonstrated that plasma-derived human activated protein C was effective in protecting the animals from coagulopathy and organ failure at a dose of 12 μg/kg/hr for nine hours. Due to the species specificity of APC, results obtained in these animals are not necessarily predictive to the treatment of humans, and the efficacious dose level of human activated protein C is extremely variable depending upon the animal model selected. For example, the serum half-life of human activated protein C in humans is 30 to 40 minutes, compared to a half-life of 8 to 10 minutes in baboons and 90 minutes in rabbits. There have been numerous recent attempts to treat sepsis in humans, for the most part using agents that block inflammatory mediators associated with the pathophysiology of this disease. However, clinical studies with a variety of agents that block inflammatory mediators have been unsuccessful (reviewed in Natanson et al., Ann. Intern. Med 120:771-783, 1994, and Gibaldi, Pharmacotherapy 13:302-308, 1993). Since many of the mediators in- volved in inflammation are compensatory responses, and therefore have salutary effects, some investigators have suggested that blocking their action may not be appropriate (see, e.g., Parrillo, N. Engl. J Med. 328:1471-1477, 1993).
More recently, phase III trials for the treatment of sepsis (Bernard et al., NEnglJ Med, 344, 699-709, 2001) were completed. Patients suffering from severe sepsis were given doses of 24 μg/kg/h for a total duration of 96 hours as infusion. A total of 1520 persons were involved in the trial and it was found that the 28 days mortality rate was reduced from 31% to 25%.
A "patient" for the purposes of the present invention includes both humans and other mammals. Thus the methods are applicable to both human therapy and veterinary ap- plications.
The variant is administered to patients in an effective dose. By "effective dose" herein is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose will depend on the disorder to be treated, and will be ascertainable by one skilled in the art using known techniques. As mentioned above, in the treatment of severe sepsis 24 μg/kg/h of hAPC is administered for 96 hours as continous infusion, which conesponds to a total amount of protein of about 230 mg for a patient having a body weight of about 100 kg. Those variants disclosed herein that have an increased functional half-life are contemplated to be more efficient, in particular in the treatment of sepsis, than the corresponding hAPC. The increased efficacy means that the effective dose needed to obtain the desired effect for a particular disorder can be smaller (less protein need to be administered) than the effective dose of hAPC or, alternatively, that by using the same dose as presently being used for hAPC, the treatment of patients with the variants disclosed herein will be more efficient in that a decrease in the mortality rate, as compared to a similar treatment with hAPC, will be observed. Moreover, such variants permit less frequent administration, such as bolus injections, of the variants. For example, the variants may be administered by a either a bolus or infusion or as a combination thereof with doses which range from 1 μg/kg body weight as a bolus every 2nd hour for several days (e.g. for 96 hours) to 1 mg/kg body weight as a bolus once every 4th day. Preferably, as low a dose as possible is administered as infrequently as possible, e.g. 1-500 μg/kg body weight, preferably 1-250 μg/kg body weight, such as 1-100 μg/kg body weight, more preferably 1-50 μg/kg body weight is administered as a bolus every 4-96 hours, e.g. every 8-96 hours, such as every 16-96 hours, every 24-96 hours, every 40-96 hours, every 48-96 hours, every 56-96 hours, or every 72-96 hours.
Preferred variant are those where the ratio between the AUC (area under the curve) of the variant, in its activated form, and the AUC of hAPC is at least 1.25 when tested in the "Human Plasma Inactivation Assay II" described in Example 11 herein. Preferably, the ratio is at least 1.5, such as at least 2, e.g. at least 3, more preferably the ratio is at least 4, such as at least 5, e.g. at least 6, even more preferably the ratio is at least 7, such as at least 8, e.g. at least 9, most preferably the ratio is at least 10.
In a further aspect the present invention relates to a pharmaceutical composition comprising a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2, with the proviso that the variant is not human protein C or hAPC comprising the following modifications: R306A, E307A, K308A, E309A, K311A, R312A, R314A, R306A+R312A, R306A+K311A+R312A+R314A, H303*+S304*+S305*+K308*+E307D+A310T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, K308N+A310S, K308N+A310T, E309N+K311S, E309N+K311T,
A310N+R312S, A310N+R312T, R312N+R314S, R312N+R314T, R306C, E307C, K308C, E309C, A310C and R312C, and that the variant does not contain a mutation in position 313, and a pharmaceutically acceptable carrier or excipient. In the present context, the term "pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the patients to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceuti- cal Sciences, 19th edition, A. R. Gennaro, Ed., Mack Publishing Company [1995]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000] ; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]).
The variant can be used "as is" and/or in a salt form thereof. Suitable salts include, but are not limited to, salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, as well as e.g. zinc salts. These salts or complexes may by present as a crystalline and/or amorphous structure.
The pharmaceutical composition of the invention may be administered alone or in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the variant, either concurrently or in accordance with another treatment schedule. In addition, the variant or pharmaceutical composition may be used as an adjuvant to other therapies.
The pharmaceutical composition of the invention may be formulated in a variety of forms, e.g. as a liquid, gel, lyophilized, or as a compressed solid. The preferred form will depend upon the particular indication being treated and will be readily able to be determined by one skilled in the art.
The administration of the formulations of the present invention can be performed in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, in- tracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other acceptable manner. The formulations can be administered continuously by infusion, although bolus injection is acceptable, using techniques well known in the art, such as pumps or implantation. In some instances the formulations may be directly applied as a solution or spray.
Parenteral compositions
An example of a pharmaceutical composition is a solution designed for parenteral administration. Although in many cases pharmaceutical solution formulations are provided in liquid form, appropriate for immediate use, such parenteral formulations may also be provided in frozen or in lyophilized form. In the former case, the composition must be thawed prior to use. The latter form is often used to enhance the stability of the active compound contained in the composition under a wider variety of storage conditions, as it is recognized by those skilled in the art that lyophilized preparations are generally more stable than their liquid counterparts. Such lyophilized preparations are reconstituted prior to use by the addition of one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.
In case of parenterals, they are prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the polypeptide having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers typi- cally employed in the art (all of which are termed "excipients"), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives.
Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are typically present at a concentration ranging from about 2 mM to about 50 mM Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate- disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid- monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fu- marate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium gly- conate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyucon- ate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid- sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid- potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additional possibilities are phosphate buffers, histidine buffers and trimethylamine salts such as Tris.
Preservatives are added to retard microbial growth, and are typically added in amounts of e.g. about 0.1%-2% (w/v). Suitable preservatives for use with the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octade- cyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g. benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.
Isotonicifiers are added to ensure isotonicity of liquid compositions and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in an amount between 0.1% and 25% by weight, typically 1% to 5%, taking into account the relative amounts of the other ingredients.
Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, omithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e. <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpynolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffi- nose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on the active protein weight.
Non-ionic surfactants or detergents (also known as "wetting agents") may be present to help solubilize the therapeutic agent as well as to protect the therapeutic polypeptide against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the polypeptide. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers (Tween®-20, Tween®-80, etc.).
Additional miscellaneous excipients include bulking agents or fillers (e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) and cosolvents.
The active ingredient may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example hydroxy- methylcellulose, gelatin or poly-(methylmethacylate) microcapsules, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra. Parenteral formulations to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.
Sustained release preparations
Suitable examples of sustained-release preparations include semi-permeable matri- ces of solid hydrophobic polymers containing the variant, the matrices having a suitable form such as a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethyl- ene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the ProLease® technology or Lupron Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethyl ene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for long periods such as up to or over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated polypeptides remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio- disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, ly- ophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
The invention is further illustrated by the following non-limiting examples.
EXAMPLES
Example 1 - Construction of protein C expression vector
A gene encoding the human protein C precursor is constructed by assembly of synthetic oligonucleotides by PCR using methods similar to the ones described in Stemmer et al. (1995) Gene 164, pp. 49-53. The native protein C signal sequence is maintained in order to allow secretion of the gene product. The synthetic gene is designed with a Nhel site at the 5 '-end and a Xbal site at the 3 '-end and subcloned behind the CMV promoter in pcDNA3.1/Hygro (Invifrogen) using these sites. The sequence encoding the protein C pre- cursor in the resulting plasmid, termed pCR4, is given in SEQ ID NO: 1.
Furthermore, in order to test for a higher gene expression, the synthetic gene is cloned into the Kpnl-Xbal sites of pcDNA3.1 Hygro containing an intron (from pCI-Neo (Promega)) in the 5' untranslated region of the gene.
Example 2 - Site directed mutasenesis
Mutants of protein C are constructed using Quick-Change (Stratagene). Primers are e.g. purchased from TAG Technology (Copenhagen, Denmark) containing the appropriate mutations. PCR reactions are performed according to the manufacturer's manual and the plasmids are transformed into TGI competent cells. Plasmid preparations are made on sin- gle clones and the sequences are verified using a DNA sequencer 3100 genetic Analyser (ABI)
Example 3 - Production
Transfection ofCHO Kl cells for expression of protein C and variants thereof Typically, CHO Kl cells are grown to near confluence in T-25 tissue culture flasks.
On the day of transfection the medium on the cells is changed with 5 ml of fresh medium (e.g. MEMα (Invifrogen Gibco #32571-028), 10% FCS, 100 U/ml of penicillin, 100 μg/ml streptomycin and 5 μg/ml Vitamin K).
Using a derivative of the pcDNA3.1/Hygro (Invifrogen) expression plasmid, en- coding the appropriate protein C variant, the cells are transfected using the Lipofec- tamin2000 (Invifrogen #116687-019) transfection agent according to the manufactures instructions. After 24 hours, 360 μg/ml Hygromycin B selection is applied to the cells. During the next few days the selective medium is changed daily until cell shedding stops. At confluence in T-25 tissue culture flask the cells are passed to a T-175 tissue culture flask and allowed to propagate under selection until confluence. The cells in the resulting pool of stable transfectants are dilution cloned and cryo-preserved for future use.
Dilution cloning Cells are trypsinised and counted in a hemacytometer. A dilution with approximately 10 cells/ml is made and 100 μl aliquots are dispensed into the wells of a 96 well tissue culture plate. After a week wells with single clones are identified by microscopy and marked. At confluence productive clones are screened by ELISA analysis of 24 hours cul- ture supematants. The expression level of productive clones are quantified by ELISA analysis of supematants from confluent T-25 tissue culture flasks. The best clones are then transferred to T-175 tissue culture flasks, propagated to confluence and cryo-preserved.
Production of protein C and variants thereof Typically, the clone with the highest expression level is chosen for production purposes. After thawing, the clone is propagated to confluence in MEMα (Invifrogen Gibco #32571-028), 10% FCS, 100 U/ml of penicillin, 100 μg/ml streptomycin and 5 μg/ml Vitamin K in a T-175 tissue culture flask and used as inoculum for one tissue culture roller bottle. The cells are propagated in roller bottles until confluent in MEMα (Invifrogen Gibco #32571 -028), 10% FCS, 100 U/ml of penicillin, 100 μg/ml streptomycin and 5 μg/ml Vitamin K. Then the medium is changed to UltraCHO (BioWhittaker #12-724Q), 0,1% ExCyte (Serologicals Proteins Inc. # 81-129-1), 100 U/ml of penicillin, 100 μg/ml streptomycin and 5 μg/ml Vitamin K for four days with a medium shift after 2 days. Finally, the medium is changed to the production medium, DMEM F-12 (Invifrogen # 11039-021), 1 : 100 ITS- A, 0,1 % ExCyte (Serologicals Proteins Inc. # 81-129-1), 100 U/ml of penicillin, 100 μg/ml sfreptomycin and 5 μg/ml Vitamin K and the medium harvested daily for purification purposes.
Example 4- Purification Frozen supematants (10-20 liters) are thawed at 4°C. The supematants are concentrated and diafiltrated into 20 mM tris-HCl, pH 7.4, 150 mM NaCl (equilibration buffer) using a TFF-system (Benchscale, Millipore) equipped with pellicon Biomax lOkDa mwco membranes. The diafiltrated supernatant is then applied onto 20 ml of equilibrated Q- sepharose FF matrix packed in a XK26 column at 20 ml/min. The column is washed with the equilibration buffer until a stable baseline (measured by the absorbance at 280 nm) is obtained. Elution of protein C is performed by using 20 mM tris-HCl, pH 7.4, 120 mM NaCl and 15mM CaCl2 at a flow rate of lOml/min.
Fractions are analysed by SDS-page, and protein C-containing fractions are pooled. The conductivity is adjusted by the addition of NaCl either as a solid or as a stock solution for optimal binding onto a phenyl sepharose 6 FF (Pharmacia).
The sample is then applied onto 10 ml matrix packed in a XK16/10 column equilibrated with 20 mM tris-HCl, pH 7.4, 1 M NaCl, lOmM CaCl2. The column is washed until stable base line with the equilibration buffer. By applying a step gradient of 20 mM tris-HCl, pH 7.4, 1 M NaCl, 5 mM EDTA, protein C is specifically eluted from the column. Protein C-containing fractions are pooled and prepared for activation.
Example 5 - Activation
Protein C polypeptides are activated using the venom protein C activator, ACC-C (Nakagaki et al., Thrombosis Research 58:593-602, 1990). The zymogen forms are incubated at 37°C for about 60 min in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, using a final concentration of 1 ng/ml of ACC-C. The activation process is checked using the APC amidolytic activity assay and polyacrylamide gel electrophoresis analysis.
Example 6 -Production of Endothelial Protein C Receptor (EPCR)
The extracellular part (amino acid 1 to 210) of endothelial protein C receptor (EPCR) is cloned from a brain cDNA library into the pCDNA3.1/Hugro(+) plasmid (Invi- trogen). Transient expression of EPCR is performed using the Fugene transfection reagent (Roche) in Cos-7 cells grown in DMEM (Gibco 21969-035) supplemented with 10% fetal Serum, 2 mM L-glutamine, 100 U/ml of penicillin, 100 μg/ml streptomycin and 5 μg/ml vitamin K. On the day of transfection the medium is substituted with fresh medium 4-5 hours before transfection. The day after transfection the medium is substituted with serum- free production medium based on DMEM (Gibco 31053-028) supplemented with 2 mM L- glutamine, 1 mM Sodium Pyruvate, 1/500 Ex-cyte (serologicals) 1/100 ITS A (Gibco 51300- 044), 100 U/ml of penicillin, 100 μg/ml streptomycin and 5 μg/ml vitamin K. After two days of incubation the medium is harvested and expressed EPCR are analysed for production and activity
Example 7 - Determination of amidolytic activity APC Amidolytic Assay Amidolytic activity is determined using the peptide substrate SPECTROZYME PCa with the formula H-D-Lys(γ-Cbo)-Pro-Arg-pNA.2AcOH (American Diagnostica Inc., product # 336) at a final concentration of 0.5 mM. Assays are performed at 23°C in 50 mM Tris-HCl (pH 8.3), 100 mM NaCl, 5 mM CaCl2. The rate of hydrolysis is recorded for 3 min at 405 nm as the change in absorbance units/min in a plate reader.
Example 8 — Determination of anticoagulant activity APC Clotting Assay
Anticoagulant activity is assessed by monitoring the prolongation of clotting time in the activated partial thromboplastin time (APTT) assay using Nycoplastin (Nycomed, product no. 1002448) together with Normal Hemostasis Reference Plasma (American Diagnostica Inc., catalogue no. 258N). Coagulation is started by mixing the APTT reagent containing hAPC with the normal hemostasis reference plasma at 37°C and measuring the clotting time by manual mixing.
Example 9 — Inactivation by alpha- 1-antitrypsin Alpha- 1-Antitrypsin Inactivation Assay hAPC is incubated with 16.6 or 42.3 μM human alpha- 1-antitrypsin (Sigma) in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM CaCl2 containing 0.1% BSA at 37°C. After 20 hours incubation a 15 μl sample of the incubated mixtures is added to 110 μl 50 mM Tris-HCl (pH 8.3), 100 mM NaCl, 5 mM CaCl2 in microplates and assayed for APC amidolytic activity as described in the "APC Amidolytic Assay" . The remaining activity is calculated by normalizing with the activity obtained in samples lacking alpha- 1-antitrypsin but otherwise incubated under identical conditions.
Example 10 -Inactivation by human plasma Human Plasma Inactivation Assay I hAPC is incubated in 90% normal human plasma (Sigma Diagnostics, Accuclot™ reference plasma) containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM CaCl2 at 37°C. Aliquots are removed after 200 min and assayed for APC amidolytic activity as described in the "APC Amidolytic Assay ". The residual APC activity after 200 min is expressed in percentage of the APC activity measured at the start of the experiment. Example 11 — In vitro half-life in human plasma Human Plasma Inactivation Assay II hAPC is incubated in 90% normal human plasma (Sigma Diagnostics, Accuclot™ reference plasma) containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM CaCl2 at 37°C. Aliquots are removed at various time-points and assayed for APC amidolytic activity as described in the "APC Amidolytic Assay ". The residual APC activity at the various time- points is expressed in percentage of the APC activity measured at the start of the experiment. The functional in vitro half-life (expressed in minutes) is calculated as the time at which 50% of the APC activity is still present.
Example 12 — Determination of anti-inflammatory effect APC Anti-inflammatory Assay I
The anti-inflammatory properties of the variants are investigated using recombinant tumor necrosis factor α (TNFα) (catalogue number: 210-TA, R&D Systems, Minneapolis, USA) stimulated human umbilical vein endothelial cells (HUVEC) (catalogue number: CC- 2519, Clonetics, San Diego, USA). HUVEC is stimulated using 1 ng/ml TNFα for about 7 hours. Various concentrations (0 - 200 nM) of hAPC and the variants are then incubated for up to 20 hours. The cells are removed by trypsination and analysed using flow cytometry measuring the surface expression of ICAM-1, VCAM-1 and/or E-selectin. For E-selectin quantification a FITC-conjugated anti-human E-selectin monoclonal antibody (CD62E) (catalog number: BBA21, R&D Systems, Minneapolis, USA) is used. The anti- inflammatory effect of hAPC and the variants is determined by calculating the APC concentration needed to suppress the TNFα stimulation of E-selectin to 50% compared to the effect of TNFα obtained without hAPC. This hAPC concentration is used to indicate the half maximum inhibitory concentration (IC50), and these values are determined for hAPC and each individual variant.
Example 13 - Determination of anti-inflammatory effect APC Anti-inflammatory Assay II As an alternative to the assay disclosed in Example 12 above, one may assess the anti-inflammatory property of hAPC and the variants by the inhibition of TNF alpha production from LPS stimulated monocytes. In this assay monocytic THP-1 cells (105) (ATCC, Rockville MD) are pre-incubated with various concentrations (0-1600 nM) of the variants for 16 hours. Cells are then stimulated using 0.5ug/ml LPS (Cat# 314, List for 16 hours. Cells are then stimulated using 0.5ug/ml LPS (Cat# 314, List Biological la- barotories) for 4 hours. TNF alpha production in the cell culture supernatant is subsequently measured using a TNF alpha ELISA (Cat# DY210, R and D systems). The anti- inflammatory effect of hAPC and the variants is determined by calculating the hAPC con- 5 centration needed to obtain 50% reduction in LPS induced TNF alfa production as compared to the effect of LPS alone. This hAPC concentration is used to indicate the half maximum inhibitory concentration (IC50), and these values are determined for human hAPC and each individual polypeptide.
l o APC Anti-inflammatory Assay III
As described above, but the THP-1 cells are pre-incubated with the polypeptide for 30 min.
Example 14- Determination of binding to the EPCR
15 EPCR Binding Assay I
The off-rate constant (koff) and the on-rate constant (kon) of wild-type human protein C and the protein C variants are measured using surface plasmon resonance (SPR). The measurements are performed on a Biacore 2000 (Biacore AB, Uppsala, Sweden). The extracellular part of EPCR is immobilized on a sensor chip SA (Biacore AB) and the dissocia-
20 tion constants (Ka = kofi/ on) are calculated from the obtained resonance units using the software program included with the instrument. The obtained Kd values are used to evaluate the affinity of each obtained protein C variant. The assay may be conducted using either the zymogen form of wild-type human protein C (and the corresponding zymogen forms of the variants) or the activated form of wild-type human protein C (and the conesponding acti-
25 vated forms of the variants).
Example 15 — Determination of binding to the EPCR EPCR Binding Assay II
Stably transfected EPCR human 293 cells are used to measure the binding of wild- 30 type human protein C and the variants of protein C to cellular membrane bound EPCR. Increasing concentrations of 125I-labeled human protein C or protein C variants are incubated with the human 293 cells in suspension and washed to remove excess of radio labeled APC. The Kd value for each variant is determined by Scatchard analysis. A similar analysis can be performed using competition binding between the protein C variants and 125I-labeled human protein C. The assay may be conducted using either the zymogen form of wild-type human protein C (and the conesponding zymogen forms of the variants) or the activated form of wild-type human protein C (and the conesponding activated forms of the variants).
Example 16 - Display and selection of APC variants
Protein C libraries may be displayed on the surface of COS cells using the protoplast fusion technique (Tan et al. (1998) PNAS 95:4247-4252). Protoplast fusion is a technique that enables the expression of bacterial DNA in a eukaryotic system.
The protoplast fusion protocol has two distinct manipulations: the formation of bacterial protoplasts and the fusion of bacterial and recipient cells. The cell wall of the bacteria must first be sufficiently degraded to enable fusions. The formation of these bacterial protoplasts is accomplished by exposure to lysozyme, followed by short periods of incubation. Fusions are facilitated by exposure to polyethylene glycol. An advantage of protoplast fusions, compared to SuperFect® (Qiagen) transfections, is that fusions may be carried out immediately following transformation; fransfected DNA does not have to be isolated and prepared. More important, the fusions are nearly clonal, allowing for the expression of single plasmid constructs. The following protocol has been optimized for a specific plasmid, and rates of transfection will vary with changes in the protocol.
The fransfected E. coli cells containing the appropriate plasmids with the protein C library are grown at 37°C in Luria Broth containing appropriate selection antibiotic (i.e. 100 μg/ml ampicillin or 40 μg/ml kanomycin) to an absorbance of 0.6-0.7 measured at 666 nm. Chloramphenicol is added to 200 μg/ml and the culture is incubated at 37°C for 12-16 hours to amplify plasmid copy numbers.
1. Bacteria from 25 ml of culture are pelleted by centrifugation at 3500 rpm for 15 minutes at room temperature. The culture supernatant is removed by aspiration.
2. The bacterial pellet is resuspended in 1.25 ml of chilled 20% sucrose/0.05 M Tris-HCl, pH 8.
3. T4 Lysozyme is prepared immediately before as a 5 mg/ml in 0.25 M Tris-HCl, pH 8 stock. 0.25 ml of lysozyme is added to the bacterial suspension and incubated on ice for six minutes.
4. 0.5 ml of 0.25 M EDTA, pH 8, is added to the mixture and incubated for 5 minutes on ice. 5. 0.5 ml of 0.5 M Tris-HCl, pH 8, is added to the mixture and incubated for 10 minutes in a 37°C water bath.
6. The bacterial suspension is then diluted with 10 ml warm (37°C) DMEM containing 10% sucrose and 10 mM MgCl2. 7. This is incubated for 10 minutes at room temperature. Protoplasts are now ready for fusion. 8. Mammalian cell lines (Cos) are cultured in Cos Medium (DMEM, 10% FCS, 1% Pen-Strep and Glutamine mix). Do not let cells grow greater than 80% confluent. Higher densities will greatly compromise transfection efficiencies. 9. Seed 1 x 106 cells per T75 flask 24 hrs before fusion (should achieve -70-80% confluence).
10. Media overlaying the cells is removed by aspiration.
11. To remove all traces of Pen-Strep, the flasks are washed thoroughly with 2 x 10ml PBS. 12. 12 ml protoplast suspension are added to each flask (~10,000-fold excess over cells), and flasks are centrifuged at 1500 rpm for 10 min at 25°C.
13. Supematants are carefully removed by suction.
14. Eight ml pre-warmed (37°C) 45% PEG1500* is added at room temperature, incubated for 7 min, and removed by suction. 15. Cells are washed three times using 10 ml serum-free DMEM
16. 20 ml DMEM containing 10% FCS, 1% Pen-Strep and Glutamine mix are added to each flask. (Choice of medium may vary if working with different cell lines.)
17. Medium is changed after 24 hrs.
18. Plasmid expression may be analyzed and sorted using FACS at 24, 48, or 72 hours.
Using the COS surface display system displaying the protein C libraries and a Fluorescent Activated Cell Sorting (FACS) system, cells can be isolated based on the expression of protein C on their cell surface, using the V5-tag, as well as the affinity of the displayed protein C variants for soluble EPCR. Soluble EPCR is FITC-labeled in order to identify high affinity binders. The EPCR binding affinity of each surface-displayed protein C variant can be determined from equilibrium binding tifration curves. Cells displaying the protein C variants are incubated in varying concentrations of FITC-labeled soluble EPCR. A flow cytometer (e.g. a FACSCalibur™) is used to measure the mean fluorescence of the cell populations. The equilibrium dissociation constant, Kd, can be fitted using a suitable model (e.g. non-linear least-squares curve fit). Variants with high affinity are subjected to DNA sequencing to determine the amino acid sequence in each particular protein C variant that binds with high affinity to EPCR.
Example 17 - Determination of PAR-1 cleavage activity of APC variants Introduction
Cleavage of PAR-1 by APC was determined by incubating the endothelial cell line EaHy926 with serially diluted APC and then examining the residual expression of intact PAR-1 by flow cytometry. In order to compare the PAR-1 cleavage activity of the tested variants and hAPC, equal amounts - based on amidolytic activity - of the variants and hAPC were used in the assay.
Methods Serially diluted ( 10 U/ml - 0.005 U/ml) human APC (Xigris®) or several APC substitution variants were incubated with EaHy926 cells for 30 minutes in 96 well microtiter plates at 37°C. Following a washing step, cells were stained with a PE-conjugated monoclonal antibody (3 μg/ml) against intact PAR-1 (SPAN- 12, Immunotech, Cat. No. IM2583) or an isotype matched control antibody (PE-conjugated IgGl, Pharmingen, Cat. No. 559320) for 30 minutes at 4°C. Receptor expression was determined using a FACSCalibur (BD) with standard settings. The Mean Fluorescence Intensity (MFI) of bound SPAN 12 was plotted as a function of APC concentration using nonlinear regression. The PAR-1 cleavage activity was then calculated using Xigris® as a standard.
Results
Preliminary results using the method described above showed that cleavage by APC 1) is EPCR dependent, 2) requires an APC active site, and 3) is not due to thrombin contamination. Of particular interest is the fact that the APC variants were able to maintain PAR-1 cleavage activity independently of the anticoagulant activity.

Claims

1. Use of a human protein C variant comprising at least one amino acid modification in position 306-314 relative to SEQ ID NO:2 for the manufacture of a medicament for the treat- ment of a hypercoagulable state or acquired protein C deficiency.
2. Use according to claim 1, wherein the hypercoagulable state or protein C deficiency is associated with sepsis.
3. Use according to claim 1 or 2, wherein the at least one amino acid modification is an amino acid substitution.
4. Use according to claim 3, wherein at least one positively charged amino acid residue has been substituted with an uncharged or a negatively charged amino acid residue.
5. Use according to claim 4, wherein at least one positively charged amino acid residue has been substituted with an uncharged amino acid residue.
6. Use according to any of claims 3-5, wherein at least one negatively charged amino acid residue has been substituted with an uncharged amino acid residue.
7. Use according to claim 3, wherein at least one amino acid residue selected from the group consisting of R306, E307, K308, E309, K311, R312 and R314 has been substituted with an uncharged amino acid residue.
8. Use according to claim 7, wherein the variant comprises at least one substitution selected from the group consisting of: a) R306A, R306V, R306L, R306I, R306F, R306W, R306P, R306G, R306S, R306T, R306Y, R306N and R306Q; or b) E307A, E307V, E307L, E307I, E307F, E307W, E307P, E307G, E307S, E307T, E307Y, E307N and E307Q; or c) K308A, K308V, K308L, K308I, K308F, K308W, K308P, K308G, K308S, K308T, K308Y, K308N and K308Q; or d) E309A, E309V, E309L, E309I, E309F, E309W, E309P, E309G, E309S, E309T, E309Y, E309N and E309Q; or e) K311A,K311V,K311L,K311I,K311F,K311W,K311P,K311G,K311S, K311T,K311Y,K311NandK311Q;or f) R312A,R312V,R312L,R312I,R312F,R312W,R312P,R312G,R312S,
R312T, R312Y, R312N and R312Q; or g) R314A, R314V, R314L, R314I, R314F, R314W, R314P, R314G, R314S, R314T, R314Y, R314N andR314Q.
9. Use according to claim 8, wherein the variant comprises at least one substitution selected from the group consisting of: a) R306A, R306V, R306L and R306I; or b) E307A, E307V, E307L and R307I; or c) K308A, K308V, K308L and K308I; or d) E309A, E309V, E309L and R309I; or e) K311A,K311V,K311LandK311I;or f) R312A,R312V,R312LandR312I;or g) R314A,R314V,R314LandR314I.
10. Use according to any of claims 1-9, wherein the variant comprises at least two amino acid substitutions in positions 306-314.
11. Use according to claim 10, wherein the variant comprises two, three, four or five amino acid substitutions in positions 306-314.
12. Use according to claim 10 or 11, wherein the variant comprises substitutions in positions K308andR314.
13. Use according to claim 12, wherein the substitutions are selected from the group consist- ing of K308A+R314A, K308A+R314V, K308A+R314L, K308A+R314I, K308V+R314A,
K308V+R314V, K308V+R314L, K308V+R314I, K308L+R314A, K308L+R314V, K308L+R314L, K308L+R314I, K308I+R314A, K308I+R314V, K308I+R314L and K308I+R314I.
14. Use according to claim 13, wherein the substitutions are K308A+R314A.
15. Use according to any of claims 1-14, wherein the variant comprises at least three amino acid substitutions in position 306-314.
16. Use according to claim 15, wherein the variant comprises substitutions in positions K308, R314 and at least one further residue selected from the group consisting of R306, E307, E309, K311 and R312.
17. Use according to claim 16, wherein the variant comprises substitutions in positions K308, R314 and at least one further residue selected from the group consisting of R306, K311 and R312.
18. Use according to claim 17, wherein the variant comprises substitutions in positions R306, K308 and R314.
19. Use according to claim 18, wherein the substitutions are R306A/V/L/I+K308A/V/L/I+R314A/V/L/I.
20. Use according to claim 19, wherein the substitutions are R306A+K308A+R314A.
21. Use according to claim 17, wherein the variant comprises substitutions in positions K308, K311 and R314.
22. Use according to claim 21, wherein the substitutions are K308A/V/L/I+K311 A/V/L/I+R314A/V/L/I.
23. Use according to claim 22, wherein the substitutions are K308A+K311A+R314A.
24. Use according to claim 17, wherein the variant comprises substitutions in positions K308, R312 and R314.
25. Use according to claim 24, wherein the substitutions are K308A V/L/I+R312A/V/L/I+R314A/V/L/I.
26. Use according to claim 25, wherein the substitutions are K308A+R312A+R314A.
27. Use according to any of claims 1-26, wherein the variant comprises at least four amino acid substitutions in position 306-314.
28. Use according to claim 27, wherein the variant comprises substitutions in positions K308, R314 and at least two further residues selected from the group consisting of R306, E307, E309, K311 and R312.
29. Use according to claim 28, wherein the variant comprises substitutions in positions K308, R314 and at least two further residues selected from the group consisting of R306, K311 and R312.
30. Use according to claim 29, wherein the substitutions are selected from the group consisting of R306A V/L/I+K308A/V/L/I+K311A/V/L/I+R314A/V/L/I, R306A/V/L/I+K308A/V/L/I+R312A/V/L/I+R314A V/L/I and K308 A/V/L/I+K311 A/V/L/I+R312A/V/L/I+R314A/V/L/I.
31. Use according to claim 30, wherein the substitutions are selected from the group consisting of R306A+K308A+K311A+R314A, R306A+K308A+R312A+R314A and K308A+K311 A+R312A+R314A.
32. Use according to any of claims 1-31, wherein the variant comprises at least five amino acid substitutions in position 306-314.
33. Use according to claim 32, wherein the variant comprises substitutions in positions K308, R314 and at least three further residues selected from the group consisting of R306, E307, E309, K311 and R312.
34. Use according to claim 33, wherein the variant comprises substitutions in positions K306, K308, K311, R312 and R314.
35. Use according to claim 34, wherein the substitutions are
R306A/V/L/I+K308A/V/L/I+K311A V/L/I+R312A V/L/I+R314A/V/L/I.
36. Use according to claim 35, wherein the substitutions are R306A+K308A+K311A+R312A+R314A.
37. Use according to any of claims 1-36, wherein N313 and T315 are not modified.
38. Use according to any of claims 1-37, wherein the variant further comprises at least one amino acid modification in the active site region as defined herein.
39. Use according to claim 38, wherein the amino acid modification in the active site region comprises at least one amino acid substitution at a position which is occupied by an amino acid residue having at least 25% of its side chain exposed to the surface.
40. Use according to claim 39, wherein the at least one substitution in the active site region comprises introduction of an amino acid residue comprising an attachment group for a non- polypeptide moiety.
41. Use according to claim 40, wherein the introduced amino acid residue comprising an attachment group for a non-polypeptide moiety creates an in vivo N-glycosylation site.
42. Use according to claim 40, wherein the introduced amino acid residue comprising an attachment group for a non-polypeptide moiety is a cysteine residue.
43. Use according to any of claims 1-42, wherein the variant is in its activated form.
44. A method of treating a patient with a hypercoagulable state or acquired protein C deficiency, comprising administering to the patient an effective amount of a variant as defined in any of claims 1 -43.
45. A variant as defined in any of claims 1-43, with the proviso that the variant is not human protein C or hAPC comprising the following modifications: R306A, E307A, K308A, E309A, K311A, R312A, R314A, R306A+R312A, R306A+K311A+R312A+R314A, H303*+S304*+S305*+K308*+E307D+A310T, S304N+R306S, S304N+R306T, S305N+E307S, S305N+E307T, R306N+K308S, R306N+K308T, E307N+E309S, E307N+E309T, K308N+A310S, K308N+A310T, E309N+K311S, E309N+K311T, A310N+R312S, A310N+R312T, R312N+R314S, R312N+R314T, R306C, E307C, K308C, E309C, A31 OC and R312C, and that the variant does not contain a mutation in position 313.
46. The variant of claim 45 for use as a medicament.
47. A pharmaceutical composition comprising a variant as defined in claim 45 and a phar- maceutically acceptable carrier or excipient.
48. A nucleotide sequence encoding the variant as defined in claim 45.
49. An expression vector comprising a nucleotide sequence as defined in claim 48.
50. A host cell comprising a nucleotide sequence as defined in claim 48 or an expression vector as defined in claim 49.
51. The host cell of claim50, which is selected from the group consisting of COS, CHO, BHK and HEK293 cells.
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