WO1991009951A2 - Proteine c recombinee a chaine legere tronquee - Google Patents

Proteine c recombinee a chaine legere tronquee Download PDF

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Publication number
WO1991009951A2
WO1991009951A2 PCT/US1990/007617 US9007617W WO9109951A2 WO 1991009951 A2 WO1991009951 A2 WO 1991009951A2 US 9007617 W US9007617 W US 9007617W WO 9109951 A2 WO9109951 A2 WO 9109951A2
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Prior art keywords
amino acid
protein
lys
acid number
arg
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PCT/US1990/007617
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English (en)
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WO1991009951A3 (fr
Inventor
Donald C. Foster
Richard D. Holly
Masahiko Suzuki
Kenji Wakabayashi
Anur Ashok Kumar
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Zymogenetics, Inc.
Teijin Limited
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Publication of WO1991009951A2 publication Critical patent/WO1991009951A2/fr
Publication of WO1991009951A3 publication Critical patent/WO1991009951A3/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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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 generally to plasma proteins and DNA sequences encoding those proteins, and more specifically to the expression of proteins having substantially the same structure and biological activity as human activated protein C.
  • APC activated protein C
  • Protein C is a vitamin K-dependent glycoprotein that contains approximately nine residues of gamma- carboxyglutamic acid (Gla) and one equivalent of beta- hydroxyaspartic acid, which are formed by post- translational modifications of glutamic acid and aspartic acid residues, respectively.
  • Gla gamma- carboxyglutamic acid
  • beta- hydroxyaspartic acid beta- hydroxyaspartic acid
  • activated protein C acts as a regulator of the coagulation process through the inactivation of factor Va and factor Villa by limited proteolysis.
  • the inactivation of factors Va and Villa by protein C is dependent upon the presence of acidic phospholipids and calcium ions. Protein S has been reported to regulate this activity by accelerating the APC-catalyzed proteolysis of factor Va (Walker, J. Biol. Chem. 255:5521-5524. 1980).
  • Protein C has also been implicated in the action of tissue-type plasminogen activator (Kisiel and Fujikawa, Behrin ⁇ Inst. Mitt. 23:29-42, 1983). Infusion of bovine APC into dogs results in increased plasminogen activator activity (Comp and Esmon, J. Clin. Invest. 68:1221-1228. 1981) . Other studies (Sakata et al., Proc. Natl. Acad. Sci.
  • protein C is useful in treating thrombotic disorders, such as venous thrombosis (Smith et al., PCT Publication No. WO 85/00521)
  • Activated protein__C may be preferred over the zymogen for the treatment of thrombosis.
  • the use of activated protein C bypasses the need for in vivo activation of protein C, thus providing a faster acting therapeutic agent.
  • protein C may be purified from clotting factor concentrates (Marlar et al.. Blood 59:1067-1072. 1982) or from plasma (Kisiel, J. Clin. Invest. 64:761-769. 1979) and activated in vitro. it is a complex and expensive process, in part due to the limited availability of the starting material and the low concentration of protein C in plasma. Furthermore, the therapeutic use of products derived from human blood carries the risk of disease transmission by, for example, hepatitis virus, cytomegalovirus, or human immunodeficiency virus (HIV) . For these reasons, it is preferable to produce human protein C and human activated protein C by genetic engineering techniques.
  • the present invention provides DNA molecules encoding activated protein C precursors, wherein the DNA molecules can be expressed by transfected mammalian cells to produce activated protein C having a heavy chain and a light chain, the light chain consisting essentially of the amino acid sequence of Figure 1 from alanine, amino acid number 1, to any one of glutamic acid, amino acid number 149, lysine, amino acid number 150, lysine amino acid number 151 or arginine, amino acid number 152.
  • the molecules encode the amino acid sequence Pre-pro-L-X ⁇ -H, wherein Pre-pro is the pre-pro peptide of protein C, or' is wholly or partially replaced with the pre-pro peptide of a vitamin K-dependent plasma protein such as protein S, factor VII, factor IX, factor X or prothrombin;
  • L is the light chain of activated protein C from alanine, amino acid number 1, to any one of glutamic acid, amino acid number 149, lysine, amino acid number 150, lysine, amino acid number 151 or arginine, amino acid number 152, as shown in Figure 1;
  • X3. is a sequence of three to ten amino acid residues selected from the group consisting of lysine and arginine; and H is the heavy chain of activated protein C.
  • the molecule encodes the amino acid sequence Pre-pro-L-R ⁇ -R2- 3-X2-( 4)n ⁇ R 5" *R 6 ⁇ R7-H
  • Pre-pro is the pre-pro peptide of protein C or is wholly or partially replaced with the pre-pro peptide of a protein selected from the group consisting of protein S, factor VII, factor IX, factor X and prothrombin
  • L is the light chain of activated protein C from alanine amino acid number 1 to any one of glutamic acid, amino acid number 149, lysine, amino acid number 150, lysine, amino acid number 151 or arginine, amino acid number 152
  • X is a sequence of three of four amino acid residues at least two or three of which are non-acidic amino acid residues
  • n 0, 1, 2, or 3
  • R1-R7 are Lys or Arg
  • H is the heavy chain of activated protein C.
  • the molecule encodes the amino acid sequence Pre-pro-L-R ⁇ _-R2-R3-R4-X3-R5-R6 ⁇ R 7 ⁇ g-H, wherein Pre-pro is the pre-pro peptide of a protein selected from the group consisting of protein C, protein S, factor VII, factor IX, factor X and prothrombin; L is the light chain of activated protein C from alanine, amino acid number 1, to any one of glutamic acid, amino acid number 149, lysine, amino acid number 150, lysine, amino acid number 151 or arginine, amino acid number 152; R ⁇ f
  • R 2' R 3' R 4' R 5' R 6r R 7 and R 8 are amino acid residues selected from the group consisting of lysine and arginine;
  • X 3 is a sequence of four non-acidic amino acid residues; and
  • H is the heavy chain of activated protein C.
  • X3 is preferably Asn-Ile-Leu-Asn.
  • cultured mammalian cells transfected with a DNA construct comprising a transcriptional promoter operably linked to a DNA molecule encoding an activated protein C precursor as described above are provided.
  • the cells may further be transfected to express the Saccharomyces cerevisiae KEX2 gene.
  • the invention provides methods of preparing activated protein C, comprising (a) transfecting cultured mammalian cells with an expression vector comprising a DNA molecule encoding an activated protein C precursor as described above operably linked to transcriptional promoter, transcriptional terminator and polyadenylation sequences, wherein, upon expression of the DNA sequence by the cells, the precursor is processed by the cells to produce activated protein C.
  • the cells may further be transfected to express the Saccharomyces cerevisiae KEX2 gene.
  • the invention further provides recombinant activated protein C having a heavy chain and a light chain, wherein the light chain consists essentially of the amino acid sequence of Figure 1 from alanine, amino acid number 1, to any one of glutamic acid, amino acid number 149, lysine, amino acid number 150, lysine, amino acid number 151 or arginine, amino acid number 152.
  • Figure 1 illustrates the nucleotide sequence of the complete protein C cDNA and the deduced amino acid sequence of human protein C.
  • the arrow indicates the junction of the activation peptide and the heavy chain.
  • the heavy chain of activated protein C extends from amino acid number 170, leucine, to amino acid number 419, proline.
  • Figure 2 illustrates the construction of the vector pD3. Symbols used are 0-1, the adenovirus 5 0-1 map unit sequence; E, the SV40 enhancer; MLP, the adenovirus 2 major late promoter; Ll-3, the adenovirus 2 tripartite leader; 5', 5' splice site; 3', 3' splice site; p(A), polyadenylation signal; DHFR, dihydrofolate reductase gene.
  • Figure 3 illustrates the construction of the vector pDX. Symbols are used as set forth in Figure 2.
  • Figure 4 illustrates the expression vectors PDX/PC962 and PC229/962.
  • Figure 5 illustrates the anticoagulant activity of protein C prepared according to certain embodiments of the present invention.
  • Figure 6 illustrates the construction of plasmids containing s ⁇ . cerevisiae KEX2 gene.
  • Figure 7 illustrates the plasmids pZMB-1 and pZMB-2. Symbols are used as set forth in Figure 2 and also include neo, neomycin resistance gene; SV40 term, SV40 terminator; SV40 prom, SV40 promoter.
  • Biological Activity A function or set of functions performed by a molecule in a biological context (i.e., in an organism or an i vitro facsimile thereof). Biological activities of proteins may be divided into catalytic and effector activities. Catalytic activities of vitamin K-dependent plasma proteins generally involve specific proteolytic cleavages of other plasma proteins, resulting in activation or deactivation of the substrates. Effector activities include specific binding of the biologically active molecule to calcium, phospholipids or other small molecules, to macromolecules, such as proteins, or to . cells.
  • activated protein C biological activity is characterized by its anticoagulant and fibrinolytic properties. Activated protein C inactivates factor Va and factor Villa in the presence of acidic phospholipids and calcium. Protein S appears to be involved in the regulation of this function (Walker, ibid.). Activated protein C also enhances fibrinolysis, an effect believed to be mediated by the lowering of plasminogen activator inhibitors levels (van Hinsbergh et al.. Blood 65: 444- 451, 1985) . The catalytic activities of activated protein C reside in the heavy chain. A protein having substantially the same biological activity as protein C will be essentially free of this activity until activated.
  • Activated Protein C A protein having the activity of activated protein C as defined above.
  • the protein will include a catalytic heavy chain and an effector light chain containing a calcium binding gla domain.
  • the light chain may be the 149-152 amino acid light chain of native human protein C, or may include amino acid substitutions, deletions or additions that do not substantially alter its effector activities.
  • the heavy chain of Protein C does not include an activation peptide.
  • Pre-Pro Peptide An amino acid sequence that occurs at the amino terminus of some proteins and is generally cleaved from the protein during translocation through the secretory pathway.
  • Pre-pro peptides comprise sequences directing the protein into the secretory pathway of the cell (signal peptides) that are characterized by the presence of a core of hydrophobic amino acids.
  • Pre-Pro peptides may also comprise processing signals.
  • pre-pro peptide may also mean a functional portion of a naturally occurring pre-pro peptide.
  • Expression Vector A DNA molecule which contains, inter alia, a DNA sequence encoding a protein of interest together with a promoter and other sequences, such as a transcription terminator and polyadenylation signal, that facilitate expression of the protein. Expression vectors further contain genetic information that provides for their replication in a host cell, either by autonomous replication or by integration into the host genome. Examples of expression vectors commonly used for recombinant DNA are plasmids and certain viruses, although they may contain elements of both. They also may include a selectable marker.
  • Cultured mammalian cells Cells that have been isolated from a mammal and are able to be propagated in vjjtrq.
  • DNA construct A DNA molecule, or a clone of such a molecule, which has been constructed through human intervention to contain sequences arranged in a way that would not otherwise occur in nature.
  • protein C is initially produced as a single chain polypeptide, which undergoes extensive processing to yield activated protein C.
  • This processing includes the formation of specific gamma-carboxyglutamic acid residues in the amino-terminal region of the light chain, beta-hydroxylation of an aspartic acid residue and proteolytic cleavage.
  • human activated protein C is generally believed to contain a light chain consisting of 155 amino acid residues (Foster et al., ibid.; Bang et al., ibid.).
  • the inventors have learned that the light chain of activated protein C purified from human plasma is heterogeneous and contains an active species consisting of anywhere from 149 to 152 amino acid residues.
  • the inventors have constructed certain novel DNA molecules that, when transfected into cultured mammalian cells, are expressed to yield activated protein C having a 149-152 amino acid light chain.
  • the carboxyl- terminal residue of this 149-152 amino acid light chain is glutamic acid, lysine or arginine.
  • the present invention provides methods of producing a protein that is gamma-carboxylated and has the biological activity of human activated protein C through the use of cultured mammalian cells transfected to express the protein. These methods rely in part on the use of novel cleavage sites to direct the proteolytic processing of activated protein C precursors.
  • These precursors may have the amino acid sequence Pre-pro-L-X ⁇ H, wherein Pre- pro is the pre-pro peptide of a vitamin K-dependent plasma protein; L is the light chain of activated protein C from alanine, amino acid number 1, to any one of glutamic acid, amino acid number 149, lysine, amino acid number 150, lysine, amino acid number 151 or arginine, amino acid number 152, as shown in Figure 1; X ⁇ is a sequence of three to ten amino acid residues selected from the group consisting of lysine and arginine; and H is the heavy chain of activated protein C.
  • Pre- pro is the pre-pro peptide of a vitamin K-dependent plasma protein
  • L is the light chain of activated protein C from alanine, amino acid number 1, to any one of glutamic acid, amino acid number 149, lysine, amino acid number 150, lysine, amino acid number 151 or arginine, amino acid number 152, as shown in
  • Vitamin K-dependent plasma proteins are those plasma proteins that contain gamma- carboxylated glutamic acid residues, including factor VII, factor IX, factor X, prothrombin, protein C and protein S.
  • X is the sequence Lys- Lys-Arg or the sequence Lys-Arg-Lys-Arg or any one of the sequences Lys-Lys-Arg-Arg, Lys-Lys-Arg-Lys-Arg, Lys-Lys- Arg-Arg-Arg-Lys-Arg, Lys-Lys-Arg-Lys-Lys-Lys-Arg-Arg-Lys- Arg, Lys-Lys-Arg-Lys-Lys-Lys-Lys-Arg and Lys-Lys- Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-
  • a second group of activated protein C precursors have the amino acid sequence Pre-pro-L-R 1 -R 2 -R3-(R4) n -X2 ⁇ (R5) m -Rg-R7-Rg-H, wherein Pre-pro is the pre-pro peptide of a protein selected from the group consisting of protein
  • L is the light chain of activated protein C from alanine, amino acid number 1, to any one of glutamic acid, amino acid number 149, lysine, amino acid number
  • R ⁇ , R2, R3, R4, R5, Re, R7 and Rg are amino acid residues selected from the group consisting of lysine and arginine;
  • X comprises a peptide bond or spacer peptide of 1-12 amino acids;
  • n, M 0, 1, 2 or 3; and
  • H is the heavy chain of activated protein C.
  • a ourth group of activated protein C precursors have the amino acid sequence Pre- ro-L- ⁇ -R2-R3- 4-X3- s- R6-R7-R8, wherein Pre-pro is the pre-pro peptide of a protein selected from the group consisting of protein C, protein S, factor VII, factor IX, factor X and prothrombin; L is the light chain of activated protein C from alanine, amino acid number 1, to any one of glutamic acid, amino acid number 149, lysine, amino acid number 150, lysine, amino acid number 151 or arginine, amino acid number 152; R ⁇ , R 2 , R3, R4, R5, e , R7 and R 8 are amino acid residues selected from the group consisting of lysine and arginine; X3 is a sequence of four non-acidic amino acid residues; and H is the heavy chain of activated protein C.
  • a particularly preferred sequence of non- acidic amino acids is Asn-Ile-
  • the present invention also provides a group of human activated protein C analogs that have the protein C amino-terminal portion (gla domain) substituted with the gla domain of a vitamin-K dependent plasma protein selected from the group consisting of factor VII, factor IX, factor X, prothrombin or protein S.
  • a vitamin-K dependent plasma protein selected from the group consisting of factor VII, factor IX, factor X, prothrombin or protein S.
  • the amino- terminal portions of vitamin K-dependent plasma proteins are responsible for at least part of their respective calcium binding activities. It has been found that, as a result of this functional homology, the gla domains of these molecules may be interchanged and the resulting chimeric proteins still retain the activity specific to the catalytic domain.
  • IX may be joined to factor VII at amino acid 38 to produce a protein having the activity of factor VTI.
  • Factor VII, factor IX, factor X, prothrombin, and protein S share this amino-terminal sequence homology with protein C.
  • This region of homology spans approximately 35-45 amino acid residues, with a C-terminal boundary generally corresponding to an exon-intron boundary in the respective gene.
  • the gla domain of jiuman protein C extends from amino acid number 1 of the mature light chain to approximately amino acid number 37 as shown in Figure 1.
  • a cloned sequence comprising the 5'-coding region of the gene for any of these proteins may be substituted for the corresponding sequence of the protein C gene.
  • activated protein C analogs consist essentially of a hybrid light chain including a gla domain operably joined to the gla domain-less light chain of activated protein C terminating at any one of glutamic acid, amino acid 149, lysine, amino acid number 150, lysine, amino acid number 151 or arginine, amino acid number 152 of native human protein C, wherein the hybrid light chain is disulfide bonded to the heavy chain of activated protein C.
  • These analogs are encoded by DNA sequences encoding Pre-pro-Gla- L-X-H, wherein Pre-pro and Gla are the pre-pro peptide and gla.
  • L is the gla domain-less light chain of activated protein C terminating at any one of glutamic acid, amino acid number 149, lysine, amino acid number 150, lysine, amino acid number 151 or arginine, amino acid number 152,
  • X is a sequence jof three to ten amino acids selected from the group consisting of lysine and arginine and H is the heavy chain of activated protein C.
  • X is the sequence Pre-pro-L-R 1 -R 2 -R3-Ala-Asn-Ser-R4-R5-R 6 -R 7 -H, wherein Pre-pro is the pre-pro peptide of a protein selected from the group consisting of protein C, protein S, factor VII, factor IX, factor X and prothrombin; L is the light chain of activated protein C from alanine, amino acid number 1, to glutamic acid, amino acid number 149;
  • R 6 and R 7 are amino acid residues selected from the group consisting of lysine and arginine; and H is the heavy chain of activated protein C.
  • these analogs are encoded by DNA sequences encoding Pre-pro-Gla-L-R 1 -R 2 -R3-X-(R4) n -R5-Rg- R 7 -H, wherein Pre-pro-L-R 1 -R 2 -R3-X-(R4)n-R5-R 6 -R 7 -H wherein Pre-pro is the pre-pro peptide of protein C or is wholly or partially replaced—with the pre-pro peptide of a protein selected from the group consisting of protein S, factor VII, factor IX, factor X and prothrombin; L is the light chain of activated protein C from alanine amino acid number 1 to any one of glutamic acid, amino acid number 149, lysine, amino acid number 150
  • these analogs are encoded by DNA sequences encoding Pre-pro-Gla-L-R 1 -R -R3-R4-X-R5-R6-R7-Rg-H, wherein each of ⁇ through Rg is Lys or Arg and X is a sequence of four non-acidic amino acid residues, preferably the sequence Asn-Ile-Leu-Asn. It is preferred that the pre-pro sequence and gla domain of an activated protein C precursor be derived from the same protein.
  • cDNA sequences are preferred for carrying out the present invention due to their lack of intervening sequences.
  • Complementary DNAs encoding protein C may be obtained from libraries prepared from liver cells according to standard laboratory procedures. It will be understood, however, that suitable DNA sequences can also be obtained from genomic clones or can be synthesized de novo according to conventional procedures. Techniques for producing synthetic nucleotide sequences are well known in the art. For example, a set of overlapping oligonucleotides may be synthesized and annealed in pairs to yield double-stranded fragments with overlapping adhesive termini.
  • fragments are then ligated as necessary to provide a complete coding sequence.
  • genomic sequences it is generally desirable to remove introns. If partial clones are obtained, it is necessary to join them in proper reading frame to produce a full length clone, using such techniques as endonuclease cleavage, ligation, and loop- out mutagenesis.
  • the coding sequence will further encode a pre-pro peptide at the amino-terminus of the protein in order to obtain proper post-translational processing (e.g. gamma-carboxylation of glutamic acid residues) and secretion from the host cell.
  • the pre-pro peptide may be that of protein C or wholly or partially replaced by the pre-pro peptide of another vitamin K- dependent plasma protein, such as factor VII, factor IX, factor X, prothrombin or protein S.
  • the cloned DNA sequence is then modified to encode an activated protein C precursor of the present invention. Modification may be achieved by site-specific mutagenesis.
  • the protein C sequence may be enzymatically cleaved to remove the native activation peptide sequence and adjacent light chain amino acids, and the sequences encoding the heavy and native light chains joined to a synthesized linker peptide containing a cleavage site.
  • Expression vectors for use in carrying out the present invention will comprise a promoter capable of directing the transcription of a cloned gene or cDNA.
  • Preferred promoters include both viral promoters and cellular promoters.
  • Viral promoters useful in this regard include the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981) and the CMV promoter (Boshart et al.. Cell 4 ⁇ :521-530, 1985).
  • a particularly preferred viral promoter is the major late promoter from adenovirus 2 (Kaufman and Sharp. Mol.
  • Cell. Biol. 2:1304-1319, 1982 Cellular promoters include the mouse kappa gene promoter (Bergman et al., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983) and the mouse VH promoter (Loh et al.. Cell 33:85- 93, 1983).
  • a particularly preferred cellular promoter is the metallothionein I promoter (Palmiter et al., Science 222:809-814. 1983).
  • Expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the insertion site for the protein C sequence or within the protein C sequence itself.
  • RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes.
  • transcription termination and polyadenylation signals located downstream of the insertion site.
  • Particularly preferred polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 Elb region, the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9_:3719-3730, 1981) and the protein C gene polyadenylation signal.
  • the expression vectors may also include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites and enhancer sequences, such as the SV40 enhancer and the sequences encoding the adenovirus VA RNAs.
  • a noncoding viral leader sequence such as the adenovirus 2 tripartite leader
  • enhancer sequences such as the SV40 enhancer and the sequences encoding the adenovirus VA RNAs.
  • a gene that confers a selectable phenotype is generally introduced into the cells along with the gene or cDNA of interest.
  • Preferred selectable markers include genes that confer resistance to drugs such as neomycin, hygromycin, and ethotrexate.
  • the selectable marker may be an amplifiable selectable marker.
  • a preferred amplifiable selectable marker is the DHFR gene.
  • Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and Simonsen, U.S. Patent 4,713,339 and U.S. Patent Application Serial No. 07/226,173). It may also be advantageous to add additional DNA, known as "carrier DNA,” to the mixture that is introduced into the cells.
  • carrier DNA additional DNA
  • the cells After the cells have taken up the DNA, they are cultured to produce activated protein C.
  • the cells are cultured according to standard methods in a growth medium containing nutrients required for the growth of mammalian cells.
  • suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.
  • the medium will generally also contain vitamin K at a concentration of about 0.1 ⁇ g/ml to about 5 ⁇ g/ml.
  • Drug selection is then applied to select for the growth of cells that are expressing the selectable marker in a stable fashion.
  • the drug concentration may be increased to select for an increased copy number of the cloned sequences, thereby increasing expression levels. Clones of stably transfected cells are then screened for expression of activated protein C.
  • Preferred cultured mammalian cells for use in the present invention include the COS-1 (ATCC CRL 1650) ,
  • a preferred BHK cell line is the tk ⁇ tsl3 BHK cell line (Waechter and Baserga, Proc.
  • NCTC 1469 ATCC CCL 9.1
  • CHO ATCC CCL 61
  • DUKX cells Urlaub and Chasin, Proc. Natl. Acad. Sci. USA
  • the activated protein C precursors of the present invention are activated by proteolytic cleavage and removal of the linker peptide in the secretory pathway of the host cell. It has been found that proteins lacking the native activation peptide and the C-terminal three to six amino acids of the light chain are nevertheless properly processed by genetically engineered host cells, resulting in secretion of activated protein C.
  • Processing of activated protein C precursors to the two-chain form may be enhanced by modifying the host cell.
  • Processing of protein C by cleavage after a sequence of two or more basic amino acids may be enhanced by introducing the Saccharomyces cerevisiae KEX2 gene into the host cell.
  • the KEX2 gene encodes an endopeptidase that cleaves after a dibasic amino acid sequence (Fuller et al., in Leive, ed.. Microbiology. 1986, 273-278, 1986).
  • Human activated protein C produced according to the present invention is isolated from the host cells by harvesting the culture media.
  • the isolated protein may be purified using conventional techniques of protein chemistry, such as by affinity chromotography on an anti- protein C antibody column.
  • Additional purification of the column eluate may be achieved by conventional chemical purification means, such as high-performance liquid chromatography (HPLC) .
  • HPLC high-performance liquid chromatography
  • the activated protein C of the present invention may be used in pharmaceutical compositions for topical or intravenous administration, generally in combination with a physiologically acceptable carrier or diluent.
  • Preferred carriers and diluents include water, buffered water, 0.4% saline, 0.3% glycine and the like.
  • Pharmaceutical compositions may also contain stabilizers and adjuvants. These compositions may be sterilized by conventional, well- known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a serile aqueous solution prior to administration.
  • the compositions may further contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium lactate, solium chloride, potassium chloride, calcium chloride, etc.
  • concentration of protein C in these formulations can vary widely, i.e., from less than about 0.5% to as much as 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution and 10 mg of protein C.
  • Actual methods for preparing parenteraliy administrable compounds will be known or apparent to those skilled in the art and are described in more detail in, for example. Remington's Pharmaceutical Science. 16th ed.. Mack Publishing Company, Easton, PA (1982) , which is incorporated herein by reference.
  • compositions containing protein C can be administered for prophylactic and/or therapeutic treatments.
  • the compositions are administered in an amount sufficient to cure or at least partially arrest the disease and its complications. Amounts effective for this use will depend on the severity of the disease or injury and the general state of the patient, but generally range from about 1 mg to about 300 mg of protein C per day, with dosages of from about 5 mg to about 25 mg of protein C per day being more commonly used. It must be kept in mind that the materials of the present invention may generally be employed in serious disease or injury states, that is, life- threatening or potentially life-threatening situations. In such cases, it is possible and may be felt desireable by the treating physician to administer substantial excesses of these protein C compositions.
  • compositions containing the hybrid protein C are administered to a patient susceptible to or otherwise at risk of a disease state or injury to enhance the patient's own anticoagulative or fibrinolytic capabilities.
  • the precise amounts again depend on the patient's state of health and general level of endogenous protein C, but generally range from about 0.5 mg to about 250 mg per 70 kilogram patient, especially about 1 mg to about 25 mg per 70 kg of body weight.
  • Restriction endonucleases and other DNA modification enzymes e.g., T4 polynucleotide kinase, calf alkaline phosphatase, DNA polymerase I [Klenow fragment] , T4 polynucleotide ligase
  • T4 polynucleotide kinase calf alkaline phosphatase
  • DNA polymerase I [Klenow fragment] e.g., T4 polynucleotide ligase
  • a cDNA coding for a portion of human protein C was prepared as described by Foster and Davie (ibid.). Briefly, a ⁇ gtll cDNA library was prepared from human liver mRNA by conventional methods. Clones were screened using an 1251-labeled affinity-purified antibody to human protein C, and phage were prepared from positive clones by the plate lysate method (Maniatis et al., ibid.), followed by banding on a cesium chloride gradient. The cDNA inserts were removed using Eco RI and subcloned into plasmid pUC9 (Vieira and Messing, Gene 19:259-268, 1982).
  • Restriction fragments were subcloned in the phage vectors M13mpl0 and M13mpll (Messing, Meth. in Enzymology 101:20- 77, 1983) and sequenced by the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467. 1977).
  • a clone was selected that contained DNA corresponding to the known partial sequence of human protein C (Kisiel, ibid., 1979) and encoded protein C beginning at amino acid 64 of the light chain and extending through the heavy chain and into the 3' non-coding region. This clone was designated AHC1375.
  • a second cDNA clone coding for protein C from amino acid 24 was also identified.
  • the insert from the larger clone was subcloned into pUC9 and the plasmid was designated pHCk6L.
  • This clone encodes a major portion of protein C, including the heavy chain coding region, termination codon, and 3' non-coding region.
  • the cDNA insert from AHC1375 was nick translated using ⁇ - P dNTP's and used to probe a human genomic library in phage ⁇ Charon 4A (Maniatis et al., Cell 15:687- 702, 1978) using the plaque hybridization procedure of Benton and Davis (Science 196:181-182. 1977) as modified by Woo (Meth. Enzymol. 68.:381-395, 1979). Positive clones were isolated and plaque-purified (Foster et al., Proc. Natl. Acad. Sci. USA 82:4673-4677, 1985, herein incorporated by ⁇ reference) . Phage DNA prepared from positive clones (Silhavy et al.
  • the cDNA insert of pHC ⁇ 6L was nick translated and used to probe the phage ⁇ Charon 4A library.
  • One genomic clone was identified that hybridized to probes made from the 5' and 3' ends of the cDNA.
  • This phage clone was digested with Eco RI, and a 4.4 kb fragment, corresponding to the 5' end of the protein C gene, was subcloned into pUC9.
  • the resultant recombinant plasmid was designated pHCR4.4.
  • Complete DNA sequence analysis revealed that the insert in ⁇ pHCR4.4 comprised two exons of 70 and 167 base pairs separated by an intron of 1263 bp. The first exon encodes amino acids -42 to -19; the second encodes amino acids -19 to 37. Sequence analysis confirmed the DNA sequence of the entire protein C gene.
  • a genomic fragment containing an exon corresponding to amino acids -42 to -19 of the pre-pro peptide of protein C was isolated, nick translated, and used as a probe to screen a cDNA library constructed by the technique of Gubler and Hoffman (Gene 25:263-269. 1983) using mRNA from Hep G2 cells. This cell line was derived from human hepatocytes and was previously shown to synthesize protein C (Fair and Bahnak, Blood 64:194-204. 1984) . Ten positive clones comprising cDNA inserted into the Eco RI site of phage ⁇ gtll were isolated and screened with an oligonucleotide probe corresponding to the 5' non- coding region of the protein C gene.
  • the cDNA contained 70 bp of 5' untranslated sequence, the entire coding sequence for human pre-pro-protein C, and the entire 3' non-coding region corresponding to the second polyadenylation site ( Figure 1) .
  • the vector pDX was derived from pDHFRIII (Berkner and Sharp, Nuc. Acids Res. 12:841-857, 1985) as shown in Figures 2 and 3.
  • the Pst I site immediately upstream from the DHFR sequence in pDHFRIII was converted to a Bel I site by digesting 10 ⁇ g of plasmid with 5 units of Pst I for 10 minutes at 37 ⁇ C in 100 ⁇ l restriction buffer A (10 mM Tris pH 8, 10 mM MgC12, 6 mM NaCl, 7 mM ,9-MSH) .
  • restriction buffer A 10 mM Tris pH 8, 10 mM MgC12, 6 mM NaCl, 7 mM ,9-MSH
  • the DNA was phenol extracted, ethanol precipitated, and resuspended in 40 ⁇ l polymerase buffer (50 mM Tris pH 8, 7mM MgCl2, 7 mM 0-MSH) containing 10 mM dCTP and 16 units T4 DNA polymerase and incubated at 12°C for 60 minutes.
  • the DNA was ligated to 2.5 ⁇ g kinased Bel I linkers in 14 ⁇ l ligase buffer (10 mM Tris pH 8, 10 mM MgCl 2 , 1 mM DTT, 1.4 mM ATP) containing 400 units T4 polynucleotide ligase for 12 hours at 12 ⁇ C.
  • 14 ⁇ l ligase buffer 10 mM Tris pH 8, 10 mM MgCl 2 , 1 mM DTT, 1.4 mM ATP
  • T4 polynucleotide ligase for 12 hours at 12 ⁇ C.
  • the DNA was resuspended in 120 ⁇ l restriction buffer B (75 mM KCI, 6 mM Tris pH 7.5, 10 mM MgCl2, 1 mM DTT), digested with 80 units Bel I for 60 minutes at 50 ⁇ C, then electrophoresed through agarose.
  • Form III plasmid DNA (10 ⁇ g) was isolated from the gel and ligated in 10 ⁇ l buffer C containing 50 units T4 polynucleotide ligase for 2 hours at 12°C, then used to transform JL. coli HB101. Positive colonies were identified by rapid DNA preparation analysis, and plasmid DNA (designated pDHFR') prepared from positive colonies was transformed into dam- E ⁇ . coli.
  • Plasmid pD2' was then generated by cleaving pDHFR' (15 ⁇ g) and pSV40 (comprising Bam HI digested SV40 DNA cloned into the Bam HI site of pML-1) (25 ⁇ g) in 100 ⁇ l restriction buffer B with 25 units Bel I for 60 minutes at 50°C, followed by the addition of 50 units of Bam HI and additional incubation at 37 ⁇ C for 60 minutes. DNA fragments were resolved by agarose gel electrophoresis, and the 4.9 kb pDHFR' fragment and 0.2 kb SV40 fragment were isolated.
  • Plasmid pD2' was modified by deleting the "poison" sequences in the pBR322 region (Lusky and Botchan, Nature 293:79-81. 1981). Plasmids pD2' (6.6 ⁇ g) and pML-1 (Lusky and Botchan, ibid.) (4 ⁇ g) were incubated .in 50 ⁇ l restriction buffer A with 10 units each Eco RI and Nru I for 2 hours at 37°C, followed by agarose gel electrophoresis.
  • the 1.7.kb pD2' fragment and the 1.8 kb pML-1 fragment were isolated and ligated together (50 ng each) in 20 ⁇ l ligase buffer containing 100 units T4 polynucleotide ligase for 2 hours at 12°C, followed by transformation into J . coli HB101. Colonies containing the desired construct (designated pD2) were identified by rapid preparation analysis. Ten ⁇ g of pD2 was then digested with 20 units each Eco RI and Bgl II in 50 ⁇ l restriction buffer A for 2 hours at 37°C. The DNA was electrophoresed through agarose, and the desired 2.8 kb fragment, comprising the pML-1, 3' splice site and poly (A) sequences, was isolated. Plasmid pDHFRIII was modified to convert the Sac
  • the resultant plasmids were digested with 50 units Hind III or Kpn I, as appropriate, and electrophoresed through agarose.
  • Gel- isolated DNA 250 ng was ligated in 30 ⁇ l ligase buffer containing 400 units T4 DNA ligase for 4 hours at 12 ⁇ C and used to transform J s . coli RR1.
  • the resultant plasmids were designated pDHFRIII(Hind III) and pDHFRIII(Kpn I).
  • a 700 bp Kpn I-Bgl II fragment was then purified from pDHFRIII(Kpn I) by digestion with Bgl II and Kpn I followed by agarose gel electrophoresis.
  • the SV40 enhancer sequence was inserted into pDHFRIII(Hind III). Fifty ⁇ g SV40 DNA was incubated in 120 ⁇ l restriction buffer A with 50 units Hind III for 2 hours at 37 ⁇ C, and the Hind III SV40 fragment (5089-968 bp) was gel purified. Plasmid pDHFRIII(Hind III) (10 ⁇ g) was treated with 250 ng calf intestinal phosphatase for 1 hour at 37 ⁇ C, phenol extracted and ethanol precipitated.
  • the linearized plasmid (50 ng) was ligated with 250 ng of the SV40-Hind III fragment in 16 ⁇ l ligase buffer for 3 hours at 12°C, using 200 units T4 polynucleotide ligase, and transformed into E ⁇ . coli HB101. A 700 base pair Eco RI-Kpn I fragment was then isolated from this plasmid.
  • Plasmid pD3 was then constructed.
  • the 700 bp Kpn I-Bgl II fragment and the 700 bp Eco RI-Kpn I fragment (50 ng each) were ligated with 10 ng of the 2.8 kb pML-1, 3' splice site, poly(A) fragment with 200 units T4 polynucleotide ligase for 4 hours at 12°C, followed by transformation of IL. coli RR1. Positive colonies were detected by rapid preparation analysis, and a large-scale preparation of pD3 (Figure 2) was made.
  • the vector pD3' was constructed in a similar manner, except that the SV40 polyadenylation signal (i.e., the SV40 Bam HI [2533 bp] to Bel I [2770 bp] fragment was inserted in the late orientation.
  • pD3' contains a Bam HI site as the site of gene insertion ( Figure 3) .
  • the vector pDX was then generated from pD3 and pD3' as shown in Figure 3.
  • the Eco RI site in pD3' was converted to a Bel I site by Eco RI cleavage, incubation with SI nuclease, and subsequent ligation with Bel I linkers.
  • DNA was prepared from a positively identified colony, and the 1.9 kb Xho I-Pst I fragment containing the altered restriction site was prepared via agarose gel electrophoresis.
  • Bel I-cleaved pD3 was ligated with kinased Eco RI-Bcl I adapters (constructed from oligonucleotides ZC525, 5'GGA ATT CT 3'; and ZC526, 5'GAT CAG AAT TCC 3') in order to generate a unique Eco RI site for inserting a gene into the expression vector.
  • a positive colony was identified by restriction endonuclease analysis, and DNA from this colony was used to isolate a 2.3 kb Xho I-Pst I fragment containing the modified restriction site.
  • the two above- described DNA fragments were incubated together with T4 DNA ligase, transformed into E ⁇ . coli HB101, and positive colonies were identified by restriction analysis. Plasmid DNA was isolated and designated pDX ( Figure 3) . This plasmid contains a unique Eco RI site for insertion of foreign genes.
  • the protein C cDNA was inserted into pDX as an Eco RI fragment. Recombinant plasmids were screened by restriction analysis to identify those having the protein C insert in the correct orientation with respect to the promoter elements, and plasmid DNA (designated pDX/PC) was prepared from a correct clone.
  • pDX/PC contains an ATG codon in the 5' non-coding region (see Figure 1) .
  • deletion mutagenesis was performed on the cDNA prior to transfection and- expression experiments. Deletion of the three base pairs was performed according to standard procedures of oligonucleotide-directed mutagenesis.
  • the pDX-based vector containing the modified cDNA was designated p594. Plasmid p594 was transfected into COS-1 (ATCC
  • the protein C secreted into the culture media was assayed by enzyme-linked immunosorbent assay (ELISA) using the same affinity-purified polyclonal antibody that was used in the initial identification of the cDNA clones, and/or a monoclonal antibody directed against the heavy chain of protein C.
  • ELISA enzyme-linked immunosorbent assay
  • the affinity-purified antibody to human protein C 100 ⁇ g/ml in 0.1 M Na2C03, pH 9.6 was added to each well of 96-well microtiter plates, and the plates were incubated overnight at 4°C.
  • the wells were washed three times with PBS (5 mM phosphate buffer, pH 7.5, 0.15 M NaCl) containing 0.05% Tween-20 to remove unbound antibody and were incubated with 100 ⁇ l of 1% bovine serum albumin, 0.05% Tween 20 in PBS at 4°C overnight.
  • the plates were rinsed several times with PBS, air dried, and stored at 4°C.
  • 100 ⁇ l of each sample was incubated for 1 hour at 37°C in the coated wells, and the wells were rinsed with 0.05% Tween-20 in PBS.
  • the plates were then incubated for 1 hour at 37 ⁇ C with a biotin-conjugated sheep polyclonal antibody to protein C (30 ng/ml) in PBS containing 1% bovine serum albumin and 0.05% Tween-20.
  • the wells were rinsed with PBS and incubated for 1 hour at 37°C with avidin- conjugated alkaline phosphatase in PBS containing 1% bovine serum albumin and 0.05% Tween-20.
  • the wells were rinsed with PBS, and alkaline phosphatase activity was measured by the addition of 100 ⁇ l of phosphatase substrate (Sigma 104; 600 ⁇ g/ml in 10% diethanolamine, pH 9.8, containing 0.3 mM MgCl2) .
  • the recombinant protein C was assayed for anticoagulant activity by measuring its ability to prolong coagulation. Dialyzed media samples were treated with Protac C (American Diagnostica) to activate the protein C. The activated samples were then added to an in vitro clotting assay (Sugo et al., J. Biol. Chem. 260:10453. 1985) . Briefly, 50 ⁇ l each of normal pooled human plasma, rabbit brain cephalin (10 mg/ml in TBS [50mM Tris pH 7.5, 150 mM NaCl]) and kaolin suspension (5 mg/ml in TBS) were mixed in a siliconized glass tube.
  • Protein C produced by transfected tk ⁇ tsl3 BHK and 293 cells was further analyzed by Western blotting. Media samples were electrophoresed on denaturing gels, and blots were prepared and probed with radiolabeled antibody to protein C. Results indicated that about 20% of the protein C from BHK cells was in the two-chain form, while about 90% of that from 293 cells was processed to the two- chain form.
  • the resultant mutant precursor of protein C designated PC962
  • PC962 contains the sequence Ser-His-Leu-Arg-Arg-Lys-Arg-Asp at the cleavage site between the light and heavy chains (Table 2; the amino acids that have been added to the sequence encoding wild-type (594) protein C appear in bold, and spaces between amino acids are used solely for aligning the light and heavy chain sequences) . Processing at the Arg-Asp bond results in a two-chain protein C molecule.
  • Table 2 Amino Acid Sequences of Cleavage-Site Mutants 594
  • the mutant molecule was generated by altering the cloned cDNA by site-specific mutagenesis (essentially as described by Zoller and Smith, DNA 3:479-488, 1984) using the mutagenic oligonucleotide ZC962 (5' AGT CAC CTG AGA AGA AAA CGA GAC A 3') and oligonucleotide ZC550 (5' TCC CAG TCA CGA CGT 3') .
  • Plasmid p594 was digested with Sst I, the approximately 870 bp fragment was cloned into M13mpll, and single-stranded template DNA was isolated. Following mutagenesis, a correct clone was identified by sequencing.
  • Replicative form DNA was isolated and digested with Sst I, and the mutagenized fragment was recovered. This mutagenized fragment was joined with Sst I-cut p594 in a two-part ligation. Clones having the Sst I fragment inserted in the desired orientation were identified by restriction enzyme mapping. The resulting expression vector was designated pDX/PC962 ( Figure 4) .
  • Plasmid pDX/PC962 was co-transfected into tk ⁇ tsl3 BHK cells with pSV2-DHFR (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981) by the calcium phosphate procedure (essentially as described by Graham and van der Eb, ibid.). The transfected cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, lx PSN antibiotic mix (Gibco 600-5640) , 2.0 mM L-glutamic acid and vitamin K (5 ⁇ g/ml).
  • DMEM Dulbecco's modified Eagle's medium
  • the cells were selected in 250 nM ethotrexate (MTX) for 14 days, and the resulting colonies were screened by the immunofilter assay (McCracken and Brown, BioTechniques. 82-87, March/April 1984). Plates were rinsed with PBS or No Serum medium (DMEM plus lx PSN antibiotic mix, 5 ⁇ g/ml vitamin K) . TeflonTM mesh (Spectrum Medical Industries, Los Angeles, CA) was then placed over the cells. Nitrocellulose filters were wetted with PBS or No Serum medium, as appropriate, and placed over the mesh.
  • MTX nM ethotrexate
  • the filters were removed and placed in filter buffer (50 mM Tris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150 mM NaCl, 0.25% gelatin) for 30 minutes at room temperature.
  • the filters were incubated for 1 hour at room temperature, with shaking, in biotin-labeled sheep anti-protein C polyclonal antibody (1 ⁇ g/ml in filter buffer) . Filters were then washed in the same buffer and incubated 1 hour at room temperature, with shaking, in avidin-conjugated horseradish peroxidase (Boehringer- Mannheim) (diluted 1:1000 in the filter buffer).
  • the clone BHK/962-1 was grown in larger scale culture, and several hundred micrograms of protein C was purified by affinity chromatography on a column prepared by coupling 7 mg of polyclonal sheep antibody against human protein C to 2 grams of CNBr-activated Sepharose 4B (Pharmacia Inc. , Piscataway, NJ) . Cell culture medium was applied to the column and the column was washed with 100 ml TBS. The protein C was eluted with TBS containing 3 M KSCN or with pH 11.5 buffer (25 mM potassium phosphate, pH 11.5, 0.2 M NaCl, 2% Tween-80, 0.5% NaN 3 ). Western blot analysis demonstrated that the mutant protein C was approximately 95% in the two-chain form, compared to about 20% two-chain protein C obtained from tk ⁇ " tsl3 BHK cells transfected with the native sequence.
  • the BHK-produced PC962 protein was assayed for its ability to be activated to a form that shows both amidolytic and anticoagulant activities.
  • Affinity- purified protein samples were exhaustively dialyzed against TBS, then activated by incubation at 37 ⁇ C for 1 hour with 0.1 volume of 1 unit/ml Protac C (American Diagnostica) .
  • Amidolytic activity was measured by adding aliquots of the activation mixture to 100 ⁇ l of 1 mM protein C substrate (Spectrozyme PCa, American Diagnostica) in a microtiter well and measuring the change in A4 0 5 over time using a microtiter plate reader.
  • Anticoagulant activity of the activated protein C was assayed as described by Sugo et al.
  • Milligram quantities of protein C were purified from either stable tk ⁇ tsl3 BHK cell clones expressing the PC962 mutant protein or stable 293 cell clones expressing the wild-type protein C (p594 transfected cells) using a monoclonal antibody column specific for the calcium- induced conformation of protein C.
  • Cell culture media were applied to the column in the presence of 5 mM CaCl2, and protein C was eluted from the column with TBS containing 10 mM EDTA.
  • the use of this purification method permitted purification of completely active protein C without exposure to denaturing conditions.
  • the purified protein C was analyzed by SDS/PAGE followed by silver staining and was shown to be >95% pure.
  • a second plasmid designated PC229/962, was constructed by inserting the PC962 cDNA into plasmid Zem229.
  • Zem229 is a pUCl ⁇ -based expression vector containing a unique Bam HI site for insertion of foreign DNA between the mouse metallothionein-I promoter and SV40 transcription terminator.
  • Zem229 also contains an expression unit comprising the SV40 early promoter, mouse dihydrofolate reductase gene, and SV40 terminator.
  • Plasmid PC229/962 was transfected into tk " tsl3 BHK cells by the calcium phosphate method. Cells were cultured in DMEM containing 5% fetal calf serum and 5 ⁇ g/ml vitamin K. The 48-hour transient expression level from this transfection was approximately 25 ng/ml. After 2 days, the transfected cells were split into selective media containing 1 ⁇ M MTX and cultured for an additional 14 days. Three plates from this transfection, containing approximately 200 colonies each, were screened by the immunofilter assay, and the 24 most intensely reacting colonies were picked by cylinder cloning. The twenty-four colonies were grown individually in 10-cm plates, and their protein C production levels were measured.
  • Colonies producing between 1.1 and 2.3 pg/cell/day were used for the production of stable protein C-producing cell lines. Subsequent analysis by SDS/PAGE followed by silver staining showed that the mutant protein was essentially completely processed to two chains. N-terminal sequence analysis showed that both the light and heavy chains of recombinant wild-type and BHK/PC962 proteins were properly processed. In addition, PC962 from tk ⁇ tsl3 BHK cells showed full amidolytic activity.
  • a DNA sequence encoding an activated protein C precursor with the cleavage site sequence Arg-Arg-Lys-Arg was constructed by mutagenesis of the wild-type protein C sequence.
  • the resultant sequence (designated 1058) was analogous to that encoding PC9 2, but lacked the portion encoding the activation peptide.
  • the amino acid sequence at the junction between the light and heavy chains of the 1058 protein is presented in Table 2.
  • the protein C sequence present in plasmid p594 was altered in a single mutagenesis to delete the codons for the activation peptide and insert the Arg-Arg codons at the processing site.
  • a mutagenesis was performed on the 870 bp Sst I fragment from p594 essentially as described in Example 3.A. using oligonucleotides ZC1058 (5' CGC AGT CAC CTG AGA AGA AAA CGA CTC ATT GAT GGG 3') and ZC550.
  • the mutagenized sequence was used to construct expression vector pDX/PC1058 (analogous to pDX/PC962) , and the vector was co-transfected into tk ⁇ tsl3 BHK cells as described in Example 3.B. - The protein was purified on a polyclonal antibody column eluted with pH 11.5 buffer.
  • the activity of the PC1058 protein was compared to that of activated plasma protein C and activated PC962.
  • Plasma protein C and PC962 (5 ⁇ g/ml) were activated by treatment with 1/10 volume Protac C (American Diagnostica) for 2 hours.
  • Anticoagulant activity was assayed by combining 50 ⁇ l human plasma with 50 ⁇ l of the samples containing activated protein C and incubating the mixtures at 37°C for 150 seconds.
  • To the mixtures was added 50 ⁇ l activated cephaloplastin (American Scientific Products, McGaw Park, IL) , and the mixtures were incubated at 37°C for 300 seconds.
  • One hundred ⁇ l of 20 mM CaCl 2 was added and the clotting times were recorded.
  • the data, presented in Figure 5, show that the PC1058 protein is an active anitcoagulant.
  • the Saccharomyces cerevisiae KEX2 gene was isolated from a yeast genomic library by screening transformed kex2 mutant cells for producton of an ⁇ -factor halo on a lawn of a suitable tester cells. One clone was obtained that complemented all reported defects of kex2 mutants (mating, ⁇ -factor production, maturation of killer toxin and sporulation in a homozygous diploid strain) . The cloned gene was subcloned into a pUC vector under the control of the yeast GAL1 promoter. The resultant plasmid, designated pl515, has been deposited with . American Type Culture Collection under accession number 67569.
  • pl5l5 was digested with Hind III, and a 2.1 kb fragment was recovered. This fragment was ligated to Hind Ill-cut pUC18 to construct plasmid pUC18/KEX2.
  • the KEX2 fragment (2.1 kb) was then isolated from pUC18/KEX2 by digesting the plasmid partially with Hind III and to completion with Bam HI. The remainder of the KEX2 sequence was then isolated as a 0.43 kb fragment from a Bam HI + Hind III digest of pl515. The two KEX2 fragments were then ligated into the Bam HI sites of the vectors Zem228 and Zem229.
  • Zem228 is similar to Zem229 but contains a neomycin resistance gene in place of the DHFR gene.
  • the inserted gene is under the control of the methallothionein-1 promoter and SV40 terminator, and the vector can be selected with the antibiotic G418.
  • the resulting plasmids were designated KEX2/Zem228 and KEX2/Zem229, respectively.
  • a high protein C producing pDX/PC1058- transfected tk ⁇ tsl3 BHK clone (pDX/PC1058-3//BHK) was selected and transfected with KEX2/Zem228 by the calcium phosphate procedure. Transfected cells were selected with 500 ⁇ g/ml G418 and 250 nM methotrexate.
  • a selected clone designated KEX2-1058//BHK, was pulse-labeled with 35 S-cysteine in cysteine-free DMEM (Gibco) containing 1% fetal calf serum and 5 ⁇ g/ml vitamin K for 24 hours.
  • the culture media were collected and assayed for the presence of single-chain and two-chain protein C by immunoprecipitation with a monoclonal antibody to protein C.
  • Two hundred and fifty ⁇ l of media was combined with 10 ⁇ g of antibody, and the mixture was incubated at 37°C for one hour.
  • One hundred ⁇ l of Staph A cell suspension (Pharmacia, Piscataway, NJ) was added, and the mixture was incubated at 37°C for one hour.
  • the cells were pelleted by centrifugation, and the pellet was resuspended in 60 ⁇ l of gel buffer containing 1% ⁇ - ercaptoethanol. The suspension was heated to 100°C for three minutes, then electrophoresed on an SDS- polyacrylamide gel. Proteins were visualized by autoradiography.
  • the KEX2-1058//BHK clone showed approximately 100% cleavage of the protein into the two- chain form.
  • the carboxy-terminal sequence of the light chain of KEX2-1058 activated protein C was determined using CNBr cleavage at the unique methionine residue of the light chain to liberate a peptide that was sequenced in its entirety by N-terminal sequence analysis.
  • Affinity- purified protein C from KEX2-1058//BHK cells grown in DMEM supplemented with 1% fetal calf serum, 250 nM ethotrexate, 500 ⁇ g/ml G418 and 5 ⁇ g/ml vitamin K was reduced by the addition of a 10-fold molar excess per Cys residue of dithiolthreitol (DTT) in 0.2 M Tris-HCl, pH 8.3, and guanidine-HCl to a final concentration of 6.0 M. The mixture was incubated at 65°C for 4-6 hours.
  • DTT dithiolthreitol
  • Iodoacetic acid pH 7.0 or iodoacetic amide was added to the reduced protein in a four-fold molar excess over the molar concentration of DTT, and the mixture was incubated for 30 minutes at 37°C.
  • the solution was dialyzed against 0.1 M NH4HC03, pH 8.5 for 24 hours at 22 ⁇ C.
  • the dialyzed solution was applied to an HPLC Poly-F column (DuPont) to isolate the light chain.
  • a 500-fold molar excess per methionine residue of CNBr was added to the purified light chain in 70% formic acid under nitrogen for 30 hours at room temperature in the dark.
  • the CNBr digest was applied to an American Biosystems Inc. Model 470A sequenator (American Biosystems Inc.
  • Oligonucleotide-directed mutagenesis was carried out on a template comprising the Sst I fragment of p594 inserted, in the proper orientation, into the Sst I site of M13mpl0. Single-stranded template DNA was prepared from the 594/mplO phage clone. Oligonucleotide-directed mutagenesis was carried out on the template using the synthetic oligonucleotides ZC1962 (5' GAG AAG AAG CGC CTC ATT GAT GGG 3') and ZC550. Positive phage clones were sequenced to confirm the mutagenesis. A positive phage clone was designated 1962.
  • Replicative form DNA was prepared from phage clone 1962 and digested with Sst I and Pst I to isolate the approximately 0.4 kb mutagenized fragment.
  • Plasmid PC229/962 was digested with Eco RI and Pst I, and the 592 bp protein C fragment was isolated.
  • a 700 bp Sst I-Eco RI protein C fragment was obtained from PC1869/229R (a plasmid comprising a protein C coding sequence similar to p594, but with the Arg codon [residue 157] substituted with a Lys codon, inserted into the Eco RI site of Zem229R.
  • Zem229R was derived from Zem229 by partial digestion with Eco RI, blunting with DNA polymerase I [Klenow fragment] and dNTPs, religation, digestion with Bam HI and ligation with Bam HI-Eco RI adapters.) Plasmid pZMB-2 ( Figure 7) was linearized by digestion with Eco RI.
  • Plasmid pZMB-2 is similar to Zem229R but contains the SV40 enhancer, adenovirus 2 major late promoter, adenovirus 2 tripartite leader, and 5' and 3' splice sites substituted for the MT-1 promoter using an Sst I-Hind III adapter.
  • the approximately 0.4 kb Pst I-Sst I fragment from phage clone 1962, the 700 bp Sst I-Eco RI fragment from PC1869/229R, the 592 bp Pst I-Eco RI fragment from PC229/962 and the linearized pZMB-2 were joined in a four- part ligation.
  • a plasmid with the insert in the correct orientation was designated pPC1962/ZMB-2.
  • Plasmid pPC1962/ZMB-2 was transfected into tk" tsl3 BHK cells by calcium phosphate co-precipitation.
  • Transfected cells were grown in DMEM containing 10% fetal calf serum, lx PSN antibiotic mix (Gibco) , 2.0 mM L- glutamic acid and 5 ⁇ g/ml vitamin K.
  • the cells were selected in 500 nM ethotrexate for 15 days, and the resulting colonies were screeened by an immunofilter assay (Example 3.B.). The most intensely reacting colonies were picked by cylinder cloning and were grown individually in 10 cm plates.
  • KEX2/ZMB-1 comprises the KEX2 coding sequence inserted into the vector ZMB-1 at the unique Bam HI site.
  • the KEX2 sequence was obtained from plasmids pUC18/KEX2 and pl515 as described above for the construction of KEX2/Zem 228.
  • ZMB-1 as shown in Figure 7, is similar to ZMB-2, but contains a neo ycin resistance gene as the selectable marker
  • Co-transfected cells were selected and media samples were collected. Activated protein C was detected in media samples from pPC1962/ZMB-2, KEX2/ZMB-1 co- transfected cells.
  • An activated protein C precursor sequence was constructed in which the sequence encoding the activation peptide was removed and an Arg codon was inserted between amino acid codons 150 and 151 of native protein C.
  • the amino acid sequence at the light-heavy chain junction of the encoded protein (designated 2043) is shown in Table 2. Processing to remove the C-terminal basic amino acid residues form the light chain yields the 149-152 amino acid forms of the light chain.
  • Expression vector ZMB-3 was constructed from Zem228R and pDX (Hagen et al., U.S. Patent No. 4,784,950). Plasmid Zem228R was derived from Zem228 by partial digestion with Eco RI, blunting with DNA polymerase I (Klenow fragment) and dNTPs, religation, digestion with Bam HI and ligation with Bam HI - Eco RI adapters. Zem228R was digested with Hind III and Eco RI, and the 520 bp fragment containing the SV40 and MT-1 promoters was removed.
  • the large fragment of Zem228R was then joined to the -1100 bp Hind III-Eeo- RI fragment of pDX, which contains the SV40 promoter/enhancer, the adenovirus major late promoter, and a set of splicing signals.
  • the resultant vector was designated ZMB-3.
  • Single stranded template DNA was prepared from phage clone 1962 and subjected to site-directed in vitro mutagenesis using the synthetic oligonucleotides ZC2043 (5' AGC CGG ATG GAG AAG AGG AAG CGC CTC ATT GC 3') and ZC550. Positive clones were sequenced to confirm the mutagenesis.
  • Replicative form DNA was prepared from a confirmed phage clone and digested with Sal I and Sst I to isolate the approximately 0.4 kb mutagenized fragment. This fragment was joined to the 5' coding sequence for activated protein C (Eco RI - Sal I fragment from the PC962 sequence), the 3' activated protein C sequence (696 bp Sst I - Eco RI fragment from PC962) and Eco RI - digested ZMB-3. A plasmid containing the insert in the correct orientation is designated pPC2043/ZMB-3.
  • Plasmid pPC2043/ZMB-3 was transfected into tk " tsl3 BHK cells (ATCC CRL 1632) . Transfected cells were selected with G418. A positive clone was transfected with KEX2/Zem229, and- ransfectants were selected with G418 and methotrexate. Positive clones were analyzed for production of APC and were found to produce up to 70% of the protein in the 2-chain form.
  • Expression vector ZMB-4 was constructed from Zem229R and pDX (Hagen et al., U.S. Patent No. 4,784,950). Zem229R was digested with Hind III and Eco RI, and the 520 bp fragment containing the SV40 and MT-1 promoters was removed. The large fragment-of Zem229R was then joined to the -1100 bp Hind III-Eco RI fragment of pDX, which contains the SV40 promoter/enhancer, the adenovirus major late promoter, and a set of splicing signals.
  • the Sst I fragment of the PC1058 DNA was inserted into M13mpl0 and mutagenized according to standard procedures with the oligonucleotide ZC2274 (5' GAG AAG AAG CGC GCC AAC TCC AGA AGA AAA CGA CT 3') .
  • the mutagenized sequence was isolated from replicative form DNA as a 378 bp Pst I-Sst I fragment.
  • DNA sequences encoding an activated protein C precursor having the linker sequence Lys-Lys-Arg-Arg-Arg- Lys-Arg, Lys-Lys-Arg-Lys-Arg, and Lys-Lys-Arg-Arg between the light (amino acids 1-149) and heavy chains were constructed. These constructs were designated PC9111, PC9112, and PC9113, respectively (Table 2).
  • Expression vector TZM-2 was constructed from ZMB-3.
  • the KEX2 transcription unit was excised from
  • Plasmid pPC2043/TZM-2 was derived from plasmid pPC2043/ZMB-3.
  • Figure 1 was replaced with synthetic DNA encoding a part of pre-pro peptide of factor VTI (5' GTC TCC CAG GCC CTC
  • the Sst I fragment of PC2274 DNA was inserted into M13mpl0 and mutagenized according to standard procedures with the following oligonucleotides.
  • the mutagenized sequences were isolated from replicative form DNA as Sac I fragments.
  • Sac I fragments The corresponding Sac I fragment in plasmid pPC2043/TZM-2 was replaced by these fragments.
  • Plasmids containing the insert in correct orientation are designated PPC9111/TZM-2, pPC9112/TZM-2, and pPC9113/TZM-2, respectively.
  • Plasmid pPC9111/TZM-2 was transfected into 293 cells. Transfected cells were selected with G418. Positive clone were analyzed for production of APC and were found to produce up to 90% of the protein in two- chain form.

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Abstract

Procédés de production d'une protéine présentant l'activité biologique de la protéine C activée humaine. La protéine est produite par des cellules hôtes mammifères auxquelles on a transmis un plasmide capable d'une intégration dans l'ADN des cellules hôtes mammifères. Le plasmide comprend un activateur suivi en aval par une séquence de nucléotides, laquelle code un précurseur de protéine C activée, lequel est traité afin de produire une protéine présentant l'activité de la protéine C activée humaine, la chaîne légère de la protéine active contenant 149 résidus d'acides aminés.
PCT/US1990/007617 1989-12-22 1990-12-21 Proteine c recombinee a chaine legere tronquee WO1991009951A2 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0266190A2 (fr) * 1986-10-29 1988-05-04 Zymogenetics, Inc. Expression de protéine C
WO1988003926A1 (fr) * 1986-11-17 1988-06-02 New England Medical Center Amelioration de la gamma-carboxylation de proteines recombinantes dependantes de la vitamine k
EP0296413A2 (fr) * 1987-06-12 1988-12-28 Hoechst Japan Limited Protéine C hybride et sa méthode de préparation
EP0319944A2 (fr) * 1987-12-08 1989-06-14 Zymogenetics, Inc. Co-expression dans des cellules eucaryotes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0266190A2 (fr) * 1986-10-29 1988-05-04 Zymogenetics, Inc. Expression de protéine C
WO1988003926A1 (fr) * 1986-11-17 1988-06-02 New England Medical Center Amelioration de la gamma-carboxylation de proteines recombinantes dependantes de la vitamine k
EP0296413A2 (fr) * 1987-06-12 1988-12-28 Hoechst Japan Limited Protéine C hybride et sa méthode de préparation
EP0319944A2 (fr) * 1987-12-08 1989-06-14 Zymogenetics, Inc. Co-expression dans des cellules eucaryotes

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WO1991009951A3 (fr) 1991-08-22
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