WO1996017062A1 - Low molecular weight thrombopoietin - Google Patents
Low molecular weight thrombopoietin Download PDFInfo
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- WO1996017062A1 WO1996017062A1 PCT/US1995/014929 US9514929W WO9617062A1 WO 1996017062 A1 WO1996017062 A1 WO 1996017062A1 US 9514929 W US9514929 W US 9514929W WO 9617062 A1 WO9617062 A1 WO 9617062A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/524—Thrombopoietin, i.e. C-MPL ligand
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- He atopoiesis is the process by which blood cells develop and differentiate from pluripotent stem cells in the bone marrow. This process involves a complex interplay of polypeptide growth factors (cytokines) acting via membrane-bound receptors on their target cells. Cytokine action results in cellular proliferation and differentiation, with response to a particular cytokine often being lineage-specific and/or stage-specific. Development of a single cell type, such as a platelet, from a stem cell may require the coordinated action of a plurality of cytokines acting in the proper sequence.
- cytokines polypeptide growth factors
- the known cytokines include the interleukins, such as IL-1, IL-2, IL-3, IL-6, IL-8, etc.; and the colony stimulating factors, such as G-CSF, M-CSF, GM-CSF, erythropoietin (EPO) , etc.
- the interleukins act as mediators of immune and inflammatory responses.
- the colony stimulating factors stimulate the proliferation of marrow-derived cells, activate mature leukocytes, and otherwise form an integral part of the host's response to inflammatory, infectious, and immunologic challenges.
- erythropoietin which stimulates the development of erythrocytes
- erythropoietin which stimulates the development of erythrocytes
- Several of the colony stimulating factors have been used in conjunction with cancer chemotherapy to speed the recovery of patients' immune systems.
- Interleukin-2, o-interferon and ⁇ -interferon are used in the treatment of certain cancers.
- An activity that stimulates megakaryocytopoiesis and thrombocytopoiesis has been identified in body fluids of thrombocytopenic animals and is referred to in the literature as "thrombopoietin" (recently reviewed by McDonald, Exp. Hematol.
- thrombopoietin thrombopoietin
- this protein has been shown to stimulate platelet production in vivo (Kaushansky et al., ibid.), it appears to be subject to proteolysis and was isolated in heterogeneous or degraded form (Bartley et al., ibid.; de Sauvage et al., ibid.).
- Preparations of thrombopoietin reported in the scientific literature are therefore not well characterized as to composition and the relative activities of the various molecular species, although at least some of the proteolytic products are biologically active.
- an isolated mammalian thrombopoietin characterized by an amino acid sequence selected from the group consisting of (a) a sequence of amino acids as shown in SEQ ID NO:2 having an amino terminus at Ser(45) and a carboxyl terminus between Ser(208) and Asn(216) , inclusive; (b) a sequence of amino acids as shown in SEQ ID NO:4 having an amino terminus at Ser(22) and a carboxyl terminus between Arg(185) and Asn(193) , inclusive; (c) a sequence of amino acids as shown in SEQ ID NO:4 having an amino terminus at Ser(22) and a carboxyl terminus at Arg(198); (d) a sequence of amino acids as shown in SEQ ID NO:4 having an amino terminus at Ser(22) and a carboxyl terminus at Phe(207); and (e) allelic varaints of (a)-(d).
- the thrombopoietin is further characterized by a peptide backbone mass of 17,490 to 19,045 amu as determined by mass spectro etry, an absence of N-linked carbohydrate, a presence of O- linked carbohydrate, and being essentially free of thrombopoietin species having a molecular weight greater than 18,885 amu as determined by mass spectrometry.
- the thrombopoietin is a mixture of polypeptides having different carboxyl termini.
- the present invention provides a composition of mammalian thrombopoietin consisting essentially of one or more polypeptides having an amino acid sequence consisting of (a) the sequence shown in SEQ ID NO:4 from an amino-terminus at Ser, residue number 22, to a carboxyl-terminus between Arg, residue number 185, and Asn, residue number 193, inclusive; and (b) allelic variants of (a) .
- the composition is further characterized by an absence of N-linked carbohydrate and a presence of O- linked carbohydrate on the one or more polypeptides.
- the present invention provides a composition of mammalian thrombopoietin consisting essentially of one or more polypeptides having an amino acid sequence selected from the group consisting of (a) a sequence of amino acids as shown in SEQ ID NO:4 having an amino terminus at Ser(22) and a carboxyl terminus at Arg(198) , (b) a sequence of amino acids as shown in SEQ ID NO:4 having an amino terminus at Ser(22) and a carboxyl terminus at Phe(207) , and (c) allelic variants of (a) and (b) .
- a pharmaceutical composition comprising a thrombopoietin polypeptide as disclosed above in combination with a pharmaceutically acceptable vehicle. Also provided is a method of stimulating platelet production in a mammal in need thereof comprising administering to the mammal a therapeutically effective amount of the composition.
- an isolated DNA molecule encoding a mammalian thrombopoietin as disclosed above.
- the DNA molecule may be used in the construction of an expression vector.
- the vector comprises a DNA segment encoding a thrombopoietin as disclosed above operably linked to one or more additional DNA segments that provide for its transcription.
- a cultured eukaryotic cell containing the disclosed expression vector, such as a yeast cell or a cultured mammalian cell. The cultured cell may be used within methods for producing a mammalian thrombopoietin.
- These methods comprise the steps of culturing a eukaryotic cell containing an expression vector comprising a first DNA segment encoding a mammalian thrombopoietin as disclosed above, wherein the first DNA segment is operably linked to a second DNA segment encoding a secretory peptide and one or more additional DNA segments that provide for transcription of the first and second DNA segments, and wherein the cell expresses the first and second DNA segments and the thrombopoietin is secreted from the cell and selectively recovered.
- Fig. 1 illustrates the effects of a representative thrombopoietin composition of the present invention on platelet levels in mice.
- Fig. 2 illustrates the dose-response relationship of platelet levels in mice treated with a representative thrombopoietin composition of the present invention.
- Allelic variant An alternative form of a gene that arises through mutation, or an altered polypeptide encoded by the mutated gene. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
- cDNA Complementary DNA, prepared by reverse transcription of a messenger RNA template, or a clone or amplified copy of such a molecule. Complementary DNA can be single-stranded or double-stranded. Essentially free: At least 95% free of a specified contaminant. When applied to a protein, level of contamination is determined by Edman degradation and amino acid sequencing.
- Expression vector A DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that e provide for its transcription.
- additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
- Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
- the term "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator.
- Gene A segment of chromosomal DNA that encodes a polypeptide chain.
- a gene includes one or more regions encoding amino acids, which in some cases are interspersed with non-coding "intervening sequences" ("introns") , together with flanking, non-coding regions which provide for transcription of the coding sequence.
- Isolated When applied to the protein the term “isolated” indicates that the protein is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is prefered to provide the proteins in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When applied to a polynucleotide molecule the term “isolated” indicates that the molecule is removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
- Peptide backbone mass The molecular mass of a polypeptide or protien in the absence of glycosylation, generally determined by mass spectrometry of deglycosylated protein or calculated from amino acid sequence.
- Promoter The portion of a gene at which RNA polymerase binds and mRNA synthesis is initiated.
- Secretory peptide An amino acid sequence that acts to direct the secretion of a mature polypeptide or protein from a cell.
- Secretory peptides are characterized by a core of hydrophobic amino acids and are typically (but not exclusively) found at the amino termini of newly synthesized proteins. Very often the secretory peptide is cleaved from the mature protein during secretion. Such secretory peptides contain processing sites that allow cleavage of the secretory peptides from the mature proteins as they pass through the secretory pathway.
- a DNA sequence encoding a secretory peptide is referred to as a signal sequence, leader sequence, prepro sequence or pre sequence.
- Thrombopoietin (TPO) proteins are characterized by their ability to specifically bind to MPL receptor from the same species and to stimulate platelet production in vi vo. In normal test animals, TPO is able to increase platelet levels by 100% or more within 10 days after beginning daily administration.
- TPO Thrombopoietin polypeptide encompasses full-length thrombopoietin molecules and biologically active portions thereof, that is fragments of a thrombopoietin that exhibit the qualitative biological activities of the intact molecule (receptor binding and in vi vo stimulation of platelet production) .
- the present invention is based in part on the discovery of a class of defined and well-characterized, biologically active thrombopoietin polypeptides having unexpectedly high specific activity, members of which were initially obtained by fractionation of recombinant TPO.
- TPO compositions of the present invention exhibit an in vi tro specific activity (units of activity per mole of protein) five to ten times that of conventional TPO preparations that comprise a mixture of glycosylated polypeptide species ranging in size from about 30 kD to about 70 kD as determined by electrophoresis on SDS- polyacrylamide gels under reducing conditions.
- the TPO compositions of the present invention have also been found to be fully active in vi vo.
- a recombinant mouse TPO preparation that contained a significant amount of protein with a molecular weight in the 18-22 kD range was fractionated by a combination of affinity chromatography and anion exchange chromatography. This low molecular weight species was analyzed by mass spectrometry and found to have a heterogeneous carboxyl terminus, with the longest and most prevalent form ending at amino acid residue 216 of SEQ ID NO:2, and the shortest form ending at residue 208 of SEQ ID NO:2. The peptide backbone mass of the longest form, taking into consideration two disulfide bonds, was determined to be 18,449 atomic mass units (amu) by mass spectrometry.
- This low molecular weight TPO contained no N-linked carbohydrate as determined by the absence of a molecular weight shift after digestion with peptide-N- glycosidase F, which is specific for the hydrolysis of ⁇ - aspartyl-glycosylamine bonds between asparagine and the innermost N-acetylglucosamine of the glycan moiety; and by mass spectrometry, which indicated that the C-terminus of the polypeptide was upstream of the first N-linked glycosylation site of the full-length TPO molecule.
- the presence of 0-linked carbohydrates on this TPO was determined by a shift in molecular weight after deglycosylation with O-glycosidase.
- the peptide backbone mass of the shortest polypeptide in T this preparation (terminating at residue 208 of SEQ ID NO:2) was 17,492 amu.
- the thrombopoietin compositions of the present invention may be a single polypeptide species or a mixture of polypeptides differing at their carboxyl termini but having a common amino terminus.
- a representative group of such polypeptides derived from mouse TPO includes molecules having a sequence of amino acids as shown in SEQ ID NO:2 from an amino-terminal Ser residue, amino acid residue no. 45, to a carboxyl terminus between residue no. 208 (Ser) and residue no. 216 (Asn) , inclusive.
- the present invention also provides related TPO polypeptides from species other than mouse.
- TPO polypeptides from species other than mouse.
- a group of human TPO polypeptides the amino acid sequences of which begin at Ser, residue 22 of SEQ ID NO:4 and terminate between residue 185 (Arg) and residue 198 (Arg), inclusive, of SEQ ID NO:4.
- human TPO polypeptides shown in Table 1 are particularly relevant to human TPO polypeptides shown in Table 1 (with reference to SEQ ID NO:4) , as well as intermediate forms, such as polypeptides having C-termini between Arg (185) and Asn (193) .
- TPO polypeptides are characterized by a peptide backbone mass of approximately 17,593 to 19,045 amu as determined by mass spectrometry.
- the polypeptide terminating at Asn (193) has a peptide backbone mass of 18,435 amu.
- These polypeptides are free of N-linked carbohydrate attachment sites, but contain O- linked carbohydrate attachment sites.
- Protein molecular weights are determined by conventional procedures. See, for example, Laemmli, Nature 227:680-685. 1970; McEwan and Larsen, eds.. Mass Spectrometry of Biological Materials. Marcel Decker, Inc. , New York, 1990; and Carr et al., Analytical Chem. ⁇ l:2802-2824, 1991, which are incorporated herein by reference. Proteins can be deglycosylated enzymatically using conventional methods. In a typical procedure, the protein is desalted by high performance liquid chromatography prior to deglycosylation. N-linked carbohydrate is removed using peptide-N-glycosidase F.
- Sialic acid residues are removed using sialidase (neuraminidase) .
- O-linked carbohydrate is removed using O-glycosidase (endo- ⁇ -N-acetylgalactosaminidase) following removal of terminal neuraminic acid moieties with sialidase.
- Glycosidases and other enzymes are available from commercial suppliers, such as Oxford Glycosystems (Rosedale, NY) and Boehringer Mannheim, Inc. (Indianapolis, IN) .
- the present invention provides isolated, biologically active, mammalian TPO polypeptides, including human, mouse, rat, porcine, canine, ovine, bovine and equine TPO polypeptides.
- primate TPO polypeptides in particular human TPO polypeptides.
- Non-human TPO polypeptides of the present invention are, in general, at least 50% identical in amino acid sequence to corresponding portions of the mouse (SEQ ID NO:2) or human (SEQ ID NO:4) sequences disclosed herein. Percent sequence identity of amino acid sequences is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad.
- allelic variants are characterized by one or more amino acid substitutions, deletions or additions.
- changes in amino acid sequence can be introduced in TPO molecules through the application of genetic engineering.
- Aromatic phenylalanine tryptophan tyrosine
- TPO glycine alanine serine threonine methionine
- essential amino acids in TPO may be identified according to procedures known in the art, such as site- directed utagenesis or alanine-scanning utagenesis (Cunningham and Wells, Science 244. 1081-1085, 1989) .
- site- directed utagenesis or alanine-scanning utagenesis (Cunningham and Wells, Science 244. 1081-1085, 1989) .
- single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. receptor binding, in vi tro or in vivo proliferative activity) to identify amino acid residues that are critical to the activity of the molecule.
- Sites of ligand-receptor interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al., Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 211:899-904, 1992; Wlodaver et al., FEBS Lett. 3_09_:59-64, 1992.
- cytokines are predicted to have a four-alpha helix structure, with the first and fourth helices being most important in ligand-receptor interactions and more highly conserved among members of the family.
- TPO amino acid sequence shown in SEQ ID NO:4 alignment of cytokine sequences suggests that these helices are bounded by amino acid residues 29 and 53, 80 and 99, 108 and 130, and 144 and 168, respectively (boundaries are + 4 residues) .
- Helix boundaries of the mouse (SEQ ID NO:2) and other non- human TPOs can be determined by alignment with the human sequence.
- Other important structural aspects of TPO include the cysteine residues at positions 51, 73, 129 and 195 of SEQ ID NO:2 (corresponding to positions 28, 50, 106 and 172 of SEQ ID NO:4).
- TPO polypeptides of the present invention by production in genetically engineered cells or organisms, although isolation from '5 natural sources of TPO (e.g. blood or other biological fluid) is within the scope of the invention.
- TPO tissue-derived neuropeptide
- a DNA segment encoding at least the polypeptide of interest is expressed in the host, and the polypeptide is recovered.
- the DNA segment may encode additional sequences, for example additional TPO amino acid residues C-terminal to the carboxyl terminus of a TPO polypeptide or a secretory signal sequence.
- a DNA segment encoding a full-length TPO molecule is expressed, and the resulting TPO is recovered and fractionated to provide a purified preparation of the truncated polypeptide(s) .
- a stop codon is introduced (e.g. by site-specific mutagenesis) into the TPO-encoding DNA segment immediately 3' to the terminal codon of the sequence of interest.
- a DNA segment encoding a TPO polypeptide further encodes one or more non-TPO amino acid residues, such as an amino-terminal methionine, a small amino- or carboxyl-terminal extension that provides an antigenic epitope or other binding domain to facilitate purification of the polypeptide, or a heterologous secretory signal sequence.
- non-TPO amino acid residues such as an amino-terminal methionine, a small amino- or carboxyl-terminal extension that provides an antigenic epitope or other binding domain to facilitate purification of the polypeptide, or a heterologous secretory signal sequence.
- a DNA segment encoding TPO is operably linked to a transcription promoter and terminator within an expression vector.
- the vector will commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
- a signal sequence is provided in the expression vector.
- the signal sequence is joined to the DNA sequence encoding TPO in the correct reading frame.
- Signal sequences are commonly positioned 5" to the DNA sequence encoding the protein or polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
- the signal sequence may be one normally associated with TPO, or may be from a gene encoding another secreted protein, such as tissue-type plasminogen activator (t-PA) .
- tissue-type plasminogen activator t-PA
- Yeast cells particularly cells of the genus Saccharomyces, are a preferred host for use in producing recombinant TPO.
- Methods for transforming yeast cells with exogenous DNA and producing recombinant proteins therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075, which are incorporated herein by reference.
- Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g. leucine) .
- a preferred vector system for use in yeast is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
- a preferred secretory signal sequence for use in yeast is that of the S. cerevisiae MF ⁇ l gene (Brake, ibid. ; Kurjan et al., U.S. Patent No. 4,546,082).
- Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092, which are incorporated herein by reference) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454, which are incorporated herein by reference. Transformation systems for other yeasts, including Hansenula polymorpha , Schizosaccharomyces pombe, Kl uyveromyces lactis,
- Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986; Cregg, U.S. Patent No. 4,882,279; and Stroman et al., U.S. Patent No. 4,879,231.
- fungal cells are also suitable as host cells.
- Aspergill us cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349, which is incorporated herein by reference.
- Methods for transforming Acre-Tioniu-T. chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228, which is incorporated herein by reference.
- Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533, which is incorporated herein by reference.
- Cultured mammalian cells are also preferred hosts within the present invention.
- Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14.:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 2:603, 1981: Graham and Van der Eb, Virology 5.2:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., eds., Current Protocols in Molecular Biology.
- CRL 1650 COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 3_6:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978, which are incorporated herein by reference) and the adenovirus major late promoter.
- Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
- a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like.
- Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
- a preferred a plifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
- Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
- hygromycin resistance multi-drug resistance
- puromycin acetyltransferase can also be used.
- eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign proteins therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222; Bang et al., U.S. Patent No. 4,775,624; and WIPO publication WO 94/06463, which are incorporated herein by reference.
- the use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.
- Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
- suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
- the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
- Transgenic animal technology may also be employed to produce TPO. It is preferred to produce the protein 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 well characterized biochemically. Furthermore, the major milk proteins are present in milk at high concentrations (from about 1 to 15 g/1) .
- mice and rats can be used (and are preferred at the proof-of-concept stage)
- 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 WIPO Publication WO 88/00239 for a comparison of factors influencing the choice of host species.
- Milk protein genes include those genes encoding caseins (see U.S. Patent No. 5,304,489, incorporated herein by reference) , beta-lactoglobulin, ⁇ -lactalbumin, and whey acidic protein.
- the beta-lactoglobulin (BLG) promoter is preferred.
- 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.
- Techniques for introducing foreign DNA sequences into mammals and their germ cells were originally developed in the mouse. See, e.g., Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380- 7384, 1980; Gordon and Ruddle, Science 214: 1244-1246, 1981; Palmiter and Brinster, Cell 41: 343-345, 1985; Brinster et al., Proc. Natl. Acad. Sci.
- Production in transgenic plants may also be employed.
- Expression may be generalized or directed to a particular organ, such as a tuber. See, Hiatt, Nature 344:469-479, 1990; Edelbaum et al., J. Interferon Res. 12:449-453, 1992; Sijmons et al., Bio/Technology 8_:217- 221, 1990; and European Patent Office Publication EP 255,378.
- low molecular weight TPO is selectively recovered from cell culture media using a combination of ultrafiltration, affinity chromatography and ion exchange chromatography. Additional purification procedures may be employed, such as hydrophobic interaction chromatography.
- conditioned medium from cells expressing recombinant TPO which contains a mixture of TPO species from about 17.5 kD to about 70 kD, is concentrated. Suitable means of concentration include binding to a dye resin and ultrafiltration, the latter being preferred. The degree of concentration will be determined by such factors as initial volume and downstream processing capacity. In general, approximately 20-fold concentration is preferred, with a practical upper limit of about 50- fold in most cases.
- the concentrated medium is then combined with an immobilized MPL receptor polypeptide comprising at least the ligand-binding domain of the receptor.
- the ligand binding domain of the mouse MPL receptor is contained within the extracellular portion of the protein (residues 27 to 480 of SEQ ID NO: 9), with residues 293-297, 358- 361, and 398-419 believed to be of particular importance for ligand binding.
- concentrated medium is adjusted to 0.5 M NaCl and a slightly alkaline pH (preferably about pH 8.0), and the column is equilibrated with a similar buffer. The medium is then applied to the column, and bound TPO polypeptide is eluted with 3 M KSCN, pH 8.0, or 3 M KSCN, 1.0 M NaCl in 0.1 M borate, pH 10.0.
- Material eluted from the immobilized receptor is then dialyzed to remove salt, such as in 3-4 changes of 20 mM Tris pH 8.5.
- the dialyzed sample is then fractionated 2.3 on a strong anion exhange medium.
- Suitable anion exchange media for use in this procedure include polymeric (e.g., agarose, dextran or polystyrene) beads derivatized with quaternary amino groups, used in a fast pressure liquid chromatography format.
- a preferred anion exchanger is Mono-Q Sepharose (available from Pharmacia Biotech, Piscataway, NJ) .
- Bound TPO is eluted from the anion exchange medium by applying a salt gradient. When using Mono-Q Sepharose, the 18.5 kD species typically elutes at a salt concentration of approximately 0.15 M NaCl.
- a truncated TPO DNA is expressed in an engineered host cell.
- the resulting TPO polypeptide is secreted into the culture media, the media and cells are separated, and the TPO polypeptide is selectively recovered from the media.
- the TPO polypeptide can be recovered by a combination of affinity purification and other techniques as disclosed above.
- the TPO of the present invention can be used therapeutically wherever it is desirable to increase proliferation of cells in the bone marrow, such as in the treatment of cytopenia, such as that induced by aplastic anemia, myelodisplastic syndromes, chemotherapy or congenital cytopenias.
- the proteins are also useful for increasing platelet production, such as in the treatment of thrombocytopenia.
- Thrombocytopenia is associated with a diverse group of diseases and clinical situations that may act alone or in concert to produce the condition. Lowered platelet counts can result from, for example, defects in platelet production, abnormal platelet distribution, dilutional losses due to massive transfusions, or abnormal destruction of platelets.
- chemotherapeutic drugs used in cancer therapy may suppress development of platelet progenitor cells in the bone marrow, and the resulting thrombocytopenia limits the chemotherapy and may necessitate transfusions.
- certain malignancies can impair platelet production and platelet distribution.
- Radiation therapy used to kill malignant cells also kills platelet progenitor cells.
- Thrombocytopenia may also arise from various platelet autoimmune disorders induced by drugs, neonatal alloimmunity or platelet transfusion alloimmunity.
- the proteins of the present invention can reduce or eliminate the need for transfusions, thereby reducing the incidence of platelet alloimmunity.
- Abnormal destruction of platelets can result from: (1) increased platelet consumption in vascular grafts or traumatized tissue; or (2) immune mechanisms associated with, for example, drug-induced thrombocytopenia, idiopathic thrombocytopenic purpura (ITP) , autoimmune diseases, he atologic disorders such as leukemia and lymphoma, or metastatic cancers involving bone marrow.
- ITP idiopathic thrombocytopenic purpura
- Other indications for the proteins of the present invention include aplastic anemia and drug-induced marrow suppression resulting from, for example, chemotherapy or treatment of HIV infection with AZT.
- TPO Thrombocytopenia is manifested as increased bleeding, such as mucosal bleedings from the nasal-oral area or the gastrointestinal tract, as well as oozing from wounds, ulcers or injection sites.
- TPO is formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours.
- pharmaceutical formulations will include TPO in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like.
- Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
- TPO can be combined with other cytokines, 2.S particularly early-acting cytokines such as stem cell factor, IL-3, IL-6, IL-11 or GM-CSF. When utilizing such a combination therapy, the cytokines may be combined in a single formulation or may be administered in separate formulations. Methods of formulation are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences. Gennaro, ed. , Mack Publishing Co., Easton PA, 1990, which is incorporated herein by reference.
- Therapeutic doses of the TPO of the present invention will generally be in the range of 0.1 to 100 ⁇ g/kg of patient weight per day, preferably 0.5-50 ⁇ g/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. In certain cases, such as when treating patients showing increased sensitivity or requiring prolonged treatment, doses in the range of 0.1- 20 ⁇ g/kg per day will be indicated. Determination of dose is within the level of ordinary skill in the art. TPO will commonly be administered over a period of up to 28 days following chemotherapy or bone-marrow transplant or until a platelet count of >20,000/mm 3 , preferably >50,000/mm 3 , is achieved.
- TPO will be administered over one week or less, often over a period of one to three days.
- a therapeutically effective amount of TPO is an amount sufficient to produce a clinically significant increase in the proliferation and/or differentiation of lymphoid or myeloid progenitor cells, which will be manifested as an increase in circulating levels of mature cells (e.g. platelets or neutrophils) .
- Treatment of platelet disorders will thus be continued until a platelet count of at least 20,000/mm 3 , preferably 50,000/mm 3 , is reached.
- TPO can also be administered in combination with other cytokines such as IL-3, -6 and -11; stem cell factor; erythropoietin; G-CSF and GM-CSF.
- EPO EPO
- GM-CSF 5-15 ⁇ g/kg
- IL-3 1-5 ⁇ g/kg
- G-CSF 1-25 ⁇ g/kg.
- Combination therapy with EPO is indicated in anemic patients with low EPO levels, wherein EPO is administered in an amount sufficient to increase erythropoiesis. Increased erythropoiesis is manifested as a subsequent increase in he atocrit.
- TPO is also a valuable tool for the in vi tro study of the differentiation and development of hematopoietic cells, such as for elucidating the mechanisms of cell differentiation and for determining the lineages of mature cells, and may also find utility as a proliferative agent in cell culture.
- TPO can also be used ex vivo, such as in autologous marrow culture. Briefly, bone marrow is removed from a patient prior to chemotherapy and treated with TPO, optionally in combination with one or more other cytokines. The treated marrow is then returned to the patient after chemotherapy to speed the recovery of the marrow.
- TPO can be used for the ex vi vo expansion of marrow or peripheral blood progenitor (PBPC) cells.
- PBPC peripheral blood progenitor
- marrow Prior to chemotherapy treatment, marrow can be stimulated with stem cell factor (SCF) or G-CSF to release early progenitor cells into peripheral circulation.
- SCF stem cell factor
- G-CSF G-CSF
- TPO T-cell activator-like effector
- cytokines including but not limited to SCF, G-CSF, IL-3, GM-CSF, IL-6 or IL-11, to differentiate and proliferate into high-density megakaryocyte cultures, which can then be returned to the patient following high-dose chemotherapy.
- Plasmid pZGmpl-1081 (deposited under the terms of the Budapest Treaty with American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD on Fegruary 14, 1994 as an E. coli DH5 ⁇ transformant and assigned accession number 69566) was digested with Eco RI and Not I, and the TPO DNA segment was recovered. This DNA was inserted into Eco RI-digested, alkaline phosphatase-treated plasmid Zem229R with a Not I/Eco RI linker. (Zem229R was deposited under the terms of the Budapest Treaty with American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD on September 23, 1993 as an E.
- coli HB101 transformant and assigned accession number 69447.
- the resulting plasmid designated mpl.229R, was transfected into BHK 570 cells (ATCC CRL 10314) .
- the transfectants were grown in 10-layer cell factories (Nunc, Inc. ; obtained from VWR Scientific, Seattle, WA) in serum-free medium and selected 1 ⁇ M methotrexate. Sixteen liters of conditioned culture medium was collected.
- TPO was purified from the conditioned medium by affinity chromatography on immobilized MPL receptor. Eighty mg of purified mouse MPL receptor extracellular domain (see Example 7) was immobilized on 8 ml of Affi- Prep 10 acrylic polymer support (Bio-Rad Laboratories, Inc.) using procedures specified by the manufacturer. The receptor-support matrix was packed into an 8 ml column.
- the concentrated media was cycled over the affinity column for several hours or overnight in the cold.
- the column was eluted with 3 M KSCN (Fluka Chemical Corp. , Ronkonkoma, NY) . Eluate fractions were pooled and dialyzed against 20 mM Tris pH 8.5.
- the dialyzed protein was applied to a Mono-Q
- Sepharose column (Pharmacia Biotech, Piscataway, NJ) at pH 8.5. Bound TPO was eluted from the column with a NaCl gradient (0 to 0.5 M) . Peak fractions were identified by monitoring absorbance at 220 nm and "western" blotting.
- TPO activity was determined by assaying mitogenic activity on a TPO-dependent cell line.
- a BHK 570 cell line transfected with the mouse TPO expression vector pZGmpl-1081 was grown in serum-free medium. Conditioned culture medium was collected, and an asymptotic mitogenic activity curve was generated using this standard solution.
- the target cells were BaF3/MPLRl.l cells (IL-3-dependent cells expressing a stably transfected Type I mouse MPL receptor; deposited September 28, 1994 under the terms of the Budapest Treaty with American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD and assigned accession number CRL 11723) .
- the point of 1/2 maximal activity was assigned the value of 50 U/ml.
- the original standard solution was calculated to contain 26,600 U/ml mouse TPO.
- a culture supernatant or purified protein preparation was diluted in RPMI 1640 medium supplemented with 57 ⁇ M 2-mercaptoethanol, 2 mM L- glutamine, 1 mM sodium pyruvate, PSN antibiotic mixture, 10 mM HEPES and 10% heat inactivated fetal bovine serum, generally using 8-24 dilutions. Briefly, 100 ⁇ l of diluted test sample or standard sample and 100 ⁇ l BaF3 cells (final cell number added about 10,000 cells/well) were combined in wells of a 96 well plate.
- each well of each plate were transferred to a filter/plate using a Packard apparatus.
- the filters were washed 8 times with water, and the filters were dried and counted. Units of TPO activity in each sample well were determined by comparison to the standard curve.
- the activity of the protein was determined to be 14 x 10 6 U/ml. Protein concentration was 30 ⁇ g/ml.
- Solvent A 2% CH 3 CN / 0.01% TFA/ H20.
- Solvent B 10% H2O / 90% CH 3 CN / 0.09% TFA.
- Wavelength 215 nm; Flow 0.5 ml/minute.
- Sequential digestion of the sample was performed with peptide-N-glycosidase F (PNGase F) , sialidase, and O- glycosidase to determine if N- or/and O-linked glycosylation were present. Deglycosylation was carried out in three steps.
- PNGase F (purchased from Oxford Glycosystems, Rosedale, NY at a concentration of 150-200 U/ml) cleaves the beta aspartyl-glycosylamine bond between asparagine and the innermost N-acetylglucosamine of the glycan.
- 1 ug of a mouse TPO known to contain N-linked sugars and migrate at approximately 70 kDa on a SDS PAGE reducing gel 3 ⁇ was used. After 18 hours, 2 X 5 ⁇ l of the PNGase F digested material was removed and stored at -20°C for gel analysis.
- sialidase (purchased from Oxford Glycosytems at a concentration of 5 mU per ⁇ l) was added to the remainder of the PNGase F-digested material, and the mixture and incubated for two hours at 37°C. After the incubation was complete, two 5 ⁇ l aliquots of the digest were stored at -20°C for gel analysis.
- Sialidase is an exoglycosidase that specifically cleaves the terminal neuraminic acid. This reaction is required prior to cleaving 0-linked sugars in mammalian proteins.
- O-glycosidase (Oxford Glycosystems; 300 mU/ml) was added to the remainder of the digested sample and then incubated for 18 hours at 37°C.
- O-glycosidase is specific for liberating GalBi ⁇ 3GalNAc from serine or threonine.
- Mass spectral analysis was performed on 28 ⁇ g of the polypeptide in three samples: with O-linked sugars present; after removal of sialic acid; and after sequential deglycosylation. The samples were all desalted prior to analysis and reconstituted in 100 ⁇ l of a 1:1 ratio of 0.1% formic acid/H2 ⁇ and 0.08% formic acid in ethanol/propanol (5:2) . Electrospray mass spectra were recorded on a Sciex (Thornhill, Ontario) API III triple quadrupole mass spectrometer fitted with an articulated ionspray plenum and an atmospheric pressure ionization source.
- the mass spectrometer was tuned and calibrated using a mixture of polypropylene glycols (PPG) 425, 1,000, and 2,000 (3.3 x 10 ⁇ 5 M, 1 X 10 -4 M, and 2 X 10" 4 M, respectively), in 50/50/0.1 H2O/methanol/formic acid (v/v/v) , 1 mM NH4OAC.
- Normal scan ESMS were recorded at instrument conditions sufficient to resolve isomers of the PPG/NH4 + doubly charged ion at m/z 520 (85% valley definition) .
- Each sample was infused at 5 ⁇ l per minute with up to 81 scans averaged.
- the mass spectrometer was scanned over a range of m/z 1200-2400, with a dwell time of 2 mSec and a step size of 0.1 amu.
- the orvice potential was set at 150 V.
- Reconstructed ion chromatogram mass spectra of the fully deglycosylated sample showed a heterogeneous C- terminus, starting at serine residue 208 and ending at asparagine residue 216.
- the masses found take into consideration that 4 amu are subtracted from the predicted mass due to disulfide bond formation and 1 amu is subtracted from the final MH + mass shown in the MacBiospec sequence analysis.
- each of the O-linked carbohydrate chains on the sample had one of the following compositions, with the addition of up to six sialic acids on the N-terminal sugar of each chain: 5 HexNAc, 5 Hexose; 4 HexNAc, 4 Hexose; 3 HexNAc, 3 Hexose.
- TPO 711 Recombinant TPO, prepared as disclosed in Example 1 (TPO 711) , was tested for thrombopoietic activity in mice.
- TPO 711 Female Balb/c mice (obtained from Jackson
- mice Five treatment groups and one control group of four mice each.
- 50 ⁇ l of blood was collected by retro- orbital bleed in a Microtainer tube (Becton Dickinson,
- Recombinant TPO was administered by intraperitoneal injection on days 1 through 10.
- Group I received vehicle (prepared by combining 2 ml 1 M Tris pH 8.0 stock, 98 ml USP water for injection, 250 mg rabbit serum albumin and 0.93 g NaCl) only.
- Group II received 20 kU/day recombinant TPO from cells expressing a full-length mouse TPO cDNA (designated TPO 525) . This primary TPO species in this preparation had apparent molecular weights of approximately 30 and 70 kD.
- Groups III-VI received 5, 10, 20 or 50 kU/day, respectively, of recombinant TPO 711. All mice were bled and blood counts were performed on days 3, 7, 10 and 14.
- TPO 711 results of the study are presented in Table 5. Data for days 3, 7, 10 and 14 are shown as percent of basal (day 0) platelet counts. TPO 711 at 50 kU/day increased platelet levels 2.7 fold on day 10, similar to the peak response seen with TPO 525 on day 7. These results indicate that the truncated TPO 711 is at least as active as TPO 525. A dose-response relationship was seen for TPO 711 on days 10 and 14.
- vehicle prepared by combining 2 ml 1 M Tris pH 8.0 stock, 98 ml USP water for injection, 250 mg rabbit serum albumin and 0.93 g NaCl
- Group III received twice daily i.p. injections of 25 kU TPO (50 kU/day) .
- mice Female Balb/c mice weighing about eighteen grams each (Jackson Laboratories, Bar Harbor, ME) were divided into seven groups as shown in Table 6. Groups I-V received the indicated dose (determined as disclosed in Example 1) administered as two equal intraperitoneal injections per day. Group VI received a single, daily i.p. injection. Group VII received a single, daily sub ⁇ cutaneous injection.
- a vector was constructed for expression of a TPO polypeptide ending at amino acid residue 193 of SEQ ID NO:4.
- the human TPO DNA sequence was mutagenized by PCR to introduce a stop codon and an EcoRI site following the codon for amino acid 193.
- Ten ng of template DNA was combined with 5 ⁇ l of 2 mM dNTPs, 5 ⁇ l lOx Tag buffer (Boehringer Mannheim, Indianapolis, IN), 0.2 ⁇ l Taq DNA polymerase (Boehringer Mannheim) , 40 pmole of each primer ZC8045 (SEQ ID N0:5) and ZC7878 (SEQ ID N0:6), and H 2 0 to 50 ⁇ l.
- the mixture was incubated for 30 cycles of 95°C, 1 minute; 50°C, 2 minutes; and 72°C, 1 minute, with a final ten minute incubation at 72°C.
- DNA was isolated from the reaction mixture and digested with PstI and Eco RI, and a 204 bp fragment encoding amino acid residues 127-193 of J7 SEQ ID NO:4 was recovered by electrophoresis and centrifugal extraction from a gel slice.
- PCR product was ligated with an EcoRI-PstI fragment encoding a modified human t-PA leader and amino acid residues 22-126 of SEQ ID NO:4, and Zem229R (deposited under the terms of the Budapest Treaty with American Type
- E. coli HB101 transformant September 28, 1993 as an E. coli HB101 transformant and assigned Accession Number 69447) that had been digested with EcoRI and treated with alkaline phosphatase.
- the ligated DNA was used to transform competent E. coli DHlObTM cells (GIBCO BRL, Gaithersburg, MD) .
- the plasmid was designated TPO202.229R.
- BHK cells were transfected with TPO202.229R using a 3:1 liposome formulation of 2,3-dioleyloxy-N- [2(sperminecarboxyamido)ethyl]-N,N-dimethy1-1- propanaminiumtrifluoroacetate and dioleoly- phosphatidylethanolamine in water (LipofectamineTM reagent, GIBCO) . Transfectants were selected in 500 nM methotrexate (MTX) . Pooled cells produced 13,110 U/ml TPO. After amplification in 5 ⁇ M MTX, pooled cells produced 20,850 U/ml/day TPO.
- MTX methotrexate
- the 646 bp EcoRI insert was removed from TPO202.229R and ligated to the vector pDX (disclosed in U.S. Patent No. 4,959,318) that had been linearized by digestion with EcoRI and treated with alkaline phosphatase.
- the resulting vector designated TPO202.pDX, was cotransfected into BHK 570 cells with Zem229R. Cells amplified in 500 nM MTX produced 17,000 U/ml/day TPO.
- ZC6091 (SEQ ID NO:7) containing a Not I restriction site.
- First strand cDNA synthesis was initiated by the addition of 8 ⁇ l of 250 mM Tris-HCl, pH 8.3, 375 mM KC1, 15 mM MgCl 2 (5x SUPERSCRIPTTM buffer; GIBCO BRL) , 4 ⁇ l of 100 mM dithiothreitol and 3 ⁇ l of a deoxynucleotide triphosphate solution containing 10 mM each of dATP, dGTP, dTTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) to the RNA-pri er mixture.
- the reaction mixture was incubated at 45° C for 4 minutes followed by the addition of 10 ⁇ l of 200 U/ ⁇ l RNase H" reverse transcriptase (GIBCO BRL, Gaithersburg, MD) .
- the efficiency of the first strand synthesis was analyzed in a parallel reaction by the addition of 10 ⁇ Ci of 32 P- ⁇ dCTP to a 10 ⁇ l aliquot of the reaction mixture to label the reaction for analysis.
- the reactions were incubated at 45° C for 1 hour followed by an incubation at 50° C for 15 minutes.
- Unincorporated 32 P- ⁇ dCTP in the labeled reaction was removed by chromatography on a 400 pore size gel filtration column (CHROMA SPIN + TE-400TM; Clontech Laboratories Inc., Palo Alto, CA) .
- Unincorporated nucleotides in the unlabeled first strand reaction were removed by twice precipitating the cDNA in the presence of 8 ⁇ g of glycogen carrier, 2.5 M ammonium acetate and 2.5 volume ethanol.
- the unlabeled cDNA was resuspended in 50 ⁇ l water for use in second strand synthesis.
- the length of the labeled first strand cDNA was determined by agarose gel electrophoresis.
- Second strand synthesis was performed on first strand cDNA under conditions that promoted first strand priming of second strand synthesis resulting in DNA hairpin formation.
- the reaction mixture was assembled at room temperature and consisted of 50 ⁇ l of the unlabeled first strand cDNA, 16.5 ⁇ l water, 20 ⁇ l of 5x polymerase I buffer (100 mM Tris: HC1, pH 7.4, 500 mM KC1, 25 mM MgCl2, 50 mM (NH )2S ⁇ 4), 1 ⁇ l of 100 mM dithiothreitol, 2 ⁇ l of a solution containing 10 mM of each deoxynucleotide triphosphate, 3 ⁇ l of 5 mM /3-NAD, 15 ⁇ l of 3 U/ ⁇ l E.
- Unincorporated 32 P- ⁇ dCTP in the labeled reaction was removed by chromatography through a 400 pore size gel filtration column (Clontech Laboratories, Inc.) before analysis by agarose gel electrophoresis.
- the unlabeled reaction was terminated by two extractions with phenol/chloroform and a chloroform extraction followed by ethanol precipitation in the presence of 2.5 M ammonium acetate.
- the single-stranded DNA of the hairpin structure was cleaved using mung bean nuclease.
- the reaction mixture contained 100 ⁇ l of second strand cDNA, 20 ⁇ l of lOx mung bean nuclease buffer (Stratagene Cloning Systems, La Jolla, CA) , 16 ⁇ l of 100 mM dithiothreitol, 51.5 ⁇ l of water and 12.5 ⁇ l of a 1:10 dilution of mung bean nuclease (Promega Corp.; final concentration 10.5 U/ ⁇ l) in mung bean nuclease dilution buffer.
- the reaction was incubated at 37° C for 15 minutes.
- the reaction was terminated by the addition of 20 ⁇ l of 1 M Tris-HCl, pH 8.0 followed by sequential phenol/chloroform and chloroform extractions as described above. Following the extractions, the DNA was precipitated in ethanol and resuspended in water.
- the resuspended cDNA was blunt-ended with T4 DNA polymerase.
- the cDNA which was resuspended in 190 ⁇ l of water, was mixed with 50 ⁇ l 5x T4 DNA polymerase buffer (250 mM Tris-HCl, pH 8.0, 250 mM KC1, 25 mM MgCl2) , 3 ⁇ l 0.1 M dithiothreitol, 3 ⁇ l of a solution containing 10 mM of each deoxynucleotide triphosphate and 4 ⁇ l of 1 U/ ⁇ l T4 DNA polymerase (Boehringer Mannheim Corp., Indianapolis, IN) .
- the reaction was terminated by the addition of 10 ⁇ l of 0.5 M EDTA followed by serial phenol/chloroform and chloroform extractions as described above.
- the DNA was chromatographed through a 400 pore size gel filtration column (Clontech Laboratories Inc.) to remove trace levels of protein and to remove short cDNAs less than - 00 bp in length.
- the DNA was ethanol precipitated in the presence of 12 ⁇ g glycogen carrier and 2.5 M ammonium acetate and was resuspended in 10 ⁇ l of water.
- cDNA was estimated to be -2 ⁇ g from a starting mRNA template of 12.5 ⁇ g.
- Eco RI adapters were ligated onto the 5' ends of the cDNA to enable cloning into a lambda phage vector.
- a 10 ⁇ l aliquot of cDNA ( ⁇ 2 ⁇ g) and 10 ⁇ l of 65 pmole/ ⁇ l of Eco RI adapter (Pharmacia LKB Biotechnology Inc.) were mixed with 2.5 ⁇ l lOx ligase buffer (Promega Corp.), 1 ⁇ l of 10 mM ATP and 2 ⁇ l of 15 U/ ⁇ l T4 DNA ligase (Promega Corp.).
- the reaction was incubated overnight ( _ 18 hours) at a temperature gradient of 0° C to 18° C. The reaction was further incubated overnight at 12° C. The reaction was terminated by the addition of 75 ⁇ l of water and 10 ⁇ l of 3 M Na acetate, followed by incubation at 65° C for 30 minutes. After incubation, the cDNA was extracted with phenol/chloroform and chloroform as described above and precipitated in the presence of 2.5 M ammonium acetate and 1.2 volume of isopropanol. Following centrifugation, the cDNA pellet was washed with 70% ethanol, air dried and resuspended in 89 ⁇ l water.
- the cDNA was digested with Not I, resulting in a cDNA having 5' Eco RI and 3' Not I cohesive ends.
- the Not I restriction site at the 3' end of the cDNA had been previously introduced through primer ZG6091 (SEQ ID NO:7) .
- Restriction enzyme digestion was carried out in a reaction mixture containing 89 ⁇ l of cDNA described above, 10 ⁇ l of 6 mM Tris-HCl, 6 mM MgCl2 150 mM NaCl, 1 mM DTT (lOx D buffer; Promega Corp., Madison, WI) and 1 ⁇ l of 12 U/ ⁇ l Not I (Promega Corp.). Digestion was carried out at 37° C for 1 hour. The reaction was terminated by serial phenol/chloroform and chloroform extractions.
- the cDNA was ethanol precipitated, washed with 70% ethanol, air dried and resuspended in 20 ⁇ l of lx gel loading buffer (10 mM Tris- HCl, pH 8.0, 1 mM EDTA, 5% glycerol and 0.125% bromphenol blue) .
- the resuspended cDNA was heated to 65°C for 5 minutes, cooled on ice and electrophoresed on a 0.8% low melt agarose gel (SEA PLAQUE GTGTM low melt agarose; FMC Corp.). Unincorporated adapters and cDNA below 1.6 kb in length were excised from the gel. The electrodes were reversed, and the cDNA was electrophoresed until concentrated near the lane origin. The area of the gel containing the concentrated cDNA was excised and placed in a microfuge tube, and the approximate volume of the gel slice was determined. An aliquot of water (300 ⁇ l) approximately three times the volume of the gel slice was added to the tube, and the agarose was melted by heating to 65° C for 15 minutes.
- SEA PLAQUE GTGTM low melt agarose FMC Corp.
- the phosphorylated cDNA was ethanol precipitated in the presence of 2.5 M ammonium acetate, washed with 70% ethanol, air dried and resuspended in 12.5 ⁇ l water. The concentration of the phosphorylated cDNA was estimated to be _ 40 fmole/ ⁇ l.
- the resulting cDNA was cloned into the lambda phage vector ⁇ ExCellTM (Pharmacia LKB Biotechnology Inc.), purchased predigested with Eco RI and Not I and dephosphorylated. Ligation of cDNA to vector was carried out in a reaction containing 2 ⁇ l of 20 fmole/ ⁇ l prepared ⁇ ExCellTM phage arms, 4 ⁇ l of water, 1 ⁇ l lOx ligase buffer (Promega Corp.), 2 ⁇ l of 40 fmole/ ⁇ l cDNA and 1 ⁇ l of 15 U/ ⁇ l T4 DNA ligase (Promega Corp.). Ligation was carried out at 4° C for 48 hours. Approximately 50% of the ligation mixture was packaged into phage using GIGAPACK®
- the resulting cDNA library contained over 1.5 x 10 7 independent recombinants with background levels of insertless phage of less than 1.5%.
- a 32 P-labeled human MPL-K receptor cDNA probe was used to isolate mouse MPL receptor cDNA from the mouse spleen cDNA phage library.
- the cDNA library was plated on SURE® strain of E. coli cells (Stratagene Cloning Systems) at a density of 40,000 to 50,000 PFU/150 mm diameter plate. Phage plaques from thirty-three plates were transferred onto nylon membranes (Hybond NTM; Amersham Corp., Arlington Heights, IL) and processed according to the directions of the manufacturer.
- the processed filters were baked for 2 hours at 80° C in a vacuum oven followed by several washes at 70° C in wash buffer (0.25 x SSC, 0.25% SDS, 1 mM EDTA) and prehybridized overnight at 65° C in hybridization solution (5x SSC, 5x Denhardt's solution, 0.1% SDS, 1 mM EDTA and 100 ⁇ g/ml heat denatured salmon sperm DNA) in a hybridization oven (model HB-2; Techne Inc., Princeton, NJ) .
- hybridization solution was discarded and replaced with fresh hybridization solution containing approximately 2 x 10 6 cpm/ml of 32 P-labeled human MPL-K cDNA prepared by the use of a commercially available labeling kit (MEGAPRIMETM kit; Amersham Corp., Arlington Heights, IL) .
- the probe was denatured at 98° C for 5 minutes before being added to the hybridization solution.
- Hybridization was at 65° C overnight.
- the filters were washed at 55° C in wash buffer (0.25 x SSC, 0.25% SDS, 1 mM EDTA) and were autoradiographed with intensifying screens for 4 days at - 70° C on XAR-5 film (Eastman Kodak Co., Rochester, NY).
- wash buffer 0.25 x SSC, 0.25% SDS, 1 mM EDTA
- intensifying screens for 4 days at - 70° C on XAR-5 film (Eastman Kodak Co., Rochester, NY).
- agar plugs were recovered from regions of the plates corresponding to primary signals and were soaked in SM (0.1 M NaCl; 50 mM Tris-HCl, pH 7.5, 0.02% gelatin) to elute phage for plaque purification. Seven plaque-purified phages were isolated that carried inserts hybridizing to the human MPL-K receptor probe.
- the phagemids contained within the ⁇ ExCellTM phage were recovered using the in vivo recombination system in accordance with the directions of the vendor.
- the identity of the cDNA inserts was confirmed by DNA sequencing.
- the isolated clones encoded a protein exhibiting a high degree of sequence identity to human MPL-P receptor and to a recently reported mouse MPL receptor (Skoda et al., EMBO J. 12: 2645-2653, 1993).
- the seven clones fell into two classes differing from each other by three clones having a deletion of sequences encoding a stretch of 60 amino acid residues near the N-terminus.
- the cDNA encoding the protein without the deletion was referred to as mouse Type I MPL receptor cDNA.
- Type II receptor cDNA lacked sequences encoding Type I receptor residues 131 to 190 of SEQ ID NO:8.
- Type I and II receptors differed from the reported mouse MPL receptor sequence (Skoda et al., ibid.) by the presence of a sequence encoding the amino acid residues Val-Arg-Thr-Ser-Pro-Ala- Gly-Glu (SEQ ID NO:9) inserted after amino acid residue 222 and by a substitution of a glycine residue for serine at position 241 (positions refer to the Type I mouse receptor) .
- Type I and II mouse MPL receptor cDNAs were subcloned into the plasmid vector pHZ-1 for expression in mammalian cells.
- Plasmid pHZ-1 is an expression vector that may be used to express protein in mammalian cells or in a frog oocyte translation system from mRNAs that have been transcribed in vi tro.
- the pHZ-1 expression unit comprises the mouse metallothionein-1 promoter, the bacteriophage T7 promoter flanked by multiple cloning banks containing unique restriction sites for insertion of coding sequences, the human growth hormone terminator and the bacteriophage T7 terminator.
- pHZ-1 contains an E.
- coli origin of replication a bacterial beta lactamase gene; a mammalian selectable marker expression unit comprising the SV40 promoter and origin, a neomycin resistance gene and the SV40 transcription terminator.
- a polymerase chain reaction employing appropriate primers was used to create an Eco RI site upstream from the translation initiation codon and a Xho I site downstream from the translation termination codon.
- the polymerase chain reaction was carried out in a mixture containing 10 ⁇ l lOx ULTMATM DNA polymerase buffer (Roche Molecular Systems, Inc., Branchburg, NJ) , 6 ⁇ l of 25 mM MgCl2, 0.2 ⁇ l of a deoxynucleotide triphosphate solution containing 10 mM each of dATP, dGTP, dTTP and dCTP (Pharmacia LKB Biotechnology Inc.), 2.5 ⁇ l of 20 pmole/ ⁇ l primer ZC6603 (SEQ ID NO:10), 2.5 ⁇ l of 20 pmole/ ⁇ l primer ZC5762 (SEQ ID NO:ll), 32.8 ⁇ l of water, 1 ⁇ l of an early log phase bacterial culture harboring either a Type I or a Type II mouse MPL receptor plasmid and 1 ⁇ l of 6 U/ ⁇ l DNA polymerase (ULTMATM polymerase; Roche Molecular Systems, Inc., Branchburg, NJ) .
- AmpliWaxTM (Roche Molecular Systems, Inc.) was employed in the reaction according to the directions of the vendor. The polymerase chain reaction was run for 25 cycles (1 minute at 95° C, 1 minute at 55° C and 3 minutes at 72° C) followed by a 10 minute incubation at 72° C. The amplified products were serially extracted with phenol/chloroform and chloroform, then ethanol precipitated in the presence of 6 ⁇ g glycogen carrier and 2.5 M ammonium acetate.
- the pellets were resuspended in 87 ⁇ l of water to which was added 10 ⁇ l of 10 x H buffer (Boehringer Mannheim, Inc.), 2 ⁇ l of 10 U/ ⁇ l Eco RI (Boehringer Mannheim, Inc.) and 1 ⁇ l of 40 U/ ⁇ l Xho I (Boehringer Mannheim, Inc.). Digestion was carried out at 37° C for 1 hour. The reaction was terminated by heating to 65° C for 15 minutes and chromatographed through a 400 pore size gel filtration column (CHROMA SPIN + TE-400TM; Clontech Laboratories Inc.).
- the isolated receptor inserts described above were ligated into Eco RI and Xho I digested and dephosphorylated pHZ-1 vector.
- the ligation reaction contained 1 ⁇ l of 50 ng/ ⁇ l prepared pHZ-1 vector, 5 ⁇ l of 5 ng/ ⁇ l cDNA insert, 2 ⁇ l of lOx ligase buffer (Promega Corp.), 11.75 ⁇ l water and 0.25 ⁇ l of 4 U/ ⁇ l T4 DNA ligase (Stratagene Cloning Systems) . Ligation was carried out at 10° C overnight.
- the ligated DNAs were transfected into E. coli (MAX EFFICIENCY DH10BTM competent cells; GIBCO BRL) in accordance with the vendor's directions.
- Type I and Type II mouse MPL and human MPL-P receptor inserts in pHZ-1 were confirmed by DNA sequencing.
- the resulting plasmids pSLmpl-8 and pSLmpl-9 carried the mouse Type II and Type I MPL receptor cDNAs, respectively.
- Plasmid pSLmpl-44 carried the human MPL-P cDNA insert.
- a mammalian expression plasmid encoding soluble mouse Type I MPL receptor (pLDmpl-53) was produced by combining DNA segments from pSLmpl-9, a mammalian expression plasmid containing the cDNA encoding full- length mouse Type I MPL receptor described above, with a DNA segment from pSLmpl-26, an expression plasmid constructed to produce the soluble mouse Type I MPL receptor in bacteria.
- a cDNA segment encoding mouse Type I MPL soluble receptor was isolated by PCR employing primers ZC6704 (SEQ ID NO:12) and ZC6703 (SEQ ID NO:13) using full-length receptor plasmid pSLmpl-9 as template.
- primers ZC6704 and ZC6703 incorporated Eco RI and Xho I restriction sites at their respective 5' ends.
- Primer ZC6703 also encoded an inframe consensus target sequence for protein kinase to enable in vi tro labeling of the purified soluble receptor with 32 P ⁇ - ATP (Li et al., Proc. Natl. Acad. Sci. U.S.A. 86: 558-562, 1989) .
- the PCR was carried out in a mixture containing 10 ⁇ l lOx ULTMATM DNA polymerase buffer (Roche Molecular Systems, Inc.), 6 ⁇ l of 25 mM MgCl2, 0.2 ⁇ l of a deoxynucleotide triphosphate solution containing 10 mM each of dATP, dGTP, dTTP and dCTP (Pharmacia LKB Biotechnology Inc.), 11 ⁇ l of 4.55 pmole/ ⁇ l primer ZC6704
- the polymerase chain reaction was run for 3 cycles (1 minute at 95° C, 1 minute at 50° C and 2 minutes at 72° C) followed by 11 cycles at increased hybridization stringency (1 minute at 95° C, 30 seconds at 55° C and 2 minutes at 72° C) followed by a 10 minute incubation at 72° C.
- the amplified product was serially extracted with phenol/chloroform and chloroform followed by chromatography through a 400 pore size gel filtration column (Clontech Laboratories, Inc.).
- the PCR product was ethanol precipitated in the presence of 20 ⁇ g glycogen carrier and 2.5 M ammonium acetate.
- the pellet was resuspended in 32 ⁇ l of water.
- 2 ⁇ l lOx H buffer Boehringer Mannheim, Inc.
- 1 ⁇ l of 10 U/ ⁇ l Eco RI Boehringer Mannheim, Inc.
- 1 ⁇ l of 40 U/ ⁇ l Xho I Boehringer Mannheim, Inc.
- Digestion was carried out at 37° C for 1 hour. Digestion was terminated by heating to 65° C for 15 minutes, and DNA was purified on a 0.7% low-melt agarose gel. Fragment recovery from low-melt agarose was done by digestion of the gel matrix with J-agarase I (New England Biolabs) .
- the resulting PCR product encoded the N-terminal extracellular domain of mouse Type I MPL receptor (residues 27 to 480 of SEQ ID N0:8). In the absence of the putative receptor trans-membrane domain (residues 483 to 504 of SEQ ID NO:8) the expressed protein is expected to be secreted in the presence of a suitable signal peptide.
- a mouse Type II soluble MPL receptor encoding cDNA was obtained using the PCR conditions described above except that pSLmpl-8 was used as template. The validity of both receptor fragments was confirmed by DNA sequencing.
- Plasmid pOmpA2-5 is a modification of pOmpA2 (Ghrayab et al., EMBO J. 1: 2437- 2442, 1984), a bacterial expression vector designed to target the recombinant protein to the periplasmic space.
- pOmpA2-5 was constructed by replacement of a 13 bp sequence between the Eco RI and Bam HI sites of pOmpA2 with a synthetic 42 bp sequence.
- the sequence was created by annealing of two 42 nucleotide complementary oligonucleotides (ZC6707, SEQ ID NO:14; ZC 6706, SEQ ID NO:15), which when base paired formed Eco RI and Bam HI cohesive ends, facilitating directional cloning into Eco RI and Bam HI digested pOmpA2.
- Xho I site inframed with respect to a bacterial leader sequence and to the mouse MPL soluble receptor encoding cDNAs described above, as well as an inframe tract of 6 histidine codons located 3' of the Xho I site to enable the recombinant protein to be purified by metal chelation affinity chromatography (Houchuli et al., Bio/Technol. j5: 1321-1325, 1988) . Following the sequence encoding the histidine tract was an inframe termination codon. The validity of the pOmpA2-5, pSLmpl-26 and pSLmpl-27 was confirmed by DNA sequencing.
- pLDmpl-53 a mammalian expression plasmid producing soluble mouse Type I MPL receptor, was constructed by combining DNA segments from pSLmpl-9 and pSLmpl-26 into expression vector pHZ-200 (pHZ-1 in which a dihydrofolate reductase sequence was substituted for the neomycin resistance gene) .
- the 1164 bp Eco RI/Bam HI cDNA fragment from pSLmpl-9 replaced the mammalian signal sequence deleted during the construction of bacterial *- 9 expression plasmid pSLmpl-26.
- the 416 bp Bam HI fragment from pSLmpl-26 supplied the coding sequence for the carboxy-terminal portion of the soluble MPL receptor, the kinase labeling domain, the poly-histidine tract and the translation terminator.
- the two fragments were gel purified and cloned into the Eco RI/Bam HI sites of pBluescript® KS+ (Stratagene Cloning Systems) to yield plasmid pBS8.76LD-5.
- pLDmpl-53 plasmid Twenty micrograms of purified pLDmpl-53 plasmid was transfected into BHK 570 cells using the calcium phosphate precipitation method. After 5 hours, the cells were shocked with 15% glycerol for 3 minutes to facilitate uptake of DNA. Fresh growth media was added overnight. The following day the cells were split at various dilutions, and selection media containing 1 ⁇ M methotrexate was added. After approximately two weeks, discrete, methotrexate-resistant colonies were visible. Resistant colonies were either pooled or maintained as distinct clones. Spent media from the pooled colonies was immediately tested for presence of soluble MPL receptor protein.
- Soluble MPL receptor protein was isolated through the interaction of the poly-histidine tract present on the carboxy-terminal of the protein with a metal chelation resin containing immobilized Ni 2+ (HIS- BINDTM; Novagen, Madison, WI) . Serum-free spent culture so media from the pLDmpl-53 pool was passed over the resin, and bound protein was eluted with 1 M imidazole. SDS-PAGE analysis revealed a single band at -67 kDa. This protein was subjected to N-terminal amino acid analysis and confirmed to be mouse MPL receptor.
- HIS- BINDTM immobilized Ni 2+
- AAG ATT CAG GGG AGA GGC CCC ATA CAG GGA GCC ACT TCA GTT
- AAC GAG CTC CCA AAC AGG ACT TCT GGA TTG TTG GAG ACA AAC TTC ACT 624 Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr 195 200 205
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Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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AU41631/96A AU4163196A (en) | 1994-11-30 | 1995-11-15 | Low molecular weight thrombopoietin |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US34699994A | 1994-11-30 | 1994-11-30 | |
US08/346,999 | 1994-11-30 |
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WO1996017062A1 true WO1996017062A1 (en) | 1996-06-06 |
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PCT/US1995/014929 WO1996017062A1 (en) | 1994-11-30 | 1995-11-15 | Low molecular weight thrombopoietin |
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WO (1) | WO1996017062A1 (en) |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996025498A2 (en) * | 1995-02-15 | 1996-08-22 | Amgen Inc. | Mpl ligand analogs |
WO1996040750A1 (en) * | 1995-06-07 | 1996-12-19 | Glaxo Group Limited | Peptides and compounds that bind to a thrombopoietin receptor |
US5756083A (en) * | 1995-02-15 | 1998-05-26 | Amgen Inc. | Mpl ligand analogs |
US5869451A (en) * | 1995-06-07 | 1999-02-09 | Glaxo Group Limited | Peptides and compounds that bind to a receptor |
US5989538A (en) * | 1995-02-15 | 1999-11-23 | Amgen Inc. | Mpl ligand analogs |
WO2000000612A1 (en) * | 1998-06-30 | 2000-01-06 | Daewoong Pharmaceutical Co., Ltd. | A novel human thrombopoietin mutein |
US6251864B1 (en) | 1995-06-07 | 2001-06-26 | Glaxo Group Limited | Peptides and compounds that bind to a receptor |
US7037491B2 (en) | 1998-06-30 | 2006-05-02 | Daewoong Pharmaceutical Co., Ltd. | Human thrombopoietin comprising glycosylation sited at residues 157 and 164 |
US7091311B2 (en) | 1996-06-07 | 2006-08-15 | Smithkline Beecham Corporation | Peptides and compounds that bind to a receptor |
US7576056B2 (en) | 2003-08-28 | 2009-08-18 | Ortho-Mcneil Pharmaceutical, Inc. | Peptides and compounds that bind to a receptor |
US7723295B2 (en) | 2003-08-28 | 2010-05-25 | Ortho-Mcneil Pharmaceutical, Inc. | Peptides and compounds that bind to a receptor |
US8067367B2 (en) | 2002-09-18 | 2011-11-29 | Janssen Pharmaceutica, N.V. | Methods of increasing platelet and hematopoietic stem cell production |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5641655A (en) * | 1994-11-30 | 1997-06-24 | Zymogenetics, Inc. | Methods for producing thrombopoietin polypeptides using a mammalian tissue plasminogen activator secretory peptide |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0675201A1 (en) * | 1994-03-31 | 1995-10-04 | Amgen Inc. | Compositions and methods for stimulating megakaryocyte growth and differentiation |
-
1995
- 1995-11-15 AU AU41631/96A patent/AU4163196A/en not_active Abandoned
- 1995-11-15 WO PCT/US1995/014929 patent/WO1996017062A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0675201A1 (en) * | 1994-03-31 | 1995-10-04 | Amgen Inc. | Compositions and methods for stimulating megakaryocyte growth and differentiation |
Non-Patent Citations (2)
Title |
---|
KATO, TAKASHI ET AL: "Purification and characterization of thrombopoietin", J. BIOCHEM. (TOKYO) (1995), 118(1), 229-36 CODEN: JOBIAO;ISSN: 0021-924X * |
SI LOK ET AL.: "Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo", NATURE, vol. 369, no. 6481, 16 June 1994 (1994-06-16), LONDON GB, pages 565 - 568 * |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5989538A (en) * | 1995-02-15 | 1999-11-23 | Amgen Inc. | Mpl ligand analogs |
WO1996025498A2 (en) * | 1995-02-15 | 1996-08-22 | Amgen Inc. | Mpl ligand analogs |
WO1996025498A3 (en) * | 1995-02-15 | 1997-03-20 | Amgen Inc | Mpl ligand analogs |
US5756083A (en) * | 1995-02-15 | 1998-05-26 | Amgen Inc. | Mpl ligand analogs |
US6121238A (en) * | 1995-06-07 | 2000-09-19 | Glaxo Wellcome Inc. | Peptides and compounds that bind to a receptor |
US6506362B1 (en) | 1995-06-07 | 2003-01-14 | Glaxo Group Limited | Labeled compounds that bind to a thrombopoietin receptor |
US5869451A (en) * | 1995-06-07 | 1999-02-09 | Glaxo Group Limited | Peptides and compounds that bind to a receptor |
US6083913A (en) * | 1995-06-07 | 2000-07-04 | Glaxo Wellcome Inc. | Peptides and compounds that bind to a thrombopoietin receptor |
WO1996040750A1 (en) * | 1995-06-07 | 1996-12-19 | Glaxo Group Limited | Peptides and compounds that bind to a thrombopoietin receptor |
US6251864B1 (en) | 1995-06-07 | 2001-06-26 | Glaxo Group Limited | Peptides and compounds that bind to a receptor |
US6465430B1 (en) | 1995-06-07 | 2002-10-15 | Smithkline Beecham Corporation | Peptides and compounds that bind to a thrombopoietin receptor |
US7091311B2 (en) | 1996-06-07 | 2006-08-15 | Smithkline Beecham Corporation | Peptides and compounds that bind to a receptor |
US8227422B2 (en) | 1996-06-07 | 2012-07-24 | Glaxosmithkline Llc | Peptides and compounds that bind to a receptor |
WO2000000612A1 (en) * | 1998-06-30 | 2000-01-06 | Daewoong Pharmaceutical Co., Ltd. | A novel human thrombopoietin mutein |
US7037491B2 (en) | 1998-06-30 | 2006-05-02 | Daewoong Pharmaceutical Co., Ltd. | Human thrombopoietin comprising glycosylation sited at residues 157 and 164 |
US8067367B2 (en) | 2002-09-18 | 2011-11-29 | Janssen Pharmaceutica, N.V. | Methods of increasing platelet and hematopoietic stem cell production |
US8283313B2 (en) | 2002-09-18 | 2012-10-09 | Janssen Pharmaceutica, Nv | Methods of increasing platelet and hematopoietic stem cell production |
US7576056B2 (en) | 2003-08-28 | 2009-08-18 | Ortho-Mcneil Pharmaceutical, Inc. | Peptides and compounds that bind to a receptor |
US7723295B2 (en) | 2003-08-28 | 2010-05-25 | Ortho-Mcneil Pharmaceutical, Inc. | Peptides and compounds that bind to a receptor |
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