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WO2004113385A1 - Variants du propeptide de la proteine c - Google Patents

Variants du propeptide de la proteine c Download PDF

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Publication number
WO2004113385A1
WO2004113385A1 PCT/DK2004/000392 DK2004000392W WO2004113385A1 WO 2004113385 A1 WO2004113385 A1 WO 2004113385A1 DK 2004000392 W DK2004000392 W DK 2004000392W WO 2004113385 A1 WO2004113385 A1 WO 2004113385A1
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Prior art keywords
protein
precursor
amino acid
residue
sequence
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PCT/DK2004/000392
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English (en)
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Bobby Soni
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Maxygen Holdings Ltd.
Maxygen Aps
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Publication of WO2004113385A1 publication Critical patent/WO2004113385A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6464—Protein C (3.4.21.69)
    • C—CHEMISTRY; METALLURGY
    • 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 novel propeptide variants of human protein C hav- ing improved processing of the propeptide.
  • Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually give rise to a fibrin clot.
  • 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 ⁇ 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 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 ⁇ -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 ⁇ -terminus of the heavy chain (positions 158-169), producing the ac- tivated protein C (APC).
  • a dodecapeptide the activation peptide
  • APC ac- tivated protein C
  • 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.
  • 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 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
  • 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.
  • APC is inhibited in plasma by the protein C inhibitor as well as by alpha- 1 - antitrypsin and alpha-2-macroglobulin.
  • 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
  • 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.
  • 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.
  • Foster et al. Biochemistry 30(2):367-72, 1991
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • zymogen protein C is intended to refer to both the one-chain form and the two-chain form of the zymogen protein C.
  • activated protein C activated human protein C
  • APC human APC
  • human APC human APC
  • 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.
  • 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,
  • 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), 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) residue
  • 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.
  • A39 AS Insertion of a serine residue after A39 is indicated by A39 AS.
  • a deletion of an amino acid residue is indicated by an asterix.
  • deletion of the alanine residue of position 39 is indicated by A39*.
  • 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.
  • the protein C polypeptide can comprise other substitutions, insertions or deletions which are not related to this substitution.
  • 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.
  • conservative substitutions include amino acids within the respective groups listed in the table below.
  • Protein C polypeptides that may be produced according to the present invention thus include not only human protein C but also variants thereof.
  • variant of a parent polypeptide
  • 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).
  • modified or “modification” includes a substitution, an insertion or a deletion.
  • 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.
  • 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
  • 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.
  • the basic amino acid residue in position -4 is an arginine or lysine residue, more preferably an arginine residue.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • non-polypeptide moiety refers to a non-polypeptide molecule that is capable of conjugating to an attachment group of the polypeptide.
  • 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.
  • 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.
  • the protein C polypeptide may thus include at least one intro-ucked 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+R222S, L2
  • 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
  • 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.
  • 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/C ⁇ 7N/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 ⁇ N/Q, K218G/S/T/C ⁇ 7N/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/T/T/C/Y/N/Q, K308G/S/
  • 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.
  • the insertion is Arg- Arg.
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • 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-mPEG), preferably or- thopyridyl-disulfide-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.
  • 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.
  • 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.
  • 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.
  • the polypeptide variant of the present invention 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • yeast cells include the 2 ⁇ 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.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including ⁇ BR322, 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
  • 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., US 4,470,461; Kaufman et ., Mol. Cell. Biol, 2, pp. 1304-
  • GS glutamine synthetase
  • 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.
  • 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.
  • DHFR dihydrofolate reductase
  • Schizosaccharomyces pombe TPI gene described by P.R. Russell, Gene 40, 1985, pp. 125-130
  • a drug e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • selectable markers include ura3 and leu2.
  • 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,
  • 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.
  • 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-l ⁇ ) 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).
  • adenovirus 2 major late promoter e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovirus immediate-early
  • 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 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.
  • 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)).
  • CHO Chinese hamster ovary
  • COS Green Monkey cell lines
  • BHK Baby Hamster Kidney
  • 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.
  • 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.
  • the nucleo- tide sequence encoding the variant polypeptide must be inserted in a glycosylating, eu- karyotic expression host.
  • 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).
  • PACE paired basic amino acid converting enzyme
  • 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.
  • the cells are cultivated in a nutrient medium suitable for production of the variant polypeptide using methods known in the art.
  • 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).
  • 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 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).
  • 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
  • extraction see, e.g., Protein Purification (2nd Edition), Janson and Ryden, editors, Wiley, New York, 1998).
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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
  • 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
  • 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 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.
  • colloidal drug delivery systems 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 matrices of solid hydrophobic polymers containing the variant of the invention, 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 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.
  • polyesters for example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)
  • polylactides
  • 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.
  • 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, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • 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.
  • A 20 mM Tris (Trizma-Base), 300 mM NaCl, 5 mM CaCl 2 , pH 7.5
  • B 20 mM Tris (Trizma-Base), 100 mM NaCl, 10 mM EDTA, pH 7.5
  • the purified samples were analysed for purity by SDS-PAGE and 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.

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Abstract

L'invention concerne un précurseur de la protéine C humaine contenant une séquence propeptidique, le résidu isoleucine en position 4 de ladite séquence ayant été remplacé par un résidu acide aminé basique. Les précurseurs de la protéine C humaine selon l'invention permettent un traitement amélioré du propeptide.
PCT/DK2004/000392 2003-06-20 2004-06-08 Variants du propeptide de la proteine c WO2004113385A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013072504A1 (fr) * 2011-11-16 2013-05-23 Centre National De La Recherche Scientifique (Cnrs) Structure cristalline du trimère des c-propeptides de procollagène iii et ses applications
WO2023119230A1 (fr) 2021-12-22 2023-06-29 L'oreal Compositions de modulation de la voie de coagulation et de la voie de nicotinamide-adénine dinucléotide et leurs procédés d'utilisation

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989012685A1 (fr) * 1987-05-18 1989-12-28 Integrated Genetics, Inc. Molecules de proteine c ameliorees et procede de production et d'activation de telles molecules
US4959318A (en) * 1985-06-27 1990-09-25 Zymogenetics, Inc. Expression of protein C
US5516650A (en) * 1985-06-27 1996-05-14 Zymogenetics, Inc. Production of activated protein C
WO2002032461A2 (fr) * 2000-10-18 2002-04-25 Maxygen Aps Molecules de proteine c ou de type proteine c activee
WO2003035861A2 (fr) * 2001-10-19 2003-05-01 Institut National De La Sante Et De La Recherche Medicale-Inserm Proteines chimeriques clivables par la thrombine
WO2003106666A2 (fr) * 2002-06-14 2003-12-24 Maxygen Aps Variants de la proteine c presentant des proprietes modifiees

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4959318A (en) * 1985-06-27 1990-09-25 Zymogenetics, Inc. Expression of protein C
US5516650A (en) * 1985-06-27 1996-05-14 Zymogenetics, Inc. Production of activated protein C
WO1989012685A1 (fr) * 1987-05-18 1989-12-28 Integrated Genetics, Inc. Molecules de proteine c ameliorees et procede de production et d'activation de telles molecules
WO2002032461A2 (fr) * 2000-10-18 2002-04-25 Maxygen Aps Molecules de proteine c ou de type proteine c activee
WO2003035861A2 (fr) * 2001-10-19 2003-05-01 Institut National De La Sante Et De La Recherche Medicale-Inserm Proteines chimeriques clivables par la thrombine
WO2003106666A2 (fr) * 2002-06-14 2003-12-24 Maxygen Aps Variants de la proteine c presentant des proprietes modifiees

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013072504A1 (fr) * 2011-11-16 2013-05-23 Centre National De La Recherche Scientifique (Cnrs) Structure cristalline du trimère des c-propeptides de procollagène iii et ses applications
WO2023119230A1 (fr) 2021-12-22 2023-06-29 L'oreal Compositions de modulation de la voie de coagulation et de la voie de nicotinamide-adénine dinucléotide et leurs procédés d'utilisation

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