AU725159B2 - Methods for stimulating erythropoiesis using thrombopoietin - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990

ORIGINAL

COMPLETE SPECIFICATION

S

S

4.

Name of Applicant: Address of Applicant: Actual Inventor(s): Address for Service: University of Washington Seattle, Washington 98195, United States of America Kenneth Kaushansky DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete Specification for the invention entitled: "Methods for stimulating erythropoiesis using thrombopoietin" The following statement is a full description of this invention, including the best method of performing it known to us: -1- Description Methods for stimulating erythropolesis using thrombopoletin.

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 15 differentiation, with response to a particular cytokine often being lineage-specific and/or stage-specific.

Development of a single cell type, such as a platelet or erythrocyte, from a stem cell may require the coordinated action of a plurality of cytokines acting in the proper 20 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. 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 7-interferon are used in the treatment of certain cancers. EPO, which stimulates the development of erythrocytes, is used in the treatment of anemia arising from renal failure. Factors responsible for stimulation of megakaryocytopoiesis and thrombocytopoiesis resisted definitive characterization, due in part to lack of a good source, a lack of good assays, and a lack of knowledge as to the site(s) of production until recently, despite three decades of work to isolate and characterize them. The megakaryocytopoietic factor, referred to in the literature as "thrombopoietin" (recently reviewed by McDonald, Exp.

Hematol. 16:201-205, 1988; and McDonald, An. J. Ped.

Hematol. Oncol. 4I:8-21, 1992) has now been identified and isolated (see copending U. S. Patent Application Serial No. 08/252,491; Lok et al., Nature 369:565-568, 1994; and Kaushansky et al., Nature 269:568-571, 1994; all herein incorporated by reference).

15 Mild bleeding disorders (MBDs) associated with platelet dysfunctions are relatively common (Bachmann, Seminars in Hematology 29'-305, 1980), as are a number of congenital disorders of platelet function, including Bernard-Soulier syndrome (deficiency in platelet GPIb), Glanzmann's thrombasthenia (deficiency of GPIIb and GPIIIa), congenital afibrinogenemia (diminished or absent levels of fibrinogen in plasma and platelets), and gray platelet syndrome (absence of a-granules) In addition there are a number of disorders associated with platelet 25 secretion, storage pool deficiency, abnormalities in platelet arachidonic acid pathway, deficiencies of platelet cyclooxygenase and thromboxane synthetase and defects in platelet activation (reviewed by Rao and Holmsen, Seminars in Hematology 23: 102-118, 1986). At present, the molecular basis for most of these defects is not well understood.

Anemias are deficiencies in the production of red blood cells (erythrocytes) and result in a reduction in the level of oxygen transported by blood to the tissues of the body. Hypoxia may be caused by loss of large amounts of blood through hemorrhage, destruction of red blood cells from exposure to autoantibodies, radiation or chemicals, reduction in oxygen intake due to high altitudes or prolonged unconsciousness. When hypoxia is present in tissue, EPO production is stimulated and increases red blood cell production. EPO promotes the conversion of primitive precursor cells in the bone marrow into pro-erythrocytes which subsequently mature, synthesize hemoglobin and are released into the circulation as red blood cells. When the number of red blood cells in circulation is greater than needed for normal tissue oxygen requirements, the level of EPO in circulation is decreased.

Severe reductions in both megakaryocyte and erythrocyte levels can be associated with the treatment of 15 various cancers with chemotherapy and radiation and diseases such as AIDS, aplastic anemia and myelodysplasias. Levels of megakaryocytss and/or erythrocytes that become too low, for example, platelet S' counts below 25,000 to 50,000 and hematocrits of less than :20 25 are likely to produce considerable morbidity and in certain circumstances these levels are life-threatening.

In addition to treating the underlying disease, specific treatments include platelet transfusions for thrombocytopenia (low megakaryocyte levels) and 25 stimulation of erythropoiesis using EPO or transfusion of red blood cells for anemia.

Recent advances in molecular biology have greatly increased our understanding of hematopoiesis, but at the same time have shown the process to be extremely complex. While many cytokines have been characterized and some have proven clinical applications, there remains a need in the art for additional agents that stimulate proliferation and differentiation of myeloid and lymphoid precursors and the production of mature blood cells.

There is a particular need for agents that stimulate the development and proliferation of cells of the megakaryocytic and erythroid lineages, including platelets and red blood cells. There is a further need in the art for agents that can be used in the simultaneous treatment of cytopenias and anemias such as those caused by destruction of hematopoietic cells in bone marrow such as in the treatment of cancer with chemotherapy and radiation, and pathological conditions such as myelodysplasia, AIDS, aplastic anemia, autoimmune disease or inflammatory conditions. The present invention fulfills these needs and provides other, related advantages.

Summary of the Invention It is an object of the present invention to S 15 provide methods for stimulating erythropoiesis by culturing bone marrow or peripheral blood cells in the presqnce of TPO and EPO in amount sufficient to produce an increase in the number of erythrocytes or erythrocyte precursors as compared to cells cultured without TPO.

20 It is a further object of the invention to provide methods for stimulating erythropoiesis by culturing bone marrow or peripheral blood cells in the presence of a composition comprising TPO in an amount sufficient to produce an increase in the number of 25 erythrocytes or erythrocyte precursors as compared to cells cultured without TPO.

It is a further object of the invention to provide methods for stimulating erythropoiesis in a mammal by administering a composition comprising TPO in a pharmaceutically acceptable vehicle to produce an increase in proliferation or differentiation of erythroid cells.

It is a further object of the invention to provide methods for stimulating erythropoiesis in a mammal by administering a composition comprising EPO and TPO in a pharmaceutically acceptable vehicle to produce an increase in proliferation or differentiation of erythroid cells.

It is a further object of the invention to provide methods for stimulating erythropoiesis in a patient by administering a composition comprising EPO and TPO in amount sufficient to increase reticulocyte counts and erythroid colony formation.

It is a further object of the invention to provide methods for stimulating erythropoiesis in a patient by administering a composition comprising TPO in an amount sufficient for increasing reticulocyte counts at least 2-fold over baseline reticulocyte counts.

It is a further object of the invention to provide methods for stimulating erythropoiesis in a patient by administering a composition comprising TPO and EPO in an amount sufficient for increasing reticulocyte 15 counts at least 2-fold over baseline reticulocyte counts.

Within one aspect, the present invention provides that the TPO is human TPO. In another embodiment, the TPO comprises of a sequence of amino acids selected from group consisting of: the sequence of amino 20 acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 172; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 175; the sequence of amino acids 25 shown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue 353; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 353; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 172; the sequence of amino acids shown in SEQ ID. NO:2 from amino acid residue 22 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 175; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 172; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 175; and the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 353.

Within another aspect, the invention provides methods where a mammal is administered TPO of 1 x 105 to 100 x 105 units TPO/kg/day, preferably 5 x 105 to 50 x 105 units TPO/kg/day.

In another embodiment, the invention provides methods where a mammal is administered TPO of 1 x 105 to 100 x 105 units TPO/kg/day, preferably 5 x 105 to 50 x 105 units TPO/kg/day and EPO of 1 to 150 units EPO/kg/day.

Brief Description of the Drawinas Figure, 1 illustrates that following the addition 15 of TPO and EPO to cultured bone marrow cells, erythroid colony formation is enhanced relative to addition of EPO alone.

Figure 2 illustrates that following the addition of TPO to animals made pancytopenic with prior irradiation 20 and chemotherapy, the decline in red blood cell count is not as severe, and returns to normal sooner in animals given TPO.

*e Detailed Description of the Invention Prior to describing the present invention in detail, it may be helpful to define certain terms used herein: Allelic variant: An alternative form of a gene that arises through mutation, or an altered polypeptide encoded by the mutated gene. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.

cDNA: Complementary DNA, prepared by reverse transcription of a messenger RNA template, or a clone or amplified copy of such a molecule. Complementary DNA can be single-stranded or double-stranded.

Expression vector: A DNA molecule, linear or 15 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, and may also include one or more origins of repllcation, one or more 20 -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 25 intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator.

Gene: A segment of chromosomal DNA that encodes a polypeptide chain. A gene includes one or more regions encoding amino acids, which in some cases are interspersed with non-coding "intervening sequences" ("introns"), together with flanking, non-coding regions which provide for transcription of the coding sequence.

Molecules complementary to: Polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to CCCGTGCAT 3'.

Promoter: The portion of a gene at which RNA polymerase binds and mRNA synthesis is initiated.

As noted above, the present invention provides methods for stimulating thrombopoiesis and erythropoiesis using proteins having hematopoietic activity. As used herein, the term "hematopoietic" denotes the ability to stimulate the proliferation and/or differentiation of myeloid or lymphoid precursors as determined by standard assays. See, for example, Metcalf, Proc.,Natl. Acad. Sci.

USA 77: 5327-5330, 1980; Metcalf et al., J. Cell. Physiol.

11: 198-206, 1983; and Metcalf et al., Exp. Hematol. 288-295, 1987. Typically, marrow cells are incubated in 15 the presence of a test sample and a control sample. The cultures are then scored for cell proliferation and differentiation by visual examination and/or staining. A particularly preferred assay is the MTT colorimetric assay *of Mosman Immunol. Meth. 65: 55-63, 1983; incorporated 20 herein by reference).

As used herein, the term "erythropoiesis" denotes the proliferation and/or differentiation of erythroid precursor cells. Standard measures of erythroid cell proliferation and differentiation include hematocrit 25 and reticulocyte counts. Hematocrit is a measurement of red blood cells, and is commonly expressed as the percentage of total blood volume which consists of erythrocytes. Reticulocyte counts measure 1-2 day-old cells that contain mRNA (absent in mature erythrocytes) and aggregates of ribosomes as demonstrated by staining (Erslev, "Reticulocyte Enumeration", in Hematology, McGraw-Hill, NY, 1990). A reticulocyte count is the percentage of such cells per 500 or 1000 cells counted.

An average range for reticulocyte counts is 0.8% to 1.2%.

EPO is commercially available (R D Systems, Minneapolis, MN and Amgen, Thousand Oaks, CA) and activity is measured V Y.11LaI"* I I 11 -1- 9 by calibration against the second international reference preparation of erythropoietin (Annable et al., Bull. Wld.

Hlth. Org. 4.7:99, 1972) using an in vivo assay which measures the incorporation of 56 Fe into red blood cells of exhypoxic polycythemic mice (Cotes et al., Nature 121:1065, 1961) or by in vitro cell proliferation assay that uses a factor-dependent human erythroleukemic cell line, TF-1 (Kitamura et al., J. Cell. Phvsiol. 140:323, 1989).

The present invention is based in part upon the discovery that thrombopoietin (TPO) stimulates erythroid cell growth. When the present inventors administered TPO to thrombocytopenic mammals, in addition to an increase in platelets, surprisingly TPO was found to augment the 15 recovery of red blood cells and produce a rapid increase in hematocrit levels.

SThe sequences of cDNA clones encoding representative human and mouse TPO proteins are shown in SEQ ID NO:1 and SEQ ID NO:3, respectively and the 20 corresponding amino acid sequence are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively. Those skilled in the art will recognize that the sequences shown in SEQ ID NOS: 1 oo* and 2, and the human genomic sequence shown in SEQ ID and 6, correspond to single alleles of the human 25 gene, and that allelic variation is expected to exist. It will also be evident that one skilled in the art could engineer sites that would facilitate manipulation of the nucleotide sequence using alternative codons.

The present invention provides methods for stimulating erythropoiesis using proteins that are substantially homologous to the proteins of SEQ ID NO: 2 and their species homologs. By "isolated" is meant a protein which is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the proteins in a highly purified form, i.e. greater than pure, more preferably greater than 99% pure. The term "substantially homologous" is used herein to denote proteins having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO: 2 or their species homologs. Such proteins will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO: 2 or their species homologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 82:10915-10919, 1992.

Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a 'gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the standard one-letter S* codes). The percent identity is then calculated as: 20 Total number of identical matches x 100 ([length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two 25 sequences] Table 1 AR N DC Q EGH IL KM F PST WY V A 4 R -1 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 E -1 0 0 2 -4 2 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 1 -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3-2 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 F -2 -3 -3-3 -2 -3-3-3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2-2 0 -1 -2-1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2-2 -3 -2-1 -2-3 2 -1 -1-2 -1 3 -3 -2-2 2 7 V 0 -3-3 -3-1-2 -2-3 -3 3 1 -2 1 -1-2 -2 0 -3 -1 4 Substantially homologous proteins are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain. See, in general Ford et al., Protein Expression and Purification 2: 107, 1991, which is incorporated herein by reference.

Table 2 Conservative amino acid substitutions Basic: Acidic: Polar: Hydrophobic: arginine lysine histidine glutamic acid aspartic acid glutamine asparagine leucine isoleucine valine phenylalanine tryptophan tyrosine glycine alanine serine threonine methionine Aromatic: Small: Essential amino acids in TPO and EPO may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 24A, 1081-1085, 1989). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity receptor binding, in vitro or in vivo proliferative activity) to identify amino acid residues that are critical to the activity of the molecule. Sites of ligand-receptor interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography *or photoaffinity labeling. See, for example, de Vos et 15 al., Science 255:306-312, 1992; Smith et al., J. Mol.

.ioL. 22 :899-904, 1992; Wlodaver et al., FEBS Lett.

309:59-64, 1992.

Biologically active muteins of EPO based on elucidation of structure-function relationships have recently been identified (Boissel et al., J. of Biol.

Chem 268:15983-15993, 1993 and Higuchi et al., J. Biol.

Chem. 267:7703-7709, 1992). EPO isoforms having different sialic acid compositions are disclosed by Strickland et Sal. EP 0428267.

25 In general, cytokines are predicted to have a four-alpha helix structure, with the first and fourth helices being most important in ligand-receptor interactions and more highly conserved among members of the family. Referring to the human TPO amino acid sequence shown in SEQ ID NO:2, alignment of cytokine sequences suggests that these helices are bounded by amino acid residues 29 and 53, 80 and 99, 108 and 130, and 144 and 168, respectively (boundaries are 4 residues).

Helix boundaries of the mouse and other non-human TPOs can be determined by alignment with the human sequence. Other important structural aspects of TPO include the cysteine residues at positions 28, 50, 106 and 172 of SEQ ID NO:2.

In addition to the hematopoietic proteins disclosed above, the methods. of the present invention include utilization of fragments of these proteins and isolated polynucleotide molecules encoding the fragments.

Of particular interest are fragments of at least 10 amino acids in length that bind to an MPL receptor, and polynucleotide molecules of at least 30 nucleotides in length encoding such polypeptides. Polypeptides of this type are identified by known screening methods, such as by digesting the intact protein or synthesizing small, overlapping polypeptides or polynucleotides (and expressing the latter), optionally in combination with the 15 techniques of structural analysis disclosed above. The resultant polypeptides are then tested for the ability to specifically bind the MPL receptor and stimulate cell proliferation via the MPL receptor. Binding is determined by conventional methods, such as that disclosed by Klotz, Science 217: 1247, 1982 ("Scatchard analysis"). Briefly, a radiolabeled test polypeptide is incubated with MPL receptor-bearing cells in the presence of increasing concentrations of unlabeled TPO. Cell-bound, labeled polypeptide is separated from free labeled polypeptide by centrifugation through phthalate oil. The binding affinity of the test polypeptide is determined by plotting the ratio of bound to free label on the ordinate versus bound label on the abscissa. Binding specificity is determined by competition with cytokines other than TPO.

Receptor binding can also be determined by precipitation of the test compound by immobilized MPL receptor (or the ligand-binding extracellular domain thereof). Briefly, the receptor or portion thereof is immobilized on an insoluble support. The test compound is labeled, e.g. by metabolically labeling of the host cells in the case of a recombinant test compound, or by conventional, in vitro labeling methods radio-iodination). The labeled compound is then combined with the immobilized receptor, unbound material is removed, and bound, labeled compound is detected. Methods for detecting a variety of labels are known in the art. Stimulation of proliferation is conveniently determined using the MTT colorimetric or 3Hthymidine incorporation assay with MPL receptor-bearing cells. Polypeptides are assayed for activity at various concentrations, typically over a range of 1 nm to 1 hM.

Larger polypeptides of up to 50 or more residues, preferably 100 or more residues, more preferably about 140 or, more residues, up to the size of the entire mature protein are also provided. For example, analysis and modeling of the amino acid sequence shown in SEQ ID 15 NO:2 from residue 28 to residue 172, inclusive, suggest that these portions of the molecules are cytokine-like domains capable of self assembly. Also of interest are molecules containing this core cytokine-like domain plus one or more additional segments or domains of the primary translation product. Thus, other polypeptides of interest include those shown in Table 3.

o *o• 16 Table 3 Mouse TPO (SEQ ID NO:4): Cys (residue 51)--Val (residue 196) Cys (51) Pro (206) Cys (51)--Thr (379) Ser (45)--Cys (195) Ser (45)--Val (196) Ser (45)--Pro (206) Ser (45)--Thr (379) Met (24)--Cys (195) Met (24)--Val (196) Met (24)--Pro (206) :.*Met (24)--Thr (379) Met (l)--Cys (195) Met (1)--Val (196) Met (1)--Pro (206) Met (1)--Thr (379) Human TPO (SEQ ID 110:2) 20 Cys (28)--Val (173) Cys (28)--Argj (175) Cys (28)--Gly (353) *Ser (22)--Cys (172) Ser (22)--Val (173) Ser (22)--Arg (175) Ser (22)--Gly (353) Met (1)--Cys (172) Met (I)--Val (173) Met (1)--Arg (175) Met (1)--Gly (353) Those skilled in the art will recognize that intermediate forms of the molecules (e.g those having Ctermini between residues 196 and 206 of SEQ ID NO:4 or those having N-termini between residues 22 and 28 of SEQ ID NO:2) are also of interest, as are polypeptides having one or more amino acid substitutions, deletions, insertions, or N- or C-terminal extensions as disclosed above. Thus, the present invention provides hematopoietic polypeptides of at least 10 amino acid residues, preferably at least 50 residues, more preferably at least 100 residues and most preferably at least about 140 residues in length, wherein said polypeptides are substantially homologous to like-size polypeptides of SEQ ID NO:2.

The proteins used in the present invention for stimulating erythropoiesis can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that 15 can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., ibid., which are incorporated herein by reference. Production of recombinant EPO has been described in Lin et al., EP 25 014805; Fritsch et al., EP 0411678; Fritsch et al., EP 0205564; Hegwick et al., EP 0209539; Lin et al., WO 85/02610; U.S. Patent No. 4,677,195 and U.S. Patent No.

4,703,008. Production of recombinant TPO has been described in Lok et al. Nature 369:565-568, 1994; Bartley et al., Cell 77:1117-1124, 1994 and Sauvage et al., Nature 369:533-538, 1994.

In general, a DNA sequence encoding a cytokine is operably linked to a transcription promoter and terminator within an expression vector. The vector will commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

To direct a protein into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence is joined to the DNA sequence encoding a protein .15 of interest in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the protein of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No.

.5,143,830). The secretory signal sequence may be that normally associated with a protein of interest, or may be from a gene encoding another secreted protein.

Yeast 'cells, particularly cells of the genus Saccharoniyces, are a preferred host for producing cytokines for use within the present invention. Methods for transforming yeast cells with exogenous DNA and producing recombinant proteins therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al. U.S. Patent No. 4,931,373; Brake, U.S. Patent No.

4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075, which are incorporated herein by reference. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient 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. A preferred secretory signal sequence for use in yeast is that of the S. cerevisiae MFal gene (Brake, ibid.; Kurjan et al., U.S. Patent No. 4,546,082). Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, 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 15 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.

13:3459-3465, 1986 and Cregg, U.S. Patent No. 4,882,279.

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 25 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.

Cultured mammalian cells are also preferred hosts. 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 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982) and DEAE-dextran mediated transfection (Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), 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 No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134, 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 15 et al., J. Gen. Virol. 26:59-72, 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.

Sr* Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes Patent Nos.

4,579,821 and 4,601,978, which are incorporated herein by I 25 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 21 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 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 15 foreign proteins therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222; Bang et al., U.S. Patent No.

4,775,624; and WIPO publication WO 94/06463, which are incorporated herein by reference. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J.

Biosci. (Bangalore) 11:47-58, 1987.

Preferred prokaryotic host cells are strains of the bacteria Escherichia coli, although Bacillus and other genera are also useful. Techniques for transforming these 25 hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, Sambrook et al., ibid.). When expressing the proteins in bacteria such as E. coli, the protein may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate. The denatured protein is then refolded by diluting the denaturant. In the latter case, the protein can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein.

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 S: 15 an essential nutrient which is complemented by the selectable marker carried on the expression vector or cotransfected into the host cell.

Transgenic animal technology may be employed to produce TPO and EPO for use in the present invention. It is preferred to produce the proteins within the mammary glands of a host female mammal. Expression in the mammary 2* gland and subsequent secretion of the protein of interest into the milk overcomes many difficulties encountered in isolating proteins from other sources. Milk is readily 25 collected, available in large quantities, and well characterized biochemically. Furthermore, the major milk proteins are present in milk at high concentrations (from about 1 to 15 g/1).

From a commercial point of view, it is clearly preferable to use as the host a species that has a large milk yield. While smaller animals such as mice and rats can be used (and are preferred at the proof-of-concept stage), it is preferred to use livestock mammals including, but not limited to, pigs, goats, sheep and cattle. Sheep are particularly preferred due to such factors as the previous history of transgenesis in this species, milk yield, cost and the ready availability of equipment for collecting sheep milk. See WIPO Publication WO 88/00239 for a comparison of factors influencing the choice of host species. It is generally desirable to select a breed of host animal that has been bred for dairy use, such as East Friesland sheep, or to introduce dairy stock by breeding of the transgenic line at a later date.

In any event, animals of known, good health status should be used.

To obtain expression in the mammary gland, a transcription promoter from a milk protein gene is used.

Milk protein genes include those genes encoding caseins (see U.S. Patent No. 5,304,489, incorporated herein by reference), beta-lactoglobulin, a-lactalbumin, and whey 15 acidic protein. The beta-lactoglobulin (BLG) promoter is preferred. In the case of the ovine beta-lactoglobulin gene, a region of at least the proximal 406 bp of flanking sequence of the gene will generally be used, although larger portions of the 5' flanking sequence, up to about 5 kbp, are preferred, such as a -4.25 kbp DNA segment encompassing the 5' flanking promoter and honcoding portion of the beta-lactoglobulin gene. See Whitelaw et al., Biochem J. 286: 31-39, 1992. Similar fragments of promoter DNA from other species are also 25 suitable.

Other regions of the beta-lactoglobulin gene may also be incorporated in constructs, as may genomic regions of the gene to be expressed. It is generally accepted in the art that constructs lacking introns, for example, express poorly in comparison with those that contain such DNA sequences (see Brinster et al., Proc. Natl. Acad. Sci.

USA 85: 836-840, 1988; Palmiter et al., Proc. Natl. Acad.

Sci. USA 88: 478-482, 1991; Whitelaw et al., Transqenic Res. 1: 3-13, 1991; WO 89/01343; WO 91/02318). In this regard, it is generally preferred, where possible, to use genomic sequences containing all or some of the native introns of a gene encoding the protein or polypeptide of interest, thus the further inclusion of at least some introns from, e.g, the beta-lactoglobulin gene, is preferred. One such region is a DNA segment which provides for intron splicing and RNA polyadenylation from the 3' non-coding region of the ovine beta-lactoglobulin gene. When substituted for the natural 3' non-coding sequences of a gene, this ovine beta-lactoglobulin segment can both enhance and stabilize expression levels of the protein or polypeptide of interest. Within other embodiments, the region surrounding the initiation ATG of the cytokine sequence is replaced with corresponding sequences from a milk specific protein gene. Such replacement provides a putative tissue-specific initiation S: 15 environment to enhance expression. It is convenient to replace the entire cytokine pre-pro and 5' non-coding sequences with those of, for example, the BLG gene, although smaller regions may be replaced.

For expression of cytokines in transgenic animals, a DNA segment encoding the cytokine is operably linked to additional DNA segments required for its expression to produce expression units. Such additional segments include the above-mentioned promoter, as well as S* sequences which provide for termination of transcription 25 and polyadenylation of mRNA. The expression units will further include a DNA segment encoding a secretory signal sequence operably linked to the segment encoding the cytokine. The secretory signal sequence may be a native cytokine secretory signal sequence or may be that of another protein, such as a milk protein. See, for example, von Heinje, Nuc. Acids Res. 14: 4683-4690, 1986; and Meade et al., U.S. Patent No. 4,873,316, which are incorporated herein by reference.

Construction of expression units for use in transgenic animals is conveniently carried out by inserting a cytokine-encoding sequence into a plasmid or phage vector containing the additional DNA segments, although the expression unit may be constructed by essentially any sequence of ligations. It is particularly convenient to provide a vector containing a DNA segment encoding a milk protein and to replace the coding sequence for the milk protein with that of the cytokine of interest, thereby creating a gene fusion that includes the expression control sequences of the milk protein gene. In any event, cloning of the expression units in plasmids or other vectors facilitates the amplification of the cytokine sequence. Amplification is conveniently carried out in bacterial E. coli) host cells, thus the vectors will typically include an origin of replication and a selectable marker functional in bacterial host 15 cells.

The expression unit is then introduced into fertilized eggs (including early-stage embryos) of the chosen host species. Introduction of heterologous DNA can :'be accomplished by one of several routes, including microinjection U.S. Patent No. 4,873,191), retroviral infection (Jaenisch, Science 240: 1468-1474, 1988) or site-directed integration using embryonic stem (ES) cells (reviewed by Bradley et al., Bio/Technoloay 534-539, 1992). The eggs are then implanted into the 25 oviducts or uteri of pseudopregnant females and allowed to develop to term. Offspring carrying the introduced DNA in their germ line can pass the DNA on to their progeny in the normal, Mendelian fashion, allowing the development of transgenic herds.

General procedures for producing transgenic animals are known in the art. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al., Bio/Technology 6: 179-183, 1988; Wall et al., Biol.

Reprod. 32: 645-651, 1985; Buhler et al., Bio/Technology 8: 140-143, 1990; Ebert et al., Bio/Technology 2: 835-838, 1991; Krimpenfort et al., Bio/Technology 9: 844-847, 1991; Wall et al., J. Cell. Biochem. A2: 113-120, 1992; U.S.

Patents Nos. 4,873,191 and 4,873,316; WIPO publications WO 88/00239, WO 90/05188, WO 92/11757; and GB 87/00458, which are incorporated herein by reference. Techniques for introducing foreign DNA sequences into mammals and their germ cells were originally developed in the mouse. See, Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380- 7384, 1980; Gordon and Ruddle, Science 214: 1244-1246, 1981; Palmiter and Brinster, Cell 41: 343-345, 1985; Brinster et al., Proc. Natl, Acad. Sci. USA 82: 4438-4442, 1985; and Hogan et al. (ibid.). These techniques were subsequently adapted for use with larger animals, including livestock species (see WIPO publications 15 WO 88/00239, WO 90/05188, and WO 92/11757; and Simons et al., Bio/Technolov 6: 179-183, 1988). To summarize, in the most efficient route used to date in the generation of transgenic mice or livestock, several hundred linear molecules of the DNA of interest are injected into one of the pro-nuclei of a fertilized egg according to techniques which have become standard in the art. Injection of DNA into the cytoplasm of a zygote can also be employed.

Production in transgenic plants may also be employed. Expression may be generalized or directed to a 25 particular organ, such as a tuber. See, Hiatt, Nature 344:469-479, 1990; Edelbaum et al., J. Interferon Res.

12:449-453, 1992; Sijmons et al., Bio/Technoloav 8:217- 221, 1990; and European Patent Office Publication EP 255,378.

TPO and EPO are purified 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 protein 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 on concanavalin A or other lectin, thereby making use of the carbohydrate present on the protein. TPO can also be purified using 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 S. the affinity tag. EPO has been purified from uremic patients exhibiting elevated EPO levels, see U.S. Patent Nos. 4,397,840, 4,303,650 and 3,865,801 and Miyake et al.

J. Biol. Chem. 252:5558, 1977. EPO obtained from both 15 uremic patients and recombinant methods have been purified using reverse-phase HPLC (Hewick et al. U.S. Patent No.

4,677,195).

TPO proteins can be used therapeutically wherever it is desirable to increase proliferation of hematopoietic cells in the bone marrow, such as in the treatment of cytopenia and anemia, such as that induced by aplastic anemia, myelodysplastic syndromes, autoimmune diseases, AIDS, chemotherapy or radiation.

Compositions containing TPO will have useful application in the treatment of disorders characterized by low red blood cell production (anemia), particularly when accompanied by low platelet production (thrombocytopenia).

Various chemotherapeutic treatments of cancers and disease states are known to result in a combination of low platelet and erythrocyte levels in patients.

Compositions of TPO have been found effective for increasing the level of circulating erythrocytes and erythrocyte precursor cells. Reduction in the circulating levels of these cells are known as anemia. The erythrocyte level in blood is measured as the amount of hemoglobin per 100 ml or as the volume of packed red blood cells per 100 ml of blood. Patients are diagnosed as anemic if their hematocrit levels fall below 11-13 gm/100 ml of blood (depending upon the age and sex of the patient). The methods of the present invention are particularly useful for treatment of anemias associated with bone marrow failure, where a decrease in blood cell formation is associated with, for example, the toxic effects of chemotherapy.

TPO proteins have been found useful for simultaneous treatment of thrombocytopenia and anemia by increasing platelet production with a concurrent increase erythroid cell levels. Anemia and thrombocytopenia are associated with a diverse group of diseases and clinical situations that may act alone or in concert to produce the 15 condition. Lowered platelet counts may be associated with anemia, for example, by dilutional losses due to massive transfusions, or abnormal destruction of bone marrow. For example, chemotherapeutic drugs used in cancer therapy may suppress development of platelet and erythroid progenitor 20 cells in the bone marrow, and the resulting thrombocytopenia and anemia limit the chemotherapy and may necessitate transfusions. In addition, certain malignancies can impair platelet and erythrocyte production and distribution. Radiation therapy used to 25 kill malignant cells also kills platelet and erythroid progenitor cells. Abnormal destruction of platelets and erythrocytes can result from hematologic disorders such as leukemia and lymphoma or metastatic cancers involving bone marrow. Other indications for the proteins of the present invention to treat concurrent anemia and thrombocytopenia 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. Symptoms of anemia include dyspnea with exertion, dizziness, fatigue, and pallor of the skin and mucous membranes. When associated with thrombocytopenia, retinal hemorrhage can be present.

EPO has been used for stimulating erythrocyte production. EPO is a an acidic glycoprotein of approximately 34,000 dalton molecular weight and may occur in three forms: a, f, and asialo. The a and 0 forms differ slightly in carbohydrate components, but have the same potency, biological activity and molecular weight. The asialo form is an a or f form with the terminal carbohydrate (sialic acid) removed. Erythropoietin is present in very low concentrations in plasma when the body is in a healthy state and tissues are receiving sufficient 15 oxygenation from the existing number of erythrocytes.

See, for example, Lin et al., U.S. Patent 4,703,008; Lin et al., WO 85/02610; Fritsch et al. EP 0411678; Hewick et al., EP 0209539 and Hewick et al., U.S. Patent 4,677,195, which are incorporated herein by reference.

In normal individuals, red blood cel- production is precisely controlled to sufficiently oxygenate tissue without producing an overabundance of red blodd cells and impeding circulation. A reduction in red blood cell o production, resulting in tissue hypoxia, stimulates EPO expression and increases endogenous EPO found in plasma.

EPO increases red blood cell production by stimulating the conversion of primitive precursor cells in the bone marrow into pro-erythroblasts which subsequently mature, synthesize hemoglobin and are released into the circulation as red blood cells.

To provide for the stimulatory effect of TPO and EPO for erythropoiesis, the present invention does not always require the administration of exogenous EPO. As stated previously, a reduction in the level of red blood cells will in some cases result in an elevation in the endogenous levels of EPO (greater than 500 mU/ml of plasma) and administration of TPO alone may be sufficient.

In cases where expression of erythropoietin is not elevated, then erythropoietin is advantageoulsy administered with compositions of TPO.

As a therapeutic, EPO is administered to uremic patients where the hemoglobin concentration is less than gi/100 ml of blood. The route of administration can be either intravenous (IV) or subcutaneous (SC) and frequency varies from daily to weekly depending upon patient's physical condition (De Marchi et al. Clin. and Experim.

Rheumatol. 11:429-444, 1993; Miller et al., N. Eng. J. of Med. :322:1689-1692, 1990; Nissenson et al., Annals of Int.

Med. .114:402-416, 1991; Erslev, Sem. Oncol. 19'(8) Suppl.

:14-18, 1992 and PROCIT Epotin-alfa package insert, 15 Amgen, Thousand Oake, CA).

For pharmaceutical use, TPO and EPO are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.

Intravenous administration will be by bolus injection or 20 infuslon over a typical period of one to several rhours.

S In general, pharmaceutical formulations will include the hematopoietic proteins in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or .the5 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 and EPO may 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 1 x 105 to 100 x 105 units/kg of patient weight per day, preferably 5 x 105 to 50 x 105 units/kg per day. Therapeutic doses of EPO will generally be in the range of 10-150 U/kg of patient weight per day, preferably 50-150 U/kg per day. For both TPO and EPO, the exact dose will be determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. The proteins will commonly be administered over a period of up to 28 days following chemotherapy, radiation therapy or bone-marrow transplant or until a platelet count of >20,000/mm 3 preferably >50,000/mm 3 a hematocrit of 30-33% and reticulocyte counts that are at least 2-fold over baseline are achieved. More commonly, the proteins will be administered over one week or more, often over a period of seven to fourteen days. In general, a therapeutically effective amount of TPO or EPO 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.

25 platelets or erythrocytes) 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.

Treatment of anemias will continued until hematocrit levels of 30-33% and a reticulocyte count of at least 2fold over baseline, a level that adequate to have a significant impact upon hematocrit, are reached. As stated previously, a normal range for reticulocyte counts is 0.8% to TPO and EPO can also be administered in combination with other cytokines such as IL-3, -6 and -11; stem cell factor; G-CSF and GM-CSF. Within regimens of combination therapy, daily doses of other cytokines will in general be: GM-CSF, 5-15 pg/kg; IL-3, 1-5 Ag/kg; and G- CSF, 1-25 Mg/kg. Combination therapy with GM-CSF, for example, is indicated in patients with low neutrophil levels.

TPO and EPO can also be used ex vivo, such as in autologous marrow culture. Briefly, bone marrow is removed from a patient prior to chemotherapy and treated with TPO, optionally in combination with EPO, optionally in combination with one or more additional cytokines. The treated marrow is then returned to the patient after chemotherapy to speed the recovery of the marrow. In addition, TPO, alone and in combination with EPO, can also be used for the ex vivo expansion of marrow or peripheral blood progenitor (PBPC) cells. Prior to chemotherapy 15 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 TPO and EPO, optionally in 20 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 Smegakaryocyte cultures, which can then be returned to the patient following high-dose chemotherapy.

25 The invention is further illustrated by the following non-limiting examples.

Example I. Induction of Red Blood Cell Colony Formation At physiological levels of EPO, the addition of TPO stimulates the production of erythroid colony forming units (CFU-E) above levels of production seen with EPO alone.

Bone marrow cells were isolated from BDF 1 mice (Jackson Labs, Bar Harbor, ME) by femoral flushing. The cells (2 x 104/100 il clot) were resuspended in medium containing a medium (Flow Laboratories, McLean, VA) supplemented with 30% fetal calf serum (Hyclone, Logan, UT), 1% bovine serum albumin, 5 x 10 5 M P-mercaptoethanol; and 2 x 10 5 M CaC1 2 One hundred-twenty U/ml recombinant mouse TPO were added to select for early erythroid progenitors (BFU-E) and late erythroid progenitor (CFU-E) colonies.

Units of TPO activity were determined using the following assay. A crude BHK/pZGmpl-1 transfectant cell line that produces mouse TPO as described in copending U.S. Patent Application No. 08/252,491, filed June 1, 1994, was grown in serum-free medium. An asymptotic mitogenic activity curve was generated using this standard solution (conditioned culture medium) and BaF3/MPLRl.1 cells (IL-3-dependent cells expressing a stably 15 transfected Type I mouse MPL receptor). The point of 1/2 maximal activity (average of 16 curves) was assigned the value of 50 U/ml. The original standard solution was calculated to contain 26,600 U/ml mouse TPO.

For test samples, a culture supernatant or purified protein preparation was diluted in RPMI 1640 m. edium supplemented with 57 pM 2-mercaptoethanol, 2 mM Lglutamine, 1 mM sodium pyruvate, PSN, 10 mM HEPES and heat inactivated fetal bovine serum, generally using 8-24 dilutions. Briefly, 100 pl of diluted test sample or standard sample and 100 pl BaF3 cells (final cell number added about 500-10,000 cells/well) were combined in wells of a 96 well plate. Internal standards included eight 2-fold dilutions of 100 U/ml mouse TPO for mouse TPO assays, or eight 2-fold dilutions of 150 U/ml mouse TPO for human TPO assays. To each well was added 2 jtl 3

H-

thymidine (1 pCi/pl; Amersham), and the plates were incubated overnight at 37*C.

The contents of each well of each plate were transferred to a filter/plate using a Packard apparatus.

The filters were washed 8 times with water, and the filters were dried and counted. Units of TPO activity in each sample well were determined by comparison to the standard curve.

Human EPO (Amgen Inc., Thousand Oaks, CA) was added at varying concentrations in the range from 0 to 300 mUnits/ml with or without 120 units TPO. Clotting was initiated by the addition of 10% citrated bovine plasma.

The bone marrow cultures were incubated for two days at 37'C in a fully humidified atmosphere containing C0 2 Erythroid colonies contained greater than 40 cells.

After incubation, the clots were harvested, dried, stained w ith benzidine and erythroid colonies were counted (Broudy et al. Arch. of Biochem. and Biophys. 265:329-336, 1988).

The results have been indexed to that of the maximal colony growth and represent the mean of at least three separate experiments of two to three replicate plates.

Figure 1 shows that at physiological concentrations of EPO, in the range of 0-100 mUnits/ml, the addition of 120 U/ml TPO results in a significant increase the number of erythroid progenitor cell colonies.

Example II. TPO-Induced Increase in Reticulocvte Counts o* ~TPO-treated animals have elevated reticulocyte counts when compared to untreated animals.

*o ~Ten male BALB/c mice (Simonsen Labs, Gilroy, CA; 25 approximately 8 weeks old) were divided into a TPO-treated group of five animals and a sham group of five animals. A 12.5 kU dose of mouse recombinant TPO was prepared in mM Tris (pH 0.9% NaC1 and 0.25% rabbit serum albumin (RSA). The sham animals were treated with buffer alone.

Each animal was given a 0.2 ml intraperitoneal injection once daily with either 12.5 kU TPO or buffer for six consecutive days. On day=0, the animals were bled, and complete blood counts (CBC), including reticulocyte counts, were determined for each animal. On day=6, the animals were bled and sacrificed, and CBCs and reticulocyte counts were measured. For the sham treated animals, the reticulocyte counts went from a baseline at d=0 of 4.5% to 8.7% at d=6, and for the TPO-treated animals, the reticulocyte counts went from a baseline at d=0 of 5.3% to 12.0% at d=6.

Example III. Increase in Ervthropoiesis in TPOand EPO-Treated Animals TPO administered to animals that had been treated with radiation and a chemotherapeutic drug showed increased erythropoietic recovery when compared to untreated animals.

Four to six-week old, female BDF 1 mice (Simonsen Labs) were irradiated by exposure to 1 37 Cs using a Gammacell 40 irradiator (Nordion International Inc., 15 Kanata, Ontario, Canada) and treated with 1.2 mg of carboplatin (Bristol Laboratories, Princeton, NJ) injected intraperitoneally on day=0. The mice were treated either with TPO or TPO buffer only. TPO or TPO buffer was administered on day=l through day=14. The mice were divided to three groups as follows: Group 1: 8 mice treated with 500 cGy radiation 1.2 mg carboplatin TPO buffer Group 2: 8 mice treated with 500 cGy radiation 1.2 mg carboplatin 25 kU TPO/day for 14 days 25 Group 3: 8 mice treated with 500 cGY radiation 1.2 mg carboplatin 75 kU TPO/day for 14 days TPO was prepared in a buffer containing 20 mM Tris (pH 0.9% NaCI and 0.25% RSA. The mice were bled and CBCs were measured on days 0 (to establish baseline), 4, 6, 8, 10, 11 (CBC and reticulocyte counts), 13 (CBC and reticulocyte counts), 15, 18, 20, 22 and 25 (CBC and reticulocyte counts) and then sacrificed.

Figure 2 demonstrates that Group 2 and Group 3, TPO-treated animals, had a statistically shorter period of red blood cell nadir and their red blood cell levels P\opcrjcA2186866 rsI.dOC-44/(0X/l)O -36recovered to baseline significantly faster than animals treated with buffer only.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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 Sinvention. Accordingly, the invention is not limited except as by the appended claims.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

S.

*g SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: University of Washington Seattle

WA

98195 (ii) TITLE OF INVENTION: Methods of Stimulating Erythropoiesis Using Hematopoietic Proteins.

(iii) NUMBER OF SEQUENCES: 6 (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 01.0, Version #1.25 e (vi) CURRENT APPLICATION DATA: APPLICATION

NUMBER:

FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: Parker, Gary E REGISTRATION NUMBER: 31-648 REFERENCE/DOCKET NUMBER: 94-09PC (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 206-442-6673 TELEFAX: 206-442-6678 INFORMATION FOR SEQ ID NO:I: SEQUENCE CHARACTERISTICS: LENGTH: 1062 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE NAME/KEY: CDS LOCATION: 1059 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:I: AIG GAG CTG ACT GMA TG CTC CIC, GIG, GTC ATG CII CTC CIA ACT GCA 48 Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Ihr Ala 1 5 10 AGG CIA ACG CTG TCC AGC CC C C CTGTTTGA T TC 9 Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Al a Cys Asp Leu Arg Val 25 :CIC AGI AMA CIG CII CGI GAC ICC CAT GIC CTI CAC AGC AGA CTG AGC 144 Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser 40 CAG TGC CCA GAG GTT CAC CCT IIG CCI ACA CET GIC CIG CTG CCT GCT 192 Gin Cys Pro Giu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala 55 GIG, GAC ITT AGC TIG GGA GMA TGG MAA ACC CAG AIG GAG GAG ACC MAG 240 Val Asp Phe Ser Leu Gly Giu Irp Lys Ihr Gin Met Giu Giu Thr Lys 70 75 GCA CAG GAC All CTG GGA GCA GIG, ACC CTT CIG CTG GAG GGA GTG ATG 288 Ala Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met 90 GCA GCA CGG GGA CMA CIG GGA CCC ACT TGC TC ICA ICC ECt CTG GGG 336 Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly 100 105 110 CAG CIT TCT Gin Leu Ser 115 GGA CAG GIC CGI CC Gly Gin Val Arg Leu 120 CTC CTT GGG GCC CIG CAG AGC CIC Leu Leu Gly Ala Leu Gin Ser Leu 125 384 CIT GGA Leu Gly 130 ACC CAG CIT CCT CCA Thr Gin Leu Pro Pro 135 CAG GGC AGG ACC ACA GCT CAC MAG GAT Gin Gly Arg Thr Thr Ala His Lys Asp 140 432 MAT GCC AIC TIC CTG Asn Ala Ile Phe Leu 150 AGC TTC CM CAC CTG Ser Phe Gin His Leu 155 GGA GGG 1CC ACC CIC Gly Gly Ser Thr Leu 170 CTC CGA GGA MAG GIG Leu Arg Gly Lys Val 160 TGC GTC AGG CGG GCC Cys Val Arg Arg Ala 175 480 CGT TIC CIG AIG Arg Phe Leu Met

CT

Leu 165 5i8 S* 55 S

S

55 5

S.

S S *55* *55*5* CCA CCC ACC Pro Pro Thr AC, GAG CTC Asn Glu Leu 195

ACA

Thr 180

CCA

Pro GCT GTC CCC AGC Ala Val Pro Ser MAC AGG ACT ICT Asn Arg Thr Ser 200 ACC TCI CIA GTC CTC, Thr Ser Leu Val Leu 190 ACA CTIG Thr Leu GGA TTG TTG GAG Gly Leu Leu Glu ACA MAC TIC ACT Thr Asn Phe Thr 205 TGG CAG CAG GGA Trp Gin Gin Gly 624 GCC TCA Al a Ser 210 GCC AGA ACT ACT Ala Arg Ihr Thr GGC ICT GGG CII CTG Gly Ser Gly Leu Leu 215 GGT CIG CTG MC CAA Gly Leu Leu Asn Gin 235

MAG

Lys 220 672 TIC AGA GCC MAG AlT Phe Arg Al a Lys Ile 225 GAC CAA AIC CCC GGA Asp Gin Ile Pro Gly 245 ACC ICC AGG ICC Thr Ser Arg Ser- 720 768 TAC dIG MAC AGG Tyr Leu Asn Arg AlA Ile 250 CAC GMA CTC TTG MIT GGA His Glu Leu Leu Asn Gly 255 ACT CGI GGA Thr Arg Gly GAC ATT ICC Asp Ile Ser 275

CIC

Leu 260 TII CCI GGA CCC Phe Pro Gly Pro

TCA

Se r 265 CGC AGG ACC CIA Arg Arg Ihr Leu GGA GCC CCG Giy Ala Pro 270 CCC MAC dTd Pro Asn Leu 816 IdA GGA ACA IdA GAC Ser Gly Ihr Ser Asp 280 ACA GGC ICC CIG Ihr Gly Ser Leu

CCA

Pro 285 864 CAG CCT Gi n Pro 290 GGA TAT TCT CCT TCC Gly Tyr Ser Pro Ser 295 CCA ACC CAT CCI CCI Pro Thr His Pro Pro 300

ACG

Thr 305 CTC TTC CCI CIT CCA Leu Phe Pro Leu Pro 310 CCC ACC TIG CCC ACC CCI Pro Ihr Leu Pro Thr Pro 315 CCI TCT GCT CCA ACG CCC Pro Ser Ala Pro Thr Pro 330 ACT GGA CAG TAT Thr Gly Gin Tyr GIG GTC GAG CIC Val Val Gin Leu 320 ACC CCI ACC AGC Thr Pro Thr Ser 335 960 CAC CCC GIG CIT CCI His Pro Leu Leu Pro 325 1008 1056 CCI CTT CIA Pro Leu Leu

MAC

Asn 340 ACA ICC TAC ACC CAC Ihr Ser Tyr Thr His 345 ICC CAG MIT CTG TCT CAG GMA Ser Gin Asn Leu Ser Gin Glu 350

S

GGG TMA Gly -INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 353 amino acids (B TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:?: 1062 Glu Leu Thr Glu Leu Leu Leu Val Val 10 Met Leu Leu Leu Thr Ala Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro 25 Ala Cys Asp Leu Arg Val Arg Leu Ser Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Gin Cys Pro Glu Val His Pro Leu Pro Thr 55 Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys 10 75 s0

S.

Al a Al a Gin Leu Pro 145 Arg Pro' Asn Ala Phe 225.

Asp( Thr I Asp Gi r Al a Leu Sly 130 Asn Phe Pro Gl u Ser 210 krg ;Iin ~rg Ilie Asp Arg Ser 115 Thr Al a Leu Thr Leu 195 Al a Al a Ile Gly Ser 275 Ile Sly 100 Gly Gin Ile Met Thr 180 Pro Arg Lys Pro LeuI 260 Ser Leu Gin Gin Leu Phe Leu 165 Al a Asn Thr Ile ;i y 24 5 Phe ;ly Gly Leu Val Pro Leu 150 Val Val Arg Thr Pro 230 Ty r Pro Thr Al a Gly Arg Pro 135 Ser Gly Pro Thr Gly 215 GlyI Leu GlyI Ser Val Pro Leu 120 Gin Phe Sly Ser Ser 200 Se r Leu ksn Pro %sp 280 Thr Thr 105 Leu Gly Gin Ser Arg 185 Sly Gly Leu Arg Ser 265 Thr Leu 90 Cys Leu Arg His Thr 170 Thr Leu Leu ksn Ile 2 50 krg ILeu Leu Sly Thr Leu 155 Leu Ser Leu Leu Gin 235 His Arg Se r ILeu Ser Al a Thr 140 Leu Cys Leu Glu Lys 220 Thr GluI ThrI LeuI Glu Ser Leu 125 Al a Arg Val1 Val Thr 205 Irp Ser Leu eu Pro 285 Gly Leu 110 Gin His Gly Arg Leu 190 Asn Gin Arg Leu Gly 270 Pro Val Leu Ser Lys Lys Arg 175 Thr Phe Gin SerI Asn 255 AlaI Asn Met Sly Leu Asp Val 160 Al a Leu Thr Giy Leu 240 Pro .eu Gin Pro Sly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gin Tyr 290 295 300 Thr 305 Leu Phe Pro Leu Pro Thr Leu Pro Thr 315 Pro Val Val Gin Leu 320 His Pro Leu Leu Pro 325 Asp Pro Ser Ala Pro 330 Thr Pro Thr Pro Thr Ser 335 Pro Leu Leu Asn 340 Thr Ser Tyr Thr Ser Gin Asn Leu Ser Gin Glu 350 Gly a a.

a a INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1486 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vii) IMMEDIATE SOURCE: CLONE: 1081 (ix) FEATURE: NAME/KEY: COS LOCATION: 105. .1241 (xi) SEQUENCE DESCRIPTI ON: SEQ ID NO:3: CCTCGTGCCG GTCCTGAGGC CCTTCTCCAC CCGGACAGAG TCCTTGGCCC ACCTCTCTCC CACCCGACTC 1GCCGAAAGA AGCACAGAAG CTCAAGCCGC CTCC AIG GCC CCA GGA Met Ala Pro Gly AAG AlT CAG GGG AGA GGC CCC ATA CAG GGA 6CC ACT ICA Gil AGA CAC Lys Ile Gin Gly Arg Gly Pro Ile Gin Gly Ala Thr Ser Val Arg His 10 15

S

S

S

*SS*

CIG

Leu

GCA

Al a

CCC

Pro

CGA

Arg

CTG

Leu 85

CAG

Gin

GGA

Gly

CIC

Leu

CAG

Gin

CAC

His 165

GGA

Gly

GCC

Al a

GTG

Val

AGA

Arg

CTG

Leu

CCT

Pro

AGC:

*Ser

GTG

Val

CTG

Leu

GGC

Gly 150

MAG

Ly s

MAG

Ly s

AGA

Arg

GCA

Al a

CTC

Leu

AGI

Ser

GCT

Al a

MAG

Lys

ATG

Met

GGA

Gly 135

CTC

Leu

GAC

Asp

GIG

Val

AIG

Met

AGA

Arg

CIA

Leu

CAG

Gin

GTG

Val

GCA

Al a

GCA

Al a 120

CAG

Gin

CIA

Leu

CCC

Pro

CGC

Arg

GAG

Gi u

CTA

Leu

AAI

Asn

TGI

Cys

GAC

Asp

CAG

Gin 105

GCA

Al a

CII

Leu

GGA

Gly

AAI

Asn

TTC

Phe 185

CTG

Le u

ACT

Th r

A

Lys

CCC

Pro

TTT

Phe 90

GAC,

Asp

CGA

Arg

TCI

Ser

ACC

Thr

GCC

Al a 170

CIG

Leu

ACT

Th r

CIG

Leu

CIG

Leu

GAC

Asp.

75

AGC:

Ser AlT Ile

GGA

Gly

GGG

Gly

CAG

Gin 155

CTC

Leu

CII

Leu

GAT

Asp

ICC

Ser

CIG

Leu 60

GIC

Val

CIG

Leu

CIA

Leu

CAG

Gin

CAG

Gin 140

CIT

Leu

TIC

Phe

CIG

I eu

TG

Leu

AGC,

Ser 45

CGT

Arg

GAC

Asp

GGA

Gly

GGG

Gly

TTG

Leu 125

GTT

Val

CCI

Pro

TTG

Leu

GIA

Val CtC Leu 30

CCC

Pro

GAC

Asp

CCT

Pro

GMA

Gi u

GCA

Al a 110

GMA

Gi u

CGC

Arg

CTA

Leu

AGC

Ser

GMA

Gi u 190

CIG

Leu

GIA

Val

ICC

Ser

TTG

Leu

IGG

Trp 95

GTG

Val

CCC

Pro

CTC

Leu

CAG

Gin

TTG

Leu 175

GGT

Gi y

GCG

Al a

GCT

Al a

CAC

His iT Ser

AMA

Lys

ICC

Ser

TCC

Ser dCT Leu

GGC

Gi y 160

CAA

Gin

CCC

Pro

GCC

Al a

CCI

Pro

CIC

Leu

AIC

Ile

ACC

Thr

CTT

Leu

TGC

Cys

TTG

Leu 145

AGG

Arg

CAA

Gi n

ACC.

Thr

ATG

Met

GCC

Al a Cli Leu

CCT

Pro

CAG

Gin

CIA

Leu

CTC

Leu 130

GGG

Gly

ACC

Thr

CTG,

Leu CiT Leu

CTT

Leu

TGI

Cy s

CAC

His GTl Val

ACG

Thr dCT Leu 115

TCA

Ser

GCC

Al a

ACA

Thr

CII

Leu

TGT

Cys 195

CTT

Leu

GAC

Asp

AGC

Se r

CIG

Leu

GMA

51 u 100

GAG

51 u

TCC

Ser

CTG

Leu

GCI

Ala

CGG

Arg 180

GIC

Val 212 260 308 356 404 452 500 548 596 644 692 AGA CGG ACC CTG CCA ACC ACA GCT GIC CCA Arg Arg Thr Leu Pro Thr Thr Ala Val Pro 200 205 AGC AGI ACT ICI Ser Ser Thr Ser CAA CIC Gln Leu 740 CTC ACA CIA Leu Thr Leu 215 MAC MAG TIC, CCA MAC Asn Lys Phe Pro Asn 220 AGG ACT ICT GGA Arg Thr Ser Sly TIG TS GAG ACG Leu Leu Glu Thr 225 788 MAC TIC Asn Phe 230 AGT GTC ACA GCC Ser Val Thr Al a ACT GCT GGC CCT Thr Ala Gly Pro GSA CTT CTG AGC AGG Sly Leu Leu Ser Arg 240 CAG CIA AAI CAA ACC Gin Leu Asn Gin Thr 260

CTT

Leu 245 CAS GSA ITC AGA SIC Gi n Gly Phe Arg Val 250 MAG ATT ACT CCT GGT Lys Ile Thr Pro Gly 255 836 884 932 980 9.

*9 9 ICC AGG TCC CCA Ser Arg Ser Pro CAA ATC IdT GGA Gin Ile Ser Sly

TAC,

Tyr 270 CTG MAC AGG ACA Leu Asn Arg Ihr .CAC GSA His Sly 275 CCT GTG Pro *Val AAT GSA Asn Sly 280 ACT CAT GGG C~T Thr His Gly Leu SAC A1C, TCG CCC Asp Ilie Ser Pro 300 111 Phe 285 GCT GGA ACC TCA Al a Gly ThrSer CUT CAG ACC teu in Thr 290 CTG GMA qCC, CA Leu Glu Ala Ser 295 GSA GCT T7C AC.AMA GGC. ICC dIG Sly Ala Phe Asn Lys Gly Ser Leu 305 1028 9 9 999* 1GCA TIC Al a Phe 310 AAC CTC CAG GGI GSA Asri Leu Gin Gly Gly 315 CIT CCI CCT TCT CCA AGC CTI GCT CCI Leu Pro Pro Ser Pro.Ser Leu Ala Pro 320 1076 SAT GSA CAd ACA CCC Asp Sly His Ihr Pro 325 GGA ICI CCA CCC CAG Gly Ser Pro Pro Gin 345 TIC CCI CUT ICA CCT Phe Pro Pro Ser Pro 330 CTC CAd CCC CTG lT Leu His Pro Leu Phe 350 T(C CCC Leu Pro ACC AC CAT Ihr Thr His 340 1124 1172 CCI GAC CCI ICC Pro Asp Pro Ser ACC, ACC Ihr Thr 355 ATG CCI MAC Met Pro Asn

ICT

Ser 360 ACC GCC CCI CAT Thr Ala Pro His CCA SIC ACA Pro Val Ihr 365 AIG TAC Met Tyr CCI CAT CCC Pro His Pro 370 1220 *1 %j 7.1 A AGG MAT TTG TCT CAG GMA ACA TAGCGCGGGC ACTCGCCCAG TGAGCGTCTG Arg Asn Leu Ser Gin Glu Thr 375 CAGCTTCTCT CGGGGACMAG CTTCCCCAGG A.AGGCTGAGA GGCAGCTGCA TCTGCTCCAG A1GTTCTGCT TTCACCTAAA AGGCCCTGGG GAAGGGATAC ACAGCACTGG AGATTGTAAA ATTITAGGAG CTATTTTTTT 'rTACCTATC AGCAATATTC ATCAGAGCAG CTAGCGATCT TIGGICTAIT TTCGGTATMA ATTTGAAAAT CACTA INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 379 amino acids TYPE: amino acid TOPOLOGY: linear 1271 1331 1391 1451 .1486 4

B

*4*4 .4 4. 9* 4 4*44 4 4 (iiy 14OLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:4: Met

I

Al a Pro Gly Lys 5 Ile Gin Gly Arg Gly 10 Leu Pro- Ilie Gin Gly, Ala. Thr Ser Val Arg Ala Met Leu 35 Pro Ala Cys His Leu Ala 20 Leu Ala Val Asp Pro Arg Arg Met Gi u 25 Leu Thr Asp Leu Leu Leu Ala Pro Val Ala Al a Leu 55 Thr Leu Ser Ser Leu Asn Lys Leu Leu Arg Asp Ser His Leu His Ser Arg Ser Gin Cys Pro Val Asp Pro Leu Ser Pro Val Leu Pro Ala Val Asp Phe Ser Leu Gly Giu Trp Lys Thr Gin Thr Glu Gin Ser Lys Ala Gin Asp Ile Leu Gly Ala Val Ser 100 105 110 0* S. S S. S S

S

S. .5 S S

S

S..

S

S. S

S

a *5*S

*SSS

S

Leu Cy s Leu 145 Arg Gin Thr Thr Leu 225 Leu Leu Arg Ser Lys 305 Se r Leu Leu 130 Gly Thr Leu Leu Ser 210 'Leu Leu Asn Thr Leu 290 Gi y Leu Leu 115 Ser Al a Thr Leu Cy s 195 Gi n 61 u Ser Gi n His 275 Gin Ser Al a Gi u Ser Leu Al a Arg 180 Val Leu Thr Arg Th r 260 Gly Thr Leu Pro Gly Leu Gin His 165 Gi y Arg Leu Asn Leu 245 Ser Pro Leu Al a Asp 325 Val Met Al a 120 Leu Gly Gin 135 Gly Leu Leu 150 Lys Asp Pro Lys Val Arg Arg Thr Leu 200 Thr Leu Asn 215 Phe Ser Val 230 Gin Gly Phe Arg Ser Pro Val Asn Gly 280 Giu Ala Ser 295 Phe Asn Leu 310 Gly His Thr Al a Le u Giy Asn Phe 185 Pro Lys Thr Arg, Val 265 Thr Asp Gin Pro Arg Se r Thr Al a 170 Leu Thr Phe Al a Val 250 Gin His- Ile Gly Phe 330 Gly Gly Gin 155 Leu Leu Thr Pro Arg 235 Lys Ile Gly Ser Gly 315 Pro Gin Gin 140 Leu Phe Leu Al a Asn 220 Thr Ile, Ser Leu Pro 300 I eu Pro Leu 125 Val Pro Leu Val Val 205 Arg Al a Thr Gi y Phe 285 Gly Pro Ser Arg Leu Ser Gi u 190 Pro Thr Gi y Pro Tyr 270 Al a Al a Pro Pro Leu 61 n Leu 175 Gly Ser Ser Pro Gly 255 Leu 61 y Phe Se r Al a 335 Leu 61 y 160 Ci n Pro Ser Ci y Gl y 240 Oin Asn Thr Asn Pro 320 Leu Glu Pro Ser Pro Thr Thr His Gly Ser Pro Pro Gin Leu His Pro Leu Phe Pro Asp 340 345 Pro Ser Thr Thr Met Pro Asn Ser Thr Ala Pro His Pro Val Thr Met 355 350 365 Tyr Pro His Pro Arg Asn Leu Ser Gin Glu Thr .370 375 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 4823 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY:,linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: Joln(632..644, 876..1003, 1290..1376, 3309..3476, 3713. .4375) (xi) SEQUENCE DESCRIPTION: SEQ ID I III S

I.

Sb..

Ob S* S 59 S p S. 55 5

C

S S S. S *6p*

*S

~S S

S*

5 9

S-S.

9 5 *555

S

55.

5.55.5 5 CTCTTGCT T1CTTTCTT1 CTTTCTTTCT TTCTTTTTT

TCTTATTGCC

CAGGTACAAG

ACCACACCCT

C1GGTGGCGA

TTACAGGCAT

AGAATTCAGG

TTCACCCTGC

TTTCAGGATA

CAGGCTGGAG

CGATTCTCCT

GCTAGTTTTT

ACTCCTGACC

GAGCCACTGC

GCTTTGGCAG

CAGGCAGTCT

GATICTTCAC

TGCAATG%'TG

GT CTCAGC CT

TTGTATTTCG

TCAGGTGATC

ACCCGGCACA

IIC CAGGCTI CTTC CTAGAA

CCTTGGTCCG

CGATCTCGGC

CCCAAGTAGC

IAGAGCCGGG

CACCCGCCIT

CCAIATGCTT

GICAGCAICT

ACTTGGTIAA

CCTTIGCCCC

T1TTTGAGAC

TCACCACAAC

TIGGATTACA

GTTTCACCAT

GGACTCCCAA

TCATCACAAG

CAAGCCCTCC

AICTCACTC

AC CC TAC TCT

GGAGTTTCAC

CTCCGCCTCC

GGCAIGAACC

GTTAG1GAGG

AGTGCTGGGA

AAAATGT GAG

CCAGCATCIG

TTCTCAC

GCCCAGAAGT

120 180 240 300 360 420 480 540 GCAAGAGCCT AAGCCGCCTC CATGGCCCCA GGAAGGATIC AGGGGAGAGG CCCCAAACAG GGAGCCACGC CAGCCAGACA CCCCGGCCAG, A AIG GAG CIG ACT G GTGAGAACAC Met Glu Leu Thr 654

ACCIGAGGGG

TGCAGGGGGC

ATTCCCIGGG

ATTIC CTCCT

CTAGGGCCAT

AGGAAGCTGG

TIICAGGTCT

CATCTTTCAA

ATGGAAACAT

GGGAACCCAI

GGGTCCTGAA

CCTCACCTCT

GACAGAAGGG

TCICCCAAAA

TGGGAATTCC

CCTCATCTAA

GAGAGAGAAA GGAGACACGC ATAAGGGGTC IGAGGGGTGG TGATACCA GCTGACAATG G AA TIG CTC CIC Glu Leu Leu Leu CTG, ICC AGC CCG GCT Lou Ser Ser Pro Ala CTG CIT CG1 GAC TCC Leu Leu Arg Asp Ser 714 74 834 886

Z

GTG GIC AIG CTI CTC CIA ACT GCA AGG CIA ACG Val Val Met Leu Leu Leu Thr Ala Arg Leu Thr 15 CCT CCI GCT IGI GAd CTC CGA GTC CTC AGT AAA Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys 982 CAT SIC CTI CAC AGE AGA dIG GTGAGAACTC CCAACATTAI CCCCTI1ATC His Val Leu His Ser Arg Leu

CGCGTAACTG

GACCCAATGA

TTATTCITCA

GACIAGCCIG

CAATCTTTI

GTAAGACACC

CIATICIICC

CMTACAGCC

CTTAIIAGGC

CAACAG AGC Ser EAIACTCCCA GGAAGACACCE ATEACTECT CTAACTCCTT CATATIGIEC CCACC1ACTG ATCACACIET CIGACAAGGA CGCATIIAAA AGCTCTCGIC. TAGAGATAGI ACICAIGGAG TACCAIAGCT CTCTCTATIT CAGCTCCCII CTCCCCCCAC CAG TGC CCA GAG IGil CAC CCI TTG CCT ACA Gin Cys Pro Glu Val His Pro Leu Pro Thr 1033 1093 1153 1213 1273 1322 CCI GTE CIG ETG, CCT GCT GIG, GAC ITT AGE TIC GGA GAA TGG AAA ACC Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Irp Lys Ihr 65 CAG AIG GTAAGAAAGC CAICCCTAAC ETIGGETTCC CIA.AGTCCTG ICTICAGIII Gin Met 1370 1426 6 *6

S

*S

S

5 6

S

S

CCCACTGCTT

GCTTGGCCAC

GGCTTGCAGG

AATCGCTCAT

AAGCAAGACT

AAAAGACTGA

AGAGATATAA

AGGCCGAGGC

GAAACCCCGT

CCCAGCTACT

GTGAGCTGAG

AAAAGAAAAA

GCCACAATGC

TCTGAGAGAA

AAAGCTAGTA

GAACTCTATT

GAAGACATAT

GCAGCCTGAA

ATCTATCCTC

CAGTTCCTAT

CTCATACCTA

CCCATGGATT

CCTAACCCMA

TCCAATATGT

GGCCATGCCT

CATAIGICAT

ATCAAGATTC

ACTICTACAT

AGGCAGATCA

CTCTACTAAG

TGGAAGGC1G

ATCATGCCAA

AAAATTCTAC

CCTGCTTCCA

TTAAATTGCC

ATTCTTGTCT

CC GAGTGGAC

GCTAATTTAT

CAGAMAGAGA

AAGAACCCTA

GGGTC CCTTC

CATTTAGTTT

CTCCAACATT

TCTACATTCA

GAATAGATTT

TTGACCTATT

CCACAGATGA

AAATCACTGA

GTGGGCCGGG

CCTGAGGGCA

AATACAGAAT

AAGCAGGAGA

TGCACTCCAG

ATGTGTAAA1

TCATTTAAGC

CCCAAACTTA

GTT1GATGTT

TACACTTAAA

TAAGAGGGAC

CTAGMAGCAT

GCGT CCCTTC

TAGTCCTTTC

ATTTATTAIT

CTTGAGCTTT

CCTATGAIGA

GAAGCTGAAC

CCCGTTCAGT

CACAAAGCTG

AAGACTAGGT

GGCTCACGCC

GGAGTTTGAG

TAGCCGGGCA

ATCCCTTGAA

CCTGGGTGAC

TAATGAGTAA

CTCTGGCCCT

CCATGTAACA

IAGCATCCCC

TATACTGGCC

CATATTAAAC

GTTTTATGGG

TIC CTTCAGG

TTTTCATCCT

ATI ATTi GAG

ITAAAAATAT

TAGCCTGTGG

ACCATGAAAA

CTTCTTAAAT

GGAAGTACCA

CAAAAACAAG

TGTAATCCCA

AGCAGCCTGG

TGGTAGTGCA

CCCAGGAGGT

AAGAGCAAAA

AGTCCTATTC

AGCACTTCCT

TTACTGAAGC

ATTGIGGAMA

TGAACACCGG

TAACATGTGT

CAATAGTTTA

ACT GAGICAG

TATGATCATT

ACGGAGTCTC

CTCACCTTCA

ATAAGATGAT

GCTGGAGAGA

TGGCATGAAG

CTAAAATMAC

GTGAMACAAC

GCACTTTGGG

CCAACATGGC

TGCC1GTAAT

GGAGGTTGTA

CTCCGTCICA

CAGCTTTCAG

ACGAAAAGGA

TGCTATIC1T,

TGCTCGTACA

ACATCCCCCT

CTAGAAAGCA

AAAAAC TAAA

GGAAGAAGGG

ATGGIAGAGT

ACT CTAT CCC 1486 1546 1606 1666 .1726 1786 1846 1906 1966 2026 2086' 2146 2206 2266 2326' 2386 2446 2506 2566 2626 2686 2746 CCAGGCTGGA GTGCAGTGGC ATGATCTCAA CTCACTGCAA CCTCAGCCTC CCGGATTCAA GC GAT CTIC C

AGCIAATITG

AACTCCTGAC

TGAGCCACTG

CAGAAAGAGT

CAGCAACGTA

TAGAGGACAC

GAATTCCTGC

TGCTGGCTAC

CTCTCTTCCA

TGTCTCAGTC

TGTAITTGIG

CICAGGTGAT

CACCCAGCCT

AAAITIGCAG

AGAAAAAAGG

GGGAGTITTT

CCTGGGTGGG

TCCTAAGGCT

TCTCTTICTC

TCCCAAGIAG

GTAGAGATGG

CCACCTGCCT

ICAITCAGTT

CACIAGAACC

AGCTCTTCTC

GAAGCAGAGG

ACCITGGTCC

C.CCCACCCGC

AG GAG GAG Glu Glu CT GGGATTAC GGTTTCACC A

CAGCCICCCA

TAAAAATCAA

AAGAGGIAAA

ACTGAAACCA

CTGATGACCA

TGTCCAGTTC

TTTAGTGTG

AGGTGCCCAC

1GTTGGGCAG

AAGTGCTGGG

ATGATCCTAA

AGCTGTAACA

AGTGTAAGAC

GCTGT CGGGA

TCAGCCTGTA

CACCAIGCCC

GCIGATCTTG

ATTACAGGCG

GGTTTIGCAG

GGGCAGATTT

CAGGCTGGAC

GACTGIGAAG

TGATICACIC

2806 2866 2926 2986 3046 3106 3166 3226 3286 3338

S

.e

S

S q

S

S

*5t* CCCTTTGAGG CAGTGCGCTT ACC AAG GCA CAG GAC ATI CTG GGA Thr Lys Ala Gin Asp Ile Leu Gly GCA GIG ACC CIT CIG CIG GAG GGA GIG ATG GCA Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala 95 GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG CAG "Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin 105 110 CGT CTC CTC CTI GGG GCC CTG CAG AGC CIC CT Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu 120 125 GIAAGICCCC AGTCAAGGGA TCTGTAGAAA CTGTTCTTT GACCTGAGGG AAGAAGGGCT CTTCCAGGGA GCICAAGGGC AGTGCICCCI GCCAGCCACA ATGCCTGGGT ACITGGCATCC GGGAGGCCTG AGAICIGGCC CTGGIGTG GCCTCAGGAC CIT CCI CCA CAG GGC AGG ACC ACA GCT CAC MAG Leu Pro Pro Gin Gly Arg.Thr Ihr Ala His Lys 135 140 GCA CGG GGA CM CTG, Ala Arg Gly Gin Leu 100 CIT TCT GGA CAG GTC Leu Ser Gly Gin Val 115 GGA ACC CAG Gly Ihr Gin 130 CIGACICAGI CCCCCIAGAA AGAAGAGCTG ATCTACTAAG IGTCTITCCI ACTTAGACAA CATCCICTGC CCICAG GAT CCC AAI GCC ATC Asp Pro Asn Ala Ile 145 3386 3434 3476 3536 3596 3656 3712 3760 TIC CIG Phe Leu 150 AGC TIC CAA CAC Ser Phe Gin His CTC CGA GGA MAG GTG CGT TIC CTG AIG Leu Arg Gly Lys Val Arg Phe Leu Met 160 3808

CTT

Leu 165 GIA GGA GGG ICC Val Gly Gly Ser ACC CTC TGC GTC AGG CGG Ihr Leu Cys Val Arg Arg 170 175 ACC ICT CIA GTC CTC ACA Thr Ser Leu Val Leu Thr 190 GCC CCA CCC ACC ACA Ala Pro Pro Thr Thr 180 CIG MAC GAG CTC CCA Leu Asn Glu Leu Pro 195 3856 3904 GCI GIC CCC AGC AGA Ala Vai Pro Ser Arg 185 9* .9 9 3 9 909 MAC AGG ACT ICT GGA Asn Arg Ihr Ser Gly 200 ACT ACT GGC ICI 666 Thr Ihr Gly Ser Gly 215 116 TTG GAG Leu Leu Glu

ACA

Thr 205 MC TIC ACT 6CC Asn Phe Thr Ala ICA GCC AGA Ser Ala Arg 210 AGA GCC MAG Arg Ala Lys CII CIG MAG IGG CAG CAG GGA TIC Leu Leu Lys Trp Gin Gin Gly Phe 220 225 ATl CCT Ile Pro 230 GGT CTG CIG MAC Gly Leu Leu Asn

CAA

Gin 235 ACC ICC AGG ICC Thr Ser Arg Ser CIG GAC CAA AIC CCC Leu Asp Gin Ile Pro 240 GGA ACT CGI GGA CTC Gly Thr Arg Gly Leu 260 3952 4000 4048 4096 4144 4192

GGA

Gly 245 TAC CIG MAC AGG Tyr Leu Asn Arg ATA CAC GMA CTC 116 Ilie His Glu Leu Leu 250 CGC AGG ACC CIA GGA Arg Arg Ihr Leu Gly 270

A

Asn 255 III CCI GGA CCC ICA Phe Pro Gly Pro Ser 265 GGA ACA ICA GAd ACA Gly Thr Ser Asp Ihr 280 GCC CCG GAd All Ala Pro Asp Ilie 1CC ICA Ser Ser 275 GGC ICC CIG Gly Ser Leu CCA CCC Pro Pro 285 MAC CTC CAG CCI GGA TAT Asn Leu Gin Pro Gly Tyr 290 TCT CCI ICC Ser Pro Ser 295 CCA ACC CAT CCT Pro Thr His Pro

CCI

Pro 300 ACT GGA CAG TAT Thr Gly Gin Tyr CIC TIC CCI Leu Phe Pro 4240 CIT CCA Leu Pro 310 CCC ACC 116 CCC ACC Pro Thr Leu Pro Thr 315 CCI GIG GIC CAG Pro Vai Val Gin CIC CAC CCC CTG CTI Leu His Pro Leu Leu 320 4288

S

S

5*

S.

S

S

5555.5 CCT GAC CCT ICT GCT CCA ACG CCC ACC CCT ACC AGC CCT CIT CTA MAC Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn 325 330 335 340 ACA TCC TAC ACC CAC ICC CAG AAT CIG TCT CAG GMA GGG TAAGGTTCTC Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin Glu Gly 345 350 AGACACTGCC GACATCAGCA TTGTCTCGTG TACAGCTCCC TTCCCTGCAG GGCGCCCC1 GGAGACAACT GGACAAGATT ICCTACTTTC ICCTGAAACC CAAAGCCCTG GTAAAAGG( TACACAGGAC TGAAAAGGGA ATCATTTTTC ACTGTACATT ATAAACC1IC AGAAGCTA1 TTTTTAAGCT ATCAGCAATA CTCATCAGAG, CAGCTAGCTC TTTGGTCTAT TTTCTGCA( AATTTGCAAC TCACTGATTC TCAACATGCT CTTTTTCTGT GATAACTCTG CAAAGACC1 GGCTGGCCTG GCAGTTGAAC AGAGGGAGAG ACTAACCTTG AGTCAGAAMA CAGAGGAAC GTAATTTCCT TTGCTTCAAA TTCAAGGCCT TCCAACGCCC CCATCCCCTT TACTATCAl CTCAGTGGGA CTCTGATC INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 353 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala 1 5 10 Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val 25 Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser 40 rG rT rG Gr 4336 4385 4445 4505 4565 4625 4685 4745 4805 4823 Gin Cys Pro Giu Val His

C

C. 9.

9 C. C

S.C.

Val Al a Al a 61 n Leu Pro 145 Arg Pro Asn Al a Phe 225 Asp Thr Asp 61 n Al a Leu Gly 130 Asn Phe Pro 61 u Ser 210 Arg 61 n Arg Phe Asp Arg Ser 115 Thr Al a Leu Thr Leu 195 Al a Al a Ile Gly Ser Ile Gi y 100 Gly Gin Ile Met Thr 180 Pro Arg Ly s Pro Leu 260 Leu Leu Gin Gin Leu Phe Leu 165 Al a Asn Thr Ile Gi y 245 Phe G1y 70 Gly Leu Val Pro Leu 150 Val Val Arg Thr Pro 230 Tyr Pro Pro Leu Pro Thr 55 Giu Trp Lys Thr Ala Val Thr Leu 90 Gly Pro Thr Cys 105 Arg Leu Leu Leu 120 Pro Gin Gly Arg 135 Ser Phe Gin His Gly Gly Ser Thr 170 Pro Ser Arg Thr 185 Thr Ser Gly Leu 200 Gly Ser Gly Leu 215 Gly Leu Leu Asn Leu Asn Arg Ilie 250 Gly Pro Ser Arg 265 Pro Gin 75 teu Leti Giy Thr Leu 155 Leu Ser Leu Leu Gin 235 His Arg Val Met Leu Ser Al a Thr 140 Leu Cys Leu 61 u Lys 220 Thr Gi u Thr Leu Leu Giu Giu Giu Gly Ser Leu 110 Leu Gin 125 Ala His Arg Gly Val Arg Val Leu 190 Thr Asn 205 Trp Gi n Ser Arg Leu Leu Leu Gly 270 Pro Ala Thr Lys Val Net Leu Gly Ser Leu Lys Asp Lys Val 160 Arg Al a 175 Thr Leu Phe Thr Gin Gly Ser Leu 240 Asn Gly 255 Ala Pro Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu 275 280 GI n Pro 290 Gly Tyr Ser Pro Pro Thr His Pro Thr Gly Gin Tyr Thr 305 Leu Phe Pro Leu Pro 310 Pro Thr Leu Pro Th r 315 Pro Val Val GIn Leu 320 His Pro Leu Leu Pro 325 Asp Pro Ser Al a Pro 330 Thr Pro Thr Pro Thr Ser 335 Pro Leu Leu Asn 340 Thr Ser Tyr Thr His 345 Ser GI n Asn Leu Ser Gin Glu 350 Gi y 9.

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Claims (23)

1. A method for stimulating in vitro erythropoiesis comprising culturing bone marrow or peripheral blood cells with a composition comprising an amount of thrombopoietin (TPO) and erythropoietin (EPO) sufficient to produce an increase in the number of erythrocytes or erythrocyte precursors as compared to cells cultured in the absence of TPO.

2. The method of claim 1, wherein the TPO is human or mouse TPO.

3. The method of claim 1, wherein the TPO comprises a sequence of amino acids selected from group consisting of: the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 172; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 175; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue 353; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 353; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 172; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 175; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 172 the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 175; and the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 353.

4. A method of stimulating in vitro erythropoiesis comprising culturing bone marrow or peripheral blood cells with an amount of TPO sufficient to produce an increase in the number of erythrocytes or erythrocyte precursors as compared to cells cultured in the absence of TPO.

5. The method of claim 4, wherein the TPO is human or mouse TPO.

6. The method of claim wherein .the TPO comprises a sequence of amino acids selected from group consisting of: the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 172; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 173; the sequence of amino acids shown from amino acid residue 1 to residue 175; the sequence of amino acids shown from amino acid residue 1 to amino acid residue the sequence of amino acids shown from amino acid residue 22 to residue 353; the sequence of amino acids shown from amino acid residue 22 to residue 172; the sequence of amino acids shown from amino acid residue 22 to residue 173; the sequence of amino acids shown from amino acid residue 22 to residue 175; the sequence of amino acids shown from amino acid residue 28 to residue 172; in SEQ ID NO:2 in SEQ 353; in SEQ ID NO:2 ID NO:2 in SEQ ID NO:2 in SEQ ID NO:2 in SEQ ID NO:2 in SEQ ID NO:2 57 the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 175; and the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 353.

7. A method for stimulating erythropoiesis comprising administering to a mammal in need thereof a composition comprising TPO in combination with a pharmaceutically acceptable vehicle in an amount sufficient to produce an increase in proliferation or differentiation of erythroid cells.

8. The method of claim 7, wherein the TPO is human TPO.

9. The method of claim 7, wherein the TPO comprises a sequence of amino acids selected. from group consisting of: the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 172; the sequence of amino acids shown in SEQ ID NO: 2 from amino acid residue 1 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 175; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue 353; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 353; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 172; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 22 to residue 175; P:\opcr\jeU21 86XA rsLdIdoc)4/A)mX) -58- the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 172; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 175; and the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 28 to residue 353. The method of claim 7, wherein of 1 x 105 to 100 x units TPO/kg/day is administered to said mammal.

11. A method for stimulating erythropoiesis comprising administering to a mammal in need thereof a composition **,comprising TPO and EPO in combination with a pharmaceutically acceptable vehicle in an amount sufficient to produce an increase in proliferation or differentiation of erythroid cells as compared to mammals not administered TPO.

12. The method of claim 11, wherein the TPO is human TPO.

13. The method of claim 11, wherein the EPO is human EPO. *go

14. The method of claim 11, wherein the TPO comprises a sequence of amino acids selected from group consisting of: the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 172; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 173; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to residue 175; the sequence of amino acids shown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue 353; P \opr\jeh2 I 66 rs I .doc- 4/)418A) -59- the sequence of amino acids shown amino acid residue 22 to residue 353; the sequence of amino acids shown amino acid residue 22 to residue 172; the sequence of amino acids shown amino acid residue 22 to residue 173; the sequence of amino acids shown amino acid residue 22 to residue 175; the sequence of amino acids shown amino acid residue 28 to residue 172; the sequence of amino acids shown amino acid residue 28 to residue 173; the sequence of amino acids shown amino acid residue 28 to residue 175; and the sequence of amino acids shown amino acid residue 28 to residue 353. in SEQ ID NO:2 from in SEQ ID NO:2 from in SEQ ID NO:2 from in SEQ ID NO:2 from in SEQ ID NO:2 from in SEQ ID NO:2 from in SEQ ID NO:2 from in SEQ ID NO:2 from 0 0 *0 0S 0 0 0*

15. The method of claim 11, wherein of 1 x 105 to 100 x 5 units TPO/kg/day. and 1 to 150 units EPO/kg/day is administered to said mammal.

16. A method for stimulating erythropoeisis comprising administering to a patient in need thereof a composition comprising TPO and EPO, in combination with a pharmaceutically acceptable vehicle, in an amount sufficient for increasing reticulocyte counts as compared to patients treated in the absence of TPO and at least 2-fold over baseline reticulocyte counts.

17. A method for stimulating erythropoiesis comprising administering to a patient in need thereof a composition comprising TPO, in combination with a pharmaceutically acceptable vehicle, in an amount sufficient for increasing P:\OPER\JEH2188666 rs .doc) 7 /l/K) reticulocyte counts at least 2-fold over baseline reticulocyte counts.

18. A method of stimulating erythropoiesis and thrombopoiesis comprising administering to a patient in need thereof a composition comprising TPO and EPO, in combination with a pharmaceutically acceptable vehicle, in an amount sufficient for increasing reticulocyte counts as compared to a patient not administered TPO and at least 2-fold over baseline reticulocyte counts and platelet levels to at least 20,000/mm e*

19. A use of a TPO and EPO in the preparation of a medicament for increasing erythropoiesis as compared to the use of EPO in the absence of TPO. A use according to claim 19, wherein reticulocyte counts are increased at least 2-fold over baseline reticulocyte sees counts.

21. A use of TPO and EPO in the preparation of a medicament for increasing erythropoiesis and thrombopoiesis as compared to the use of EPO in the absence of TPO, wherein S reticulocyte counts are increased at least 2-fold over base- line reticulocyte counts and platelet levels are increased to at least 20,000/mm 3

22. A use of a TPO in the preparation of a medicament for stimulating erythropoiesis.

23. A use according to claim 22 wherein reticulocyte counts are increased at least 2-fold over baseline reticulocyte counts. 14 P:\oper\ch\2186866 -61

24. A method according to any one of claims 1 to 18, or a use according to any one of claims 19 to 23 as hereinbefore described with reference to the figures and/or examples. DATED this 4th day of August 2000 UNIVERSITY OF WASHINGTON By its Patent Attorneys DAVIES COLLISION CAVE sees 0.0. .0

49.. 1* S 0 S0 S