# Patent

## Title
Compositions and methods for producing gamma-carboxylated proteins

## Abstract

The present invention relates a host cell comprising an expression vector comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences and a nucleic acid molecule encoding a vitamin K epoxido reductase and associated expression control sequences and a nucleic acid molecule encoding a γ-glutamyl carboxylase and associated control sequences. The invention further relates to a method of producing a protein requiring gamma-carboxylation in high yields.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a national phase application under 35 U.S.C. §371 of PCT International Application No. PCT/SE2006/000426, filed Apr. 10, 2006, which claims priority to Swedish Application Serial No. 0500831-3, filed Apr. 13, 2005.
TECHNICAL FIELD
The present invention relates a host cell comprising an expression vector comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences and a nucleic acid molecule encoding a vitamin K epoxido reductase and associated expression control sequences, and a γ-glutamyl carboxylase and associated control sequences. The invention further relates to a method of producing a protein requiring gamma-carboxylation in high yields.
BACKGROUND TO THE INVENTION
Bleeding is a common clinical problem. It is a consequence of disease, trauma, surgery and medicinal treatment. It is imperative to mechanically stop the bleeding. This may be difficult or even impossible due to the location of the bleeding or because it diffuses from many (small) vessels. Patients who are bleeding may thus require treatment with agents that support haemostasis. This may be blood-derived products (haemotherapy), agents that cause the release of endogenous haemostatic agents, recombinant coagulation factors (F), or agents that delay the dissolution of blood clots.
The first line treatment among the blood derived products, often obtained from the local hospital, are whole blood for volume substitution and support of haemostasis, packed red cells for the improvement of oxygen transporting capacity, platelet concentrates to raise the number of platelets (if low or defective) and fresh frozen plasma for support of the haemostasis (blood coagulation and platelet aggregation). Second line plasma derived products that support haemostasis are plasma cryoprecipitate, prothrombin complex concentrates, activated prothrombin complex concentrates and purified coagulation factors. Several coagulation factors are today available as human recombinant proteins, inactive (coagulation factors VIII and IX) and activated (coagulation factor VIIa).
Haemophilia is an inherited or acquired bleeding disorder with either abnormal or deficient coagulation factor or antibodies directed towards a coagulation factor which inhibits the procoagulant function. The most common haemophilias are haemophilia A (lack coagulation factor VIII) and haemophilia B (factor IX). The purified or recombinant single coagulation factors are the main treatment of patients with haemophilia. Patients with inhibitory antibodies posses a treatment problem as they may also neutralise the coagulation factor that is administered to the patient.
The active form of Protein C (APC) is an inhibitor of plasma coagulation by degradation of the activated coagulation factors Va and VIIIa. Recombinant APC has been shown to be an effective treatment of undue plasma coagulation in patients with sepsis.
Coagulation factors for therapeutic use can be obtained from human plasma, although the purification process is not simple and requires many steps of which several aim at eliminating contaminating viruses. But even with extensive safety measures and testing of blood-derived products, contamination with infectious viruses or prions cannot be ruled out. Because of this risk it is highly desirable to produce human therapeutic proteins from recombinant cells grown in media without animal derived components. This is not always straightforward as many proteins require a mammalian host to be produced in a fully functional form, i.e. be correctly post-translationally modified. Among the coagulation factors commercially produced in recombinant cells are FVII (NovoSeven), FVIII (Kogenate, Recombinate, Refacto) and FIX (BeneFix) (Roddie and Ludlam. Blood Rev. 11:169-177, 1997) and Active Protein C (Xigris). One of the major obstacles in obtaining large amounts of fully functional recombinant human coagulation factors lies in the Gla-domain present in FII, FVII, FIX, FX, Protein S and Protein C. This domain contains glutamic acid residues that are post-translationally modified by addition of carboxyl groups. The production of these factors are hampered by the fact that over-expression of them result in under-carboxylated, and hence inactive, protein. The Gla modifications are a result of the action of a vitamin K-dependent enzyme called γ-glutamyl carboxylase (GGCX). This enzyme has been extensively studied by many scientists, particularly those involved in coagulation factor research (WO-A-8803926; Wu et al. Science 254(5038):1634-1636, 1991; Rehemtulla et al, Proc Natl Acad Sci USA 90:4611-4615, 1993; Stanley J. Biol. Chem. 274(24):16940-16944, 1999; Vo et al., FEBS letters 445:256-260, 1999; Begley et al, The Journal of Biological Chemistry 275(46):36245-36249, 2000; Walker et al., The Journal of Biological Chemistry 276(11):7769-7774, 2001; Bandyopadhyay, et al. Proc Natl Acad Sci USA 99(3):1264-1269, 2002; Czerwiec et al., Eur J Biochem 269:6162-6172, 2002; Hallgren et al, Biochemistry 41(50):15045-15055,2002; Harvey et al., The Journal of Biological Chemistry 278(10):8363-8369, 2003). Attempts to co-express GGCX with coagulation factor FIX has been tried by at least two scientific groups but were not successful (Rehemtulla, et al. 1993, ibid; Hallgren et al. 2002, ibid). Considering the large interest in GGCX enzymes, it may be assumed that many more trials have failed and thus have not been reported. GGCX requires reduced vitamin K as a cofactor. The reduced vitamin K is by GGCX converted to vitamin K epoxide, which is recycled to reduced vitamin K by Vitamin K epoxidoreductase (VKOR). Thus for efficient vitamin K dependent carboxylation of proteins two enzymes are required, GGCX and VKOR. Cloning and identification of VKOR was reported 2004 (Li et al, Nature 427:541-543, 2004, Rost et al., Nature 427:537-541, 2004). The VKOR protein is a 163 amino acid polypeptide with at least one predicted transmembrane region. From recombinant cells expressing VKOR activity is localized to the microsomal subcellular fraction.
For human FII (prothrombin) at least 8 out of 10 Glu residues have to be correctly modified in order to obtain fully functional prothrombin (Malhotra, et al., J. Biol. Chem. 260:279-287, 1985; Seegers and Walz Prothrombin and other vitamin K proteins', CRC Press, 1986). Similarity, human coagulation factor IX clotting activity require γ-carboxylation of at lest 10 out of 12 glutamic residues in the Gla-domain (White et al, Thromb. Haemost. 78:261-265, 1997). Extensive efforts to obtain high production levels of rhFII have been made using several different systems such as CHO cells, BHK cells, 293cells and vaccinia virus expression systems, but have all failed or resulted in an under-carboxylated product and thus functionally inactive prothrombin (Jørgensen et al., J. Biol. Chem. 262:6729-6734, 1987; Russo et al., Biotechnol Appl Biochem 14(2):222-233, 1991; Fischer et al, J Biotechnol 38(2):129-136, 1995; Herlitschka et al. Protein Expr. Purif. 8(3):358-364, 1996; Russo et al, Protein Expr. Purif. 10:214-225, 1997; Vo et al. 1999, ibid; Wu and Suttie Thromb Res 96(2):91-98, 1999). Earlier reported productivities for carboxylated recombinant human prothrombin are low; 20 mg/L for mutant prothrombin (Côte et al., J. Biol. Chem 269:11374-11380, 1994), 0.55 mg/L for human prothrombin expressed in CHO cells (fully carboxylated, Jøergensen et al. 1987, ibid), 25 mg/L in CHO cells (degree of carboxylation not shown, Russo et al. 1997, ibid).
As far as known co-expression of a protein requiring γ-carboxylation and VKOR has not been reported earlier.
WO 92/19636 discloses the cloning and sequence identification of a human and bovine vitamin K dependent carboxylase. The application suggests co-expressing the vitamin K dependent carboxylase and a vitamin K dependent protein in a suitable host cell in order to prepare the vitamin K dependent protein. No co-expression of the carboxylase and vitamin K dependent protein is exemplified.
WO 92/19636 discloses the cloning and sequence identification of a human and bovine vitamin K dependent carboxylase. The application suggests co-expressing the vitamin K dependent carboxylase (GGCX) and a vitamin K dependent protein in a suitable host cell in order to prepare the vitamin K dependent protein. No co-expression of the carboxylase and vitamin K dependent protein is exemplified.
WO 2005/038019 claims a method of increasing the overall productivity of γ-carboxylated protein by a controlled co-expression of γ-carboxylated protein and GGCX. The invention is exemplified with improved productivity of coagulation factors II and FIX.
WO 2005/030039 suggests co-expression of vitamin K dependent proteins with Vitamin K epoxide reductase (VKOR) in order to improve γ-carboxylation. However, no such co-expression expression is exemplified.
Co-expression of coagulation factor X (FX) and VKOR has been shown to improve the share of γ-carboxylated protein by Sun et al. (Blood 106: 3811-3815, 2005). Wajih et al. (JBC 280:31603-31607, 2005) has in addition demonstrated improved share of γ-carboxylated coagulation factor IX (FIX) by co-expression with VKOR. Both publications reported that VKOR incresed the share of γ-carboxylated protein but VKOR co-expression did not improve the overall productivity of coagulation factor.
There is a need for improved methods to produce activated blood clotting factors in high yields. The present invention sets out to address this need.
SUMMARY OF THE INVENTION
According to a first aspect of the invention there is provided a host cell comprising an expression vector comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences, and an expression vector comprising a nucleic acid molecule encoding a vitamin K epoxidoreductase and associated expression control sequences, wherein the host cell further comprises a nucleic acid molecule encoding a γ-glutamyl carboxylase and associated expression control sequences.
In another aspect, a cell is provided which is engineered to express (i) a protein which requires gamma-carboxylation, and (ii) vitamin K epoxidoreductase, wherein the proteins (i) and (ii) are expressed in a ratio between 10:1 and 500:1.
According to a further aspect a genetically modified eukaryotic host cell is provided comprising: (i) a polynucleotide encoding vitamin K epoxidoreductase protein wherein said vitamin K epoxidoreductase protein encoding sequence is operably linked to expression control sequences permitting expression of vitamin K epoxidoreductase protein by said cell; (ii) a polynucleotide encoding a protein requiring carboxylation by the γ-glutamyl carboxylase protein operably linked to expression control sequences permitting expression of said protein requiring carboxylation by said cell, and (iii) a polynucleotide encoding gamma-glutamyl carboxylase
According to yet another aspect a vector is provided comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences and a nucleic acid molecule encoding a vitamin K epoxidoreductase and associated expression control sequences.
According to another aspect a method is provided for producing gamma-carboxylated protein comprising:(i) culturing a cell expressing a recombinant protein which requires gamma-carboxylation, vitamin K epoxidoreductase and a γ-glutamyl carboxylase and (ii) isolating gamma-carboxylated protein.
According to another aspect a method is provided of producing a pharmaceutical composition suitable for inducing blood clotting or promoting increased or decreased coagulation, comprising purifying active carboxylated protein produced according to the above methods and admixing the purified carboxylated protein with one or more pharmaceutically acceptable carriers or excipients.
According to a further aspect a method is provided of promoting increased or decreased coagulation in a subject comprising administering a pharmacologically effective amount of an isolated gamma-carboxylated protein obtained by the above methods to a patient in need thereof.
The protein requiring gamma-carboxylation produced by the methods of the present invention can be used in haemostatic or antithrombothic therapy.



BRIEF DESCRIPTION OF FIGURES
FIG. 1 shows a plasmid map of F10NopA (factor X+GGCX) co-expression vector and a plasmid map of VKORzeo (VKOR) expression vector.
FIG. 2 shows plasmid maps of vectors used for co-expression of FII, GGCX and VKOR



DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of the invention there is provided a host cell comprising an expression vector comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences, and an expression vector comprising a nucleic acid molecule encoding a vitamin K epoxidoreductase and associated expression control sequences, wherein the host cell further comprises a nucleic acid molecule encoding a γ-glutamyl carboxylase and associated expression control sequences.
In one embodiment said nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences comprises a first promoter, and said nucleic acid molecule encoding a vitamin K epoxidoreductase and associated expression control sequences comprises a second promoter. In another embodiment the first promoter is sufficiently stronger than the second promoter so that the protein requiring gamma-carboxylation and the vitamin K epoxidoreductase are expressed in a ratio of at least 10:1. In another embodiment the first promoter is sufficiently stronger than the second promoter so that the protein requiring gamma-carboxylation and the vitamin K epoxidoreductase are expressed in a ratio of at least 5:1.
In another embodiment the cell further comprises a nucleic acid molecule encoding a γ-glutamyl carboxylase and associated expression control sequences. In one embodiment, the nucleic acid molecule encoding a γ-glutamyl carboxylase and associated expression control sequences further comprises a third promoter, wherein the first promoter is sufficiently stronger than the third promoter so that the protein requiring gamma-carboxylation and the γ-glutamyl carboxylase are expressed in a ratio of at least 10:1. In another embodiment the first promoter is sufficiently stronger than the second promoter so that the protein requiring gamma-carboxylation and the vitamin K epoxidoreductase are expressed in a ratio of at least 5:1.
The first promoter can be human cytomegalovirus (hCMV) immediate-early promoter and the second and third promoter can be SV40 early promoter.
In one particular embodiment, both the nucleic acid molecule encoding the protein requiring gamma-carboxylation and associated expression control sequences, and the nucleic acid molecule encoding the Vitamin K epoxidoreductase, and optionally the γ-glutamyl carboxylase, and associated expression control sequences are located on the same expression vector. In another embodiment these two or optionally three nucleic acid molecules are located on two or more separate expression vectors.
In another aspect a cell is provided which is engineered to express (i) a protein which requires gamma-carboxylation, and (ii) vitamin K epoxidoreductase, wherein the proteins (i) and (ii) are expressed in a ratio between 10:1 and 500:1. In another embodiment, the proteins (i) and (ii) are expressed in a ratio between 5:1 and 500:1
The protein which requires gamma-carboxylation is selected from the group consisting of: coagulation factor VII, coagulation FVII, coagulation factor IX, coagulation FIX, prothrombin, coagulation factor II, coagulation FII, coagulation factor X, coagulation FX, and their activated forms FVIIa, FIXa, FXa, Protein C, Protein S, Protein Z, Bone Gla protein, Matrix Gla protein, Growth arrest-specific protein 6, snake venom proteases similar to coagulation factors such as Factor X-like snake venom proteases, and Acanthophiinae FXa-like protein.
In one embodiment, the protein which requires gamma-carboxylation is a vitamin K dependent coagulation factor. In another embodiment, the protein which requires gamma-carboxylation is Factor FX. In a third embodiment, the protein which requires gamma-carboxylation is prothrombin. In a forth embodiment, the protein which requires gamma-carboxylation is Factor X. In a fifth embodiment, the protein which requires gamma-carboxylation is factor VII.
The protein which requires gamma-carboxylation is preferably a human protein but all eukaryotic proteins is encompassed by the invention. Vitamin K epoxidoreductase is preferably a human protein but all eukaryotic Vitamin K epoxidoreductases can be used in the present invention, γ-glutamyl carboxylase is preferably a human protein but all eukaryotic γ-glutamyl carboxylases can be used in the present invention.
According to a further aspect a genetically modified eukaryotic host cell is provided comprising:

    (i) a polynucleotide encoding vitamin K epoxidoreductase protein wherein said vitamin K epoxidoreductase protein encoding sequence is operably linked to expression control sequences permitting expression of vitamin K epoxidoreductase protein by said cell; and
    (ii) a polynucleotide encoding a protein requiring carboxylation by the γ-glutamyl carboxylase protein operably linked to expression control sequences permitting expression of said protein requiring carboxylation by said cell.
    (iii) a polynucleotide encoding gamma-glutamyl carboxylase wherein said gamma-glutamyl carboxylase protein encoding sequence is operably linked to expression control sequences permitting expression of gamma-glutamyl carboxylase protein by said cell


In one embodiment, the cell is capable of expressing the vitamin K epoxidoreductase protein and the protein requiring carboxylation in the ratio of at least 1:10. In another embodiment, said ratio is at least 1:5.
The host cell is preferably a eukaryotic cell. Typical host cells include, but are not limited to insect cells, yeast cells, and mammalian cells. Mammalian cells are particularly preferred. Suitable mammalian cells lines include, but are not limited to, CHO, HEK, NS0, 293, Per C.6, BHK and COS cells, and derivatives thereof. In one embodiment the host cell is the mammalian cell line CHO-S.
According to yet another aspect a vector is provided comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences and a nucleic acid molecule encoding a vitamin K epoxidoreductase and associated expression control sequences. In one embodiment the nucleic acid molecule encodes a protein requiring gamma-carboxylation and associated expression control sequences comprises a first promoter, and the nucleic acid molecule encoding a vitamin K epoxidoreductase and associated expression control sequences comprises a second promoter. The first promoter can be sufficiently stronger than the second promoter so that the protein requiring gamma-carboxylation and the vitamin K epoxidoreductase are expressed in a ratio of at least 10:1. In another embodiment this ratio is 5:1. The vector could also comprise a nucleic acid molecule encoding a γ-glutamyl carboxylase and associated expression control sequences. Said nucleic acid molecule encoding a γ-glutamyl carboxylase and associated expression control sequences could comprise a third promoter, wherein the first promoter is sufficiently stronger than the third promoter so that the protein requiring gamma-carboxylation and γ-glutamyl carboxylase are expressed in a ratio of at least 10:1. In another embodiment this ratio is 5:1. The protein which requires gamma-carboxylation can be selected from the group consisting of: coagulation factor VII, coagulation FVII, coagulation factor IX, coagulation FIX, prothrombin, coagulation factor II, coagulation FII, coagulation factor X, coagulation FX, and their activated forms FVIIa, FIXa, Fxa, snake venom proteases similar to coagulation factors such as Factor X-like snake venom proteases and Acanthophiinae FXa-like protein, Protein C, Protein S, Protein Z, Bone Gla protein, Matrix Gla protein, Growth arrest-specific protein 6.
According to another aspect a method is provided for producing gamma-carboxylated protein comprising: (i) culturing a cell expressing a recombinant protein which requires gamma-carboxylation, vitamin K epoxidoreductase and a γ-glutamyl carboxylase and (ii) isolating gamma-carboxylated protein.
Said cell expresses the protein which requires gamma-carboxylation and vitamin K epoxidoreductase in a ratio of at least 10:1, under conditions suitable for expression of both proteins.
The vitamin K dependent coagulation factors (FII, FVII, FIX, FX and their activated forms FIIa or thrombin, FVIIa, FIXa, FXa) produced by the present method of co-expression with VKOR alone or in combination with GGCX can be expected to be useful in the prevention and treatment of bleeding following trauma, surgery or diseases of the liver, kidneys, platelets or blood coagulation factors (haemophilia). Likewise the coagulation factor Protein C and its activated form APC can be expected to be useful in the prevention and treatment of disorders of increased coagulation with or without decreased levels of Protein C. The method is also applicable to other proteins that require post-translational carboxylation.
The present invention will be applicable to improve the productivity of any protein that is dependent on γ-carboxylation, such proteins include, but are not limited to: prothrombin, coagulation factor II (FII), coagulation factor VII (FVII), coagulation factor IX (FIX), coagulation factor X (FX), Protein C, Protein S, Protein Z, Bone Gla protein (also known as: BGP or osteocalcin), Matrix Gla protein (MGP), proline rich Gla polypeptide 1(PRRG1), proline rich Gla polypeptide 2 (PRRG2), Growth arrest-specific protein 6 (Gas 6). Other suitable proteins are: FXa-like protein in venom of elapid snake (subfamily Acanthophiinae) and cone snail venom (Conus textile).
Each of these proteins, including their nucleic acid and amino acid sequences, are well known. Table 1 identifies representative sequences of wild-type and mutant forms of the various proteins that can be used in the present invention.











TABLE 1






CDNA
SPLICE VARIANTS

GENE EMBL


DESCRIPTION
EMBL ACC#
(PROTEIN)
MUTATIONS
ACC#







Glutamate gamma
BC013979
2; BC013979; AF253530
1 SNP (EMBL#
U65896


carboxylase


U65896); 2 SNPs





(OMIM# 137167)


Prothrombin
V00595
1; V00595
approx. 100 SNP's
AF478696





(EMBL#





AF478696)


Factor VII
AF466933
4; AF466933; AF272774;
21 SNPs (OMIM#
J02933




AR030786; AAN60063
277500)


Factor IX
A01819
3; A01819; A34669;
5 SNPs (EMBL#
AF536327




M19063
AF536327); 108





SNPs (OMIM#





306900)


Factor X
BC046125
4; BC040125; M57285;
118 SNPs
AF503510




AR095306; AB005892
(EMBL#





AF503510); 14





SNPs (OMIM#





227600)


Protein C
BC034377
7; AB083690; AB083693;
57 SNPs (EMBL#
AF378903




I09623; S50739; S72338
AF378903); 25





SNPs (OMIM#





176860)


Osteocalcin
AF141310
5; AF141310; AF141310;

X04143




BC033656; X04143;




X51699


Matrix GLA protein
BC005272
1; BC005272


Growth arrest-
BC038984
1; BC038984


specific 6; AXL


stimulatory factor


Protein Z
M55670
2; AB033749; AB033749


Proline-rich Gla (G-
AF009242
2; AF009242; BC030786


carboxyglutamic


acid) polypeptide 1


Proline-rich Gla (G-
AF009243
2; AF009243; BC026032


carboxyglutamic


acid) polypeptide 2


Vitamin K-
BC015801
1; BC015801
approx. 100
AY308744


dependent protein


SNPs (EMBL#


S precursor


AY308744); 8





SNPs (OMIM#





176880)


Snake venom FX-
AY769963
Add more?


like proteases
AAT42490



AAT42491



AAX37260



AAX37261



AAX37262



AAX37263



AAX37264



AAV34695









It will be appreciated that the invention is not restricted to a particular protein or protein encoding sequence of one of these proteins to be co-expressed. Moreover, and in particular with respect to blood coagulation factors, numerous mutant forms of the proteins have been disclosed in the art. The present invention is equally applicable to these mutant forms, including naturally occurring allelic variants, of the proteins as it is to wild-type sequence. In one embodiment the invention can be undertaking with any wild-type protein or one with at least 90%, preferably at least 95% sequence identity thereto.
The sequence identity between two sequences can be determined by pair-wise computer alignment analysis, using programs such as, BestFit, Gap or FrameAlign. The preferred alignment tool is BestFit. In practise, when searching for similar/identical sequences to the query search, from within a sequence database, it is generally necessary to perform an initial identification of similar sequences using suitable software such as Blast, Blast2, NCBI Blast2, WashU Blast2, FastA, Fasta3 and PILEUP, and a scoring matrix such as Blosum 62. Such software packages endeavour to closely approximate the “gold-standard” alignment algorithm of Smith-Waterman. Thus, the preferred software/search engine program for use in assessing similarity, i.e., how two primary polypeptide sequences line up is Smith-Waterman. Identity refers to direct matches, similarity allows for conservative substitutions.
The term vitamin K epoxidoreductase or “VKOR”, as used herein, refers to an enzyme that catalyses reduction of vitamin K epoxide and vitamin K to form reduced vitamin K.
Vitamin K reductases are widely distributed, and have been cloned from several different species such as mouse (Mus musculus), rat (Rattus norveigicus), chicken (Gallus gallus) and cow (Bos taurus). Homolgous proteins can be predicted from sequences from organsims of widely dispersed phylogenetic origin such as mammals, birds, amphibians, bony fishes, flies, kinetoplastids and bacteria. Table 2 represents a non-limiting list of representative sequences of predicted proteins homologous to human VKOR (sorted after species origin) that can be used in the present invention.








TABLE 2





Species
Data base accession #/ID







Homo sapiens (man)
NP_775788



NP_996560



AAR28759



AAQ13668



AAQ88821



CAH10673


Bos taurus (bovine)
NP_001003903


Mus musculus (mouse)
NP_848715



BAB26325



NP_001001327


Rattus norveigicus (rat)
NP_976080



NP_976083



AAQ91028


Gallus gallus (chicken)
NP_001001328



NP_996530


Xenopus laevis (clawed frog)
AAH43742



AAH77384


Xenopus tropicalis (amphibians)
AAH76993


Tetraodon nigroviridis (bony fishes)
CAF98534



CAG07588


Takifugo rubripes (torafugo)
AAR82913



AAR82912


Anopheles gambiae (mosquito)
XP_310541



EAA06271


Drosophila melanogaster (fruit fly)
DAA02561


Trypanosoma brucei (protozoa)
XP_340583


Corynebacterium efficiens (high GC Gram+
NP_737490


bacteria)


Corynebacterium glutamicum (high GC
NP_600038


Gram+ bacteria)


Mycobacterium leprae (high GC Gram+
NP_302145


bacteria)









The term “γ-glutamyl carboxylase” or “GGCX”, as used herein, refers to a vitamin K dependent enzyme that catalyses carboxylation of glutamic acid residues.
GGCX enzymes are widely distributed, and have been cloned from many different species such as the beluga whale Delphinapterus leucas, the toadfish Opsanus tau, chicken (Gallus gallus), hagfish (Myxine glutinosa), horseshoe crab (Limulus polyphemus), and the cone snail Conus textile (Begley et al., 2000, ibid; Bandyopadhyay et al. 2002, ibid). The carboxylase from conus snail is similar to bovine carboxylase and has been expressed in COS cells (Czerwiec et al. 2002, ibid). Additional proteins similar to GGCX can be found in insects and prokaryotes such as Anopheles gambiae, Drosophila melanogaster and Leptospira with NCBI accession numbers: gi 31217234, gi 21298685, gi 24216281, gi 24197548 and (Bandyopadhyay et al., 2002, ibid), respectively. The carboxylase enzyme displays remarkable evolutionary conservation. Several of the non-human enzymes have shown, or may be predicted to have, activity similar to that of the human GGCX we have used, and may therefore be used as an alternative to the human enzyme.
Table 3 identifies representative sequences of predicted proteins homologous to human GGXC (sorted after species origin) that can be used in the present invention.










TABLE 3







Species
Data base accession #/ID









Homo sapiens (man)
NM_000821.2




HUMGLUCARB




HUMHGCA




BC004422




HSU65896




AF253530.1



Papio hamadryas (red baboon)
AC116665.1



Delphinapterus leucas (white whale)
AF278713



Bos taurus (bovine)
NM_174066.2




BOVCARBOXG




BOVBGCA



Ovis aries (domestic sheep)
AF312035



Rattus norvegicus (brown rat)
NM_031756.1




AF065387



Mus musculus (mouse)
NM_019802.1




AP087938



Opsanus tau (bony fishes)
AF278714.1



Conus textile (molluscs)
AY0044904.1




AP382823.2



Conus imperialis (molluscs)
AF448234.1



Conus episcopatus (molluscs)
AF448233.1



Conus omaria (molluscs)
AF448235.1



Drosophila melanogaster (fruit fly)
NM_079161.2



Anopheles gambiae (mosquito)
XM_316389.1



Secale cereale (monocots)
SCE314767



Triticum aestivum (common wheat)
AF280606.1



Triticum urartu (monocots)
AY245579.1



Hordeum vulgare (barley)
BLYHORDCA



Leptospira interrogans (spirochetes)
AE011514.1



Streptomyces coelicolor (high GC
SCO939109



Gram+ bacteria)
SCO939124




AF425987.1



Streptomyces lividans (high GC
SLU22894



Gram+ bacteria)



Streptomyces viginiae (high GC
SVSNBDE



Gram+ bacteria)



Micrococcus luteus (high GC Gram+
MLSPCOPER



bacteria)



Chlamydomonas reinhardtii (green
AF479588.1



algae)



Dictyostelium discoideum (slime
AC115612.2



mold)



Coturnix coturnix (birds)
AF364329.1



Bradyrhizobium japonicum
AP005937.1



(α-protoebacteria)



Rhodobacter sphaeroides
RSY14197



(α-proteobacteria)



Sinorhizobium meliloti
RME603647



(α-proteobacteria)
AF119834



Mesorhizobium loti
AP003014.2



(α-proteobacteria)



Chromobacterium violaceum
AE016910.1



(β-proteobacteria)
AE016918.1



Pseudomonas aeruginosa
AE004613.1



(γ-proteobacteria)
AF165882



Xanthomonas axonopodis
AE011706.1



(γ-proteobacteria)



Human herpesvirus 8
KSU52064




KSU75698




AF305694




AF360120




AF192756










Each of the above-identified GGCX proteins can be used as the carboxylase enzyme in the present invention.
One way to effect the co-expressed proteins is to use different promoters as part of the respective expression control sequences. The art is replete with examples of different cloning vectors, promoters and other expression control sequences that are capable of expressing heterologous proteins to differing degrees or extents. Recombinant expression technology is suitably advanced such that a person skilled in the art of protein expression is able to select promoters and other control sequences to bring about co-expression of the protein requiring carboxylation, vitamin K epoxidoreductase and, optionally, the γ-carboxylase. The selection of which particular promoters and other expression control sequences to use is a matter of individual choice
In one embodiment, the control sequences associated with the protein requiring gamma-carboxylation comprise a strong promoter. In one embodiment this is the human cytomegalovirus (hCMV) immediate-early promoter. A strong promoter is here defined as a promoter giving rise to at least 5-fold higher numbers of mRNA transcripts than a weak promoter used in the same cell under similar conditions.
In another embodiment, the control sequences associated with the vitamin K epoxido reductase, and when present the γ-glutamyl carboxylase, comprises a weak promoter. In one embodiment this is SV40 early promoter. In another embodiment the protein requiring gamma-carboxylation and the vitamin K epoxido reductase, and optionally the γ-glutamyl carboxylase, are under the control of different promoter elements with the promoter controlling expression of the vitamin K epoxido reductase, and optionally the γ-glutamyl carboxylase, being weaker that the promoter controlling expression of the protein requiring gamma-carboxylation.
The invention has been exemplified by use of the strong CMV promoter (Boshart et al. Cell 41:521-530, 1985) to over-express Factor X and the weaker SV40 promoter (Wenger et al. Anal Biochem 221:416-418, 1994) to control the expression of vitamin K epoxido reductase and optionally the GGCX expression. Other strong promoter that could be used according to the present invention include, but are not limited to, pEF-1α [human elongation factor-1α subunit gene) (Mizushima and Nagata, Nuc Acids Res 18:5322,1990; Goldman et al., BioTechniques 21:1013-1015, 1996)], pRSV [Rous sarcoma virus (Gorman et al ., Proc Natl Acad Sci USA 79:6777-6781 ,1982)] and pUbC [human ubiquitin (Schorpp et al, Nuc Acids Res 24:1787-1788,1996)].
The invention also extends to purified gamma carboxylated protein produced by the methods of the present invention and their use in coagulant therapy.
According to yet another aspect of the invention there is provided a method of promoting increased or decreased coagulation in a subject comprising administering a pharmacologically effective amount of an isolated gamma-carboxylated protein obtained by the above-described methods to a patient in need thereof.
According to a further aspect of the invention there is provided a method of producing a pharmaceutical composition suitable for inducing blood clotting, comprising purifying active carboxylated protein expressed from a host cell adapted to express a protein requiring gamma-carboxylation and γ-glutamyl carboxylase in a ratio of at least 5:1 and admixing the purified carboxylated protein with one or more pharmaceutically acceptable carriers or excipients.
The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art, but will most likely be in a form suitable for injection, either parenterally or directly into the wound site.
Powders suitable for preparation of an aqueous preparation for injection, by the addition of a suitable diluent, generally contain the active ingredient together with suitable carriers and excipients, suspending agent and one or more stabilisers or preservatives. The diluent may contain other suitable excipients, such as preservatives, tonicity modifiers and stabilizers.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
The pharmaceutical compositions of the invention may also be in the form of a sterile solution or suspension in a non-toxic parenterally acceptable diluent or solvent, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.
For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990; or, Volume 99 of Drugs and the pharmaceutical sciences; Protein formulation and delivery (Eugen J. McNally, executive editor), Marcel Dekker Inc 2000.
The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for injection to humans will generally contain, for example, from 0.2 mg to 6 g or from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 0.2 mg to about 10 g or about 1 mg to about 500 mg of the active ingredient. Proteinaceous therapeutics are usually stored frozen or freeze-dried. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The size of the dose for therapeutic or prophylactic purposes of a compound will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. In using a compound for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 20 μg to 75 mg per kg body or from 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, 20 μg to 30 mg per kg body weight or from 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 20 μg to 30mg per kg or from 0.5 mg to 25 mg per kg body weight will be used. As an alternative the compound can be administered as an infusion of 1 μg-10 mg per kilo body weight and hour during a time period of a few hours to several days.
EXPERIMENTAL SECTION
The invention will be further described by the following non-limiting examples.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art.
Such techniques are explained fully in the literature. See, e.g., Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (3rd ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. (2002); Glover & Hames, eds., DNA Cloning 3: A Practical Approach, Vols. I, II, & III, IRL Press, Oxford (1995); Colowick & Kaplan, eds., Methods in Enzymology, Academic Press; Weir et al., eds., Handbook of Experimental Immunology, 5th ed., Blackwell Scientific Publications, Ltd., Edinburgh, (1997).
Example 1
To investigate the importance of VKOR in expression of carboxylated proteins we have expressed human coagulation factor X (FX) in CHO cells. Fully functional FX has been expressed earlier by Camire et al. 2000 (Biochemistry 39:14322-14329) who obtained approximately 1 μg carboxylated FX per million cells and day, and by Himmelspach et al 2000 (Thromb Res 97:51-67) who claimed obtaining up to 25% (19.5 μg) active FX per million cells and day using a CHO cell line that has been subjected to DHFR amplification. Himmelspach et al. reported incomplete processing of recombinant FX and the maximal productivity of active FX actually shown was 5 μg/ml culture medium In both publications cells were grown as adherent cells in serum-containing medium. Cells were grown to desired confluence, the medium replaced with serum free medium and incubation continued to allow accumulation of product. The amount of product was then estimated from this “serum-free” medium. This culture procedure is not suitable for large scale protein production as the cells will only produce product for a short period. In addition the product will be contaminated with serum proteins such as bovine FX which is highly undesirable as serum proteins will be difficult to remove and may cause antibody formation if present in a product injected to patients The obtained cell lines have thus not been shown suitable for commercial production of pharmaceutical FX.
Establisment of Stable Cell Lines Producing Recombinant Human Factor X
The FX coding sequence was PCR amplified from human liver cDNA using primers:










F10F1:
5′-CACACCATGGGGCGCCCACT-3′
(SEQ ID NO: 2)



 


F10R1:
5′-GAGTGGGATCTCACTTTAATGGA-3′
(SEQ ID NO: 3)






Cloning of the PCR product was first done by TA-TOPO cloning into pCDNA3.1-V5His (Invitrogen). Clones containing the correct FX sequence were identified by DNA sequencing and by transient expression in COS-7 cells. A blunt-end fragment containing the FX encoding sequence was then cloned into the EcoRV-digested and phosphatase—treated expression vector nopA. Obtained F10nopA (SEQ ID NO:6) clones were verified by DNA sequencing of the inserted sequence and by transient expression in COS-7.
The VKOR coding sequence was PCR amplified from human liver cDNA using primers:










VF1:
5′-CACCATGGGCAGCACCTGGGGGA-3′
(SEQ ID NO: 4)



 


VR1:
5′-GCTCAGTGCCTCTTAGCCTT-3′
(SEQ ID NO: 5)






Cloning of the PCR product was first done by TA-TOPO cloning into pCDNA3.1-V5His (Invitrogen). Clones containing the correct VKOR encoding sequence (SEQ ID NO:1) were identified by DNA sequencing. A HindIII-NotI fragment containing the VKOR sequence was then transferred to the expression vector pZeoSV2+ (Invitrogen) digested with the same enzymes. VKORzeo clones (SEQ ID NO:7) obtained were verified by DNA sequencing.
CHO-S cells (Invitrogen) were grown in DMEM F12 medium containing Glutamax I and 9% heat treated FBS, essentially as recommended by Invitrogen. Transfection of CHO-S was done with PvuI-digested (linearized) F10nopA, SspI-digested VKORzeo and Lipofectamine 2000 essentially as recommended by Invitrogen. The DNA transfection mix contained a 1.6-fold molar excess of F10NopA compared to VKORzeo. On the day after transfection, transfected cells were seeded in selection medium; growth medium plus 400 μg/ml G418, to 96-well plates. The VKORzeo construct was thus not selected for, but was randomly integrated in the G418-resistant transfectants. Following days plates were inspected to confirm that a suitable number of clones per well (5-10) were obtained. Six days post transfection the selection medium was replaced by growth medium supplemented with Vitamin K (1 μg/ml). The next day plates were sampled and assayed for FX activity using an assay based on Russels' Viper Venom (RVV-X), which activates FX to FXa. FXa activity was then measured using a chromogenic substrate (S2765, Chromogenix, Mölndal, Sweden). The RVV-X assay is eqivalent to the assay used by Himmelspach et al. for the same purpose. Wells with the highest activity were identified and the clones contained were expanded and subjected to limiting dilution cloning. After limiting dilution cloning and selection of the best clones, chosen clones were expanded and transferred to growth in protein-free medium (CD-CHO supplemented as recommended by Invitrogen plus 1 μg/ml vitamin K). Productivity of recombinant FX was estimated from T-flask cultures. The expression of VKOR was assayed by ReatTime PCR analyses. It was found that all selected clones expressing fully active FX also expressed VKOR. From this we conclude that co-expression of VKOR improves the expression of fully active human coagulation Factor X. The obtained cell lines grow well in protein and animal component free medium and produce FX in the absence of antibiotic selection pressure. Obtained cell lines are therefore considered suitable for large scale protein production and are capable of producing high amounts of active FX. The share of fully active FX is also significantly higher than previously reported.
Example 2
Analyses of Productivity and mRna Ratios for Co-Expression of FX, VKOR and GGCX
Clones obtained in Example 1 were grown in T-flasks in protein free chemically defined CHO medium without antibiotics (Invitrogen). Samples were collected from 4 day cultures for preparation of cDNA and samples for productivity estimates were collected from cultures 5 days after routine split. Control samples were also prepared from the parent non-transfected CHO-S cell line grown in the same medium and analyses of the control samples gave the expected results. Spinner cultures were grown in CD-CHO with or without supplementation of animal component free additives. The amount of active rhFX was estimated by an assay based on RVV-X as in example 1, and a standard of serially diluted purified plasma derived human Factor X (Haematologic Technologies Inc., Vermont, USA). RNA was isolated with TrizolTM according to the protocol supplied by the vendor, Invitrogen. The isolated RNA was DNaseI treated with the kit DNA-freeTM from Ambion. cDNA synthesis was carried out using hexamer primers and kit contents from SuperscriptTM First-Strand Synthesis System for RT-PCR (Invitrogen). Primers and Vic-labeled probes for Real-Time RT-PCR were selected using the software Primer ExpressTM from Applied Biosystems.
Human γ-Carboxylase Oligonucleotides









5′ ACACCTCTGGTTCAGACCTTTCTT 3′
(SEQ ID NO: 8)



Forward primer


 


5′ AATCGCTCATGGAAAGGAGTATTT 3′
(SEQ ID NO: 9)


Reverse primer


 


5′ CAACAAAGGCTCCAGGAGATTGAACGC 3′
(SEQ ID NO: 10)


Probe






Human Factor X Oligonucleotides

Primers were manufactured by Operon/Qiagen and the probes were ordered from Applied Biosystems.









5′ CCGCAACAGCTGCAAGCT-3′
(SEQ ID NO: 11)



Forward primer


 


5′ TGTCGTAGCCGGCACAGA-3′
(SEQ ID NO: 12)


Reverse primer


 


5′ CAGCAGCTTCATCATCACCCAGAACATG
(SEQ ED NO: 13)


Probe






Human VKOR Oligonucleotides










5′ GCTGGGCCTCTGTCCTGAT-3′
Seq(SEQ ID NO: 14)



Forward primer


 


5′ ATCCAGGCCAGGTAGACAGAAC-3′
Se(SEQ ID NO: 15)


Reverse primer


 


5′ CTGCTGAGCTCCCTGGTGTCTCTCG
S(SEQ ID NO: 16)


Probe






Rodent GAPDH control primers and probe were also used (Applied Biosystems; ABI #4308318 TaqMan® Rodent GAPDH Control Reagents Protocol)—Amplicon length 177 bp. The Real-Time RT-PCR reactions were performed on the 7500 Real Time PCR System, Applied Biosystems. The expected length of the amplified PCR products was confirmed on agarose gels.







TABLE 4







Results from Real-Time PCR analyses of FX expressing clones.











Clone
FX
VKOR
GGCX
GAPDH


name
mRNA/cell
mRNA/cell
mRNA/cell
mRNA/cell














FX1-5
137
0.62
1
1553


FX2-5
13
0.48
0.26
2985


FX3-9
3
0.03
0.17
1891


FX6
267
3
11
2289


FX17-2
319
2
37
2381
















TABLE 5







Productivity estimates and mRNA ratios. Ratios are calculated


from data in table 4. Productivity was estimated from


activity assays of diluted culture samples.












Ratio
Ratio
Active FX
Active FX


Clone name
FX:VKOR
FX:GGCX
μg/ml T-flask
μg/ml spinner





FX1-5
221:1
137:1 
0.5
Not done


FX2-5
 27:1
50:1
2.4
Not done


FX3-9
100:1
18:1
0.8
Not done


FX6
 89:1
24:1
6.9
14


FX17-2
160:1
 9:1
8.7
21









The productivities listed in Table 5 are all above those previously obtained from non-amplified cell lines. Esimates of total FX concentration, including inactive FX, was done using a Biacore assay and by SDS-PAGE and Western blotting.
Biacore Assay for the Estimation of the Concentration of Total rhFX
The BIAcore3000TM analytical systems, the running buffer (10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA and 0.05% P20, pH 7.4), rabbit anti-mouse Fc in 0.15 M NaCl (RAM Fc, lot no. 610) and the CM5 sensor chips were purchased from Biacore AB (Uppsala, Sweden). The procedure was run at 5 μl/min at 25° C. A standardised amine coupling procedure (35 μl activation time) at 25° C. was used to covalently couple 11000 RU of the capturing antibody RAMFc (35 μl, 30 μg/ml in 10 mM sodium-acetate, pH 5.0) to channel 4 of the CMS chip. After immobilisation the surface was regenerated with 5 μl 10 mM glycine buffer pH 1.8 and further equilibrated with the running buffer. With a 20 μl flow of the mouse anti-FX monoclonal IgG antibody N77121M (Biodesign, Maine, USA) (diluted 1/100 in running buffer) 660 RU was captured. Binding of FX in medium resulted in a very stable complex with negligible dissociation. For each new sample of FX the RAM Fc surface was reproducibly regenerated for multiple sandwich experiments. The difference in RU between channel 4 with coupled RAM Fc and channel 3 with a clean surface was used to quantify the binding of 5 μl FX. A standard (2, 4,6, 8, 10, 15 and 20 μg/ml in medium) of pdFX from Haematologic Technologies Inc. (Vermont, USA) was run and the difference in RU was plotted against the concentration of phFX and the equation for one binding site was fitted to the data. The difference in RU of the unknown samples was used to calculate the concentration of rhFX from the standard curve.







TABLE 6







Share of fully active rhFX produced. Total amount of


rhFX was estimated from spinner culture samples using


a Biacore assay and amount of active rhFX was estimated


by an RVV-X assay. All samples are from spinner cultures


in animal component free growth medium.











Total FX μg/ml
Active FX μg/ml



Clone/sample
(Biacore assay)
(RVV-X)
% active FX













FX17-2/p050131
18.6
10.1
54


FX17-2/p050202
20.6
9.6
47


FX17-2/p050225
11.8
12.1
103


FX17-2/sp2050309
38.3
16.3
43


FX17-2/sp1050309
25.1
13.6
54


FX17-2/sp2050310
39.7
21.1
53


FX17-2/sp1050310
28.1
13
46









Results in table 6 indicates that co-expression of VKOR enhances the expression of fully active rhFX. The high share (43-103%) of fully active rhFX is in agreement with data from SDS-PAGE, Western blot and protein purification.
Example 3
Co-expression of Human Prothrombin, GGCX and VKOR
To obtain a cell line capable of producing high levels of fully active human prothrombin (hFII) we have earlier co-expressed the vitamin K dependent modification enzymeγ-glutamyl carboxylase (GGCX) and hFII. Using this strategy we obtained the P1E2 clone. P1E2 is a highly productive clone expressing rhFII, but, although expression of correctly modified rhFII is vastly improved compared to other FII-producing clones, still only 20-60% (depending on the culture conditions) of the total amount of this rhFII produced is fully γ-carboxylated. In an attempt to further improve the level of fully γ-carboxylated rhFII and hence lower the production costs of rhFII, a new expression strategy was tested using vitamin K epoxide reductase (VKOR). We have cloned VKOR into two different vectors under the control of two different promoters; pCMV in the pHygro vector and pSV40 in the pZeo vector. In CHO-cells, the pCMV promoter is estimated to have an  ̃6x higher promoter activity than the pSV40 promoter. Both constructs were used in two separate co-transfections to obtain rhFII producing cell lines.
Cell Line Development and Productivity Estimates
Cell line development was initiated by cotransfecting CHO-S with the PP6 construct (encoding hFII and hGGCX) (SEQ ID NO: 20) and either of the VKOR constructs (FIG. 1-2). Molar ratios used in the transfections were 2:3 (PP6:VKOR). After seeding and selection of transfectants in 96-well plates totally 5500-8500 clones per transfection were then screened using an ecarin based chromogenic-assay. 18 clone pools were selected after the initial screen. After the second screen 9 clone pools were selected, expanded and subjected to a third screening assay. For each transfection the best producing clone pool was selected for limiting dilution. Six 96-well plates were seeded with 0.5 cells/well. 24 clones was selected and upscaled after screening. As the vectors encoding VKOR have not been selected for, Taqman analyses were done to verify VKOR expresssion. In all the three top clones selected; A3F4 (PP6/pZeoVKOR), B11E8 and B9A12 (PP6/pHygroVKOR), VKOR mRNA was detected. Four runs of spinner experiments were done to evaluate and compare the productivity of rhFII for the PP6/VKOR clones compared to the P1E2 clone.







TABLE 7







Production of rhFII in spinner flasks.














Experi-
Active

Share of active rhFII


Cell
Sample
ment
rhFII
SPR
(active rhFII/total


line
ID
run
(μg/mL)
(pg/cell/day)
rhFII in %)















A3F4
050317
A
10.3
1.38
100


A3F4
050415
B
27.6
1
100


A3F4
050425
C
23
2.6
100


B9A12
050317
A
10.3
0.84
68


B9A12
050413
B
27.4
1
100


B9A12
050424
C
14.4
6.06
79


B11E8
050317
A
13
1.74
75


B11E8
050414
B
27.4
2.2
80


B11E8
050424
C
22.2
4
78


P1E2
050415
B
37.6
2.1
61


P1E2
050424
C
25.4
2.5
21









The novel approach to co-express VKOR, GGCX and rhFII resulted in several rhFII-expressing clones producing a much higher share (60-100%) of fully active rhFII compared to the P1E2 clone (20-60%, see table 1). For two of the clones, B11E8 and B9A12 (both PP6/pHygroVKOR cotransfection) a higher specific productivity rate (amount of active protein produced per cell and day, SPR) than the P1E2 clone was obtained under some culture conditions. The A3F4 clone produced 100% fully carboxylated rhFII in all the culture experiments run. This clone has the highest mRNA ratio of both modification enzymes (GGCX and VKOR) to FII compared with the other two clones. However, A3F4 does not produce more fully active rhFII than the other clones.
Example 4
Improved γ-Carboxylation by Supertransfection with VKOR
In a second attempt to use VKOR for improvement of rhFII production, the P1E2 clone (example 3) was modified to co-express VKOR. The pHygroVKOR construct (SEQ ED NO: 22) was in this case used to transfect P1E2 (see Appendix, FIG. 1) and clones were screened for improved productivity by a prothrombinase activity assay. Totally 7000-8000 clones were screened using an end-point prothrombinase assay adapted to 96-well format. Sixteen clone pools were selected after the initial screen. Chosen clone pools were expanded and screened both with both ecarin and prothrombinase assay in order to estimate the share of active rhFII. After this screen 6 clone pools were selected and expanded. The three best producing clone pools were selected for limiting dilution cloning. Twenty-eight clones originating from all three pools were selected and up-scaled after the initial prothrombinase screen. After a second screen, eight clones were selected and up-scaled. Taqman analyses were done to verify VKOR expression in one clone from each cloned pool. In all the three top clones selected (M3F6, P4A4 and O3G3), VKOR mRNA was detected. Three runs of shaker or spinner cultures were done to evaluate the productivity of rhFII for the P1E2/VKOR clones compared to the parent P1E2 clone.
Taqman analyses were done to verify VKOR expression in one clone from each cloned pool. In all three top clones selected; M3F6, P4A4 and O3G3, VKOR mRNA was detected. Because of the selection and screening procedures used to obtain these clones, they are considered to express an optimal level of VKOR expression. This optimal expression level is further characterized in example 5.







TABLE 8







Production of rhFII at peak productivity in spinner/shaker


cultures using animal component free media.





















SPR;


Sample

Viable



Active/
specific


(Clone

cell

Total
Active
total
productivity


and
Experiment
densities
Viability
rhFII
rhFII
rhFII
rate


Date)
series
(cells/ml)
(%)
mg/L
mg/L
(%)
pg/cell/day

















P1E2
A
1700000
87
211.9
40
19
7.2


050622


P1E2
B
3866666
93
94.7
48.4
51
1.7


050610


P1E2
C
2500000
nd
47.9
38.4
80
nd


051011


M3F6
A
2550000
83
181.6
74.7
41
21.5


050622


M3F6
B
1950000
59
80.2
55
69
1.8


050609


O3G3
A
3525000
>95
194.6
63.7
33
3.1


050621


O3G3
B
3200000
85
128.7
68.6
53
4.2


050609


O3G3
C
6400000
nd
72.8
76.8
105
nd


051010


P4A4
A
2700000
>95
176.9
34.1
19
3.3


050621


P4A4
B
2233333
81
101.6
64.2
63
3.8


050610





The P1E2 parent cell line not containing the VKOR construct was grown in paralell under the same conditions as a control.






Results from the culture experiments showed that the amount and share of active rhFII during different culture conditions varied, but for most runs the amount of fully active rhFII produced was better for the novel P1E2/VKOR clones than for the original P1E2 cell line.
Example 5
Establishment of Optimal mRNA Expression Ratios
Messenger RNA prepared from the cell lines in Example 4 and 5 was analysed with Real-Time PCR similarly as in Example 3. For GGCX and VKOR the same oligonucleotides as in Example 3 were used.
Oligonucleotides for prothrombin were:









5′ TGGAGGACAAAACCGAAAGAGA 3′
(SEQ ID NO: 17)



Forward primer


 


5′ CATCCGAGCCCTCCACAA 3′
(SEQ ID NO: 18)


Reverse primer


 


5′ CTCCTGGAATCCTACATCGACGGGC 3′
(SEQ ID NO: 19)


Probe













TABLE 9







Analyses of mRNA ratios at peak expression


of human prothrombin (rhFII)















Best






productivity



mRNA
mRNA
mRNA
obtained *



ratio
ratio
ratio
(active rhFII


Cell line
FII/GGCX
FII/VKOR
VKOR/GGCX
mg/L)














A3F4
5
13
0.4
27.6


B9A12
86
32
3
27.4


B11E8
29
33
0.9
27.4


M3F6
304
15
20
74.7


O3G3
218
17
13
76.8


P4A4
255
128
2
64.2


P1E2 (control
221
No VKOR
No VKOR
48.4


without

detected
detected


VKOR)





* Measurement of productivity done under similar conditions in spinner or shake flasks.






Results in table 9 indicates that there is an optimal expression level of GGCX and VKOR in relation to the γ-carboxylated protein produced. Clones M3F4, O3G3 and P4A4 were obtained by transfecting P1E2 (earlier obtained by transfection with a construct containing rhFII+GGCX) with a construct containing VKOR under the control of the strong CMV promoter. Screening was perfoimed with an assay specifically detecting clones with an improved productivity of fully active rhFII. Clones with an optimal expression level of VKOR in relation to rhFII and GGCX have thus been selected.
Messenger RNA prepared from the cell lines in Example 4 and 5 was analysed with Real-Time PCR similarily as in Example 3. All analyses included a GAPDH control reaction as in Example 3.

## Claims

1. An in vitro host cell comprising:
a first recombinant DNA comprising a sequence encoding a protein requiring gamma-carboxylation operably linked to a first expression control sequence;
a second recombinant DNA comprising a sequence encoding a vitamin K epoxidoreductase (VKOR) operably linked to a second expression control sequence; and
a third recombinant DNA comprising a sequence encoding a γ-glutamyl carboxylase operably linked to a third expression control sequence, wherein the protein requiring gamma-carboxylation and the VKOR are expressed, in the cell in a ratio of at least 10:1.

2. The host cell of claim 1, wherein mRNA encoding the protein requiring gamma-carboxylation and mRNA encoding a VKOR are expressed in the cell in a ratio of at least 10:1.

3. The host cell of claim 1, wherein mRNA encoding the protein requiring gamma-carboxylation and mRNA encoding γ-glutamyl carboxylase are expressed in the cell in a ratio of at least 10:1.

4. The host cell of claim 1, wherein the first recombinant DNA encoding a protein requiring gamma-carboxylation and the DNA encoding a γ-glutamyl carboxylase are located on a single expression vector in the cell.

5. The host cell of claim 1, wherein the first recombinant DNA encoding a protein requiring gamma-carboxylation, the second recombinant DNA encoding a VKOR, and the DNA encoding a γ-glutamyl carboxylase are located on a single expression vector in the cell.

6. The host cell of claim 1, wherein the first expression control sequence comprises a first promoter, the second expression control sequence comprises a second promoter, and the activity of the first promoter in the host cell is greater than the activity of the second promoter.

7. The host cell of claim 1, wherein the first promoter is selected from the group consisting of human cytomegalovirus (hCMV) immediate-early promoter, human elongation factor-1 α subunit gene promoter (pEF-1α), Rous sarcoma virus promoter (pRSV), and human ubiquitin promoter (pUbC).

8. The host cell of claim 6, wherein the first promoter is hCMV immediate-early promoter, and the second promoter is SV40 early promoter.

9. The host cell of claim 6, wherein the third expression control sequence comprises a third promoter, and the activity of the first promoter in the host cell is greater than the activity of the third promoter.

10. The host cell of claim 1, wherein the protein requiring gamma-carboxylation is selected from the group consisting of: coagulation factor VII, coagulation factor IX, prothrombin, coagulation factor X, Factor X-like snake venom proteases, Protein C, Protein S, Protein Z, osteocalcin, Matrix Gla protein, and Growth arrest-specific protein 6.

11. The host cell of claim 1, wherein the protein requiring gamma-carboxylation is a vitamin K dependent coagulation factor.

12. The host cell of claim 1, wherein the protein requiring gamma-carboxylation is coagulation factor IX.

13. The host cell of claim 1, wherein the protein requiring gamma-carboxylation is coagulation factor X.

14. The host cell of claim 1, wherein the protein requiring gamma-carboxylation is a Factor X-like snake venom protease.

15. The host cell of claim 1, wherein the protein requiring gamma-carboxylation is prothrombin.

16. The host cell of claim 1, wherein the protein requiring gamma-carboxylation is coagulation factor VII.

17. The host cell of claim 1, wherein the protein requiring gamma-carboxylation is Protein C.

18. The host cell of claim 1, wherein the cell is a mammalian cell.

19. The host cell of claim 1, wherein the cell is a yeast cell or an insect cell.

20. The host cell of claim 1, wherein the cell is a CHO cell, a HEK cell, a NS0 cell, a Per C.6 cell, a BHK cell, or a COS cell.

21. An in vitro cell engineered to express (i) a protein that requires gamma-carboxylation, and (ii) a VKOR, wherein the cell expresses (i) and (ii) in a ratio between 10:1 and 500:1.

22. A method for producing a composition, the method comprising:
(a) providing a recombinant cell comprising a first nucleic acid sequence encoding a protein requiring gamma-carboxylation operably linked to a first expression control sequence, a second and heterologous nucleic acid sequence encoding a VKOR operably linked to a second expression control sequence, and a third nucleic acid sequence encoding a γ-glutamyl carboxylase operably linked to a third expression control sequence;
(b) culturing the cell in vitro under conditions suitable for expressing each nucleic acid sequence, wherein (i) the protein requiring gamma-carboxylation and the VKOR are expressed in the cell in a ratio of at least 10:1, and (ii) the protein requiring gamma-carboxylation is carboxylated in the cell, thereby producing a gamma-carboxylated protein; and
(c) isolating the gamma-carboxylated protein or an activated form thereof.

23. The method of claim 22, further comprising:
(d) preparing a pharmaceutical composition comprising the isolated gamma-carboxylated protein or an activated form thereof.

24. The method of claim 22, wherein mRNA encoding the protein requiring gamma-carboxylation and mRNA encoding VKOR are expressed in the cell in a ratio of at least 10:1.

25. The method of claim 22, wherein mRNA encoding the protein requiring gamma-carboxylation and mRNA encoding γ-glutamyl carboxylase are expressed in the cell in a ratio of at least 10:1.

26. The method of claim 22, wherein both the first and third nucleic acid sequences are on the same expression vector in the cell.

27. The method of claim 22, wherein the first, second and third nucleic acid sequences are on the same expression vector in the cell.

28. The method of claim 22, wherein the first expression control sequence comprises a first promoter, the second expression control sequence comprises a second promoter, and the activity of the first promoter in the cell is greater than the activity of the second promoter.

29. The method of claim 28, wherein the first promoter is selected from the group consisting of human cytomegalovirus (hCMV) immediate-early promoter, human elongation factor-1α subunit gene promoter (pEF-1α), Rous sarcoma virus promoter (pRSV), and human ubiquitin promoter (pUbC).

30. The method of claim 28, wherein the first promoter is hCMV immediate-early promoter and the second promoter is SV40 early promoter.

31. The method of claim 22, wherein the first expression control sequence comprises a first promoter, the second expression control sequence comprises a second promoter, the third expression control sequence comprises a third promoter, and the activity of the first promoter in the cell is greater than the activity of the third promoter.

32. The method of claim 31, wherein the first promoter is selected from the group consisting of hCMV immediate-early promoter, pEF-1α, pRSV, and pUbC.

33. The method of claim 31, wherein the first promoter is hCMV immediate-early promoter, and the third promoter is SV40 early promoter.

34. The method of claim 31, wherein the activity of the first promoter in the recombinant cell is greater than the activity of each of the second and third promoters.

35. The method of claim 34, wherein the first promoter is selected from the group consisting of hCMV immediate-early promoter, pEF-1α, pRSV, and pUbC.

36. The method of claim 34, wherein the first promoter is hCMV immediate-early promoter, and each of the second and third promoters is SV40 early promoter.

37. The method of claim 22, wherein the cell is a mammalian cell.

38. The method of claim 22, wherein the cell is a yeast cell or an insect cell.

39. The method of claim 22, wherein the cell is a CHO cell, a HEK cell, a NS0 cell, a Per C.6 cell, a BHK cell, or a COS cell.

40. The method of claim 22, wherein the protein requiring gamma-carboxylation is selected from the group consisting of: coagulation factor VII, coagulation factor IX, prothrombin, coagulation factor X, Factor X-like snake venom proteases, Protein C, Protein S, Protein Z, osteocalcin, Matrix Gla protein, and Growth arrest-specific protein 6.

41. The method of claim 22, wherein the protein that requires gamma-carboxylation is a vitamin K dependent coagulation factor.

42. The method of claim 22, wherein the protein that requires gamma-carboxylation is coagulation factor IX.

43. The method of claim 22, wherein the protein that requires gamma-carboxylation is coagulation factor X.

44. The method of claim 22, wherein the protein that requires gamma-carboxylation is a Factor X-like snake venom protease.

45. The method of claim 22, wherein the protein that requires gamma-carboxylation is prothrombin.

46. The method of claim 22, wherein the protein that requires gamma-carboxylation is coagulation factor VII.

47. The method of claim 22, wherein the protein that requires gamma-carboxylation is Protein C.

48. The host cell of claim 1, wherein the protein requiring gamma-carboxylation and the VKOR are expressed in the cell in a ratio between 10:1 and 500:1.

49. The method of claim 22, wherein the protein requiring gamma-carboxylation and the VKOR are expressed in the cell in a ratio between 10:1 and 500:1.