WO2005068620A1 - Procede de production de proteines de recombinaison - Google Patents

Procede de production de proteines de recombinaison Download PDF

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Publication number
WO2005068620A1
WO2005068620A1 PCT/EP2005/050060 EP2005050060W WO2005068620A1 WO 2005068620 A1 WO2005068620 A1 WO 2005068620A1 EP 2005050060 W EP2005050060 W EP 2005050060W WO 2005068620 A1 WO2005068620 A1 WO 2005068620A1
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fvii
l305vλ
factor vii
cells
cell
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PCT/EP2005/050060
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English (en)
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Jan Nehlin
Ivan Svendsen
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Novo Nordisk Health Care Ag
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Publication of WO2005068620A1 publication Critical patent/WO2005068620A1/fr
Priority to US11/481,169 priority Critical patent/US20070072271A1/en
Priority to US12/464,973 priority patent/US20090221800A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/647—Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
    • 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)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • 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)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)

Definitions

  • the present invention relates to methods for the production of recombinant proteins.
  • the blood coagulation cascade consists of a series of enzymatic reactions leading to the conversion of soluble plasma fibrinogen to fibrin clot.
  • the coagulation factors are primarily synthesized in the liver and are either enzyme precursors (FXII, FXI, FX, thrombin) or cofactors (FV and FVIII).
  • Coagulation is initiated by binding of activated factor VII (FVIIa) in plasma to tissue factor (TF), a glycoprotein which is expressed on the surface of cells in response to injury.
  • TF tissue factor
  • the main role in vivo of the TF:FVIIa complex is to activate FIX, which together with FVIII can activate FX.
  • Activated FX amplifies the generation of thrombin that induces the formation of fibrin.
  • the blood components which participate in what has been referred to as the coagulation "cascade” are proenzymes or zymogens, enzymatically inactive proteins which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor.
  • Coagulation factors that have undergone such a conversion are generally referred to as “active factors,” and are designated by the addition of a lower case “a” suffix (e.g., activated factor VII (FVIIa)).
  • active factors e.g., activated factor VII (FVIIa)
  • the clotting proteins are subject to a variety of co- and post-translational modifications, including, e.g., asparagine-linked (N-linked) glycosylation; O-linked glycosylation; and ⁇ - carboxylation of glu residues. For this reason, it is preferable to produce them in higher eukaryotic cells, which are able to modify the recombinant proteins appropriately.
  • FVII is normally synthesized in the liver but it has lately been found to be expressed in a number of other cell types and tissues including smooth muscle cells, macrophages, fibroblasts, keratinocytes and atherosclerotic plaques.
  • the present invention provides this alternative source of FVII production, where FVII is expressed from leukocyte cells.
  • the present invention relates in a broad aspect to the expression of recombinant FVII polypeptides in cells from the lymphoid lineage especially those cells that differentiate into B-cells or cancerous derivatives thereof , e.g.
  • CLL chronic lymphocytic leukemia
  • ALL Acute [ymphoblastic leukemia
  • CML chronic myeloid leukemia
  • pre B-cell leukemia Burkitts lymphoma, Multiple myeloma.
  • the present invention relates to a method for the production of a purified Factor VII polypeptide the method comprising: (i) transfecting a leukocyte cell with a vector comprising a promoter sequence and a polynucleotide sequence coding for the Factor VII polypeptide; (ii) cultivating the transformed host cell expressing the Factor VII polypeptide in a culture medium under conditions appropriate for expression of the Factor VII polypeptide; (iii) recovering all or part of the culture medium comprising the Factor VII polypeptide; and (iiii) purifying the Factor VII polypeptide from the culture medium.
  • the present invention relates to a leukocyte cell transformed with a vector comprising a promoter sequence and a polynucleotide sequence encoding a Factor VII polypeptide.
  • the present invention relates to a purified Factor VII polypeptide obtained by a method comprising: (i) transfecting a leukocyte cell with a vector comprising a promoter sequence and a polynucleotide sequence coding for the Factor VII polypeptide; (ii) cultivating the transformed host cell expressing the Factor VII polypeptide in a culture medium under conditions appropriate for expression of the Factor VII polypeptide; (iii) recovering all or part of the culture medium comprising the Factor VII polypeptide; and (iiii) purifying the Factor VII polypeptide from the culture medium.
  • the leukocyte cell is a lymphoid cell.
  • FIG. 1 Protein blots of FVII-secreted samples run under non-reducing conditions ( Figure 1 A) or under reducing conditions (Figure 1 B) on 12% NuPage Bis-Tris polyacrylamide gels (Invitrogen Corp.). Lane 1 is a molecular size marker (Magic marker). Lane 2 is a sample derived from a control producer cell line, FVII-expressing hamster CHO-K1. Lanes 3-11 are samples derived from different FVII-expressing SP/0 myeloma cells selected with 800 micrograms/ml G418. Lane 13 is a sample derived from a FVII-expressing X63 myeloma cell line selected with 600 microgram/ml G418. Lanes 14 and 15 represent negative control samples derived from pcDNA3.1-transfected SP2/0 myeloma cells.
  • FIG. 1 Protein blot of FVII-Fc secreted samples run under non-reducing conditions ( Figure 2A) or under reducing conditions (Figure 2B) on 12% NuPage Bis-Tris polyacrylamide gels (Invitrogen Corp.). Lane 1 is a molecular size marker (magic marker). Lane 2 is a FVII-Fc (FVII analogue)-expressing myeloma cell line.
  • FIG. 3 Protein blot of FVII-secreted samples treated with N-glycosidase F (PNGase).
  • Panel A Non-reducing conditions.
  • Panel B reducing conditions.
  • Lanes 2-7 represent supematants from diverse FVII-expressing cells, untreated (2,4,6) or treated with PNGase (3,5,7).
  • Lane 1 is a molecular size marker (Magic marker).
  • Lanes 2 and 3 derive from FVII- expressing myeloma SP2/0 cells.
  • Lanes 3 and 4 derive from FVII-expressing hamster CHO- K1 producer cell line.
  • Lanes 5 and 6 derive from a FVII-mutated expressing hamster CHO-K1 cell line devoid of glycans.
  • the samples were all run on 12% NuPage Bis-Tris polyacrylamide gels (Invitrogen Corp.).
  • FVII INVENTION Factor VII
  • TF tissue factor
  • FVII-Fc FVII fusion protein
  • High-producing clones were isolated that expressed active wild type human FVII and FVII-Fc fusion protein, both in serum-containing media and in media without serum, in the presence of vitamin K. Further increases in FVII polypeptide production may be obtained with cell fusions between the myeloma cell lines expressing FVII and FVII analogues and several other high-protein producing industrial cell lines. Production of FVII for therapeutic use has been obtained in baby hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells cultured with or without the presence of serum. Serum-free production of FVII or other recombinant proteins often leads to productivity and cell viability losses, resulting in high costs and inefficient production rates.
  • the inventors of the present invention have found that myeloma cells are highly applicable for the production of FVII polypeptides.
  • Myeloma cells expressing FVII polypeptides showed to be robust cell lines that can withstand growth in media without serum, allowing production of high levels of recombinant FVII without losses of cell viability.
  • the term "purified Factor VII polypeptide” as used herein, means a Factor VII polypeptide that has been separated from at least about 50 percent by weight of polynucleotides, lipids, carbohydrates and any other contaminating polypeptides or other contaminants that are found in the culture medium following expression in a eukaryotic host cells which would interfere with its therapeutic, diagnostic, prophylactic or research use.
  • the purified Factor VII polypeptide has been separated from at least about 60 such as 80, such as 90, such as 95, such as 99 percent by weight of polynucleotides, lipids, carbohydrates and any other contaminating polypeptides or other contaminants that are found in the culture medium following expression in a eukaryotic host cells.
  • the Factor VII polypeptide can be purified to be substantially free of natural contaminants from the culture medium through the use of any of a variety of methodologies. Standard chromatographic separation technology for the purification of the Factor VII polypeptide may also be used in some of the purification steps.
  • purifying a polypeptide from a composition comprising the polypeptide and one or more contaminants is meant increasing the degree of purity of the polypeptide in the composition by removing (completely or partially) at least one contaminant from the composition.
  • a “purification step” may be part of an overall purification process resulting in a "homogeneous" composition, which is used herein to refer to a composition comprising at least about 70% by weight of the polypeptide of interest, based on total weight of the composition, preferably at least about 80% by weight.
  • leukocyte cell means any cell existing or derived from nucleated cells that occur in blood or tissue fluid, exclusive of erythrocytes and erythrocyte precursors.
  • the term includes hybridomas of leucocytes as well as the major clases of leukocytes including lymphoid cells such as B-, T- and NK (Natural killer) cells, monocytes including macrophages, and neutrophils, eosinophils and basophils.
  • lymphoid cells such as B-, T- and NK (Natural killer) cells
  • monocytes including macrophages
  • neutrophils eosinophils and basophils.
  • myeloma cells lymphoma cells, leukaemia cells and lymphoma cells.
  • the term also includes the parental cells, including but not restricted to hematopoeitic stem cells (HSC) giving rise to the hematopoeitic cell lineages as described in Wagers and Weissman, 2004 Cell 116,639-648.
  • HSC hematopoeitic stem cells
  • leukemia cell as used herein means any cell derived from a malignant leukocyte or derivatives thereof, any cell from the parental lineage leading to leukocyte formation, including but not restricted to any of several nucleated cells that naturally occur in blood or tissue fluid, such as lymphocytes, monocytes, granulocytes hereunder neutrophils, eosinophils, basophils and precursors of these cells.
  • lymphoid cell means any cell derived from the lymphoid lineage.
  • the term comprises all parental cells, and derivatives and hybridomas thereof, either primary or established cell lines, derived from human, non-human, non-primate species, including but not restricted to avian, amphibian, mammalian, reptile species, and/or derived from non-vertebrate species, including but not restricted to marine/aquatic organisms, insects, plants, lichens, moss, fungi.
  • the term includes stem cells, differentiated cells, virus- transformed cells, cancerous cells, lymphoma cells, myeloma cells, leukemia cells and cell hybrids, any cell whose origin could be related with the lymphoid lineage, either in vertebrate or invertebrate species.
  • the term includes lymphoid stem cells that give rise to the pre-B and pre-T cell lineages, the pre-B cells that give rise to B cells and thereafter actively Ig- producing plasma cells, as well as the pre-T cells that give rise to T cells and thereafter T helper, T suppressor and NK cells. Included within the term is any cancerous derivatives thereof, e.g.
  • lymphocytic leukemia chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • CML chronic myeloid leukemia
  • pre B-cell leukemia Burkitts lymphoma
  • Multiple myeloma The term "lymphoma cell” as used herein means any cell derived from a malignant neoplasm primarily affecting lymph nodes.
  • myeloma cell means any malignant cell of bone marrow origin, including but not restricted to cells of B-lymphocyte lineage, such as CLL (chronic lymphocytic leukemia), ALL (Acute lymphoblastic leukemia), CML (chronic myeloid leukemia), pre B-cell leukemia, Burkitts lymphoma, Multiple myeloma, primary tumor cells, cells from established myeloma cell lines, hybrid cells produced from myeloma cells that retain the characteristic growth properties of myeloma cells, multiple myeloma, plasma cell myeloma, peripheral plasmacytoma, solitary plasmacytoma, and plasmoma.
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • CML chronic myeloid leukemia
  • pre B-cell leukemia Burkitts lymphoma
  • Multiple myeloma primary tumor cells
  • the myeloma cell-line may be a rat, mouse, human or any other mammalian species myeloma or hybridoma cell-line, such as the rat YB2/3.0 Ag20 hybridoma cell-line, the mouse NS/O, NS-1 myeloma cell-lines or the mouse SP2/0-Ag14 hybridoma cell-line, MOPC-31C, P3X63Ag8.653, P3XAg8U.1, MPC-11 , FO, Fox-NY, NS1 , Human: RPMI 8226.IM-9, HS-Sultan, SKO-007, MC/CAR, HuNS1 , NCI-H929, Human/Mouse: SHM-D33, A6, 36, mouse J558L myeloma cells, etc.
  • any other mammalian species myeloma or hybridoma cell-line such as the rat YB2/3.0 Ag20 hybrido
  • Rat hybridoma cell-line YB2/3.0 Ag20 is described in British patent specification 2079313 and is on deposit at the American Type Culture Collection (as YB2/O or YB2/3HL. P2. G11. 16Ag.20) under Accession Number CRL1662.
  • Mouse hybridoma cell-line SP2- OAg14 is on deposit at the American Culture Collection under Accession Number CRL1581.
  • Mouse hybridoma cell-line P3/NS1/1 Ag4.0 (the NS-1 cell-line) is on deposit at the American Culture Collection under Accession Number T1 B18.
  • Mouse myeloma P3X63Ag8.653 cell line is on deposit at the American Culture Collection under Accession Number CRL1580.
  • the myeloma cell is selected from the group consisting of YB2/3.0 Ag20, SP2-OAg14, P3/NS1/1 Ag4.0, P3X63Ag8.653, mouse J558L myeloma cells, and mouse NS/O, NS-1 myeloma cell-lines.
  • suitable cells include but are not limited to hormone-secreting cells, whether normal ortumorigenic, derived from blood, body fluids and tissues including but not restricted to pancreas, prostate gland, mammary gland, pituitary gland, hypothalamus, kidney, endocrine and exocrine glands, skin, muscle, vessels, either of human, primate, cow, pig, goal, sheep origin, and other vertebrate species.
  • the invention relates to a transgenic animal containing a transformed cell of the invention.
  • the transformed cell is a mammary gland epithelial cell.
  • the invention relates to a method for producing the Factor VII polypeptide, the method comprising recovering the Factor VII polypeptide from milk produced by the transgenic animal.
  • epithelial cells of mammary gland origin and their derivatives such as MCF10A (ATCC number CRL-10317), L612 (ATTC number CRL-10724), MCF-12A (ATCC number CRL-10782), MCF-7 (ATCC number HTB-22), BT-20 (ATCC number HTB-19), BT- 474 (ATCC number HTB-20), MDA-MB-231 (ATCC number HTB-26), MDA-MB-436, SK-BR- 3 (ATCC number HTB-30), MDA-MB-361 (ATCC number HTB-27), MDA-MB-157 (ATCC number HTB-24), MDA-MB-175-VII (ATCC number HTB-25), T-47D (ATCC number HTB- 133), MDA-MB-468 (ATCC number HTB-132), MDA-
  • the list comprises all wild-type epithelial cells of mammary gland origin that either are capable of secreting b-casein and/or lactoferrin in their differentiated state, or epithelial cells of mammary gland origin that no longer express markers of differentiation typical of a mammary epithelial cell but are dedifferentiated, and could be defined as actively dividing, with increased expression of ld-1 (Singh et al. Oncogene, 2002, 21 (12):1812-1822) and/or with mutations in BRCA1 , with positive expression of estrogen and progesterone receptors.
  • the list includes all cytokeratin 19 positive cells.
  • the list comprises also stem cells giving rise to the breast epithelial cell lineage including all cells expressing sca-1 (Stem-cell-antigen 1 ).
  • the method of the invention is a method, wherein the promoter is selected from the list consisting of cytomegalovirus promoter, metallothionein promoter, and adenovirus major late promoter.
  • the method of the invention is a method, wherein the lymphoid cell is selected from the group consisting of CLL (chronic lymphocytic leukemia) cells, ALL (Acute lymphoblastic leukemia) cells, CML (chronic myeloid leukemia), pre B-cell leukemia cells, Burkitts lymphoma cells, Multiple myeloma cells, mouse myeloma cells, rat myeloma cells, human myeloma cells, fusion cell lines and transgenic myeloma cell lines.
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • CML chronic myeloid leukemia
  • pre B-cell leukemia cells Burkitts lymphoma cells
  • Multiple myeloma cells mouse myeloma cells, rat myeloma cells, human myeloma cells, fusion cell lines and transgenic myeloma cell lines.
  • the method of the invention is a method, wherein the lymphoid cell is selected from the group consisting of YB2/3.0 Ag20, SP2-OAg14, P3/NS1/1 Ag4.0, P3X63Ag8.653, mouse J558L myeloma cells, and mouse NS/O, NS-1 hybridoma cell- lines, and transgenic myeloma cell lines with increased copy number of genes encoding proteins required for elevated protein expression, including mutated myeloma cell lines with enhanced productivity.
  • the method of the invention is a method, wherein the lymphoid cell is selected from the group consisting of mouse myeloma cells, rat myeloma cells and human myeloma cells.
  • the method of the invention is a method, wherein the lymphoid cell is selected from the group consisting of YB2/3.0 Ag20, SP2-OAg14, P3/NS1/1 Ag4.0, P3X63Ag8.653, mouse J558L myeloma cells, and mouse NS/O, NS-1 hybridoma cell- lines.
  • the method of the invention is a method, wherein the Factor VII polypeptide is a compound having the formula A-(LM)-C, wherein A is a FVIIa polypeptide; LM is an optional linker moiety; C comprises an immunostimulatory effector domain; and wherein the compound binds to TF, as described in International patent application DK03/00481 , which is hereby incorporated by reference in its entirety.
  • the method of the invention is a method, wherein the transformed host cell expressing the Factor VII polypeptide is cultivated in a culture medium under conditions appropriate for expression of the Factor VII polypeptide and in the absence of serum.
  • the cell cultures are cultivated in a medium lacking any animal derived components.
  • the methods of the present invention are particularly useful for large-scale production processes.
  • large-scale is typically meant methods wherein the volume of the liquid Factor VII polypeptide compositions is at least 100 L, such as at least 500 L, e.g. at least 1000 L, or at least 5000 L
  • the Factor VII polypeptide is wild-type human factor VII.
  • the Factor VII polypeptide has a proteolytic activity higher than wild type human FVIIa.
  • the Factor VII polypeptide is selected from the group consisting of: L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, V158D/E296V/M298Q/
  • K316H/L305V/V158T-FVII K316H/L305V/K337A/V158T-FVII, K316H/L305V/K337A/M298Q- FVII, K316H/L305V/K337A/V158D-FVII, K316H/L305V/V158D/M298Q-FVII, K316H/L305V/V158D/E296V-FVII, K316H/L305V V158T/M298Q-FVII, K316H/L305V/V158T/E296V-FVII, K316H/L305V/V158T/E296V-FVII, K316H/L305V/E296V/M298Q-FVII, K316H/L305V/V158D/E296V/M298Q-FVII, K316H/L305V/V158D/E296V/M298Q-F
  • K316H/L305V/V158T/E296V/M298Q-FVII K316H/L305V/V158T/K337A M298Q-FVII, K316H/L305V/V158T/E296V/K337A-FVII, K316H/L305V/V158D/K337A/M298Q-FVII, K316H/L305V/V158D/E296V/K337A -FVII, K316H/L305V/V158D/E296V/M298Q/K337A- FVII, K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII, K316Q/L305V/V158D-FVII, K316Q/L305V/V158D-FVII, K316Q/L305V/V158D-FVI
  • K316Q/L305V/V158T-FVII K316Q/L305V/K337A/V158T-FVII, K316Q/L305V/K337A/M298Q- FVII, K316Q/L305V/K337A/M298Q- FVII, K316Q/L305V/K337A/V158D-FVII, K316Q/L305V ⁇ /158D/M298Q-FVII, K316Q/L305V/V158D/E296V-FVII, K316Q/L305V/V158T/M298Q-FVII, K316Q/L305V/V158T/E296V-FVII, K316Q/L305V/V158T/E296V-FVII, K316Q/L305V/E296V/M298Q-FVII, K316Q/L305V/V158D/E296V/M298Q-FVII,
  • K316Q/L305V/V158T/E296V/M298Q-FVII K316Q/L305V/V158T/K337A/M298Q-FVII, K316Q/L305V/V158T/E296V/K337A-FVII, K316Q/L305V/V158D/K337A/M298Q-FVII, K316Q/L305V/V158D/E296V/K337A -FVII, K316Q/L305V/V158D/E296V/M298Q/K337A- FVII, K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII, F374Y/V158D- FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII, F374Y/V158
  • Fractor VII polypeptide encompasses wild-type Factor VII (i.e., a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950), as well as variants of Factor VII exhibiting substantially the same or improved biological activity relative to wild-type Factor VII, Factor Vll-related polypeptides as well as Factor VII derivatives, Factor VII conjugates, and FVII fusion proteins.
  • the term “Factor VII” is intended to encompass Factor VII polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated Factor Vila.
  • Factor VII is cleaved between residues 152 and 153 to yield Factor Vila.
  • variants of Factor VII may exhibit different properties relative to human Factor VII, including stability, phospholipid binding, altered specific activity, and the like.
  • Factor Vll-related polypeptides encompasses polypeptides, including variants, in which the Factor Vila biological activity has been substantially modified or reduced relative to the activity of wild-type Factor Vila. These polypeptides include, without limitation, Factor VII or Factor Vila into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide.
  • the term includes conjugates of chemically inactivated wt-FVIIa with Fc domain as described in International patent application DK03/00481 , which is incorporated by reference in its entirety.
  • the term also includes dimers of FVII polypeptides, including variants, wherein the dimer is catalytically inactive as disclosed in International patent application 03/076461 , which is incorporated by reference in its entirety.
  • the term "Factor VII derivative" as used herein, is intended to designate wild-type Factor VII, variants of Factor VII exhibiting substantially the same or improved biological activity relative to wild-type Factor VII and Factor Vll-related polypeptides, in which one or more of the amino acids of the parent peptide have been chemically modified, e.g.
  • FVII fusion proteins means a FVII polypeptide, which is conjugated to another functional polypeptide.
  • FVII-Fc a FVII-Fc, wherein the FVII polypeptide part of the protein is conjugated to the Fc portion of an antibody.
  • PEGylated human Factor Vila means human Factor Vila, having a PEG molecule conjugated to a human Factor Vila polypeptide.
  • the PEG molecule may be attached to any part of the Factor Vila polypeptide including any amino acid residue or carbohydrate moiety of the Factor Vila polypeptide.
  • the term "cysteine- PEGylated human Factor Vila” means Factor Vila having a PEG molecule conjugated to a sulfhydryl group of a cysteine introduced in human Factor Vila.
  • the biological activity of Factor Vila in blood clotting derives from its ability to (i) bind to tissue factor (TF) and (ii) catalyze the proteolytic cleavage of Factor IX or Factor X to produce activated Factor IX or X (Factor IXa or Xa, respectively).
  • Factor Vila biological activity may be quantified by measuring the ability of a preparation to promote blood clotting using Factor Vll-deficient plasma and thromboplastin, as described, e.g., in U.S. Patent No. 5,997,864. In this assay, biological activity is expressed as the reduction in clotting time relative to a control sample and is converted to "Factor VII units" by comparison with a pooled human serum standard containing 1 unit/ml Factor VII activity.
  • Factor Vila biological activity may be quantified by (i) measuring the ability of Factor Vila to produce Factor Xa in a system comprising TF embedded in a lipid membrane and Factor X. (Persson et al., J.
  • Factor VII variants having substantially the same or improved biological activity relative to wild-type Factor Vila encompass those that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75% and most preferably at least about 90% of the specific activity of Factor Vila that has been produced in the same cell type, when tested in one or more of a clotting assay, proteolysis assay, or TF binding assay as described above.
  • Factor VII variants having substantially reduced biological activity relative to wild-type Factor Vila are those that exhibit less than about 25%, preferably less than about 10%, more preferably less than about 5% and most preferably less than about 1% of the specific activity of wild-type Factor Vila that has been produced in the same cell type when tested in one or more of a clotting assay, proteolysis assay, or TF binding assay as described above.
  • Factor VII variants having a substantially modified biological activity relative to wild-type Factor VII include, without limitation, Factor VII variants that exhibit TF- independent Factor X proteolytic activity and those that bind TF but do not cleave Factor X.
  • Variants of Factor VII include, without limitation, polypeptides having an amino acid sequence that differs from the sequence of wild-type Factor VII by insertion, deletion, or substitution of one or more amino acids.
  • Non-limiting examples of Factor VII variants having substantially the same or increased proteolytic activity compared to recombinant wild type human Factor Vila include S52A-FVIIa, S60A-FVIIa ( Lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); FVIIa variants exhibiting increased proteolytic stability as disclosed in U.S. Patent No.
  • Non-limiting examples of FVII variants having increased biological activity compared to wild-type FVIIa include FVII variants as disclosed in WO 01/83725, WO 02/22776, WO 02/077218, PCT/DK02/00635 (corresponding to WO 03/027147), Danish patent application PA 2002 01423 (corresponding to WO 04/029090), Danish patent application PA 2001 01627 (corresponding to WO 03/027147); WO 02/38162 (Scripps Research Institute); and FVIIa variants with enhanced activity as disclosed in JP 2001061479 (Chemo-Sero- Therapeutic Res Inst).
  • variants of factor VII include, without limitation, L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A- FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII, V158
  • the terminology for amino acid substitutions used are as follows.
  • the first letter represents the amino acid naturally present at a position of human wild type FVII.
  • the following number represents the position in human wild type FVII.
  • the second letter represent the different amino acid substituting for (replacing) the natural amino acid.
  • An example is M298Q, where a methionine at position 298 of human wild type FVII is replaced by a glutamine.
  • V158T/M298Q the valine in position 158 of human wild type FVII is replaced by a threonine and the methionine in position 298 of human wild type FVII is replaced by a Glutamine in the same Factor VII polypeptide.
  • the factor VII polypeptide is a polypeptide, wherein the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 1.25. In one embodiment the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 2.0. In a further embodiment the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 4.0.
  • the factor VII polypeptide is a polypeptide, wherein the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 1.25 when tested in a Factor Vila activity assay. In one embodiment the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 2.0 when tested in a Factor Vila activity assay. In a further embodiment the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 4.0 when tested in a Factor Vila activity assay.
  • the Factor Vila activity may be measured by the assays described under "assays".
  • the factor VII polypeptide is a polypeptide, wherein the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 1.25 when tested in the "In Vitro Hydrolysis Assay". In one embodiment the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 2.0 when tested in the "In Vitro Hydrolysis Assay”. In a further embodiment the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 4.0 when tested in the "In Vitro Hydrolysis Assay".
  • the factor VII polypeptide is a polypeptide, wherein the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 1.25 when tested in the "In Vitro Proteolysis Assay". In one embodiment the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 2.0 when tested in the "In Vitro Proteolysis Assay”. In a further embodiment the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 4.0 when tested in the "In Vitro Proteolysis Assay".
  • the ratio between the activity of the Factor VII polypeptide and the activity of the wild type human Factor Vila is at least about 8.0 when tested in the "In Vitro Proteolysis Assay".
  • the present invention is suitable for Factor Vll/Vlla variants with increased activity compared to wild type.
  • Factor Vll/Vlla variants with increased activity may be found by testing in suitable assays described in the following. These assays can be performed as a simple preliminary in vitro test. Thus, the section “assays” discloses a simple test (entitled “In Vitro Hydrolysis Assay") for the activity of Factor Vila variants of the invention.
  • Factor Vila variants which are of particular interest are such variants where the ratio between the activity of the variant and the activity of wild type Factor VII is above 1.0, e.g. at least about 1.25, preferably at least about 2.0, such as at least about 3.0 or, even more preferred, at least about 4.0 when tested in the "In Vitro Hydrolysis Assay".
  • the activity of the variants can also be measured using a physiological substrate such as factor X ("In Vitro Proteolysis Assay”) (see under “assays”), suitably at a concentration of 100-1000 nM, where the factor Xa generated is measured after the addition of a suitable chromogenic substrate (eg. S-2765).
  • the activity assay may be run at physiological temperature.
  • the ability of the Factor Vila variants to generate thrombin can also be measured in an assay comprising all relevant coagulation factors and inhibitors at physiological concentrations (minus factor VIII when mimicking hemophilia A conditions) and activated platelets (as described on p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547 which is hereby incorporated as reference).
  • the Factor VII polypeptides described herein are produced by means of recombinant nucleic acid techniques.
  • a cloned wild-type Factor VII nucleic acid sequence is modified to encode the desired protein.
  • This modified sequence is then inserted into an expression vector, which is in turn transformed or transfected into host cells.
  • the complete nucleotide and amino acid sequences for human Factor VII are known (see U.S. 4,784,950, where the cloning and expression of recombinant human Factor VII is described).
  • the bovine Factor VII sequence is described in Takeya et al., J. Biol. Chem. 263:14868- 14872 (1988)).
  • the amino acid sequence alterations may be accomplished by a variety of techniques.
  • Modification of the nucleic acid sequence may be by site-specific mutagenesis.
  • Techniques for site-specific mutagenesis are well known in the art and are described in, for example, Zoller and Smith (DNA 3:479-488, 1984) or "Splicing by extension overlap", Horton et al., Gene 77, 1989, pp. 61-68.
  • Zoller and Smith DNA 3:479-488, 1984
  • Splicing by extension overlap Horton et al., Gene 77, 1989, pp. 61-68.
  • using the nucleotide and amino acid sequences of Factor VII one may introduce the alteration(s) of choice.
  • procedures for preparing a DNA construct using polymerase chain reaction using specific primers are well known to persons skilled in the art (cf. PCR Protocols, 1990, Academic Press, San Diego, California, USA).
  • the nucleic acid construct encoding the Factor VII polypeptide of the invention may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).
  • the nucleic acid construct encoding the Factor VII polypeptide may also be prepared synthetically by established standard methods, e.g.
  • oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in suitable vectors.
  • the nucleic acid construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleic acid construct, in accordance with standard techniques.
  • the nucleic acid construct is preferably a DNA construct.
  • DNA sequences for use in producing Factor VII polypeptides according to the present invention will typically encode a pre-pro polypeptide at the amino-terminus of Factor VII to obtain proper posttranslational processing (e.g. gamma-carboxylation of glutamic acid residues) and secretion from the host cell.
  • the pre-pro polypeptide may be that of Factor VII or another vitamin K-dependent plasma protein, such as Factor IX, Factor X, prothrombin, protein C or protein S.
  • Factor VII another vitamin K-dependent plasma protein
  • additional modifications can be made in the amino acid sequence of the Factor VII polypeptides where those modifications do not significantly impair the ability of the protein to act as a coagulant.
  • the Factor VII polypeptides can also be modified in the activation cleavage site to inhibit the conversion of zymogen Factor VII into its activated two-chain form, as generally described in U.S. 5,288,629.
  • Expression vectors for use in expressing Factor Vila variants will comprise a promoter capable of directing the transcription of a cloned gene or cDNA causing gene expression in animal cells (e.g., a SV40 promoter, a BPV promoter, a metallothionein promoter, a dhfr promoter, various long terminal repeat of retrovirus or LTRs all of which are well known).
  • a promoter capable of directing the transcription of a cloned gene or cDNA causing gene expression in animal cells e.g., a SV40 promoter, a BPV promoter, a metallothionein promoter, a dhfr promoter, various long terminal repeat of retrovirus or LTRs all of which are well known.
  • Preferred promoters include viral promoters and cellular promoters.
  • Viral promoters include the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 :85
  • 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 mouse metallothionein-l 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 Factor VII sequence itself. Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes. Also contained in the expression vectors is a polyadenylation signal located downstream of the insertion site. Particularly preferred polyadenylation signals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 Elb region, the human growth hormone gene terminator (DeNoto et al. Nucl. Acids Res.
  • 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.
  • Cloned DNA sequences are introduced into cultured myeloma cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603-616, 1981 ; Graham and Van der Eb, Virology 52d:456-467, 1973), cationic liposome-mediated transfection (Feigner et al., Proc. Natl. Acad. Sci. 84:7413-7417), electroporation (Neumann et al., EMBO J. 1 :841 -845, 1982) or infection by retroviral or viral-based expression vectors.
  • IRES element Internal ribosome entry sequence
  • a gene that confers a selectable phenotype is generally introduced into cells along with the gene or cDNA of interest.
  • Preferred selectable markers include genes that confer resistance to drugs such as neomycin, hygromycin, and methotrexate.
  • the selectable marker may be an amplifiable selectable marker.
  • a preferred amplifiable selectable marker is a dihydrofolate reductase (DHFR) sequence.
  • Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, MA, incorporated herein by reference). The person skilled in the art will easily be able to choose suitable selectable markers. 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.
  • 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. 4,713,339). It may also be advantageous to add additional DNA, known as "carrier DNA,” to the mixture that is introduced into the cells. After the cells have taken up the DNA, they are grown in an appropriate growth medium, typically for 1-2 days, to begin expressing the gene of interest.
  • the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g.
  • Growth media generally include a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, proteins and growth factors.
  • the medium will contain vitamin K, preferably at a concentration of about 0.1 mg/ml to about 5 mg/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 the desired Factor VII polypeptide.
  • Transgenic animal technology may be employed to produce the Factor VII polypeptides of the invention.
  • the proteins within the mammary glands of a host female mammal It is preferred to produce the proteins within the mammary glands of a host female mammal. Expression in the mammary gland and subsequent secretion of the protein of interest into the milk overcomes many difficulties encountered in isolating proteins from other sources. Milk is readily collected, available in large quantities, and biochemically well characterized. Furthermore, the major milk proteins are present in milk at high concentrations (typically from about 1 to 15 g/l). From a commercial point of view, it is clearly preferable to use as the host a species that has a large milk yield. While smaller animals such as mice and rats can be used (and are preferred at the proof of principle stage), it is preferred to use livestock mammals including, but not limited to, pigs, goats, sheep and cattle.
  • Sheep are particularly preferred due to such factors as the previous history of transgenesis in this species, milk yield, cost and the ready availability of equipment for collecting sheep milk (see, for example, WO 88/00239 for a comparison of factors influencing the choice of host species). It is generally desirable to select a breed of host animal that has been bred for dairy use, such as East Friesland sheep, or to introduce dairy stock by breeding of the transgenic line at a later date. In any event, animals of known, good health status should be used. To obtain expression in the mammary gland, a transcription promoter from a milk protein gene is used. Milk protein genes include those genes encoding caseins (see U.S.
  • beta lactoglobulin a region of at least the proximal 406 bp of 5' flanking sequence of the gene will generally be used, although larger portions of the 5' flanking sequence, up to about 5 kbp, are preferred, such as a ⁇ 4.25 kbp DNA segment encompassing the 5' flanking promoter and non coding portion of the beta lactoglobulin gene (see Whitelaw et al., Biochem. J. 286: 31 39 (1992)). Similar fragments of promoter DNA from other species are also suitable.
  • beta lactoglobulin gene may also be incorporated in constructs, as may genomic regions of the gene to be expressed. It is generally accepted in the art that constructs lacking introns, for example, express poorly in comparison with those that contain such DNA sequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836 840 (1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478482 (1991); Whitelaw et al., Transgenic Res. 1 : 3 13 (1991); WO 89/01343; and WO 91/02318, each of which is incorporated herein by reference).
  • genomic sequences containing all or some of the native introns of a gene encoding the protein or polypeptide of interest thus the further inclusion of at least some introns from, e.g, the beta lactoglobulin gene, is preferred.
  • One such region is a DNA segment that provides for intron splicing and RNA polyadenylation from the 3' non coding region of the ovine beta lactoglobulin gene. When substituted for the natural 3' non coding sequences of a gene, this ovine beta lactoglobulin segment can both enhance and stabilize expression levels of the protein or polypeptide of interest.
  • the region surrounding the initiation ATG of the variant Factor VII sequence is replaced with corresponding sequences from a milk specific protein gene.
  • Such replacement provides a putative tissue specific initiation environment to enhance expression. It is convenient to replace the entire variant Factor VII pre pro and 5' non coding sequences with those of, for example, the BLG gene, although smaller regions may be replaced.
  • a DNA segment encoding variant Factor VII is operably linked to additional DNA segments required for its expression to produce expression units.
  • additional segments include the above mentioned promoter, as well as sequences that provide for termination of transcription and polyadenylation of mRNA.
  • the expression units will further include a DNA segment encoding a secretory signal sequence operably linked to the segment encoding modified Factor VII.
  • the secretory signal sequence may be a native Factor VII secretory signal sequence or may be that of another protein, such as a milk protein (see, for example, von Heijne, Nucl. Acids Res. 14: 4683 4690 (1986); and Meade et al., U.S. 4,873,316, which are incorporated herein by reference).
  • Construction of expression units for use in transgenic animals is conveniently carried out by inserting a variant Factor VII sequence into a plasmid or phage vector containing the additional DNA segments, although the expression unit may be constructed by essentially any sequence of ligations.
  • a vector containing a DNA segment encoding a milk protein and to replace the coding sequence for the milk protein with that of a variant Factor VII polypeptide; thereby creating a gene fusion that includes the expression control sequences of the milk protein gene.
  • cloning of the expression units in plasmids or other vectors facilitates the amplification of the variant Factor VII sequence. Amplification is conveniently carried out in bacterial (e.g. E. coli) host cells, thus the vectors will typically include an origin of replication and a selectable marker functional in bacterial host cells.
  • the expression unit is then introduced into fertilized eggs (including early stage embryos) of the chosen host species.
  • heterologous DNA can be accomplished by one of several routes, including microinjection (e.g. U.S. Patent No. 4,873,191), retroviral infection (Jaenisch, Science 240: 1468 1474 (1988)) or site directed integration using embryonic stem (ES) cells (reviewed by Bradley et al.,
  • the Factor VII polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction
  • IEF isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • the Factor VII polypeptides of the invention is purified to at least about 90 to 95% homogeneity, preferably to at least about 98% homogeneity. Purity may be assessed by e.g. gel electrophoresis and amino-terminal amino acid sequencing.
  • the Factor VII polypeptide is cleaved at its activation site in order to convert it to its two-chain form. Activation may be carried out according to procedures known in the art, such as those disclosed by Osterud, et al., Biochemistry 11 :2853-2857 (1972); Thomas, U.S. Patent No. 4,456,591 ; Hedner and Kisiel, J. Clin. Invest.
  • Factor VII may be activated by passing it through an ion-exchange chromatography column, such as Mono Q (Pharmacia fine Chemicals) or the like. The resulting activated Factor VII polypeptide may then be formulated and administered as described below.
  • Factor Vila Wild type (native) Factor Vila and Factor Vila variant (both hereafter referred to as "Factor Vila") are assayed in parallel to directly compare their specific activities.
  • the assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark).
  • the absorbance at 405 nm is measured continuously in a SpectraMax ® 340 plate reader (Molecular Devices, USA).
  • the absorbance developed during a 20-minute incubation, after subtraction of the absorbance in a blank well containing no enzyme, is used to calculate the ratio between the activities of vari-ant and wild-type Factor Vila:
  • Ratio (A405 nm Factor Vila variant)/(A405 nm Factor Vila wild-type).
  • Wild type (native) Factor Vila and Factor Vila variant both hereafter referred to as "Factor Vila" are assayed in parallel to directly compare their specific activities. The assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). Factor Vila (10 nM) and Factor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 5 mM CaCI 2 and 1 mg/ml bovine serum albumin, are incubated for 15 min.
  • Factor X cleavage is then stopped by the addition of 50 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 20 mM EDTA and 1 mg/ml bovine serum albumin.
  • the amount of Factor Xa generated is measured by addition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final concentration 0.5 mM.
  • the absorbance at 405 nm is measured continuously in a SpectraMax ® 340 plate reader (Molecular Devices, USA).
  • Ratio (A405 nm Factor Vila variant)/(A405 nm Factor Vila wild-type).
  • FVII and its related analogues can be successfully expressed in cells that normally do not express FVII, that is, in cells whose origin and function is not involved in the FVII coagulation cascade. Moreover, it is shown that FVII and its analogues can be expressed abundantly and in their active form in a manner that is comparable to existing producer cell lines.
  • the practice of the present invention is based on conventional techniques that are within the skill of the art. Unless otherwise indicated, the methods in molecular biology necessary to generate plasmids, hereunder recombinant DNA cloning and microbiology techniques, are described in detail in Sambrook & Russell, Molecular Cloning, A Laboratory manual (2001); Ausubel et al.
  • Example 1 Expression of human FVII and FVII analogues in myeloma cells. Plasmids were first purified using the Qiagen Maxi Prep plasmid purification kits. Transfection of plasmids containing cDNAs encoding human FVII (pTS8) and FVII analogues (eg. pTS72) into myeloma cell lines P3X63Ag8.653 and SP2-OAg14 (also referred to as X63 and SP2/0) was performed. Two myeloma cell lines, SP2/0 and X63 were stably transfected using Lipofectamine
  • Example 2 Clonal selection of high-FVII and FVII analogues- producing cell lines Limited dilution of stable transfectants was performed in 96-well plates, reaching a concentration of 1 -10 cells/well. The highest producing cell populations (>300 ng/ml FVII) were chosen. Clones from myeloma cell lines SP2/0 and X63 have the ability to express both recombinant human FVII and the fusion protein FVII-Fc, as judged from ELISA-based assays and Western blotting. Relevant cell pools (from the 96-well plates) were expanded to 6-well plates, confirmed as positive by ELISA, and expanded further to 25 cc flasks and afterwards to 175 cc flasks. Secreted FVII amounts was measured in the order of >1 mg/ml.
  • the assay is based on how efficient FVII can promote coagulation in plasma using thromboplastin as described in U.S. Patent No. 5,997,864. Lack of vitamin K in the cell media resulted in non-detectable active FVII, whereas myeloma cells grown with vitamin K (5 microgram/ml), express active FVII and active FVII analogues (FVII- Fc). Sp2/0 cells express more active, higher levels of FVII, than X-63 cells. Cells expressing plasmid controls (pcDNA3.1 and plRESneo-2b) do not have any detectable FVII activity, as expected.
  • Myeloma cells expressing FVII and FVII analogues were grown in media without serum, in the presence of vitamin K, and initial measurements suggest that myeloma cells can efficiently express active FVII and FVII analogues (FVII-Fc).
  • Example 4 Fusion of myeloma cells with themselves or with other cell types.
  • Myeloma cells expressing FVII and/or FVII analogues (FVII-Fc) were electrofused (cell fusion chamber/multiporator Eppendorf) with themselves or to liver cells and/or other organ specific cells, and stable polyploid cell clones were generated. The resulting clones were screened for their ability to express active human FVII and FVII analogues (FVII-Fc), in the presence of vitamin K.
  • FVII-Fc FVII analogues
  • Myeloma cells may be fused either by classical fusion methods or by electrofusion (Langonem J.J. and van Vunakis, H. editors, Immunochemical techniques, Methods in Enzymology, Volume 121 , Academic Press, 1986; Bartal, A.H. and Hirshaut, Y. editors, hybridoma formation: methods and mechanisms, Humana Press, 1987).
  • FVII analogues eg. pTS72
  • control plasmids pcDNA3.1 Invitrogen Corp,
  • plRESneo-2b Stratagene
  • Myeloma cells were seeded out onto 96-well plates, cultured in DMEM media with Glutamax (Invitrogen), 10% fetal calf serum and containing a range of G418 (Geneticin; Invitrogen Corp.) concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 ⁇ g/ml. Their survival ability was followed during at least 10 days. Visual inspection of the surviving clones led us to conclude that a concentration of 600 ⁇ g/ml G418 is an appropriate amount that could kill all existing cells.
  • Glutamax Invitrogen
  • G418 Geneticin; Invitrogen Corp.
  • any selection of exogenously introduced expression vectors carrying a G418 antibiotic marker would have to be done at a minimum of 600 ⁇ g/ml, for the chosen myeloma cells, to be able to eliminate any non-desired untransfected cells.
  • Plasmid preparation and transfection The following plasmids were first purified using the Maxi Prep plasmid purification kits (Qiagen): plasmid pTS8 containing a cDNA encoding human FVII, plasmid pTS72 containing a cDNA encoding FVII-Fc, a FVII analogue, control/parental plasmid pcDNA3.1 (Invitrogen Corp.) of pTS8 and control/parental plasmid plRESneo-2b (Stratagene) of pTS72 .
  • Qiagen Maxi Prep plasmid purification kits
  • the two myeloma cell lines, SP2/0 and X63 were stably transfected using Lipofectamine 2000 (Invitrogen Corp) according to the manufacturer's instructions with the above purified plasmids. Each transfection was performed in 5 ml Opti-Mem medium without fetal calf serum and antibiotics during 48 h in 6-well dishes with a cell density of 1.0 x 10 6 c/ml.
  • Opti- MEM is a modification of Eagle's minimum essential medium supplemented with hypoxanthine, thymidine, sodium pyruvate, L-Glutamine, trace elements and growth factors (Invitrogen Corp.).
  • the transfection media was carefully discarded and new DMEM media with 10% fetal calf serum, Glutamax, 5 microgram/ml vitamin K and antibiotic G418 was added.
  • the selection of stable transfectants was done at both 600 and 800 micrograms/ml G418 (Geneticin, Invitrogen Corp.). Media changes were performed once every Monday, Wednesday and Friday. A higher concentration of G418 (800 micrograms/ml) would result in a stronger selection of stable clones expressing FVII, while restricting the growth of clones whose plasmid copy number and resulting FVII expression is not high enough, as would be expected from the selection of clones in media containing 600 microgram/ml G418.
  • An initial FVII-quick test assay was performed on the transfectants and it was confirmed that FVII can be expressed in myeloma cells (Table 1).
  • Example 6 Clonal selection of high-FVII and FVII analogues- producing cell lines Approximately 2 weeks after transfection, the transfected cells were subject to limited dilution, whereby cells are counted and diluted out in order to attempt getting approximately 1-10 cell clones/well in 96-well plates.
  • pTS8 (FVII-expressing) plasmid-transfected SP2/0 cells were seeded out onto 4 96-well plates and selected with 600 and 800 micrograms/ml G418 (2 plates each). The same was done for pTS72 (FVII-Fc-expressing) transfected cells.
  • pTS8 (FVII-expressing) plasmid-transfected X63 cells were seeded onto 8 96-well plates and selected with 600 and 800 micrograms/ml G418 (4 plates each). Control plates carrying single-transfected cell clones of the parental/control plasmids were also tested. Approximately 10 days after performing the limited dilution procedure, 100 microliters of cell supernatants from wells where growth could be detected were transferred to 96-well assay plates and subject to a FVII-quick Elisa test, to measure the concentration of secreted FVII. It was possible to detect FVII (up to 390 ng/ml) in a number of wells.
  • clones from myeloma cell lines SP2/0 and X63 have the ability to express both recombinant human FVII and the fusion protein FVII-Fc, as judged from ELISA-based assays and Western blotting (see Figures 1 A, 1 B and Table 2).
  • Relevant cell pools (from the 96-well plates) were expanded to 6-well plates, confirmed as positive by ELISA, and expanded further to 25 cc flasks and afterwards to 175 cc flasks.
  • Secreted FVII amounts were measured in the order of >1 mg/ml. It could also be concluded that FVII expression was higher in SP2/0 myeloma cells than in X63 myeloma cells as judged from FVII Elisa assays (data not shown).
  • Protein characterization A Western blot procedure (protein blot) was run under reducing and non-reducing conditions to detect FVII.
  • One single band was obtained that is similar to a positive control, a FVII-analogue expressed in CHO-K1 cells.
  • the patterns are different under reducing conditions. This could be due to the fact that vitamin K was not added in the original media.
  • vitamin K addition did not change the resulting profiles in a retest of the above mentioned Western under reducing or non- reducing conditions.
  • FIG. 1 A A protein gel was run under reducing and non-reducing conditions to detect FVII by the Western blot procedure ( Figures 1 A and B). A single band of the same size as a positive control was obtained ( Figure 1 A,B). Myeloma cell pools expressing the FVII-Fc analogue were also harvested and protein extracts were subject to FVII-protein blots. A representative example is shown in Figure 2A (non-reducing conditions) and in Figure 2B (reducing conditions).
  • Protein samples derived from cell culture supernatants (20 microliters) were prepared either under non-reducing or reducing conditions, and denatured at 72 degrees C during 10 min before loading onto NuPage 12% Bis-Tris acrylamide gels in a Novex XCell II MiniCell system (Invitrogen Corp.) and electrophoresed at 200 volts during 0,5-1 hour. A molecular size marker, Magic Marker, was used in the runs. The protein ladders were subsequently transferred to a nitrocellulose membrane using the Blot module of the Novex XCell II MiniCell system at 24-28 volts during 1 ,5 hours at room temperature.
  • nitrocellulose membrane was blocked with wash buffer containing 2% Tween 20 during 2 min and incubated with rabbit anti-human FVII polyclonal IgG primary antibody at a concentration of 0.2 microgram/ml, and a goat anti-mouse IgG-HRP conjugated secondary antibody at a 1 :2000 dilution. Chemiluminescence was detected by using a Fuji luminescence scanner.
  • Coagulation assay An activity assay was carried out on myeloma clones expressing plasmid controls or FVII and/or FVII analogues (FVII-Fc), in the absence or presence of vitamin K (5 microgram/ml) in the media.
  • the assay is based on how efficient FVII can promote coagulation in plasma using thromboplastin as described in U.S. Patent No. 5,997,864. Lack of vitamin K in the cell media resulted in non-detectable active FVII, whereas myeloma cells grown with vitamin K (5 microgram/ml), express active FVII and active FVII analogues (FVII- Fc).
  • Sp2/0 cells express more active, higher levels of FVII, than X-63 cells.
  • Cells expressing plasmid controls do not have any detectable FVII activity, as expected.
  • Supernatants from the 25 cc flasks were used to measure the clotting activity of the
  • FVII being produced (Table 1 ).
  • Normal human plasma containing 0.5 microgram/ml FVII is expected to give 1-1.1 U/ml FVII clotting activity.
  • Myeloma SP2/0 cells expressing 0.5 microgram/ml of FVII showed a clotting activity of up to 0.51 U/ml (Table 1), indicating that myeloma cells are indeed capable of expressing functionally active FVII, although to a lesser degree than normal human plasma.
  • the contribution of vitamin K to the activity of FVII has been reported previously (see reviews by Berkner, 2000, J. Nutr. 130:1877-1880; Suttie, 1992, J. Am. Diet Assoc. 92:585-590).
  • FVII-Fc Coagulation activity of expressed FVII-Fc, a FVII analogue was lower than for FVII, (Table 1).
  • Myeloma cells expressing FVII and FVII analogues (FVII-Fc) were grown in media without serum, in the presence of vitamin K, and initial measurements suggest that myeloma cells can efficiently express active FVII and FVII analogues (FVII-Fc).
  • the one-step coagulation assay for measurement of FVII activity (FVII:C) in human plasma was performed according to standard operating procedures at Novo Nordisk, using an ACL300/3000 research instrument, as described in Broze & Majerus, Human Factor VII, Methods Enzymol. 80:228-237 (1981).
  • test sample's ability to normalize coagulation time is measured in a one-step system consisting of Factor Vll-deprived plasma (Helena Labs) and rabbit thromboplastin (Manchester reagent). Coagulation is started by addition of thromboplastin-Ca ++ reagent.
  • Glvcosidation assay Supernatants from various FVII-expressing myeloma cell lines were obtained directly and analyzed by Western blotting ( Figure 3A and B). The cell supernatants were loaded onto Nupage 12% Bis-Tris acrylamide gels, separated by electrophoresis, transferred to PVDF membranes (Invitrogen Corp.) and incubated with a rabbit anti-human FVII polyclonal antibody. FVII expressed in myeloma cells is glycosylated, as well as FVII expressed in CHO-K1 cells (lanes 2,4, Figure 3A and B).
  • a mutant FVII than cannot undergo glycation exhibits the lower molecular weight band, even in the presence of PNGase ( Figure 3 A,B, lanes 6,7).

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Abstract

La présente invention concerne un procédé de production d'un polypeptide de recombinaison.
PCT/EP2005/050060 2004-01-07 2005-01-07 Procede de production de proteines de recombinaison WO2005068620A1 (fr)

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US11/481,169 US20070072271A1 (en) 2004-01-07 2006-07-05 Method for the production of recombinant proteins
US12/464,973 US20090221800A1 (en) 2004-01-07 2009-05-13 Method for the Production of Recombinant Proteins

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9476037B2 (en) 2008-04-11 2016-10-25 Catalyst Biosciences, Inc. Factor VII polypeptides that are modified and uses thereof
US11266724B2 (en) 2019-08-15 2022-03-08 Catalyst Biosciences, Inc. Modified factor VII polypeptides for subcutaneous administration and on-demand treatment

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0870859A (ja) * 1994-06-27 1996-03-19 Takeda Chem Ind Ltd 動物細胞培養用の無血清培地、および生理活性ペプチドまたは蛋白質の製造法
WO2002029084A2 (fr) * 2000-10-02 2002-04-11 Novo Nordisk A/S Production exempte de serum, a l'echelle industrielle, de facteur vii de recombinaison dans des cellules mammaliennes

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Publication number Priority date Publication date Assignee Title
JPH0870859A (ja) * 1994-06-27 1996-03-19 Takeda Chem Ind Ltd 動物細胞培養用の無血清培地、および生理活性ペプチドまたは蛋白質の製造法
WO2002029084A2 (fr) * 2000-10-02 2002-04-11 Novo Nordisk A/S Production exempte de serum, a l'echelle industrielle, de facteur vii de recombinaison dans des cellules mammaliennes

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BROAD D ET AL: "PRODUCTION OF RECOMBINANT PROTEINS IN SERUM-FREE MEDIA", CYTOTECHNOLOGY, KLUWER ACADEMIC PUBLISHERS, DORDRECHT, NL, vol. 5, no. 1, 1991, pages 47 - 55, XP001073869, ISSN: 0920-9069 *
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; 1992, NIKOLAENKO N S ET AL: "[The cultivation of mouse and human lymphoid cells on serum-free media]", XP002328245, Database accession no. NLM1293879 *
PATENT ABSTRACTS OF JAPAN vol. 1996, no. 07 31 July 1996 (1996-07-31) *
PETERSEN L C ET AL: "Characterization of recombinant murine factor VIIa and recombinant murine tissue factor: a human-murine species compatibility study", THROMBOSIS RESEARCH, TARRYTOWN, NY, US, vol. 116, no. 1, 2005, pages 75 - 85, XP004858144, ISSN: 0049-3848 *
TSITOLOGIIA. 1992, vol. 34, no. 8, 1992, pages 88 - 95, ISSN: 0041-3771 *
YOO E M ET AL: "Myeloma expression systems", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 261, no. 1-2, 1 March 2002 (2002-03-01), pages 1 - 20, XP004341264, ISSN: 0022-1759 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9476037B2 (en) 2008-04-11 2016-10-25 Catalyst Biosciences, Inc. Factor VII polypeptides that are modified and uses thereof
US10160961B2 (en) 2008-04-11 2018-12-25 Catalyst Biosciences, Inc. Factor VII polypeptides that are modified and uses thereof
US11203749B2 (en) 2008-04-11 2021-12-21 Catalyst Biosciences, Inc. Factor VII polypeptides that are modified and uses thereof
US11266724B2 (en) 2019-08-15 2022-03-08 Catalyst Biosciences, Inc. Modified factor VII polypeptides for subcutaneous administration and on-demand treatment

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