AU7589898A - Novel administration of thrombopoietin - Google Patents

Description

WO 98/52598 PCT/US98/10475 NOVEL ADMINISTRATION OF THROMBOPOIETIN Field of the Invention: The present invention relates to a new method of using thrombopoietin, and biologically active 5 derivatives and isoforms thereof, for the treatment of immune and/or hematopoietic disorders including thrombocytopenia. The use contemplates the co-administration of such materials together with a cytokine, especially a colony stimulating factor or interleukin. The use includes and is included within a method for treating a mammal having or at risk for thrombocytopenia by administering to the mammal in need of such treatment a therapeutically effective amount of the material(s). 10 Background of the Invention: The hematopoietic system produces the mature highly specialized blood cells known to be necessary for survival of all mammals. These mature cells include erythrocytes, specialized to transport oxygen and carbon dioxide, T- and B-lymphocytes, responsible for cell- and antibody mediated immune responses, platelets or thrombocytes, specialized to form blood clots, and 15 granulocytes and macrophages, specialized as scavengers and as accessory cells to combat infection. All of these specialized mature blood cells are derived from a single common primitive cel type referred to as the pluripotent stem cell found primarily in bone marrow. The mature highly specialized blood cells must be produced in large numbers continuously throughout the life of a mammal. The vast majority of these specialized blood cells are destined to 20 remain functionally active for only a few hours to weeks. Thus, continuous renewal of these mature blood cells, the primitive stem cells themselves, as well as any intermediate or lineage, committed progenitor cell lines lined between the primitive and mature cells, is necessary in order to maintain the normal steady state blood cell needs for continued life of the mammal. At the heart of the hematopoietic system lies the pluripotent stem cell(s). These cells are 25 relatively few in number and undergo self-renewal by proliferation to produce daughter stem cells, or they are transformed in a series of differentiation steps into increasingly mature lineage-restricted progenitor cells, ultimately forming the highly specialized mature blood cell(s). The underlying principal of the normal hematopoietic cell system appears to be decreased capacity for self-renewal as multipotency is lost and lineage-restriction and maturity is acquired. Thus, 30 at one end of the hematopoietic cell spectrum lies the pluripotent stem cell possessing the capacity for self-renewal and differentiation into all the various lineage-specific committed progenitor cells. At the other end of the spectrum lie the highly lineage-restricted progenitors and their progeny which have lost the ability of self renewal but have acquired mature functional activity. The proliferation and development of stem cells and lineage-restricted progenitor cells are 35 carefully controlled by a variety of hematopoietic growth factors or cytokines. Thus, hematopoietic growth factors may influence growth and differentiation of one or more lineages, may overlap with WO 98/52598 PCT/US98/10475 other growth factors in affecting a single progenitor cell-line, or may act synergistically with other factors. It will be appreciated from the foregoing that novel hematopoietic growth factors that effect survival, proliferation, differentiation or maturation of any of the blood cells or predecessors thereof 5 would be useful, especially to assist in the re-establishment of a diminished hematopoietic system caused by disease or after radiation- or chemo-therapy. Platelets are critical elements of the blood clotting mechanism. Depletion of the circulating level of platelets, called thrombocytopenia, occurs and is manifested in various clinical conditions and disorders. Clinical thrombocytopenia is commonly defined as a condition wherein the platelet count is 10 below about 150 X 109 per liter. The major causes of thrombocytopenia can be broadly divided into three categories on the basis of platelet life span, namely: 1) impaired production of platelets by the bone marrow, e.g., thrombocytopenia brought about by chemo- and radiation-therapy, 2) platelet sequestration in the spleen (splenomegaly) and 3) increased destruction of platelets in the peripheral circulation, e.g., thrombocytopenia brought about by autoimmune disorders. Additionally, in patients 15 receiving large volumes of rapidly administered platelet-poor blood products, thrombocytopenia may develop due to dilution factors. A more detailed description of thrombocytopenia and its causes, may be found in Schafner, "Thrombocytopenia and Disorders of Platelet Disfunction", Internal Medicine, John J. Hutton et al. Eds., Little, Brown & Co., Boston/Toronto/London, Third Ed. (1990) as well as International Patent Application No. PCT/US94/14553 (International Publication No. WO95/18858). 20 The therapeutic approach to the treatment of patients with thrombocytopenia is dictated by the severity and urgency of the clinical situation. The treatment is similar for HIV-associated and non HIV-related thrombocytopenia, and although a number of different therapeutic approaches have been used, the therapy remains clinically controversial. It will be appreciated from the foregoing that one way to treat thrombocytopenia would be to 25 obtain an agent capable of accelerating the differentiation and maturation of megakaryocytes or precursors thereof into the platelet-producing form. Considerable efforts have been expended on identifying such an agent. One commonly referred to is thrombopoietin (TPO), the subject of the present application. Other names for TPO commonly found in the literature at this time include: thrombocytopoiesis stimulating factor (TSF); megakaryocyte colony-stimulating factor (MK-CSF), 30 megakaryocyte growth and development factor (MGDF), megakaryocyte stimulating factor, megakaryocyte potentiator and mpl ligand. The cited International Patent Application PCT/US94/14553 describes the identification, isolation, production and use of an isolated mammalian megakaryocytopoietic proliferation and maturation promoting protein denominated the "MPL ligand" (ML), or more commonly, 35 "thrombopoietin" (TPO), which has been found capable of stimulating proliferation, maturation and/or differentiation of megakaryocytes into the mature platelet-producing form. 2 WO 98/52598 PCT/US98/10475 Attention is directed as well to International Patent Application Publications Nos. WO95/26746, WO95/21919 and WO95/21920. The PCT/US94/14553 application includes various aspects of associated embodiments of TPO, including a method of treating a mammal having or at risk for a hematopoietic disorder, notably 5 thrombocytopenia, comprising administering a therapeutically effective amount of TPO materials to the mammal. Optionally, TPO is administered as such or in combination with a cytokine, especially a colony stimulating factor or interleukin. For purposes disclosed in the International Patent Application, TPO is broadly defined as including TPO itself or various variants, derivatives or isoforms thereof, including fragments that share at least one biological property in common with intact TPO for the 10 treatment ofthrombocytopenia. "Biological property", when used in conjunction with the definition of the various TPO materials useful as described in the patent application, means that they have thrombopoietic activity or an in vivo effector or antigenic function or activity that is directly or indirectly caused or performed by the TPO material. With respect to the therapeutic use of thrombopoietin materials, as described in the 15 International Patent Application No. PCT/US94/14553, the TPO materials are therein described for administration in admixture with a pharmaceutically acceptable carrier via any of several administrative modes. The daily regimen is described as ranging from about 0.1 to 100 microgram/kilogram body weight, preferably from about 0.1 to 50 microgram/kg body weight, preferably at an initial dosage ranging from about 1 to 5 microgram/kg per day. Implicit within the 20 teachings of the patent application is a regimen of administering such a dosage rate over a period of several to many days following a projected or actual state of reduced platelet count. Published clinical studies of clinically administered thrombopoietin indicates a dosage and administration regimen consisting of the administration of thrombopoietin, subcutaneously at dosages of 0.03 to 5.0 microgram/kg body weight once per day over a period of ten days for a condition marked 25 by thrombocytopenia. See Abstract 1977, Blood 86 (1995). See also Abstracts 1012, 1014 and 1978, Blood 86 (1995). A single injection of pegylated (PEG) murine megakarocyte growth and development factor (mMGDF) into mice is sufficient to produce a stimulation of megakaryocyte frequency, size and ploidization. The PEG-mMGDF was administered to mice at a dose of 25 micrograms/kg as a single 30 intravenous injection. Blood, Feb. 1, 1997, 89(3):823-33). The in vivo effects of PEG-rhMGDF on hematopoiesis in normal mice is reported in Stem Cells, Nov. 1996, 14(6):651-60. See also Blood, Jul. 15, 1996, 88(2):511-21 and Blood, Jun. 15, 1996, 87(12):5006-15. The effects of rhTPO on myelosuppressive chemotherapy-induced thrombocytopenia in monkeys is reported in Brit. J. Haematol., Sept. 1996, 94(4):722-8. After treatment with nimustine on 35 day 0, monkeys intravenously received rhTPO at a dose of 0.04, 0.2 or 1.0 microgram/kg/d. Administration of rhTPO following nimustine treatment reduced the severity of thrombocytopenia and accelerated the rate of platelet recovery in a dose dependent fashion. The thrombopoietic effects of 3 WO 98/52598 PCT/US98/10475 PEG-rhMGDF in human patients with advanced cancer are reported in Lancet, Nov. 9, 1996, 348(9037):1279-81. In this study, PEG-rhMGDF was given by subcutaneous injection at a dose of 0.03, 0.1, 0.3 or 1.0 microgram/kg body weight before chemotherapy. Further, the effects of PEG rhMGDF on platelet counts after chemotherapy for lung cancer are reported in New Engl. J. Med., Feb. 5 6, 1997, 336(6):404-9 and the effects of this compound injected subcutaneously into rhesus macaques receiving intense marrow suppression by hepsulfam treatment is reported in Blood, Jan. 1, 1997, 89(1):15565. Likewise, the compound epoetin alfa, which is a given name for erythropoietin (marketed as EPOGEN by Amgen, Inc.), is a glycoprotein indicated for stimulation of red blood cell production. It 10 is indicated in a dosage and administration regimen consisting of starting doses over a range of 150 to 300 units per kg three times weekly for a period of many weeks in order to stimulate the proliferation of red blood cells in patients suffering from a depletion however realized. G-CSF and GM-CSF are cytokines which induce cycling and increase proliferation of myeloid progenitor cells. The pharmacokinetics of these cytokines have been studied and, different 15 administration regimens have been proposed for each of these drugs when used in conjunction with chemotherapy. Filgastrium, marketed as NEUPOGEN by Amgen, Inc., is a granulocyte colony stimulating factor (G-CSF). Its indicated regimen is the administration of from 5 to 10 microgram/kg subcutaneously daily for two weeks following chemotherapy. G-CSF is contra-indicated for 20 prechemotherapy administration. Clinical trials in which G-CSF was administered after chemotherapy and also both before and after chemotherapy have shown that preadministration worsened the toxic effects of the chemotherapeutic agent on the bone marrow. J. Nat. Canc. Inst.,1996, vol. 88, No. 19 and Exp. Hemat., 1994, 22:100-102. GM-CSF has also been studied clinically for use in conjunction with chemotherapy. In 25 contrast to G-CSF, GM-CSF has a relatively short effective half-life. Administration of GM-CSF is followed by a rapid increase in the proliferative activity of the hematopoietic precursors. However, within 72 hours after suspension of administration, a negative feedback is established resulting in a reduction of the proliferative activity of the marrow to values below baseline. The short half-life of GM-CSF has enabled this cytokine to be administered prior to chemotherapy. Cancer, 1993, vol. 72, 30 No. 10. The conventional regimen in administering materials for the proliferation of red blood cells or other primary blood cells to reverse the effects of thrombocytopenia, is continuous administration of therapeutically effective amounts of the biological material daily over a period of many days to patients in need of such therapy following chemotherapy resulting in thrombocytopenia. While GM-CSF may 35 have limited effectiveness when administered prior to chemotherapy, G-CSF worsens patient thrombocytopenia when administered prior to chemotherapy. The administration of cytokines having a relatively long half-life and which start hematopoietic progenitor cells cycling and proliferating, prior 4 WO 98/52598 PCT/US98/10475 to treatment with radiation or a chemotherapeutic agent has generally been contra-indicated since these cytoreductive treatments kill not only malignant cells, but also the proliferating progenitor cells as well. Another approach to the treatment of patients with thrombocytopenia or who are at risk of 5 thrombocytopenia as a result of a medical procedure, e.g. radiation and/or chemotherapy, is to rescue the patient with an autologous hematopoietic implant. In this approach, patients are administered a compound which mobilizes peripheral blood hematopoietic progenitor cells prior to the medical treatment which will induce thrombocytopenia. The mobilized progenitor cells are harvested by known leukapheresis procedures and then retransplanted into the patient after the onset of 10 thrombocytopenia in order to reestablish the patients autologous hematopoietic cells in the bone marrow. Unfortunately, many patients undergoing mobilization have very low numbers of progenitor cells at the time of harvest, necessitating multiple leukopheresis procedures which is painful and inconvenient for the patient. A method which improves the mobilization of progenitor cells thereby reducing the number of leukopheresis procedures is therefore highly desirable. 15 For convenience to physicians and patients alike, there exists an objective of developing alternative dosage/administration regimens of cytokines materials that would be advantageous and therapeutically equivalent or superior to reverse the effects of thrombocytopenia. Summary of the Invention One object of the present invention, therefore, is to provide a method of administering a 20 thrombopoietin which provides improved recovery from thrombocytopenia and overcomes the deficiencies noted above for existing methods of administering cytokines. Another object is to provide a method of administering a thrombopoietin to a mammal or patient receiving radiation and/or chemotherapy treatment which minimizes thrombocytopenia associated with such treatment and reduces the need for platelet transfusions in the mammal. 25 These and other objects which will become apparent during the course of the following descriptions of exemplary embodiments have been achieved by the method of the present invention. The present invention is based upon the unexpected and surprising finding that biologically active thrombopoietin materials can produce therapeutic effect by administering a single or low multiple daily dose of a therapeutically effective amount to a patient having or in need of treatment for 30 thrombocytopenia. This finding is based on a finding that thrombopoietin materials are growth factors for and are believed to act directly on early bone marrow stem cells and megakaryocyte progenitor cells, in contrast to G-CSF and GM-CSF which are thought to act on progenitor cells later in the hematopoietic cell lineage. The materials of the invention are capable of causing megakaryocyte differentiation of stem cells and increasing platelet count following administration. They induce 35 proliferation and differentiation of bone marrow hematopoietic cells, increasing the number of mature megakaryocytes, which yield increased numbers of circulating platlets. 5 WO 98/52598 PCT/US98/10475 Thus, the present invention is directed to a method of treating a mammal having or at risk for thrombocytopenia comprising administering to a mammal in need of such treatment a single or low multiple daily dose of a therapeutically effective amount of a thrombopoietin. In one aspect, the present invention is directed to the single administration of a therapeutically effective amount of a 5 thrombopoietin to such a mammal. In another aspect of this embodiment, the invention concerns the administration of a thrombopoietin to a mammal which receives at least one cycle of radiation and/or chemotherapeutic agent in need of such a cycle. Typically, the mammal will need one or more of such a cycle for the treatment of a tumor, malignancy, etc. In another aspect, the invention is directed to a method of 10 reducing the number of platelet transfusions in a thrombocytopenic patient. In a further aspect, the invention is directed to a method of mobilizing progenitor cells by the administration of a single or low-multiple daily dose of an effective amount of a thrombopoietin. Brief Description of the Drawings Figure 1 - Animals rendered pancytopenic, by a combination of 5.0 Gy of 15 y-irradiation and carboplatin (1.2 mg), were injected subcutaneously with 0.1 microgram rmTPO(335) for 1,2, 4, or 8 days. Panel A shows the platelet response to the treatment regimens while panels B and C represent the erythrocyte and leukocyte responses respectively over a 28 day period. The key set forth in panel B refers to all three panels. Figure 2 - Animals rendered pancytopenic, by a combination of 5.0 Gy of 20 y-irradiation and carboplatin (1.2 mg), were injected subcutaneously with a single dose at various levels of rmTPO(335) 24 hours after the initiation of the experiment. Panel A shows the platelet response to the treatment regimens while panels B and C represent the erythrocyte and leukocyte responses respectively over a 28 day period. The key set forth in panel B refers to all three panels. Figure 3 - Log-linear representations of the platelet (panel A) and erythrocyte (panel B) 25 responses to single administrations of rmTPO(335) given either subcutaneously or intravenously in animals rendered pancytopenic by a combination of 5.0 Gy of y-irradiation and carboplatin (1.2 mg). The cell numbers plotted are those measured on day 14 after initiation of the experiment. is base line zero level. Figure 4 - Animals rendered pancytopenic, by a combination of 5.0 Gy of 30 y-irradiation and carboplatin (1.2 mg), were injected intravenously with a single dose at various levels of rmTPO(335) 24 hours after the initiation of the experiment. Panel A shows the platelet response to the treatment regimens while panels B and C represent the erythrocyte and leukocyte responses respectively over a 28 day period. The key set forth in panel B refers to all three panels. Figure 5 - Animals rendered pancytopenic, by a combination of 5.0 Gy of 35 y-irradiation and carboplatin (1.2 mg), were injected subcutaneously with a single dose at 24 hours after the initiation of the experiment with various forms of rmTPO(153) conjugated to polyethylene 6 WO 98/52598 PCT/US98/10475 glycol (peg) of either 20K or 40K molecular weight. Panel A shows the platelet response to the treatment regimens while panels B and C represent the erythrocyte and leukocyte responses respectively over a 28 day period. The key set forth in panel B refers to all three panels. Figure 6 - Animals rendered pancytopenic, by a combination of 5.0 Gy of 5 y-irradiation and carboplatin (1.2 mg), were injected subcutaneously with a single dose at 24 hours after the initiation of the experiment with either rmTPO(335) or rmTPO(153) conjugated to polyethylene glycol (peg) of 40K molecular weight. Panel A shows the platelet response to the treatment regimens while panels B and C represent the erythrocyte and leukocyte responses respectively over a 28 day period. The key set forth in panel B refers to all three panels. 10 Figure 7 - Animals rendered pancytopenic, by a combination of 5.0 Gy of y-irradiation and carboplatin (1.2 mg), were injected intravenously with a single dose at 24 hours after the initiation of the experiment with either rmTPO(335) or rmTPO(153) conjugated to polyethylene glycol (peg) of 40K molecular weight. Panel A shows the platelet response to the treatment regimens while panels B and C represent the erythrocyte and leukocyte responses respectively over a 28 day 15 period. The key set forth in panel B refers to all three panels. Figure 8 - Figure 8 shows the thrombocyte level of 6 Gy irradiated mice at the time of nadir in placebo controls as a function of the time of administration of a single i.p. dose (0.3 microgram) of TPO at each of the various time points indicated in the Figure. Figure 9 - Figure 9 shows the thrombocyte level of 6 Gy irradiated mice at the time of nadir in 20 placebo controls as a function of the time of administration of a single i.p. dose (30 microgram) of TPO at two hours before (-2h) irradiation. Figure 10 - In order to model a protracted form of cytoreductive treatment very similar to radiation or chemotherapy, total body irradiation (TBI) was given to mice in three equal fractions of 3 Gy separated by 24 hours each. TPO was given in a total dose of 0.9 microgram in three different 25 dosing regimen; 3 x 0.3 microgram at +2h from irradiation, 0.9 microgram at +2h from irradiation, and 0.9 microgram at -2h from irradiation. The resulting thrombocyte levels are shown vs a placebo. Figure 11 - This Figure shows hemopoietic progenitor cell data of the femur for the regimen of Figure 10. Figure 12 - This Figure shows hemopoietic progenitor cell data of the spleen for the regimen 30 of Figure 10. Figure 13 - Figure 13 shows pharmacokinetic data following three doses of 0.3 microgram or a single dose of 0.9 microgram of TPO. Figure 14 - Figure 14 shows median platelet counts averaged over dose levels by study arm for Example 7, cycle 1 (chemotherapy alone). 35 Figure 15 - Figure 15 shows median platelet counts averaged over dose levels by study arm for Example 7, cycle 2 (chemotherapy and rhTPO). 7 WO 98/52598 PCT/US98/10475 Figure 16 - Figure 16 shows median platelet counts averaged over dose levels by study arm for Example 7, cycle 3 (chemotherapy and rhTPO). Figure 17 - Figure 17 shows median platelet counts averaged over dose levels by study arm for Example 7, cycle 4 (chemotherapy and rhTPO). 5 Figure 18 - Figure 18 shows median platelet count by rhTPO dose level for arm C, cycle 2 of Example 7. Figure 19 - Figure 19 shows median platelet count by rhTPO dose level for arm D, cycle 2 of Example 7. Figure 20 - Platelet counts in mice exposed to a single inhalation of rhTPO; see 10 Example 8. Figure 21 - Platelet counts in mice exposed to multiple inhalations of rhTPO; see Example 8. Figure 22 - Expansion of CD34 + cells by TPO/FL/KL. In Figure 22 is shown the expansion of CD34 cells over 8 weeks in cultures containing TPO, Flt-3 and c-kit ligand. An expansion of over a 10e6 fold is observed; see Example 14. 15 Figure 23 - Expansion of CD34+CD38- cells by TPO/KL/FL. As shown in Figure 23 the subpopulation ofCD34 CD38 cells is also expanded. At week one this subpopulation only makes up only 8% of the culture but by week 8 comprises 33% of the culture, indicating a 4 fold expansion. This demonstrates both an expansion and maintanence of a primitive progenitor population in these expanded cultures; see Example 14. 20 Figure 24 - Expansion of multilineage activity by TPO/KL/FL. In Figure 24 the ability of the expanded cultures to give rise to multilineage colonies in vitro is shown. The number of colonies generated increases porportionally with the expansion of CD34 + cells in the culture. This indicates that the expanded cells are maintaining their multipotential activity; see Example 14. Definitions 25 As used herein, a "mammal having or at risk for thrombocytopenia" means a mammal, including a human, which is experiencing thrombocytopenia, that is, a platelet count which is below the platelet count for average normal individuals in the mammal population. In humans, thrombocytopenia is defined as a condition where the platelet count is below about 150 x 10 9 per liter of blood. The mammal may, however, also be at risk for thrombocytopenia, meaning that the mammal 30 may forseeably experience a thrombocytopenic condition as a result of a specific treatment which is known to cause thrombocytopenia. For example, a mammal is at risk for thrombocytopenia if the mammal will be administered a radiation and/or chemotherapeutic treatment which is known to induce thrombocytopenia in the treated mammal. In other words, it is clearly forseeable that the mammal is at risk for or has a high probability of experiencing thrombocytopenia as a result of the treatment which is 35 known to induce thrombocytopenia. Such mammals at risk for thrombocytopenia may be treated with the method of the present invention. Included within the scope of this invention are mammals having or 8 WO 98/52598 PCT/US98/10475 at risk for thrombocytopenia as a result of a disfunctional liver, e.g. liver cirrhosis, and mammals undergoing progenitor cell mobilization therapy and apheresis, generally prior to radiation and/or chemotherapy treatment. The term "cytokine" is a generic term for proteins released by one cell population which act on 5 another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone, insulin-like growth factors, human growth hormone including N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and 10 leutinizing hormone (LH), hematopoietic growth factor, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factors (TNF-a and TNF-P3), mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, nerve growth factors (NGFs) such as NGF-P, insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factors, interferons (IFNs) such as interferon-a, -3 and -y, colony 15 stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM CSF), and granulocyte-CSF (G-CSF), interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL 6, IL-7, IL-8, IL-9, IL-11, IL-12 and other polypeptide factors including LIF, SCF, FLT-3 ligand and kit-ligand (KL). As used herein the foregoing terms are meant to include proteins from natural sources or from recombinant cell culture. Similarly, the terms are intended to include biologically active 20 equivalents; e.g., differing in amino acid sequence by one or more amino acids or in type or extent of glycosylation. The term "biologically active" when used in conjunction with a thrombopoietin (TPO) means thrombopoietin or a thrombopoietic polypeptide that exhibits thrombopoietic activity or shares an effector function of the mpl ligand isolated from aplastic porcine plasma or expressed in recombinant 25 cell culture. A principal known effector function of the mpl and stimulating the incorporation of labeled nucleotides ( 3 H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected with human mpl P. Another known effector function of the mpl ligand or polypeptide herein is the ability to stimulate the incorporation of 35S into circulating platelets in a mouse platelet rebound assay. Yet another known effector function of mpl ligand is the ability to stimulate in vitro human 30 megakaryocytopoiesis that may be quantitated by using a radio labeled monoclonal antibody specific to the megakaryocyte glycoprotein GPIIbIIIa. The terms "mpl ligand", mpl ligand polypeptide", "ML", "thrombopoietin" or "TPO" are used interchangeably herein and include any polypeptide that possesses the property of binding to mpl, a member of the cytokine receptor superfamily, and having a biological property of mpl ligand. An 35 exemplary biological property is the ability to stimulate the incorporation of labeled nucleotides (e.g. 3 H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected with human mpl. Another 9 WO 98/52598 PCT/US98/10475 exemplary biological property is the ability to stimulate the incorporation of 3 5 S into circulating platelets in a mouse platelet rebound assay. This definition encompasses a polypeptide isolated from a mpl ligand source such as aplastic porcine plasma described herein or from another source, such as another animal species, including humans, or prepared by recombinant or synthetic methods. 5 Examples include TPO(332) and rhTPO 332 . Also included in this definition is the thrombopoietic ligand described in WO 95/28907 having a molecular weight of about 31,000 daltons (31 kd) as determined by SDS gel under reducing conditions and 28,000 daltons (28kd) under non-reducing conditions. The term "TPO" includes variant forms, such as fragments, alleles, isoforms, analogues, chimera thereof and mixtures of these forms. For convenience, all of these ligands will be referred to 10 below simply as "TPO" recognizing that all individual ligands and ligand mixtures are referred to by this term. Preferably, the TPO is a compound having thrombopoietic activity or being capable of increasing serum platelet counts in a mammal. The TPO is preferably capable of increasing endogenous platelet counts by at least 10%, more preferably by 50%, and most preferably capable of 15 elevating platelet counts in a human to greater than about 150 X 109 per liter of blood. The TPO of this invention preferably has at least 70% overall sequence identity with the amino acid sequence of the highly purified substantially homogeneous porcine mpl ligand polypeptide and at least 80% sequence identity with the "EPO-domain" of the porcine mpl ligand polypeptide. Alternatively, the TPO of this invention may be a mature human mpl ligand (hML), or a variant or post 20 transcriptionally modified form thereof or a protein having about 80% sequence identity with mature human mpl ligand. Alternatively, the TPO may be a fragment, especially an amino-terminus or "EPO domain" fragment, of the mature human mpl ligand. Preferably, the amino terminus fragment retains substantially all of the human ML sequence between the first and fourth cysteine residues but may contain substantial additions, deletions or substitutions outside that region. According to this 25 embodiment, the fragment polypeptide may be represented by the formula: X-hTPO(7-151 )-Y Where hTPO(7-151) represents the human TPO (hML) amino acid sequence from Cys 7 through Cys 15 1 inclusive; X represents the amino group of Cys 7 or one or more of the amino-terminus amino acid residue(s) of the mature TPO or amino acid residue extensions thereto such as Met, Lys, 30 Tyr or amino acid substitutions thereof such as arginine to lysine or leader sequences containing, for example, proteolytic cleavage sites (e.g. Factor Xa or thrombin); and Y represents the carboxy terminal group of Cys 15 1 or one or more carboxy-terminus amino acid residue(s) of the mature TPO or extensions thereto. A "TPO fragment" means a portion of a naturally occurring mature full length mpl ligand or 35 TPO sequence having one or more amino acid residues or carbohydrate units deleted. The deleted amino acid residue(s) may occur anywhere in the peptide including at either the N-terminal or C 10 WO 98/52598 PCT/US98/10475 terminal end or internally, so long as the fragment shares at least one biological property in common with mpl ligand. Mpl ligand fragments typically will have a consecutive sequence of at least 10, 15, 20, 25, 30 or 40 amino acid residues that are identical to the sequences of the mpl ligand isolated from a mammal including the ligand isolated from aplastic porcine plasma or the human or murine ligand, 5 especially the EPO-domain thereof. Representative examples of N-terminal fragments are TPO(153), hML 153 or TPO(Met- 1 1-153). The terms "TPO isoform(s)" and "TPO sequence isoform(s)" or the term "derivatives" in association with TPO, etc. as used herein means a biologically active material as defined below having less than 100% sequence identity with the TPO isolated from recombinant cell culture, aplastic porcine 10 plasma or the human mpl ligand. Ordinarily, a biologically active mpl ligand or TPO isoform will have an amino acid sequence having at least about 70% amino acid sequence identity with the mpl ligand/TPO isolated from aplastic porcine plasma or the mature murine, human mpl ligand or fragments thereof, preferably at least about 75%, more preferably at least about 80%, still more preferably at least about 85%, even more preferably at least about 90%, and most preferably at least 15 about 95%. In one isoform embodiment, the TPO may have the formula: SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer HisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProXaaPro ValLeuLeuProAlaValAspXaaXaaLeuGlyGluTrpLysThrGlnMetGluGlu 20 ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnXaaXaaXaa XaaGlyArgThrThrAlaHisXaaAspProAsnAlaIlePheLeuSerPheGlnHis LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal 25 ArgArgAlaProProThrThrAlaValProSerArgThrSerLeuValLeuThrLeu AsnGluLeuProAsnArgThrSerGlyLeuLeuGluThrAsnPheThrAlaSerAla ArgThrThrGlySerGlyLeuLeuLysXaaGlnGlnGlyPheArgAlaLys IlePro GlyLeuLeuAsnGlnThrSerArgSerLeuAspGlnI leProGlyTyrLeuAsnArg I leHisGluLeuLeuAsnGlyThrArgGlyLeuPheProGlyProSerArgArgThr 30 LeuGlyAlaProAspIleSerSerGlyThrSerAspThrGlySerLeuProProAsn LeuGlnProGlyTyrSerProSerProThrHisProProThrGlyGlnTyrThrLeu PheProLeuProProThrLeuProThrProValVaGlnLeuHisProLeuLeuPro AspProSerAlaProThrProThrProThrSerProLeuLeuAsnThrSerTyrThr HisSerGlnAsnLeuSerGlnGluGly 35 where: 11 WO 98/52598 PCT/US98/10475 Xaa at position 37 is Thr, Asp or Glu; Xaa at position 46 is Phe, Ala, Val, Leu, Ile, Pro, Trp, or Met; Xaa at position 47 is Ser, Asp or Glu; Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or Met; 5 Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn; Xaa at position 122 is Lys, Arg, His, Glu, or Asp; Xaa at position 200 is Trp, Ala, Val, Leu, Ile, Pro, Phe, Met, Arg and Lys, or His. 10 where from 1 to 179 amino acids can be deleted from the C-terminus and with the proviso that at least one of the amino acids designated by Xaa are different from the corresponding amino acids of the native TPO (1-332). 15 This embodiment also includes a TPO fragment with the following formula: SerProAlaProProAlaCysAspLeuArgValLeuSerLysLeuLeuArgAspSer HisValLeuHisSerArgLeuSerGlnCysProGluValHisProLeuProXaaPro ValLeuLeuProAlaValAspXaaXaaLeuGlyGluTrpLysThrGlnMetGluGlu 20 ThrLysAlaGlnAspIleLeuGlyAlaValThrLeuLeuLeuGluGlyValMetAla AlaArgGlyGlnLeuGlyProThrCysLeuSerSerLeuLeuGlyGlnLeuSerGly GlnValArgLeuLeuLeuGlyAlaLeuGlnSerLeuLeuGlyThrGlnXaaXaaXaa XaaGlyArgThrThrAlaHisXaaAspProAsnAlal lePheLeuSerPheGlnHis LeuLeuArgGlyLysValArgPheLeuMetLeuValGlyGlySerThrLeuCysVal 25 Arg where: Xaa at position 37 is Thr, Asp or Glu; Xaa at position 46 is Phe, Ala, Val, Leu, Ile, Pro, Trp, or Met; 30 Xaa at position 47 is Ser, Asp or Glu; Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or Met; Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; Xaa at position 115 is deleted or Gln, Gly, Ser, Thr, Tyr, or Asn; 35 Xaa at position 122 is Lys, Arg, His, Glu, or Asp; 12 WO 98/52598 PCT/US98/10475 and with the proviso that at least one of the amino acids designated by Xaa is different from the corresponding amino acids of the native TPO (1-332). These variants may have an improved biological profile, such as increased proliferative activity and/or decreased side-effects, and/or improved physical properties, such as improved half-life, stability, and/or re-fold efficiencies. The 5 preparation of the polypeptides of this embodiment is described in WO96/23888. TPO "analogues" include covalent modification of TPO or mpl ligand by linking the TPO polypeptide to one of a variety of nonproteinaceous polymers, e.g. polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. TPO polypeptides covalently linked to the forgoing 10 polymers are referred to herein as pegylated TPO. A "chimeric polypeptide" or "chimera" as used herein is a polypeptide containing full length parent ligand (TPO or mpl ligand) or one or more fragments thereof fused or bonded to a second heterologous polypeptide or one or more fragments thereof. The chimera will share at least one biological property in common with TPO. The second polypeptide will typically be a cytokine, for 15 example the cytokines noted above, immunoglobin or fragment thereof. The two polypeptides may be directly bonded together or may be bonded together through a linker, for example a peptide linker which may have 2-50, generally 2-20 amino acid units. Specific examples include TPO/G-CSF, TPO/GM-CSF, TPO/IL-3, TPO/IL-6, etc. Preparation of chimeric proteins may be accomplished using methods well-known in the art. 20 The term "biological property" when used in conjunction with either the "mpl ligand" or "isolated mpl ligand" or "TPO" means having thrombopoietic activity or having an in vivo effector or antigenic function or activity that is directly or indirectly caused or performed by a mpl ligand or "TPO" (whether in its native or denatured conformation) or a fragment thereof. Effector functions include mpl binding and any carrier binding activity, agonism or antagonism of mpl, especially 25 transduction of a proliferative signal including replication, DNA regulatory function, modulation of the biological activity of other cytokines, receptor (especially cytokine) activation, deactivation, up-or down regulation, cell growth or differentiation and the like. An antigenic function means possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against the native mpl ligand or TPO. The principal antigenic function of a mpl ligand or TPO polypeptide is that it binds 30 with an affinity of at least about 106 I/mole to an antibody raised against the mpl ligand or TPO isolated from aplastic porcine plasma. Ordinarily, the polypeptide binds with an affinity of at least about 107 I/mole. Most preferably, the antigenically active mpl ligand or TPO polypeptide is a polypeptide that binds to an antibody raised against the mpl ligand or TPO having one of the above described effector functions. The antibodies used to define "biological property" are rabbit polyclonal 35 antibodies raised by formulating the mpl ligand or TPO isolated from recombinant cell culture or aplastic porcine plasma in Freund's complete adjuvant, subcutaneously injecting the formulation, and 13 WO 98/52598 PCT/US98/10475 boosting the immune response by intraperitoneal injection of the formulation until the titer of mpl ligand or TPO antibody plateaus. "Thrombopoietic activity" is defined as biological activity characterized by accelerating the proliferation, differentiation and/or maturation of megakaryocytes or megakaryocyte precursors into 5 the platelet producing form of these cells. This activity may be measured in various assays including an in vivo mouse platelet rebound synthesis assay, induction of platelet cell surface antigen assay as measured by an anti-platelet immunoassay (anti-GPIbIIIa) for a human leukemia megakaryoblastic cell line (CMK), and induction of polyploidization in a megakaryoblastic cell line (DAMI). By the term "low-multiple" in connection with the dosing is meant the administration of 10 multiple doses of therapeutically effective amounts over a short period of time. A low-multiple dose may include 2 to about 6 doses per day, preferably 2-4 doses per day. Thus, the present invention is directed to the mere single administration of a therapeutically effective amount of a thrombopoietin. It has been found that a single administration produces a therapeutic effect equivalent to that realized when a therapeutically effective amount of the same material is administered over the conventional 15 multiple many day regimen suggested and taught by the art. The term "treatment cycle" as used herein means a course of radiation and/or chemotherapeutic agent administration (treatment phase) in which a mammal or patient is treated with radiation and/or chemotherapeutic agent, generally followed by a period of observation and recovery (recovery phase). The treatment phase may include a single administration of radiation and/or 20 chemotherapeutic agent or multiple administrations, preferably separated by a period of time which is usually chosen so as to minimize discomfort to the mammal or patient and allow recovery of neutrophil and platelet counts to about pretreatment levels. The time period is generally determined by the tolerance of the mammal or patient for the particular radiation and/or chemotherapeutic agent. A typical treatment phase may run 1-10 days, preferably 1-6 or 1-4 days, during which time the radiation 25 or chemotherapeutic agent is administered continuously or portion-wise. A typical recovery phase may run 5-60 days, preferably 14-24 days, during which time the mammal or patient is observed, evaluated and allowed to recover from the treatment. Optionally, more than one treatment cycle may be given, typically 2 to about 6 cycles depending on the particular treatment regimen and the purpose of the treatment. 30 Detailed Description of the Preferred Embodiments It has now been discovered that TPO has pharmacokinetic properties which are suprisingly different than the properties of cytokines such as G-CSF and GM-CSF and which allow TPO to be administered prior to and/or concurrently with radiation and/or chemotherapy. Prior and/or concurrent administration of TPO has been found to reduce the depth of the nadir of thrombocytopenia and to 35 shorten the time for platelet titer recovery in patients receiving radiation and/or chemotherapeutic treatment. This difference in properties of TPO is believed to derive from the effect of TPO on early 14 WO 98/52598 PCT/US98/10475 progenitor cells in the hematopoietic lineage. This effect appears to result in a delay in the appearance of more mature cells in the lineage following administration of TPO, allowing radiation and/or chemotherapy treatments to be given with little or clinically insignificant loss of proliferating cells during cytoreductive treatments. The discovery of these unique properties of TPO is part of the present 5 invention. This discovery is significant since patients receiving radiation and/or chemotherapy frequently require platelet transfusions. Frequent platelet transfusions may result in alloimmunization. This results in the need for HLA-matched donors and more frequent transfusions. The provision of platelet transfusions is an important and often difficult medical problem. Reducing the need and frequency of 10 such transfusions improves patient care and mitigates complications associated with transfusions, such as blood antigen incompatibilities, lack of suitable platelet donors, contamination of donated materials, e.g. contamination with a virus, etc. Any treatment that prevents or shortens the duration of prolonged thrombocytopenia represents an important medical advance. The present invention reduces the necessity for platelet transfusions in patients experiencing thrombocytopenia. 15 The method of the invention hastens the recovery of platelet titers to baseline levels and even to substantially elevated levels following radiation and/or chemotherapy. The generation of elevated platelet titers is useful in preparing a patient for subsequent cycles of radiation and/or chemotherapy treatment. A patient entering a subsequent cycle of treatment with elevated platelet levels is better able to withstand the cytoreductive effects of the treatment. The invention, therefore, is effective to increase 20 patient tolerance to a radiation and/or chemotherapeutic regimen relative to the patient tolerance for the regimen without administration of TPO according to the invention. The method of the invention is also useful in mobilization therapy. In mobilization therapy, the peripheral blood progenitor cells are mobilized from the bone marrow to reduce or eliminate neutropenia and/or thrombocytopenia. In the method of the invention, TPO is administered as a single 25 or low-multiple daily dose to mobilize peripheral blood progenitor cells. Typically, the mammal is a human patient having or at risk for thrombocytpenia, for example, as a result of radiation and/or chemotherapy or liver disease. According to the invention, TPO is administered to the patient prior to or concurrent with radiation or chemotherapy treatment. Of course, TPO may also be administered subsequent to radiation or chemotherapy treatment to restore platelet blood titer in conjunction with the 30 prior or concurrent administration noted above. The TPO may also be administered together with another cytokine, e.g. G-CSF, IL-3, IL-6, GM-CSF, etc. The progenitor cells which are mobilized by the method of the invention may be collected by standard leukapheresis, optionally frozen, and retransplanted into the patient after radiation and/or chemotherapy. The additional cytokine is generally administered in an amount similar to the amount of TPO. For example, in mobilization 35 therapy the TPO might be administered in an amount from about 0.1-10 microgram/kg alone or together with an additional cytokine in a similar amount. For heterologous bone marrow transplants, TPO optionally in combination with another cytokine as discussed above, may be administered to a 15 WO 98/52598 PCT/US98/10475 mammal including a human patient, for the purpose of mobilizing peripheral blood progenitor cells which may then be harvested by leukapheresis, optionally frozen and transplanted into a mammal having or at risk for thrombocytopenia. The mammal or patient donating the heterologous bone marrow graft (progenitor cells) and the transplant recipient may be tissue-typed according to known 5 procedures. The TPO and other cytokine are generally administered in the amounts discussed above for autologous transplants. Additionally, it is well known that repeated cycles of radiation and/or chemotherapy result in a cumulative myelosuppression which limits the dose intensity of individual chemotherapeutic agents, particularly when used in combination therapy. Commercially available myeloid growth factors, such 10 as G-CSF, have helped to reduce neutropenia; however cumulative thrombocytopenia remains a problem. The present invention significantly reduces neutropenia during combination chemotherapy thereby increasing the number of treatment cycles a patient will tolerate during chemotherapy. A larger number of treatment cycles or stronger doses of the chemotherapeutic agents improves the cancer kill rate. 15 The method of the invention also reduces the likelihood of formation of anti-TPO antibodies. Immunogenicity is reduced or eliminated due to less frequent dosing, including single doses, relative to continuous daily dosing known in the art. Dosing is preferably intravenous. However, hybrid regimen in which IV dosing is combined with SC dosing is also contemplated by this invention. For example, it may be desirable to administer an initial IV dose of TPO before or shortly after treatment with 20 radiation and/or chemotherapy in order to reduce the platelet nadir associated with such treatment and to accelerate recovery of platelet titers. This initial dose of TPO might be followed by one or more SC doses of TPO after the treatment to maintain the platelet levels. An alternative to parenteral or subcutaneous delivery of TPO is aerosol delivery. The pulmonary route of administration is an attractive alternative to intravenous (IV) or subcutaneous (SC) 25 delivery because of the ease of administration and the large surface area of the lung for absorption. However, the barrier to absorption of proteins is formidable and the mechanism of absorption is unclear. Despite the potential limitations, several proteins (e.g. insulin, hGH, BSA, and LHRH) have been delivered successfully via the lungs to target the systemic circulation. See Adjei et al, International J Pharm., 61:135-144, 1990; Colthorpe et al, Pharm. Res., 9:764-768, 1992; Folkesson et 30 al, Acta Physiologica Scandinavia, 139:347-54, 1990; Patton et al, Biotech. Therapeut., 1:213-228, 1989-90; and Niven et al, Pharm. Res., 12:1343-9, 1995. The table shown below indicates proteins which have been tested for delivery via the lung. 16 WO 98/52598 PCT/US98/10475 Protein* Molecular Weight Bioavailability' Reference (kDa) (%) LHRH 1.067 4-18 Adjei, et al, 1990 Insulin 5.7 5.6 (IT) Colthorpe et al, 1992 Insulin 5.7 57 (aerosol) Colthorpe et al, 1992 rhG-CSF 18.8 66 Niven et al, 1995 hGH 22.0 35 Patton et al, 1989 BSA 67.0 4.3 Folkesson et al, 1990 * LHRH=Leutinizing hormone releasing hormone, rhG-CSF--recombinant human granulocyte-colony stimulating factor, hGH= human growth hormone, and BSA= bovine serum albumin + IT= intratracheal instillation 5 In an interesting embodiment of this invention, it has been discovered that a TPO, e.g. recombinant human thrombopoietin (rhTPO), can be administered via aerosol inhalation to target the systemic circulation. rhTPO is an about 80 kD glycoprotein. Therapeutic serum concentrations can be achieved by delivering rhTPO to the lungs as a liquid or powder aerosol. Solutions of TPO may be 10 nebulized using conventional nebulizers and administered to a mammal or human patient through the nose or mouth as an aerosol. TPO may also be dried to a powder, e.g. by spray-drying, and administered using a conventional dry powder inhaler. A higher dose of rhTPO is required to achieve a similar therapeutic effect when given as an aerosol as compared to IV. Generally, the dose by aerosol should be about 100-fold higher for aerosol administration as compared to IV administration. A 15 suitable dose range for aerosol administration is about 5-1000 microgram/kg, preferably 50-750 microgram/kg, as a single inhalation dose or as multiple inhalations on a single day or on multiple days, preferably 2-10 or 2-6, sequential or non-sequential days. The method of the invention may be used with any radiation and/or chemotherapy regimen in which a mammal or human patient is having or is at risk of thrombocytopenia. The method may be 20 used with conventional chemotherapeutic agents used in conventional amounts including, but not limited to asparaginase, bleomycin, calcium leucovorin, carmustine, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin, epirubicin, etoposide, fluorouracil, fluoxymesterone, flutamide, hexamethylmelamine, hydroxyurea, ifosfamide, leuprolide, levamisole, leuprolide depot, lomustine, mechlorethamine, melphalan, mercaptopurine, 25 methotrexate, methyl-CCNU (semustine), methylprednisolone, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, streptozocin, tamoxifen, thioguanine, triethylene thiophosphoramide, vinblastine, vincristine, and combinations thereof. These compounds may, optionally, be given with a known uroprotecting compound such as mesna, etc. where appropriate and indicated. Mesna is commercially available and is routinely give to counteract the urinary tract 30 irritation and hemorragic cystitis due to chemotherapeutic agent metabolites, e.g. ifosfamide 17 WO 98/52598 PCT/US98/10475 metabolites. Typically, the chemotherapeutic agents are given in combination to maximize tumor cell kill with minimal or at least acceptable toxicity to the mammal or patient. The method of the invention further reduces this toxicity. Suitable non-limiting chemotherapy regimen with which the method of the invention may be used are listed below using conventional acronyms and indicating specific 5 tumors/cancers for which the regimen is contemplated. The chemotherapeutic agents may be administered in conventional amounts and according to conventional treatment times and regimen. See, for example, "The Cerenex Handbook", Robert S. Benjamin, Ed., Cerenex Pharmaceuticals, Research Triangle Park, N.C. (1993); "Combination Cancer Chemotherapy Regimens", Roger W. Anderson and William J. Dana, Eds., Laderley Laboratories (1991). Any cytoreductive regimen which 10 induces thrombocytopenia is considered to within the scope of the invention, however. Breast Cancer CAF - cyclophosphamide, doxorubicin, fluorouracil CFM - cyclophosphamide, doxorubicin, mitoxantrone CFPT - cyclophosphamide, fluorouracil, prednisolone, tamoxifen 15 CMF - cyclophosphamide, methotrexate, fluorouracil CMFP - cyclophosphamide, methotrexate, fluorouracil, prednisolone CMFVP - cyclophosphamide, methotrexate, fluorouracil, vincristine, prednisolone FAC - fluorouracil, doxorubicin, cyclophosphamide IMF - ifosfamide, methotrexate, fluorouracil, mesna 20 VATH - vinblastine, doxorubicin, thiotepa, fluoxymesterone CEP - cyclophosphamide, etoposide, cisplatin ICE - ifosfamide, cyclophosphamide, etoposide AC - doxorubicin, cyclophosphamide FLAC - fluorouracil, calcium leucovorin, doxorubicin, cyclophosphamide 25 Colon Cancer F-CL - Fluorouracil, calcium leucovorin FLe - levamisole, fluorouracil FMV - fluorouracil, methyl-CCNU, vincristine Gastric Cancer 30 FAM - fluorouracil, doxorubicin, mitomycin C FAME - fluorouracil, doxorubicin, methyl-CCNU FCE - fluorouracil, cisplatin, etoposide Genitourinary Cancer CAP - cisplatin, doxorubicin, cyclophosphamide 35 CISCA - cyclophosphamide, doxorubicin, cisplatin CVEB - cisplatin, vinblastine, etoposide, bleomycin FL - flutamide, leuprolide acetate or flutamide, leuprolide acetate depot 18 WO 98/52598 PCT/US98/10475 L-VAM - leuprolide acetate, vinblastine, doxorubicin, mitomycin C MVAC - methotrexate, vinblastine, doxorubicin, cisplatin VAB - vinblastine, dactinomycin, bleomycin, cisplatin, cyclophosphamide VB - vinblastine, methotrexate 5 VBP - vinblastine, bleomycin, cisplatin Gestational Trophoblastic Disease DMC - dactinomycin, methotrexate, cyclophosphamide Head and Neck Cancer CF - cisplatin, fluorouracil CFL - cisplatin, fluorouracil, calcium leucovorin 10 COB - cisplatin, vincristine, bleomycin MAP - mitomycin C, doxorubicin, cisplatin MBC - methotrexate, bleomycin, cisplatin MF - methotrexate, fluorouracil, calcium leucovorin Leukemias 15 Acute Lymphocytic Leukemia (A.L.L.) DVP - daunorubicin, vincristine, prednisone MM - mercaptopurine, methotrexate AVDP - asparaginase, vincristine, daunorubicin, prednisone Acute Myelogenous Leukemia (A.M.L.) 20 AA - cytarabine, doxorubicin COAP - cyclophosphamide, vincristine, cytarabine, prednisone MV - mitoxantrone, etoposide Acute Non-Lymphocytic Leukemia (A.N.L.L.) DCT - daunorubicin, cytarabine, thioguanine 25 MC - mitoxantrone, cytarabine CD - cytarabine, daunorubicin TC - thioguanine, cytarabine Chronic Lymphatic Leukemia (C.L.L.) CVP - cyclophosphamide, vincristine, prednisone 30 Lung Cancer Small Cell COPE - cyclophosphamide, cisplatin, etoposide, vincristine CV - cisplatin, etoposide VAC - vincristine, doxorubicin, cyclophosphamide 35 VC - etoposide, carboplatin ICE - ifosfamide, cyclophosphamide, etoposide CEP - cyclophosphamide, etoposide, cisplatin 19 WO 98/52598 PCT/US98/10475 Non-small Cell BACON - bleomycin, doxorubicin, lomustine, vincristine, mechlorethamine CAMP - cyclophosphamide, doxorubicin, methotrexate, procarbazine CAP - cyclophosphamide, doxorubicin, cisplatin 5 CV - cisplatin, etoposide CVI - cisplatin, etoposide, ifosfamide, mesna FAM - fluorouracil, doxorubicin, mitomycin C FOMi/CAP - fluorouracil, vincristine, mitomycin C, cyclophosphamide, doxorubicin, cisplatin 10 MACC - methotrexate, doxorubicin, cyclophosphamide, lomustine ICE - ifosfamide, cyclophosphamide, etoposide CEP - cyclophosphamide, etoposide, cisplatin Lymphoma Hodgkin's 15 ABVD - doxorubicin, bleomycin, vincristine, dacarbazine B-CAVe - bleomycin, lomustine, doxorubicin, vinblastine B-DOPA - bleomycin, dacarbazine, vincristine, prednisone, doxorubicin CVPP - lomustine, vinblastine, procarbazine, prednisone MOPP - mechlorethamine, vincristine, procarbazine, prednisone 20 MVPP - mechlorethamine, vinblastine, procarbazine, prednisone NOVP - mitoxantrone, vinblastine, prednisone, vincristine Non-Hodgkin's ASHAP - doxorubicin, cisplatin, cytarabine, methylprednisolone BACOP - bleomycin, doxorubicin, cyclophosphamide, vincristine, prednisone 25 CHOP - cyclophosphamide, doxorubicin, vincristine, prednisone CHOP-Bleo - cyclophosphamide, doxorubicin, vincristine, prednisone, bleomycin COMLA - cyclophosphamide, vincristine, methotrexate, calcium leucovorin, cytarabine COP - cyclophosphamide, vincristine, prednisone 30 COPP - cyclophosphamide, vincristine, procarbazine, prednisone CVP - cyclophosphamide, vincristine, prednisone E-SHAP - etoposide, cisplatin, cytarabine, methylprednisolone IMVP-16 - ifosfamide, mesna, methotrexate, etoposide m-BACOD - bleomycin, doxorubicin, cyclophosphamide, vincristine, 35 dexamethasone, methotrexate, calcium leucovorin m-BACOS - doxorubicin, vincristine, bleomycin, cyclophosphamide, methotrexate, calcium leucovorin 20 WO 9N/52598 PCT/US98/10475 MINE - mesna, ifosfamide, mitoxantrone, etoposide OPEN - etoposide, mitoxantrone, vincristine, prednisone Pro-MACE - prednisone, methotrexate, calcium leucovorin, doxorubicin, cyclophosphamide, etoposide 5 AI - doxorubicin, ifosfamide AC - doxorubicin, cyclophosphamide ICE - ifosfamide, cyclophosphamide, etoposide CEP - cyclophosphamide, etoposide, cisplatin Malignant Melanoma 10 BHD - carmustine, hydroxyurea, dacarbazine DTIC-ACTD - dacarbazine, dactinomycin VBC - vinblastine, bleomycin, cisplatin VDP - vinblastine, dacarbazine, cisplatin Multiple Myeloma 15 AC - doxorubicin, carmustine BCP - carmustine, cyclophosphamide, prednisone MeCP - methyl-CCNU, cyclophosphamide, prednisone MP - melphalan, prednisone M-2 - vincristine, carmustine, cyclophosphamide, melphalan, prednisone 20 VAD - vincristine, doxorubicin, dexamethasone VBAP - vincristine, carmustine, doxorubicin, prednisone VCAP - vincristine, cyclophosphamide, doxorubicin, prednisone Ovarian Cancer Epithelial 25 C - carboplatin AP - doxorubicin, cisplatin CDC - carboplatin, doxorubicin, cyclophosphamide CHAD - cyclophosphamide, doxorubicin, cisplatin, hexamethylmelamine CHAP - cyclophosphamide, hexamethylmelamine, doxorubicin, cisplatin 30 CP - cyclophosphamide, cisplatin PAC - cisplatin, doxorubicin, cyclophosphamide AC - doxorubicin, cyclophosphamide ICE - ifosfamide, cyclophosphamide, etoposide CEP - cyclophosphamide, etoposide, cisplatin 35 Germ Cell VAC - vincristine, dactinomycin, cyclophosphamide 21 WU 98/5295 PCT/US98/10475 Endometrial Cancer C - carboplatin AC - doxorubicin, cyclophosphamide Pancreatic Cancer 5 FMS - fluorouracil, mitomycin C, streptozocin SD - streptozocin, doxorubicin Pediatric Tumors A.L.L. DVP - daunorubicin, vincristine, prednisone 10 VAP - daunorubicin, asparaginase, prednisone CT - cytarabine, thioguanine DCPM - daunorubicin, cytarabine, prednisolone, mercaptopurine A.N.L.L. DC - daunorubicin, cytarabine 15 Bony sarcoma AC - doxorubicin, cisplatin HDMTX - methotrexate, calcium leucovorin T-2 - dactinomycin, doxorubicin, vincristine, cyclophosphamide Hodgkin's disease 20 ACOPP - doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone MOPP - mechlorethamine, vincristine, procarbazine, prednisone Soft Tissue Sarcoma VAC - vincristine, dactinomycin, cyclophosphamide Sarcoma 25 Bony Sarcoma AC - doxorubicin, cisplatin CYVADIC - cyclophosphamide, vincristine, doxorubicin, dacarbazine HDMTX - methotrexate, calcium leucovorin IMAC - ifosfamide, mesna, doxorubicin, cisplatin 30 Soft Tissue CYADIC - cyclophosphamide, doxorubicin, dacarbazine CYVADIC - cyclophosphamide, vincristine, doxorubicin, dacarbazine ID - ifosfamide, mesna, doxorubicin VAC - vincristine, dactinomycin, cyclophosphamide 35 AI - ifosfamide, doxorubicin MAID - mesna, doxorubicin, ifosfamide, dacarbazine 22 WO 98/52598 PCT/US98/10475 Selected regimens particularly associated with thrombocytopenia which may be treated with the method of the invention include: Regimens Malignancy 5 * carboplatin/paclitaxel Ovary NSCLC * FUDR/leucovorin/doxorubicin/cisplatin HBV+/HCV+ Hepatoma * cisplatin/alpha interferon/5-FU/ HBV+/HCV+ Hepatoma doxorubicin 10 * ifosfamide/mesna/carboplatin/etoposide NSCLC * cisplatin/ifosfamide/mesna/etoposide Testicle * High-dose carboplatin/etoposide Testicle (salvage) * methotrexate/vinblastine/doxorubicin/ Bladder cisplatin 15 * BCNU (carmustine) Glioblastoma * procarbazine/CCNU/vincristine Brain * cyclophaphamide/etoposide +/- Salvage Hodgkin's disease, breast, cisplatin or carboplatin ovary, non-Hodgkin's lymphoma, head and neck, lung, myeloma, sarcoma 20 * dexamethasone/HIDAC/cisplatin salvage non-Hodgkin's lymphoma * mesnalifosfamide/doxorubicin/+ DTIC Soft tissue sarcoma * cyclophophamide/vincristine/DTIC Soft tissue sarcoma doxorubicin 25 The single or low multiple dose of the invention may be given prior to the first treatment time of radiation and/or chemotherapeutic agent in a treatment cycle, during or concurrent with a treatment time in a treatment cycle or following one or more individual treatment times of radiation or chemotherapeutic agent in a treatment cycle. For example, a cycle may constitute a single treatment time of radiation or chemotherapeutic agent. In the invention, the single or low-multiple dose of TPO 30 would be administered before, during or after this treatment time. Alternatively, the cycle may constitute multiple treatment times, for example 2-10 or more treatment times, of radiation or chemotherapeutic agent. Here, the invention contemplates administering TPO before, during or after any one treatment time or before, during or after each individual treatment time. For example, the cycle may constitute three treatments with a chemotherapeutic agent. In the method of the invention, 35 TPO might be administered before each of the three treatment times or might be administered after each of the three treatment times. Of course, the invention also includes administration of a single 23 WO 98/52598 PCT/US98/10475 daily dose of TPO before the first treatment time of the chemotherapeutic agent in the cycle and after the last treatment time in the cycle. In a preferred embodiment, the mammal receives at least one treatment cycle of radiation and/or chemotherapeutic agent, where the treatment cycle has a first treatment time T O and a last 5 treatment time TF for administering radiation and/or chemotherapeutic agent. The dose of TPO is preferably administered at T O plus or minus 24 hours, more preferably T o plus or minus 10 hours, still more preferably T o plus or minus 6 hours, and most preferably T o plus or minus 2 hours. In alternative embodiments, the dose is administered at T o or prior to To, but not more than seven days prior to T 0 , preferably not more than one day prior to To. For single dose cycles, TO = TF. 10 In another preferred embodiment, the dose is administered prior to TF, but not more than seven days prior to TF. As noted above, TPO may also be administered after TF. When a second dose of TPO is administered after TF, the dose is preferably administered not more than 24 hours after TF. The mammal or patient may, of course, receive multiple treatment cycles, generally 2-6 cycles, but as many cycles as is medically necessary to reduce the size of or to completely irradicate a cancer or tumor. In 15 some treat regimen, a tumor is reduced in size relative to the size of the tumor prior to radiation and/or chemotherapy treatment, and then surgery is utilized to remove the remaining malignant tissue of the tumor. The method of the invention may be used in these regimen as well. The invention also includes co-administering a therapeutically effective amount of a cytokine, a colony stimulating factor and an interleukin, generally after administration of the TPO dose, 20 preferably after administration of the last TPO dose in a treatment cycle. The cytokine is preferably KL, LIF, G-CSF, GM-CSF, M-CSF, EPO, FLT-3, IL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 or IL 11, in particular, G-CSF or GM-CSF. It has been found in accord with the present invention that the single or low multiple administration regimen of the present invention is effective at daily dosage rates on the order of about 25 0.1 to 50, preferably about 0.1 to 10, more preferably about I to 5, or preferably about 1 to 3 microgram/kg body weight of the patient. In single dosing, preferred would be the total administration of about 2 ±1.5 microgram/kg of body weight. In low-multiple dosing, preferred would be the administration of from about 0.5 to 1.5 microgram/kg body weight per dose. The above dosages are predicated on preferred intravenous administration. In administration via the subcutaneous route, the 30 total amount administered would be in the range of about one to three times the amount administered via the intravenous route, preferably about two times. Further, the doses for administration via the lung are higher as noted above. Specific therapeutically effective dosages for individual patients may be determined by conventional methods. The method of the invention also preferably provides a dose of TPO sufficient to maintain a 35 blood TPO level in the mammal of 35 x 10

-

12 M or greater during the radiation and/or chemotherapy 24 -WO 98/52598 PCT/US98/10475 treatment cycle. Preferably, the dose is sufficient to maintain a blood TPO level of 100 x 10-12 M or greater, more preferably about 35 x 10-12 M to about 3500 x 10-12 M during the treatment cycle. The optimal dosage rate and regimen will be determined by the attending physician taking into consideration various factors known to modify the action of drugs including severity and type of 5 disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. It will be understood that although a single daily administration of a thrombopoietin to a patient has been found to be therapeutically effective for the treatment of thrombocytopenia, it can be appreciated that a low-multiple (daily) regimen may be employed. It has been found that a single dose 10 stimulates the onset of therapeutic response, and although multiple dosing is contemplated herein, termination of dosing after a single or low-multiple administration is independent of therapeutic response. The biologically active thrombopoietin materials of the present invention can be administered, in accord herewith, in various routes including via the nose or lung, subcutaneously, and preferably 15 intravenously. In all events, depending upon the route of administration, the biologically active thrombopoietin materials of the present invention are preferably administered in combination with an appropriate pharmaceutically acceptable carrier or excipient. When administered systemically, the therapeutic composition should be pyrogen-free and in a parenterally acceptable solution having due regard for physiological pH isotonicity and stability. These conditions are generally well known and 20 accepted to those of skill in the appropriate art. Briefly, dosage formulations of the materials of the present invention are prepared for storage or administration by mixing the compound having the desired degree of purity with physiologically acceptable carriers, excipients and/or stabilizers. Such materials are non-toxic to the recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, acetate and other !5 organic acid salts; antioxidants such as ascorbic acid; low molecular weight peptides such as polyarginine, proteins such as serum albumen, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates including cellulose or its derivatives, glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohol such as mannitol or sorbitol; 0 counter-ions such as sodium; and/or non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol. The biologically active thrombopoietin materials hereof can be administered as the free acid or base form or as a pharmaceutically acceptable salt and are compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc. as called for 5 by accepted pharmaceutical practice. 25 - WO 98/52598 PCT/US98/10475 sterile compositions for injection can be formulated according to conventional pharmaceutical or pharmacological practice. For example, dissolution or suspension of the active material in a vehicle such as water or naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Again, buffers, preservatives, anti-oxidants 5 and the like can be incorporated according to accepted pharmaceutical practice. The biologically active thrombopoietin materials of the present invention may be employed alone or administered in combination with other cytokines, hematapoietins, interleukins, growth factors, or antibodies in the treatment of the above identified disorders and conditions marked by thrombocytopenia. Thus, the present active materials may be employed in combination with other protein or peptide having 10 thrombopoietic activity including: G-CSF, GM-CSF, LIF, M-CSF, IL-2, IL-3, erythropoietin (EPO), Kit ligand, IL-6, IL-11, FLT-3 ligand, and so forth. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels 15 (e.g. poly(2-hydroxyethyl-methacrylate) as described by Langer et al. J Biomed. Mater. Res., 15:167 277 (1981) and Langer, Chem. Tec., 12:98-105 (1982) or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,779,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.Biopolymers, 22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer et al., supra), degradable lactic acid-glycolic acid copolymers such as the LUPROM DEPOT' (injectable 20 microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-) 3-hydroxybutyric acid (EP 133,988). While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of 5 exposure to moisture at 37 0 C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for protein stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S S bond formation through disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, 0 and developing specific polymer matrix compositions. Sustained-release thrombopoietic protein compositions also include liposomally entrapped megakaryocytopoietic protein. Liposomes containing megakaryocytopoietic protein are prepared by methods knew per se: DE, 3,218,121; Epstein et al.Proc. Natl. Acad. Sci. USA, 82:3688-3698 (1985); Hwang et al.Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 5 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800 Angstroms) 26 - WO 98/52598 PCT/US98/10475 unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal megakaryocytopoietic protein therapy. A type of covalent modification of TPO or mpl ligand comprises linking the TPO polypeptide to one of a variety of nonproteinaceous polymers, e.g. polyethylene glycol, polypropylene glycol, or 5 polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. TPO polypeptides covalently linked to the forgoing polymers are referred to herein as pegylated TPO. It will be appreciated that some screening of the recovered TPO variant will be needed to select the optimal variant for binding to a mpl and having the immunological and/or biological activity 10 defined above. One can screen for stability in recombinant cell culture or in plasma (e.g., against proteolytic cleavage), high affinity to a mpl member, oxidative stability, ability to be secreted in elevated yields, and the like. For example, a change in the immunological character of the TPO polypeptide, such as affinity for a given antibody, is measured by a competitive-type immunoassay. Other potential modifications of protein or polypeptide properties such as redox or thermal stability, 15 hydrophobicity, or susceptibility to proteolytic degradation are assayed by methods well known in the art. Methods of Making Isolation of the Human mpl Ligand (TPO) Gene Human genomic DNA clones of the TPO gene were isolated by screening a human genomic 20 library in 8-Gem 12 with pR45, under low stringency conditions or under high stringency conditions with a fragment corresponding to the 3N half of human cDNA coding for the mpl ligand. Two overlapping lambda clones spanning 35 kb were isolated. Two overlapping fragments (BamH1 and EcoRI) containing the entire TPO gene were subcloned and sequenced. The structure of the human gene is composed of 6 exons within 7 kb of genomic DNA. The 25 boundaries of all exon/intron junctions are consistent with the consensus motif established for mammalian genes (Shapiro, M.B. et al., Nucl. Acids. Res. 15:7155 (1987)). Exon 1 and exon 2 contain SN untranslated sequence and the initial four amino acids of the signal peptide. The remainder of the secretory signal and the first 26 amino acids of the mature protein are encoded within exon 3. The entire carboxyl domain and 3N untranslated as well as -50 amino acids of the erthropoietin-like 30 domain are encoded within exon 6. The four amino acids involved in the deletion observed within hML-2 (hTPO-2) are encoded at the 5N end of exon 6. Analysis of human genomic DNA by Southern blot indicated the gene for TPO is present in a single copy. The chromosomal location of the gene was determined by fluorescent in situ hybridization (FISH) which mapped to chromosome 3q27-28. 35 27 WO 98/52598 PCT/US98/10475 Expression and Purification of TPO from 293 Cells Preparation and purification of ML or TPO from 293 cells is described in detail in Example 1. Briefly, cDNA corresponding to the TPO entire open reading frame was obtained by PCR using pRK5 hmpl I. The PCR product was purified and cloned between the restriction sites Clal and XbaI of the 5 plasmid pRK5tkneo.ORF (a vector coding for the entire open reading frame). A second vector coding for the EPO homologous domain was generated the same but using different PCR primers to obtain the final construct called pRK5-tkneoEPO-D. These two constructs were transfected into human embryonic kidney cells by the CaPO 4 method and neomycin resistant clones were selected and allowed to grow to confluency. Expression of 10 ML 153 or ML 332 in the conditioned media from these clones was assessed using the Ba/F3-mpl proliferation assay. Purification ofrhML 332 was conducted as described in Example 1. Briefly, 293-rhML 332 conditioned media was applied to a BLUE-SEPHAROSE (Pharmacia) column that was subsequently washed with a buffer containing 2M urea. the column was eluted with a buffer containing 2M urea and 15 1M NaC1. The BLUE-SEPHAROSE elution pool was then directly applied to a WGA-SEPHAROSE column, washed with 10 column volumes of buffer containing 2M urea and IM NaCl and eluted with the same buffer containing 0.5M N-acetyl-D-glucosamine. The WGA-SEPHAROSE eluate was applied to a C4-HPLC column (Synchrom, Inc.) and eluted with a discontinuous propanol gradient. By SDS-PAGE the purified 293-fhML 332 migrates as a broad band in the 68-80 kDa region of the gel. 20 Purification ofrhML 5 3 was also conducted as described in Example 1. Briefly, 293-rhML 153 conditioned media was resolved on BLUE-SEPHAROSE as described for rhML 332 . The BLUE SEPHAROSE eluate was applied directly to a mpl-affinity column as described above. RhML 153 eluted from the mpl-affinity column was purified to homogeneity using a C4-HPLC column run under the same conditions used for rhML 332 . By SDS-PAGE the purified rhML 153 resolves into 20 major 25 and 2 minor bands with Mr of- 18,000-22,000. Expression and Purification of TPO from Chinese Hamster Ovary (CHO) Cells The expression vectors used to transfect CHO cells are designated: pSVI5.ID.LL.MLORF (full length of TPO 332 ), and pSVI5.ID.LL.MLEPO-D (truncated or TPO 153) . 30 cDNA corresponding to the entire open reading frame of TPO was obtained by PCR. The PCR product was purified and cloned between two restriction sites (Clal and Sall) of the plasmid pSVI5.ID.LL to obtain the vector pSVI5.ID.LL.MLORF. A second construct corresponding to the EPO homologous domain was generated the same way but using a different reverse primer (EPOD.Sal). The final construct for the vector coding for the EPO homologous domain of TPO is 35 called pSVI5.ID.LL.MLEPO-D. 28 WO 98/52598 PCT/US98/10475 These two constructs were linearized with Notl and transfected into Chinese Hamster Ovary cells (CHO-DP12 cells, EP 307,247 published 15 March 1989) by electroporation. 10 7 cells were electroporated in a BRL electroporation apparatus (350 Volts, 330 mF, low capacitance) in the presence of 10, 25 or 50 mg of DNA as described (Andreason, G.L. J.Tissue Cult. Meth., 15:56 5 (1993)). The day following transfection, cells were split in DHFR selective media (High glucose DMEM-F12 50:50 without glycine, 2mM glutamine, 2-5% dialyzed fetal calf serum). 10 to 15 days later individual colonies were transferred to 96 well plates and allowed to grow to confluency. Expression of ML 153 or ML 332 in the conditioned media from these clones was assessed using the Ba/F3-mpl proliferation assay. 10 The process for purifying and isolating TPO from harvested CHO cell culture fluid is described in Example 2. Briefly, harvested cell culture fluid (HCCF) is applied to a BLUE-SEPHAROSE column (Pharmacia) at a ratio of approximately 100L of HCCF per liter of resin. The column is then washed with 3 to 5 column volumes of buffer followed by 3 to 5 column volumes of a buffer containing 2.0M urea. TPO is then eluted with 3 to 5 column volumes of buffer containing both 2.0M 15 urea and 1.0M NaCl. The BLUE-SEPHAROSE eluate pool containing TPO is then applied to a wheat germ lectin SEPHAROSE column (Pharmacia) equilibrated in the BLUE-SEPHAROSE eluting buffer at a ratio of from 8 to 16 ml of BLUE-SEPHAROSE eluate per ml of resin. The column is then washed with 2 to 3 column volumes of equilibration buffer. TPO is then eluted with 2 to 5 column volumes of a buffer 20 containing 2.0M urea and 0.5M N-acetyl-D-glucosamine. The wheat germ lectin eluate containing TPO is then acidified and C 1 2

E

8 is added to a final concentration of 0.04%. The resulting pool is applied to a C4 reversed phase column equilibrated in 01% TFA, 0.04% C 1 2

E

8 at a load of approximately 0.2 to 0.5 mg protein per ml of resin. The protein is eluted in a two phase linear gradient of acetonitrile containing 0.1% TFA and 25 0.04% C 12

E

8 and a pool is made on the basis of SDS-PAGE. The C4 Pool is then diluted and diafiltered versus approximately 6 volumes of buffer on an AMICON YM or like ultrafiltration membrane having a 10,000 to 30,000 Dalton molecular weight cut off. The resulting diafiltrate may be then directly processed or further concentrated by ultrafiltration. The diafiltrate/concentrate is usually adjusted to a final concentration of 0.01% TWEEN-80. 30 All or a portion of the diafiltrate/concentrate equivalent to 2 to 5% of the calculated column volume is then applied to a SEPHACRYL S-300 HR column (Pharmacia) equilibrated in a buffer containing 0.01% TWEEN-80 and chromatographed. The TPO containing fractions which are free of aggregate and proteolytic degradation products are then pooled on the basis of SDS-PAGE. The resulting pool is filtered and stored at 2-8 0 C. 29 WO 98/52598 PCT/US98/10475 Methods for Transforming and Inducing TPO Synthesis in a Microorganism and Isolating, Purifying and Refolding TPO Made Therein Construction ofE. coli TPO expression vectors is described in detail in Example 3. Briefly, 5 plasmids pMP21, pMPl 51, pMP41, pMP57 and pMP202 were all designed to express the first 155 amino acids of TPO downstream of a small leader which varies among the different constructs. The leaders provide primarily for high level translation initiation and rapid purification. The plasmids pMP210-1, -TS, -21, 22, -24, -25 are designed to express the first 153 amino acids of TPO downstream of an initiation methionine and differ only in the codon usage for the first 6 amino acids of TPO, while 10 the plasmid pMP251 is a derivative ofpMP210-1 in which the carboxy-terminal end of TPO is extended by two amino acids. All of the above plasmids will produce high levels of intracellular expression of TPO in E. coli upon induction of the tryptophan promoter (Yansure, D. G. et al., Methods in Enzymology, 185:54-60 (Goeddel, D.V., Ed.) Academic Press, San Diego (1990)). The plasmids pMP I and pMP 172 are intermediates in the construction of the above TPO intracellular 15 expression plasmids. The above TPO expression plasmids were used to transform the E. coli using the CaCI 2 heat shock method (Mandel, M. et al., J. Mol. Biol, 53:159-162, (1970)) and other procedures described in Example 3. Briefly, the transformed cells were grown first at 37 0 C until the optical density (600 nm) of the culture reached approximately 2-3. The culture was then diluted and, after growth with aeration, acid was added. The culture was then allowed to continue growing with aeration 20 for another 15 hours after which time the cells were harvested by centrifugation. The isolation, purification and refolding procedures given below for production of biologically active, refolded human TPO or fragments thereof is described in Example 4 can be applied for the recovery of any TPO variant including N and C terminal extended forms. Other procedures suitable for refolding recombinant or synthetic TPO can be found in the following patents: Builder et al., US 4,511,502; 25 Jones et al., US 4,512,922; Olson, US 4,518,526 and Builder et al., US 4,620,948; for a general description of the recovery and refolding process for a variety of recombinant proteins expressed in an insoluble form in E. coli. Methods for Measurement of Thrombopoietic Activity 30 Thrombopoietic activity may be measured in various assays including the Ba/F3 mpl ligand assay. An in vivo mouse platelet rebound synthesis assay, induction of platelet cell surface antigen assay as measured by an anti-platelet immunoassay (anti-GPIIbIIIa) for a human leukemia megakaryoblastic cell line (CMK) (see Sato et al., Brit. J. Heamatol., 72:184-190 (1989)) and induction of polyploidization in a megakaryoblastic cell line (DAMI) (see Ogura et al., Blood, 35 72(1):49-60 (1988)). Maturation of megakaryocytes from immature, largely non-DNA synthesizing cells, to morphologically identifiable megakaryocytes involves a process that includes appearance of 30 WO 98/52598 PCT/US98/10475 cytoplasmic organelles, acquisition of membrane antigens (GPIIbIIIa), endoreplication and release of platelets as described in the background. A lineage specific promoter (i.e. the mpl ligand) of megakaryocyte maturation would be expected to induce at least some of these changes in immature megakaryocytes leading to platelet release and alleviation of thrombocytopenia. Thus, assays were 5 designed to measure the emergence of these parameters in immature megakaryocyte cell lines, i.e., CMK and DAMI cells. The CMK assay measures the appearance of a specific platelet marker, GPIIbIIIa, and platelet shedding. The DAMI assay measures endoreplication since increases in ploidy are hallmarks of mature megakaryocytes. Recognizable megakaryocytes have ploidy values of 2N, 4N, 8N, 16N, 32N, etc. Finally, the in vivo mouse platelet rebound assay is useful in demonstrating that 10 administration of the test compound (here the mpl ligand) results in elevation of platelet numbers. Two additional in vitro assays have been developed to measure TPO activity. The first is a kinase receptor activation (KIRA) ELISA in which CHO cells are transfected with a mpl-Rse chimera and tyrosine phosphorylation of Rse is measured by ELISA after exposure of the mpl portion of the chimera to mpl ligand. The second is a receptor based ELISA in which ELISA plate coated rabbit anti 15 human IgG captures human chimeric receptor mpl-IgG which binds the mpl ligand being assayed. A biotinylated rabbit polyclonal antibody to mpl ligand (TPO 155 ) is used to detect bound mpl ligand which is measured using streptavidin-peroxidase. Therapeutic Use of Thrombopoietin Materials 20 The biologically active thrombopoietic protein (TPO) may be used in a sterile pharmaceutical preparation or formulation to stimulate megakaryocytopoietic or thrombopoietic activity in patients suffering from thrombocytopenia due to impaired production, sequestration, or increased destruction of platelets. Thrombocytopenia-associated bone marrow hypoplasia (e.g. aplastic anemia following chemotherapy or bone marrow transplant) may be effectively treated with the compounds of this 25 invention as well as disorders such as disseminated intravascular coagulation (DIC), immune thrombocytopenia (including HIV-induced ITP and non HIV-induced ITP), chronic idiopathic thrombocytopenia, congenital thrombocytopenia, myelodysplasia, and thrombotic thrombocytopenia. Additionally, these megakaryocytopoietic proteins may be useful in treating myeloproliferative thrombocytotic diseases as well as thrombocytosis from inflammatory conditions and in iron 30 deficiency. The method of the invention is also useful to treat mammals or human patients which have suffered from exposure to ionizing radiation sufficient to cause thrombocytopenia, for example, persons exposed to nuclear accidents such as the well-known accident which occurred at Chernobyl. TPO is well tolerated by patients and this may justify the rapid administration of TPO within a few 35 hours after a nuclear accident to all persons affected by radiation. As noted in more detail below, the TPO responsiveness of progenitor cells appears to be very large shortly after exposure of a person to 31 WO 98/52598 PCT/US98/10475 radiation and/or chemotherapy. the method of the invention may also be used as a radioprotective procedure by administering TPO prophylactically to a person who will be exposed to ionizing radiation. For example, an emergency worker may be required to enter highly contaminated areas in cases of a major nuclear accident. Administration of a prophylactic dose of TPO prior to exposure 5 according to the dosing methods of the present invention will ensure that the worker has high levels of early multilineage progenitor cells in order to reduce the degree of thrombocytopenia induced by the exposure to radiation. Preferred uses of the thrombocytopoietic protein (TPO) of this invention are in: myelotoxic chemotherapy for treatment of leukemia or solid tumors, myeloablative chemotherapy for autologous 10 or allogeneic bone marrow transplant, myelodysplasia, idiopathic aplastic anemia, congenital thrombocytopenia, and immune thrombocytopenia. Still other disorders usefully treated with the thrombopoietin proteins of this invention include defects or damage to platelets resulting from drugs, poisoning or activation on artificial surfaces. In these cases, the instant compounds may be employed to stimulate "shedding" or new "undamaged" 15 platelets. Examples EXAMPLE 1 - Expression and Purification of TPO from 293 Cells 20 Preparation of 293 Cell Expression Vectors A cDNA corresponding to the TPO entire open reading frame was obtained by PCR using the following oligonucleotides as primers: TABLE 1 293 PCR Primers Cla.FL.F:5N ATC GAT ATC GAT CAG CCA GAC ACC CCG GCC AG 3N (SEQ ID NO: 1) hmpII-R: 5N GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA 3N (SEQ ID NO:2) prk5-Hmpl was used as a template for the reaction in the presence of pfu DNA polymerase 25 (Stratagene). Initial denaturation was for 7 min. at 94oC followed by 25 cycles of amplification (1 min. at 94oC, 1 min. at 55 0 C and 1 min. at 72 0 C). Final extension was for 15 min. at 72 0 C. the PCR product was purified and cloned between the restriction sites Clal and XbaI of the plasmid pRK5tkneo, 32 WO 98/52598 PCT/US98/10475 a pRK5 derived vector modified to express a neomycin resistance gene under the control of the thymidine kinase promote, to obtain the vector pRK5tkneo.ORF. A second construct corresponding to the epo homologous domain was generated the same way but using Cla.FL.F as forward primer and the following reverse primer: 5 Arg. STOP.Xba: 5NTCT AGA TCT AGA TCA CCT GAC GCA GAG GGT GGA CC 3N (SEQ ID NO: 3) The final construct is called pRK5-tkneoEPO-D. The sequence of both constructs was verified. 10 Transfection of Human Embryonic Kidney cells These 2 constructs were transfected into human embryonic kidney cells by the CaPO 4 method. 24 hours after transfection selection of neomycin resistant clones was started in the presence of 0.4 mg/ml G418. 10 to 15 days later individual colonies were transferred to 96 well plates and allowed to 15 grow to confluency. Expression ofML 153 or ML 332 (TPO153 or TPO 332) in the conditioned media from these clones was assessed using the Ba/F3-mpl proliferation assay. Purification of rhML 332 392-rhML 332 conditioned media was applied to a BLUE-SEPHAROSE (Pharmacia) column 20 that was equilibrated in 10 mM sodium phosphate pH 7.4 (buffer A). The column was subsequently washed with 10 column volumes each of buffer A and buffer A containing 2M urea. The column was then eluted with buffer A containing 2M urea and IM NaCI. The BLUE-SEPHAROSE elution pool was then directly applied to a WGA-SEPHAROSE column equilibrated in buffer A. The WGA SEPHAROSE column was then washed with 10 column volumes of buffer A containing 2M urea and I 25 M NaCI and eluted with the same buffer containing 0.5M N-acetyl-D-glucosamine. The WGA SEPHAROSE eluate was applied to a C4-HPLC column (Synchrom, Inc.) equilibrated in 0.1% TFA. The C4-HPLC column was eluted with discontinuous propanol gradient (0-25%, 25-35%, 35-70%). rhML 332 was found to elute in the 28-30% propanol region of the gradient. by SDS-PAGE the purified rhML 332 migrates as a broad band in the 68-8- kDa region of the gel. 30 Purification ofrhML 153 392-rhML 153 conditioned media was resolved on BLUE-SEPHAROSE as described for rhML 332 . The BLUE-SEPHAROSE eluate was applied directly to a mpl-affinity column as described above. RhML 153 eluted from the mpl-affinity column was purified to homogeneity using a C4-HPLC 33 WO 98/52598 PCT/US98/10475 column run under the same conditions as described for rhML 332 . By SDS-PAGE the purified rhML 153 resolves into 2 major and 2 minor bands with Mr of 18,000-21,000. EXAMPLE 2 - Expression and Purification of TPO from CHO 5 1. Description of CHO Expression Vectors The expression vectors used in the electroporation protocols described below have been designated: pSV15.ID.LL.MLORF (full length or hTPO332), and 10 pSV I5.ID.LL.MLEPO-D (truncated or hTPO 153 )* 2. Preparation of CHO Expression Vectors A cDNA corresponding to the hTPO entire open reading frame was obtained by PCR using the oligonucleotide primes of the following Table. 15 CHO Expression Vector PCR Primers Cla.FL.F2 5' ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G 3' (SEQ ID NO:4) ORF.Sal 5' AGT CGA CGT CGA CGT CGG CAG TGT CTG AGA ACC 3' (SEQ ID NO:5) PRK5-hmpl I was used as template for the reaction in the presence of pfu DNA polymerase (Stratagene). Initial denaturation was for 7 min. at 94 0 C followed by 25 cycles of amplification (1 min. 20 at 94oC, 1 min. at 55 0 C and 1 min. at 72oC). Final extension was for 15 min. at 72 0 C. The PCR product was purified and cloned between the restriction sites Clal and Sall of the plasmid pSV15.ID.LL to obtain the vector pSV15.ID.LL.MLORF. A second construct corresponding to the EPO homologous domain was generated the same way but using Cla.FL.F2 as forward primer and the following reverse primer: EPOD.Sal 5' AGT CGA CGT CGA CTC ACC TGA CGC AGA GGG TGG ACC 3' (SEQ ID 25 NO:6). The final construct is called pSV15.ID.LL.MLEPO-D. The sequence of both constructs was verified. In essence, the coding sequences for the full length and truncated ligand were introduced into the multiple cloning site of the CHO expression vector pSV15.ID.LL. This vector contains the SV40 early promoter/enhancer region, a modified splice unit containing the mouse DHFR cDNA, a multiple 30 cloning site for the introduction of the gene of interest (in this case the TPO sequences described) an 34 WO 98/52598 PCT/US98/10475 SV40 polyadenylation signal and origin of replication and the beta-lactamase gene for plasmid selection and amplification in bacteria. 3. Methodology for Establishing Stable CHO Cell Lines Expressing Recombinant Human TP0 332 and 5 TPO 15 3 a. Description of CHO parent cell line The host CHO (Chinese Hamster Ovary) cell line used for the expression of the TPO molecules described herein is known as CHO-DP12 (see EP 307,247 published 15 March 1989). This mammalian cell line was clonally selected from a transfection of the parent line (CHO-K1 DUX-B 11 10 (DHFR-)- obtained from Dr. Frank Lee of Stanford University with the permission of Dr. L. Chasin) with a vector expressing preproinsulin to obtain clones with reduced insulin requirements. These cells are also DHFR minus and clones can be selected for the presence of DHFR cDNA vector sequences by growth on medium devoid of nucleoside supplements (glycine, hypoxanthine, and thymidine). This selection system for stably expressing CHO cell lines is commonly used. 15 b. Transfection method (electroporation)

TPO

332 and TP0 153 expressing cell lines were generated by transfecting DP12 cells via electroporation (see e.g. Andreason, G.L. J. Tiss. Cult. Meth., 15, 56 (1993) with linearized pSVI5.ID.LL.MLORF or pSVI5.ID.LL.MLEPO-D plasmids respectively. Three (3) restriction enzyme reaction mixtures were set up for each plasmid cutting; 10 micrograms, 25 micrograms and 50 20 micrograms of the vector with the enzyme NOTI by standard molecular biology methods. This restriction site is found only once in the vector in the linearization region 3' and outside the TPO ligand transcription units. The 100 microliter reactions were set up for overnight incubation at 37 degrees. The next day the mixes were phenol-chloroform-isoamyl alcohol (50:49:1) extracted one time and ethanol precipitated on dry ice for approximately one hour. The precipitate was then collected by a 15 25 minute microcentrifugation and dried. The linearized DNA was resuspended into 50 microliters of Ham's DMEM-F12 1:1 medium supplemented with standard antibiotics and 2mM glutamine. Suspension growing DP 12 cells were collected, washed one time in the medium described for resuspending the DNA and finally resuspended in the same medium at a concentration of 107 cells per 750 microliters. Aliquots of cells (750 microliters) and each linearized DNA mix were incubated 30 together at room temperature for one hour and then transferred to a BRL electroporation chamber. Each reaction mix was then electroporated in a standard BRL electroporation apparatus at 350 volts set at 330 micro F and low capacitance. After electroporation, the cells were allowed to sit in the apparatus for 5 minutes and then on ice for an additional 10 minute incubation period. The electroporated cells were transferred to 60mm cell culture dishes containing 5 ml of standard, complete 35 growth medium for CHO cells (High glucose DMEM-F12 50:50 without glycine supplemented with 35 WO 98/52598 PCT/US98/10475 lX GHT, 2mM glutamine, and 5% fetal calf serum) and grown overnight in a 5% CO 2 cell culture incubator. c. Selection and screening method The next day, cells were trypsinized off the plates by standard methods and transferred to 5 150mm tissue culture dishes containing DHFR selective medium (Ham's DMEM-F12, 1:lmedium described above supplemented with either 2% or 5% dialyzed fetal calf serum but devoid of glycine, hypoxanthine and thymidine this is the standard DHFR selection medium we use). Cells from each 60mm dish were subsequently replated into 5/150 mm dishes. Cells were then incubated for 10 to 15 days( with one medium change) at 37 degrees/1 5% CO 2 until clones began to appear and reached sizes 10 amenable to transfer to 96 well dishes. Over a period of 4-5 days, cell lines were transferred to 96 well dishes using sterile yellow tips on a pipettman set at 50 ml. The cells were allowed to grow to confluency (usually 3-5 days) and then the trays were trypsinized and 2 copies of the original tray were reproduced. Two of these copies were short term stored in the freezer with cells in each well diluted into 50 microliter pf 10% FCS in DMSO. 5 day conditioned serum free medium samples were assayed 15 from confluent wells in the third tray for TPO expression via the Ba/F cell based activity assay. The highest expressing clones based on this assay were revived from storage and scaled up to 2 confluent 150mm T-flasks for transfer to the cell culture group for suspension adaptation, re-assay and banking. d. Amplification Protocol 20 Several of the highest titer cell lines from the selection described above were subsequently put through a standard methotrexate amplification regime to generate higher titer clones. CHO cell clones are expanded and plated in 10cm dishes at 4 concentrations of methotrexate (i.e 50 nM, 100 nM, 200 nM and 400 nM) at two or three cell numbers (105, 5x1 05, and 106 cells per dish). These cultures are then incubated at 37 degree/5% CO 2 until clones are established and amenable to transfer to 96 well 25 dishes for further assay. Several high titer clones from this selection were again subjected to greater concentrations of methotrexate (i.e. 600 nM, 800 nM, 1000 nM and 1200 nM) and as before resistant clones are allowed to establish and then transferred to 96 well dishes and assayed. 4. Culturing Stable CHO Cell Lines Expressing Recombinant Human TPO 3 3 2 and TP0 15 3 Banked cells are thawed and the cell population is expanded by standard cell growth methods 30 in either serum free or serum containing medium. After expansion to sufficient cell density, cells are washed to remove spent cell culture media. Cells are then cultured by any standard method including; batch, fed-batch or continuous culture at 25-40 0 C, neutral pH, with a dissolved 02 content of at least 5% until the constitutively secreted TPO is accumulated. Cell culture fluid is then separated from the cells by mechanical means such as centrifugation. 35 5. Purification of Recombinant Human TPO from CHO Culture Fluids 36 WO 98/52598 PCT/US98/10475 Harvested cell culture fluid (HCCF) is directly applied to a BLUE-SEPHAROSE 6 FAST FLOW column (Phamacia) equilibrated in 0.01 M Na phosphate pH 7.4, 0.15M NaCI at a ratio of approximately 100 L of HCCF per liter of resin and at a linear flow rate of approximately 300 ml/hr/cm 2 . The column is then washed with 3 to 5 column volumes of equilibration buffer followed by 5 3 to 5 column volumes of 0.01 M Na phosphate pH 7.4, 2.0 M urea. The TPO is then eluted with 3 to 5 column volumes of 0.01 M Na phosphate pH 7.4, 2.0M urea, 1.0M NaCl. The BLUE-SEPHAROSE pool containing TPO is then applied to a wheat germ lectin SEPHAROSE 6 MB column (Pharmacia) equilibrated in 0.01 M Na phosphate pH 7.4, 2.0M urea, and 1.0M NaCI at a ratio of from 8 to 16 ml of BLUE-SEPHAROSE pool per ml of resin at flow rate of approximately 50 ml/hr/cm 2 . The column is 10 then washed with 2 to 3 column volumes of equilibration buffer. The TPO is then eluted with 2 to 5 column volumes of 0.01 M Na phosphate pH 7.4, 2.0M urea, 0.5 M N-acetyl-D-glucosamine. The wheat germ lectin pool is then adjusted to a final concentration of 0.04%C 1 2

E

8 and 0.1% trifluroacetic acid (TFA). The resulting pool is applied to a C4 reverse phase column (Vydac 214TP1022) equilibrated in 0.1% TFA, 0.04% C 12

E

8 at a load of approximately 0.2 to 0.5 mg protein 15 per ml of resin at a flow rate of 157 ml/hr/cm 2 . The protein is eluted in a two phase linear gradient of acetonitrile containing 0.1% TFA, 0.04%

C

12

E

8 . The first phase is composed of a linear gradient from O to 30% acetonitrile in 15 minutes, the second phase is composed of a linear gradient from 30 to 60% acetonitrile in 60 minutes. The TPO elutes at approximately 50% acetonitrile. A pool is made on the basis of SDS-PAGE. 20 The C4 pool is then diluted with 2 volumes of 0.01 M Na phosphate pH 7.4, 0.15 M NaCI and diafiltered versus approximately 6 volumes of 0.01 M Na phosphate pH 7.4, 0.15 M NaCI on an AMICOM YM or like ultrafiltration membrane having a 10,000 to 30,000 Dalton molecular weight cut-off. The resulting diafiltrate may be then directly processed or further concentrated by ultrafiltration. The diafiltrate/concentrate is adjusted to a final concentration of 0.01% TWEEN-80. 25 All or a portion of the diafiltrate/concentrate equivalent to 2 to 5% of the calculated column volume is then applied to a SEPHACRYL S-300 HR column (Pharmacia) equilibrated in 0.01 M Na phosphate pH 7.4, 0.15M NaCI, 0.01% TWEEN-80 and chromatographed at a flow rate of approximately 17 ml/hr/cm 2 . The TPO containing fractions which are free of aggregate and proteolytic degradation products are pooled on the basis of SDS-PAGE. The resulting pool is filtered 30 on a 0.22 micron filter, MILLEX-GV or like, and stored at 2-8 0 C. EXAMPLE 3 - Transformation and Induction of TPO Protein Synthesis In E. coli 1. Construction of E. coli TPO expression vectors The plasmids pMP21, pMP151, pMP41, pMP57 and pMP202 are all designed to express the 35 first 155 amino acids of TPO downstream of a small leader which varies among the different constructs. The leaders provide primarily for high level translation initiation and rapid purification. The 37 WO 98/52598 PCT/US98/10475 plasmids pMP210-1, -T8, -21, -22, -24, -25 are designed to express the first 153 amino acids of TPO downstream of an initiation methionine and differ only in the codon usage for the first 6 amino acids of TPO, while the plasmid pMP251 is a derivative ofpMP210-1 in which the carboxy terminal end of TPO is extended by two amino acids. All of the above plasmids will produce high levels of 5 intracellular expression of TPO in E. coli upon induction of the tryptophan promoter (Yansura, D. G. et. al. Methods in Enzymology (Goeddel, D. V., Ed.) 185:54-60, Academic Press, San Diego (1990)). The plasmids pMP1 and pMP172 are intermediates in the construction of the above TPO intracellular expression plasmids. (a) Plasmid pMP1 10 The plasmid pMP1 is a secretion vector for the first 155 amino acids of TPO, and was constructed by ligating together 5 fragments of DNA. The first of these was the vector pPho21 in which the small Mlul-BamHI fragment had been removed. pPho21 is a derivative of phGH1 (Chang, C. N. et. al., Gene 55:189-196 (1987) in which the human growth hormone gene has been replaced with the E. coli phoA gene, and a Mlul restriction site has been engineered into the coding sequence 15 for the STII signal sequence at amino acids 20-21. The next two fragments, a 258 base pair Hinfl-Pstl piece of DNA from pRK5-hmpl encoding TPO amino acids 19-103, and the following synthetic DNA encoding amino acids 1-18 5'-CGCGTATGCCAGCCCGGCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCG TG (SEQ ID NO: 7) 20 ATACGGTCGGGCCGAGGAGGACGAACACTGGAGGCTCAGGAGTCATTTGACGAAGCACT GA-5' (SEQ ID NO:8) were preligated with T4-DNA ligase, and second cut with Pstl. The fourth was a 152 base pair Pstl-HaelII fragment from pRK5hmplI encoding amino acids 104-155 of TPO. The last was a 412 base pair Stul-BamHI fragment from pdh 108 containing the lambda to transcriptional terminator as 25 previously described (Scholtissek, S. et. al., NAR 15:3185 (1987)). (b) Plasmid pMP21 The plasmid pMP21 is designed to express the first 155 amino acids of TPO with the aid of a 13 amino acid leader comprising part of the STII signal sequence. It was constructed by ligating together three (3) DNA fragments, the first of these being the vector pVEG31 in which the small 30 Xbal-Sphl fragment had been removed. The vector pVEG31 is a derivative of pHGH207-1 (de Boer, H. A. et. al., in Promoter Structure and Function (Rodriguez, R. L. and Chamberlain, M. J., Ed), 462, Praeger, New York (1982)) in which the human growth hormone gene has been replaced by the gene for vascular endothelial growth factor ( this identical vector fragment can be obtained from this latter plasmid). 35 The second part in the ligation was a synthetic DNA duplex with the following sequence: 5'-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAA (SEQ ID NO:9) 38 WO 98/52598 PCT/US98/10475 TTAATACTTTTTCTTATAGCGTAAAGAAGAATTGCGC-5' (SEQ ID NO:10) The last piece was a 1072 base pair Mlul-Sphl fragment from pMPI encoding 155 amino acids of TPO. (c) Plasmid pMP 151 5 The plasmid pMP151 is designed to express the first 155 amino acids of TPO downstream of a leader comprising 7 amino acids of the STII signal sequence, 8 histidines, and a factor Xa cleavage site. pMP151 was constructed by ligating together three DNA fragments, the first of these being the previously described vector pVEG31 from which the small Xbal-Sphl fragment had been removed. The second was a synthetic DNA duplex with the following sequence: 10 5'-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACATCGAA GGTCGTAGCC (SEQ ID NO: 11)

TTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTCCAGCAT

5' (SEQ ID NO:12) The last was a 1064 base pair BgLI-Sphl fragment from pMP 11 encoding 154 amino acids of 15 TPO. The plasmid pMPI 1 is identical to pMPI with the exception of a few codon changes in the STII signal sequence( this fragment can be obtained from pMP 1). (d) Plasmid pMP202 The plasmid pMP202 is very similar to the expression vector pMP 151 with the exception that the factor Xa cleavage site in the leader has been replaced with a thrombin cleavage site. As shown in 20 Fig. 36, pMP202 was constructed by ligating together three DNA fragments. The first of these was the previously described pVEG31 in which the small Xbal-Sphl fragment had been removed. The second was a synthetic DNA duplex with the following sequence: 5'-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACATCGAA CCACGTAGCC (SEQ ID NO:13) 25 TTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTTGGTGCA T-5' (SEQ ID NO: 14) The last piece was a 1064 base pair BglI-Sphl fragment from the previously described plasmid pMP 11. (e) Plasmid pMP172 30 The plasmid pMP172 is a secretion vector for the first 153 amino acids of TPO, and is an intermediate for the construction of pMP210. pMP 172 was prepared by ligating together three DNA fragments, the first of which was the vector pLS321 amB in which the small EcoRI-Hindl section had been removed. The second was a 946 base pair EcoRI-Hgal fragment from the previously described plasmid pMP 11. The last piece was a synthetic DNA duplex with the following sequence: 35 5'-TCCACCCTCTGCGTCAGGT (SEQ IDNO:15) GGAGACGCAGTCCATCGA-5' (SEQ ID NO:16) 39 WO 98/52598 PCT/US98/10475 (f) Plasmid pMP210 The plasmid pMP210 is designed to express the first 153 amino acids of TPO after a translational initiation methionine. This plasmid was actually made as a bank of plasmids in which the first 6 codons of TPO were randomized in the third position of each codon, and was constructed by the 5 ligation of three DNA fragments. The first of these was the previously described vector pVEG31 in which the small Xbal-Sphl fragment had been removed. The second was a synthetic DNA duplex shown below treated first with DNA polymerase (Klenow) followed by digestion with Xbal and Hinl, and encoding the initiation methionine and the randomized first 6 codons of TPO. 5'-GCAGCAGTTCTAGAATTATGTCNCCNGCNCCNCCNGCNTGTGACCTCCGAACACTGGAG 10 GCTGTTCTCAGTAAA (SEQ ID NO:17) CAAGAGTCATTTGACGAAGCACTGAGGGTACAGGAAG-5' (SEQ ID NO: 18) The third was a 890 base pair Hinfl-Sphl fragment from pMP 172 encoding amino acids 19-153 of TPO. The plasmid pMP210 bank of approximately 3700 clones was retransformed onto high 15 tetracycline (50 microgram/ml) LB plates to select out high translational initiation clones (Yansura, D.G. et al., Methods: A Companion to Methods in Enzymology 4:151-158 (1992)). Of the 8 colonies which came up on high tetracycline plates, five of the best in terms of TPO expression were subject to DNA sequencing. (g) Plasmid pMP41 20 The plasmid pMP41 is designed to express the first 155 amino acids of TPO fused to a leader consisting of 7 amino acids of the STII signal sequence following by a factor Xa cleavage site. The plasmid was constructed by ligating together three pieces of DNA, the first of which was the previously described vector pVEG31 in which the small Xbal-Sphl fragment had been removed. The second was the following synthetic DNA duplex: 25 5'-CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (SEQ ID NO: 19) TTAATACTTTTTCTTATAGCGTAAATAGCTTCCAGCAT-5N (SEQ ID NO:20) The last piece of the ligation was the 1064 base pair Bgll-Sphl fragment from the previously described plasmid pMP 11. (h) Plasmid pMP57 30 The plasmid pMP57 expresses the first 155 amino acids of TPO downstream of a leader consisting of 9 amino acids of the Stll signal sequence and the dibasic site Lys-Arg. This dibasic site provides for a means of removing the leader with the protease ArgC. This plasmid was constructed by ligating together three DNA pieces. The first of these was the previously described vector pVEG31 in which the small XbaI-SphI fragment had been removed. The second was the following synthetic DNA 35 duplex: 5'-NCTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAAACGTAGCC (SEQ ID NO:21) 40 WO 98/52598 PCT/US98/10475 TTAATACTTTTTCTTATAGCGTAAAGAAGAATTTGCAT-5N (SEQ ID NO:22) The last part of the ligation was the 1064 base pair Bgil-Sphl fragment from the previously described plasmid pMP 11. (i) Plasmid pMP251 5 The plasmid pMP251 is a derivative of pMP210-1 in which two additional amino acids of TPO are included on the carboxy terminal end. This plasmid was constructed by ligating together two pieces of DNA, the first of these being the previously described pMP21 in which the small Xbal-Apal fragment had been removed. The second part of the ligation was a 316 base pair Xbal-Apal fragment from pMP210-1. 10 2. Transformation and Induction of E. co/i with TPO expression vectors The above TPO expression plasmids were used to transform the E. coli strain 44C6 (w3110 tonA A rpoHts lon A cip A galE) using the CaCl 2 heat shock method (Mandel, M. et al., J Mol. Biol., 53:159-162, (1970)). The transformed cells were grown first at 37 0 C in LB media containing 50 pg/ml carbenicillin until the optical density (600nm) of the culture reached approximately 2-3. The LB 15 culture was then diluted 20x into M9 media containing 0.49% casamino acids (w/v) and 50 pg/ml carbenicillin. After growth with aeration at 30 0 C for 1 hour, indole-3-acrylic acid was added to a final concentration of 50 llg/ml. The culture was then allowed to continue growing at 30 0 C with aeration for another 15 hours at which time the cells were harvested by centrifugation. 20 EXAMPLE 4 - Production of Biologically Active TPO (Met - 1 1-153) in E. coli. The procedures given below for production of biologically active, refolded TPO (Metl-153) can be applied in analogy for the recovery of other TPO variants including N and C terminal extended forms. 1. Recovery of non-soluble TPO (Met- 1 1-153) 25 E. coli cells expressing TPO (Met- 1 1-153) encoded by the plasmid pMP210-1 are fermented as described above. Typically, about 100g of cells are resuspended in 1 (10 volumes) of cell disruption buffer (10 mM Tris, 5 mM EDTA, pH 8) with a Polytron homogenizer and the cells centrifuged at 5000 x g for 30 minutes. The washed cell pellet is again resuspended in 1 L cell disruption buffer with the Polytron homogenizer and the cell suspension is passed through an LH 30 CELL DISRUPTER (LH Inceltech, Inc.) or through a MICROFLUIDIZER (Microfluidics International) according to the manufactures' instructions. The suspension is centrifuged at 5,000 x g for 30 min. and resuspended and centrifuged a second time to make a washed refractile body pellet. The washed pellet is used immediately or stored frozen at -70 0 C 35 2. Solubilization and purification ofmonomeric TPO Met -1 1-153) 41 WO 98/52598 PCT/US98/10475 solubilization of the TPO protein. High concentrations of urea (6-8M) are also useful but generally result in lower yields compared to guanidine. After solubilization, the solution is centrifuged at 30,000 x g for 30 min. to produce a clear supernatant containing denatured, monomeric TPO protein. The supernatant is then chromatographed on a SUPERDEX 200 gel filtration column (Pharmacia, 2.6 x 60 5 cm) at a flow rate of 2 ml/min. and the protein eluted with 20 mM Na phosphate, pH 6.0, with 10 mM DTT fractions containing monomeric, denatured TPO protein eluting between 160 and 200 ml are pooled. The TPO protein is further purified on a semi-preparative C4 reversed phase column (2 x 20 cm VYDAC). The sample is applied at 5 ml/min. to a column equilibrated in 0.1% TFA(trifluoroacetic acid) with 30% acetonitrile. The protein is eluted with a linear gradient of acetonitrile (30-60% in 60 10 min.). The purified reduced protein elutes at approximately 50% acetonitrile. This material is used for refolding to obtain biologically active TPO variant. 3. Generation of biologically active TPO (Met- 1 1-153) Approximately 20 mg of monomeric, reduced and denatured TPO protein in 40 ml 0.1% TFA/50% acetonitrile is diluted into 360 ml of refolding buffer containing optimally the following 15 reagents: 50 mM Tris 0.3 M NaCl 5 mM EDTA 2% CHAPS detergent 20 25% glycerol 5 mM oxidized glutathione 1 mM reduced glutathione pH adjusted to 8.3 After mixing, the refolding buffer is gently stirred at 4 0 C for 12-48 hr to effect maximal 25 refolding yields of the correct disulfide-bonded form of TPO (see below). The solution is then acidified with TFA to a final concentration of 0.2%, filtered through a 0.45 or 0.22 micron filter, and 110 volume of acetonitrile added. This solution is then pumped directly onto a C4 reversed phase column and the purified, refolded TPO (Met- 1 1-153) eluted with the same gradient program as above. Refolded, biologically active TPO is eluted at approximately 45% acetonitrile under these conditions. 30 Improper disulfide-bonded versions of TPO are eluted earlier. The final purified TPO (Met - 1 1-153) is greater than 95% pure as assessed by SDS gels and analytical C4 reversed phase chromatography. For animal studies, the C4 purified material was dialyzed into physiologically compatible buffers. Isotonic buffers (10 mM Na acetate, pH 5.5, 10 mM Na succinate, pH 5.5 or 10 mM Na phosphate, pH 7.4) containing 150 mM NaCI and 0.01% TWEEN-80 were utilized. 35 Because of the high potency of TPO in the Ba/F3 assay (half maximal stimulation is achieved at approximately 3 pgml), it is possible to obtain biologically active material utilizing many different 42 WO 98/52598 PCT/US98/10475 buffer, detergent and redox conditions. However, under most conditions only a small amount of properly folded material (<10%) is obtained. For commercial manufacturing processes, it is desirable to have refolding yields at least 10%, more preferably 30-50% and most preferably >50%. Many different detergents (TRITON X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, 5 SARKOSYL, TWEEN-20 and TWEEN-80, ZWITTERGENT 3-14 and others) were assessed for efficiency to support high refolding yields. Of these detergents, only the CHAPS family (CHAPS and CHAPSO) were found to be generally useful in the refolding reaction to limit protein aggregation and improper disulfide formation. Levels of CHAPS greater than 1% were most useful. Sodium chloride was required for best yields, with the optimal levels between 0.1 M and 0.5M. The presence of EDTA 10 (1-5 mM) limited the amount of metal-catalyzed oxidation (and aggregation) which was observed with some preparations. Glycerol concentrations of greater than 15% produced the optimal refolding conditions. For maximum yields, it was essential to have both oxidized and reduced glutathione or oxidized and reduced cysteine as the redox reagent pair. Generally higher yields were observed when the mole ratio of oxidized reagent is equal to or in excess over the reduced reagent member of the 15 redox pair pH values between 7.5 and about 9 were optimal for refolding of these TPO variants. Organic solvents (e.g. ethanol, acetonitrile, methanol) were tolerated at concentrations of 10-15% or lower. Higher levels of organic solvents increased the amount of improperly folded forms. Tris and phosphate buffers were generally useful. Incubation at 4oC also produced higher levels of properly folded TPO. 20 Refolding yields of 40-60% (based on the amount of reduced and denatured TPO used in the refolding reaction) are typical for preparations of TPO that have been purified through the first C4 step. Active material can be obtained when less pure preparations (e.g. directly after the Superdex 200 column or after the initial refractile body extraction) although the yields are less due to extensive precipitation and interference of non-TPO proteins during the TPO refolding process. 25 Since TPO (Met- 1 1-153) contains 4 cysteine residues, it is possible to generate three different disulfide versions of this protein: version 1: disulfides between cysteine residues 1-4 and 2-3 version 2: disulfides between cysteine residues 1-2 and 3-4 version 3: disulfides between cysteine residues 1-3 and 2-4. 30 During the initial exploration in determining refolding conditions, several different peaks containing the TPO protein were separated by C4 reversed phase chromatography. Only one of these peaks had significant biological activity as determined using the Ba/F3 assay. Subsequently, the refolding conditions were optimized to yield preferentially that version. Under these conditions, the misfolded versions are less than 10-20% of the total monomer TPO obtained. 35 The disulfide pattern for the biologically active TPO has been determined to be 1-4 and 2-3 by mass spectrometry and protein sequencing (i.e. version 1). Aliquots of the various C4-resolved peaks 43 WO 98/52598 PCT/US98/10475 (5-10 nmoles) were digested with trypsin (1:25 mole ratio of trypsin to protein). The digestion mixture was analyzed by matrix assisted laser desorption mass spectrometry before and after reduction with DTT. After reduction, masses corresponding to most of the larger tryptic peptides of TPO were detected. In the un-reduced samples, some of these masses were missing and new masses were 5 observed. The mass of the new peaks corresponded basically to the sum of the individual tryptic peptides involved in the disulfide pair. Thus it was possible to unequivocally assign the disulfide pattern of the refolded, recombinant, biologically active TPO to be 1-4 and 2-3. This is consistent with the known disulfide pattern of the related molecule erythropoietin. D. Biological activity of recombinant, refolded TPO (met 1-153) 10 Refolded and purified TPO (Met- 1 1-153) has activity in both in vitro and in vivo assays. In the Ba/F3 assay, half-maximal stimulation of thymidine incorporation into the Ba/F3 cells was achieved at 3.3 pg /ml (0.3 pM). In the mpl receptor-based ELISA, half-maximal activity occurred at 1.9 ng/ml (120 pM). In normal and myelosuppressed animals produced by near-lethal X-radiation, TPO (Met- 1 1-153) was highly potent (activity was seen at doses as low as 30 ng/mouse) to stimulate the 15 production of new platelets. EXAMPLE 5 - Myelosuppressed (Carboplatin/Irradiation) Mouse Data Animals All animal studies were approved by the Institutional Care and Use Committee of Genentech 20 Inc. Prior to the start of the experiment all animals were ear-tagged for identification and a base-line complete blood count (CBC) obtained. Groups of 10 female C57BL/6 mice were irradiated with 5.0 Gy of gamma irradiation from a 137 Cs source. Within 6 hours, the animals were given 1.2 mg carboplatin as a 200 microliter intraperitoneal injection. The following are the protocols and results using recombinant murine thrombopoietin 25 (rmTPO) in a standard mouse model. It will be understood that one skilled in the art considers this model to correlate with treatment in human beings. Human thrombopoietin has been tested in the same mouse model and was found to show relevant activity, albeit at a lesser level because of the species specificity. Therefore, the following protocol was chosen using the proper murine TPO counterpart for that species so that relevant effect could be demonstrated. Again, use of human TPO in the mouse 30 protocol would provide similar results differing only in degree. Procurement of Blood Samples Prior to the experiment and at time points throughout the study, 40 microliter of blood was taken from the orbital sinus and immediately diluted into 10 mL of diluent to prevent clotting. The 35 complete blood count (CBC) from each blood sample was measured on a SERRANO Baker system 44 WO 98/52598 PCT/US98/10475 9018 blood analyzer within 60 min of collection. Only half of the animals in each dose group were bled on a given day; thus, each animal was bled on alternate time points. Treatment Regimens 5 Experiment 1: In order to determine the response to recombinant murine thrombopoietin (rmTPO335aa) in animals rendered thrombocytopenic, groups of animals were treated for 1, 2, 4, or 8 consecutive days with 0.1 microgram/day (5 microgram/kg/day approx.). Treatment with rmTPO (335aa) was started 24 hours after the initiation of the model and was given as a daily 100 microliter subcutaneous injection. 10 Experiment 2: In order to determine the nature of the dose-response relationship for rmTPO(335) in this model, animals were given a single injection of rmTPO (335) 24 hours after the initiation of the model. Groups of animals received one of 0.01, 0.03, 0.1 or 0.3 microgram of rmTPO (335) as a single 100 microliter subcutaneous injection. In order to compare two routes of administration, a contemporaneous experiment used 4 groups of animals receiving identical doses of rmTPO (335) but 15 via an intravenous route (lateral tail vein). Experiment 3: This series of experiments was done to compare the efficacy of various pegylated truncated rmTPO molecules (rmTPO(153) coupled to polyethylene glycol (PEG). i. In this experiment thrombocytopenic animals were injected (0.1 microgram subcutaneous) with one of the following pegylated rmTPO(153) molecules: no PEG, one 20K PEG or one 40K PEG. 20 ii. In the final experiment there was compared the effects of administering a single 40K PEG rmTPO(153) molecule by giving 0. microgram either subcutaneously or intravenously to animals rendered thrombocytopenic. rmTPO(335) (0.1 microgram) was used as a positive control. Results 25 The combination of sublethal irradiation and carboplatin resulted in a reproducible response giving consistent thrombocytopenia in 100% of the animals. The nadir for the thrombocytopenia occurred at day 10 with a gradual recovery of platelet numbers by day 21 to day 28. Accompanying this thrombocytopenia was a pronounced anemia with the nadir occurring slightly later on day 14 to 17 and recovery to control red blood cell counts by day 28. White blood cell counts were also depleted 30 during the course of the experiment. Experiment 1: A single dose of 0.1 microgram rmTPO(335) given 24 hours after the initiation of the model accelerated the recovery of platelet numbers in this murine model. This single administration of rmTPO(335) elevated the nadir of the response from 196x10 3 ± 33x10 3 /microliter on day 10 to 35 434x10 3 ± 7x10 3 /microliter on day 7. The initial rate of decline in the platelet numbers remained unchanged but the recovery phase was much more rapid with platelet numbers returning to normal by 45 WO 98/52598 PCT/US98/10475 day 14 as opposed to day 21 in the control group. Some further improvement in the rate of recovery was seen by giving 0.1 microgram/day on day 1 and day 2 but this was marginal. No further improvement could be seen by giving rmTPO(335) for 4 or 8 consecutive days (Fig. la). In addition to the accelerated recovery in platelet numbers, the anemia which develops in these animals was also 5 attenuated by a single dose of rmTPO(335) given on day 1. As with the platelet counts, no further advantage could be gained by giving rmTPO(335) more than once (Fig. lb). rmTPO(335) had no effect on the leukocytopenia that accompanies the falls in platelet and red blood cell counts. (Fig. I c). Experiment 2: The response to single subcutaneous doses of rmTPO(335) given 24 hours after the 10 initiation of the model was dose dependent. The lowest dose tested (0.01 microgram) had no effect on the platelet recovery compared to controls. However, the response is almost maximal when 0.03 microgram was given (Fig. 2a). This extremely steep dose response curve is better appreciated when the platelet numbers on day 14 are plotted on a log-linear plot (Fig. 3a). A similar steep dose response is seen for erythrocyte repopulation in this model (Fig. 3b). Intravenous administration of rmTPO(335) 15 gave a similar dose dependent response. However, the lowest dose tested (0.01 microgram) was effective when given IV, (Fig. 4a) suggesting that the dose response curve is shifted to the left. This increase in potency is small since the shift is less than half an order of magnitude (Fig. 3a). What is more important is that both routes of administration have the comparable maxima (Fig. 3a). The subcutaneous and intravenous route of administration also augmented the recovery from the anemia in 20 a dose-dependent fashion (Figs. 2a, 3b, 4b). However, neither the subcutaneous nor the intravenous route of administration had an effect on the leukocytopenia over the dose range tested (Figs. 2c,4c). Experiment 3: A. Pegylation of the rmTPO(153) with either a single 20K PEG or a single 40K PEG had a 25 greater effect on the platelet recovery than the un-pegylated molecule. Unlike the full-length molecule, neither of the pegylated rmTPO(153) molecules affected the nadir of the thrombocytopenia but greatly accelerated the recovery phase of the model when given as a single 0.1 microgram SC dose 24 hours after initiation of the model (Fig 5a). This is very evident on day 14 when the platelet counts are 80x10 3 + 15x10 3 /microliter, 268x10 3 ± 67x10 3 /microliter, 697x10 3 + 297x10 3 /microliter, and 30 878x10 3 ± 31x10 3 /microliter for controls, rmTPO(153) no PEG, rmTPO(153) + 20K PEG and rmTPO(153) + 40K PEG respectively (Fig. 5a). The same profile was also evident on the erythrocyte response (Fig. 5b). None of these rmTPO(153)-based molecules had any effect on the leukocytopenia in this model. (Fig. 5c). B. rmTPO(153) + 40K PEG (0.1 microgram) gave a consistent response when administered as 35 either a single intravenous or subcutaneous injection. In this experiment, the subcutaneous route slightly altered the nadir on day 10 and returned platelets to control levels by day 14 as compared to 46 WO 98/52598 PCT/US98/10475 day 28 in the control group (Fig. 6a). In the animals given the drug intravenously, there was a similar effect on the nadir and rate of recovery (Fig. 7a). The response to this 40K pegylated truncated rmTPO(153) molecule is almost identical to the response to the rmTPO(335) on both platelet and erythrocyte recovery when given either subcutaneously (Fig. 6b) or intravenously (Fig. 7b). As with 5 all of the other experiments rmTPO(153) + 40K PEG given either subcutaneously or intravenously had no effect on the circulating levels of white blood cells (Figs. 6c, 7c). In parallel experiments, the use of 10K-pegylated versions of this molecule did not modify the response to rmTPO(153) on either platelet or erythrocyte repopulation. 10 EXAMPLE 6 The following are protocols and results using single-dose therapy with recombinant human thrombopoietin (rhTPO 332 ) in human patients receiving cytotoxic chemotherapy: Single-dose therapy with recombinant human thrombopoietin (rhTPO) in patients receiving cytotoxic chemotherapy. 15 Preclinical models of intensive chemoradiotherapy demonstrated that a single dose of rhTPO raises the platelet nadir and shortens the period of severe thrombocytopenia. Interim results of two Phase I studies in which single doses of rhTPO were administered to cancer patients receiving chemotherapy are presented. 20 Patients and Methods: Both studies began with 21-day, pre-chemotherapy periods (cycle 0) for assessment of rhTPO safety and platelet response after single IV bolus injections of 0.3, 0.6, or 1.2 meg/kg (3 patients per group in each study). Patients then received the same dose of rhTPO after chemotherapy in selected subsequent cycles. The first study population consisted of patients with advanced malignancies who 25 received rhTPO the day following salvage thiotepa chemotherapy (65 mg/m 2 q28d) in each of two consecutive chemotherapy cycles. The second study included chemotherapy naive patients with sarcoma undergoing induction treatment with AI chemotherapy (doxorubicin 90 mg/m 2 , 10 g/m 2 q2Id. Following cycle 0, patients in this study were monitored during the first chemotherapy cycle and received a single rhTPO injection the day following completion of chemotherapy (d5) during the 30 second and subsequent cycles. Results: 14 patients have been treated to date. rhTPO was well tolerated with no reported serious adverse events attributed to study drug. Antibodies to rhTPO have not been observed. In cycle 0 the 35 lowest (0.3 mcg/kg) dose was weakly active, with increased activity at higher doses as shown below. 47 WO 98/52598 PCT/US98/10475 RhTPO Patients Mean Baseline Median Maximum Median % dose N Patients (1 l) (SD) Platelet (1 g1) (Range) Increase (mcg/kg) 0.3 7 339 (133) 510 (277-628) 40 0.6 5 235 (69) 486 (386-509) 103 1.2 2 203 (46) 523 (437, 608) 158 The maximum platelet count during cycle 0 occurred on median day 11 (range 7-14). No significant changes were found in WBC or HCT. FACS analysis of bone marrow showed increases in all CD34+ subsets in 2/2 patients following 0.6 mcg/kg. Increases in peripheral blood CD34+ cells 5 were also seen in these patients, suggesting that TPO might have stem cell mobilizing activity. Dose calculation and post-chemotherapy treatment are ongoing. Together these phase 1 studies suggest that single dose administration of rhTPO is safe and well tolerated. The 0.3, 0.6. and 1.2 mcg/kg. dose levels show increasing thrombopoietic activity. The ongoing treatment of patients at higher dose levels will test the hypotheses that a single dose of rhTPO 10 is efficacious in ameliorating thrombocytopenia following intensive chemotherapy. EXAMPLE 7A - A Phase I Study To Determine The Safety, Tolerance, Pharmacokinetics And Pharmacodynamics Of Recombinant Human Thrombopoietin (rhTPO) In Subjects With Sarcoma Receiving Adriamycin And Ifosfamide 15 Treatment Plan This was a single-center, open-label, dose-escalation, study of single and multiple IV doses of rhTPO with major safety endpoints. rhTPO was administered to subjects with histologically diagnosed sarcoma to determine whether its administration helped to prevent, delay, ameliorate, or shorten the duration of the known thrombocytopenic effects of doxorubicin and ifosfamide. 20 At present, 71 subjects have been enrolled on to this study which addresses the safety and the activity, of various rhTPO dosing schedule in conjuction with G-CSF and GM-CSF. The study begins with a 21-day pre-chemotherapy cycle (cycle 0) to assess safety, activity, and pharmacokinetics in patients with cancer. The data indicates a dose-dependent increase in peripheral blood platelet counts and bone marrow megakaryocytes in response to either a single or multiple IV doses of rhTPO, with 25 doses ranging from 0.3-3.6 mg/kg. Thus rhTPO dosing regimens in which all of the rhTPO was administered after the completion of chemotherapy were safe, but demonstrated only modest activity. One patient with a normal platelet count developed an uncomplicated deep venous thrombosis in the leg which resolved with conservative therapy, and neutralizing antibodies have not been observed. Subjects had either metastatic or unresectable sarcoma. 30 In patients who have received a single dose of rhTPO prior to the chemotherapy have experienced a benefit in terms of reducing the depth and duration of the platelet nadir when compared 48 WO 98/52598 PCT/US98/10475 to patients in other dosing arms or historical controls, and this effect has been exhibited throughout the six cycles of planned chemotherapy (of note, none of the historical control patients (n= 18) received all six cycles, and only two received five cycles). The prechemotherapy dose has been well-tolerated. As dosing through chemotherapy is safe and potentially more efficacious, this rhTPO dosing regimen is 5 being incorporated into the protocol. Dose Levels Five dose levels of rhTPO and six regimens (Arms A-F) were evaluated in this study. Dose levels and the number of subjects per dose level are as shown in the Table below. 10 Dose Levels, Doses and Number of Subjects LEVEL DOSE N Dose lA 0.3 gg/kg X 1 3 2A 0.6 gg/kg X 1 3 3A 1.2 jig/kg X 1 3 4A 2.4 jg/kg Xl 1 4 5A 3.6 gg/kg X 1 3 IB 0.3 jig/kg X 2 6 2B 0.6 jig/kg X 2 3 3B 1.2 gg/kg X 2 4 4B 2.4 jg/kg X 2 3 5B 3.6 jig/kg X 2 3 3C 1.2 jg/kg QDX7 3 4C 2.4 jg/kg QDX7 3 3D 1.2 jg/kg pre/post 6 4D 2.4 jig/kg pre/post 6 OBD*-Chemo 1.2 jig/kg pre/post 6 OBD-TPO 1.2 jg/kg pre/post 6 3E 1.2 jg/kg pre/post 6 TPO +GM-CSF 3Fa 1.2 jg/kg d -1,1, 4 6 + G-CSF 3Fb 1.2 gg/kg d -1,1, 4 6 + GM CSF 83 *OBD--Optimal Biologic Dose 49 WO 98/52598 PCT/US98/10475 Peak elevations in platelet count after single- or multiple-dose administration in preclinical studies have been observed within several days after initiation of dosing. Therefore, a peak platelet response would be expected within 7-14 days after initiation of dosing in this study. Current clinical experience has supported these findings. 5 Study Design Subjects were assigned to one of the dose groups described in the Table above. Study schemata for Arms A-F are shown below. All subjects received doxorubicin and ifosfamide in Cycle 1 (Days 0, 1, 2, and 3) and in additional cycles. The rhTPO dosing regimens for this study are as follows: 10 Arm A Cycle 0: rhTPO on Day 0 Cycle 1: no rhTPO Cycle 2+: rhTPO on Day 4 Arm B 15 Cycle 0: rhTPO on Days 0 and 3 Cycle 1: no rhTPO Cycle 2+: rhTPO on Days 4 and 7 Arm C Cycle 1: no rhTPO 20 Cycle 2+: daily rhTPO on Days 4 through 10, or until the post-nadir platelet count is 3 100,000/ L Arm D Cycle 1: no rhTPO Cycle 2+: rhTPO on Days D 1, 4, and 7 25 Arm E Cycle 1: no rhTPO Cycle 2+: rhTPO as per Arm D regimen, but with GM-CSF replacing G-CSF Cycle 0: This phase of the study evaluates safety, pharmacokinetics, and pharmacodynamics of single and multiple dosing. Subjects in Arm A received a single IV injection of rhTPO (Day 0). 30 Subjects in Arm B received an IV injection of rhTPO on Days 0 and 3. Cycle 1: Subjects received doxorubicin and ifosfamide. There was no rhTPO administered during Cycle 1. This provides a control cycle for comparison of each subject's response to subsequent cycles with rhTPO. This control cycle also clarifies chemotherapy-related adverse events in the absence of rhTPO dosing. 35 Cycle 2+: Subjects received doxorubicin and ifosfamide. Subjects in Arm A received a single IV injection of rhTPO (Day 4). Subjects in Arm B received IV injections of rhTPO on Days 4 and 7. 50 WO 98/52598 PCT/US98/10475 Subjects in Arm C received up to seven daily IV injections of rhTPO on Days 4-10, or until the post-nadir platelet count was greater than or equal to 100,000/iL. Subjects in Arm D received IV injections of rhTPO on Day -1 (1 day prior to chemotherapy) and on Days 4 and 7. To explore a possible synergistic relationship of rhTPO with GM-CSF in humans while 5 minimizing subject risk, GM-CSF replaced G-CSF in Arm E. GM-CSF was administered in Cycles 1 and 2, and in subsequent cycles where there is proven benefit. Subjects in Arm E received rhTPO according to the Arm D regimen. Study Schema ARM A 0 21 0m 0 1 2 3 21 0 1 2 3 4 21 2 10 ARM B 0 1 2 3 21 0 oM IrM I 0 1 2 3 21 0 1 2 3 4 5 6 7 21 2 J ARM C 0 1 2 3 21 Iamlm i I I 0 1 2 3 4 5 6 7 8 910 21 2 ARM D and E 0 1 2 3 21 -1 0 1 2 3 4 5 6 7 21 2 ARM F 0 1 2 3 21 -1 0 1 2 3 4 21 2 *rhTPO MA Chemotherapy 15 51 WO 98/52598 PCT/US98/10475 Doxorubicin Doxorubicin was obtained from commercial sources and stored and administered in accordance with the manufacturer's guidelines. Doxorubicin (a total of 90 mg/m 2 ) was given as a continuous infusion for the first 3 days of each chemotherapy cycle (30 mg/m 2 daily for 3 days, (Days 5 0-2). Ifosfamide Ifosfamide was obtained from commercial sources and stored and administered in accordance with the manufacturer's guidelines. Ifosfamide (a total of 10 g/m 2 ) was given as four separate daily 10 infusions during the first 4 days of each chemotherapy cycle (2.5 g/m 2 over 3 hours daily for 4 days, (Days 0-3). Mesna Mesna was obtained from commercial sources and stored and administered in accordance with 15 the manufacturer's guidelines. Mesna (500 mg/m 2 or 20% of the dose of ifosfamide) was administered IV over 3 hours with the initial dose of ifosfamide on Day 0 of each chemotherapy cycle. Mesna administration was maintained as a continuous IV infusion (1500 mg/m 2 /day for a total of 6 g/m 2 ) until 24 hours after the final ifosfamide administration of each chemotherapy cycle (Days 0-4). 20 G-CSF G-CSF was obtained from commercial sources and stored and administered in accordance with the manufacturer's guidelines. G-CSF (5 gg/kg) was administered daily beginning on Day 4 of Cycle I and any subsequent chemotherapy cycles. Subjects were instructed to self-administer G-CSF. All injections of G-CSF should be administered at -8:00 p.m. Injections on the same day as rhTPO 25 administration should be given -12 hours after rhTPO administration to help define any injection-related phenomena. G-CSF administration continued on a daily basis until the absolute neutrophil count was > 1500/gL for at least two consecutive measurements post-nadir. GM-CSF 30 GM-CSF is obtained from commercial sources and stored and administered in accordance with the manufacture's guidelines. GM-CSF (250 mg/m 2 ) is administered subcutaneously daily beginning on Day 4 of Cycle 1 and any subsequent chemotherapy cycles. Subjects are instructed to self-administer GM-CSF. All injections of GM-CSF should be administered at -8:00 p.m. Injections on the same day as rhTPO administrations should be given -12 hours after rhTPO administration to 52 WO 98/52598 PCT/US98/10475 help define any injection-related phenomena. GM-CSF administration continued on a daily basis until the post-nadir absolute neutrophil count is >1500/ptL for at least two consecutive measurements. The results of this study are shown in Figures 14 - 19. 5 EXAMPLE 7B - A Phase I Trial Of Recombinant Human Thrombopoietin (rhTPO) Given Via Subcutaneous Injection To Subjects With Advanced Gynecologic Malignancy Receiving Carboplatin This study addresses the safety of rhTPO administered subcutaneously and the activity of rhTPO dosing in an every other day schedule. The study begins with a 21-day pre-chemotherapy cycle (cycle 0) to assess safety, activity, and pharmacokinetics in patients with cancer. At present, 16 10 subjects have been enrolled on to this trial. The data indicates a dose-dependent increase in peripheral blood platelet counts in cycle 0, though the platelet responses, and the pharmacokjinetics, are blunted when compared to similar doses of IV rhTPO. Thus far, two antibodies to the truncated form of rhTPO (erythropoietin-like domain) have been observed; one was preformed, and neither was neutralizing in a bioassay, or clinically. Subjects had recurrent or advanced gynecologic neoplasms and were receiving 15 carboplatin. Schema 1 21 0M oW1 I 1 21 1 2 3 4 5 6 7 8 21 20 N rhTPO SCarboplatin chemotherapy Dose Levels, Doses and Number of Subjects DOSE LEVEL DOSE N IA 0.6 pg/kg QOD X4 3 2A 1.2 pg/kg QOD X4 3 3A 2.4 jg/kg QOD X4 3 4A 3.6 gg/kg QOD X4 3 Control* 1.2 jg/kg QOD X4 6 18 25 *Control--patients receive rhTPO only in cycles after experiencing a platelet count < 30/000/pl in the preceding cycle 53 WO 98/52598 PCT/US98/10475 EXAMPLE 8 Animals: 76 CD-1 female mice BW= 19.7-26.3 g Aerosol Exposure: Mice were exposed to rhTPO in a nose-only inhalation (CH Technologies) with simultaneous 5 measurement of breathing pattern (body plethysmograph) for a total duration of 60 minutes. A PARI IS2 nebulizer was used to aerosolize rhTPO at 22 psi and a flow rate of 5.1 LPM. Filter samples were taken to estimate the aerosol concentration for each exposure. A vehicle control group and 3 different concentrations (0.05, 0.5, and 5.0 mg/ml rhTPO) were nebulized. The dose groups are expressed as the estimated amount per kg that deposited in the mouse lungs for each group. Half of the animals were 10 exposed only once (Single exposure) while the other half were exposed to rhTPO 3 times on days 0, 2 and 6. Anti-rhTPO antibodies were measured in only the highest dose group, but these antibodies were not neutralizing. The dose groups are shown in the table below. Dose groups: (rhTPO) Nebulized (mg/ml) n Estimated Lung Dose (mg/kg) 0 17 0 0.05 17 6.4 0.5 17 64 5.0 16 640 15 Data Endpoints: Serum and blood were collected at -4, 3, 6, 8, 10, 14, 21, 30, and 43 days post aerosol inhalation for hematology (platelet counts) and measurement of serum anti-TPO antibodies. 20 Estimation of Deposited rhTPO Lung Dose: Deposited dose (gg/kg) = Chamber concentration (gg/ml) x Minute volume (ml/min) x time of exposure (min) x Deposition fraction / BW (kg) where: Chamber concentration = 0.000839, 0.00839, or 0.0839 ig/ml Minute volume = 30 ml/min 25 Time of exposure = 60 min Deposition fraction = 0.1 Body weight = .023 kg Deposited dose = 6.4, 64, or 640 gg/kg (for 0.05, 0.5, or 5 mg/ml solutions). Platelet counts for mice 30 exposed to a single inhalation (dose) of rhTPO are shown in Figure 20. Platelet counts for mice exposed to multiple inhalations (dose) of rhTPO are shown in Figure 21. 54 WO 98/52598 PCT/US98/10475 EXAMPLE 9 Animals: Female (C57BLxCBA)F1 (BCBA) mice approximately 12 weeks old were bred at the Experimental Animal Facility of the Erasmus University, Rotterdam, The Netherlands, and maintained under SPF conditions. Housing, experiments and all other conditions were approved by an 5 ethical committee in accordance with legal regulations in The Netherlands. Experimental setup: TBI was given at day 0 using a two opposing 137 Cs sources (Gammacell 40, Atomic Energy of Canada, Ottawa, Canada) at a dose rate between 0.92 and 0.94 Gy/min. Doses used were 6 Gy for the single dose irradiation and a total dose of 9 Gy was split in three doses of 3 Gy given with 24 hour intervals. For each data point groups of three mice were killed. All parameters 10 were collected for individual mice. Test Drug: Recombinant full length murine TPO produced by CHO cells (Genentech Inc., South San Francisco, CA was used throughout the experiments, diluted in PBS/0.01% TWEEN-20 and administered intraperitoneally in a volume of 0.5 ml. TPO levels: Data for characterization plasma TPO pharmacokinetics were generated at 15 Genentech, Inc. as previously described. In short, mice were injected i.p. with 1 2 5 I-rmTPO either with a single dose of 0.9 microgram/mouse (ca 45 microgram/kg) or with three doses of 0.3 microgram/mouse (ca 15 microgram/kg) separated by 24 hours. Citrated blood was collected immediately after dosing and at intervals thereafter (n=3 mice per timepoint), centrifugated at 2950 x g for 10 minutes, plasma harvested, and TCA- precipitable radioactivity determined. Pharmacokinetic 20 parameters were estimated after converting TCA-precipitable cpm/mL and fitting the concentration versus time data to a two-compartment model with first order absorption using nonlinear least-squares regression analysis (WIN-NONLIN; Statistical Consultants, Lexington, KY). Area under the concentration time curves (AUC), maximum concentration (Cmax), terminal half-lives (tl/2), and clearance (mL/hr/kg) were calculated using coefficients and exponents obtained from the model fits. 25 Hematological examinations: After either-anesthesia the mice were bled by retro-orbital puncture and killed by cervical dislocation. Blood was collected in EDTA tubes. Complete blood cell counts were measured using a SYSMEX F-800 hematology analyzer (Toa Medical Electronics Co., LTD., Kobe, Japan). Differential white blood cell counts were performed after May-Grunwald Giemsa staining. 30 Colony assays: Serum free methylcellulose cultures were used in this study. Appropriate numbers of bone marrow cells were suspended in Dulbecco's modified Eagle's medium (Dulbecco's MEM) obtained from GIBCO (Life Technologies LTD, Paisley, Scotland) supplemented with the amino acids L-alanine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid and L-proline (Sigma), vitamin B 12, biotin, Na-pyruvate, glucose, NaHCO 3 , and antibiotics (penicillin and 35 streptomycin) at an osmolarity of 300 mOsm/l (c-medium). Appropriate numbers of cells in cc medium containing 0.8% methylcellulose (Methocel A4M Premium Grade, Dow Chemical Co., 55 WU 9M/52598 PCT/US98/10475 Barendrecht, The Netherlands), 1% bovine serum albumin (BSA, fraction V, Sigma), 2x1 0-6 mol/l iron saturated human transferrin (Intergen Company N.Y., NY), 10 -7 mol/l N 2 SeO 3 (Merck), 10 -4 mol/Il 3-mercapto-ethanol (Merck, linoleic acid (Merck), and Cholesterol (Sigma) at a final concentration between 7.5 x 10-6 mol/l and 1.5 x 10 -5 mol/l for both, depending on the kind of 5 progenitor cell colony cultured and 10 -3 g/l nucleosides (cytidine, adenosine, uridine, guanosine, 2' deoxycytidine, 2'-deoxyadenosine, thymidine and 2'-deoxyguanosine obtained from Sigma) were plated in 35 mm Falcon 1008 Petri dishes (Bercton Dickinson Labware) in 1 ml. aliquots. Granulocyte/macrophage colony formation was stimulated by a saturating concentration of M CSF purified from pregnant mouse uteri extract (PMUE) essentially as described before, supplemented 10 with 100 ng/ml murine stem cell factor (SCF, a kind gift from Immunex Corporation, Seattle, WA) and 10 ng/ml murine IL-3 (R&D, Minneapolis, MN). GM-CFU colonies were counted after 7 days of culture. BFU-E growth was stimulated by 100 ng/ml DCF and 4 U/ml murine erythropoietin (EPO, Behringwerke, Marburg, F.R.G.) purified from the serum of phenylhydrazine treated mice, titrated to 15 an optimal concentration. Colonies were counted after 10 days of culture. The culture medium of the erythroid progenitors also contained hermine (bovine, type I, Sigma) at a concentration of 2x 10 - 4 mol/l. Megakaryocyte progenitor cells (CFU-Meg) were cultured in 0.25% agar cultures. Colony formation was stimulated by 100 ng/ml SCF, 10ng/ml IL-3 and 10 ng/ml murine TPO (Genentech, Inc., 20 San Francisco, CA). After 10 days colonies were dried, stained for acetylcholinesterase positive cells and counted. All cultures were grown in duplicate at 37 C in a fully humidified atmosphere with 10%

CO

2 in the air. Colony numbers represent the mean ± standard deviation of bone marrow samples of individual mice. The spleen colony assay: This assay was performed as described by Till and McCulloch. 25 Briefly, mice were injected with 5 x 10 4 BM cells or 5 x 105 spleen cells in HH one day after TBI. Thirteen days later, mice were sacrificed, and spleens were excised and fixed in Tellyesniczsky's solution (64% ethanol, 5% acetic acid and 2% formaldehyde) in H 2 0. Statistics: Standard deviations were calculated and are given in the text and the figures on the assumption of a normal distribution. The significance of a difference was calculated by one way 30 analysis of variance followed by a non-paired Student's t test using STATVIEW, Abacus Concepts Inc., Berkeley, CA. All colony assays were done in duplicate for individual mice. The results of the colony assays are expressed as the means ± ISD per femur or spleen for at least three mice per group. Experimental Design 1: BCBA F 1 mice were subjected to 3 Gy total body irradiation at time 0. 0.3 microgram TPO/mouse was administered i.p. at various time points as indicated in Figure 8 35 or 30 microgram TPO/mouse i.p. at -2 h. 56 WO 98/52598 PCT/US98/10475 Figure 8 shows the thrombocyte level of 6 Gy irradiated mice at the time of the nadir in the placebo controls as a function of the time of administration of a single dose of TPO. Based on the kinetics observed, it appears that the peak levels of TPO reached shortly after TPO administration were the most relevant for efficacy. This was directly confirmed by administration of a very high dose of 5 TPO 2 h before TBI (Fig. 9). The effect originated from multilineage cells, as shown in the peripheral blood by equivalent effects of TPO on red cells, white cells (mainly neutrophils) and platelets. See Table I below. Table 1 - Major peripheral blood cell counts 10 days after 6 GY TBI and TPO administration Time TPO relative to dose (jg) N red cells white cells platelets TBI x1012/1 x 109/1 x 109/1 - - 8 7.1 ± 0.6 0.4 ± 0.2 156 + 68 +2 h 0.3 12 9.1 ± 0.6 1.1 ± 0.3 742 ± 92 -2 h 0.3 3 9.0 + 0.1 0.7 ± 0.2 532 ± 54 -2 h 30 3 9.0 + 0.3 0.8 ± 0.2 718 ± 86 +24 h 0.3 15 7.5 ± 0.5 0.4 ± 0.1 465 ± 41 normal mice - 19 10.2 ± 0.6 5.3 ± 1.9 1,123 ± 89 10 Experiment design 2: BCBA Fl mice were subjected to 6 Gy total body irradiation (TBI) at time -2 d, -1 d, 0 and 0.9 Microgram TPO/mouse was administered i.p. at various time points as indicated in the legend of Fig. 10, i.e., 2 hours before the first fraction of TBI, 2 hours after the last fraction of TBI, or 3 fractions of 0.3 micrograms TPO 2 h after each fraction of TBI. 15 To simulate a protracted form of cytoreductive treatment more similar to chemotherapy, TBI was given in three equal fractions of 3 Gy separated by 24 h each (experimental design 2). TPO was given in a total dose of 0.9 microgram as indicated in the legend of Fig. 10. The thrombocyte response was optimal when this dose of TPO was given in three equal fractions of 0.3 microgram TPO, 2 h after each fraction of TBI. Similar effects were shown for red cells and white cells. Table 2 shows that in 20 this experimental setting also, a very high dose of TPO 2 h before the first fraction of TBI was equally effective. Fig. 11 and Fig. 12 show the hemopoietic progenitor cell data of bone marrow and spleen, respectively which demonstrate that the most optimal dose schedule for TPO, i.e., 3 x 0.3 microgram 2 h after each fraction of TBI, also rapidly normalized progenitor cell levels without major fluctuations seen in the other treatment groups. 25 Figure 13 shows the pharmacokinetic data following three doses of 0.3 microgram of TPO or a single dose of 0.9 microgram. Peak levels relevant occur about 2 h after i.p. injection. An effective level is 30 ng/ml plasma. However, the minimum effective TPO level has not yet been determined by titration experiments. From the data, it can be seen that maintaining a high level of TPO during cytoreductive 30 treatment results in multilineage stimulation and peripheral blood cell recovery. 57 WO 98/52598 PCT/US98/10475 Table 2 - Major peripheral blood cell counts 10 days after 3 x 3 Gy TBI and TPO administration TPO time TPO dose N red cells white cells platelets relative to (pg) x10 1 2 /1 x10 9 /1 x 10 9 /1 TBI - 9 5.8 ± 0.8 0.3 ± 0.1 51 ± 25 3x +2 h 0.3 6 8.6 ± 0.2 1.0 + 0.2 883 ± 163 + 2 h 0.9 3 6.6 ± 0.7 0.4 ± 0.1 527 ± 135 -2 h 0.9 3 7.2 ± 0.2 0.5 ± 0.1 215 ± 80 -2 h 30 3 8.4 ± 1.0 0.9 ± 0.1 791 ± 156 normal mice - 19 10.2 ± 0.6 5.3 ± 1.9 1123 ± 89 5 TPO appeared to be highly effective in mice exposed to a total dose of 9 Gy TBI in three equal fractions separated by 24 h each, if administered intraperitoneally 2 h after each TBI fraction. TPO administration prevented the severe reduction of thrombocyte numbers observed in the placebo control group, stimulated the recovery of granulocytes and also fully prevented the development of anemia. Similar to the 6 Gy single TBI experiments, the effect appeared to be mediated by accelerated 10 reconstitution of progenitor cells of multiple blood cell differentiation lineages. This implies that the efficacy of TPO is dependent on residual immature multilineage cells, which need to be stimulated by TPO in a time window following TBI. We approached the question as to whether immature cells were involved by simple short-term quantitative transplantation assay. Following transplantation of bone marrow into lethally irradiated 15 recipients, the number of spleen colonies at day 13 (CFU-S-13) is a meausure for relatively immature repopulating stem cells associated with the initial short-term wave of hemopoietic reconstitution, which lasts for several months. By this assay, the effect of TPO treatment on immature progenitor cells could be directly demonstrated. In mice exposed to 9 Gy TBI in three equal fractions, the number of detectable CFU-S-13 of TPO treated mice were approximately 14-fold increased at 24 h after the last 20 fraction of TBI compared to the placebo control mice (Table 3). Similar increases were observed in progenitor cell numbers along the three lineages examined. These results provided a strong indication of a major effect of TPO on the most immature multilineage cells detectable by a clonogenic assay, consistent with the presence of TPO receptors on such immature cells. The small time window to achieve optimal efficacy of TPO is peculiar in view of the slow 25 phase of the wash-out of TPO levels, which has a terminal half-life of approximately 20 h and would result in approximately similar levels at all time points within the first twelve h after administration with the exception of the initial rise due to the distribution in plasma. This led us to speculate that the high levels of TPO in the first few hours after administration would be of decisive importance. The 58 WO 98/52598 PCT/US98/10475 latter hypothesis was tested in two ways, i.e., by pharmacokinetic measurements and by administration of a very high dose of TPO before TBI to examine whether an efficacy could be reached similar to that obtained by TPO administration after each fraction of radiation. By superimposing the pharmacokinetic data on the efficacy data, it can be derived that effective levels are larger than 20 5 ng/ml and occur approximately 2 h after i.p. administration. From the latter observation, we concluded that in these mice a high level of TPO approximately 4 h after TBI is required to alleviate the radiation induced bone marrow syndrome by stimulation of immature target cells. This was directly tested by administration of a dose of 30 micrograms TPO i.p. (calculated on the basis of the initial distribution) 2 h before the first radiation fraction. The results demonstrated that indeed such very high levels of TPO 10 during the fractionated TBI regime are decisive to prevent thrombocytopenia. Thrombocyte counts 10 days after the last dose of TBI in mice treated with 30 micrograms before the first dose of TBI were not significantly differently from those of the mice treated with the most effective schedule of 0.3 microgram TPO 2 h after each TBI fraction. A similar efficacy was obtained by the high dose of TPO 2 h before a single TBI dose of 6 Gy. 15 Table 3: Effects of TPO treatment following 9 Gy TBI (3x3Gy, 24 hours apart) on CFU-S day 13 and progenitor cell content of bone marrow 24 hours after the last dose of TBI in mice treated with TPO 2 hours after each dose of TBI vs control CFU-S d13 GM-CFU BFU-E CFU-Meg Treatment (colonies per /femur /femur /femur femur) Placebo 1.9 597 ± 229 8.3 ± 3 22 ± 10 TPO3*0.3 27.3 2026 131 558 282 430 ± 135 20 EXAMPLE 10 - Study of Patients receiving rhTPO and undergoing Autologous Bone Marrow Transplant (ABMT). After harvest of the bone marrow harvest, all patients received cyclophosphamide 180 mg/kg, thiotepa 900 mg/m 2 , ± carboplatin 600 mg/m 2 . After infusion of the unmanipulated graft (day 0), patients began IV rhTPO on day 1, either qd or q3d, until either day 21 or a patient count > 50 k/pl (see 25 Table below). The rhTPO was administered to patients cohorts in dose-escalating fashion. All patients received G-CSF until the absolute neutrophil count was _ 500/pl. Platelets were transfused for a platelet count < 20 K/Ipl, or as clinically indicated. Platelet recovery was defined as the first day of an unsupported platelet count > 25,000/pl (simple definition) or, more rigorously, the first of > 2 consecutive days of untransfused platelet counts 30 > 25,000/pl with the second count being greater than or equal to the preceding value (stable or rising definition). Neutrophil recovery was defined as the first of 2 consecutive days with an absolute 59 WO 98/52598 PCT/US98/10475 neutrophil count > 500/pl. Patients were compared to 15 historical controls undergoing autologous bone marrow transplant using the same transplant regimens. Table Median Number of Median Day Median Day of Number Platelet of Platelet Neutrophil Dose Level (N) Transfusions Recovery Recovery (range) (range) (>500/ml) (range) 1 (0.3 tg/kg) 7 6(2-11) 18(11-25) 10.5(9-13) 2 (0.6 tg/kg) 6 7(3-15) 16.5 (12-40) 11(10-28) 3 (1.2 jig/kg) 7 6(3-11) 21(15-28) 11 (10-13) 4 (2.4 jig/kg) 3 11(6-16) 24.5 (16-33) 15.5 (11-20) 5 (0.6 gg/kg) 3 6(5-10) 17(15-21) 11(10-11) 6(4.8 pg/kg) 6 8(4-18) 19(14-34) 11.5 (10-14) All Study Patients 32 6 (2-18) 18 (11-40) 11(9-28) Historical Controls 15 Not Available 19 10 5 rhTPO in all Dose Levels was administered Q3D with the exception of Dose Level 5 where the rhTPO was administered QD. Thirty-three patients with a median age 49 years (23-59 years), were enrolled in this study; all evaluable for safety and hematologic response. Seven patients received rhTPO at the 0.3 jg/kg dose 10 level, 7 at the 0.6 pg/kg level; 7 at 1.2 ptg/kg; 3 at 2.4 jtg/kg; and 6 at 4.8 jig/kg (see Table above). All levels were safely tolerated, and rhTPO has not been associated with any study-drug-related adverse events. Chemotherapy-related adverse effects occurred as expected. One patient experienced prolonged pancytopenia due to human herpes virus-6 infection. Only two hemorhagic episodes during the period of thrombocytopenia, epistaxis and a subdural hematoma, were observed, both when platelet 15 counts were < 25,000tL. Both resolved without long-term sequelae. There were no episodes of thrombosis or veno-occluusive disease noted. Over the entire group of patients in this study, the median time to plt recovery (stable and rising definintion) was 17.5 days. In summary, rhTPO in combination with G-CSF appears to be safe and well-tolerated in patients undergoing myeloablative therapy and rescue with autologous bone marrow. 20 EXAMPLE 11 - Peripheral Blood Progenitor Cell (PBPC) mobilization study in Patients with Breast Cancer Patients were treated in cohorts of 3 evaluable patients at a thrombopoietin (TPO) dose of 0.6,1.2 or 2.4 ig/kg IV with dose escalation in the successive cohorts if toxicities related to 60 WO 98/52598 PCT/US98/10475 thrombopoietin did not occur. No serious toxicity related to TPO occurred. The optimal biologic dose is considered the lowest dose which will maximize CD34+ cells/kg/liter of blood processed in the PBSC collection. Secondary endpoints are the intervals to recovery of granulocytes and platelets after CVP and after CBT/PBPC transplant. 5 Treatment Remimen CVP: Cyclophosphamide 1.5 mg/m 2 /d IV+ mesna; etoposide 250 mg/m 2 dl-3; cisplatin 40 mg/m 2 dl-3, followed by single dose thrombpoietin 0.6-2.4 gg/kg IV d4 G-CSF 6pg/kg ql2h. Upon recovery of WBC > 1.0, PBPC collection by large volume apheresis 3x BV, target > 10 3 x 106 CD34+ cells/kg. CBT: Cyclophosphamide2.0 gm/m 2 IV, Thiotepa 240 mg/m 2 /d, BCNU 150 mg/m 2 /d days -8, -7, -6 with reinfusion of the cryopreserved cells on day 0. G-CSF 5 pg/kg/d SC until recovery of granulocytes. Table Independent t-test against Parameters G-CSF only G-CSF + TPO null hypothesis no difference at a.05 Apheresis Collection Parameters (mean ± std) Patient 6 patients 12 patients n/a Population 4/6 (67%) single 10/12 (83%) single collection collections 2/6 (33%) two 2/12 (17%) two collections collections Patient 71.5 ± 12.0 72.5 ± 15.1 p = .4908 Weight (kg) Blood Processed Processed 13.298 ± 1.255 13.300 ± 1.825 p = .3626 (L) per Apheresis Collection nd 60% nd Efficiency of (median) CD34 cells Transplant Dose (106) per kg (mean ± std) CD34 15.268 ± 18.867 34.479 ± 32.235 p =.1999 CD34 t THY 5.653 ± 7.267 23.417 ± 22.411 p = .0802 CD34' THydim 4.842 ± 6.447 13.671 ± 14.941 p = .1900 CD34 t CD41+ 2.511 ± 3.107 1.592 ± 1.345 p =. 3864 CD34 * CD41+ 0.537 ± 0.383 0.753 ± 0.639 p = .4617

THY

+ CD34' CD41+ 0.444 ± 0.364 0.450 ± 0.372 p =. 9713 THYdim 1 61 WO 98/52598 PCT/US98/10475 Engraftment: days to reach cell target (mean ± std)

ANC

500 10 ± 1 9 ± 1 nd

PLT

20 11 ± 2 9 ± 2 nd

PLT

50 12 ± 1 12 ± 3 nd Thrombopoietin was well tolerated when given with G-CSF following CVP chemotherapy for PBPC mobilization. Mobilization was enhanced compared to historical controls. A median of one apheresis was 5 required to reach target cell dose. Hematopoietic recovery post transplant was rapid. EXAMPLE 12 - PBPC Study of Patients with Breast Cancer A phase I clinical trial was conducted to assess the feasibility and possible efficacy of single and repeated doses of thrombopoietin (TPO) together with G-CSF 10 microgram/kg for peripheral 10 blood progenitor cell (PBPC) mobilization followed by high dose chemotherapy (HDCT) with cisplatin, VP-16 and cyclophosphamide (Cy) in patients with high-risk and responsive stage IV breast cancer. The HDCT treatment scheme was as follows: Day -12 Cisplatin 125 mg/m 2 VP-16 30 mg/kg Day -5 Cisplatin 125 mg/m 2 VP-16 30 mg/kg Day -3 Cy 100 mg/kg Day -2 25% of PBPC (transplant) Day 0 75% of PBPC (transplant) 15 The TPO dosing scheme was as follows: Day TPO Dose # of Patients Arm A 1 0.3 microgram/kg 3 1.2 microgram/kg 3 2.4 microgram/kg 4 Arm B -3, -1, 1 0.6 microgram/kg 6 1.2 microgram/kg 0 0.3 microgram/kg 3 62 WO 98/52598 PCT/US98/10475 Mobilization consisted of TPO and G-CSF 10 microgram/kg (5 microgram/kg BID) in group A. G-CSF 10 microgram/kg once a day in group B. G-CSF 10 microgram/kg (5 microgram/kg BID) in group C. Apheresses were carried out processing ~ 10 liter of blood via a CS-3000 Fenwall or COBE 5 Spectra cell separator. PBPC-s were cryopreserved in a solution containing 5% DMSO and were frozen by simple immersion into a -130 oC freezer. Group A vs B vs C comparisons were performed using the Kruskal-Wallis test. Group A vs B were compared using Wilcoxon rank-sum test. Data on PBPC apheresis, hematopoietic recovery and transfusion requirements are presented as median (range). Platelet (PLT) transfusions were provided for : 20,000 PLT/ul or as clinically indicated. 10 Platelet independence is defined as the day after the last platelet transfusion. Single pheresis products and pooled platelet products are counted as 1 PLT transfusion. PATIENT CHARACTERISTICS Group A Group B Group C Age 46 (34-61) 44 (28-62) 45 (36-57) Prior Regimens 1(1-4) 1.5 (1-5) 1 (1-2) Prior ChemoRx cycles 6 (3-23) 5 (3-18) 4 (3-7) N (%) N (%) N (%) Stage 11/111 12 (73) 14 (54) 11(100) Stage IV 7(37) 12(46) 0 Prior Rt 3(16) 6(23) 0 15 COMPARISON OF APHERESIS REQUIREMENTS, CD34 and MNC YIELD Group A B C p-value N 19 26 11 Aphereses 2 (2) 4 (2-6) 3 (3-5) .0001 MNC* 4.1 (.5-6.5) 2.3 (1.2-4.3) NA .0001 CD34 t 5 (2-12.7) 0.9 (0.1-4.3) 2.4 (0.4-7-7) .0001 * Mononuclear cell yield in 10 8 /kg; + CD34 yield in 10 6 /kg 63 WO 98/52598 PCT/US98/10475 HEMATOPOIETIC RECOVERY and TRANSFUSION REQUIREMENTS Group A B p-value N 14 26 AGC _ 500/ul 7.5 (6-9) 8 (7-11) .0001 PLT independence 9 (7-11) 10.5 (5-21) .0001 PLT transfusions 4 (1-10) 6 (2-52) .002 RBC transfusions 3 (2-7) 4 (2-6) .009 RBC = red blood cell; AGC = TPO in the dose ranges tested is well tolerated. TPO in combination with G-CSF increases the 5 efficacy of PBPC mobilization and CD34' yield. TPO and G-CSF mobilized PBPC accelerate both platelet and granulocyte recovery. EXAMPLE 13 - Other Embodiments The invention specifically contemplates treatment cycles in which the radiation or 10 chemotherapy agent is administered on multiple consecutive days, for example on 4, 5, 6 or 7 consecutive days, where the TPO dose is administered prior to the first of the consecutive days and/or concurrent with one or more of the consecutive days of the treatment cycle. For a treatment cycle of 5 consecutive days, TPO might be given on day -1 and on days 6, 9, 12, 15, etc. In another example, for a treatment cycle of 7 consecutive days, TPO might be given on day -1 and on days 2, 4, 6, 8, 10, 12, 15 etc. or on day -1 and on days 4, 6, 8, 10, 12, etc. In another embodiment, the radiation or chemotherapy agent will be given on alternate days of the treatment cycle, e.g. days 1, 3, 5. In this embodiment, TPO might be given on day -1 and on days 2, 4, 6, 8, 10, etc. EXAMPLE 14 20 A further use of TPO according to the invention is for ex vivo expansion of progenitor cells obtained, for example, from mammalian bone marrow, peripheral blood, or umbilical cord blood. Progenitors expanded in such a manner can be used for allogeneic stem cell transplant for patients who have been treated with chemotherapy or radiation treatment. A progenitor population that is enriched for stem cell activity is the CD34 population and this population can be futher enriched by selecting 25 for CD34 CD38" cells. This population has the ability to generate multilineage hematopoietic colonies in vitro and multipotential hematopoietic engraftment in vivo. Progenitor stem cells for expansion can be obtained from peripheral blood by pheresis and from bone marrrow aspirates by standard techniques. Hematopoietic progenitors can also be obtained from umbilical cord blood. Hematopoietic stem cells having the CD34 phenotype may then be isolated from the blood or marrow using an 30 immunomagnetic enrichment column. The isolated cells may then be cultured in a cell growth medium 64 WO 98/52598 PCT/US98/10475 containing a cocktail of the growth factors TPO, Flt-3 and c-kit ligand in order to expand or increase the number of cells in the stem cell population. The growth factors are added in amounts sufficient to stimulate growth of the progenitor stem cells. Preferably, each of the growth factors is added in an amount of about 10 to about 100 ng/ml to growth media containing about 102 to about 106 stem 5 cells/ml. The cells are cultured using standard culture techniques, for example, about 35 - 40'C in aproximately 5% CO 2 for about 1 - 8 weeks. The cultures are exchanged into fresh media containing growth factors every week. The growth media may contain other conventional nutrients, fetal serum, etc in standard amounts. The expanded cells are then readministered as an allogeneic stem cell transplant according to known procedures. 10 In the following table is shown the ability of the CD34' cultures after 8 weeks expansion to generate lymphohematopoiesis in vivo using the SCID-hu bone assay. In bone grafts injected with cells from the expanded cultures these cells contributed to the lymphoid, myeloid and progenitor hematopoietic compartments. This shows that the expanded cells maintained their in vivo multipotent engraftment potential. 15 TABLE: Engraftment of TPO/KLIFL expanded progenitors in SCID-hu mice. Condition Study % of mice % donor % donor % donor J engrafted HLA/CD34 IHLA/CD33 HLA/CD19 TPO/FL/KL 1 80% 4.9 +/- 1.5 8.8 +/- 2.1 55.3 +/- 8.9 TPO/FL/KL 2 100% 6.9 +/- 2.4 4.9 +/- 1.9 59.6 +/- 14.1 20 MATERIALS AND METHODS Isolation of human hematopoietic stem cell population: Hematopoietic progenitor cell populations were isolated from human bone marrow. Briefly, the mononuclear fraction was enriched for CD34+ cells using animmunomagnetic enrichment column. (Miltenyi Biotech, Auburn, CA). Purity was routinely >90% by FACS. 25 Suspension culture assays: Hematopoietic stem cell populations were seeded at 2e4 cells/mL in IMDM Gibco BRL (Grand Island, NY), plus 10 % fetal bovine serum (Gibco BRL), 10 - 5 M 2 mercaptoethanol, 10

-

6 M hydrocortisone, and 2 mM L-glutamine (Gibco BRL). Growth factors were added at the following concentrations: Flt-3 ligand (Immunex, Seattle WA) 50 ng/mL, TPO (Genentech, S. San Francisco, CA) 50 ng/mL, and c-kit ligand (R and D Systems) 50 ng/mL. Cultures 30 were incubated at 37 0 C/5% CO2 for seven days. On day 7 all wells were harvested and all cells were 65 WO 98/52598 PCT/US98/10475 counted by hemacytometer. For subsequent platings, 2e4 cells/mL were added to fresh media and growth factors and incubated for an additional seven days. All conditions were done in duplicate. Flow cytometric analysis: For FACS analysis, cells were resuspended in PBS/2% FBS at le6 cells /mL and stained with mouse anti human CD34 FITC, CD38 PE (Becton Dickenson). Viable cells 5 were selected by propidium iodide exclusion and analyzed on a FACscan (Becton Dickenson). Colony Assays: Methylcellulose colony assays were performed using "complete" myeloid methylcellulose media (Stem Cell Technologies, Vancouver, B.C.). Cells were seeded in methylcellulose at 1,000 cells/mL and plated in 4 x 35 mm gridded dishes. Colonies were counted and visually phenotyped on an inverted phase contrast microscope after 14 days in culture. 10 SCID-hu mouse reconstitution assay: CB-17 scid/scid mice were implanted with fetal bone marrow as described previously. Mice were used so that the grafts and cells were mismatched for major histocompatibility complex (MHC) class I antigens. Mice received 250 rads whole body 'y irradiation and then followed by injection of 30,000 cultured human bone marrow cells into the bone graft. Eight weeks after the injection of the cells, the bone grafts were harvested and analyzed for 15 donor HLA contribution to FITC conjugated anti human CD34 (progenitor), anti human CD33 (myeloid), and anti human CD19 (lymphoid). Donor HLA positive cells were then sorted by FACS. Thirty thousand donor HLA positive cells were then injected into secondary recipients in the same manner as the primary recipients. Eight weeks after injection the secondary bone grafts were removed and analyzed for engraftment of CD34, CDI9, and CD33. 20 The foregoing description details specific methods which can be employed to practice the present invention. Having detailed such specific methods, those skilled in the art will well enough know how to devise alternative reliable methods at arriving at the same information in using the fruits of the present invention. Thus, however detailed the foregoing may appear in test, it should not be 25 construed as limiting the overall scope thereof; rather, the ambit of the present invention is to be determined only be the lawful construction of the appended claims. All documents cited herein are hereby expressly incorporated by reference. 66 WO 98/52598 PCT/US98/10475 SEQUENCE LISTING (1) GENERAL INFORMATION: 5 (i) APPLICANT: Genentech, Inc. Cohen, Robert Eaton, Dan L Jones, Andrew JS Jones, Dennie V 10 Powell, Michael F Sweeney, Theresa D Thomas, Griffith R and Wagemacher, Gerard 15 (ii) TITLE OF INVENTION: Novel Administration of Thrombopoietin (iii) NUMBER OF SEQUENCES: 24 (iv) CORRESPONDENCE ADDRESS: 20 (A) ADDRESSEE: Genentech, Inc. (B) STREET: 1 DNA Way (C) CITY: South San Francisco (D) STATE: California (E) COUNTRY: USA 25 (F) ZIP: 94080 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 3.5 inch, 1.44 Mb floppy disk (B) COMPUTER: IBM PC compatible 30 (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: WinPatin (Genentech) (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: 35 (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/859767 40 (B) FILING DATE: 21-May-1997 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 09/015016 (B) FILING DATE: 28-Jan-1998 45 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Schwartz, Timothy R. (B) REGISTRATION NUMBER: 32171 (C) REFERENCE/DOCKET NUMBER: PO989P4PCT 50 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 650/225-7467 (B) TELEFAX: 650/952-9881 (2) INFORMATION FOR SEQ ID NO:1: 55 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single 60 (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 67 WO 98/52598 PCT/US98/10475 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: 5 ATCGATATCG ATCAGCCAGA CACCCCGGCC AG 32 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: 10 (A) LENGTH: 36 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GCTAGCTCTA GACAGGGAAG GGAGCTGTAC ATGAGA 36 20 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs 25 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCTAGATCTA GATCACCTGA CGCAGAGGGT GGACC 35 35 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: Nucleic Acid 40 (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 45 ATCGATATCG ATAGCCAGAC ACCCCGGCCA G 31 (2) INFORMATION FOR SEQ ID NO:5: 50 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single 55 (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 60 AGTCGACGTC GACGTCGGCA GTGTCTGAGA ACC 33 68 WO 98/52598 PCT/US98/10475 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: 5 (A) LENGTH: 36 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AGTCGACGTC GACTCACCTG ACGCAGAGGG TGGACC 36 15 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 62 base pairs 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CGCGTATGCC AGCCCGGCTC CTCCTGCTTG TGACCTCCGA GTCCTCAGTA 50 30 AACTGCTTCG TG 62 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: 35 (A) LENGTH: 61 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AGTCACGAAG CAGTTTACTG AGGACTCGGA GGTCACAAGC AGGAGGAGCC 50 45 GGGCTGGCAT A 61 (2) INFORMATION FOR SEQ ID NO:9: 50 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear 55 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 60 CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTAA 37 69 WO 98/52598 PCT/US98/10475 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs 5 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CGCGTTAAGA AGAAATGCGA TATTCTTTTT CATAATT 37 15 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 base pairs (B) TYPE: Nucleic Acid 20 (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 25 CTAGAATTAT GAAAAAGAAT ATCGCATTTC ATCACCATCA CCATCACCAT 50 CACATCGAAG GTCGTA 66 30 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 64 base pairs 35 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TACGACCTCG ATGTGATGGT GATGGTGATG GTGATGAAAT GCGATATTCT 50 45 TTTTCATAAT TCCG 64 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: 50 (A) LENGTH: 65 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 55 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CTAGAATTAT GAAAAAGAAT ATCGCATTTC ATCACCATCA CCATCACCAT 50 60 CACATCGAAC CACGT 65 70 WO 98/52598 PCT/US98/10475 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: 5 (A) LENGTH: 66 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TACGTGGTTC GATGTGATGG TGATGGTGAT GGTGATGAAA TGCGATATTC 50 15 TTTTTCATAA TTCCGA 66 (2) INFORMATION FOR SEQ ID NO:15: 20 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear 25 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 30 TCCACCCTCT GCGTCAGGT 19 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: 35 (A) LENGTH: 18 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AGCTACCTGA CGCAGAGG 18 45 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 62 base pairs 50 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 55 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GCAGCAGTTC TAGAATTATG TCNCCNGCNC CNCCNGCNTG TGACCTCCGA 50 60 ACACTGGAGG CT 62 71 WO 98/52598 PCT/US98/10475 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 base pairs 5 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GAAGGACATG GGAGTCACGA AGCAGTTTAC TGAGAACAAA TGACTCTTG 49 15 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: Nucleic Acid 20 (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 25 CTAGAATTAT GAAAAAGAAT ATCGCATTTA TCGAAGGTCG TAGCC 45 (2) INFORMATION FOR SEQ ID NO:20: 30 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single 35 (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 40 TACGACCTTC GATAAATGCG ATATTCTTTT TCATAATT 38 (2) INFORMATION FOR SEQ ID NO:21: 45 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear 50 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 55 CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTAAACG TAGCC 45 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: 60 (A) LENGTH: 38 base pairs (B) TYPE: Nucleic Acid 72 WO 98/52598 PCT/US98/10475 (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TACGTTTAAG AAGAAATGCG ATATTCTTTT TCATAATT 38 10 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 332 amino acids (B) TYPE: Amino Acid 15 (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu 20 1 5 10 15 Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro 20 25 30 25 Glu Val His Pro Leu Pro Xaa Pro Val Leu Leu Pro Ala Val Asp 35 40 45 Xaa Xaa Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala 50 55 60 30 Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met 65 70 75 Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu 35 80 85 90 Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln 95 100 105 40 Ser Leu Leu Gly Thr Gln Xaa Xaa Xaa Xaa Gly Arg Thr Thr Ala 110 115 120 His Xaa Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu 125 130 135 45 Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu 140 145 150 Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr 50 155 160 165 Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly 170 175 180 55 Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser 185 190 195 Gly Leu Leu Lys Xaa Gln Gln Gly Phe Arg Ala Lys Ile Pro Gly 200 205 210 60 Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly Tyr 73 WO 98/52598 PCT/US98/10475 215 220 225 Leu Asn Arg Ile His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe 230 235 240 5 Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser 245 250 255 Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly 10 260 265 270 Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu 275 280 285 15 Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His 290 295 300 Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser 305 310 315 20 Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln 320 325 330 Glu Gly 25 332 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: 30 (A) LENGTH: 153 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 35 Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu 1 5 10 15 Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro 40 20 25 30 Glu Val His Pro Leu Pro Xaa Pro Val Leu Leu Pro Ala Val Asp 35 40 45 45 Xaa Xaa Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala 50 55 60 Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met 65 70 75 50 Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu 80 85 90 Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln 55 95 100 105 Ser Leu Leu Gly Thr Gln Xaa Xaa Xaa Xaa Gly Arg Thr Thr Ala 110 115 120 60 His Xaa Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu 125 130 135 74 WO 98/52598 PCT/US98/10475 Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu 140 145 150 5 Cys Val Arg 153 75

Claims (48)

1. A method for treating a mammal having or at risk for thrombocytopenia, comprising administering to a mammal in need of such treatment a single or low-multiple daily dose of a 5 therapeutically effective amount of a thrombopoietin (TPO).

2. The method of Claim 1, wherein the mammal receives at least one treatment cycle of radiation and/or chemotherapeutic agent, the treatment cycle having a first treatment time T o and a last treatment time TF for administering radiation and/or chemotherapeutic agent. 10

3. The method of Claim 2, wherein the dose is administered at T o plus or minus 24 hours.

4. The method of Claim 3, wherein the dose is administered at T o plus or minus ten hours.

5. The method of Claim 4, wherein the dose is administered at T o plus or minus six hours.

6. The method of Claim 5, wherein the dose is administered at T o plus or minus two hours

7. The method of Claim 2, wherein the dose is administered at T o or prior to TO, but not more 15 than seven days prior to T 0 .

8. The method of Claim 7, wherein the dose is administered prior to T o , but not more than one day prior to T o .

9. The method of Claim 2, wherein T O = TF.

10. The method of Claim 2, wherein the treatment cycle comprises multiple treatments of 20 radiation and/or chemotherapeutic agent.

11. The method of Claim 10, wherein the dose is administered prior to TF, but not more than seven days prior to TF.

12. The method of Claim 11, wherein the treatment cycle comprises 2-10 treatments.

13. The method of Claim 2, further comprising administering a second dose of TPO after T 0 . 25

14. The method of Claim 13, wherein the second dose is administered after TF.

15. The method of Claim 14, wherein the second dose is administered not more than 24 hours after TF.

16. The method of Claim 2, further comprising administering a dose of TPO concurrent with a treatment time. 30

17. The method of Claim 2, wherein the mammal receives multiple treatment cycles.

18. The method of Claim 21, wherein the mammal receives 2-6 treatment cycles.

19. The method of Claim 1, wherein the TPO is administered in a single therapeutically effective daily dose. 76 WO 98/52598 PCT/US98/10475

20. The method of Claim 1, wherein the TPO is administered in a low-multiple therapeutically effective daily dose.

21. The method of Claim 20, wherein the low-multiple dose comprises 2-6 doses per day.

22. The method of Claim 1, further comprising co-administering a therapeutically effective 5 amount of an agent selected from the group consisting of a cytokine, a colony stimulating factor and an interleukin.

23. The method of Claim 22, wherein the agent is selected from the group consisting of KL, LIF, G-CSF, GM-CSF, M-CSF, EPO, FLT-3, IL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 and IL-11. 10

24. The method of Claim 1, wherein the dose is administered together with a carrier or excipient.

25. The method of Claim 24, wherein the carrier or excipient contains a chelating agent.

26. The method of Claim 25, wherein the chelating agent is EDTA.

27. The method of Claim 1, wherein the TPO is selected from the group consisting of 15 a) a TPO fragment polypeptide; b) a TPO isoform polypeptide; c) a TPO chimeric polypeptide; and d) a pegylated TPO polypeptide.

28. The method of Claim 27, wherein the pegylated polypeptide is prepared with 20 polyethylene glycol.

29. The method of Claim 1, wherein the TPO is selected from the group consisting of a) a TPO polypeptide that is isolated from a mammal; b) a TPO polypeptide that is made by recombinant means; and c) a TPO polypeptide that is made by synthetic means. 25

30. The method of Claim 1, wherein the TPO is selected from the group consisting of a) a polypeptide that is human; and b) a polypeptide that is non-immunogenic in a human.

31. The method of Claim 1, wherein the TPO is represented by the formula: X-hTPO(7-151)-Y 30 where hTPO(7-151) is a human TPO (hML) amino acid sequence from Cys 7 through Cys 15 1 inclusive; X is the amino group of Cys 7 or one or more of the amino-terminus amino acid residue(s) of mature TPO or amino acid residue extensions thereto including Met, Lys, Tyr or amino acid substitutions thereof including arginine to lysine or thrombin); and Y is the carboxy terminal group of Cys 1 5 1 or one or more carboxy-terminus amino acid residue(s) of 35 mature TPO or extensions thereto.

32. The method of Claim 1, wherein the TPO is human TPO. 77 WO 98/52598 PCT/US98/10475

33. The method of Claim 32, wherein the TPO is human TPO(153).

34. The method of Claim 32, wherein the TPO is human TPO(332).

35. The method of Claim 32, wherein the TPO is rhTPO 332 . 5

36. The method of Claim 1, wherein the TPO is administered intravenously.

37. The method of Claim 1, wherein the TPO is administered subcutaneously.

38. The method of Claim 1, wherein the TPO is administered by inhalation.

39. The method of Claim 1, wherein the dose is sufficient to maintain a blood level of TPO in the mammal of 35 x 10- 12 M or greater during the treatment cycle. 10

40. The method of Claim 39, wherein the dose is sufficient to maintain a blood level of TPO of 100 x 10- 1 2 M or greater during the treatment cycle.

41. The method of Claim 41, wherein the dose is sufficient to maintain a blood level of TPO of about 35 10- 12 M to about 3500 x 10- 12 M during the treatment cycle.

42. The method of Claim 1, wherein the dose ranges from about 0.1-10 microgram/kg of body 15 weight.

43. The method of Claim 42, wherein the dose ranges from about 1 to about 5 microgram/kg of body weight.

44. The method of Claim 38, wherein the dose is about 5-1000 microgram/kg in an aerosol.

45. A method for increasing the number of CD34+ cells in a mammalian blood sample, comprising 20 adding an effective amount of TPO and a cytokine selected from the group consisting of FLT 3, KL and a mixture thereof, to CD34+ cells in the sample.

46. The method of Claim 45, comprising adding TPO, FLT-3 and KL.

47. The method of Claim 45, further comprising administering the sample to a mammal.

48. The method of Claim 46, further comprising administering the sample to a mammal. 78