WO1998006849A1 - Expression vectors, cells, and methods for preparing thrombopoietin polypeptides - Google Patents

Abstract

Thrombopoietin polypeptides and vectors, cells, and methods for making the polypeptides are disclosed. The polypeptides are 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 5 to 18, and wherein up to 35 % of the amino acid residues of B are individually replaced by other amino acid residues. The polypeptides are useful with methods of stimulating proliferation or development of hematopoietic cells in vitro and in vivo.

Description

Description EXPRESSION VECTORS, CELLS, AND METHODS FOR PREPARING

THROMBOPOIETIN POLYPEPTIDES

Background of the Invention

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 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 m 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 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 22:1117-1124, 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 . , Ceil 27:1117-1124, 1994) . Molecular species as small as 25 kD have been found to be active in vi tro (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 m v i tro , as has the product of expression of the full-length human cDNA, which encodes a primary translation product of 353 ammo acids (Bartley et al . , ibid) .

There remains a need in the art for methods of producing thrombopoietin in large amounts and in a cost- effective manner. There is also a need for methods of producing thrombopoietin in eukaryotic microorganisms, such as yeast . There is a further need for methods or the efficient production of lower molecular weight forms of thrombopoietin, which may be less subject to proteolysis than the full-length molecule. The present 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) a transcription promoter; (b) a first DNA segment encoding a secretory leader; (c) 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 m 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 ammo acid residues of B are individually replaced by other am o acid residues; and (d) 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 ammo 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 5 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 platelet number a mammal comprising administering to the mammal a TPO polypeptide as disclosed above combination with a pharmaceutically acceptable vehicle.

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 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. 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. "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 m 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-term al cytokme domain and a carboxyl-terminal ( "C-terminal " ) domain. Referring to SEQ ID NO:2, the cytokine domain is bounded by cysteme 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 vi vo stimulation of platelet production) .

A representative cDNA encoding full length human thrombopoietin is shown SEQ ID NO : 1 Those skilled m the art will recognize that the DNA of SEQ ID NO : 1 and the encoded ammo 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-termmal (e.g , residues 1-6 of SEQ ID NO : 2 ) and/or C-termmal (e.g., residue 152 of SEQ ID NO: 2) extensions that do not destroy the essential biological activity of the molecule. Considerable sequence variation is allowed withm these short extensions. The C-termmal 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-lmked 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 (e.g., 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 (i.e. the amino acid residue corresponding to residue 155 of SEQ ID NO: 2) . The portion of the C- terminal 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 (e.g., de Sauvage et al . , ibid.), this dipeptide has been found to inhibit secretion. The present invention thus provides TPO 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 wild- type thrombopoietins (e.g. 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-termmal 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 ammo acid residues. The TPO cytokine domain may also include from one to about 15, 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), 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 8.6 : 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 (e.g., Lowman et al . , Bioche . 3_.0.10832-10837, 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 25% of the residues of B are replaced as compared to the corresponding naturally occuring sequence. Exemplary C- terminal domain sequences are shown in SEQ ID NO: 4 through SEQ ID NO: 7. While not wishing to be bound by theory, the Thr-Thr dipeptide may provide an attachment site for an 0- linked carbohydrate chain. Thus withm one embodiment of the present invention, B comprises a Thr-Thr dipeptide. Within a particularly preferred embodiment, the N- terminal 5 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 (e.g., Saccharomyces cerevisiae) .

Those skilled in the art will recognize that the effect of certain amino acid substitutions may be host- cell-specific. It is therefore preferred to assay the effects of such substitutions m 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. Withm 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 ammo- terminal 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 polypeptide may undergo post-translational processing wherein additional moieties, such as carbohydrate chains, are added to it. The exact nature of such post- translational 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. col i DHlOb 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 Saccharo yces cerevisiae mating pheromone - 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 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

Sa ccharomyces , 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 reco binant 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 (e.g. leucine) . A preferred vector system for use yeast is the POTl 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. 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; K gsman 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 , Schi zosa ccharomyces pombe , Kl uyveromyces la ct is ,

Kl υyveromyces fragilis , Us tilago maydi s , Pichia pas tori s , 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.

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, Aspergil l 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 Acremom um chrysogenum are disclosed by Sumino et al . , U.S. Patent No. 5,162,228, which is incorporated herein py 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, 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, John Wiley and Sons, Inc.,

NY, 1987) , and cationic lipid-mediated transfection

(Hawley-Nelson et al . , Focus 1^:73-79, 1993), which are incorporated herein by reference. The production of recombinant proteins in cultured mammalian cells is disclosed, for example, by Levmson et al . , U.S. Patent 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. 3_.6: 59-72, 1977) and Chinese hamster ovary (e.g. CHO-Kl; 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 metallothione genes (U.S. 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 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 amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate . Other drug resistance genes (e.g. 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 . , ___. 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. 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 ammo 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 . 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 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 (e.g. 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 polypeptides 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, hematologic disorders such as leukemia and lymphoma, or etastatic 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, 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. In addition, TPO polypeptides can be combined with other cytokines, 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 TPO will generally be in the range of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5-50 μg/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 μg/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/mm³, preferably >50,000/mm³, 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/mm³, preferably 50,000/mm³, 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 μg/kg; IL-3, 1-5 μg/kg; and G-CSF, 1-25 μg/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 vi tro study of the differentiation and development of he atopoietic cells, such as for elucidating the mechanisms of cell differentiation and for determining the lineages of mature cells, and may also f nd 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 . cerθvisiae of truncated human TPO polypeptides differing m 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 . cerevisia e triose phosphate isomerase ( TPI1 ) gene promoter (see U.S. Patent No. 4,599,311, incorporated herein by reference), S. cerevisiae MFαl pre-pro sequence, TPO sequence, and S . cerevisiae TPI1 terminator. The vectors further included a Schizosa ccharomyces pombe triose phosphate isomerase (POT) gene, allowing selection in media containing glucose, an ampicillin resistance gene for selection in E . col i , 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 MFαl 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°C, 1 minute; 53°C, 1 minute; and 72°C, 1.5 minute. The modified TPO sequence was isolated as a 479 bp Hind III- Xba I fragment and was ligated into pUCiβ 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 95°C, 1 minute; 68°C, 4 minutes ; followed by one cycle of 95°C, 1 minute; 68°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 pTP0GN2.

The variant TPO sequence was then constructed by combining a 457 bp Hind III -Sal I fragment from pTPOGN2 and a synthetic fragment constructed from oligonucleotides ZC8488 (SEQ ID NO:13) and ZC8489 (SEQ ID NO : 1 ) in a three-part ligation with Hind III + Xba I digested pUC19. The resultant plasmid was designated pTP0GN6. A 539 bp Hind III -Xba I fragment encoding the last few residues of the α- factor pre-pro peptide and the truncated TPO polypeptide was isolated from pTP0GN6. 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 TPIl promoter and MFαl pre-pro sequences, and a 680 bp Xba I -Bam HI fragment comprising the TPIl 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 -MFαl fragment, a 540 bp fragment encoding last few residues of the α-factor pre-pro peptide and the truncated TPO polypeptide, and the 680 bp Xba I-Bam HI TPIl terminator fragment. The resultant plasmid was designated pD85.

Plasmids pD79 and pD85 were transformed m Saccharomyces cerevisiae strain JG134 (MATα Δtpi l : : URA3 ura3 - 52 I eu2 -Δ2 pep4 -Δl [ cir°] ) 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 5% glucose. The cells were separated from the culture medium by centrifugation. Media samples were diluted 1:100 TPO dilution buffer (RPMI 1640 supplemented with 10% fetal bovine serum, 2 M L-glutamine, 1 mM sodium pyruvate, 50 μg/ l penicillin, 50 μg/ml streptomycin, 100 μg/ml neomycin, 0.00033% β-mercaptoethanol , 25 mM Hepes . 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 81=5640-5644, 1992) as target cells. BaF3 is an mterleukm-3 dependent pre- lymphoid cell line derived from mur e 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 m the presence of ³H- thymidine for 16 to 19 hours at 37°C. The amount of ³H- 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 Table 1.

Table 1

TPO

Experiment Plasmid (units/ml media)

1 pD85 40 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-termmal 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 C- term al segment refer to SEQ ID NO : 3. Table 2

Plasmid Arg-Arg C- Term. Sequence pD117 - 1-5 pD119 - 1-9 pD121 - 1-13 pD123 - 1-18 pD125 + 1-18

Plasmid pTPOGNβ 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 Ill-Sal I fragment of pTPOGN2 (Example 1) comprising coding sequence for a portion of the α- factor secretory leader and part of the human TPO cytokine domain; and a Sal I -Xba I adapter constructed from oligonucleotides ZC8486 (SEQ ID NO: 15) 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 TPIl promoter and α - factor secretory leader joined to the coding sequence for the cytokine domain of human TPO in the plasmid backbone of pMVRl (disclosed U.S. Patent No. 5,155,027)], which contained the S . cerevisiae TPIl promoter and α- factor secretory leader; a 540 bp Hind III -Xba I fragment of pTPOGN8 , which contained a portion of the α-factor secretory leader coding sequence and the coding sequence of a TPO variant consisting of the cytokine domain joined at its C-termmus to the 18-resιdue polypeptide of SEQ ID NO: 17; and a 680 bp Xba I -Bam HI S . cerevisiae TPIl terminator fragment .

Piasmids shown in Table 2 were constructed by first inserting the Bglll-EcoRI fragment of pD83, comprising TPIl terminator and vector sequences, nto pUC19 (cut with Sail and EcoRI ■ with one of the pairs of oligonucleotides shown Table 3. The resulting piasmids were designated pTPOMIl, 2, 3, 4, and 5 as shown in Table 3. Two pD83 fragments, Bgll-Sall, comprising the TPIl promoter, MFal secretory leader, 5' TPO coding region, and vector sequences; and EcoRI-Bgll, comprising vector sequences, were joined to Sall-EcoRI fragments from pTPOMIl-5 (comprising 3' TPO coding sequences, the TPIl terminator, and vector sequences) to construct pD117, pD119, pD121, pD123, and pD125, respectively.

Table 3

Plasmid Oligonucleotides pTPOMIl ZC10086 (SEQ ID NO:18)

ZC10087 (SEQ ID NO:19) pTPOMI2 ZC10095 (SEQ ID NO: 20) 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 NO:25) pTPOMI5 ZC10108 (SEQ ID NO:26)

ZC10109 (SEQ ID N0:27)

Piasmids were transformed into S . cerevi siae JG134 or M35. M35 was derived from pD79-transformed JG134 by UV mutagenesis of an overnight culture. Cells were diluted 1:1000, and 50 μl of the dilution was plated on YEPD (1% 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°C. Each plate was then replica-plated onto a fresh YEPD plate, covered with nitrocellulose, and incubated overnight at 30°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 the primary screen were further assayed by Western blotting and an activity assay. One mutant, designated GN35, 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 kana ycin resistance gene and the S . cerevisiae TPIl 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% D- galactose, 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. Piasmids pD79 and pD85 were included as controls. In addition, piasmids pHB109

(encoding the human TPO cytokine domain alone) and pBJllδ

(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.

Tab] .e 4 Host Plamid TPO (ng/ml)

JG134 pD117 26 pD119 51 pD121 53 pD123 35 pD125 3 pD79 70 pD85 3 pHB109 below limit pBJllδ 8 M35 pD79 180 Example 3

Using conventional molecular biology techniques, a series of pDPOT-based piasmids 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 C- terminal domain of human TPO. The sequences of the C- terminal segments of these polypeptides are shown below in Table 5. Amino acids are designated using the conventional one-letter code.

Table 5 Plasmid C-Terminal Domain pD91 A P P D T A V P S R T S L V L T L N (SEQ ID NO:5)

pD93 A P P D T A V P S E T S L V L T L N

(SEQ ID NO:6)

pD95 A P P T T A V P S E T S L V L T L N

(SEQ ID N0:7)

Piasmids 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

TPO

Plasmid {units/ml ) pDPOT 0 pD79 700 pD85 100 pD91 200 pD93 300 pD95 600 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.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: ZymoGenetics. Inc.

1201 Eastlake Avenue East Seattle, Washington 98102 United States of America

(n) TITLE OF INVENTION: EXPRESSION VECTORS. CELLS, AND METHODS FOR PREPARING THROMBOPOIETIN POLYPEPTIDES

(ni) NUMBER OF SEQUENCES: 27

(iv) CORRESPONDENCE ADDRESS:

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(vi) CURRENT APPLICATION DATA

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vm) ATTORNEY/AGENT INFORMATION

(A) NAME: Parker. Gary E

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(lx) TELECOMMUNICATION INFORMATION

(A) TELEPHONE. 206-442-6673

(B) TELEFAX: 206-442-6678 (2) INFORMATION FOR SEQ ID NO.l:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1062 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(lx) FEATURE:

(A) NAME/KEY: mat_peptιde

(B) LOCATION: 64..1062

(lx) FEATURE:

(A) NAME/KEY: sιg_peptιde

(B) LOCATION: 1..63

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1. 1062

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 1

ATG GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA

48

Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala

-21 -20 -15 -10

AGG CTA ACG CTG TCC AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC 96

Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val -5 1 5 10

CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC 144

Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser 15 20 25

CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA CCT GTC CTG CTG CCT GCT 192

Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala

30 35 40 GTG GAC Tπ AGC TTG GGA GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG 240

Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys 45 50 55

GCA CAG GAC Aπ CTG GGA GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG 288

Ala Gin Asp He Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met

60 65 70 75

GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG 336

Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly 80 85 90

CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GGG GCC C1G CAG AGC CTC 384

Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu 95 100 105

Cπ GGA ACC CAG CTT CCT CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT 432

Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp 110 115 120

CCC AAT GCC ATC πC CTG AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG 480

Pro Asn Ala He Phe Leu Ser Phe Gin His Lou Leu Arg Gly Lys Val 125 130 135

CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC C1C TGC G1C AGG CGG GCC

528

Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala

140 145 150 155

CCA CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CIA GTC CTC ACA CTG 576

Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu 160 165 170

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 175 180 185 GCC TCA GCC AGA ACT ACT GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA 672

Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin Gly 190 195 200

TTC AGA GCC AAG ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG 720

Phe Arg Ala Lys He Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser Leu 205 210 215

GAC CAA ATC CCC GGA TAC CTG AAC AGG ATA CAC GAA CTC TTG AAT GGA

768

Asp Gin He Pro Gly Tyr Leu Asn Arg He His Glu Leu Leu Asn Gly

220 225 230 235

ACT CGT GGA CTC Tπ CCT GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG 816

Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro 240 245 250

GAC ATT TCC TCA GGA ACA TCA GAC ACA GGC TCC CTG CCA CCC AAC CTC 864

Asp He Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu 255 260 265

CAG CCT GGA TAT TCT CCT TCC CCA ACC CA1 CCT CCT ACT GGA CAG TAT 912

Gin Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gin Tyr 270 275 280

ACG CTC TTC CCT CTT CCA CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC 960

Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gin Leu 285 290 295

CAC CCC CTG CTT CCT GAC CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC

1008

His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser

300 305 310 315

CCT CTT CTA AAC ACA TCC TAC ACC CAC TCC CAG AAT CTG TCT CAG GAA 1056

Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin Glu 320 325 330 GGG TAA

1062

Gly

(2) INFORMATION FOR SEQ ID NO 2

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 353 ammo acids

(D) TOPOLOGY linear

(ii) MOLECULE TYPE protein

(xi) SEQUENCE DESCRIPTION SEQ ID NO 2

Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala -21 20 -15 10

Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Lys Asp Leu Arg Val -5 1 5 10

Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser 15 20 25

Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala 30 35 40

Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys 45 50 55

Ala Gin Asp He Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met 60 65 70 75

Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly 80 85 90

Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu 95 100 105

Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp 110 115 120

Pro Asn Aia He Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val 125 130 135 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 He Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser Leu 205 210 215

Asp Gin He Pro Gly Tyr Leu Asn Arg He 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 He 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

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 178 ammo acids

(B) TYPE, amino acid (C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr 1 5 10 15

Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe 20 25 30

Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin 35 40 45

Gly Phe Arg Ala Lys He Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser 50 55 60

Leu Asp Gin He Pro Gly Tyr Leu Asn Arg He His Glu Leu Leu Asn 65 70 75 80

Gly Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala 85 90 95

Pro Asp He Ser Ser Gly Thr Ser Asp Ihr Gly Ser Leu Pro Pro Asn 100 10b 110

Leu Gin Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gin 115 120 125

Tyr Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Ihr Pro Val Val Gin 130 135 140

Leu His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr 145 150 155 160

Ser Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin 165 170 175

Glu Gly

(2) INFORMATION FOR SEQ ID NO: 4- (l) SEQUENCE CHARACTERISTICS

(A) LENGTH 18 ammo acids

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE peptide

(xi) SEQUENCE DESCRIPTION SEQ ID NO 4

Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Ala Leu Thr

1 5 10 15

Leu Asn

(2) INFORMATION FOR SEQ ID NO 5

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 18 amino acids

(B) TYPE amino acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE peptide

(xi) SEQUENCE DESCRIPTION SEQ ID NO 5

Ala Pro Pro Asp Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr 1 5 10 15

Leu Asn

(2) INFORMATION FOR SEQ ID NO 6

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 18 amino acids

(C) STRANDEDNESS single

(D) TOPOLOGY linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION. SEQ ID N0:6

Ala Pro Pro Asp Thr Ala Val Pro Ser Glu Thr Ser Leu Val Leu Thr 1 5 10 15

Leu Asn

(2) INFORMATION FOR SEQ ID NO- 7:

(i) SEQUENCE CHARACTERISTICS-

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY, linear

(n) MOLECULE TYPE peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 7

Ala Pro Pro Thr Thr Ala Val Pro Ser Glu Thr Ser Leu Val Leu Thr

1 5 10 15

Leu Asn

(2) INFORMATION FOR SEQ ID NO: 8.

(i) SEQUENCE CHARACTERISTICS'

(A) LENGTH: 5 amino acids

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE- peptide

(xi) SEQUENCE DESCRIPTION SEQ ID NO-8 Ala Pro Pro Thr Thr 1 5

(2) INFORMATION FOR SEQ ID NO 9

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 49 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(vn) IMMEDIATE SOURCE (B) CLONE ZC7623

(xi) SEQUENCE DESCRIPTION SEQ ID NO 9

GCGCGCAAGC TTGGACAAGA GAAGCCCGGC TCCTCCTGCT IGTGACCTC 49

(2) INFORMATION FOR SEQ ID NO 10

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 51 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(vi l) IMMEDIATE SOURCE (B) CLONE ZC7627

(xi) SEQUENCE DESCRIPTION SEQ ID NO 10

GCGCGCAAGC TTTCTAGACT ATCAGACGCA GAGGGTGGAC CCTCCTACAA G 51

(2) INFORMATION FOR SEQ ID NO 11

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 40 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single (D) TOPOLOGY: linear

(vi i) IMMEDIATE SOURCE: (B) CLONE. ZC7868

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 11

GTGTGTGAAT TCTAGACTAT CAGACGCAGA GGGTCGACCC 40

(2) INFORMATION FOR SEQ ID NO.12

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 35 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vn) IMMEDIATE SOURCE- (B) CLONE ZC7870

(xi) SEQUENCE DESCRIPTION SEQ ID NO 12

GTGTGTAAGC TTGGACAAGA GAAGCCCGGC GCCTC 35

(2) INFORMATION FOR SEQ ID NO 13

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 82 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear

(vi l) IMMEDIATE SOURCE. (B) CLONE. ZC8488

(xi) SEQUENCE DESCRIPTION SEQ ID NO 13 TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGGCRTG 60

ACTTTGAACT GATAGAGATC TT 82

(2) INFORMATION FOR SEQ ID NO 14

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 82 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(vi l) IMMEDIATE SOURCE (B) CLONE ZC8489

(xi) SEQUENCE DESCRIPTION SEQ ID NO 14

CTAGAAGATC TCTATCAGTT CAAAGTCAAA GCCAAAGAAG RCTGGAIGG AACAGCAGTG 60

GTTGGTGGGG CGACGCAGAG GG 82

(2) INFORMATION FOR SEQ ID NO 15

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 85 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(vi i) IMMEDIATE SOURCE (B) CLONE ZC8486

(xi) SEQUENCE DESCRIPTION SEQ ID NO 15

TCGACCCTCT GCGTCGGAGC TCCACCAGAC GAAGCTGTTC CAGACAGAGA CGAATTGGTT 60 TTGGAATTGA ACTGATAGAG ATCR 85

(2) INFORMATION FOR SEQ ID NO: 16:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 85 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(vn) IMMEDIATE SOURCE: (B) CLONE: ZC8487

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.16

CTAGAAGATC TCTATCAGTT CAARCCAAA ACCAATTCGT CTCTGTCTGG AACAGCRCG 60

TCTGGTGGAG CTCCGACGCA GAGGG 85

(2) INFORMATION FOR SEQ ID NO.17.

(i) SEQUENCE CHARACTERISTICS-

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) 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 15

Leu Asn

(2) INFORMATION FOR SEQ ID NO: 18- (i) SEQUENCE CHARACTERISTICS

(A) LENGTH 37 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(vi l) IMMEDIATE SOURCE

(B) CLONE ZC10086

(xi) SEQUENCE DESCRIPTION SEQ ID NO 18

TCGACCCTCT GCGTCGCCCC ACCAACCACT TGATAGA 37

(2) INFORMATION FOR SEQ ID NO 19

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 37 base pairs

(B) TYPE nucleic aciα

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(vi i) IMMEDIATE SOURCE

(B) CLONE ZC10087

(xi) SEQUENCE DESCRIPTION SEQ ID NO 19

GATCTCTATC AAGTGGRGG TGGGGCGACG CAGAGGG 37

(2) INFORMATION FOR SEQ ID NO 20

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 49 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(vi l) IMMEDIATE SOURCE

(B) CLONE ZC10095 (xi) SEQUENCE DESCRIPTION SEQ ID NO 20

TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCTGATAGA 49

(2) INFORMATION FOR SEQ ID NO 21

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 49 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(vn) IMMEDIATE SOURCE

(B) CLONE ZC10096

(xi) SEQUENCE DESCRIPTION SLQ ID NO 21

GATCTCTATC AGGATGGAAC AGCAGTGGTT GGFGGGGCGA CGCAGAGGG 49

(2) INFORMATION FOR SEQ ID NO 22

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 61 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(vn ) IMMEDIATE SOURCE

(B) CLONE ZC10120

(xi) SEQUENCE DESCRIPTION SEQ ID NO 22

TCGACCCTCT GCGTCGCCCC ACCAACCACI GCTGTICCAT CCAGAACT7C TTTGTGATAG 60

A 61 (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 61 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY- linear

(vi l) IMMEDIATE SOURCE:

(B) CLONE: ZC10121

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 23

GATCTCTATC ACAAAGAAGT TCTGGATGGA ACAGCAGTGG TTGGTGGGGC GACGCAGAGG 60

G 61

(2) INFORMATION FOR SEQ ID NO: 24-

(i) SEQUENCE CHARACTERISTICS-

(A) LENGTH- 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY- linear

(vi i) IMMEDIATE SOURCE:

(B) CLONE: ZC10118

(xi) SEQUENCE DESCRIPTION. SEQ ID NO.24

TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGGTπTG 60

ACRTGAACT GATAGA 76

(2) INFORMATION FOR SEQ ID NO: 25:

(ι) SEQUENCE CHARACTERISTICS (A) LENGTH- 76 base pairs

(C) STRANDEDNESS si ngl e

(D) TOPOLOGY l i near

(vn) IMMEDIATE SOURCE

(B) CLONE ZC10119

(xi) SEQUENCE DESCRIPTION SEQ ID NO 25

GATCTCTATC AGTTCAAAGT CAAAACCAAA GAAGTTCTGG ATGGAACAGC AGTGGRGGT 60

GGGGCGACGC AGAGGG 76

(2) INFORMATION FOR SEQ ID NO 26

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 82 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(vi i) IMMEDIATE SOURCE

(B) CLONE ZC10108

(xi) SEQUENCE DESCRIPTION SEQ ID NO 26

TCGACCCTCT GCGTCAGGCG GGCCCCACCA ACCACTGCTG TTCCATCCAG AACRCRTG 60

GTRTGACR TGAACTGATA GA 82

(2) INFORMATION FOR SEQ ID NO 27

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 82 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear (vi l) IMMEDIATE SOURCE

(B) CLONE ZC10109

(xi) SEQUENCE DESCRIPTION SEQ ID NO 27

GATCTCTATC AGRCAAAGT CAAAACCAAA GAAGTTCTGG ATGGAACAGC AGTGGRGGT 60

GGGGCCCGCC TGACGCAGAG GG 82

Claims

Claims What is claimed is:

1. An expression vector replicable in a eukaryotic host cell and comprising the following operably linked elements:

(a) a transcription promoter;

(b) a first DNA segment encoding a secretory leader;

(c) 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

(d) 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.

5. 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.

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.

10. 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- eu-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.

15. 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.

17. A cultured eukaryotic cell containing an expression vector according to claim 1, wherein said cell produces and secretes said TPO polypeptide.

18. A yeast cell according to claim 17.

19. 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 5 to 18, and wherein up to 35% of said residues of B are individually replaced by other amino acid residues.

20. 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) a transcription promoter;

(b) a first DNA segment encoding a secretory leader;

(c) 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 ammo acid residues; and (d) 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.

21. A method of increasing platelet number in a mammal comprising administering to said animal 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 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.