AU728881B2 - Expression vectors, cells, and methods for preparing thrombopoietin polypeptides - Google Patents
Expression vectors, cells, and methods for preparing thrombopoietin polypeptides Download PDFInfo
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Description
WO 98/06849 PCT/US97/13543 1 Description EXPRESSION VECTORS, CELLS, AND METHODS FOR PREPARING THROMBOPOIETIN POLYPEPTIDES Background of the Invention Hematopoiesis 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 the target cells.
Cytokine action results in cellular proliferation and differentiation, with a 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.
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. In general, 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.
Various cytokines have been developed as therapeutic agents. For example, erythropoietin, which stimulates the development of erythrocytes, is used in the treatment of anemia arising from renal failure. Several of the colony stimulating factors have been used in conjunction with cancer chemotherapy to speed the recovery of patients' immune systems. Interleukin-2, a-interferon and c-interferon are used in the treatment of certain cancers. An activity that stimulates megakaryocytopoiesis WO 98/06849 PCT/US97/13543 2 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. 16:201-205, 1988 and McDonald, Am.
J. Ped. Hematol. Oncol. 14:8-21, 1992). Efforts to purify and characterize this activity led to the cloning of a protein that binds to the cellular Mpl receptor and stimulates megakaryocytopoiesis and thrombocytopoiesis.
See, de Sauvage et al., Nature 369:533-538, 1994; Lok et al., Nature 369:565-568, 1994; Kaushansky et al., Nature 369:568-571, 1994; Wendling et al., Nature 369:571-574, 1994; Bartley et al., Cell 77:1117-1124, 1994; and WIPO publication WO 95/21920. This Mpl receptor-binding cytokine is termed thrombopoietin.
Analysis of amino acid sequences indicates that the mature human TPO extends from amino acid residue 1 (Ser) to amino acid residue 332 (Gly) of SEQ ID NO:2. TPO is subject to proteolysis and has been isolated in heterogeneous or degraded form (de Sauvage et al., Nature 369:533-538, 1994; Bartley et al., Cell 77:1117-1124, 1994). Molecular species as small as 25 kD have been found to be active in vitro (Bartley et al., ibid), and recombinant human TPO polypeptides of 153 (de Sauvage et al., ibid) and 174 amino acids (Bartley et al., ibid) have been reported as being active in vitro, as has the product of expression of the full-length human cDNA, which encodes a primary translation product of 353 amino acids (Bartley et al., ibid).
There remains a need in the art for methods of producing thrombopoietin in large amounts and in a costeffective manner. There is also a need for methods of producing thrombopoietin in eukaryotic microorganisms, such as yeast. There is a further need for methods for the efficient production of lower molecular weight forms of thrombopoietin, which may be less subject to proteolysis than the full-length molecule. The present WO 98/06849 PCT/US97/13543 3 invention addresses these needs and provides other, related advantages.
Summary of the Invention Within one aspect, the present invention provides expression vectors replicable in a eukaryotic host cell. The vectors comprise the following operably linked elements: a transcription promoter; a first DNA segment encoding a secretory leader; a second DNA segment encoding a thrombopoietin (TPO) polypeptide consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18 and wherein up to 35% of amino acid residues of B are individually replaced by other amino acid residues; and a transcription terminator. Within one embodiment of the invention, the expression vector is replicable in yeast. Within another embodiment, the secretory leader is a Saccharomyces cerevisiae alpha-factor secretory leader.
Within another embodiment,- B does not comprise an Arg-Arg dipeptide. Within further embodiments, residue number 4 of B is Thr or Asp; y is at least 10 and residue 10 of B is Arg or Glu; and y is at least 14 and residue 14 of B is Val or Ala. Within an additional embodiment, y is at least 14, residue 4 of B is Thr or Asp, residue 10 of B is Arg or Glu, and residue 14 of B is Val or Ala. Within other embodiments, X is a peptide bond or a single amino acid residue.
Within another aspect of the invention there is provided a cultured eukaryotic cell containing an expression vector as disclosed above, wherein the cell produces and secretes the TPO polypeptide. Within a preferred embodiment the cell is a yeast cell.
Within a third aspect of the invention, there is provided a thrombopoietin polypeptide characterized by an amino acid backbone consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from to 18, and wherein up to 35% of the residues of B are individually replaced by other amino acid residues.
Within a fourth aspect of the invention there is provided a method of making a TPO polypeptide comprising culturing a eukaryotic host cell transfected or transformed with an expression vector as disclosed above, wherein the linked first and second DNA segments are expressed by the host cell to produce the TPO polypeptide, and recovering the TPO polypeptide.
Within a fifth aspect of the invention there is disclosed a method of increasing s5 platelet number in a mammal comprising administering to the mammal a TPO polypeptide as disclosed above in combination with a pharmaceutically acceptable vehicle.
Accordingly, there is provided in a first embodiment of the present invention an expression vector replicable in a eukaryotic host cell and comprising the following operably linked elements: a transcription promoter; a first DNA segment encoding a secretory leader; a second DNA segment encoding a thrombopoietin (TPO) polypeptide consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; 25 X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18 and wherein up to 35% of said residues of B are individually replaced by other amino acid residues; and a transcription terminator.
According to a second embodiment of the present invention there is provided a cultured eukaryotic cell containing an expression vector according to the first embodiment, wherein said cell produces and secretes said TPO polypeptide.
sA According to a third embodiment of the present invention there is provided a TPO polypeptide produced by a cell according to the second embodiment.
[R:\LIBZZ]03471.doc:mrr 4a According to a fourth embodiment of the present invention there is provided a thrombopoietin polypeptide characterised by an amino acid backbone consisting of C-X- B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and wherein up to 35% of said residues of B are individually replaced by other amino acid residues.
According to a fifth embodiment of the present invention there is provided a 0o method of making a TPO polypeptide comprising: culturing a host cell transfected or transformed with an expression vector replicable in the host cell and comprising the following operably linked elements: a transcription promoter; a first DNA segment encoding a secretory leader; 1 5 a second DNA segment encoding a thrombopoietin polypeptide consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18 and wherein up to 35% of said residues of B are individually replaced by other amino acid residues; and a transcription terminator, wherein the linked first and second DNA segments are expressed by the host cell to produce the TPO polypeptide; and 25 recovering the TPO polypeptide.
According to a sixth embodiment of the present invention there is provided a TPO polypeptide made by the method according to the fifth embodiment.
According to a seventh embodiment of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a TPO polypeptide according to any one of the third, fourth or sixth embodiments together with a pharmaceutically acceptable vehicle.
According to a eighth embodiment of the present invention there is provided a method of increasing platelet number in an animal comprising administering to said AL animal a thrombopoietin polypeptide characterised by an amino acid backbone S41 consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; [R:\LIBZZ]03471.doc:mrr X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and wherein up to 35% of said residues of B are individually replaced by other amino acid residues, in combination with a pharmaceutically acceptable vehicle.
According to a ninth embodiment of the present invention there is provided a thrombopoietin polypeptide characterised by an amino acid backbone consisting of C-X- B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and wherein up to 35% of said residues of B are individually replaced by other amino acid residues when used to increase platelet number.
15 According to a tenth embodiment of the present invention there is provided a thrombopoietin polypeptide characterised by an amino acid backbone consisting of C-X- B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation °o that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and wherein up to 35% of said residues of B are individually replaced by other amino acid residues when used to increase platelet number.
According to an eleventh embodiment of the present invention there is provided use of a thrombopoietin polypeptide characterised by an amino acid backbone consisting 25 of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a S* peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and wherein up to 35% of said residues of B are individually replaced by other amino acid residues, in the manufacture of a medicament for increasing platelet number.
According to a twelfth embodiment of the present invention there is provided a method of increasing platelet number in an animal comprising administering to said animal a TPO polypeptide according to the fourth or sixth embodiment or a composition according to the seventh embodiment.
[R:\LIBZZ]03471.doc:mrr 4c According to a thirteenth embodiment of the present invention there is provided a TPO polypeptide according to the fourth or sixth embodiment or a composition according to the seventh embodiment when used to increase platelet number.
According to a fourteenth embodiment of the present invention there is provided use of a TPO polypeptide according to the fourth or sixth embodiment in the manufacture of a medicament for increasing platelet number.
According to a fifteenth embodiment of the present invention there is provided a medicament manufactured according to the eleventh or fourteenth embodiment.
These and other aspects of the invention will become evident upon reference to the following detailed description.
Detailed Description of the Invention Prior to describing the invention in more detail, certain terms used herein will be defined: The term "allelic variant" is used herein to denote an alternative form of a gene 15 is 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 sequence.
*S
oo* *oo *o *o~o *oo [R:\LIBZZ]0347 1doc:mrr WO 98/06849 PCTfUS97/13543 An "expression vector" is a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences. An expression vector 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.
Replication of expression vectors in a host organism can be autonomous or through integration into the host genome.
The term "characterized by" is used to denote the limits of a feature or element. For example, a polypeptide characterized by an amino acid backbone of a given sequence contains the recited amino acid sequence but does include additional amino acid residues. Such a polypeptide may, however, further include carbohydrate chains or other post-translational modifications.
A "secretory leader" is a polypeptide that directs and facilitates the passage of a protein through the secretory pathway of a host cell. Secretory leaders are sometimes referred to as prepro sequences. Secretory leaders 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 leader is cleaved from the mature protein during secretion in one or more cleavage events. Such secretory leaders contain processing sites that allow cleavage of the secretory leaders from the mature proteins as they pass through the secretory pathway.
A "promoter" is the portion of a gene at which RNA polymerase binds and mRNA synthesis is initiated.
WO 98/06849 PCT/US97/13543 6 "Thrombopoietin" (or "TPO") is a protein characterized by the ability to specifically bind to Mpl receptor from the same species and to stimulate platelet production in vivo. In normal test animals, TPO is able to increase platelet levels by 100% or more within 10 days after beginning daily administration. Full-length
TPO
comprises an amino-terminal cytokine domain and a carboxyl-terminal ("C-terminal") domain. Referring to SEQ ID NO:2, the cytokine domain is bounded by cysteine residues at positions 7 and 151.
The term "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 vivo stimulation of platelet production).
A representative cDNA encoding full length human thrombopoietin is shown in SEQ ID NO:1. Those skilled in the art will recognize that the DNA of SEQ ID NO:1 and the encoded amino acid sequence (SEQ ID NO:2) represent a single allele of the human TPO gene and that allelic variation is expected to exist. Allelic variants of SEQ ID NO:1 can be obtained by cloning from cells, tissues, or nucleic acids prepared from different individuals and sequencing the resulting clones.
As used herein, the term "thrombopoietin cytokine domain polypeptide" refers to this core polypeptide (residues 7-151 or SEQ ID NO:2 and corresponding regions of allelic variants of SEQ ID NO:2), which may further comprise short N-terminal residues 1-6 of SEQ ID NO:2) and/or C-terminal residue 152 of SEQ ID NO:2) extensions that do not destroy the essential biological activity of the molecule.
Considerable sequence variation is allowed within these short extensions. The C-terminal domain of human TPO extends from residue 155 (Ala) to residue 332 (Gly) of SEQ ID NO:2. This domain comprises potential O-and N-linked WO 98/06849 PCT/US97/13543 7 glycosylation sites. All or part of this domain can be deleted without complete loss of biological activity. The two domains are separated by an Arg-Arg dipeptide (residues 153 to 154 of SEQ ID NO:2). Although it has been postulated that this dipeptide is a processing site that is cleaved during maturation of TPO de Sauvage et al., ibid.), studies carried out by the assignee of the present invention indicate that significant cleavage does not occur at this site.
As used herein, the phrase "a portion of a thrombopoietin C-terminal domain" includes from one amino acid of a TPO C-terminal domain up to and including a complete TPO C-terminal domain. In general, the portion of the C-terminal domain will be a contiguous segment of a naturally occuring TPO C-terminal domain, having as its first (amino-terminal) amino acid residue the first amino acid residue of the corresponding complete TPO C-terminal domain the amino acid residue corresponding to residue 155 of SEQ ID NO:2). The portion of the Cterminal domain used within the present invention is preferably from 5 to 18 amino acid residues in length, more preferably at least 9 residues in length, most preferably from 14 to 18 residues in length.
The present invention provides improved methods for preparing thrombopoietin polypeptides, together with expression vectors and cells that are useful within the methods. The invention is based in part on the discovery that certain amino acid changes in TPO polypeptides result in increased secretion by eukaryotic host cells. Within the invention, secretion of TPO polypeptides is enhanced by deleting or replacing the Arg-Arg dipeptide that separates the cytokine domain from the C-terminal domain of the native molecule. Although the TPO polypeptides are not cleaved at this Arg-Arg dipeptide as has been postulated in the literature de Sauvage et al., ibid.), this dipeptide has been found to inhibit secretion. The present invention thus provides TPO WO 98/06849 PCTIUS9713543 8 polypeptides characterized by the elimination of the dibasic amino acid pair immediately C-terminal to the cytokine domain. These TPO polypeptides are thus characterized by the structure C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide, X is a peptide bond or a linker consisting of one or two amino acid residues, and B is derived from a portion of a thrombopoietin C-terminal domain, subject to the limitation that X, alone or with C or B, does not form a pair of basic amino acid residues.
Within the proteins of the present invention, therefore, the dibasic sequence that occurs at the junction of the cytokine and C-terminal domains of wildtype thrombopoietins residues 153 and 154 of SEQ ID NO:2) is not present. It is preferred that such dibasic sequences are not present elsewhere in the molecule as well, particularly in B.
TPO polypeptides of the present invention will most commonly comprise a naturally occuring human TPO cytokine domain amino acid sequence linked via a peptide bond to a portion of a C-terminal domain of a mammalian (preferably human) thrombopoietin. Within a preferred embodiment of the invention, B consists of from 5 to 18 contiguous residues of the C-terminal domain beginning with the amino-terminal residue of the C-terminal domain, wherein up to 35% of the amino acid residues of B may be individually replaced by other amino acid residues. The TPO cytokine domain may also include from one to about preferably no more than 10, more preferably no more than 7, amino acid substitutions. Amino acid substitutions are made in non-critical amino acid residues, that is those residues whose substitution do not materially affect the biological activity of the molecule. Methods for identifying non-critical amino acid residues are known in the art, and include alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991), WO 98/06849 PCT/US97/13543 9 wherein single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity. The effects of multiple amino acid substitutions on protein activity can be assessed as disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc. Natl.
Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display Lowman et al., Biochem. 30:10832-10837, 1991; Ladner et al., U.S.
Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988). In addition to testing the mutagenized polypeptides for biological activity, expression vectors encoding the polypeptides can be transformed into cultured cells to assay the effects of mutations on polypeptide secretion.
Within B, amino acid replacements as compared to a naturally occuring TPO sequence are designed to preserve or enhance the beneficial effect of this portion of the molecule on secretion. Preferred substitutions include the replacement of charged residues with uncharged residues. Other preferred substitutions include the replacement of valine (residue 168 of SEQ ID NO: 2) with alanine, the replacement of arginine (residue 164 of SEQ ID NO: 2) with glutamic acid, and the replacement of threonine (residue 158 of SEQ ID NO: 2) with aspartic acid. Such substitutions may be made individually or in any combination. It is preferred that not more than of the residues of B are replaced as compared to the corresponding naturally occuring sequence. Exemplary Cterminal domain sequences are shown in SEQ ID NO:4 through SEQ ID NO:7.
WO 98/06849 PCTIUS97/13543 While not wishing to be bound by theory, the Thr-Thr dipeptide may provide an attachment site for an Olinked carbohydrate chain. Thus within one embodiment of the present invention, B comprises a Thr-Thr dipeptide.
Within a particularly preferred embodiment, the N-terminal amino acid residues of B are Ala-Pro-Pro-Thr-Thr (SEQ ID NO:8).
Secretion levels can be further enhanced by the addition of an N-linked carbohydrate addition site (Asn-X- Ser/Thr). However, the presence of such sequences may lead to undesired hyperglycosylation in certain host cells Saccharomyces cerevisiae).
Those skilled in the art will recognize that the effect of certain amino acid substitutions may be hostcell-specific. It is therefore preferred to assay the effects of such substitutions in the host cell type that will be used for polypeptide production.
The expression vectors of the present invention, which are replicable in a eukaryotic host cell, comprise a transcription promoter and a transcription terminator operably linked to a first DNA segment encoding a secretory peptide and a second DNA segment encoding a TPO polypeptide as disclosed above. The second DNA segment thus encodes a TPO polypeptide consisting of C-X-B, wherein C, X, and B are as defined above. Within a preferred embodiment of the invention, B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18 and wherein up to 35%, preferably not more than 25%, of said residues of B are individually replaced by other amino acid residues.
A "DNA segment encoding a TPO polypeptide consisting of C-X-B" means that the nascent polypeptide consists of the recited elements, reading from the aminoterminal end to the carboxyl-terminal end. The term "encoding" is thus used to refer to the direct product of transcription and translation of the DNA segment. It will be understood by those skilled in the art that such a WO 98/06849 PCT/US97/13543 11 polypeptide may undergo post-translational processing wherein additional moieties, such as carbohydrate chains, are added to it. The exact nature of such posttranslational modifications will be determined in part by the type of host cell in which the polypeptide is produced.
Mouse and human TPO DNAs were cloned as disclosed in WIPO publication WO 95/21920, which is incorporated herein by reference in its entirety. Plasmid pZGmpl-1081, comprising a mouse TPO DNA sequence, was deposited under the terms of the Budapest Treaty on February 14, 1994 with American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD and assigned accession number 69566. Plasmid pZGmpl-124, comprising a human TPO cDNA, was deposited with American Type Culture Collection on May 4, 1994 as an E. coli DH1Ob transformant under accession number 69615. These mouse and human cDNAs are useful as probes for isolating other TPO-encoding DNAs, including genomic DNAs, allelic variants, and DNAs from other species.
Suitable host cells for use within the present invention include any type of eukaryotic cell that can be engineered to express heterologous DNA, can be grown in culture, and has a secretory pathway.
To direct a TPO polypeptide into the secretory pathway of the host cell, a DNA sequence encoding a secretory leader is used in combination with a DNA sequence encoding a TPO polypeptide. The secretory leader may be that of a TPO or that of another secreted protein, such as tissue-type plasminogen activator (t-PA) or the Saccharomyces cerevisiae mating pheromone a-factor. When using a heterologous secretory leader in combination with a TPO polypeptide, the respective DNA segments are joined in the correct reading frame so that the joined segments encode a fusion protein. The joined secretory leader and TPO polypeptide will typically define a proteolytic cleavage site at their junction so that the secretory WO 98/06849 PCTIUS97/13543 12 leader is removed from the TPO polypeptide during secretion. However, those skilled in the art will recognize that a fusion protein can be recovered and subsequently processed to release the TPO polypeptide.
Yeast cells, particularly cells of the genus Saccharomyces, are a preferred host within the present invention. Yeast cells have a long history of use in the production of products for human consumption and are relatively inexpensive to culture. 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 a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient leucine).
A
preferred vector system for use in yeast is the POT1 vector system disclosed by Kawasaki et al. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, 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, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol.
1 WO 98/06849 PCT/US97/13543 13 132:3459-3465, 1986; Cregg, U.S. Patent No. 4,882,279; and Stroman et al., U.S. Patent No. 4,879,231.
It is preferred to use a host strain that is selected for a high level of TPO polypeptide secretion.
A
parent strain that is genotypically suited to fermentation and protein production is mutagenized by conventional methods, such as ultraviolet irradiation or chemical mutagenesis using, for example, ethyl methane sulfonate or nitrosoguanidine. Surviving cells are screened for protein secretion levels using conventional assay methods, such as a filter colony assay, wherein cells are overlayed with nitrocellulose, which is subsequently probed with an antibody in a Western blot format. Additional assays, such as activity assays, may also be used.
Other fungal cells are also suitable as host cells. For example, Aspergillus 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 Acremonium 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.
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 7:603, 1981: Graham and Van der Eb, Viroloqy 52 :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, John Wiley and Sons, Inc., NY, 1987), and cationic lipid-mediated transfection (Hawley-Nelson et al., Focus 15:73-79, 1993), which are incorporated herein by reference. The production of recombinant proteins in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent WO 98/06849 PCT/US97/13543 14 No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; Mulvihill et al., U.S. Patent No. 5,486,471; Foster et al., U.S. Patent No. 5,358,932; and Mulvihill et al., U.S. Patent No.
5,385,831, which are incorporated herein by reference.
Preferred cultured mammalian cells include the COS-1 (ATCC No. 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. 36:59-72, 1977) and Chinese hamster ovary 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, U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes Patents 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 neomycintype 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 WO 98/06849 PCTIS9713543 preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
Other drug resistance genes hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used.
Other higher 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. (Banqalore) 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. A variety of 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 cotransfected into the host cell.
TPO polypeptides prepared according to the present invention are selectively recovered using methods generally known in the art, such as affinity purification and separations based on size, charge, solubility and other properties of the protein. When the polypeptide is produced in cultured mammalian cells, it is preferred to culture the cells in a serum-free culture medium in order to limit the amount of contaminating protein. The medium WO 98/06849 PCTIUS97/13543 16 is harvested and fractionated. Preferred methods of fractionation include affinity chromatography, such as on an immobilized Mpl receptor protein or ligand-binding portion thereof or through the use of an affinity tag polyhistidine, substance P or other polypeptide or protein for which an antibody or other specific binding agent is available). A specific cleavage site may be provided between the protein of interest and the affinity tag. Other chromatographic methods can also be employed, such as cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction chromatography.
TPO polypeptide prepared according to 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.
TPO
polypeptides 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. For example, 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. In addition, 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.
TPO
WO 98/06849 PCTIUS97/13543 17 polypeptides can reduce or eliminate the need for transfusions, thereby reducing the incidence of platelet alloimmunity. Abnormal destruction of platelets can result from: increased platelet consumption in vascular grafts or traumatized tissue; or immune mechanisms associated with, for example, drug-induced thrombocytopenia, idiopathic thrombocytopenic purpura (ITP), autoimmune diseases, hematologic disorders such as leukemia and lymphoma, or metastatic cancers involving bone marrow. Other indications for TPO include aplastic anemia and drug-induced marrow suppression resulting from, for example, chemotherapy or treatment of HIV infection with AZT.
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.
For pharmaceutical use, TPO polypeptides are 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.
In general, pharmaceutical formulations will include a TPO polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 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. In addition, TPO polypeptides can be combined with other cytokines, particularly earlyacting 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 WO 98/06849 PCTIUTS97/13543 18 is incorporated herein by reference. Therapeutic doses of TPO will generally be in the range of 0.1 to 100 pg/kg of patient weight per day, preferably 0.5-50 pg/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 tg/kg per day will be indicated. Determination of dose is within the level of ordinary skill in the art. TPO polypeptides 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. More commonly, TPO .polypeptides will be administered over one week or less, often over a period of one to three days. In general, a therapeutically effective amount of a TPO polypeptide 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 polypeptides 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.
Within regimens of combination therapy, daily doses of other cytokines will in general be: EPO, 150 U/kg; GM- CSF, 5-15 jg/kg; IL-3, 1-5 g/kg; and G-CSF, 1-25 pg/kg.
Combination therapy with EPO, for example, is indicated in anemic patients with low EPO levels.
TPO polypeptides are also valuable tools for the in vitro study of the differentiation and development of hematopoietic cells, such as for elucidating the mechanisms of cell differentiation and for determining the WO 98/06849 PCT/US97/13543 19 lineages of mature cells, and may also find utility as a proliferative agent in cell culture.
TPO polypeptides 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 polypeptides, 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. In addition, TPO polypeptides can also be used for the ex vivo expansion of marrow or peripheral blood progenitor (PBPC) cells. Prior to chemotherapy treatment, marrow can be stimulated with stem cell factor (SCF) or G-CSF to release early progenitor cells into peripheral circulation. These progenitors can be collected and concentrated from peripheral blood and then treated in culture with one or more TPO polypeptides, optionally in combination with one or more other 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.
The invention is further illustrated by the following non-limiting examples.
Examples Example 1 Two expression vectors were constructed to compare expression in the yeast S. cerevisiae of truncated human TPO polypeptides differing in the presence or absence of the Arg-Arg dipeptide at positions 153-154 of SEQ ID NO:2. The two vectors were derived from plasmid pDPOT (ATCC #68001; disclosed in U.S. Patent No.
5,128,321). Both vectors included a TPO expression casette comprising the S. cerevisiae triose phosphate isomerase (TPII) gene promoter (see U.S. Patent No.
4,599,311, incorporated herein by reference),
S.
WO 98/06849 PCT/US97/13543 cerevisiae MFal pre-pro sequence, TPO sequence, and S.
cerevisiae TPI1 terminator. The vectors further included a Schizosaccharomyces pombe triose phosphate isomerase (POT) gene, allowing selection in media containing glucose, an ampicillin resistance gene for selection in E.
coli, and a leu2-d selectable marker. Vector pD85 encoded the wild-type human TPO sequence from amino acid residue 1 to residue 172 of SEQ ID NO:2 wherein residue 168 (Val) was replaced with Ala. Vector pD79 encoded a variant TPO 1-172 sequence wherein the codons for arginine residues 153 and 154 were deleted, and the valine codon (residue 168) was replaced with an alanine codon (amino acid positions refer to SEQ ID NO:2) A full-length human TPO DNA was modified by polymerase chain reaction (PCR) to add five codons of the MFal pre-pro sequence at the 5' end and a stop codon at the 3' end. PCR was carried out using primers ZC7623 (SEQ ID NO:9) and ZC7627 (SEQ ID NO:10). PCR was run using Taq polymerase and ~20 ng of template DNA with 25 cycles of 94 0 C, 1 minute; 53 0 C, 1 minute; and 72 0 C, 1.5 minute. The modified TPO sequence was isolated as a 479 bp Hind III- Xba I fragment and was ligated into pUC18 that had been cleaved with the same enzymes. The resultant plasmid was designated pHB76.
The TPO sequence in pHB76 was modified to introduce Bbe I and Sal I sites at the 5' and 3' ends, respectively, of the sequence encoding the cytokine domain. PCR was carried out using primers ZC7868 (SEQ ID NO:11) and ZC7870 (SEQ ID NO:12). PCR was run using Taq polymerase and -20 ng of template DNA, with nine cycles of 0 C, 1 minute; 68 0 C, 4 minutes; followed by one cycle of 0 C, 1 minute; 68 0 C, 10 minutes. The TPO sequence was recovered as a 482 bp Hind III-Eco RI fragment and was ligated into pUC19 that had been cleaved with the same enzymes. The resultant plasmid was designated pTPOGN2.
The variant TPO sequence was then constructed by combining a 457 bp Hind III-Sal I fragment from pTPOGN2 WO 98/06849 PCTI/US97/13543 21 and a synthetic fragment constructed from oligonucleotides ZC8488 (SEQ ID NO:13) and ZC8489 (SEQ ID NO:14) in a three-part ligation with Hind III Xba I digested pUC19.
The resultant plasmid was designated pTPOGN6. A 539 bp Hind III-Xba I fragment encoding the last few residues of the a-factor pre-pro peptide and the truncated TPO polypeptide was isolated from pTPOGN6. This fragment was joined in a four-part ligation with pDPOT (cleaved with Bam HI and treated with alkaline phosphatase), a 1230 bp Bgl II-Hind III fragment comprising the TPI1 promoter and MFal pre-pro sequences, and a 680 bp Xba I-Bam HI fragment comprising the TPI1 terminator. The resultant plasmid was designated pD79.
A control plasmid encoding residues 1 to 172 of human TPO (SEQ ID NO:2) was constructed by joining, in a four-part ligation, pDPOT (cleaved with Bam HI and treated with alkaline phosphatase), the 1230 bp Bgl II-Hind III TPIl-MFal fragment, a 540 bp fragment encoding last few residues of the a-factor pre-pro peptide and the truncated TPO polypeptide, and the 680 bp Xba I-Bam HI TPI1 terminator fragment. The resultant plasmid was designated Plasmids pD79 and pD85 were transformed in Saccharomyces cerevisiae strain JG134 (MATa Atpil::URA3 ura3-52 leu2-A2 pep4-Al [cir 0 essentially as disclosed by Hinnen et al. (Proc. Natl. Acad. Sci. USA 75:1929-1933, 1978). Transformants were selected for their ability to grow on medium containing glucose as the sole carbon source.
Transformants were grown for about 60 hours in liquid medium containing 1% yeast extract, 1% peptone, and glucose. The cells were separated from the culture medium by centrifugation. Media samples were diluted 1:100 in TPO dilution buffer (RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 jg/ml penicillin, 50 Ag/ml streptomycin, 100 tg/ml neomycin, 0.00033% -mercaptoethanol, 25 mM Hepes.
WO 98/06849 PCT/US97/13543 22 Biological activity of TPO was assayed in a mitogenesis assay using BaF3 cells transfected with an expression vector encoding the human MPL receptor (Vigon et al., Proc. Natl. Acad. Sci. USA 89:5640-5644, 1992) as target cells. BaF3 is an interleukin-3 dependent prelymphoid cell line derived from murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey- Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986).
Cells were exposed to test samples in the presence of 3
H-
thymidine for 16 to 19 hours at 37 0 C. The amount of 3
H-
thymidine incorporated into cellular DNA was quantitated by comparison to a standard curve of human TPO. 10 U/ml was defined as the amount giving half-maximal stimulation in the mitogenesis assay. Results of two experiments are shown in Table 1.
Table 1
TPO
Experiment Plasmid (units/ml media) 1 pD85 pD79 740 2 pD85 below limit pD79 160 Example 2 A series of plasmids encoding TPO polypeptides consisting of the cytokine domain of human TPO (residues 22 to 152 of SEQ ID NO:2) linked to a C-terminal segment by either a peptide bond or an Arg-Arg dipeptide was constructed. Table 2 shows the structures of the encoded polypeptides: Arg-Arg indicates presence or absence of the dipeptide; amino acid numbers for the Cterminal segment refer to SEQ ID NO:3.
WO 98/06849 PCTIUS97/13543 23 Table 2 Plasmid Arq-Arg C- Term. Sequence pD117 pD119 1-9 pD121 1-13 pD123 1-18 pD125 1-18 Plasmid pTPOGN8 was constructed in a three-part ligation using pUC19, which had been linearized by digestion with Hind III and Xba I; a 457 bp Hind III-Sal I fragment of pTPOGN2 (Example 1) comprising coding sequence for a portion of the a-factor secretory leader and part of the human TPO cytokine domain; and a Sal I-Xba I adapter constructed from oligonucleotides ZC8486 (SEQ ID and ZC8487 (SEQ ID NO:16).
Plasmid pD83 was constructed in a four-part ligation using the following fragments: Bam HI-digested, alkaline phosphatase-treated pDPOT; a 1230 bp Bgl II-Hind III fragment of pHB105-4 (a plasmid containing the S.
cerevisiae TPII promoter and a-factor secretory leader joined to the coding sequence for the cytokine domain of human TPO in the plasmid backbone of pMVR1 (disclosed in U.S. Patent No. 5,155,027)), which contained the S.
cerevisiae TPII promoter and a-factor secretory leader; a 540 bp Hind III-Xba I fragment of pTPOGN8, which contained a portion of the a-factor secretory leader coding sequence and the coding sequence of a TPO variant consisting of the cytokine domain joined at its C-terminus to the 18-residue polypeptide of SEQ ID NO:17; and a 680 bp Xba I-Bam HI S.
cerevisiae TPII terminator fragment.
Plasmids shown in Table 2 were constructed by first inserting the BglII-EcoRI fragment of pD83, comprising TPII terminator and vector sequences, into pUC19 (cut with SalI and EcoRI) with one of the pairs of oligonucleotides shown in Table 3. The resulting plasmids WO 98/06849 PCT/US97/13543 24 were designated pTPOMI1, 2, 3, 4, and 5 as shown in Table 3. Two pD83 fragments, BglI-SalI, comprising the TPII promoter, MFal secretory leader, 5' TPO coding region, and vector sequences; and EcoRI-BglI, comprising vector sequences, were joined to SalI-EcoRI fragments from (comprising 3' TPO coding sequences, the TPII terminator, and vector sequences) to construct pD117, pD119, pD121, pD123, and pD125, respectively.
Table 3 Plasmid Oligonucleotides pTPOMI1 ZC10086 (SEQ ID NO:18) ZC10087 (SEQ ID NO:19) pTPOMI2 ZC10095 (SEQ ID ZC10096 (SEQ ID NO:21) pTPOMI3 ZC10120 (SEQ ID NO:22) ZC10121 (SEQ ID NO:23) pTPOMI4 ZC10118 (SEQ ID NO:24) ZC10119 (SEQ ID pTPOMI5 ZC10108 (SEQ ID NO:26) ZC10109 (SEQ ID NO:27) Plasmids were transformed into S. cerevisiae JG134 or M35. M35 was derived from pD79-transformed JG134 by UV mutagenesis of an overnight culture. Cells were diluted 1:1000, and 50 t1 of the dilution was plated on YEPD yeast extract, 2% peptone, 2% D-glucose, 0.004% adenine, 0.006% L-leucine). Plates were exposed to UV light for 20 seconds in a darkroom, placed in a light impenetrable box, and incubated for two days at 30 0 C. Each plate was then replica-plated onto a fresh YEPD plate, covered with nitrocellulose, and incubated overnight at 0 C. The nitrocellulose was then removed and washed of any adhering yeast cells. The nitrocellulose was developed via standard Western technique using 5% milk in IX PBS for blocking and a rat anti-human TPO antibody.
Colonies exhibiting a high level of secretion in the WO 98/06849 PCTIUS97/13543 primary screen were further assayed by Western blotting and an activity assay. One mutant, designated consistently produced 2-4 times greater activity than the parent strain. GN35 was cured of pD79 by transforming with a 2-micron-based plasmid containing a prokaryotic kanamycin resistance gene and the S. cerevisiae TPII gene.
Transformants resistant to 2 mg/ml G418 were selected and cultured in YEPD containing G418, then in YEPGGE (0.004% adenine, 0.006% L-leucine, 1% yeast extract, 0.4% Dgalactose, 2% peptone, 3% glycerol, 1% ethanol). Cells were then screened for the inability to grow on glucose as a carbon source, indicating loss of the triose phosphate isomerase gene on pD79. The cured cells were designated strain M35. Transformants were grown in liquid medium containing 2% peptone, 1% yeast extract, and 5% glucose with aeration for 70 hours. Plasmids pD79 and pD85 were included as controls. In addition, plasmids pHB109 (encoding the human TPO cytokine domain alone) and pBJ118 (encoding the same polypeptide as pD85 and pD125) were assayed.
Media were collected and assayed as disclosed in Example 1. Assay results are shown in Table 4.
Table 4 Host Plamid TPO (nq/ml) JG134 pD117 26 pD119 51 pD121 53 pD123 pD125 3 pD79 3 pHB109 below limit pBJ118 8 M35 pD79 180 WO 98/06849 PCT/US97/13543 26 Example 3 Using conventional molecular biology techniques, a series of pDPOT-based plasmids encoding TPO polypeptides was constructed. Each of the encoded TPO polypeptides consisted of the cytokine domain of human TPO linked via a peptide bond to a C-terminal segment derived from the Cterminal domain of human TPO. The sequences of the Cterminal segments of these polypeptides are shown below in Table 5. Amino acids are designated using the conventional one-letter code.
Plasmid pD91 Table C-Terminal Domain APPD TAV PS R T S LVLTLN (SEQ ID APPDTAV PS E TS LVL TLN (SEQ ID NO:6) AP PT TAV PS ET S LVL TLN (SEQ ID NO:7) pD93 Plasmids were transformed into strain JG134, and transformants were cultured as disclosed in Example 2.
Media were harvested and assayed as disclosed in Example 1.
Results are shown in Table 6.
Table 6 Plasmid pDPOT pD79 pD91 pD93
TPO
(units/ml) 0 700 100 200 300 600 WO 98/06849 PCTIUS97/13543 27 From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
WO 98/06849 PCTIUS97/13543 28 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: ZymoGenetics,Inc.
1201 Eastlake Avenue East Seattle. Washington 98102 United States of America (ii) TITLE OF INVENTION: EXPRESSION VECTORS, CELLS, AND METHODS FOR PREPARING THROMBOPOIETIN
POLYPEPTIDES
(iii) NUMBER OF SEQUENCES: 27 (iv) CORRESPONDENCE
ADDRESS:
ADDRESSEE: ZymoGenetics, Inc.
STREET: 1201 Eastlake Avenue East CITY: Seattle STATE: WA COUNTRY: USA ZIP: 98102 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:
CLASSIFICATION:
(viii) ATTORNEY/AGENT
INFORMATION:
NAME: Parker, Gary E.
REGISTRATION NUMBER: 31.648 REFERENCE/DOCKET NUMBER: 95-34 (ix) TELECOMMUNICATION
INFORMATION:
TELEPHONE: 206-442-6673 TELEFAX: 206-442-6678 WO 98/06849 29 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1062 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA PCT/US97/13543 (ix) FEATURE:
NAME/KEY:
LOCATION:
(ix) FEATURE:
NAME/KEY:
LOCATION:
(ix) FEATURE:
NAME/KEY:
LOCATION:
mat_peptide 64..1062 sig_peptide 1..63
CDS
1..1062 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA
ATG
48 Met Glu -21 -20 Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala
AGG
96 Arg CTA ACG CTG TCC AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC Leu Ser Lys Leu Leu Arg Asp Ser His 20 Val Leu His Ser Arg Leu Ser
CAG
192 TGC CCA GAG GTT CAC CCT TTG CCT ACA CCT GTC CTG CTG CCT GCT Gln Cys Pro Glu Val His Pro Pro Thr Pro Val Leu Leu Pro Ala WO 98/06849 GTG GAC TTT AGC TTG GGA GAA TGG AAA A PCTUS97/13543 Icc CAG ATG GAG GAG ACC AAG Trp Lys Thr Val Asp Phe Ser Leu Gly
GCA
288 Ala
GCA
336 Ala
CAG
384 Gin
CTT
432 Leu
CCC
480 Pro
CGT
528 Arg 140
CCA
576 Pro
AAC
624
CAG
Gin
GCA
Ala
CTT
Leu
GGA
Gly
AAT
Asn 125
TTC
Phe
CCC
Pro
GAC
Asp
CGG
Arg
TCT
Ser
ACC
Thr 110
GCC
Ala
CTG
Leu
ACC
Thr
ATT
Ile
GGA
Gly
GGA
Gly
CAG
Gin
ATC
lie
ATG
Met
ACA
Thr
CTG
Leu
CAA
Gin
CAG
Gin
CTT
Leu
TTC
Phe
CTT
Leu
OCT
Ala 160
AAC
GGA
Gly 65
CTG
Leu
GTC
Va1
CCI
Pro
CTG
Leu
GTA
Va1 145
GTC
Val Glu 50
GCA
Ala
GGA
Gly
CGT
Arg
CCA
Pro
AGC
Ser 130
GGA
Gly
CCC
Pro Gin Met Glu Giu Thr Lys
GTG
Va1
CCC
Pro
CTC
Leu
CAG
Gin 115
TTC
Phe
GGG
Gly
AGC
Ser ACC CTT Thr Leu ACT TGC Thr Cys 85 CTC CTT Leu Leu 100 GGC AGG Gly Arg CAA CAC Gin His ICC ACC Sen Thr AGA ACC Arg Ihr 165 GGA TTG
CTG
Leu 70
CTC
Leu
GGG
Gly
ACC
Thr
CTG
Leu
CTC
Leu 150
TCT
Sen
CTG
Leu
TCA
Sen
GCC
Ala
ACA
Thr
CTC
Leu 135
TGC
Cys
CTA
Leu
GAG
Glu
ICC
Sen
CTG
Leu
GCT
Ala 120
CGA
Arg
GTC
Val
CTC
Val
GGA
Gly
CTC
Leu
CAG
Gin 105
CAC
His
GGA
Gly
AGG
Arg
CTC
Leu
GTG
Va1
CTG
Leu
AGC
Sen
AAG
Lys
AAG
Lys
CGG
Arg
ACA
Thr
ATG
Met
GGG
Gly
CTC
Leu
GAT
Asp
GTG
Va1
GCC
Ala 155
CTG
Leu 170 TTC ACT GAG CTC CCA AGG ACT TCT TTG GAG ACA AAC Asn Giu Leu Pro Asn Arg Thr Sen Gly Leu Leu Glu Thr Asn Phe Ihr 175 180 WO 98/06849 PCTIUS97/13543 GCC TCA GCC AGA ACT ACT GGC TCT 672 Ala Sen Ala 190 Arg Thr Thr Gly TTC AGA 720 Phe Arg 205 GAG CAA 768 Asp Gin 220 ACT CGT 816 Ihr Arg GAC ATT 864 Asp Ile CAG CCI 912 Gin Pro ACG CTC 960 Thr Leu 285 CAC CCC 1008 His Pro 300 CCI CTT 1056
GCC
Ala
ATC
Ile
GGA
Gly
ICC
Sen
GGA
Gly 270
TTC
Phe
AAG
Lys
CCC
Pro
CTC
Leu
TCA
Ser 255
TAT
Ty r
CCI
Pro
ATI
Ile
GGA
Gly
TTT
Phe 240
GGA
Gly
TCT
Sen
CTT
Leu
CCI
Pro
TAG
Tyr 225
CCI
Pro
ACA
Thn
CCI
Pro
CCA
Pro
GGI
Gly 210
CTG
Leu
GGA
Gly
ICA
Sen
ICC
Sen
CCC
Pro 290
CCI
Pro Ser 195
CTG
Leu
AAC
Asn
CCC
Pro
GAG
Asp
CCA
Pro 275
ACC
Ihr
TCT
Sen GGG CTI Gly Leu CTG AAC Leu Asn AGG ATA Arg Ile TCA CGG Sen Arg 245 ACA GGC Thn Gly 260 ACC CAT Thn His TTG CCC Leu Pro GCT GCA Ala Pro
CTG
Leu
CAA
Gin
CAC
His 230
AGG
Arg
ICC
Sen
CCI
Pro
ACC
Thr
ACG
Thr 310
GAG
AAG
Lys
ACC
Thn 215
GAA
Glu
ACC
Thr
CTG
Leu
CCI
Pro
CCI
Pro 295
CCC
Pro
TGG
Trp 200
ICC
Sen
CTC
Leu
CTA
Leu
CCA
Pro
ACT
Thr 280
GTG
Val
ACC
Thr
GAG
Gin
AGG
Arg
TTG
Leu
GGA
Gly
CCC
Pro 265
GGA
Gly
GTC
Val
CCI
Pro
GAG
Gin
ICC
Sen
AAT
Asn 0CC Ala 250
AAC
Asn
GAG
Gin
GAG
Gin
ACC
Thr
GGA
Gly
CTG
Leu
GGA
Gly 235
CCG
Pro
CTC
Leu
TAT
Tyn
CTC
Leu
AGG
Sen 315
GAA
CTG CTT CCI GAG Leu Leu Pro Asp 305 CTA AAC ACA ICCTAG ACC CAC ICC AAT CTG TCT CAG Pro Leu Leu Asn Thr Sen Tyr Thr His Sen Gin Asn Leu Sen Gin Glu 320 330 WO 98/06849 32 GGG TAA 1062 Gly INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 353 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: PCTIUS97/13543 Met Glu -21 -20 Leu Thr Glu Leu Leu Val Val Met Leu Leu Leu Thr Ala Arg Leu Thr Leu Ser Ser 1 Pro Ala Pro Pro Ala Cys Asp Leu 5 Arg Val Leu Ser Lys Gin Cys Pro Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Leu Pro Ala Glu Val His Pro Leu 35 Pro Thr Pro Val Leu Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Glu Glu Thr Lys Ala Gin Asp Ile Leu Ala Ala Arg Gly Gin Gly Ala Val Thr Leu Leu Glu Gly Val Leu Gly Pro Thr Cys 85 Leu Ser Ser Leu Leu Gly Gin Leu Ser Leu Gly Thr 110 Gly Gin Val Arg Leu Leu Gly Ala Leu Gin Ser Leu 105 His Lys Asp Gin Leu Pro Pro Gly Arg Thr Thr Pro Asn 125 Ala Ile Phe Leu Ser 130 Phe Gin His Leu Arg Gly Lys Val WO 98/06849 PCT[S97/13543 33 Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala 140 145 150 155 Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu 160 165 170 Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr 175 180 185 Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin Gly 190 195 200 Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser Leu 205 210 215 Asp Gin Ile Pro Gly Tyr Leu Asn Arg Ile His Glu Leu Leu Asn Gly 220 225 230 235 Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro 240 245 250 Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu 255 260 265 Gin Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gin Tyr 270 275 280 Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gin Leu 285 290 295 His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser 300 305 310 315 Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin Glu 320 325 330 Gly INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 178 amino acids TYPE: amino acid WO 98/06849 PCT/US97/13543 34 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Thr Ala Ser Leu Pro Asn Arg Thr Ser 25 Gly Leu Leu Glu Thr Asn Phe Trp Gln G1n Ala Arg Thr Thr Ser Gly Leu Leu Lys Gly Phe Arg Ala Lys IlePro Gly Leu Leu Asn Thr Ser Arg Ser Leu Asp Gin Ile Pro Tyr Leu Asn Arg Ile 75 His Glu Leu Leu Asn Gly Thr Arg Gly Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Leu Gin Pro 115 Ser 100 Ser Gly Thr Ser Asp 105 Thr Gly Ser Leu Pro Pro Asn 110 Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gln 120 125 Tyr Thr 130 Leu Phe Pro Leu Pro 135 Pro Thr Leu Pro Thr 140 Pro Val Val Gin Leu 145 His Pro Leu Leu Asp Pro Ser Ala Pro 155 Thr Pro Thr Pro Ser Pro Leu Leu Asn 165 Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin 175 Glu Gly INFORMATION FOR SEQ ID NO:4: WO 98/06849 SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: PCT/US97/13543 Ala 1 Pro Pro Thr Thr Ala Val Pro Ser 5 Thr Ser Leu Ala Leu Leu Asn INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Pro Pro Asp Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr 1 5 10 Leu Asn INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/06849 PCTIS97/13543 36 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Ala Pro Pro Asp Thr Ala Val Pro Ser Glu Thr Ser Leu Val 1 5 10 Leu Asn INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Leu Thr Ala 1 Pro Pro Thr Thr Ala Val Pro 5 Ser Glu Thr Ser Leu Val Leu Thr 10 Leu Asn INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: WO 98/06849 PCT/US97/13543 37 Ala Pro Pro Thr Thr 1 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 49 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC7623 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GCGCGCAAGC TTGGACAAGA GAAGCCCGGC TCCTCCTGCT TGTGACCTC 49 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC7627 (xi) SEQUENCE DESCRIPTION: SEQ ID GCGCGCAAGC TTTCTAGACT ATCAGACGCA GAGGGTGGAC CCTCCTACAA
G
51 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 98/06849 38 TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC7868 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GTGTGTGAAT TCTAGACTAT CAGACGCAGA GGGTCGACCC INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC7870 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GTGTGTAAGC TTGGACAAGA GAAGCCCGGC GCCTC INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 82 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC8488 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: PCT/US97/13543 WO 98/06849 PCT/US97/13543 39 TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGGCTTTG ACTTTGAACT-GATAGAGATC TT 82 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 82 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC8489 SEQUENCE DESCRIPTION: SEQ ID NO:14: CTAGAAGATC TCTATCAGTT CAAAGTCAAA GCCAAAGAAG TTCTGGATGG AACAGCAGTG GTTGGTGGGG CGACGCAGAG GG 82 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 85 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC8486 (xi) SEQUENCE DESCRIPTION: SEQ ID TCGACCCTCT GCGTCGGAGC TCCACCAGAC GAAGCTGTTC CAGACAGAGA CGAATTGGTT WO 98/06849 PCTfUS97/1343 TTGGAATTGA ACTGATAGAG ATCTT INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 85 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC8487 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CTAGAAGATC TCTATCAGTT CAATTCCAAA ACCAATTCGT CTCTGTCTGG AACAGCTTCG TCTGGTGGAG CTCCGACGCA GAGGG INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Ala Pro Pro Asp Glu Ala Val Pro Asp Arg Asp Glu Leu Val Leu Glu 1 5 10 Leu Asn INFORMATION FOR SEQ ID NO:18: WO 98/06849 41 SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC10086 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: TCGACCCTCT GCGTCGCCCC ACCAACCACT TGATAGA 37 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC10087 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GATCTCTATC AAGTGGTTGG TGGGGCGACG CAGAGGG 37 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 49 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC10095 PCT/US97/13543 WO 98/06849 PCT/US97/13543 42 (xi) SEQUENCE DESCRIPTION: SEQ ID TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCTGATAGA 49 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 49 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC10096 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GATCTCTATC AGGATGGAAC AGCAGTGGTT GGTGGGGCGA CGCAGAGGG 49 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC10120 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGTGATAG
A
61 WO 98/06849 PCT/US97/13543 43 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC10121 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GATCTCTATC ACAAAGAAGT TCTGGATGGA ACAGCAGTGG TTGGTGGGGC GACGCAGAGG
G
61 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 76 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC10118 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGGTTTTG ACTTTGAACT GATAGA 76 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 76 base pairs WO 98/06849 PCTIUS97/13543 44 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC10119 (xi) SEQUENCE DESCRIPTION: SEQ ID GATCTCTATC AGTTCAAAGT CAAAACCAAA GAAGTTCTGG ATGGAACAGC AGTGGTTGGT GGGGCGACGC AGAGGG 76 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 82 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vii) IMMEDIATE SOURCE: CLONE: ZC10108 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: TCGACCCTCT GCGTCAGGCG GGCCCCACCA ACCACTGCTG TTCCATCCAG AACTTCTTTG GTTTTGACTT TGAACTGATA GA 82 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 82 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 98/06849 PCTIUS97/13543 (vii) IMMEDIATE SOURCE: CLONE: ZC10109 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GATCTCTATC AGTTCMAAGT CAAMACCAAA GMAGTTCTGG ATGGMCAGC AGTGGTTGGT GGGGCCCGCC TGACGCAGAG GG 82
Claims (26)
1. An expression vector replicable in a eukaryotic host cell and comprising the following operably linked elements: a transcription promoter; a first DNA segment encoding a secretory leader; a second DNA segment encoding a thrombopoietin (TPO) polypeptide consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18 and wherein up to 35% of said residues of B are individually replaced by other amino acid residues; and a transcription terminator.
2. An expression vector according to claim 1 wherein said vector is replicable in yeast.
3. An expression vector according to claim 2 wherein said secretory leader is a Saccharomyces cerevisiae alpha-factor secretory leader.
4. An expression vector according to claim 1 wherein y is at least. 9. An expression vector according to claim 1 wherein B comprises a Thr-Thr dipeptide.
6. An expression vector according to claim 1 wherein B does not comprise an Arg-Arg dipeptide. WO 98/06849 PCTfUS97/13543 47
7. An expression vector according to claim 1 wherein up to 25% of said residues of B are individually replaced by other amino acid residues.
8. An expression vector according to claim 1 wherein residues 1 to 5 of B are Ala-Pro-Pro-Thr-Thr (SEQ ID NO:8).
9. An expression vector according to claim 1 wherein residue 4 of B is Thr or Asp. An expression vector according to claim 1 wherein y is at least 10 and residue 10 of B is Arg or Glu.
11. An expression vector according to claim 1 wherein y is at least 14 and residue 14 of B is Val or Ala.
12. An expression vector according to claim 11 wherein B is Ala-Pro-Pro-Thr-Thr-Ala-Val-Pro-Ser-Arg-Thr- Ser-Leu-Ala-Leu-Thr-Leu-Asn (SEQ ID NO:4).
13. An expression vector according to claim 1 wherein B is a sequence of amino acid residues selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.
14. An expression vector according to claim 1 wherein X is a peptide bond. An expression vector according to claim 1 wherein X is a single amino acid residue.
16. An expression vector according to claim 1 wherein C consists of residues 1 to 152 of SEQ ID NO:2. 48
17. An expression vector replicable in a eukaryotic host cell and comprising a DNA segment which encodes for the polypeptide C-X-B, substantially as hereinbefore described with reference to any one of the examples.
18. A cultured eukaryotic cell containing an expression vector according to any one of claims 1 to 17, wherein said cell produces and secretes said TPO polypeptide.
19. A yeast cell according to claim 18. A TPO polypeptide produced by the cell of claim 18 or claim 19.
21. A thrombopoietin polypeptide characterised by an amino acid backbone consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; to X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and wherein up to 35% of said residues of B are individually replaced by other amino acid residues. Is 22. A thrombopoietin polypeptide comprising the polypeptide C-X-B, substantially as hereinbefore described with reference to any one of the examples.
23. A method of making a TPO polypeptide comprising: culturing a host cell transfected or transformed with an expression vector replicable in the host cell and comprising the following operably linked elements: a transcription promoter; a first DNA segment encoding a secretory leader; a second DNA segment encoding a thrombopoietin polypeptide consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; i X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the 25 limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18 and wherein up to 35% of said residues of B are individually replaced by other amino acid residues; and a transcription terminator, wherein the linked first and second DNA segments are expressed by the host cell to produce the TPO polypeptide; and recovering the TPO polypeptide.
24. A method of making a TPO polypeptide comprising the polypeptide C-X-B, substantially as hereinbefore described with reference to any one of the examples. A TPO polypeptide made by the method of claim 23 or claim 24. [R:\LIBZZ]03471.doc:mrr 49
26. A pharmaceutical composition comprising a therapeutically effective amount of a TPO polypeptide according to any one of claims 20 to 22, and 25 together with a pharmaceutically acceptable vehicle.
27. A method of increasing platelet number in an animal comprising administering to said animal a thrombopoietin polypeptide characterised by an amino acid backbone consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and wherein up to 35% of said residues of B are individually replaced by other amino acid residues, in combination with a pharmaceutically acceptable vehicle.
28. A thrombopoietin polypeptide characterised by an amino acid backbone consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; 15 X is a peptide bond or a linker consisting of one or two amino acid residues, subject to S' the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and wherein up to 35% of said residues of B are individually replaced by other amino acid residues when used to increase platelet number.
29. Use of a thrombopoietin polypeptide characterised by an amino acid backbone consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID 25 NO:3, wherein y is an integer from 5 to 18, and wherein up to 35% of said residues of B are individually replaced by other amino acid residues, in the manufacture of a medicament for increasing platelet number. A method of increasing platelet number in an animal comprising administering to said animal a TPO polypeptide according to any one of claims 20 to 22, and 25 or a composition according to claim 26.
31. A TPO polypeptide according to any one of claims 20 to 22 and 25 or a composition according to claim 26 when used to increase platelet number. R:BZZ]047do:m 'V N-F r~ V [R:\LIBZZ]0347 1.doc:mrr
32. Use of a TPO polypeptide according to any one of claims 20 to 22 and in the manufacture of a medicament for increasing platelet number.
33. A medicament manufactured according to claim 29 or claim 32. Dated 10 November, 2000 ZymoGenetics, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON o e* a [R:\LIBZZ]03471 doc:mrr
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US69644796A | 1996-08-13 | 1996-08-13 | |
US08/696447 | 1996-08-13 | ||
PCT/US1997/013543 WO1998006849A1 (en) | 1996-08-13 | 1997-07-30 | Expression vectors, cells, and methods for preparing thrombopoietin polypeptides |
Publications (2)
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AU3823897A AU3823897A (en) | 1998-03-06 |
AU728881B2 true AU728881B2 (en) | 2001-01-18 |
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AU38238/97A Ceased AU728881B2 (en) | 1996-08-13 | 1997-07-30 | Expression vectors, cells, and methods for preparing thrombopoietin polypeptides |
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EP (1) | EP0920511A1 (en) |
JP (1) | JP2000516465A (en) |
KR (1) | KR20000029998A (en) |
CN (1) | CN1230993A (en) |
AU (1) | AU728881B2 (en) |
CA (1) | CA2262507A1 (en) |
NZ (1) | NZ334103A (en) |
WO (1) | WO1998006849A1 (en) |
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AU750803B2 (en) | 1997-10-29 | 2002-07-25 | University Of Pittsburgh | Use of vectors such as adenoviruses and/or adeno associated viruses and/or retroviruses and/or herpes simplex viruses and/or liposomes and/or plasmids as a vehicle for genetic information enabling mammal cells to produce agents for the treatment of bone pathologies |
KR19990081421A (en) * | 1998-04-29 | 1999-11-15 | 성재갑 | Method for producing human platelet promoter (TPO) using animal cells |
AU2001280161A1 (en) * | 2000-08-24 | 2002-03-04 | Kirin Beer Kabushiki Kaisha | C-mpl ligand-containing medicinal compositions for increasing platelets and erythrocytes |
WO2004078780A1 (en) * | 2003-03-04 | 2004-09-16 | Pepharm R&D Limited | Pharmaceutical composition containing l-seryl-l-leucine |
Citations (1)
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WO1995018858A1 (en) * | 1994-01-03 | 1995-07-13 | Genentech, Inc. | Thrombopoietin |
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EP0668352A1 (en) * | 1994-02-14 | 1995-08-23 | Kirin Brewery Company, Ltd. | Protein having TPO activity |
-
1997
- 1997-07-30 AU AU38238/97A patent/AU728881B2/en not_active Ceased
- 1997-07-30 JP JP10509794A patent/JP2000516465A/en active Pending
- 1997-07-30 CA CA002262507A patent/CA2262507A1/en not_active Abandoned
- 1997-07-30 CN CN97197984A patent/CN1230993A/en active Pending
- 1997-07-30 EP EP97935253A patent/EP0920511A1/en not_active Withdrawn
- 1997-07-30 NZ NZ334103A patent/NZ334103A/en unknown
- 1997-07-30 KR KR1019997001270A patent/KR20000029998A/en not_active Application Discontinuation
- 1997-07-30 WO PCT/US1997/013543 patent/WO1998006849A1/en not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1995018858A1 (en) * | 1994-01-03 | 1995-07-13 | Genentech, Inc. | Thrombopoietin |
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WO1998006849A1 (en) | 1998-02-19 |
KR20000029998A (en) | 2000-05-25 |
AU3823897A (en) | 1998-03-06 |
EP0920511A1 (en) | 1999-06-09 |
CN1230993A (en) | 1999-10-06 |
CA2262507A1 (en) | 1998-02-19 |
JP2000516465A (en) | 2000-12-12 |
NZ334103A (en) | 2000-09-29 |
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