WO2004113385A1 - Protein c propeptide variants - Google Patents

Abstract

A precursor of human protein C comprising a propeptide sequence, wherein the isoleucine residue in position -4 of the propeptide sequence has been substituted with a basic amino acid residue. Precursors of human protein C according to the invention exhibit improved processing of the propeptide.

Description

PROTEIN C PROPEPTIDE VARIANTS

FIELD OF THE INVENTION

The present invention relates to novel propeptide variants of human protein C hav- ing improved processing of the propeptide.

BACKGROUND OF THE INVENTION

Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually give rise to a fibrin clot. Generally, blood compo- nents participating in the coagulation "cascade" are proenzymes or zymogens, i.e. enzy- matically 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 K- dependent 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 K- dependent carboxylation of nine glutamic acid residues of the light chain resulting in nine gamma-carboxyglutamic acid residues in the Ν-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 Ν-terminus of the heavy chain (positions 158-169), producing the ac- tivated protein C (APC).

Protein C is activated by limited proteolysis by throrribin 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 influence 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 LX) 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 con- tains 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-Uke 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 included a covalently bound inhibitor (D-Phe-Pro-Arg chloro- methylketone, 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 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 glycosylation 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. Although properly processed recombinant human protein C has been expressed from HEK 293 cells (Yan et al, Bio/Technology 8:655-661, 1990), it is known in the art that human protein C is poorly processed in many mammalian cell lines. For example, Foster et al. (Biochemistry 30(2):367-72, 1991) describe co-expression of human protein C together with the yeast KEX2 endopeptidase in order to obtain cleavage of the propeptide as well as cleavage of the dibasic Lys-Arg in position 156-157 between the light and heavy chains. Cleavage of the light and heavy chains has also been obtained in an alternative manner by introduction of the residues Arg- Arg between residues 155 and 156 (US 4,959,318 and US 5,516,650). However, the problem of obtaining proper cleavage of the propeptide from the light chain of recombinantly produced human protein C or variants thereof, without the need for e.g. co-expressing an endopeptidase, has yet to be sufficiently addressed.

BRIEF DISCLOSURE OF THE INVENTION

The object of the present invention is thus to provide precursors of human protein C with improved processing of the propeptide. This object is achieved by providing a human protein C precursor comprising a propeptide sequence wherein the isoleucine residue in position -4 of the propeptide sequence, relative to SEQ ID NO:l, has been substituted with a basic amino acid residue.

Other aspects of the invention relate to a nucleotide sequence encoding the protein C precursor of the invention, expression vectors comprising the nucleotide sequence, host cells comprising the expression vector or the nucleotide sequence, and a method of preparing a protein C polypeptide by expressing the protein C precursor. Still other aspects relate to pharmaceutical compositions comprising a protein C polypeptide produced by the method of the invention as well as use of such polypeptides for the treatment of certain diseases.

DETAILED DISCLOSURE OF THE INVENTION Definitions In the context of the present application the following definitions apply:

As used herein, the term "protein C precursor" refers to the DNA-encoded form of protein C that includes the propeptide (residues -18 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), as shown in SEQ ID NO:l. The protein C precur- sor may also include a signal peptide, e.g. the native signal peptide of human protein C (residues -A2 to -19 of SEQ ID NO: 1), or alternatively an altered version of the human protein C signal peptide or a heterologous signal peptide selected according the particular expression system used.

The term "propeptide sequence" refers to the 18 amino acid propeptide sequence of human protein C shown as residues -18 to -1 of SEQ ID NO: 1.

The term "one-chain zymogen protein C" refers to the one-chain inactive form of protein C, which includes 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:l or 2. The term "two-chain zymogen 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) (but without the Lys-Arg dipeptide between the light chain and the heavy chain), shown in SEQ ID NO:l or 2. The term "zymogen protein C" is intended to refer 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 "human APC" 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:l or 2. The amino acid sequence of activated protein C may be referred to as "the APC part" of the amino acid sequence of SEQ ID NO: 1 or 2.

The term "protein C" encompasses all of the above-mentioned forms of protein C, i.e. the "protein C precursor" form, the "zymogen protein C" form (the one-chain form as well as the two-chain form) and the "activated protein C form". The "Gla domain" comprises amino acid residues 1-45 of SEQ ID NO:l or 2.

The "EGF domains" comprise amino acid residues 55-134 of SEQ ID NO:l or 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, VI 85, 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.

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), ethionine (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. A39S indicates that the alanine residue of position 39 is substituted with a serine residue. Alternative 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. 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 A39 AS. 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:l. The term "differs" or "differs from" when used in connection with specific mutations is intended to allow for additional differences being present apart from the specified amino acid difference. For instance, in addition to the substitution of the isoleucine residue in the -4 position of the propeptide disclosed herein, the protein C polypeptide can comprise other substitutions, insertions or deletions which are not related to this substitution. Thus, in addition to the amino acid alterations disclosed herein aiming at improving processing of the propeptide, it will be understood that the molecule may, if desired, contain other alterations that need not be related to this effect. Such alterations, e.g. with the aim of introducing at least one site for conjugation to a non-polypeptide moiety, may e.g. be performed with the aim of increasing the anti-inflammatory effect, increased the half-life and/or lowering the anticoagulant activity of the variant. Additional alterations may further include, for example, 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)

Protein C polypeptides that may be produced according to the present invention thus include not only human protein C but also variants thereof.

The term "variant" (of a parent polypeptide) is intended to cover a polypeptide which, in addition to the mutation in the propeptide described herein, differs in one or more amino acid residues from its parent polypeptide, normally 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, 1-8, 1-6, 1-5, 1-4 or 1-3 amino acid residues, e.g. one or two amino acid residues. The parent polypeptide in the present context is generally human protein C (SEQ ID NO:2), in particu- lar a human protein C precursor comprising the propeptide sequence (SEQ ID NO: 1).

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 with another amino acid residue, but may also mean deletion (without substitution) of the amino acid residue to be removed.

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. Variants of the invention

As explained above, the protein C precursor of the invention includes a substitution of the isoleucine residue in position -4 ("I(-4)") of the propeptide sequence shown in SEQ ID NO:l with a basic amino acid residue. Basic amino acid residues are understood to in- elude arginine, lysine and histidine residues. Preferably, the basic amino acid residue in position -4 is an arginine or lysine residue, more preferably an arginine residue.

For purposes of simplicity, the propeptide sequence is discussed in the context of the present specification as comprising residues -18 to -1 of SEQ ID NO:l, but with the substitution as defined herein in the -4 position. Persons skilled in the art will be aware, however, that one or more additional alterations may optionally be performed in the propeptide sequence, e.g. one or more amino acid substitutions, insertions and/or deletions relative to SEQ ID NO:l, such as .1-8 such alterations, e.g. 1-6 such alterations. For example, the propeptide sequence may, in addition to the substitution in I(-4), comprise 1, 2, 3, 4 or 5 additional substitutions. Such substitutions are preferably conservative substitutions or sub- stitutions with amino acid residues found in corresponding positions in other vitamin Independent propeptides.

As will be explained in more detail below, the present invention is in particular directed to expression of protein C polypeptides in mammalian cells, e.g. in CHO cells, COS cells BHK cells or HEK 293 cells. A preferred cell type is a CHO cell, e.g. CHO-K1. As explained above, protein C polypeptides produced according to the invention may, in addition to the alteration of the propeptide, include one or more additional mutations, e.g. at least one substitution, insertion or deletion, typically at least one substitution. Alternatively, the protein C polypeptide may have the sequence of human protein C (SEQ ID NO:2). When additional mutations are present, these may e.g. be aimed at increasing the anti-inflammatory effect, increasing the half-life and/or lowering the anticoagulant activity of the resulting polypeptide. Typically, such mutations are aimed at introducing and/or at removing at least one amino acid residue comprising an attachment group for a non- . polypeptide moiety.

The term "non-polypeptide moiety" refers to a non-polypeptide molecule that is capable of conjugating to an attachment group of the polypeptide. Examples of such non- polypeptide moieties include polymer molecules, sugar moieties, lipophilic compounds and organic derivatizing agents. The non-polypeptide moiety can be directly covalently joined to the attachment group or it can be indirectly covalently joined to the attachment group through an intervening moiety, such as a bridge, spacer or linker moiety or moieties. Pre- ferred examples of non-polypeptide moieties are a polymer molecule, in particular a linear or branched polyethylene glycol or other polyalkylene glycol, and a sugar moiety, in particular an N- or O-linked oligosaccharide generally attached by in vivo glycosylation.

In one embodiment, the protein C polypeptide may thus include at least one intro- duced in vivo N-glycosylation site created by a substitution 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, T315N+V317S, T315N+V317T, F316N+L318S, F316N+L318T, 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.

Preferred substitutions for introduction of an in vivo N-glycosylation site are 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; preferably 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; such as 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 from the group consisting of D189N+K191T, D214N, D251N, and L386N+H388T. Particularly preferred substitutions include D214N and K251N. See WO 02/32461 for firrther information on variants of protein C with introduced glycosylation sites.

As is also described in WO 02/32461, at least some of the charged residues of protein C interact with each other. For example, K251 is believed to form a salt bridge to D214. Moreover, a cluster of negatively charged amino acid residues (D214, E215 and E357) is present in the protein. Therefore, another group of amino acid substitutions of interest include substitutions where a charged amino acid residue in the active site region and having at least 25% of its side chain exposed to the surface (as defined in WO 02/32461, i.e. in particular D172, D189, K191, K192, K193, D214, E215, K217, K218, K251, D255, R306, E307, K308, E309, R312, D351, R352, E357 and E382) 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), or with an amino acid residue having an opposite charge.

Specific examples of substitutions to an amino acid residue with 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, R306D, R306E, E307K, E307R, K308D, K308E, E309K, E309R, R312D, R312E, D351K, D351R, R352D, R352E, E357K, E357R, E382K and E382R, such as D214K, D214R, E215K, E215R, K251D, K251E, E357K or E357R, e.g. D214K, D214R, K251D or K251E. Specific examples of substitutions to an amino acid residue having a polar side chain include D172G/S/T/C/Y/N/Q, D189G/S/T/C/Y/N/Q, K191G/S/T/C/Y/N/Q, K192G/S/T/CΛ7N/Q, K193G/S/T/CΛ7N/Q, D214G/S/T/C/Y/N/Q, E215G/S/T/C/Y/N/Q, K217G/S/T/CΛ N/Q, K218G/S/T/CΛ7N/Q, K251G/S/T/C/Y/N/Q, D255G/S/T/C/Y/N/Q, R306G/S/T/C/Y/N/Q, E307G/S/T/C/Y/N/Q, K308G/S/T/CΛ7N/Q, E309G/S/T/CΛ7N/Q, R312G/S/T/C/Y/N/Q, D351G/S/T/CΛ7N/Q, R352G/S/T/C/Y/N/Q, E357G/S/T/CΛ7N/Q and E382G/S/T/CΛ N/Q, such as D214G/S/T/C/Y/N/Q, E215G/S/T/C/Y/N/Q, K251G/S/T/CΛ N/Q or E357G/S/T/CΛ N/Q, e.g. D214Q, E215Q, K251Q or E357Q, in particular K251 Q.

In addition to the substitution in position -4 and possible other amino acid muta- tions discussed above or elsewhere herein, the protein C polypeptides produced according to the invention may also contain an insertion of one or two Lys and/or Arg residues between residues 155 and 156. Preferably, the insertion is Arg- Arg. As indicated above, such insertions serve to enhance cleavage between the light and heavy chains. An example of a preferred embodiment is thus a protein C polypeptide that includes, in addition to substitution in the -A position of the propeptide, an Arg- Arg insertion between residues 155 and 156 as well as one or two substitutions selected from the group consisting of D214N, D214K, K251D, K251N and K251Q. In the case of substitutions in both of positions 214 and 251, these may e.g. be K251D and D214K.

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, tiireonine or cysteine, preferably serine or threonine, and most preferably threonine.

In another embodiment, the protein C polypeptide produced according to the inven- tion includes at least one introduced amino acid residue comprising an attachment group for a non-polypeptide moiety, in particular an introduced cysteine residue. Preferably, such a cysteine residue is introduced in a position selected from the group consisting of D 172, D189, S190, K191, K192, K193, D214, E215, S216, K217, K218, L220, V243, V245, S250, K251, S252, T253, T254, L296, Y302, H303, S304, S305, T315, F316, V334, S336, N337, M338, 1348, L349, D351, R352, E357, G383, L386, L387 and H388; more preferably from the group consisting of D189, S190, K191, D214, K217, K251, S252, T253, Y302, S336, N337, M338, G383 and L386; such as from the group consisting of D189, K191, D214, K251, S252, T253, Y302, S336, N337, M338, G383 and L386; in particular from the group consisting of D189, D214, K251 and L386. The non-polypeptide moiety to be cova- lently attached to said introduced cysteine residue is preferably a polymer molecule, in particular a linear or branched polyethylene glycol or other polyalkylene oxide.

Specific examples of activated PEG polymers particularly preferred for coupling to cysteine residues include the following linear PEGs: vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG); maleimide-PEG (MAL-PEG), preferably maleimide- mPEG (MAL-mPEG) and orthopyridyl-disulfide-PEG (OPSS-PEG), preferably or- thopyridyl-disulfide-mPEG (OPSS-mPEG). Such PEG or mPEG polymers will generally have a size of from about 1 kDa to about 40 kDa, such as from about 1 kDa to about 20 kDa, e.g. from about 2 kDa to about 15 kDa, such as from about 3 kDa to about 10 kDa; for example about 5 kDa, about 6 kDa, about 10 kD, about 12 kDa or about 20 kDa. For PEGylation to cysteine residues the protein C variant is usually treated with a reducing agent, such as dithiothreitol (DDT) prior to PEGylation. The reducing agent is subsequently removed by any conventional method, such as by desalting. Conjugation of PEG to a cysteine residue typically takes place in a suitable buffer at about pH 6-9 at temperatures of about 4°C to 25°C for periods up to about 16 hours.

In a further embodiment, the protein C polypeptide may comprise at least one amino acid modification in the autolysis loop constituted by the amino acid residues in position 306-314 relative to SEQ ID NO: 1 or 2 in order to achieve a reduced anticoagulant activity. This modification may e.g. include substitution of at least one of R306, E307, K308, E309, K311 , R312 and R314 with an uncharged amino acid residue, e.g. A, V, L, I, F, W, P, G, S, T, Y, N or Q.

Further information on protein C variants comprising advantageous substitutions of the type described above as well as conjugation of such variants to one or more non- polypeptide moieties is found in WO 02/32461 and PCT/DK03/00392, which are hereby incorporated herein by reference.

Other examples of protein C variants whose production by way of a propeptide according to the invention having a substitution in position -4 is contemplated to be useful include those having mutations in one or more positions selected from 10,11, 12, 32, 33, 167, 168, 172, 194, 195, 228, 249, 254, 302, 313, 316 and 329. See, e.g., WO 00/66754, WO 01/59084, US 5,196,322, WO 01/57193 and WO 01/36462 for further details regarding variants with mutations in these positions.

Methods for preparing a polypeptide variant of the invention

The polypeptide variant of the present invention, 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 gamma-carboxylating host cell, in particular a mammalian cell.

A nucleotide sequence encoding a polypeptide precursor of the invention may be constructed by isolating or synthesizing a nucleotide sequence encoding the parent protein C precursor, such as the protein C precursor with the amino acid sequence shown in SEQ ID NO:l, 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^).

The nucleotide sequence may conveniently be modified by site-directed mutagene- sis in accordance with conventional methods. Alternatively, the nucleotide sequence may be prepared by chemical synthesis, e.g. by using an oligonucleotide synthesizer, wherein oligonucleotides 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 portions 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 and hosts for expressing the polypeptide. For example, in selecting a vector, the host must be considered because the vector must be able to replicate in it or be able to integrate into the chromosome. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the nucleotide sequence encoding the polypeptide, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the nucleotide sequence, their secretion characteristics, their ability to fold the polypeptide correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the nucleotide sequence.

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 of the invention is operably linked to additional segments required for transcription 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 cytomegalovirus. Specific vectors are, e.g., pCDNA3.1(+)\Hyg (Invitrogen, Carlsbad, CA, USA) and CI-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 YorkAcad. Sci. 782, 202-207, 1996, and pPICZ A, B or C (Invitrogen). Useful vectors for insect cells include pVL941 , pBG311 (Cate et al., Cell 45, pp.

5 685-98 (1986), pBluebac 4.5 and pMelbac (both available from invitrogen). Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including ρBR322, pET3a and pET12a (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 Ml 3 and filamentous single stranded

10 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; Kaufman et ., Mol. Cell. Biol, 2, pp. 1304-

15 19 (1982)), and by glutamine synthetase ("GS") amplification (see, e.g., US 5,122,464 and EP 0338841).

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.

20 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 Schizosaccharomyces pombe TPI gene (described by P.R. Russell, Gene 40, 1985, pp. 125-130), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. For Saccharomyces

25 cerevisiae, selectable markers include ura3 and leu2.

The term "control sequences" is defined herein to include all components which are necessary or advantageous for the expression of the variant polypeptide of the invention. 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,

30 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 SV40 and adenovirus, e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovirus immediate-early gene promoter (CMV), the human elongation factor l (EF-lα) pro- moter, 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, JMolBiol 1987;196(4):947-50).

In order to improve expression in mammalian cells a synthetic intron may be in- serted 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, WT, USA).

The nucleotide sequence of the invention encoding a protein C polypeptide precursor, whether prepared by site-directed mutagenesis, synthesis, PCR or other methods, will generally include a nucleotide sequence that encodes 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, when 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.

Suitable host cells that may be used to produce the polypeptide precursor of the invention include, in particular, mammalian cells. 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)). Mammalian cells, such as CHO cells, may be modified to express a 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. It will be understood that in order to achieve in vivo glycosylation of a protein C molecule comprising one or more glycosylation sites, the nucleo- tide sequence encoding the variant polypeptide must be inserted in a glycosylating, eu- karyotic expression host.

In order to increase secretion it may be of interest to produce the variant polypeptide of the invention together with an endoprotease, for example a PACE (paired basic amino acid converting enzyme) (e.g. as described in US 5,986,079), such as a Kex2 endo- protease (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, liposome-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 is 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, Cam- bridge 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 cells may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermenta- tions) 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). When the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium.

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 isoelectric 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).

Pharmaceutical compositions and use

Protein C polypeptides produced according to the present invention may be formulated as known in the art in a pharmaceutical composition comprising a polypeptide and at least one pharmaceutically acceptable carrier or excipient. In the present context, the term "pharmaceutically acceptable" means that the carrier or excipient, at the dosages and con- centrations 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 Pharmaceutical Sciences, 19th edition, A. R. Gen- naro, 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).

Polypeptides produced according to the invention may in particular be used for the manufacture of a medicament for treating or preventing a disease selected from the group consisting of stroke; myocardial infarction; after venous thrombosis; disseminated intravas- cular coagulation (DIG); sepsis; septic shock; emboli, such as pulmonary emboli; transplantation, such as bone marrow transplantation; burns; pregnancy; major surgery/trauma or adult respiratory stress syndrome (ARDS), in particular for the treatment of sepsis, including septic shock. The invention thus includes a method for treating or preventing such diseases or conditions by administering to a patient in need thereof an effective amount of a protein C polypeptide produced according to the invention, or of a pharmaceutical composition comprising the polypeptide.

A "patient" for the purposes of the present invention includes both humans and other mammals, i.e. the methods are applicable to both human therapy and veterinary applications. The polypeptides of the invention will be 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. The polypeptide variant of the invention 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 of the invention, either concurrently or in accordance with another treatment schedule. In addition, the variant or pharmaceutical composition of the invention 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 for- mulations 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 typically 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 cit- rate 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 inven- tion 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 rela- tive 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 sol bilizes 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, as- paragine, histidine, alanine, omithine, L-leucine, 2-phenylalanine, glutamic acid, tiireoriine, 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 polyvinylpyrrolidone; 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 sur- factants 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 matrices of solid hydrophobic polymers containing the variant of the invention, 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 ethylene-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 ethylene-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, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

All references cited herein are hereby incorporated by reference in their entirety for all purposes. The invention is further illustrated by the following non-limiting example.

Example 1 Experiments were carried out to determine the effect of the I(-4)R substitution in human protein C in promoting correct N-terminal processing. A comparison was made between two protein C variants, one of which had the substitution to arginine in position —A, while the other had the native isoleucine residue in this position. Both variants had two additional arginine residues inserted after position 157 as well as the substitution K251N.

Both variants were expressed in CHO-K1 cells, purified from roller bottle fermentation media by a single antibody-affinity chromatographic step, and subsequently analysed by SDS-PAGE and for N-terminal sequence as explained below. The relative amount of incorrect vs. correct N-terminally processed amino acids was determined for each variant.

Materials and Methods

Buffers

A: 20 mM Tris (Trizma-Base), 300 mM NaCl, 5 mM CaCl₂, pH 7.5 B: 20 mM Tris (Trizma-Base), 100 mM NaCl, 10 mM EDTA, pH 7.5

Antibody-affinity chromatography 8 ml cyanogen bromide (CNBr) activated Sepharose 4 FF matrix from Pharmacia

(lot # 285324) was reacted with 8 mg anti-human protein C (Ca-14 lot # GCII-30W) specific for the EGF domain of protein C. The matrix was packed in a Pharmacia XK 16 column tube, resulting in an approx. 8 ml column, and equilibrated with Buffer A prior to sample application.

Sample preparation

4500 ml of roller bottle fermentation media containing the variant having the I(- 4)R substitution was ultrafiltered and diafiltered against Buffer A on a Tangential Flow- Filtration system (Millipore) using filters with a molecular cut-off of 10 kDa (Pellicon, Mil- lipore) to a final volume of 900 ml. The sample was made sterile by filtering through a 0.22 μm filter and applied to a column overnight.

4500 ml of roller bottle fermentation media containing the variant without the substitution in position -4 was processed analogously, resulting in a final volume of 750 ml.

Chromatography

Sample was loaded onto the antibody-affinity column at a flow rate of 0.1 column volumes per minute (CV/min). Unbound material (flow-through) was collected as one pool for later analysis. The column was washed with 10 CV of Buffer A at a flow rate of 0.25 CV/min and flow-through was collected as one pool. Bound material was eluted from the column with 5 CV of buffer B, flow-rate 0.25 CV/min, and fractions of 0.5 CV were collected. Fractions containing protein, as determined by OD280 trace, were pooled.

Analyses

The purified samples were analysed for purity by SDS-PAGE and N-terminal sequencing.

SDS-PAGE 20 μl sample was mixed with lOμl Reducing Cocktail (250 μl NuPAGE LDS Sample Buffer (4x) and 100 μl 0.5M dithiothreitol (DTT)) and heated for 5 minutes at 95°C. Samples were applied on NuPAGE Novex Bis-Tris 4-12% gels and electrophoresed in a MES buffer system for 35 minutes at a constant 200 V. Gels were incubated in fixing solution (50% (v/v) methanol, 10% (v/v) acetic acid, 40% (v/v) deionized water), and protein was visualized by staining with Novex® Colloidal Blue using the manufacturer's protocol (invitrogen, Carlsbad, CA, USA).

N-terminal sequencing

Prior to N-terminal amino acid sequence analyses, protein samples were applied to PVDF membranes in ProSorb devices as follows (basically as recommended by the manufacturer). Initially, 10 ml of methanol was added to the sample compartment of a ProSorb device to wet the hydrophobic PVDF membrane. After the methanol had passed through the PVDF membrane, a protein sample containing 5-10 mg of protein was diluted to a total volume of 100 ml using 0.1% TFA and added to the sample compartment. After the liquid had passed through the PVDF membrane, the membrane was washed 2 times with 200 ml of 0.1 % TFA. Following the washes, the membrane was air dried and punched out. N-terminal amino acid sequence analyses were carried out in a Procise 494 Protein Sequencer operated according to the manufacturer's instructions. Results and Discussion

SDS-PAGE

SDS-PAGE analysis of the two samples leads to separation of the heavy and light chains due to reduction of the disulphide bridge that holds them together. The heavy chain gives rise to several protein bands due to glycosylation heterogeneity, while correctly processed light chain normally only gives rise to one band at approximately 22 kDa.

In this example, a band with an apparent molecular weight that is slightly higher than the light chain is seen for the variant with the wild-type isoleucine in position -A, but not for the variant of the invention with an arginine in the -4 position. This indicates that some of the light chains with the native propeptide contain additional amino acids due to an incomplete or incorrect processing of the N-terminal and that the substitution in the -A position leads to correct N-terminal processing.

N-terminal sequencing

The starting sequence for the correct N-terminal of the light chain of APC is A-N- S-F. Prior data (not shown) has shown that incorrectly processed protein C is cleaved so that it leads to an N-terminal starting with P-A-P-L, i.e. corresponding to the residues in positions -23 to -20 of the precursor. N-terminal sequencing of the samples therefore indicates that there is a presence of precursor peptide in the sample with the native isoleucine in position -4. The prevalence of this incorrectly processed form is estimated to be up to about 10% of that of intact light chain, whereas it is not detected at all in the sample with the I(-4)R substitution.

Claims

1. A precursor of human protein C comprising a propeptide sequence, wherein the isoleucine residue in position -4 of the propeptide sequence has been substituted with a basic amino acid residue.

2. The protein C precursor of claim 1 , wherein the isoleucine residue in position -A of the propeptide sequence has been substituted with an arginine or lysine residue.

3. The protein C precursor of claim 1 , wherein the isoleucine residue in position -4 of the propeptide sequence has been substituted with an arginine residue.

4. The protein C precursor of any of the preceding claims, comprising, in addition to the substitution in position -4, at least one and up to 15 additional mutations relative to SEQ ID NO:l.

5. The protein C precursor of claim 4, comprising at least one and up to 15 mutations relative to SEQ ID NO:2.

6. The protein C precursor of claim 5, comprising at least one introduced N-glycosylation site relative to SEQ ID NO:2.

7. The protein C precursor of claim 5, comprising at least one introduced cysteine residue relative to SEQ ID NO:2.

8. The protein C precursor of claim 5, comprising at least one substitution in which a charged amino acid residue in the active site region having at least 25% of its side chain exposed to the surface is substituted with an amino acid residue having no charge or an opposite charge.

9. The protein C precursor of any of the preceding claims, further comprising a signal peptide.

10. The protein C precursor of any of the preceding claims, comprising at least one additional alteration in the propeptide sequence.

11. The protein C precursor of claim 10, wherein the at least one additional alteration is a 5 conservative substitution or a substitution with an amino acid residue found in a corresponding position in another vitamin K-dependent propeptide.

12. The protein C precursor of any of claims 1-3, comprising the amino acid sequence of human protein C (SEQ ID NO:2).

10

13. The protein C precursor of any of the preceding claims, comprising insertion of at least one Arg or Lys residue between residues 155 and 156 of SEQ ID NO:2.

14. The protein C precursor of claim 13, wherein the insertion is Arg- Arg.

15

15. A nucleotide sequence encoding the protein C precursor of any of claims 1-14.

16. An expression vector comprising the nucleotide sequence of claim 15.

20 17. A eukaryotic host cell comprising the nucleotide sequence of claim 15 or the expression vector of claim 16.

18. The host cell of claim 17 which is a mammalian cell.

25 19. The host cell of claim 18, selected from group consisting of CHO cells, COS cells, BHK cells and HEK 293 cells.

20. The host cell of claim 19 which is a CHO cell.

30 21. A method for producing a protein C polypeptide, comprising culturing the host cell of any of claims 17-20 under conditions that allow expression of a protein C precursor and subsequent processing of the propeptide sequence.

22. A protein C polypeptide produced by the method of claim 21.