FR2714670A1 - Thrombopoietin polypeptide, ligand for mpl cytokine receptor - Google Patents

Abstract

An isolated homogeneous mpl ligand polypeptide (I); designated thrombopoietin (TPO) is new.

Description

The present invention relates to isolation, purification and synthesis

  by recombination or chemical synthesis of proteins which influence the survival, proliferation, differentiation or maturation of hematopoietic cells, especially platelet progenitor cells. In particular, the present invention relates to the cloning and expression of nucleic acids which encode a protein ligand capable of binding to mpl and activating mpl, which is a representative of the superfamily of cytokine receptors. The present invention further relates to the use of these proteins alone or in combination with other cytokines to treat immune or hematopoietic disorders, including thrombocytopenia. I. Himatopoietic system

  The hematopoietic system produces the highly specialized mature red blood cells known to be necessary for the survival of all mammals. These mature cells include erythrocytes, which are specialized in the transport of oxygen and carbon dioxide, T and B lymphocytes, which are responsible for immune responses mediated by cells and antibodies, platelets or thrombocytes, whose specialty is the formation of blood clots, and granulocytes and macrophages, assuming a special function of scrubbers and accessory cells to fight infections. Granulocytes are further subdivided into neutrophils, eosinophils, basophils and mast cells, which are specialized cell types with distinct functions. It should be noted that all these specialized mature blood cells are derived from a single common primitive cell type, called a pluripotent (or totipotent) stem cell, which is found mainly in the bone marrow (Dexter et al., Ann Rev Cell Biol., 3: 423-441 [(1987)).

  Highly specialized mature blood cells must be produced in large numbers continuously throughout the life of a mammal. The vast majority of these specialized blood cells are intended to remain functionally active for a period ranging from a few hours to several weeks (Cronkite et al., Blood Cells, 2: 263-284 [1976]). Therefore, the continuous turnover of mature blood cells, the primitive stem cells themselves, as well as any intermediate or ascending cell lines between the primitive cells and the mature cells, is necessary to meet the normal mammalian requirements. blood cells at steady state.

  The cell or pluripotent stem cells are at the heart of the hematopoietic system. These cells are relatively few in number and self-renewing by producing daughter stem cells or are transformed, in a series of differentiation steps, into lineage-restricted, increasingly mature progenitor cells. ultimately forming the highly specialized mature cell or blood cells. For example, some multipotent progenitor cells, called CFC-Mix, derived from stem cells undergo proliferation (self-renewal) and development by producing colonies that contain all the different myeloid cells: erythrocytes, neutrophils, megakaryocytes (platelet predecessors) , macrophages, basophils, eosinophils and mast cells. Other progenitor cells of the lymphoid line undergo proliferation and development in T and B cells.

  In addition, there is another class of progenitor cells of intermediate commitment with respect to their progeny between CFC-Mix progenitor cells and myeloid cells. These progenitor cells of restricted lineage are classified on the basis of the offspring they generate. Thus, the known immediate predecessors of myeloid cells are: erythrocyte colony forming units (CFU-E) for erythrocytes, granulocyte / macrophage colony forming cells (GM-CFC) for neutrophils and macrophages, ellules forming megakaryocyte (Meg-CFC) colonies for megakaryocytes, eosinophilic colony forming cells (Eos-CFC) for eosinophils, basophilic colony forming cells (Low-CFC) for mast cells. It is known (see below) or other intermediate cell predecessors between pluripotent and mature blood cells with varying degrees of lineage restriction and self-renewal ability can be demonstrated. .

  The underlying principle of the normal hematopoietic cellular system appears to be the reduced capacity for self-renewal because multiple potentiality is lost and lineage restriction and maturity are acquired. Thus, there is at one end of the hematopoietic cell spectrum the pluripotent stem cell having the ability to self-renew and differentiate into all the various committed progenitor cells specific to a lineage. This capability is the basis of bone marrow transplantation therapy, in which primitive stem cells repopulate the entire system of hematopoietic cells. At the other end of the spectrum are highly lineage-restricted progenitors and their offspring, which have lost the ability to self-renew and have acquired mature functional activity.

  Proliferation and development of lineage-limited stem cells and progenitor cells are carefully dominated by various hematopoietic growth factors or cytokines. The role of these growth factors in vivo is complex and incompletely understood. Certain growth factors, such as interleukin-3 (IL-3), are capable of stimulating both multipotent stem cells as well as committed progenitor cells of several lineages, including for example megakaryocytes. Other factors such as granulocyte / macrophage colony stimulating factor (GM-CSF) were first considered to be limited in their action to GM-CFC cells. Later, however, it was found that GM-CSF also influenced the proliferation and development, among others, of megakaryocytes. As a result, IL-3 and GM-CSF were found to have overlapping biological activities, albeit at different degrees. More recently, interleukin-6 (IL-6) and interleukin-11 (IL-11), although not individually influencing the formation of megakaryocyte colonies, have been shown to act synergistically with IL-3 by stimulating megakaryocyte maturation (Yonemura et al., Exp Hematol., 20: 1011-1016 [1992]).

  Thus, hematopoietic growth factors can influence the growth and differentiation of one or more lineages, may overlap with other growth factors by affecting a single progenitor cell line, or they may act synergistically. with other factors. It also appears that hematopoietic growth factors can show their effect at different stages of cell development from the totipotent stem cell to the mature blood cell, through the various committed progenitors, limited in lineage. For example, erythropoietin (epo) seems to activate only the proliferation of mature erythrocyte progenitor cells. IL-3 appears to exert its effect earlier by influencing stem cells

  primitive and intermediate progenitor cells with restricted lineage. Other growth factors such as stem cell factor (SCF) can further influence primitive cell development. From the foregoing, it will be apparent that novel hematopoietic growth factors which affect the survival, proliferation, differentiation or maturation of all blood cells or their predecessors would be useful, in particular to promote the restoration of a hematopoietic system decreased to cause of illness or after radiotherapy or chemotherapy. II. Megakaryocytopoi & se - production of platelets

  Regulation of megakaryocytopoiesis and platelet production has been studied in detail by Mazur, Exp. Hematol. 15: 248 [1987] and by Hoffman, Blood, 74: 1196-1212 [1989]. In short, pluripotent bone marrow stem cells differentiate into megakaryocytic, erythrocyte and myelocytic cell lines. It is considered that there is a hierarchy of megakaryocytic progenitor cells involved between stem cells and megacariocytes. At least three classes of megakaryocytic progenitor cells have been identified, namely: megakaryocytes (BFU-MK), cell-forming units, megakaryocytes (CFU-MK) of colony-forming units, and Low density megakaryocyte progenitor cells (LD-CFU-MK). Megakaryocyte maturation itself is a

  continuous process of development which has been subdivided into stages on the basis of normal morphological criteria. The most rapidly recognizable member of the megakaryocyte family (MK or meg) is that which belongs to megakaryoblasts. These cells initially have a diameter of 20 to 30 μm, possess a basophilic cytoplasm and have a slightly irregular nucleus with loose, slightly reticular chromatin, and several nucleoli. Later, megakaryoblasts can contain up to 32 nuclei (polyploids), but the cytoplasm remains scarce and immature. As ripening is accomplished, the core

  becomes more lobulated and pycnotic, the cytoplasm grows in quantity and becomes more acidophilic and granular. The most mature cells in this family may give the impression of releasing platelets at their periphery.

  Normally, less than 10% of megakaryocytes are in the blast cell stage and more than 50% are mature. The arbitrary morphological classifications commonly applied to the megakaryocyte series include the megakaryoblast for the earliest form; the promégacarocyte or basophilic megakaryocyte for the intermediate form; and the mature megakaryocyte (acidophilus, granular or platelet producer) for the ultimate forms. The mature megakaryocyte extends cytoplasmic filaments into sinusoidal spaces where they separate and fragment into individual platelets (Williams et al., Hematology, 1972). It is presumed that megakaryocytopoiesis involves several regulatory factors (Williams et al., Br. J. Haematol., 52: 173 [1982] and Williams et al., J. Cell Physiol., 110: 101 [1982]). . The first level of the megakaryocytopoiesis is presumed to be mitotic, with respect to cell proliferation and colony initiation from CFU-MK, but not affected by platelet counts (Burstein et al., J. Cell Physiol. 109: 333 [1981] and Kimura et al., Exp Hematol., 13: 1048 [1985]). The last stage of maturation is non-mitotic, which involves nuclear polyploidization and cytoplasmic maturation and is probably regulated in a retroactive mechanism by the number of peripheral platelets (Odell et al., Blood, 48: 765 [1976] and Ebbe et al., Blood, 32: 787 [1968]).

  The existence of a distinct and specific megakaryocyte colony stimulating factor (MK-CSF) has been controversial (Mazur, Exp Hematol., 15: 340-350 [1987]). However, most authors believe that a process as vital to survival as platelet production should be regulated by one or more cytokines that are exclusively responsible for this process. The hypothesis that there are one or more cytokines specific for megacarocytes / platelets has been the basis for more than 30 years of research - but at present no cytokine of this genus has been purified, sequenced, and established by experimental methods as a single MK-CSF (TPO) factor.

  Although it has been reported that MK-CSF factors have been partially purified starting from experimentally triggered thrombocytopenia (Hill et al., Exp Hematol., 14: 752 [1986]) and a conditioned medium of human embryonic kidney [CM] (McDonald et al., J. Lab.Clin.

  Med., 85:59 [1975]) and, in humans, from a plastic anemia and urinary extracts related to an idiopathic thrombocytopenic purpura (Kawakita et al., Blood, 6: 556 [1983] ) and plasma (Hoffman et al., J. Clin Invest, 5: 1174 [1985]), their physiological function is hitherto unknown in most cases. Mitogen pokeweed-activated spleen cell conditioned medium (PWM-SpCM) and WEHI-3 murine myelomonocytic cell line (WEHI-3CM) were used for the potentiation of megakaryocytes. The PWM-SpCM cell contains growth-promoting factors of CFU-MK (Metcalf et al., Pro Natl Acad Sci., USA, 72: 1744-1748 [1975], Quesenberry et al., Blood, 65: 214 [1985] and NN Iscove, in Hematopoietic Cell Differentiation, ICN-UCLA Symposia on Molecular and Cellular Biology, Volume 10, published under the direction of Golde et al., [New

  York, Academy Press, pp. 37-52 [1978]), one of which is interleukin-3 (IL-3), multi-branching colony stimulating factor (multi-CSF [Burstein, Blood Cells, 11: 469 [1986]). Other factors in this environment have not yet been identified or isolated. WEHI-3 is a murine myelomonocytic cell line that secretes relatively large amounts of IL-3 and smaller amounts of GM-CSF. IL-3 has been found to enhance the growth of a broad range of hematopoietic cells (Ihle et al., J. Immunol 13: 282 [1983]). It has also been found that IL-3 exerts a synergistic action together with many of the known hematopoietic hormones or growth factors (Bartelmez et al., J. Cell.

  Physiol., 122: 362-369 (1985) and Warren et al., Cell, 46: 667-674 [1988]), comprising both erythropoietin (EPO) and interleukin-1 (IL-1). , in the induction of very early multipotential precursors and the formation of very large mixed hematopoietic colonies. Other sources of megakaryocytic potentiation in conditioned media of murine lung, bone, macrophage cell lines, peritoneal exudate cells and human embryonic kidney cells have been found. Despite some contradictory data (Mazur, Hematol Exp., 15: 340-350 [19871), there is some evidence (Geissler et al., Br. J. Haematol., 60: 233-238 [1985]) that activated T cells, rather than

  monocytes play a role in favor of megakaryocytopolysis. These observations suggest that activated T-cell secretions such as interleukins may be regulatory factors in MK development (Geissler et al., Exp.Hematol., 15: 845-853 [1987]). Several studies on megakaryocytopoiesis involving purified EPO erythropoietin (Vainchenker et al., Blood, 54: 940 [1979], McLeod et al., Nature, 261: 492-4 [1976] and Williams et al., Exp. Hematol., 12: 734 [1984]) indicate that this hormone produces an activating effect on MK colony formation. This has also been demonstrated in serum-free cultures as well as in serum-containing cultures and in the absence of accessory cells (Williams et al., Exp Hematol., 12: 734 [1984]).

  It has been postulated that EPO is more involved in the single cell and bicellular stage constituting the aspects of megakaryocytopolysis as opposed to the effect of PWM-SpCM which has been implicated in the quadricellular stage of megakaryocyte development. The interaction of all these factors on the early and late phases of megakaryocyte development remains to be elucidated. Results obtained in different laboratories suggest that the only multiple lineage factors that individually possess MK colony-stimulating activity are GM-CSF and IL-3 and, to a lesser extent, IL-6 stimulating factor. B cells (Ikebuchi et al., Proc Natl Acad Sci USA 84: 9035 [1987]). More recently, several authors have reported that IL-11 and leukemia inhibitory factor (LIF) synergistically work with IL-3 by increasing the size and thickness of megakaryocites (Yonemura et al. , British Journal of Hematology, 84: 16-23 [1993], Burstein et al., J. Cell Physiol., 153: 305-312 [1992], Metcalf et al., Blood, 76: 50-56. [1990], Metcalf et al., Blood, 77: 2150-2153 [1991], Bruno et al.

  Exp. Hematol., 19: 378-381 [1991]; and Yonemura et al., Exp. Hematol., 20: 1011-1016 [1992]). Other documents of interest include: Eppstein et al., U.S. Patent No. 4,962,091; Chong, U.S. Patent No. 4,879,111; Fernandes et al., U.S. Patent No. 4,604,377; Wissler et al., U.S. Patent No. 4,512,971; Gottlieb, U.S. Patent No. 4,468,379; Bennett et al., U.S. Patent No. 5,215,895; Kogan et al., U.S. Patent No. 5,250,732; Kimura et al., Eur. J. Immunol., (9): 1927-1931 [1990]; Secor et al., J. of Immunol., 144 (4): 1484-1489 [1990]; Warren et al. J. of

  Immunol., 140 (1): 94-99 [1988]; Warren et al., Exp. Hematol., 17 (11): 1095-1099 [1989]; Bruno et al., Exp. Hematol., 17 (10): 1038-1043 [1989]; Tanikawa et al., Exp. Hematol., 17 (8): 883-888 [1989]; Koike et al., Blood, 75 (12): 2286-2291 [1990]; Lotem, Blood, 75 (5): 1545-1551 [1989]; Rennick et al., Blood, 73 (7): 1828-1835 [1989]; and Clutterbuck et al., Blood, 73 (6): 1504-1512 [1989]. III. Thrombocytopenia Platelets are the determining factors of the blood coagulation mechanism. The depletion of circulating platelets, called thrombocytopenia, accompanies various states and clinical disorders. Thrombocytopenia is commonly defined as having a platelet count of less than 150 x 109 per liter. The main causes of thrombocytopenia can be broadly divided into three categories based on platelet life, namely: (1) impairment of platelet production by bone marrow, (2) platelet sequestration in platelets. the spleen (splenomegaly), or (3) the increased destruction of platelets in the circulation

  peripheral (eg autoimmune thrombocytopenia or chemotherapy and radiotherapy). In addition, in patients who receive large volumes of platelet-poor blood products administered rapidly, thrombocytopenia may develop as a result of the dilution. Clinical haemorrhagic manifestations of thrombocytopenia depend on the severity of the thrombocytopenia, its cause, and the coagulation defects that may be associated with it. In general, patients with platelet counts between 20 and 100 x 109 per liter may experience excessive post-traumatic hemorrhage, while those with platelet counts below 20 x 109 per liter may experience spontaneous bleeding. The latter patients are candidates for platelet transfusion with the associated immune and viral risk. For everything

  given degree of thrombocytopenia, the bleeding tends to be more severe when the cause is reduced production than when it comes to an accentuation of platelet destruction. In the latter case, accelerated reconstitution of platelets circulates younger, larger and more hemostatically effective platelets. Thrombocytopenia may result from various disorders briefly described below. A more detailed description can be found in A.I. Schafner, "Thrombocytopenia and Disorders of Platelet Function", Internal Medicine, 3rd Edition, edited by John J. Hutton et al., Little

  Brown and Co. Boston / Toronto / London [1990]. (a) Thrombocytopenia due to abnormal platelet production The causes of congenital thrombocytopenia include constitutional aplastic anemia (Fanconi syndrome) and congenital amgakaryocyte thrombocytopenia, which may be associated with skeletal malformations. Acquired disorders of platelet production are caused by megakaryocyte hypoplasia or ineffective thrombocytosis. Megakaryocytic hypoplasia can result from a variety of conditions, including bone marrow suppression (including idiopathic forms or myelosuppression by chemotherapeutic agents or radiotherapy), myelofibrosis, leukemia, and bone marrow invasion by metastatic tumor or granulomas. In some cases, toxins, infectious agents or drugs may interfere relatively selectively with thrombopoiesis; examples include transient thrombocytopenia caused by alcohol and certain viral infections and moderate thrombocytopenia associated with the administration of thiazide type diuretics. Finally, inefficient thrombopoiesis accompanying side effects of megaloblastic processes (folate or B12 deficiency) can also cause thrombocytopenia, usually accompanied by anemia and leukopenia. The usual treatment of thrombocytopenia due to a reduction in platelet production is based on the identification and antecedent causes of the deficiency.

  medulla. Platelet transfusions are usually reserved for patients with severe haemorrhagic complications, or for coverage during surgical procedures, since isoimmunization may cause the patient to be refractory to subsequent platelet transfusions. Mucosal bleeding resulting from severe thrombocytopenia may be reduced by oral or intravenous administration of antifibrinolytic agents. However, thrombotic complications may develop if antifibrinolytic agents are used in patients with disseminated intravascular coagulation (DIC). (b) Thrombocytopenia due to splenic sequestration

  Splenomegaly due to any cause may be associated with thrombocytopenia ranging from mild to moderate. It is a highly passive (hypersplenic) process of splenic platelet sequestration, in contrast to active platelet destruction by the spleen in immune-mediated thrombocytopenia discussed below. Although the most common cause of hyper-splenism is congestive splenomegaly resulting from portal hypertension due to alcoholic cirrhosis, other forms of congestive, invasive or lymphoproliferative splenomegaly are also associated with thrombocytopenia. Platelet levels generally do not fall below 50 x 10 'per liter as the sole consequence of hypersplenism. (c) Thrombocytopenia due to platelet destruction without immune mediation Thrombocytopenia may result from accelerated platelet destruction by various non-immunological processes. Disorders of this type include disseminated intravascular coagulation, the presence of intravascular prostheses, the extracorporeal circulation of ang, and thrombotic microangiopathies such as thrombotic thrombocytic purpura. In all these situations, circulating platelets that are exposed to artificial surfaces or to abnormal vascular tunics

  males are consumed at these sites or are damaged and prematurely removed by the reticuloendothelial system. Disease states or disorders in which disseminated intravascular coagulation (DIC) may occur are described in great detail in Harrison's Principles of Internal Medicine, 11th Edition, page 1478, edited by Braunwald et al., McGraw Hill [ 1987]. Intravascular prostheses, including heart valves and intra-aortic balloons, may cause mild to moderate destructive thrombocytopenia, and transient thrombocytopenia in patients undergoing cardiopulmonary bypass or hemodialysis may result from the consumption or alteration of platelets in the extracorporeal circuit. (d) Drug-induced immune thrombocytopenia

  More than 100 drugs have been implicated in immunologically mediated thrombocytopenia. However, only quinidine, quinine, gold, sulfonamides, cephalothin and heparin have been well characterized. Drug-induced thrombocytopenia is frequently very severe and normally occurs precipitously in the period when patients are taking the sensitizing medication. (e) Immune thrombocytopenic purpura (autoimmune) (ITP)

  ITP in adults is a chronic disease characterized by autoimmune destruction of platelets. The autoantibody is usually IgG, although other immunoglobulins have also been reported. Although it was found that the autoantibody of the ITP was associated with the platelet membrane GPIIbIII., Specificity for platelet antigen was not identified in most cases. Extravascular destruction of sensitized platelets takes place in the reticuloendothelial system of the spleen and liver. Although more than half of the cases of ITP are idiopathic, many patients have rheumatic or autoimmune diseases (eg systemic lupus erythematosus) or lymphoproliferative disorders (eg, chronic lymphocytic leukemia) underlying them. (f) Acquired ITP-induced ITP, HIV The ITP is an increasingly common complication of HIV infection (Morris et al., Ann Intern Med, 96: 714; 717 [1982]), and can occur at all stages of the disease, both in patients with acquired immune deficiency syndrome (AIDS) and in patients with complex symptoms related to AIDS. and patients with HIV infection, but without AIDS symptoms. HIV infection is a communicable disease characterized at the final stage by a profound deficiency of HIV. immune function of cells as well as the appearance of opportunistic infection and malignancy. The primary immunological abnormality resulting from HIV infection is progressive depletion and

  functional impairment of T cells expressing CD4 cell surface glycoprotein (Lane et al., Ann Rev. Immunol., 3: 477 [1985]). The loss of function of helper / CD4-inducing T cells is probably responsible for the profound abnormalities of cellular and humoral immunity leading to opportunistic germs infections and characteristic malignancies of AIDS (H. Lane, supra). Although the mechanism of HIV-associated PI is not known, it is believed to be different from the mechanism of non-HIV infection-associated PI (Walsh et al., N. Eng., J. Med. 311: 635-639 [1984] and Ratner, Am. J. Med., 86: 194-198 [1989]).

  IV. Routine Therapy for Thrombocytopenia The therapeutic approach to treating patients with thrombocytopenia is dictated by the severity and urgency of the clinical situation. The treatment is similar for HIV-associated thrombocytopenia and unrelated thrombocytopenia, and although a number of different therapeutic approaches have been used, the therapy remains controversial. Platelet levels in patients in whom thrombocytopenia has been diagnosed have been successfully elevated by glucocorticoid therapy (eg, prednisolone), however, in most patients the response is not complete or recurrence appears when the dose of glucocorticoid is reduced or when its administration is interrupted. According to studies of patients with JTP associated with the HIV virus, some researchers have suggested that glucocorticoid therapy may lead to predisposition to AIDS. Glucocorticoids are usually given if the platelet count falls below 20 x 109 / l or when spontaneous bleeding occurs.

  For patients refractory to glucocorticoid, the compound: 4 (2-chlorophenyl) -9-methyl-2- [3- (4-morpholinyl) -3-propanone-1-yl] 6H-thieno [3,2, f] [11, 2,4] triazolo [4,3, a] [1,4] diazepine (WEB 2086) has been used successfully to treat a severe case of ITP not associated with the HIV virus. A patient with platelet counts of 37,000-58,000 / ~ 1 was treated with WEB 2086 and after one to two weeks treatment, platelet counts were 140,000-190,000. 1 (European Patent No. 361,077 and Lobman et al., Lancet, 1147 [1988]). Although the optimal treatment of acquired amakacaryocyte thrombocytopenic purpura (AATP) is random, antithymocyte globulin (ATG), a horse antiserum against thymic tissue has been shown to be effective.

  human, produced a prolonged complete remission (Trimble et al., Am., J. Hematol., 37: 126-127 (1991).) However, a recent publication indicates that the hematopoietic effects of ATG are attributable to thimerosal, there o likely the protein acts as a mercury vehicle (Pannela et al., Cancer Research, 50: 4429-4435 [1990]). Good splenectomy results have been reported Splenectomy eliminates the main site of destruction platelets and the main source of autoantibody production in many patients, which results in prolonged remissions without treatment in many patients.

  Surgical procedures should generally be avoided in patients whose immunity is impaired, splenectomy is recommended only in severe cases of thrombocytopenia (for example in cases of severe ITP associated with HIV) in patients who do not do not respond to glucocorticoid treatment for two to three weeks or who do not achieve a sustained response after discontinuation of glucocorticoid administration. In the current state of scientific knowledge, there is no evidence that splenectomy predisposes patients to AIDS. In addition to prednisolone therapy and splenectomy, certain cytotoxic agents, for example vincristine and azidothimidine (AZT, zidovudine), also show promise in the treatment of HIV-induced ITP; however, the results are preliminary. It follows from the foregoing that a means of treating thrombocytopenia would be to obtain an agent capable of accelerating the differentiation and maturation of megakaryocytes or their precursors into the lacquer producing form. Considerable efforts have been made to

  identify such an agent, commonly known as "thrombopoietin" (TPO). Other names commonly found in the literature for TPO include: thrombocytopoiesis stimulating factor (TSF), megakaryocyte colony stimulating factor (MK-CSF), megakaryocyte stimulating factor, and agent for potentiating megakaryocytes. The activity of TPO was observed as early as 1959 (Rak et al., Med Exp, 1: 125) and attempts to characterize and purify this agent have been continued to date. Although there are reports of partial purification of TPO-active polypeptides (see, for example, Tayrien et al., J. Biol Chem., 262: 3262 [1987] and Hoffman et al., J. Clin. Invest 75: 1174 [1985]), others hypothesized that TPO was not a distinct entity in itself but rather a simple polyfunctional manifestation of a known hormone (IL-3, Sparrow et al., Prog Clin Biol Res, 215: 123 [1986]). Whatever its form or origin, a molecule with thrombopoietic activity would offer an important therapeutic value. Although no protein has been unambiguously identified as TPO, considerable interest surrounds recent evidence that mpl, the suspected cytokine receptor, may be the transducer of a thrombopoietic signal.

  V. Mpl is a megakaryocytopolytic cytokine receptor It is believed that the proliferation and maturation of hematopoietic cells are closely dependent on factors that positively or negatively modulate pluripotential stem cell proliferation and differentiation between multiple lineages. These effects are mediated by high affinity binding of extracellular protein factors to specific cell surface receptors. These cell surface receptors share considerable homology and are generally classified as members of the cytokine receptor superfamily. Members of the superfamily include receptors for: IL-2 (γ and γ chains) (Hatakeyama et al., Science, 244: 551-556 [1989], Takeshita et al., Science, 257: 379-382 [1991] ]), IL-3 (Itoh et al., Science, 247: 324-328 [1990], Gorman et al., Proc Natl, Acad Sci USA 87: 5459-5463 [1990], Kitamura et al. , Cell, 66: 1165-1174 [1991a], Kitamura et al., Proc Natl Acad Sci USA, 88: 5082-5086 [1991b]), IL-4 (Mosley et al., Cell, 59: 335-348 [1989], IL-5 (Takaki et al., EMBO J. 9: 4367-4374 [1990], Tavernier et al., Cell, 66: 1175-1184 [1991]), IL-6 (Yamasaki et al., Science, 241: 825-828 [1988] Hibi et al., Cell, 63: 1149-1157 [1990]), IL-7 (Goodwin et al., Cell, 60: 941-951 [1990]). ]), IL-9 (Renault et al., Proc Natl Acad Sci USA, 89: 5690-5694 [1992]), granulocyte-macrophage colony stimulating factor (GM-SCF) (Gearing et al. EMBO J., 8: 3667-3676 [1991], Hayashida et al., Proc Natl.

  Acad. Sci. USA, 244: 9655-9659 [1990]), granulocyte colony stimulating factor (G-CSF) (Fukunaga et al., Cell, 61: 341-350 [1990a], Fukunaga et al Proc Natl Acad. Sci USA, 87: 8702-8706 (1990b), Larsen et al., J. Exp Med., 172: 1559-1570 [1990], EPO (D'Andrea et al., Cell, 57: 277-285 [ 1989], Jones et al., Blood, 76: 31-35 [1990]), leukemia inhibitory factor (LIF) (Gearing et al., EMBO J., 10: 2839-2848 [1991]), Oncostatin M ( OSM) (Rose et al., Proc Natl Acad Sci USA, 88: 8641-8645 [1991]) as well as prolactin receptors (Boutin et al., Proc Natl Acad Sci USA, 88 7744-7748 [1988] Edery et al Proc Natl Acad Sci USA 86-2112-2116 [1989]), growth hormone (GH) (Leung et al., Nature, 330: 537-543). [1987]) and ciliary neurotrophic factor (CNTF) (Davis et al., Science, 253: 59-63 [1991].) Members of the cytokine receptor superfamily can be grouped into three functional categories (for one view). overall, see Nicola et al., Cell, 67: 1 -4 [1991]). The first class includes receivers

  single chain such as the erythropoietin receptor (EPO-R) or the granulocyte colony stimulating factor receptor (G-CSF-R), which binds the high affinity ligand by the extracellular domain and thereby generates a signal intracellular. A second class of receptors, referred to as α-subunits, include the interleukin-6 receptor (IL6-R), the granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R), the interleukin-3 receptor (IL-3-Ra) and other members of the cytokine receptor superfamily. These α-subunits bind the low affinity ligand but can not perform transduction of an intracellular signal. A high affinity receptor capable of signal transduction is generated by a heterodimer between an α-subunit and a member of a third class of cytokine receptors, referred to as λ subunits, for example Oc, a common subunit 6 three subunits IL3-Ra and GM-CSF-R. The evidence that mpl is a member of the cytokine receptor superfamily comes from sequence homology (Gearing, EMBO J., 8: 3667-3676 [1988];

  Proc. Natl. Acad. Sci. USA, 87: 6834-6938 [1990]; Davis et al., Science, 253: 59-63 [1991] and Vigon et al., Proc. Natl. Acad. Sci. USA, 89: 5640-5644 [1992]) and its ability to transduce proliferative signals. The protein sequence deduced from the molecular cloning of mouse c-mpl reveals that this protein is the homologue of other cytokine receptors. The extracellular domain contains 465 amino acid residues and is composed of two secondary domains each with four highly conserved cysteines and a particular motif in the N-terminal secondary domain and the C-terminal secondary domain. The extracellular ligand-binding domains can be expected to have similar structural geometries with a double platen sheet. This doubled extracellular domain is highly homologous with the signal transduction haun common to the receptors

  IL-3, IL-5 and GM-CSF as well as low affinity binding domains of LIF (Vigon et al., Oncogene, 8: 2607-2615 [1993]). Thus, mpl may belong to the low affinity ligand binding class of cytokine receptors. A comparison of murine mpl and mature human mpl P reveals that these proteins have a sequence identity of 81%. More particularly, the extracellular subdomains of the N terminal end and the C terminal end respectively share 75% and 80% of the sequence identity. The most conserved mpl region is the cytoplasmic domain showing 91% amino acid identity, with a sequence identity of 37 residues near the transmembrane domain in both species. Accordingly, mpl is reported to be one of the most conserved members of the cytokine receptor superfamily (Vigon, supra). The evidence that mpl is a functional receptor capable of transducing a proliferative signal results from the construction of chimeric receptors containing an extracellular domain from a cytokine receptor having a high affinity for a cytokine known to the domain. cytoplasmic mpl. Since no known ligand for mpl has been reported, it was necessary to construct the chimeric extracellular domain for binding a high affinity ligand from a first class cytokine receptor such as IL-4R or G-CSFR.

  Vigon et al., Supra, fused the extracellular domain of G-CSFR with the transmembrane domain as well as the cytoplasmic domain of c-mpl. An IL-3-dependent cell line, BAF / B03 (Ba / F3), was transfected with G-CSFR / mpl at the same time as a full-length G-CSFR control. The cells transfected with the chimera grew equally well in the presence of cytokine IL-3 or G-CSF. Similarly, cells transfected with the G-CSFR receptor also grew well in IL-3 or G-CSF. All cells died in the absence of growth factors. A similar experiment was conducted by Skoda et al. EMBO J., 12 (7): 2645-2652 [1993], in which the two extracellular and transmembrane domains of human IL-4 receptor (hIL-4-R) were fused to the cytoplasmic domain of mouse mpl, and trans - fected into a murine Ba / F3 cell line dependent on

  IL-3. Ba / F3 cells transfected with the wild-type hIL-4-R receptor normally proliferated in the presence of IL-4 or species-specific IL-3. Ba / F3 cells transfected with hIL-4R / mpl have proliferated normally in

  the presence of hIL-4 (in the presence or in the absence of IL-3), thus demonstrating that in Ba / F3 cells, the cytoplasmic domain mpl contains all the elements necessary for the transduction of a proliferative signal. These chimeric experiments demonstrate the proliferative signaling ability of the cytoplasmic domain mpl but are silent as to whether or not the extracellular domain mpl may bind a ligand. These results are consistent with at least two possibilities, namely, mpl is a single-chain (class one) receptor such as EPO-R or G-CSFR or it is a signal transducing subunit 0 ( class three) requiring an α subunit such as IL-3 (Skoda et al., supra).

  VI. The mpl ligand is a thraombopoaitin (TPO) As described above, it has been suggested that the serum contains a single factor, sometimes called thrombopetin (TPO), which exerts a synergistic action with various other cytokines by activating the growth. and the maturation of megakaryocytes. No such natural factor has ever been isolated from serum or any other source, although considerable effort has been made by many groups. Although it is not known whether mpl is able or directly to fix a megakaryocyte stimulating factor, recent experiments have demonstrated that mpl was involved in proliferative signal transduction from one or more factors encountered in serum from patients with aplastic bone marrow (Methia et al., Blood, 82 (5): 1395-1401 [1993]).

  Evidence that a single serum colony-forming factor, distinct from IL-1α, IL-3, IL-4, IL-6, IL-11, SCF, EPO, G-CSF and GM-CSF accomplishes transduction of a proliferative signal via mpl results from examination of the distribution of c-mpl expression in primitive and committed hematopoietic cell lines and mpl antisense studies in one or more of these lineages. cell. Using reverse transcriptase (RT) -PCR in immunopurified human hematopoietic cells,

  Methia et al., Supra, demonstrated that mpl mRNA accented messages were only found in purified CD34 'cells, megakaryocytes, and platelets. Purified CD34 'cells from bone marrow (BM) account for about 1% of all bone marrow cells and are enriched in primitive and committed progenitors of all lineages (eg erythrocyte, granulomacrophage and megakaryocytic).

  It has been demonstrated that mpl antisense oligodeoxynucleotides suppress the formation of megakaryocytic colonies from pluripotent CD34 cells cultured in serum from patients with aplastic marrow (a source rich in megakaryocytic colony stimulating activity [MK-CSA]). . These same antisense oligodeoxynucleotides had no effect on the formation of colonies of erythrocytes or granulomacrophages. It was not yet known whether mpl directly bound to a ligand and whether the serum factor presented as the cause of megakaryocytopenia was acting via mpl.

  However, it has been suggested that if mpl binds directly to a ligand, its amino acid sequence is likely to be highly conserved and to have cross-reactivity between species because of the considerable sequence identity between extracellular domains mpl human and murine (Vigon et al., supra [1993]).

  VII. Objects In view of the foregoing indications, it should be noted that a current and permanent technical need exists to be able to isolate and identify molecules capable of stimulating the proliferation, differentiation and maturation of hematopoietic cells, especially megakaryocytes. or their predecessors for therapeutic use in the treatment of thrombocytopenia. Such a molecule is considered to be a mpl ligand and therefore there is a need to isolate such a ligand or similar ligands to evaluate their role in cell growth and differentiation.

  Accordingly, it is an object of the present invention to provide a pharmaceutically pure molecule capable of stimulating proliferation, differentiation and / or maturation of megakaryocytes into the mature platelet producing form. Another object of the present invention is to present the molecule in a form suitable for therapeutic use in the treatment of a haematopoietic disorder, including thrombocytopenia. The present invention further aims to isolate, purify and specifically identify protein ligands capable of binding in vivo to a cytokine superfamily receptor called mpl and to transduce a proliferative signal.

  Another object is to find nucleic acid molecules encoding such protein ligands and utilize these nucleic acid molecules to produce mpl binding ligands in a recombinant cell culture for diagnostic and therapeutic purposes. The invention further aims to find derivatives and modified forms of protein ligands comprising amino acid sequence variants, variant glycoprotein forms and covalent derivatives thereof. Another object of the invention is to find merged polypeptide forms combining an mpl ligand and a heterologous protein and its covalent derivatives.

  It is also an object of the invention to provide polypeptide forms of mp1 ligand-binding variants to amino acid additions and amino acid substitutions of the EPO sequence to produce a protein capable of regulating proliferation and growth. both platelets and progenitors of red blood cells. The invention further aims to prepare immunogens to create antibodies against mpl ligands or their fused forms, as well as to obtain antibodies capable of binding these ligands. Other features and advantages of the present invention will become apparent from the detailed description which follows.

  The invention achieves the goals it has set out to achieve by creating an isolated mammalian protein that activates megakaryocytopolytic proliferation and maturation, called "mpl ligand" (ML) or "thrombopoietin". (TPO) capable of stimulating proliferation, maturation and / or differentiation of megakaryocytes into the mature platelet-producing form. The term "ligand mpl" will also be used indifferently. This predominantly homogeneous protein can be purified from a natural source by a method comprising: (1) contacting starting plasma containing the mpl ligand molecules to be purified with an immobilized receptor polypeptide, more particularly an mpl polypeptide or a mpl-immobilized fused polypeptide under a condition in which the mpl ligand molecules to be purified are selectively adsorbed onto the immobilized receptor polypeptide, (2) washing the immobilized receptor polypeptide and its carrier for removing the unadsorbed material and (3) eluting the ligand mpl molecules, using an elution buffer, from the immobilized receptor polypeptide to which they are adsorbed. The natural source is advantageously mammalian plasma or urine containing the mpl ligand. Optionally, the mammal is aplastic and the immobilized receptor is an mpl-IgG fusion.

  The megakaryocytopoietic proliferation and maturation promoting protein may advantageously be a predominantly homogeneous isolated mpl ligand polypeptide obtained by synthetic means and by recombination. The "mpl # or" TPO ligand "polypeptide of the present invention advantageously has an overall sequence identity of at least 70% with the amino acid sequence of the highly purified predominantly homogeneous porcine ligand ligand polypeptide and at least 80% of the protein. Sequence identity with the "EPO domain" of the porcine mpl ligand polypeptide The mpl ligand of the present invention may be a mature human mpl ligand (hML) having the mature amino acid sequence shown in Figure 1 (SEQ ID NO : 1) or a variant or a modified form after transcription of this sequence or a protein having 80% sequence identity with the mature human mpl ligand The mpl ligand variant is optionally a fragment, in particular an amino-terminal fragment or "EPO domain" of the mature human mpl ligand (hML) The amino-terminal fragment keeps almost all of the human ML sequence between the first and fourth cysteine residues, but it may contain ons, deletions or substitutions important to this region. According to this embodiment, the fragmentary polypeptide can be represented by the formula:

  X-hML (7-151) -Y wherein hML (7-151) represents the amino acid sequence of TPO (hML) ranging from Cys' to Cys11 inclusive; X represents the amino group of Cys7 or one or more of the amino-terminal amino acid residues of the mature hML ligand or extensions of amino acid residues to that ligand, such as Met, Tyr or leader sequences containing for example proteolytic cleavage sites (for example factor Xa or thrombin); and Y represents the carboxy terminal group of Cys'51 or one or more carboxyl-terminal amino acid residues of the mature hML ligand or corresponding extensions.

  If desired, the mpl ligand polypeptide or fragment thereof can be fused with a heterologous (chimeric) polypeptide. A preferred heterologous polypeptide is a cytokine, a colony-stimulating factor or a corresponding interleukin or fragment, particularly a ligand-kit (KL), IL-1, IL-3, IL-6, IL-11, EPO , GM-CSF or LIF. A preferred optional heterologous polypeptide is an immunoglobulin chain, including IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgD, human IgM or a corresponding fragment, including in particular the constant domain of a heavy chain. IgG.

  According to another of its aspects, the invention provides a composition comprising an isolated mpl agonist which is biologically active and which is advantageously capable of stimulating the incorporation of labeled nucleotides (for example 3H-thymidine) into Ba cell DNA. / F3 dependent on IL-3 transfected with human mpl. The mpl agonist may be the biologically active mpl ligand and is advantageously capable of stimulating 35S incorporation into circulating platelets in a mouse platelet rebound assay. Suitable mpl agonists include hML153, hML (R153A, R154A), hML2, hML3, hML4, mML, mML2, mML3, pML and pML2 or fragments thereof.

  In another embodiment, the present invention provides an isolated antibody capable of binding to the mpl ligand. Isolated antibody capable of binding to the mpl ligand may optionally be fused to a second polypeptide and the antibody or its fused form may be used to isolate and purify the mpl ligand from a source of the type described above for immobilized mpl . According to another aspect of this embodiment, the invention provides a method for detecting mpl ligand in vitro or in vivo, which comprises the steps of bringing the antibody into contact with a sample, including a serum sample, suspected to contain the ligand and to detect if a link has occurred.

  In yet other embodiments, the invention provides an isolated nucleic acid nucleic acid molecule encoding the mpl ligand or fragments thereof, which nucleic acid molecule can optionally be labeled with a detectable moiety, and a nucleic acid molecule having a sequence which is complementary to a nucleic acid molecule having a coding sequence for an mpl ligand, or which hybridizes under moderate to very stringent conditions, with this nucleic acid molecule . Preferred nucleic acid molecules are those that encode human, porcine and murine mpl ligand and include both genomic RNA and DNA, and cDNA. In yet another aspect of this embodiment, the nucleic acid molecule is a DNA encoding the mpl ligand and further comprises a replicable vector to which the DNA is conveniently attached to influence sequences recognized by a host. transformed with the vector. The DNA is optionally cDNA having the sequence 5'-3 '(SEQ ID N 2), 3'-5' shown in Figure 1 or a fragment thereof. This other aspect comprises host cells, preferably CHO cells, transformed with the vector and a method of using DNA to effect the production of mpl ligand, which advantageously consists in expressing cDNA encoding the ligand. mpl in a transformed host cell culture and to collect the mpl ligand from the host cells or the host cell culture. The mpl ligand prepared in this manner is advantageously human mpl ligand.

  The invention further relates to a method of treating a mammal with a hematopoietic disorder, including thrombocytopenia, which comprises administering a therapeutically effective amount of a mpl ligand to the mammal. The mpl ligand can be administered in combination with a cytokine, such as a colony stimulating factor or interleukin. Preferred colony-stimulating factors or interleukins include ligand-kit (KL), LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-3, IL-6 and IL. -11. The invention further relates to a method for isolating and purifying TPO (ML) from a TPO-producing microorganism, which comprises the steps of: (1) disrupting or lysing TPO-containing cells, ( 2) optionally separating the soluble material from the TPO-containing insoluble material, (3) solubilizing the TPO contained in the insoluble material with a solubilizing buffer, (4) separating the solubilized TPO from the rest of the soluble and insoluble material, (5) to fold the TPO in a redox buffer, and (6) to separate the properly folded TPO from the incorrectly folded TPO.

  The process allows solubilization of insoluble material containing TPO with a chaotropic agent, wherein said chaotropic agent is selected from a guanidine salt, sodium thiocyanate or urea. The method further enables the solubilized TPO to be separated from the remaining soluble and insoluble material by one or more steps selected between centrifugation, gel filtration and reverse phase chromatography. The process folding step provides a redox buffer containing both an oxidizing agent and a reducing agent. In general, the oxidizing agent is oxygen or a compound containing at least one disulfide bond and the reducing agent is a compound containing at least one free sulfhydryl group. The oxidizing agent is advantageously chosen between oxidized glutathione (GSSG) and cystine and the reducing agent is chosen between reduced glutathione (GSH) and cysteine. Preferably, the oxidizing agent is oxidized glutathione (GSSG) and the reducing agent is glutathione

  reduced (GSH). It is also appreciated that the molar proportion of oxidizing agent is equal to or greater than that of the reducing agent. The redox buffer further contains a detergent, advantageously chosen between the substances CHAPS and CHAPSO, present in a proportion of at least 1%. The redox buffer further contains NaCl preferably in a concentration range of about 0.1 to 0.5 M, and glycerol, preferably at a concentration greater than 15 K. The pH of the redox buffer is preferably from about 7.5 to 9.0 and the refolding step is run at 4 for 12-48 hours. The folding step produces biologically active TPO in which a disulfide bond is formed between the Cys group closest to the amino terminal end and the Cys group closest to the carboxy terminal end of the EPO domain.

  The invention further relates to a method for purifying biologically active TPO from a microorganism, comprising: (1) lysing the extracellular membrane of at least one microorganism, (2) treating the lysate containing the TPO with a chaotropic agent, (3) folding of the TPO, and (4) separation of impurities and poorly folded TPO from the suitably folded TPO.

  BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the deduced amino acid sequence (SEQ ID N: 1) of human mpl ligand cDNA (hML) and the coding nucleotide sequence (SEQ ID N: 2). Nucleotides are numbered at the beginning of each line. The untranslated regions 5 'and 3' are indicated in lowercase letters. Amino acid residues are numbered above the sequence from Ser 1 of the mature mpl ligand (ML) protein sequence. The limits of the presumed exon 3 are indicated by the arrows and the potential sites of N-glycosylation are boxed. The tine residues are indicated by a dot above the sequence. The underlined sequence corresponds to the N-terminal sequence determined on purified mpl ligand from porcine plasma.

  Figure 2 illustrates the procedure used for the incorporation assay of 3H-thymidine ligand mpl. To determine the presence of mpl ligand from various sources, mpl P Ba / F3 cells were deprived of IL-3 for 24 hours in a humidified incubator at 37 C in air supplemented with 5% CO 2. After IL-3 deprivation, the cells were plated in 96-cell culture dishes with and without diluted samples and cultured for 24 hours in a cell culture incubator. 20 μl of serum-free RPMI medium containing 1 μCi of 3H-thymidine was added to each well for the last 6-8 hours. The cells were then harvested on 96-well filter plates and washed with water. Counts were then carried out on the filters. Figure 3 shows the effect of pronase, DTT and heat on the ability of APP to stimulate proliferation of Ba / F3-mpl cells. For the pronase digestion of APP, Pronase (Boehringer Mannheim) or Beef Serum Albumin was coupled to Affl gel.

  GellO (Biorad) and incubated individually with APP for 18 hours at 37 ° C. Then the resins were removed by centrifugation and the supernatants were titrated The APP was also heated to 800C for 4 minutes or adjusted to 100 SM in DTT and then dialysed against PBS Figure 4 illustrates the elution of the ligand mpl activity of Phenyl-Toyopearl, Blue-Sepharose and

  Ultralink-mpl. Fractions 4-8 of the affinity column for mpl constituted the maximum activity fractions eluted from the column. Figure 5 shows the sodium dodecyl sulfate and polyacrylamide gel electrophoresis of eluted Ultralink-mpl fractions. 1 ml of acetone containing 1 mM HCl was added at 200 ° C. to 200 ° C. of each of fractions 2-8. After 3 hours at -20 ° C the samples were centrifuged and the resulting pellets were washed twice with acetone at -20 ° C. The acetone washed pellets were then dissolved in 30 μl of SDS solubilization buffer, adjusted to 100 μM DTT and the solution was heated at 900 ° C. for 5 minutes. The samples were then resolved on a 4-20% sodium dodecyl sulfate and polyacrylamide gel and the proteins were visualized by silver staining.

  Figure 6 shows the elution of mpl ligand activity in sodium dodecyl sulfate and polyacrylamide gel electrophoresis. Fraction 6 from the affinity column for mpl was resolved on SDS-polyacrylamide gel at 4-200C under non-reducing conditions. After electrophoresis, the gel was sliced into 12 equal regions and electroeluted as described in the examples. The electroeluted samples were dialysed in PBS and titrated at a dilution of 1/20. The Mr controls used to calibrate the gel consisted of Novex Mark 12 controls.

  Figure 7 illustrates the effect of spent APP in mpl ligand on human megakaryocytopoiesis. Depleted APP ligand mpl was prepared by passing 1 ml on a 1 ml affinity column for mpl (700 μg mpl-IgG / ml NHS-superose, Pharmacia). Human peripheral stem cell cultures were adjusted to 10% APP or 10% spent APP ligand mpl and were cultured for 12 days. The megakaryocytopoiesis was quantitatively estimated as described in the examples.

  Figure 8 illustrates the effect of mpl-IgG on stimulation of human megakaryocytopoiesis by APP. Human peripheral stem cell cultures were adjusted to 10% APP and cultured for 12 days. The 2nd and 4th days were initially added to mpl-IgG (0.5 g) or ANP-R-IgG (0.5 g). After 12 days, the megakaryocytopenia was quantitatively evaluated as described in the examples. The average of duplicate samples is plotted with the two actual data in parentheses. Figure 9 reproduces the two strands of a 390 base pair fragment of human genomic DNA encoding the mpl ligand. The deduced amino acid sequence of "exon 3" (SEQ ID N: 3), the coding sequence (SEQ ID NO: 4) and its complement (SEQ ID NO: 5) are represented. Figure 10 shows the deduced amino acid sequence of mature human mpl ligand (hML) (SEQ ID N: 6) and mature human erythropoietin (hEPO) (SEQ ID N: 7). The amino acid sequence predicted for the human mpl ligand is aligned with the sequence of human erythropoietin. Identical amino acids are boxed and spaces introduced for optimal alignment are indicated by dashes. Potential N-glycosylation sites are underlined by a solid line for hML and dotted lines for hEPO. The two important cysteines for the activity of erythropoietin are indicated by a reinforced point. Figure 11 illustrates the deduced amino acid sequence of mature human mpl ligand isoforms hML (SEQ ID N: 6), hML2 (SEQ ID NO: 8), hML3 (SEQ ID N: 9) and hML4 (SEQ ID N : 10). Identical amino acids are boxed and spaces introduced for optimal alignment are indicated by dashes.

  FIGS. 12A, 12B and 12C illustrate the effect of human mpl ligand on proliferation of Ba / F3-mpl (A) human in vitro megakaryocytopoiesis cells quantitatively estimated by radioactively labeled murine IgG monoclonal antibody. , specific for the megakaryocyte glycoprotein (GPIIbIIIa (B) and murine thrombopoiesis measured in a platelet rebound assay (C).) 293 cells were transfected by the CaPO4 method (C.Gorman in DNA Cloning: A New Approach 2). 143-190 [1985]) with the vector pRK5 alone, pRK5-hML or pRK5-ML1s3 overnight (the pRK5-MLls3 vector was generated by introduction of a termination codon after residue 153 of hML by PCR) The medium was then conditioned for 36 hours and titrated to evaluate the stimulation of Ba / F3-mpl cell proliferation as described in Example 1 (A) or in vitro human megakaryocytopoiesis (B). megakaryocytopoiesis has been estimated quantitatively using 125I-labeled murine IgG monoclonal antibody (HP1-1D) against the megacarocyte-specific glycoprotein, GPIIbIIIa as described by Grant et al., in Blood 69: 1334-1339 [1987] ). The effect of the partially purified ML (rML) recombinant on in vivo platelet production (C) was determined using the rebound thrombocytosis assay described by T. P. McDonald in Proc. Soc. Exp. iol. Med. 144: 1006-10012 (1973). Partially purified rML was prepared from 200 ml of conditioned medium containing the recombinant ML. The medium was passed through a 2 ml column of PBS-balanced Sepharose blue and the column was washed with PBS and eluted with PBS containing 2M urea and as much NaCl. The active fraction was dialyzed in PBS and adjusted to 1 mg / ml with endotoxin-free beef serum albumin. The sample contained less than one endotoxin unit / ml. Mice were injected with 64,000, 32,000 or 16,000 units of rML or excised alone. Each group consisted of six mice.

  The average deviation and standard deviation of each group is shown. The values of p were determined by a 2-train T-test comparing the medians. Figure 13 compares the effect of human mpl ligand isoforms with the variants in the Ba / F3-mpl cell proliferation assay. The assay focused on hML, a blank, hML2, hML3, hML (R153A, R154A) and hMLls3 at various dilutions as described in Example 1. Figures 14A, 14B and 14C show the deduced amino acid sequence (SEQ ID NO: 1) human mpl ligand (hML) or human TPO (hTPO) and the sequence encoding human genomic DNA (SEQ ID NO: 11). Nucleotides and amino acid residues are numbered at the beginning of each line.

  Figure 15 shows sodium dodecyl sulphate gel electrophoresis and purified 293-rhML3,32 polyacrylamide and purified 293-rhML, 53. Figure 16 shows the nucleotide sequence: sequence encoding the cDNA (SEQ ID N: 12) and the deduced amino acid sequence (SEQ ID N: 13) of the open reading frame of a murine ML isoform. This mature murine mpl ligand isoform contains 331 amino acid residues, four less than the presumed full length mML, and is therefore designated mML2. Nucleotides are numbered at the beginning of each line. The amino acid residues are numbered above the sequence from Ser 1. Potential N-glycosylation sites are underlined. Cysteine residues are indicated by a dot above the sequence.

  Figure 17 shows the cDNA sequence (SEQ ID NO: 14) and the predicted protein sequence (SEQ ID NO: 15) of this murine ML isoform (mML). Nucleotides are numbered at the beginning of each line. Amino acid residues are numbered above the sequence from Ser 1. This mature murine mpl ligand isoform contains 335 amino acid residues and is considered to be full-length mpl ligand, designated as MML. The signal sequence is indicated by a dashed underline and the presumed cleavage point is indicated by an arrow. The untranslated regions 5 'and 3' are indicated by lowercase letters. The two deletions observed as a result of alternate splicing (mML2 and mML3) are underlined. The four cysteine residues are indicated by a dot. -The seven potential sites of N-glycosylation are framed. Figure 18 compares the deduced amino acid sequence of the human ML isoform hML3 (SEQ ID NO: 9) and a murine ML isoform designated mML3 (SEQ ID NO: 16). The amino acid sequence predicted for the human mpl ligand is aligned with the mpl ligand murine sequence. The identical amino acids are boxed and the spaces introduced for optimal alignment are indicated by dashes. Amino acids are numbered at the beginning of each line. Figure 19 compares predicted amino acid sequences of mature ML (SEQ ID NO: 17), porcine (SEQ ID NO: 18) and human (SEQ ID NO: 6) isoforms.

  The amino acid sequences are aligned, with the introduction of dashed spaces, for optimal alignment. Amino acids are numbered at the beginning of each line with a frame of identical residues. Potential N-glycosylation sites are indicated by a shaded box and cysteine residues are designated by a dot. The conserved diamino acid motif that has a potential protease cleavage site is underlined. The deletion of four amino acids, the appearance of which is observed for the three species (ML2), is indicated by a frame in reinforced lines. Figure 20 shows the cDNA sequence (SEQ ID N: 19) and the predicted mature protein sequence (SEQ ID N: 18) of a porcine ML isoform (pML). This porcine mpl ligand isoform contains 332 amino acid residues and is considered to be the full length porcine ligand, designated pML. Nucleotides are numbered at the beginning of each line. Amino acid residues are numbered above the sequence from Ser 1.

  Figure 21 shows the cDNA sequence (SEQ ID: 20) and the predicted mature protein sequence (SEQ ID N: 21) of a porcine ML isoform (pML2). This porcine mpl ligand isoform contains 328 amino acid residues and is a deletion form of four residues of the full length ppl ligand mpl designated pML2. Nucleotides are numbered at the beginning of each line. The amino acid residues are numbered above the sequence from Ser 1. Figure 22 compares the deduced amino acid sequence of the full length pork isoform ML, pML (SEQ ID N: 18) and an ML isoform of pork designated pML2 (SEC ID N: 21). The predicted amino acid sequence for pML is aligned with the pML2 sequence. Identical amino acids are boxed and spaces introduced for optimal alignment are indicated by dashes. Amino acids are numbered at the beginning of each line.

  Figure 23 shows the relevant characteristics of the plasmid pSV15.ID.LL.MLORF ("full length" or TP0332) used for the transfection of CHO-DP12 host cells for the production of CHO-rhTPO332. Figure 24 shows relevant features of plasmid pSV15.ID.LL.MLEPO-D ("truncated" or TPO153) used for transfection of CHO-DP12 host cells for the production of CHO-rhTPOs3. Figures 25A, 25B and 25C show the effect of E. coli-rhTPO (.. t-153 on platelets (A), red blood cells (B) and white blood cells (C) in normal mice. injected to two groups of 6 female C57 B6 mice, once a day, PBS buffer or 0.3 Mg of E. coli-rhTPO (, t1 1s3) (100 M1 subcutaneously). From day 3 to day 7, 40 μl of blood were taken from the orbital sinus, which was immediately diluted in 10 ml of commercial diluent and whole blood counts were performed on a Serrono Baker 9018 hematology analyzer. are represented by the means, the standard deviation of the mean.

  FIGS. 26A, 26B and 26C show the effect produced by E.coli-rhTPO (, tl, 53) on platelets (A), red blood cells (B) and white blood cells (C) in irradiated mice. a sublethal dose. Two groups of 10 female C57 B6 mice were exposed to sublethal irradiation with 750 cGy gamma rays emitted from a 137Cs source and injected daily with PBS buffer or 3.0 Lg E. coli-rhTPO 3. lt-153 (100 M1 subcutaneously). Initially then at intervals, 40 ml of blood were taken from the orbital sinus. This blood was immediately diluted in 10 ml of commercial diluent and whole blood counts were performed on a hematology analyzer.

  Serrono Baker 9018. The results are presented as mean standard deviations from the mean. Figures 27A, 27B and 27C show the effect of CHO-rhTPO332 on (A) platelets (thrombocytes), (B) red blood cells (erythrocytes) and (c) white blood cells (leukocytes) in normal mice. Two groups of six female C57 B6 mice, ampon PBS or 0.3 g of CHO-rhTPO332 (100 μg, subcutaneously) were injected once a day. Initially and from the 3rd to the 7th day, 40 g1 of blood were taken by the orbital sinus. This blood was immediately diluted in 10 ml of commercial diluent and whole blood counts were performed on a Serrono Baker 9018 hematology analyzer. The results are presented as mean standard deviation from average.

  Figure 28 shows dose response curves for various forms of rhTPO from various cell lines. The dose response curves were constructed for rhTPO from the following cell lines: hTPO332 from CHO (complete length of Chinese hamster ovary cells); hTPOm-153 (truncated form derived from E. coli with an N-terminal methionine group); hTPO332 (full length TPO from 293 human cells); Met-less 155 E. coli (truncated form [rhTPO5ss] without the terminal methionine of E. coli). Groups of 6 female C57B6 mice received a group-dependent daily injection of rhTPO for 7 days. Each day, 40 g of blood were taken from the orbital sinus for whole blood counts. The results presented above are the maximal effects observed with the various treatments and, with the exception of (met 153 E. coli), these effects appeared at the 7th day of treatment. In the group "met 153 E. coli" mentioned above, the maximum effect was observed on the 5th day. The results are presented as mean standard deviations from the mean.

  Figure 29 shows dose response curves that compare the activity of whole and "pinched" forms of rhTPO produced in CHO cells with the truncated form derived from E. coli. Groups of 6 female C57B6 mice, 0.3 Mg rhTPO of various types were injected daily. From the 2nd to the 7th day, 40 μl of blood were taken from the orbital sinus for whole blood counts. Treatment groups were: TPO, 53, rogue form of TPO derived from E. coli; TPO332 (mixed fraction) Whole TPO containing approximately 80-90% of the whole form and 10-20% of the pinched forms; TPO332 (fraction 30K) = purified pinch fraction from the original Nmixten preparation; TP0332 (70K fraction) = purified whole TPO fraction from the original "original" preparation The results are presented as averages + the standard deviation from the mean Figure 30 is a sketch showing the KIRA test ELISA for the assay of TPO This figure shows the MPL / Rse.gD chimera and the corresponding parts of the receivers from which it is derived as well as the final assembly (right part of the figure) and a schematic diagram (left side of Figure) showing the corresponding steps of the test.

  Figure 31 is a block diagram of the KIRA-ELISA assay showing each step of the process. Figures 32A-32L show the nucleotide sequence (SEQ ID N: 22) of the expression vector pSV117.ID.LL used for the expression of Rse.gD in Example 17. Figure 33 is a schematic representation of the preparation of plasmid pMP1. Figure 34 is a schematic representation of the preparation of plasmid pMP21. Figure 35 is a schematic representation of the preparation of plasmid pMP151. Figure 36 is a schematic representation of the preparation of plasmid pMP202. Figure 37 is a schematic representation of the preparation of plasmid pMP172. Figure 38 is a schematic representation of the preparation of plasmid pMP210.

  Figure 39 is a table of the five best TPO expression clones of the library of plasmid pMP210 SEQ ID NO: 23, 24, 25, 26, 27 and 28). Figure 40 is a schematic representation of the preparation of plasmid pMP41. Figure 41 is a schematic representation of the preparation of plasmid pMP57. Figure 42 is a schematic representation of the preparation of plasmid pMP251. Fig. 43 is a calibration curve for determining the concentration of TPO.

  DETAILED DESCRIPTION OF THE INVENTION I. Definitions In general, the following terms or expressions have the definition indicated, whenever used herein. A chaotropic agent refers to a compound which, in aqueous solution and at suitable concentrations, can cause a change in the spatial configuration or conformation of a protein by at least partial disruption of the forces responsible for maintaining the secondary and tertiary normal structure. of the protein. These compounds include, for example, urea, guanidine hydrochloride and sodium thiocyanate. High concentrations, usually 4-9 M, of these compounds are normally required to exert the conformational effect on proteins.

  The term "cytokineu" is a general term used to refer to proteins released by a population of cells that act on another cell as intercellular mediators.Examples of these cytokines are the traditional lymphokines, monokines and polypeptide hormones. number of cytokines, growth hormone, insulin-like growth factors, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein-like hormones such as follicle stimulating hormone (FSH), thyrotropin (TSH) and luteinizing hormone (LH) , hematopoietic growth factor, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α (TNF-α and TNF-O) , anti-Mullerian hormone, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, nerve growth factors such as NGF-β, the factor platelet growth, transforming growth factors (TGF) such as TGF-α and TGF-β, insulin-like growth factors I and II, erythropoietin (EPO), osteoinducer factors, interferons such as interferon-a, interferon- and interferon-T, colony stimulating factors (CSF) such as macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7. , IL-8, IL-9, IL-11, IL-12 and other polypeptide factors comprising

  LIF, SCF and ligand-kit factors. The foregoing terms and expressions used herein encompass proteins of natural origin or from recombinant cell culture. Similarly, the terms and expressions are intended to cover biologically active equivalents, for example whose amino acid sequence differs from one or more amino acids or the type or degree of glycosylation. The terms "mpl ligand" or "mpl ligand polypeptide", "ML", "thrombopoietin or" TPO "are used interchangeably herein and include any polypeptide that has the property of binding to mpl, A member of the cytokine receptor superfamily, and having a biological property of ML as defined below, an example of biological property is the ability to stimulate the incorporation of labeled nucleotides (e.g., 3H-thymidine) into DNA. of Ba / F3 cells dependent on IL-3

  transfected with human mpl P. Another example of biological property is the ability to stimulate the incorporation of 35S into circulating platelets in a rebound assay involving mouse platelets. This definition covers the polypeptide isolated from a source of mpl ligand such as aplastic pig plasma described herein or from another source, for example from another animal species, including the human species, or prepared by recombinant or synthetic methods, and covers variants, including functional derivatives, fragments, alleles, isoforms and the like. A "mpl ligand fragment" or "TPO fragment" is a portion of a mature natural full mpl ligand or TPO ligand sequence, one or more amino acid residues or carbohydrate moieties removed. The deleted amino acid residue (s) can be anywhere in the peptide including at the N-terminal or C-terminal end or in the internal position. The fragment shares at least one biological property with the ligand mpl.

  The mpl ligand fragments normally have a consecutive sequence of at least 10, 15, 20, 25, 30 or 40 amino acid residues which are identical to the sequences of the isolated mpl ligand of a mammal, including the isolated plasma ligand of aplastic pig or the human or murine ligand, in particular its EPO domain. Representative examples of N-terminal fragments are hMLIs3 or TPO (Met''1-153). "Mpl ligand variants" or "mpl ligand sequence variants" as used herein refer to biologically active mpl ligands as defined below having less than 100% sequence identity with the mpl ligand. mpl isolated from a recombinant cell culture or porcine aplastic plasma or human ligand having the deduced sequence shown in Figure 1 (SEQ ID N: 1).

  Typically, a biologically active mpl ligand variant has an amino acid sequence having at least about 70% amino acid sequence identity with the mpl ligand isolated from aplastic pig plasma or the mature murine or human ligand or fragments of this ligand (see Figure 1 [SEQ ID NO: 1]), preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about at least about 90% and especially at least about 95%. A "mpl chimeric ligand" is a polypeptide comprising an entire mpl ligand or one or more fragments thereof ligated or bound to a second heterologous polypeptide or to one or more of its fragments. The chimera shares at least one biological property with the ligand mpl.

  The second polypeptide is normally a cytokine, an immunoglobulin or a corresponding fragment. The terms "isolated mpl ligand", "highly purified mpl ligand" and "substantially homogeneous mpl ligand" are used interchangeably and refer to an mpl ligand that has been obtained by purification from a source of such ligand or that has been prepared by recombinant or synthetic methods and which is sufficiently free from other peptides or proteins (1) to obtain at least 15 and preferably 20 amino acid residues of the N-terminus or of a sequence internal amino acid using a centrifugal trough sequencing apparatus or the best commercially available amino acid sequencing apparatus or as modified by methods disclosed at the time of the present application, or (2) to acquire homogeneity by sodium dodecyl sulfate and polyacrylamide gel electrophoresis under non-reducing or reducing conditions using Coomassie blue or preferably For the purposes of this specification, homogeneity means less than about 5% contamination by proteins from other sources.

  The term "biological property used in connection with" ligan mpl "or" isolated mpl ligand "means that this ligand has in vivo thrombopoletic or effector function or antigenic function or activity that is caused or exerted directly or indirectly by an mpl ligand (in its natural or denatured conformation) or a corresponding fragment. Effector functions include mpl binding and any carrier binding activity, agonism or antagonism to mpl, including proliferative signal transduction including replication, regulatory function DNA modulation, modulation of the biological activity of other cytokines, activation of receptors (particularly cytokines), deactivation, up- or down-regulation, growth or differentiation of cells, etc. An antigenic function means the possession of an epitope or antigenic site that is capable of cross-reacting with antibodies against the natural mpl ligand. The main antigenic function of a ligand mpl polypeptide is that it attaches with an affinity of at least

  minus about 10G 1 / mole to an antibody against mpl ligand isolated from aplastic pork plasma. Ordinarily, the polypeptide binds with an affinity of at least about 107 l / mol. Preferably, the mpl ligand polypeptide having antigenic activity is a polypeptide that binds to an mp1 ligand antibody having one of the effector functions described above. The antibodies used to define a "biological activity" are polyclonal rabbit antibodies developed by formulation of the mpl ligand isolated from a recombinant cell culture or aplastic pig plasma in Freund's complete adjuvant, subcutaneous injection of the formulation and enhancement of the immune response by intraperitoneal injection of the formulation until the titer of the antibody against ligand mpl is level.

  The term "biologically active" used in conjunction with "mpl ligand" or "isolated mpl ligand" refers to a mpl ligand or polypeptide that is endowed with thrombopoietic activity and that shares an effector function of mpl ligand isolated from plasma. aplastic pig or expressed in a recombinant cell culture described herein. A known principal effector function of the mpl ligand or polypeptide referred to herein is mpl binding and stimulation of the incorporation of labeled nucleotides (3H-thymidine) into Ba / F3 cell DNA under the control of IL-3, transfected with human mpl P. Another known effector function of the mpl or polypeptide ligand referred to herein is the ability to stimulate 35S incorporation into circulating platelets in the platelet bounce assay in the mouse. The ability to stimulate megakaryapyolysis in vitro is yet another known effector function of the mpl ligand, which can be expressed quantitatively by the use of a radioactively labeled monoclonal antibody specific for the megakaryocytic glycoprotein GPIIbIIIa.

  The term "percent amino acid sequence identity" used for the mpl ligand sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the residues in the sequence. mpl ligand sequence isolated from aplastic pig plasma or in the murine or human ligand having the deduced amino acid sequence shown in Figure 1 (SEQ ID N: 1), after sequence alignment and introduction, if necessary, of spaces to obtain the maximum percentage of sequence identity, without considering any conservative substitution as part of the sequence identity. None of the N-terminal, C-terminal, or internal extensions, deletions, or insertions in the mpl ligand sequence should be interpreted as affecting sequence identity or homology. Thus, examples of biologically active ligand polypeptides considered to have identical sequences include: prepro-mpl ligand, pro-mpl ligand and mature mpl ligand.

  "Mpl ligand microsequencing" can be accomplished by any suitable standard procedure provided that this procedure is sufficiently sensitive. In such a method, a highly purified polypeptide from sodium dodecyl sulfate gels or a final high performance liquid chromatography step is sequenced directly by automated degradation of Edman (phenyl isothiocyanate) using a Applied Biosystems model 470A gas phase sequencing equipped with 120A Phenylthiohydantoin Amino Acid Analyzer (PTH). In addition, mpl ligand fragments prepared by chemical digestion (eg CNBr, hydroxylamine, 2-nitro-5-thiocyanobenzoate) or enzymatic (eg trypsin, clostripain, staphylococcal protease) followed by purification of the fragments (eg HPLC) can be sequenced in a similar way. The amino acids of PTH are analyzed using the ChromPerfect data system (Justice Innovations, Palo Alto, CA). Sequence interpretation is performed on a Digital Equipment Co. VAX 11/785 computer as described by Henzel et al. in J. Chromatography, 404: 41-52 [1987]. Where appropriate, aliquots of the CHLP moieties may be subjected to 5-20% gel electrophoresis of sodium dodecyl sulfate and polyacrylamide with electrotransport to a PVDF membrane (ProBlott, AIB, Foster City, CA) and staining. Coomassie brilliant blue (Matsurdiara, J. Biol Chem., 262: 10035-10038 [1987]). A specific protein identified by the stain is detached from the imprint by xcision and N-terminal sequencing is performed with the gas phase sequencing apparatus described above. For internal protein sequences, the fractions of

  HPLC were dried under vacuum (SpeedVac), resuspended in appropriate buffers and digested with cyanogen bromide, Lys-specific Lys-C enzyme (Vako Chemis, Richmond, VA), or with it. -N (Boehringer Mannheim, Indianapolis, IN). After digestion, the resulting peptides are sequenced as a mixture and after resolution by C4-column HPLC they are developed with a propanol gradient in 0.1% TFA prior to gas phase sequencing.

  Thrombocytopenia "is defined as platelet counts of less than 150 x 10 'per liter of blood." Thrombopoietic activity "is defined as a biological activity which consists in accelerating the proliferation, differentiation and / or maturation of megakaryocytes or their precursors to the platelet-producing form of these cells This activity can be measured in various assays including the in vivo mouse platelet bounce synthesis assay, the antigen induction assay platelet surface area as measured by an anti-platelet immunoassay (anti-GPIIbIIIa) for the human leukemic magacaryoblastic cell line (CMK) and the induction of polyploidization in a megakaryoblastic cell line (DAMI). "Thrombopoietin" (TPO) is defined as a compound with thrombopolic activity or being able to increase serum platelets in a mammal The TPO is advantageously capable of increasing endogenous platelet counts by at least 10%, preferably 50%, especially capable of elevating platelet levels in a human to over 150%. x 109 per liter of blood.

  The term "mpl solute ligand nucleic acid" refers to RNA or DNA containing more than 16 and preferably 20 or more nucleotide bases in sequence encoding the biologically active mpl ligand or fragment thereof, which is complementary to RNA or DNA, which hybridizes to RNA or DNA and remains stably bound under moderate to severe conditions. This RNA or DNA is devoid of at least one contaminating nucleic acid with which it is normally associated in the natural source, and preferably substantially free of any other mammalian RNA or DNA. The expression lacking at least one contaminating nucleic acid with which it is normally associated "encompasses the case where the nucleic acid is present in the natural source or cell, but is in a different chromosomal site or is otherwise flanked by nucleic acid sequences that are not normally encountered in the source cell An example of isolated mpl ligand nucleic acid is RNA or

  DNA that encodes a biologically active mpl ligand sharing at least 75% sequence identity, preferably at least 80%, more preferably at least 85%, more preferably 90% and preferably 95% sequence identity with the human, murine or porcine mpl ligand.

  The term "regulatory sequences" refers to DNA sequences necessary to express a coding sequence that is actively linked in a particular host organism. Suitable regulatory sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, a ribosomal binding site and, optionally, other sequences that are not yet well understood. It is known that eukaryotic cells utilize promoters, polyadenylation signals and activators.

  The term "actively linked" to nucleic acids means that the nucleic acids are placed in a functional relationship with another amino acid sequence. For example, the DNA of a presequence or secretory leader is actively bound to the DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or activator is actively linked to a coding sequence if it affects transcription of the sequence; or a ribosomal binding site is actively linked to a coding sequence if it is positioned to facilitate translation. Generally, the term "actively bound" means that the DNA sequences that are bound are contiguous and, in the case of a secretory leader, contiguous and in the reading phase. However, activators do not have to be contiguous. Linkage is accomplished by ligation at suitable restriction sites. If such sites do not exist, synthetic oligonucleotide linkers or oligonucleotide linkers are used in accordance with conventional practice.

  The term "exogenous", when referring to an element, means a nucleic acid sequence that is foreign to the cell, or homologous to the cell but in a position in the host cell nucleic acid. element is not usually found. The term "cell", the terms "cell line" and "cell culture" are used interchangeably herein and these designations include any offspring of a cell or cell line. terms and expressions such as "transformants" and "transformed cells" refer to the main cell concerned and crops derived therefrom, irrespective of the number of transfers.It should also be noted that any offspring can not be exactly identical. in terms of DNA content, due to deliberate or inadvertent mutations, the descendance of mutants having the same function or biological activity as that detected in the initially transformed ellule is included. different designations, this is clear from the context.

  "Plasmids" are self-replicating circular DNA molecules having independent origins of replication and are referred to herein as a lowercase "p" preceded and / or followed by capital letters and / or numbers. The starting plasmids referred to herein are commercially available, publicly available on an unlimited basis, or can be assembled from available plasmids according to published methods. In addition, other equivalent plasmids are known in the art and are obvious to those skilled in the art.

  The term "restriction enzyme digestion" for DNA means catalytic cleavage of internal phosphodiester bonds of DNA with an enzyme that acts only in certain locations or sites in the DNA sequence. These enzymes are called "restriction endonucleases". Each restriction endonuclease recognizes a specific DNA sequence called a "restriction site," which exhibits double symmetry. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements as established by the enzyme suppliers are used. Restriction enzymes are commonly referred to as abbreviations consisting of a capital letter followed by other letters representing the microorganism from which each restriction enzyme was initially obtained, and then a number designating the particular enzyme. . In general, about 1 μg of plasmid or DNA fragment is used with about 1-2 units of enzyme in about 20 μl of buffer solution. Buffers and amounts of substrate suitable for particular restriction enzymes are specified by the manufacturer. Incubation of about 1 hour at 370C is usually used, but incubation may vary according to the instructions of the supplier. After incubation, the protein or polypeptide

  tide is removed by phenol and chloroform extraction and the digested nucleic acid is recovered from the aqueous fraction by ethanol precipitation. Restriction enzyme digestion may be followed by hydrolysis by a bacterial alkaline phosphatase of the 5 'terminal phosphates to prevent the two cleaved ends of a DNA fragment from "taking a circular shape" or forming a closed loop that would prevent the insertion of another DNA fragment at the site of

  restriction. Unless otherwise specified, digestion of plasmids is not followed by dephosphorylation of the 5 'terminus. Reactive dephosphorylation methods are conventional, as described in Sections 1.56-1.61 of Sambrook et al. entitled Molecular Cloning: A Laboratory Manual [New York: Cold Spring Harbor Laboratory Press, 1989]. "Separation or" isolation "of a given fragment of restriction digest DNA means separation of the polyacrylamide or agarose gel digest by electrophoresis, identification of the fragment. interesting by comparison of its mobility with that of DNA fragments marker of known molecular weight, removal of the gel section containing the desired fragment and the separation of the gel from the DNA This method is generally known See, for example Lawn et al., Nucleic Acids Res., 9: 6103-6114 [1981], and Goeddel et al., Nucleic Acids Res., 8: 4057 [1980].

  "Southern analysis" or "Southern blot analysis" is a method by which the presence of DNA sequences in a restriction endonuclease digest of DNA or a composition containing DNA is confirmed by hybridization. to a known labeled oligonucleotide or a DNA fragment. Southern analysis normally involves the electrophoretic separation of DNA digests from agarose gels, denaturation of the DNA after electrophoretic separation and transfer of DNA to a nitrocellulose membrane support, nylon or another suitable membrane for analysis with a radioactivity-labeled, biotin-labeled, or enzyme-labeled as described in Sections 9.37-9.52 of Sambrook et al. , supra. The term "Northern analysis" or "Northern blot analysis" refers to a method used to identify RNA sequences that hybridize to a known probe such as an oligonucleotide, a DNA fragment, the cDNA or a fragment. cDNA, or an RNA fragment. The probe is labeled with a radioisotope such as 32p, or by binding to biotin, or with an enzyme. The RNA to be analyzed is usually separated by electrophoresis on an agarose or polyacrylamide gel, transferred to a membrane of nitrocellulose, nylon or other suitable material and hybridized with the probe, by conventional techniques. known in the prior art such as those described in Sections 7.39-7.52 of Sambrook et al., supra. The term "ligation" refers to the process of forming photodiester bonds between two nucleic acid fragments. For ligation of both fragments, the ends of the fragments must be compatible with each other. In some cases, the ends are directly compatible with endonuclease digestion. However, it may be necessary to first convert the staggered ends commonly produced after endonuclease digestion into blunt ends to make them compatible for ligation. To make the ends blunt, the DNA is treated in a suitable buffer for at least 15 minutes at 15 ° C. with about 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of the four deoxyribonucleotide triphosphates. .

  The DNA is then purified by phenol and chloroform extraction and ethanol precipitation. The DNA fragments that must be welded together are dissolved in approximately equimolar amounts. The solution also contains ATP, ligase buffer and ligase such as T4 DNA ligase at about 10 units per 0.5 g of DNA. If the DNA is to be ligated into a vector, the vector is first linearized by digestion with the endonuclease or the appropriate restriction endonucleases. The linearized fragment is then treated with bacterial alkaline phosphatase or calf intestinal phosphatase to prevent self-ligation during the ligation step.

  The "preparation" of DNA from cells means the isolation of plasmid DNA from a host cell culture. Commonly used methods for the preparation of DNA are the large and small scale plasmid preparations described in sections 1.25-1.33 of Sambrook et al., Supra. After the preparation of DNA, it can be purified by methods well known in the art as described in paragraph 1.40 of Sambrook et al., Supra. "Oligonucleotides" are short-lived, single-helical or double-helical polydeoxynucleotides that are synthesized chemically by known methods (for example, phosphotriester, phosphite or phosphoramidite chemistry using solid phase techniques). as described in European Patent No. 266,032 published on May 4, 1988 or via deoxynucleoside-

  Intermediate H-phosphonates as described by Froehler et al., In Nucl. Acids Res., 14: 5399-5407 [1986]). Other methods include the polymerase chain reaction as defined below and other methods of self priming and oligonucleotide syntheses on solid supports. All these processes are described by Engels et al., Agnew. Chem. Int., English language edition, 28: 716-734 (1989). These methods are used if the entire nucleic acid sequence of the gene is known or if the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, if the amino acid target sequence is known, potential nucleic acid sequences can be derived using known and preferred coding residues for each amino acid residue. The oligonucleotides are then purified on polyacrylamide gels. "Polymerase chain reaction" or "PCR" refers to a procedure or technique in which small amounts of a specific piece of nucleic acid, RNA and / or DNA are amplified as described in the US Pat. U.S. 4,683,195 filed July 28, 1987. In general, information on the sequence from the ends of the region of interest or beyond requires to be available, for example, oligonucleotide primers. can be designed, these primers are identi-

  or similar, in their sequence, to opposite strands of the matrix to be amplified. The terminal nucleotides of the two primers may coincide with the ends of the amplified material. The PCR reaction can be used to amplify specific RNA sequences, specific DNA sequences, from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage sequences or plasmids, etc. See generally Mullis et al., Cold Spring Harbor Symp. As. Biol., 51: 263 [1987]; published under the direction of

  Erlich, PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a method of reacting a nucleic acid polymerase to amplify a nucleic acid test sample comprising use of a nucleic acid known as a primer and a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid.

  "Strict conditions" are conditions which (1) use a low ionic strength and a high temperature for washing, for example 0.015 M NaCl / 0.0015 M sodium citrate / 0.1 * NaDodSO4 ( SDS) at 50 ° C, or (2) during the hybridization use a denaturing agent such as formamide, for example 50% formamide (v / v) with 0.1% bovine serum albumin / 0.1 % Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl and 75 mM sodium citrate at 42 C. Another example is the use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x solution of Denhardt, ultrasound-treated salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate at 420 ° C, with washes at 420 ° C in 0.2 × SSC and SDS at 0.1%. Moderately rigorous conditions are described by Sambrook et al., Supra, and include the use of a less severe washing solution and hybridization conditions (eg temperature, ionic strength, and percentage of SDS). As described above, an example of moderately stringent conditions includes conditions such as overnight incubation at 37 ° C. in a solution comprising 20% formamide, 5X SSC (150 mM NaCl) mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 X Denhardt's solution, 10% dextran sulfate, and 4 I / ml denatured sheared salmon sperm DNA. followed by washing the filters in 1 × SSC at about 37.degree.

  C. Those skilled in the art will recognize how to adjust the temperature, ionic strength, etc., as necessary to take into account factors such as probe length, etc. The terms "antibodies" (Abs) and "immunoglobulins" (Igs) refer to glycoproteins having the same structural characteristics While antibodies show binding specificity to a specific antigen, immunoglobulins include both antibodies and antibodies. Other antibody-like molecules lacking specificity for antigens, polypeptides of the latter type are produced, for example, at low levels by the lymphatic system and at high levels by myelomas. are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is bound to a heavy chain by a single bond

  covalently disulfide while the number of disulfide bonds varies between the heavy chains of different isotypes of immunoglobulins. Each heavy and light chain also has regularly spaced intracellular disulfide bridges. Each heavy chain has at one of its ends a variable domain (V.) followed by a certain number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are considered to form an interface between the variable domains of light and heavy chains (Clothia et al., J. Mol Biol., 186: 651-663 (1985), Novotny and Haber Proc Natl Acad Sci USA 82: 4592-4596 [1985]).

  The term "variable" refers to the fact that some portions of the variable domains differ considerably in their sequence among the antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed across all antibody variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light chain and heavy chain variable domains. The most heavily conserved portions of the variable domains are called framework (FR). The natural heavy and light chain variable domains each comprise four FR regions, greatly adopting the configuration of a Y-sheet, connected by three CDR regions which form loops connecting the sheet-like structure and which in some cases are part of this structure. structure. The CDR regions in each chain are kept close to each other by the FR regions and, with the CDRs of the other chain, they contribute to the formation of the antibody antigen binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD [1987]). Constant domains are not directly involved in the binding of an antibody to an antigen, but have various effector functions, for example the participation of the antibody in antibody-dependent cellular toxicity.

  The papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a unique antigen-binding site, and a residual "Fc" fragment whose name reflects its ability to to crystallize easily. Treatment with pepsin gives an F (ab ') 2 fragment which has two antigen-combining sites and is still able to cross-link an antigen.

  The term "Fv" refers to the minimal antibody fragment that contains a complete site for recognition and binding of an antigen. This region consists of a variable domain dimer with a heavy chain and a light chain in close, non-covalent association. It is in this configuration that the three CDR regions of each variable domain act together to define an antibody binding site on the surface of the V.-VL dimer. The six CDR regions collectively confer the specificity of antigen binding to the antibody. However, even a single variable domain (or half of a Fv fragment comprising only three CDR regions specific for an antigen) has the ability to recognize and read an antigen, although with a lower affinity than the entire binding site.

  The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab 'fragments differ from Fab fragments by the addition of a small number of residues at the carboxy terminus of the CH1 domain of the heavy chain comprising one or more cysteines from the antibody binding region. The term Fab'-SH is used here to denote a Fab 'fragment in which the constant domain cysteine residue (s) carry a free thiol group. F (ab ') 2 antibody fragments were initially produced as pairs of Fab' fragments showing between them cysteines as linkers. Other chemical couplings of antibody fragments are also known.

  The "light chains" of antibodies (immunoglobulins) from all vertebrate species can be attributed to one of two distinctly distinct types, called kappa and lambda (A), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and many of them can still be ivised into subclasses (isotypes), eg IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The constant domains of the heavy chain which correspond to the different classes of immunoglobulins are designated respectively by the letters a, delta, epsilon, y and p. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

  The term "antibody" is used in its broadest sense and covers especially simple monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions of polyepitopic specificity, as well as antibody fragments. (eg Fab, F (ab ') 2 and Fv) as long as they exhibit the desired biological activity. The term "monoclonal antibody" as used herein refers to an antibody obtained from a predominantly homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible natural mutations that may be present in secondary quantities. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations which normally comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The epithet "monoclonal" indicates the character of

  the antibody, namely, that it is obtained from a substantially homogeneous population of antibodies, and it should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler & Milstein, in Nature, 256: 495 (1975), or they can be prepared by recombinant methods of DNA (see, for example, the United States patent

  4,816,567 (Cabilly et al.)). The monoclonal antibodies referred to herein include especially "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical to or homologous to the corresponding sequences in antibodies derived from them. a particular species or belonging to a particular class or subclass of antibodies, while the rest of the chain or chains is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of these antibodies, as long as they show the desired biological activity (U.S. Patent No. 4,816,567 (Cabilly et al.); and Morrison et al., Proc Natl Acad Sci USA, 81: 6851-6855 [1984]).

  "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab ', F (ab') 2, or other linker sequencing antibody antigens) which contain a minimal sequence derived from non-human immunoglobulin The humanized antibodies are for the most part human immunoglobulins (antibody of a recipient) in which residues of a complementary critical region (CDR) of the The recipient is replaced by residues of a CDR region of a non-human species (donor antibody) such as mouse, rat or rabbit with the desired specificity, affinity and capacity. Human immunoglobulin may be replaced by corresponding non-human residues In addition, a humanized antibody may comprise residues which are not found in the recipient's antibody or in the sequestrants. imported from the regions

  CDR or framing. These modifications are made to better refine and optimize the performance of the antibodies. In general, the humanized antibody comprises substantially all of at least one and for example two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or almost all the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also comprises at least a portion of a constant immunoglobulin (Fc) region, normally that of a human immunoglobulin. For more details, see:

  Jones et al., Nature, 321: 522-525 [1986], Reichman et al., Nature, 332: 323-329 [1988]; and Presta, Curt. Op. Struct. Biol., 2: 593-596 [1992]. The term "non-immunogenic in a human" means that upon contact with the polypeptide in a pharmaceutically acceptable carrier and in a therapeutically effective amount with the appropriate tissue of a human, no sensitivity state or resistance to the polypeptide can be demonstrated during the second administration of the polypeptide after a suitable latency period (for example 8 to 14 days). II. Preferred Embodiments of the Invention Preferred polypeptides of the present invention are predominantly homogeneous polypeptides, referred to as mpl ligands or mpl or thrombopoietin (TPO) ligand (s), which have the property of binding to a mpl , representing the cytokine receptor superfamily, and having the biological property of stimulating the incorporation of labeled nucleotides (3H-thymidine) into the DNA of cells

  Ba / F3 under the control of IL-3 transfected with a human mpl P. More preferred mpl ligands are isolated from mammalian proteins endowed with hematopolytic activity, especially megakaryocytopolytic or thrombocytopolytic, namely capable of stimulating proliferation, maturation and / or differentiation of immature or immature megakaryocytes. from their predecessors into the mature form producing platelets. Highly preferred polypeptides according to the invention are human mpl ligands, including their fragments endowed with hematopoietic, megakaryo-cytopoietic or thrombopoietic activity. If necessary, these human mpl ligands lack glycosylation. Other preferred human mpl ligands are the "EPO domain" of hML called hML1s3 or hTPO1s3, a truncated form of hML called hML24s or hTPO24s and the mature whole polypeptide having the amino acid sequence shown in Fig. 1 (SEQ ID N: 1), called hML, hML332 or hTPO332 and the biologically active substitution variant, hML (R153A, R1S4A).

  Other preferred polypeptides according to the present invention are mpl ligand variants endowed with biological or immunological activity selected from hML2, hML3, hML4, mML, mML2, mML3, pML and pML2. Preferred optional polypeptides of the present invention are biologically active mpl ligand variants that have an amino acid sequence having at least 70% amino acid sequence identity to human mpl ligand (see FIG. 1 [SEQ ID] N: 1]), the murine mpl ligand (see FIG. 16 [SEQ ID NO: 12 and 131), the recombinant porcine mpl ligand (see FIG. 19 [SEQ ID N: 181]) or the porcine mpl ligand isolated from pig plasma aplastic, advantageously at least 75%, more preferably at least

  80 ', more preferably at least 85%, preferably at least 90% and especially at least 95%. The isolated mpl ligand of aplastic pig plasma has the following characteristics: (1) The partially purified ligand is eluted in a gel filtration column operation in PBS, PBS containing 0.1% SDS or PBS containing 4M MgCl2 with a Mr value of 60,000 to 70,000; (2) The activity of the ligand is destroyed by the pronase; (3) The ligand is stable at low pH (2.5) 0.1% SDS and 2M urea; (4) The ligand is a glycoprotein, based on its binding to various lectin columns; (5) The highly purified ligand is eluted in electrophoresis without reduction on sodium dodecyl sulphate and polyacrylamide gel with a Mr value of 25,000- 35,000. Smaller amounts of activity are also eluted with a Mr value of the order of 18,000-22,000 and 60,000; (6) The highly purified ligand resolves in sodium dodecyl sulfate and polyacrylamide gel electrophoresis with reduction to a doublet with Mr values of 28,000 and 31,000; (7) The amino-terminal sequence of 18,000-22,000, 28,000 and 31,000 bands is the same -

  SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO: 29); and (8) The ligand binds and elutes the following affinity columns: Blue Sepharose, Blue CM-Sepharose, MONO-Q, MONO-S, Lens Lectin-Sepharose, WGA-Sepharose, Con A-Sepharose, Ether 650m Toyopearl, Butyl 650m Toyopearl, Phenyl 650m Toyopearl, and

  Phenyl-Sepharose. More preferred ligand polypeptides are those encoded by human genomic DNA or cDNA having an amino acid sequence as shown in Figure 1 (SEQ ID N: 1). Other preferred natural biologically active mpl ligand polypeptides of the present invention include prepro-mpl ligand, pro-mpl ligand, mature mpl ligand, mpl ligand fragments and their glycosylation variants. Other preferred polypeptides according to the present invention include variants and constructs of the mpl ligand sequence. Typically, preferred mpl ligand sequence variants and constructs are biologically active mpl ligand variants that have an amino acid sequence having at least 70% amino acid sequence identity with the human mpl ligand or isolated mpl ligand. aplastic pig plasma, advantageously at least 75%, more preferably at least 80%, more preferably at least 85%, still more preferably at least 90% and especially at least 95%. An example of a preferred mpl ligand variant is an N-terminal domain hML variant (termed the "EPO domain" because of its sequence homology with erythropoietin).

  The preferred hML EPO domain comprises approximately the first 153 amino acid residues of mature hML and is designated hML 153. An optional preferred hML sequence variant is a variant in which one or more of the monobasic or dibasic amino acid residues in the hML. C-terminal domain is substituted with one or more non-basic amino acid residues (e.g. hydrophobic, neutral, acid, aromatic, Gly, Pro, etc.). A preferred C-terminal hML domain variant comprises a variant in which the residues Arg 153 and 154 are replaced by Ala residues. This variant is called hML332 (R153A, R154A). Another preferred hML variant includes hML332 or hML1s3 wherein amino residues 111-114 (QLPP or LPPQ) are deleted or replaced by a different tetrapeptide sequence (eg AGAG, etc.). The deletion mutants above are called A4hML332 or A4hML1s3. A preferred construct is a fusion between an mpl ligand or fragment thereof (defined below) and a heterologous polyptide or fragment thereof. For example, the hMLls3 variant can be ligated to an IgG fragment to enhance its half-life in serum or to IL-3, G-CSF or EPO to produce a molecule whose chimeric thrombopoietic or hematopoietic activity is strengthened.

  Another preferred human mpl ligand chimera is an "ML-EPO domain construct" which consists of N-terminal hML 153-157 residues substituted with one or more, but not all, residues. Approximately aligned human EPOs as shown in Figure 10 (SEQ ID N: 7). In this embodiment, the hML chimera would have a length of about 153-166 residues where individual residues or blocks of residues from the human EPO sequence are added to or substituted for the hML sequence at positions corresponding to the alignment shown in Fig. 10 (SEQ ID NO: 6). Examples of block-sequence insert segments in the N-terminal portion of hML would include one or more N-glycosylation sites at (EPO) positions 24-27, 38-40 and 83-85; one or more of the four amphipathic α-helicoidal beams predicted in positions (EPO) 9-22, 59-76, 90-107 and 132-152; and other highly conserved regions including the N-terminal and

  C-terminal and residue positions (epo) 44-52 (see, for example, Wen et al., Blood, 82: 1507-1516 [1993] and Boissel et al., J. Biol Chem., 268 (21)). ): 15983-15993 [1993]). This ML-EPOn domain construct is considered to possess mixed thrombopoietic-erythropoietic (TEPO) biological activity.

  Other preferred polypeptides of the present invention include mpl ligand fragments having a consecutive sequence of at least 10, 15, 20, 25, 30 or 40 amino acid residues which are identical to the mpl ligand sequences isolated from the aplastic pig or human mpl ligand described herein (see, e.g., Table 14, Example 24). A preferred mpl ligand fragment is the human ML [1-X] fragment where X is 153, 164, 191, 205, 207, 217, 229 or 245 (see FIG. 1 (SEQ ID N: 1) for the sequence of residues 1-X). Other preferred mpl ligand moieties include those produced as a result of the hydrolysis or chemical or enzymatic digestion of the purified ligand.

  Another preferred aspect of the invention is a method for purifying mpl ligand molecules, which comprises contacting a source of mpl ligand containing the mpl ligand molecules with an immobilized receptor polypeptide, more particularly a mpl polypeptide or a fused mpl polypeptide, under conditions in which the mpl ligand molecules to be purified are selectively adsorbed on the immobilized receptor polypeptide, washing the immobilized carrier to remove the unadsorbed material, and eluting with a buffer. elution, molecules to be purified by elimination of

  immobilized receptor polypeptide. The source containing the mpl ligand may be plasma in which the immobilized receptor is advantageously an mpl-IgG fusion. Alternatively, the mpl ligand-containing source is a recombinant cell culture in which the ligand mpl concentration in the culture medium or in the cell lysates is generally higher than in plasma or other natural sources. In this case, the mpl-IgG immunoaffinity method described above, while still useful, is not usually necessary and conventional methods of protein purification known in the art can be applied. In short, the preferred purification method for obtaining a substantially homogeneous mpl ligand comprises two steps of: removing particulate debris, represented by hasty cells or lysed fragments, e.g. by centrifugation or ultrafiltration ; optionally, the protein may be concentrated with a commercially available protein concentration filter; and then separating the ligand from the other impurities by one or more steps selected from: immunoaffinity, ion exchange (eg DEAE or matrices containing carboxymethyl or sulfopropyl groups), blue-Sepharose, CM blue-Sepharose, MONO-Q,

  MONO-S, lens lectin-Sepharose, WGA-Sepharose, Con A-Sepharose, Toypearl Ether, Butyl Toypearl, Phenyl Toylpearl, protein A-Sepharose, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse phase HPLC (eg silica gel bearing pendant aliphatic groups) or Sephadex molecular sieve or size exclusion chromatography and precipitation with ethanol or ammonium sulfate. A protease inhibitor such as methylsulfonyl fluoride (PMSF) may be included in any of the preceding steps to inhibit proteolysis. In another preferred embodiment, the present invention provides an isolated antibody capable of binding to the mpl ligand. A preferred example of isolated ligand-bound antibody is monoclonal (Kohler and Milstein, Nature, 256: 495-497 [1975]); Campbell, Laboratory Techniques in Biochemistry and Molecular Biology, edited by Burdon et al., Volume 13, Elsevier Science Publishers, Amsterdam [1985]; and Huss et al., Science 246: 1275-1281 [1989]). An isolated mpl ligand-bound antibody that is preferred is an antibody that binds to an mpl ligand with an affinity of at least 106 l / mole. The antibody binds more

  advantageously with an affinity of at least about 10 7 1 / mol. Preferably, the antibody is directed against the mpl ligand having one of the effector functions described above. The isolated antibody capable of binding to the mpl ligand may optionally be fused to a second polypeptide and the antibody or its fused form may be used to isolate and purify the mpl ligand from a source as described above for a polypeptide immobilized mpl. According to another preferred aspect of this embodiment, the invention provides a method for detecting mpl ligand in vitro and in vivo, which comprises the steps of contacting the antibody with a sample, including a suspected serum sample. to contain the ligand, and to detect if a binding has occurred. In still other preferred embodiments, the invention provides an isolated nucleic acid molecule which encodes the mpl ligand or its fragments, which nucleic acid molecule may or may not be labeled with a detectable moiety, and a nucleic acid molecule having a sequence that is complementary to a nucleic acid molecule having a mpl ligand encoding sequence, or hybridizes under rigorous or moderately stringent conditions with such a nucleic acid molecule. A nucleic acid or a preferred mpl ligand is an RNA or a

  DNA encoding a biologically active mpl ligand sharing at least 75% sequence identity, preferably at least 80%, more preferably at least 85%, more preferably 90%, and especially 95% sequence identity with mpl ligand human. More preferred isolated nucleic acid molecules are DNA sequences encoding a biologically active mpl ligand, which is selected from: (a) DNA based on the coding region of a mammalian mpl ligand gene (For example DNA comprising the nucleotide sequence proposed in Figure 1 (SEQ ID N: 2) or fragments thereof); (b) DNA capable of hybridizing with DNA of (a) under at least moderately stringent conditions; and (c) a DNA which is degenerated into a DNA defined in (a) or (b) which results from the degeneracy of the genetic code. It is believed that the novel mpl ligands described herein may be members of a family of ligands or cytokines having a suitable sequence identity that their DNA can hybridize with the DNA of Figure 1 (SEQ ID NO.

  ID N: 2) (or its complement or fragments) under conditions of moderate to moderate severity. Thus, another aspect of the present invention comprises DNA which hybridizes under low to moderate stringency conditions with the DNA encoding mpl ligand polypeptides. In another preferred embodiment of the invention, the nucleic acid molecule is a cDNA encoding the mpl ligand and further comprises a replicable vector in which cDNA is actively bound to regulate sequences recognized by haste. transformed with the vector.

  This aspect further comprises vector-transformed host cells and a method of using cDNA to effect mpl ligand production, which comprises expressing mpl ligand-encoding cDNA in cell culture. transformed hosts and isolation of the ligand mpl from the host cell culture. The mpl ligand prepared in this manner is advantageously a substantially homogeneous human mpl ligand. A preferred host cell for mpl ligand production is Chinese hamster ovary (CHO) cell. The invention further relates to a preferred method of treating a mammal with an immunological or hematopoietic disorder, including thrombocytopenia, which comprises administering a therapeutically effective amount of a mpl ligand to the mammal. Optionally, the mpl ligand is administered in combination with a cytokine,

  such as a colony stimulating factor or an interleukin. Preferred colony stimulating factors or interleukins include: kit-ligand, LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-2, IL-3 IL-5, IL-6, IL-7, IL-8, IL-9 or IL-11. II. Processes of Preparation Some authors have long thought that platelet production could be influenced by multiple humoral factors specific to lineages. It has been suggested that two distinct cytokine activities, called megakaryocyte colony stimulating factor (mega-CSF) and thrombopoietin, regulate megakaryocytopoiesis and thrombopoiesis (Williams et al., J. Cell Physiol 110: 101-104 [J. 1982], Williams et al., Blood Cells, 15: 123-133 [1989], and

  Gordon et al., Blood, 80: 302-307 [1992]). According to this hypothesis, the meg-CSF factor stimulates the proliferation of progenitor megakaryocytes, while thrombopoietin mainly affects the maturation of more differentiated cells and, finally, the release of platelets. Since the 1960s, the induction and appearance of meg-CSF and thrombopoietin activities in the plasma, serum and urine of animals and humans following thrombocytopenic episodes has been the subject of extensive documentation (Odell et al., Proc.Soc.Expl.Biol.Med., 108: 428-431 [1961], Nakeff et al., Acta Haematol., 54: 340-344 [1975]; , Proc Soc., Exp Biol., 108: 146-149 [1961], Schreiner et al., J. Clin Invest, 49: 1709-1713 [1970], Ebbe, Blood, 44: 605- 608 [1974], Hoffman et al, N. Engl J. Med., 305: 533 [1981], Straneva et al., Exp Hematol., 17: 1122-1127 [1988], Mazur et al., Exp. Hematol., 13: 1164 [1985] Mazur et al., J. Clin Invest, 68: 733-741 [1981], Sheiner et al.

  Blood, 56: 183-188 [1980]; Hill et al., Exp. Hematol. 20: 354-360 [1992]; and Hegyi et al., Int. J. Cell. Cloning, 8: 236-244 [1990]). These activities have been reported to be specific and distinct from known cytokines (RJ Hill et al., Blood 80: 346 (1992), CL Erickson-Miller et al., Brit J. Haematol 84: 197-203 ( 1993) JE Straneva et al., Exp.Hematol 20: 4750 (1992) and J. Tsukada et al., Blood 81: 866-867 [1993]). So far, attempts to purify meg-CSF or thrombopoietin from plasma or thrombocytopenic urine have not been successful.

  Consistent with the above observations describing thrombocytopenic plasma, it was found that aplastic porcine plasma (APP) from irradiated pigs stimulated human megakaryocytopenia in vitro. This stimulatory activity was found to be abrogated by the soluble extracellular domain of c-mpl, confirming that APP is a potential source of the mpl (ML) ligand presumed. We have successfully purified the mpl ligand of APP and the amino acid sequence information has been used to isolate murine, porcine and human ML cDNA. These ML ligands have sequence homology with erythropoietin and have both meg-CSF and thrombopoietin activities. 1. Purification and identification of plasma mpl ligand

  As noted above, it has been reported that aplastic plasma from various species contained activities that stimulated hematopoiesis in vitro, however, no hematopoietic stimulating factor isolated from plasma has yet been reported. Aplastic plasma source is the source of irradiated pigs. This aplastic porcine plasma (APP) stimulates human hematopoiesis in vitro. To determine if the APA contained the mpl ligand, its effect was titrated by measuring the incorporation of 3 H-thymidine into Ba / F3 cells transfected with human mpl P (Ba / F3-mpI) by the method illustrated in FIG. Figure 2. APP stimulated the incorporation of 3H-thymidine into Ba / F3-mpl cells but not into control Ba / F3 cells (i.e., not transfected with human mpl P). In addition, no such activity was observed in normal pig plasma. These results indicated that the APP contained one or more factors that transduced a proliferative signal through the mpl receptor and could therefore be the natural ligand for this receptor. This hypothesis was reinforced by demonstrating that the treatment of APP with soluble mpl-IgG blocked the stimulatory effects of APP on Ba / F3-mpl cells. APP activity appeared to be a protein because pronase, DTT, or heat destroys APP activity (Figure 3). The activity was also non-dialysable.

  However, the activity was stable at low pH (pH 2.5 for 2 hours) and was shown to bind to and elute several lectin affinity columns. which indicates that it was a glycoprotein. To further elucidate the structure and identity of this activity, it was affinity purified from APP using an mpl-IgG chimera. APP was treated according to the protocol described in Examples 1 and 2. Briefly, the mpl ligand was purified by hydrophobic interaction chromatography (HIC), immobilized dye chromatography, and mpl receptor affinity chromatography. The activity collected in each step is shown in Fig. 4 and the purification factor is shown in Table 1. The total activity collected by the affinity column for the mpl receptor was about 10% . The maximum activity fraction (F6) of the affinity column for mpl has an estimated specific value of 9.8 x 10 7 units / mg. The overall purification from 5 liters of APP reached a factor of about 4 x 10 '(0.8 units / mg to 3.3 x 10' units / mg) with a factor of 83 x 10 'of the amount of protein (from 250 g to 3 g). The specific activity of the ligand eluted from the affinity column for the mpl receptor was estimated to be about 3 x 10 5 units / mg.

  TABLE 1 Purification of the Dpl ligand Purification of m Iand I I IT T .. ~~ Activity- Sample Factor-Volume Pro-Unit / ml Unit speci-puriicatillon ml teine, purifica- - inci / fhl - night s / um tion ,. mqgl ~ l! ni & ffla2,% APP 5000 50 40 200 000 0.8 1 Phpnjle 4700 0.8 40 200 000 50 94 62 Blue-Sep.640 0.93 400 256 000 430 128 538 mpl <i1) Fractions Fr - 7n 12 5x10-4 1666 20,000 3,300,000 10 4 100,000 The protein was assayed by the Bradford method. The protein concentration of mpl elution fractions 5-7 represents values estimated on the basis of the silver stained sodium dodecylsulfate gel staining intensity. One unit is defined as the amount that causes 50% of the maximum stimulation of Ba / F3-mpl cell proliferation. Analysis of fractions eluted from the affinity column for mpl by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Novex gel, 4-20%) under reducing conditions revealed the presence of several proteins (FIG. 5). Proteins that silver stained with the highest intensity were resolved with apparent Mr values of 66,000, 55,000, 30,000, 28,000 and 18,000-22,000. To determine which of these proteins stimulated proliferation cultures of Ba / F3-mpl cells were eluted from the gel as described in

  Example 2. The results of this experiment showed that most of the activity was eluted from a slice of gel which contained M. proteins having a value of 28,000-32,000, with a lower activity eluted in the 18,000-22,000 freezing region (Figure 6). The only visible proteins in these regions had Mr values of 30,000, 28,000 and 18,000-22,000. To identify and obtain the sequence for the proteins that resolve in that region of the gel (i.e. bands at 30, 28 and 18-22 kDa), these three proteins were analyzed by electroblotting to PVDF membranes and sequenced as described in Example 3. The amino-terminal sequences obtained are shown in Table 2

  TABLE 2 Amino terminal sequences of mpl kDa ligand (S) PAPPA (C) DPRLLNKLLRDD (H / S) VLH (G) RL (SEQ ID NO: 30) 10 28 kDa 1 5 10 15 20 ( S) PAPPAXDPRLLNKLLRDD (H) VL (H) GR (SEQ ID NO: 31) 18-22 kDa 1 5 10 X PA PPA XO PR LX (N) (K) SE ID NO: 32) The computer-assisted analysis revealed that the amino acid sequences were new. Because all three sequences were the same, the 30 kDa, 28 kDa, and 18-22 kDa proteins were considered to be related and could be different forms of the same novel protein. In addition, this protein or proteins was a likely candidate as a natural mpl ligand because the activity resolved in sodium dodecyl sulfate and polyacrylamide gel electrophoresis in the same region (28,000-32,000). ) a 4-20% gel. In addition, the partially purified ligand migrated with a Mr value of 17,000-000 when it. was subjected to gel filtration column chromatography Superose 12 (Pharmacia). We

  considers that the different forms of Mr of the ligand are the result of differences in proteolysis or glycosylation or other modifications after or before translation. As described above, the antisense human mpl rRNA abrogated megakaryocytopolesis in human bone marrow cultures enriched in CD 34 'progenitor cells without affecting the differentiation of other hematopoietic cell lines (Methia et al., Supra). This result suggests that the mpl receptor may play a role in the differentiation and proliferation of megakaryocytes in vitro. To further elucidate the role of mpl ligand in megakaryocytopoiesis, the effects of APP and depleted APP in ligand mpl on

  human megakaryocytopolesis in vitro. The effect of APP on human megakaryocytopoiesis was determined from a modified version of the liquid suspension megakaryocytopoiesis assay described in Example 4. In this test, human peripheral stem cells (PSCs) were used. were treated with APP before and after affinity chromatography for mpl-IgG. The GPIIbIII stimulation of the megakaryocytopoiesis was quantitatively evaluated with an anti-IIbIII antibody, labeled with 25sI (FIG. 7). As shown in FIG. 7, 10% of APP stimulated about a factor of 3 while the ligand-depleted APP had no effect. It is important to note that the ligand-depleted APP failed to induce proliferation of Ba / F3-mpl cells.

  In another experiment, soluble human IgM mpl added at time 0 and days 2 and 4 to cultures containing 10% APP neutralized the stimulatory effects of APP on human megakaryocytopolesis (Figure 8). These results indicate that the mpl ligand plays a regulatory role in human megakaryocytopenia and thus may be useful for the treatment of thrombocytopenia. 2. Molecular Cloning of the mpl ligand According to the amino-terminal amino acid sequence derived from the 30 kDa, 28 kDa and 18-22 kDa proteins (see Table 2 above), two degenerate oligonucleotide primer-groups have were designed and used to amplify PCR genomic pig DNA. It was considered that if the amino-terminal amino acid sequence was encoded by a single exon, a correct length of 69 base pairs could be expected for the correct PCR product. A DNA fragment of this size was found and subcloned into pGEMT. The sequences of the DNA polymerase chain reaction primers of the oligonucleotides and the three clones obtained are shown in Example 5. The amino acid sequence (PRLLNKLLR [SEQ ID N: 33]) of the peptide encoded between the primers PCR was identical to that obtained by sequencing the amino-terminal protein of porcine ligand (see residues 9-17 for the porcine protein sequences of 28 and 30 kDa above).

A synthetic oligonucleotide based on the sequence of the PCR fragment was used to sort a human genomic DNA library. A 45-base oligonucleotide, called pR45, was designed and synthesized on the basis of the sequence of the PCR fragment. This oligonucleotide had the following sequence: S GCC-GTG-AAG-GAC-GTG-GTC'GTCACG-MAG-CAGITI -TAG-TAG-GAG-TCG3 (SEO ID NO: 34) This deoxyoligonucleotide was used to sort a human genomic DNA library in ygeml2 under low stringency hybridization and washing conditions according to Example 6. Positive clones were picked, plaque purified and analyzed by restriction mapping and Southern Blot analysis. EcoRI fragment

  390-base pair XbaI that had hybridized to the 45-base oligonucleotide was subcloned into pBluescript SK-. DNA sequencing of this clone confirmed that the DNA encoding the human homologue of the porcine ligand was isolated. The human DNA sequence and the deduced amino acid sequence are shown in Figure 9 (SEQ ID NOs 3 and 4). The predicted intron positions of the genomic sequence are also indicated by arrows, and define a presumed exon ("exon 3"). On the basis of the sequence of the human "exon 3" (Example 6), oligonucleotides corresponding to the 3 'and 5' ends of the exon sequence were synthesized. These two primers were used in PCR reactions using as template a complementary DNA prepared from various human tissues. The expected size of the correct PCR product was 140 base pairs. After analysis of the 12% polyacrylamide gel PCR products, a DNA fragment of expected length was detected in cDNA libraries prepared from kidney of human adults, 293 fetal kidney cells and CDNA prepared from human fetal liver.

  A fetal liver cDNA library (7 x 10 'clones) in lambda DR2 was then sorted with the same 45-base oligonucleotide that was used to treat the human genomic library and the cDNA library. fetal liver under low stringency hybridization conditions. Positive clones were picked, plaque purified and the length of the insertion segment determined by PCR. A clone with a 1.8 kilobase insertion segment was chosen for further analysis. Using the procedures described in Example 7, the nucleotide and the deduced amino acid sequence of the human mpl ligand (hML) were obtained. These sequences are presented in Figure 1 (SEQ ID N 1 and 2). 3. Structure of human mpl ligand (hML) The cDNA sequence of human mpl ligand (hML) (FIG. 1 [SEQ ID N: 2]) comprises 1774 nucleotides monitored

  a poly (A) tail. It contains 215 nucleotides of 5 'untranslated sequence and a 3' untranslated region of 498 nucleotides. The presumed initiation codon in nucleotide position (216-218) is in a consensus sequence favorable to the initiation of a eukaryotic translation. The open reading frame is 1059 nucleotides in length and encodes a 353 amino acid residue polypeptide, starting in nucleotide position 220. The N-terminus of the predicted amino acid sequence is highly hydrophobic and probably corresponds to a signal peptide. Computer analysis of the predicted sequence of amino acids (see

  Heijne et al., Eur. J. Biochem., 133: 17-21 [1983]) indicates a potential cleavage site for signal peptidase between residues 21 and 22. Cleavage in this position would result in a mature polypeptide of 332 amino acid residues starting with by the amino-terminal sequence obtained from purified mpl ligand of pig plasma. The unglycosylated molecular weight predicted of the ligand at 332 amino acid residues is about 38 kDa. There are 6 potential N-glycosylation sites and 4 cysteine residues. A comparison of the mpl ligand sequence with the genebank sequence database revealed a 23% identity between the 153 amino-terminal residues of the mature human mpl ligand and human erythropoietin (Fig. 10 [SEQ ID] NO: 6 and 7]). When conservative substitutions are taken into consideration, this region of hML shows a 50% similarity to human erythropoietin (hEPO). Erythropoietin hEPO and hML ligand both contain four cysteines. Three of these cysteines are conserved in hML, including the first and last cysteine. Site-directed mutagenesis experiments have shown that the first and last cysteines of erythro-poletine form a disulfide bond which is required for function (F.F. Wang et al., Endocrinology 116: 2286-2292 [1983]). By analogy, the first and last cysteines of hML can also form a decisive disulfide bond. None of the glycosylation sites are conserved in hML.

  All potential glycosylation sites bound to the hML nitrogen are located in the carboxy-terminal half of the hML polypeptide. Like erythropoietin hEPO, the messenger RNA of hML does not contain the AAUAAA polyadenylation consensus sequence, nor does it contain the AUUUA regulatory element that is present in many 3'-untranslated regions. cytokines and is believed to influence the stability of mRNA (Shaw et al., Cell, 46: 659-667 [1986]). Northern blot analysis reveals low levels of a single 1.8 kilobase hML RNA transcript in fetal liver and adult liver. After longer exposure, a smaller band of the same size could be detected in the adult kidney. By way of comparison, human erythropoietin is expressed in the fetal liver and, in response to a hypoxy, in the adult kidney and liver (Jacobs et al., Nature, 313: 804-809 [1985] and Bondurant et al., Molec Cell Biol., 6: 2731-2733 [1986]).

  The importance of the C-terminal region of hML remains to be elucidated. Based on the presence of the six potential sites for nitrogen-linked glycosylation and the ability of the ligand to bind to lectin affinity columns, this region of the hML ligand is likewise glycosylated. In some gel elution experiments, an activity resolution with a Mr value of about 60,000, which may represent the full-length glycosylated molecule, has been observed. The C-terminal region can therefore act by stabilizing and increasing the half-life of circulating hML. In the case of erythropoietin, the unglycosylated form has a complete biological activity in vitro, but has a substantially reduced half-life in plasma relative to glycosylated erythropoietin (Takeuchi et al., J. Biol. Chem., 265: 12127-12130 [1990], Narhi et al., J. Biol Chem., 266: 23022-23026 [1991] and Spivack et al., Blood, 7: 90-99 [19891]. The C-terminal domain of hML contains two dibasic amino acid sequences [Arg-Arg motifs in positions 153-154 and 245-246] that could serve as potential sites

  maturation. Cleavage at these sites may be responsible for generating the 30, 28 and 18-22 kDa forms of ML isolated from APP. It is important to note that the sequence Argls3-Arg, 54 appears immediately after the ML domain similar to erythropoietin. These observations indicate that the entire ML may represent a precursor protein that undergoes limited proteolysis by generating the mature ligand. 4. Isoforms and variants of human mpl ligand Isoforms or alternatively spliced forms of human mpl ligand were detected by PCR in adult human liver. Briefly, primers corresponding to each end as well as selected internal regions of the hML coding sequence were synthesized. These primers were used in an RT-PCR region to amplify adult human liver RNA as described in Example 10.

  In addition to the full-length form, called hML, three other forms, called hML2, hML3 and hML4, have been observed or deduced. The deduced mature amino acid sequences of the four isoforms are shown in Figure 11 (SEQ ID NO: 6, 8, 9 and 10). The hML3 form has a deletion of 116 nucleotides at position 700, from which amino acid deletion and frame shift result. The cDNA now encodes a mature polypeptide which is 265 amino acids long and diverges from the hML sequence at amino acid residue 139. Finally, hML 4 comprises both a 12 nucleotide deletion following the position of nucleotide 618 (which is also found in the mouse and porcine sequences (see below)) and the 116 base pair deletion observed in the hML3 form, although no clone with a deletion of only 12 base pairs (after nucleotide 619) has been isolated from humans (form designated hML2), this form is likely to exist because such an isoform has been identified in mice and in pigs (see below), and because it has been identified together with the deletion of

  116 nucleotides in hML4. A substitution variant of hML in which the dibasic Argl53-Argl54 sequence was replaced by two alanine residues and an "EPO domain" truncated form of hML were assembled to determine whether the full length ML form was necessary for biological activity. The Arg1s3-Argls4 dibasic sequence substitution variant, called hML (R153A-R154A) was assembled using the PCR reaction as described in Example 10. The "domain EPO" truncated form, hML, 53, was also performed using the PCR reaction by introducing a stop codon following Args3. 5. Expression of recombinant human mpl ligand (rhML) in (293) human embryonic kidney cells Transient transfection To confirm that the cloned human cDNA encoded a ligand for mpl, the ligand was expressed in 293 mammalian cells under the influence of the immediate cytomegalovirus early promoter using the pRK5-hML or PRK5-hML153 expression vectors. Supernatant fluids from 293 transient transfection-positive human embryonic kidney cells have been shown to stimulate 3H-thymidine incorporation into Ba / F3-mpl cells, but not into

  parental cells Ba / F3 (FIG. 12A). Media from the 293 cells transfected with the pRK vector alone did not contain this activity. The addition of mpl-IgG to these media abolished the stimulation (data not shown). These results show that the cloned cDNA encodes a functional human ML (hML). To determine whether the "EPO domain" alone could bind and activate the mpl receptor, the truncated form of hML, namely rhML, 53, was expressed in 293 cells. Supernatant fluids from transfected cells were found to have similar activity to supernatants from cells expressing full-length hML (Fig. 12A), indicating that the C-terminal domain of ML is not not needed for binding and activation of c-mpl.

  6. The mpl ligand stimulates the mycaryocytopolysis and thrombopoiesis The total length rhML form and the truncated rbhML1s3 form of recombinant hML stimulated the human megakaryocytopolysis in vitro (Figure 12B). This effect was observed in the absence of other exogenous hematoptic growth factors. With the exception of IL-3, ML was the only tested hematopoietic growth factor that exhibited this activity. This effect on megakaryocytopolysis was not observed for IL-11, IL-6, IL-1, erythro-poletine, G-CSF, IL-9, LIF, kit-ligand (LK), M -CSF, OSM and GM-CSF when tested separately in the test according to the invention (data not shown). The result demonstrates that ML exhibits stimulatory activity against megakaryocytes, and indicates a regulatory role of megakaryocytopolysis for ML. Thrombopoleetic activities in plasma of thromocytopenic animals have been shown to stimulate platelet production in a mouse thrombocytosis test (McDonald,

  Proc. Soc. Exp. Biol. Med., 14: 1006-1001 [1973] and McDonald et al., Scand. J. Haematol., 16: 326-334 [19761]. In this model, acute thrombocytopenia is triggered in mice using a specific antiplatelet serum, resulting in predictable bouncing thrombocytosis. These immuno-thrombocythemic mice are more sensitive to thrombopoietin-like exogenous activities than are normal mice (McDonald, Proc.Soc.Exp.Lab.Med., 14: 1006-1001 [1973]), as well as mice. Exhypoxics are more sensitive to erythropoietin than are normal mice (McDonald et al., J. Lab.Clin Med 77: 134-143 [1971]). To determine if rML stimulates platelet production in vivo, mice with rebound thrombocytosis received an injection of partially purified rhML. Platelet levels and 3sS incorporation into platelets were then quantitatively evaluated. Injection to mice of 64,000 or 32,000 units of rML markedly increased platelet production, as evidenced by an approximately 20% increase in platelet count (p = 0.0005 and 0.0001). , respectively) and an approximately 40% increase in 35S incorporation to platelets (p = 0.003) in treated mice, compared to control mice to which the vehicle alone had

  injected (Figure 12C). This degree of stimulation is comparable to that observed with IL-6 in this model (results not shown). Treatment with 16,000 units of rML did not significantly boost platelet production. These results indicate that ML stimulates platelet production in a dose-dependent manner and therefore has activity similar to that of thrombopoietin. 293 cells were also transfected with the other hML isoform assemblies described above, and the supernatants were titrated according to the Ba / F3-mpl proliferation assay (see Figure 13). The hML2 and hML3 forms did not show detectable activity in this assay, however, the hML (R153A, R154A) activity was similar to that of hMLs and hMLs3, indicating that maturation at the dibasic site Argls3-Argl. is neither necessary nor harmful to the activity. 7. Megacaryocytopoiesis and mpl ligand It has been presumed that megakaryocytopoiesis is regulated at multiple cell levels (Williams et al., J. Cell Physiol., 110: 101-104 [1982] and Williams et al., 2714670 84).

  Blood Cells, 15: 123-133 [1989]). This is largely based on the observation that some hematopoietic growth factors stimulate the proliferation of megakaryocyte progenitors while others appear to primarily affect maturation. The results presented here suggest that ML acts both as a proliferation and maturation factor. The fact that ML stimulates the proliferation of megakaryocyte progenitors is supported by a number of evidences. First, APP stimulates both proliferation and processing of human megakaryocytes in vitro, and this stimulation is fully inhibited by mpl-IgG (Figures 7 and 8). In addition, the inhibition of megakaryocyte colony formation by C-mpl antisense oligonucleotides (Methia et al., Blood, 82: 1395-1401 [19931)) and highlighting that c-mpl could perform transduction of a proliferation signal into cells in which it was transfected (Skoda et al., EMBO, 12: 2645-2653 [1993] and Vigon et al., Oncogene, 8: 2607-2617 (1993)) indicate It is also apparent that ML stimulates proliferation The apparent expression of this c-mpl during all stages of megakaryocyte differentiation (Methia et al., Blood, 82: 1395-1401 [1993]) and the ability of recombinant ML to rapidly stimulate platelet production in vivo indicate that ML also affects maturation The availability of recombinant ML makes it possible to carefully evaluate its role in the regulation of megakaryocytopolesis and thrombopoiesis as well as its ability to influence d other haematopoietic lineages 8. Isolation The human mpl ligand (TPO) gene was cloned. Human TPO gene genomic DNA clones were isolated by sorting a human genomic library into X-GemI2 with pR45, under low stringency conditions or under highly stringent conditions. a fragment

  corresponding to the 3 'half of the human cDNA encoding the mpl ligand. Two covering lambda clones covering 35 kb were isolated. Two overlapping fragments (BamHI and EcoRI) containing the entire TPO gene were subcloned and sequenced (see Figures 14A, 14B and 14C). The structure of the human gene is composed of 6 exons within 7 kb of genomic DNA. The boundaries of all exon / intron junctions are consistent with the consensus motif established for mammalian genes (M. B. Shapiro et al., Nucl Acids Res., 15: 7155 [1987]). Exon i and exon 2 contain the 5 'untranslated sequence and the initial 4 amino acids of the signal peptide. The remainder of the secretory signal and the first 26 amino acids of the mature protein are encoded in exon 3. The entire carboxyl domain and the 3 'untranslated sequence as well as about 50 amino acids of the erythropoietin-like domain are The four amino acids involved in the deletion observed in hML-2 (hTPO-2) are encoded at the 5 'end of exon 6. Southern human genomic DNA analysis blot indicated that the gene for TPO was present in a single copy. The chromosomal location of the gene was determined by fluorescent in situ hybridization (FISH), which indicated chromosome 3q27-28. 9. TPO Expression and Purification of 293 Cells Preparation and purification of ML or TPO from 293 cells are described in detail in Example 19. Briefly, cDNA corresponding to the entire open reading frame of TPO was obtained by PCR reaction using plasmid pRKS5-hmplI. The PCR product was purified and cloned between ClaI and XbaI restriction sites of plasmid pRK5tkneo (modified pRK5 derived vector to express a neomycin resistant gene under the influence of the thymidine kinase promoter) to obtain the vector prKStkneo.ORF (vector coding for the entire open reading frame). A second vector encoding the homologous domain of EPO was generated in the same way, but using different PCR primers to obtain the final assembly called pRK5-tkneoEPO-D.

  These two constructs were transfected into human embryonic kidney cells by the CaPO4 method, and neomycin resistant clones were selected and grown to confluence. The expression of ML1s3 or ML332 in the conditioned medium from these clones was determined using the Ba / F3mpl proliferation assay. The purification of rhML332 was conducted as described in Example 19. Briefly, 293-rhML332 conditioned media was applied to a Blue-Sepharose column (Pharmacia) which was then washed with a buffer containing The column was eluted with a buffer containing 2M urea and 1M NaCl. The eluted volume of Blue-Sepharose was then applied directly to a WGA-Sepharose column, washed with 10 volumes. of a buffer containing 2M urea and 1M NaCl and eluted with the same buffer containing 0.5M N-acetyl-D-glucosamine. The WGA-Sepharose eluate was loaded onto a C4 column of high performance liquid chromatography (Synchrom, Inc.) and eluted with a discontinuous propanol gradient. In gel electrophoresis of sodium dodecyl sulfate and polyacrylamide, the purified 293-rhML332 migrates as a broad band in the 68-80 kDa region of the gel (see Figure 15). Purification of rhML1s3 was also carried out as described in Example 19. Briefly, 293-rhML1s3-conditioned media were resolved on Blue-Sepharose as described for rhML332. The Blue Sepharose eluate was applied directly to an affinity column for mpl as described above. The rhML1s3 eluted from the mpl affinity column was purified to homogeneous consistency using a C4 HPLC column operating under the same conditions as those used for rhML332. In sodium dodecyl sulfate and polyacrylamide gel electrophoresis, the purified rhML153 is resolved into dominant bands and two secondary bands with a Mr value of about 18,000-22,000 (see Figure 15). 10. Mouse mpl ligand A DNA fragment corresponding to the coding region of the human mpl ligand was obtained by PCR, gel purified and labeled in the presence of 32P-dATP and 32P-dCTP. This probe was used to sort 10 'clones of a mouse liver cDNA library into XGT10. A murine clone (FIG. 16 [SEQ ID NOS: 12 and 13]) containing a 1443 base pair insert was isolated and sequenced. The initiation codon presumed in nucleotide position 138-141 was in a consensus sequence favorable to the initiation of eukaryotic translation (M. Kozak, J. Cell Biol., 108: 229-241 [1989] ). This sequence defines an open reading frame of 1056 nucleotides, which predicts a primary translation product of 352 amino acids. 137 nucleotides of untranslated sequence 5 'and 246 nucleotides of untranslated sequence 3' flank this open reading frame. There is no poly (A) tail following the 3 'untranslated region, indicating that the clone is probably incomplete.

  The N-terminus of the predicted amino acid sequence is highly hydrophobic and presumably represents a signal peptide. Computer analysis (G. von Heijne, Eur J. Biochem 133: 17-21 [1983]) indicated a potential cleavage site for signal peptidase between residues 21 and 22. Cleavage in this position would generate a mature polypeptide of 331 amino acids (35 kDa) identified mML331 (or mML2 for reasons described below). The sequence contains 4 cysteines, all conserved in the human sequence, and seven potential N-glycosylation sites. 5 of them are preserved in the human sequence. Again, as for hML, the seven potential N-glycosylation sites are in the C-terminal half of the protein. Compared with the human ML, considerable nucleotide identity as well as deduced amino acid sequences were observed in the "EPO domains" of these MLs. However, when the deduced amino acid sequences of human ML and mouse ML were aligned, it was found that the murine sequence exhibited a tetrapeptide deletion between residues 111 and 114 corresponding to the 12 nucleotide deletion following the nucleotide position 618 observed in both the human cDNA (see above) and the pork cDNA (see below). As a result, other clones were examined for possible murine ML isoforms. One of the clones encoded a sequence polypeptide deduced from 335 amino acids containing the "missing" tetrapeptide LPLQ. This form is assumed to be the whole length murine ML and is called mML or mML33s. The nucleotide and the deduced amino acid sequence for mML are shown in Figure 17 (SEQ ID NOS: 14 and 15). This cDNA clone consists of 1443 base pairs followed by a poly (A) tail. It has a 1068 base pair open reading frame flanked by 134 bases of 5 'untranslated sequence and 241 bases of 3' untranslated sequence. The presumed initiation codon is in the position of nucleotides 138-140. The open reading frame encodes a predicted protein of 356 amino acids, the first 21 of which are highly hydrophobic and likely to function as a secretion signal. Finally, a third murine clone was isolated, sequenced, and found to contain the 116 nucleotide deletion corresponding to hML3. This murine isoform is therefore called mML3. A comparison of the deduced amino acid sequences of these two isoforms is shown in Figure 18 (SEQ ID NO: 9 and 16). The identity of the overall amino acid sequence between human ML and mouse ML (Fig. 19 (SEQ ID N: 6 and 171)) is 72%, but this homology is not uniformly

  distributed. The region defined as "EPO domain" (amino acids 1-153 for the human sequence and 1-149 for the mouse) is better conserved (86% homology) than the carboxy-terminal region of the protein (62% d homology). This may further indicate that only the "EPO domain" is important for the biological activity of the protein. It is interesting to note that of the two dibasic amino acid motifs found in hML, only the dibasic motif immediately following the "EPO domain" (residue position 153-154) in the human sequence is present in the murine sequence. This is consistent with the possibility that full-length ML may represent a precursor protein that undergoes limited proteolysis to generate the mature ligand. Alternatively, proteolysis between Argls3-Argls4 may facilitate clearance of hML. An expression vector containing the entire mML coding sequence was transiently transfected into 293 cells as described in Example 1. Conditioned media from these cells stimulated the incorporation of 3H-thymidine into Ba cells. / F3 expressing a murine or human mpl, but had no effect on the parental cell line (mpl-less). This indicates that the cloned murine ML cDNA encodes a functional ligand that is capable of activating both the murine ML receptor and the human ML receptor (mpl). 11. Porcine mpl ligand

  Porcine ML (pML) cDNA was isolated by RACE PCR reaction as described in Example 13. A 1342 base pair PCR cDNA product was found in the kidney and subcloned. Several clones were sequenced and found to encode a porcine mpl ligand of 332 amino acid residues, called pML (or pML332) having the nucleotide sequence and the deduced amino acid sequence shown in Figure 20 (SEQ ID NO: 18 and 19). Again, a second form designated pML2, encoding a protein with a deletion of 4 amino acid residues (228 amino acid residues) was identified (see Figure 21 [SEQ ID NO: 21]). A comparison between the amino acid sequences of pML2 and pML2 shows that the latter form is identical, except that the QLPP tetrapeptide corresponding to residues 111-114 included has been removed (see FIG. 22 [SEQ ID NO: 18 and 21]). . The four amino acid deletions observed in the murine ML and porcine ML cDNA appear precisely in the same position in the predicted proteins. A comparison of the predicted amino acid sequences of the mature ML of human, mouse and pig (FIG. 19 [SEQ ID NO: 6, 17 and 18]) indicates that the identity of the overall sequence is 72. % between mouse and human, 68% between mouse and pig and 73% between pig and human.

  The homology is notably greater in the amino-terminal half of the ML (homologous domain EPO). This domain is identical to 80-84% between any two species, while the carboxy-terminal half (carbohydrate domain) is only 57-67% identical. A dibasic amino acid motif that could represent a protease cleavage site is present at the carboxyl end of the homology domain with erythropoietin. This motif is conserved between the three species in this position (Figure 19 [SEQ ID N: 6, 17 and 18]). A second dibasic site present in positions 245 and 246 in the human sequence does not exist in the mouse or pork sequences. The mouse and porcine ML sequences contain 4 cysteines, all conserved in the human sequence. There are seven sites of potential N-glycosylation in the mouse ligand and six in the pig ML, so S are conserved in the human sequence. Again, all potential N-glycosylation sites are located in the C-terminal half of the protein. 12. Expression and purification of TPO from Chinese hamster ovary cells (CHO)

  The expression vectors used for transfection of CHO cells are called pSV15.ID.LL.MLORF (total length or TPO332) and pSV15.I D.LL.MLEPO-D (truncated form or TPO153). The relevant characteristics of these plasmids are shown in FIGS. 23 and 24. The transfection operations are described in Example 20. Briefly, the cDNA corresponding to the entire open reading frame of TPO was obtained by PCR reaction. The PCR product was purified and cloned between two restriction sites (ClaI and SalI) of plasmid pSV15.ID.LL to obtain the vector pSV15.ID.LL.MLORF. A second assembly corresponding to the EPO homologous domain was generated in the same way but using a different inverse primer (EPOD.Sal). The final assembly for the vector coding for the EPO homologous domain of TPO is called pSV15.ID.LL.MLEPO-D. These two constructs were linearized with NotI and transfected into Chinese hamster ovary cells (CHO-DP12 cells, European Patent No. 307,247 published March 15, 1989) by electroporation. 107 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 (G. L. Andreason, J. Tissue Cult.

  Meth. 15, 56 [1993]). The day after the transfection, the cells were distributed in selective media DHFR (DMEM-F12 high glucose content 50:50 without glycine, 2 mM glutamine, 2-5% dialyzed fetal calf serum). Ten to fifteen days later, individual colonies were transferred to 96-well plates and allowed to grow until they were confluent. The expression of ML153 or ML332 in the conditioned media from these clones was determined from the Ba / F3-mpl proliferation method (described in Example 1).

  The process of purifying and isolating TPO from the collected CHO cell culture fluid is described in Example 20. Briefly, the collected cell culture fluid (HCCF) is loaded onto a Blue Sepharose column (Pharmacia) in a ratio of about 100 liters of HCCF per liter of resin. The column is then washed with 3 to 5 times its volume of buffer and then 3 to 5 times its volume of a buffer containing urea 2.0 M. The TPO is then

  eluted with 3 to 5 column volumes of buffer containing both 2.0 M urea and 1.0 M NaCl. The pooled Blue Sepharose eluate containing TPO is then loaded onto a Sepharose column containing wheat germ lectin (Pharmacia) equilibrated in Blue Sepharose elution buffer in a ratio of 8 to 16 ml of eluate. of Blue Sepharose per ml of resin. The column is then washed with 2 to 3 times its volume of equilibration buffer. The TPO is then eluted with 2 to 5 column volumes of a buffer containing 2.0M urea and 0.5M N-acetyl-D-glucosamine. The wheat germ lectin eluate containing TPO is then acidified and EC is added at a final concentration of 0.04%. The resulting liquid is loaded onto a reversed phase C4 column equilibrated in 0.1% TFA medium and 0.04% CI2E at a loading of about 0.2 to 0.5 mg protein per ml. of resin. The protein is eluted in a linear two-phase acetonitrile gradient containing 0.1% TFA and 0.04% C12E, and sample collection is performed based on sodium dodecyl sulfate gel electrophoresis. and polyacrylamide.

  The samples collected from column C4 are then diluted and dialyzed against about 6 volumes of buffer on an Amicon YM ultrafiltration membrane or the like having a molecular weight range of 10,000 to 30,000 daltons. The resulting diafiltrate can then be directly processed or it can be further concentrated by ultrafiltration. The diafiltrate / concentrate is usually adjusted to a final concentration of 0.01% Tween-80. The whole or a portion of the diafiltrate / concentrate equivalent to 2-5% of the calculated volume of the column is then loaded onto a Sephacryl S-300 HR (Pharmacia) column equilibrated in a buffer containing 0.01% Tween 80, and chromatographed. The TPO-containing fractions which are free of aggregates and proteolytic degradation products are then pooled on the basis of sodium dodecyl sulfate and polyacrylamide gel electrophoresis. The pooled sample thus obtained is filtered and stored at 2-8 ° C. 13. Processes of transformation and induction of TPO synthesis in a microorganism and isolation, purification and refolding of TPO therein is produced

  The construction of TPO expression vectors in E. coli is described in detail in Example 21. Briefly, plasmids pMP21, pMP151, pMP41, pMP57 and pMP202 were all designed to express the first 155 amino acids of TPO in vitro. downstream of a small leader that varies between different assemblies. Leaders primarily allow the initiation of high-level translation and rapid purification. Plasmids pMP210-1, -T8, -21, -22, 30 -24, -25 are designed to express the first 153 amino acids of TPO downstream of an initiating methionine and only differ in use. codons for the first 6 amino acids of TPO, while the plasmid pPM251 is a derivative of pMP210-1 in which the carboxy-terminal end of the TPO is extended by two amino acids. All of the above plasmids produce high levels of intracellular TPO expression in E. coli induced by the tryptophan promoter (DG Yansura et al., Methods in Enzymology (edited by DV Goeddel) 185: 54- 0, Academic Press, San Diego [1990]). Plasmids pPM1 and pMP172 are intermediates in the construction of

  TPO intracellular expression plasmids above. The above TPO expression plasmids were used to transform E. coli using the CaCl 2 heat shock method (M. Mandel et al., J. Mol Biol., 53: 159-162 [1970]). ]) and other procedures described in Example 21. Briefly, the transformed cells were first grown at 370C until the optical density (600 nm) of the culture reached about 2-3. . The culture was then diluted and, after growth with aeration, an acid was added. The culture was then allowed to continue growing under aeration for another 15 hours, after which the cells were harvested by centrifugation.

  The isolation, purification and refolding operations indicated below for the production of biologically active refolded human TPO or TPO fragments are described in Examples 22 and 23 and can be applied to the isolation of any TPO variant. comprising N-terminal or C-terminal extension forms. Other procedures that are suitable for refolding recombinant or synthetic TPO can be found in the following patents: U.S. Patent No. 4,511,502 to Builder et al., U.S. Pat. No. 4,512,922 to Jones et al., U.S. Patent No. 4,518,526 to Olson and U.S. Patent No. 4,620,948 to Builder et al. ; for a general description of the isolation and refolding operations of various recombinant proteins expressed in an insoluble form in E. coli. A. Separation of Insoluble TPO A microorganism such as E. coli expressing the

  TPO encoded by any suitable plasmid is fermented under conditions in which TPO is deposited into insoluble "refractile bodies". Optionally, cells are first washed in cell disruption buffer. For example, about 100 g of cells are resuspended in about 10 volumes of cell disruption buffer (e.g. 10 mM Tris, 5 mM EDTA, pH 8) with, for example, a Polytron homogenizer and the cells are centrifuged at 5000 xg for 30 minutes. The cells are then lysed using any conventional technique such as tonic shock, ultrasound, pressure pumping, chemical or enzymatic operations. For example, the above washed cell pellet may be resuspended in a further 10 volumes of cell disruption buffer by means of a homogenizer and the cell suspension is passed through a cell disruptor. LH cells (LH Inceltech, Inc.) or in a microfluidification apparatus (Microfluidics International) according to the manufacturer's instructions. The particulate material containing the TPO is then separated from the liquid phase and optionally washed with any suitable liquid. For example, a cell lysate suspension can be centrifuged at 5000 x g for 30 minutes, the cells resuspended and they can be centrifuged a second time to form a washed pellet of refreshing bodies. The washed pellet may be used immediately or it may be stored in a frozen state (for example at -700C).

  B. Solubilization and purification of monomeric TPO The insoluble TPO in the refractive body pellet is then solublized with a solubilizing buffer. The solubilizing buffer contains a chaotropic agent and is usually buffered to a basic pH and contains a reducing agent to improve the yield of monomeric TPO. Examples of chaotropic agents include urea, guanidine hydrochloride and sodium thiocyanate. A preferred chaotropic agent is guanidine hydrochloride. The concentration of the chaotropic agent is usually 49 M, preferably 6-8 M. The pH of the solubilizing buffer is maintained with any suitable buffer in a pH range of about 7.5-9. 5, preferably 8.0-9.0 and in particular 8.0. The solubilizing buffer also advantageously contains a reducing agent which aids in the production of the monomeric form of TPO. Suitable reducing agents include organic compounds containing a free thiol (RSH). Examples of reducing agents are dithiothreitol (DTT), dithioerythritol (DTE), mercapto-

  Ethanol, glutathione (GSH), cysteamine and cysteine. Reducing agent of choice is dithiothreitol (DTT). The solubilizing buffer may optionally contain a mild-acting oxidizing agent (eg, molecular oxygen) and sulfite to form the monomeric TPO by sulfitolysis. In this embodiment, the resulting TPO-S-sulfonate is subsequently folded in the presence of the redox buffer (eg, GSH / GSSG) to form suitably folded TPO. The TPO protein is usually further purified, for example by centrifugation, gel filtration chromatography and reverse phase column chromatography.

  By way of illustration, the following procedure gave suitable yields of monomeric TPO. The refractile pellet is resuspended in about 5 volumes by weight of the solubilizing buffer (20 mM Tris, pH 8, with 6-8 M guanidine and 25 mM DTT) and the suspension is stirred for 1-3 hours. or overnight at 4 C to solubilize the TPO protein. High concentrations of urea (6-8 M) are also useful, but generally give slightly lower yields compared to guanidine. After solubilization, the solution is centrifuged at 30,000 x g for 30 minutes to produce a clear supernatant containing the denatured monomeric TPO protein. The supernatant liquid is then chromatographed on a Superdex 200 gel filtration column (Pharmacia, 2.6 x 60 cm) at a flow rate of 2 ml per minute, and the protein is eluted with 20 mM phosphate phosphate. sodium at pH 6.0 with 10 mM DTT. The fractions containing the denatured monomeric TPO protein which are eluted between 160 and 200 ml are pooled. The TPO protein is further purified on a semi-preparative C4 reverse phase column (VYDAC 2 x 20 cm). The sample is loaded at a rate of 5 ml / min on 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 min). The purified reduced protein is eluted when acetonitrile is about 50%. This material is used for refolding to obtain a biologically active variant of TPO. C. Folding TPO to Generate the Logically Active Form After solubilization and after further TPO purification, the biologically adactive form is obtained by refolding the denatured monomeric TPO in a redox buffer. Due to the high activity of TPO (a semi-maximal stimulation in the Ba / F3 assay is achieved at about 3 μg / ml), it is possible to obtain a biologically active material using many different conditions with respect to buffer, detergent and redox. However, under most conditions, just a small amount of properly folded material (<10k) is obtained. For industrial manufacturing processes, it is desirable to obtain refolding efficiencies of at least 10%, more preferably 30 to 50%, and especially

  more than 50 %. Many different detergents including Triton X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, sarkosyl, Tween 20 and Tween 80, Zwittergent 3-14 as well as others have proved suitable for the production of at least a little matter properly folded. Among them, however, the most popular detergents were those of the CHAPS family (CHAPS and CHAPSO), which proved to be the ones that worked best in the folding reaction and which best limited aggregation. protein and the inappropriate formation of disulfide. CHAPS proportions greater than about 1% have been preferred. The presence of sodium chloride was necessary to obtain the best yields,

  optimal proportions between 0.1 M and 0.5 M. The presence of EDTA (1-5 mM) in the redox buffer was appreciated to limit the degree of oxidation (and aggregation) catalyzed by a metal , which has been observed with certain preparations. Glycerol concentrations above 15% gave optimal conditions for refolding. For peak yields, it was essential that the redox buffer contain a redox pair consisting of both oxidized and reduced organic thiol (RSH). Suitable pairs of redox include mercaptoethanol,

  glutathione (GSH), cysteamine, cysteine and their corresponding oxidized forms. Oxidoreduction pairs that were preferred were glutathione (GSH) pairs: oxidized glutathione (GSSG) or cysteine: cystine. The most favored oxidation-reduction pair was glutathione (GSH): oxidized glutathione (GSSG). In general, high yields were observed when the molar proportion of the oxidized member of the oxidation-reduction pair was equal to or greater than that of the reduced member of that pair. PH values between 7.5 and about 9 were optimal for folding of these TPO variants. Organic solvents (eg ethanol, acetonitrile, methanol) have been tolerated

  at concentrations of 10 to 15% or less. Higher proportions of organic solvents have increased the amount of improperly folded shapes. Tris and phosphate buffers have generally been useful. Incubation at 40C also produced higher proportions of properly folded TPO. Folding yields of 40-60% (based on the amount of reduced and denatured TPO used in the refolding reaction) are representative of TPO preparations that were purified by the first step on C4. Active material can be obtained when less pure preparations are used (for example directly after the Superdex 200 column or after the initial extraction of refractile bodies), although the yields are lower as a result of precipitation and precipitation. accentuated foreign protein interference with TPO during the TPO refolding process.

  Since TPO contains 4 residues of cysteine, it is possible to generate three different disulphide versions of this protein: Version 1: disulphides between cysteine residues 1-4 and 2-3 Version 2: disulfide between residues of cysteine 1-2 and 3-4 Version 3: Disulfide between cysteine residues 1-3 and 2-4. During the initial scanning to determine the 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 by Ba / F3 assay. Then, the folding conditions were optimized in order to preferentially obtain the version concerned.

  Under these conditions, the poorly folded versions represented less than 10 to 20% of the total monomeric TPO obtained in the solubilization step. The disulfide unit for biologically active TPO was determined to be 1-4 and 2-3 by mass spectrometry and protein sequencing, the cysteines being sequentially numbered from the amino-terminus. This cysteine cross-linking motif is consistent with the known disulfide-disulfide unit of erythropoietin, which is a related molecule. D. Biological activity of recombinant folded TPO

  Refolded and purified TPO has activity in both in vitro and in vivo assays. For example, in the Ba / F3 assay, a semi-maximal stimulation of the thymidine incorporation to Ba / F3 cells for TPO (Met '1-153) was obtained at a dose of 3.3 μg. / ml (0.3 μM) in the mpl receptor-based ELISA, the semi-maximal activity occurring at 1.9 ng / ml (120 μM). In normal animals rendered myelosuppressed by a sub-lethal X-ray dose, folded TPO (Met-1-153) was very potent (the activity was observed at down to 30 ng / mouse) to stimulate the production of new platelets. Similar biological activity was observed for other folded forms of TPO according to the procedures described above (see Figures 25, 26 and 28).

  14. Thrombopolocyte Activity Measurement Methods Thrombopoietic activity can be measured in a variety of assays including the mpl Ba / F3 ligand assay described in Example 1, a mouse platelet rebound synthesis assay. vivo, a platelet cell surface antigen induction assay as measured by an anti-platelet immunoassay (anti-GPIIbIIIa) for a human leukemia megakaryoblastic cell line (CMK) (see Sato et al., Brit. J. Heamotol., 72: 184-190 [1989]) (see also the megakaryocytopoiesis suspension test in a liquid described in Example 4), and the induction of plyploidisation in a megakaryoblastic cell line ( DAMI) (see Ogura et al., Blood, 72 (1): 49-60 [1988]). The processing of megakaryocytes from immature, largely non-synthesizing DNA cells into morphologically identifiable eacaryocytes involves a process that involves the appearance of cytoplasmic organelles, the acquisition of membrane antigens (GPIIbIII), a endoreplication and platelet release as described in the preamble. A specific lineage promoter (i.e., the mpl ligand) of maturation should be expected

  megakaryocytes induce at least some of these changes in immature megakaryocytes, leading to platelet release and mitral thrombocytopenia. Therefore, assays have been developed to measure the emergence of these parameters in immature megakaryocyte cell lines, namely CMK and DAMI cells. The CMK assay (Example 4) measures the appearance of a specific platelet marker, GBIIbIII, and platelet budding. The DAMI assay (Example 15) measures endoreplication since ploidy enhancements are mature megakaryocyte fingerprints. The recognizable megakaryocytes have ploidy values of 2N, 4N, 8N, 16N, 32N, etc. Finally, the in vivo mouse platelet rebound assay (Example 16) is useful to demonstrate that administration of the test compound (in this case ligand for mpl) results in elevation of platelet numbers. Two other in vitro assays were developed to measure TPO activity. The first is a kinase receptor ELISA (KIRA) assay in which CHO cells are transfected with an mpl-Rse chimera and the phosphorylation of Rse tyrosine is measured by the assay.

  ELISA after exposure of the mpl portion of the chimera to the mpl ligand (see Example 17). The second is a receptor-based ELISA in which an anti-human rabbit IgG coated ELISA plate captures the mpl-IgG human chimeric receptor that attaches to the mpl ligand being assayed. A polyclonal rabbit antibody bound to biotin, against mpl ligand (TPOls5), is used to detect the bound mpl ligand that is measured by means of streptavidin-peroxidase as described in Example 18. 15. In vivo biological response of TPU-treated sublethal and normal-dose normal and sublethal-treated mice were treated with whole truncated TPO isolated from Chinese hamster ovary (CHO) cells, E. coli, and E. coli. coli and human embryonic kidney cells (293). The two forms of TPO produced in these three hosts stimulated platelet production in mice, however, whole OPO isolated from CHO appeared to produce the greatest response in vivo. These results indicate that proper glycosylation of the carboxy-terminal domain may be required for optimal activity in vivo. (a) E. coli-rhTPO (Met - ', 153)

  The "Met" form of the EPO domain (Met in position -1 plus the first 153 residues of human TPO) produced in E. coli (see Example 23) was injected daily into normal female C57 B6 mice as described in the captions. These Figures show that the unglycosylated truncated form of TPO produced in E. coli and refolded as described above is capable of stimulating approximately twice the platelet production in mice. without affecting the population of red blood cells or white blood cells. The same molecule injected daily into C57 B6 female mice that received sublethal irradiation ('37Cs) as described in the legends of Figs. 26A, B and C stimulated platelet separation and reduced nadir, but had no effect on erythrocytes or leukocytes. (b) CHO-rhTPO332

  The whole form of TPO produced in CHO cells and injected daily into normal female C57 B6 mice as described in the legends of Figures 27 A, B and C produced an approximately five-fold increase in platelet production in normal mice without affecting the population of erythrocytes or leucocytes. (c) CHO-rhTPO332; E. coli-rhTPOt-1lls3); 293- rhTPO332; and E. coli-rhTPO5ss Dose response curves were constructed for the treatment of normal mice with rhTPO from various cell lines (CHO-rhTPO332, E. coli-rhTPO (t-1.ls3); -rhTPO332; and E. coli-rhTPO! ss) as described in the legend relating to Figure 28. This figure shows that all the tested forms of the molecule stimulate the production of platelets, however the whole form produced in CHO cells possesses the greatest activity in vivo. (d) CHO-rhTPOIs3, CHO-rhTPO. and CHO-

  rhTPO332 Dose response curves were also constructed for the treatment of normal mice with various forms of rhTPO produced in CHO (CHO-rhTPO153, CHOrhTPO.cliw.and CHO-rhTPO332) as described in the legend relating to Figure 29 This figure shows that all the tested CHO forms of the molecule stimulate platelet production, but that the entire 70 kDa form has the greatest activity in vivo.

  6. General recombinant preparation of mpl ligand and variants The mpl ligand is advantageously prepared by standard recombination methods which involve the production of the mpl ligand polypeptide by culturing cells which have been reannected to express the mpl ligand nucleic acid (normally by transformation. cells with an expression vector) and separation of the polypeptide from the cells. However, it is optionally contemplated that the mpl ligand can be produced by homologous recombination, or with recombinant production methods using elements less introduced into cells already containing mpl ligand-encoding DNA. For example, a potent promoter / enhancer element, a suppressor or an exogenous transcription modulator element may be inserted into the genome of the subject host cell with sufficient proximity and orientation to influence the transcription of the DNA encoding the mpl ligand polypeptide. longed for. The control element does not encode the ligand mpl, the DNA being rather native to the genome of the host cell. Cells producing the receptor polypeptide of the present invention, or elevated or lowered levels of expression, as the case may be, are then selected. Thus, the invention relates to a method of producing mpl ligand which comprises the steps of inserting a transcription modulator element into the genome of a cell containing the mpl ligand nucleic acid molecule with sufficient proximity and orientation. the optional nucleic acid molecule to influence its transcription, with another optional step which comprises culturing the cell containing the transcription modulator element and the nucleic acid molecule. The invention also relates to a host cell containing the native mpl ligand nucleic acid molecule actively linked to exogenous control sequences recognized by the host cell.

  A. Isolation of the DNA encoding the mpl ligand polypeptide The DNA encoding the mpl ligand polypeptide can be obtained from any cDNA library prepared from a tissue presumed to have the ligand mRNA. mpl and expresses it at a detectable level. The ligand gene for mpl can also be obtained from a genomic DNA library or by in vitro oligonucleotide synthesis from the complete sequence of nucleotides or amino acids.

  The banks are sorted with probes designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries, suitable probes include monoclonal and polyclonal antibodies that recognize the mpl ligand and bind specifically to that ligand. Suitable probes for cDNA bands include oligonucleotides of about 20-80 bases in length that encode known or suspected portions of mpl ligand cDNA of the same species or different species; and / or complementary or homologous cDNAs or fragments thereof that encode the same or a similar gene. Suitable probes for sorting genomic DNA libraries include, but are not limited to, oligonucleotides, cDNAs or fragments thereof that encode the same or a similar gene, and / or homologous genomic cDNAs or fragments thereof. Sorting of the cDNA or genomic library with the chosen probe can be conducted by standard procedures as described in Chapters 10-12 of Sambrook, et al., Supra. Another way of isolating the mpl ligand gene is to use the PCR methodology as described in Chapter 14 of Sambrook et al., Supra. This

  method requires the use of oligonucleotide probes that hybridize to DNA encoding ligand mpl. Oligonucleotide selection strategies are described below. A preferred method for practicing the present invention is to use carefully selected oligonucleotide sequences to screen cDNA libraries from various tissues, preferably human or porcine kidney (adult or fetal) cell lines or of liver. For example, cDNA libraries of

  Human fetal liver cell lines are sorted with oligonucleotide probes. Alternatively, human genomic libraries can be sorted with the oligonucleotide probes. The oligonucleotide sequences selected as probes should be of sufficient length and should be sufficiently unambiguous to minimize the false positive data. The actual nucleotide sequence or sequences are usually designated on the basis of mpl ligand regions that have the least codon redundancy. Oligonucleotides can be degenerated at one or more positions. The use of degenerate oligonucleotides is of particular importance when screening a library from a species for which the preferential use of a codon is not known.

  The oligonucleotide must be labeled so that it can be detected by DNA hybridization in the library being sorted. The preferred method of labeling is to use ATP (e.g., y32p) and a polynucleotide kinase for radiolabeling the 5 'end of the oligonucleotide. However, other methods can be used to label the oligonucleotide, including but not limited to biotin labeling or enzymatic labeling. Of particular interest is the mpl ligand nucleic acid which encodes a ligand polypeptide for whole mpl. In some preferred embodiments, the nucleic acid sequence comprises the natural signal sequence of ligand mpl. The nucleic acid having the entire protein coding sequence is obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence. B. Amino acid sequence variants of natural mpl ligand

  Mpl ligand amino acid sequence variants are prepared by introducing appropriate variations of the uucleotides into the mpl ligand DNA, or by in vitro synthesis of the desired mpl ligand polypeptide. Such variants include, for example, deletions or insertions or substitutions of residues in the amino acid sequence for porcine mpl ligand. For example, the carboxy-terminal portions of the mature whole mpl ligand can be removed by proteolytic cleavage, in vivo or in vitro, or by cloning and expressing a fragment of the DNA encoding the entire mpl ligand to produce a biologically active variant . Any combination of deletion, insertion and substitution is performed to achieve final assembly, provided that this final assembly has the desired biological activity. Amino acid variations can also alter mpl ligand processes occurring after translation, for example, changing the number or position of glycosylation sites. For the design of amino acid sequence variants of the mpl ligand, the location of the mutation site and the nature of the mutation depend on the characteristic or characteristics of the mpl ligand to be modified. The mutation sites can be modified individually or in series, for example (1) by first substituting with conservative amino acid choices and then with more radical selections depending on the results obtained, (2) deletion of the residual target or (3) insertion of residues of the same or a different class near the localized site, or combinations of options 1-3.

  A useful method of identifying certain residues or regions of the mpl ligand polypeptide which are preferred sites for mutagenesis is referred to as "alanine exploration mutagenesis" as described by Cunningham and Wells in Science, 244: 1081. 1085 [19891. In this method, a residue or group of target residues is identified (eg charged residues such as arg, asp, his, lys and glu) and replaced by any amino acid, but preferably by a neutral amino acid or negatively charged (preferably alanine or polyalanine) to effect the interaction of the amino acids with the surrounding aqueous medium in the cell or outside the cell. These domains demonstrating a functional sensitivity to substitutions are then refined by the introduction of supplemental variants or other variants at the sites or for the substitution sites. Thus, although the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation itself does not need to be determined. For example, to optimize the performance of a mutation at a given site, alanine-based mutagenesis or random codon or target region mutagenesis is performed and the mpl ligand variants expressed for the optimal combination of the desired activity.

  There are two main variables in the assembly of amino acid sequence variants: the location of the mutation site and the nature of the mutation. For example, variants of the mpl ligand polypeptide comprise variants with respect to the ligand sequence for mpl, and may represent natural alleles (which do not require manipulation of mpl ligand DNA) or predetermined mutant forms obtained by mutation of the DNA, to arrive at an allele or variant not found in nature. In general, the location and nature of the chosen mutation depend on the character of the ligand mpl to be modified. Deletions of amino acid sequences generally range from about 1 to 30 residues, preferably from about 1 to 10 residues, and are normally contiguous. Alternatively, deletions of amino acid sequences for the mpl ligand may comprise a portion or all of the carboxy-terminal glycoprotein domain. Deletions of amino acid sequences may also include one or more of the first 6 amino-terminal residues of the mature protein. Optional deletions of amino acid sequences include one or more residues in one or more loop regions that exist between "helical beams". Contiguous deletions are usually made in even numbers of residues, but single or odd deletions fall within the scope of

  the invention. Deletions can be introduced into regions of low homology among mpl ligands that share the greatest sequence identity to alter mpl ligand activity. Either deletions can be introduced into regions of low homology among mpl ligand polypeptides of humans or other mammals, which share the most sequence identity with the human mpl ligand. Deletions of the mammalian mpl ligand polypeptide in areas of high homology with other mammalian mpl ligands are more likely to alter the biological activity of the mpl ligand to a greater extent. The number of consecutive deletions is chosen so as to preserve the tertiary structure of mpl ligands in the affected domain, for example a folded beta sheet or an alpha helix. Insertions of amino acid sequences include amino-terminal and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as internal insertions to sequences relating to single or multiple amino acid residues. Insertions within sequences (i.e., insertions within the mature mpl ligand sequence) can range generally from about 1 to 10 residues, more preferably from 1 to 5,

  in particular from 1 to 3. An example of a preferred fusion is that of an mpl ligand or a fragment of this ligand and another cytokine or its fragment. Examples of terminal insertions include a mature mpl ligand with an N-terminal methionyl residue, an artifact of the direct expression of mature mpl ligand in a recombinant cell culture, and a fusion of a hetero N-terminal signal sequence. - at the N-terminus of the mature mpl ligand molecule to facilitate the secretion of mature mpl ligand by recombinant hosts. Such signal sequences are generally obtained from the desired host cell species and are therefore homologous to this cell species. Suitable sequences include STII or Ipp for E. coli, alpha factor for yeast and viral signals

  such as herpes gD for mammalian cells. Other insertion variants of the mpl ligand molecule include fusion at the N- or C-terminus of mpl ligand of immunogenic polypeptides (i.e., non-endogenous for the host to which the fusion is administered), for example, bacterial polypeptides such as a beta-lactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, and C-terminal fusions with proteins having a long half-life. such as constant regions of immunoglobulins (or other regions of immunoglobulins), albumin or ferritin, as described in WO 89/02922 released to the Public Inspection on April 6, 1989. A third group variants comprise amino acid-substituted variants. These variants show the removal of at least one amino acid residue from the ligand molecule mpl in place of which a different residue is inserted. The sites of greatest interest for substitution mutagenesis include sites identified as mpl ligand active sites and sites where amino acids encountered in other analogues are significantly different in terms of side chain congestion, charge. or hydrophobic character, but where there is also a high degree of sequence identity at the chosen site

  between various species of mpl ligands and / or in the various animal analogs of a ligand mpl member. Other sites of interest are those in which particular residues of the ligand mpl from various representatives of a family and / or various animal species of the same representative are identical. These sites, especially those that fall into a sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner. These conservative substitutions are shown in Table 3 in

  column titled substitutions. If these substitutions result in a change in biological activity, more important changes, referred to as examples of substitutions in Table 3, or as described below with respect to amino acid classes, are introduced and the products are sorted. .

  TABLE 3 Residu Exeeples of Preferred Substitutions of Original Substitutions Ala (A) Val; Leu: Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Set Ser Gin (Q) Gln Aspn Asn Asp Asp Gly Pro Pro His (H) Asn; Gin; Lys; Arg Arg binds (I) Leu; Val; Met; To the; Phe; norleucine Leu Leu (L) norleucine; island; val; Met; Ala: Phe binds Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; Leu Phe (F) Leu; Val; binds; Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tire Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) binds; Leu; Met; Phe; To the; norleucine Leu Significant changes in the function or immunological identity of the ligand mpl are accomplished by the selection of substitutions which differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region. substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the size of the side chain. The natural residues are divided into groups based on the common properties of the side chains: (1) hydrophobic residues: norleucine, Met, Ala, Val, Leu,

  Island; (2) neutral hydrophilic residues: Cys, Ser, Thr; (3) acid residues: Asp, Glu; (4) basic residues: Asn, Gln, His, Lys, Arg; (5) residues that influence the orientation of the chain: Gly, Pro; and (6) aromatic residues: Trp, Tyr, Phe. Non-conservative substitutions involve the exchange of one representative of one of these classes for another. These substituted residues may also be introduced at the conservative substitution sites or, preferably, at the remaining (non-conserved) sites. In one embodiment of the invention, it is desirable to inactivate one or more protease cleavage sites that exist in the molecule. These sites are identified by the inspection of the encoded amino acid sequence, in the case of trypsin, for example for an arginyl or lysinyl residue. When protease cleavage sites have been identified, they are rendered inactive at

  proteolytic cleavage by replacing the target residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine; by deletion of the residue; or by inserting a prolyl residue immediately after the residue. In another embodiment, any methionyl residue other than the starting methionyl residue of the signal sequence, or any residue located at less than about 3 residues from the N-terminus or C-terminus relative to each of these residues. methionyl residues, is substituted by another residue (preferably according to Table 3) or deleted. Alternatively, about 1 to 3 residues are inserted at positions adjacent to these sites. Any cysteine residue not involved in maintaining the proper conformation of the mpl ligand can also be substituted, generally with serine, to improve the oxidation stability of the molecule and to prevent aberrant crosslinking. The first and fourth cysteines in the epo domain, numbered from the amino-terminal end, were found to be necessary to maintain the correct conformation but the second and third were not. As a result, the second and third cysteines in the epo system can be substituted.

  Nucleic acid molecules encoding mpl ligand amino acid sequence variants are prepared by various methods known in the art. Such methods include, but are not limited to, isolation from a natural source (in the case of natural amino acid sequence variants) or oligonucleotide-mediated (or site-directed) mutagenesis preparation , PCR mutagenesis and cassette mutagenesis of a previously prepared variant or a non-variant version of the mpl ligand polypeptide.

  Oligonucleotide-mediated mutagenesis is a preferred method for the preparation of substitution variants, deletion and insertion of mpl ligand DNA. This technique is well known in the art as described by Adelman et al., DNA, 2: 183 [1983]. Briefly, mpl ligand DNA is modified by hybridization of an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the sequence unmodified or natural DNA of ligand mpl. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template which will thus contain the primer of the oligonucleotide and which will encode the selected modification in the ligand mpl DNA. In general, oligonucleotides with a length of at least 25 nucleotides are used. An optimal oligonucleotide comprises 12 to 15 nucleotides which are entirely complementary to the matrix on either side of the nucleotide or nucleotides coding for the mutation. This ensures that

  the oligonucleotide correctly hybridizes to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized by techniques known in the art such as that described by Crea et al., Proc. Natl. Acad. Sci. USA, 75: 5765 [1978]. The DNA template can be generated by vectors which are derived from M13 bacteriophage vectors (the commercially available M13mp18 and M13mp19 vectors are suitable) or vectors which contain as replication origin a single-stranded phage as described by Viera et al., in Meth. Enzymol., 153: 3 [1987]. Thus, the DNA to be mutated can be inserted into one of these vectors to generate a single-stranded template. Production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al., Entitled Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, NY, 1989).

  Alternatively, a single-stranded DNA template can be generated by denaturing double-stranded (or other) plasmid DNA by conventional techniques. For modification of the natural DNA sequence (to generate, for example, amino acid sequence variants), the oligonucleotide is hybridized to the single-stranded template under suitable hybridization conditions. An enzyme polymerizing the DNA, usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis.

  Thus, a heterogeneous double-helix molecule is formed so that one strand of DNA encodes the mutated form of the ligand mpl and the other strand (the original template) encodes the unmodified natural sequence of the ligand mpl. This heterogeneous double-helix molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli JM101. After the cells have grown, they are plated on agarose plates and sorted using the 32-phosphate radioactivity labeled oligonucleotide primer to identify the bacterial colonies that contain the mutated DNA. The mutation region is then removed and placed in a suitable vector for the production of proteins, generally an expression vector of the type normally used for the transformation of a suitable host. The method just described can be modified such that a homo-duplex molecule is created in which both strands of the plasmid contain the mutation or mutations. The modifications are as follows: the single-stranded oligonucleotide is annealed to the single-stranded template as described above. A mixture of three deoxyribonucleotides, namely deoxyribo-adenosine (dATP), deoxyriboguanosine (dGTP) and deoxyribonucleotide

  thymidine (dTTP) is combined with a modified thiodesoxyribocytosine called dCTP- (aS) (available from Amersham Corporation). This mixture is added to the matrix-oligonucleotide complex. When DNA polymerase is added to this mixture, a DNA strand identical to the template, except for the mutated bases, is generated. In addition, this new strand of DNA contains dCTP- (aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion.

  After the template strand of the heterogeneous double helix has been cut with a suitable restriction enzyme, the template strand can be digested with the ExoIII nuclease or other appropriate nuclease beyond the region containing the site or sites to undergo mutagenesis. The reaction is then stopped leaving a molecule that is only partially rendered single-stranded. A complete double-stranded DNA omoduplex is then formed using DNA polymerase in the presence of the four deoxyribonucleotide triphosphates, ATP and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell such as E. coli JM101 as described above. DNA encoding mpl ligand mutants with more than one amino acid to be substituted can be generated according to one of several methods. If the amino acids occupy close positions in the polypeptide chain, they can be mutated simultaneously by the use of an oligonucleotide which encodes all of the desired amino acid substitutions. However, if the amino acids are at a distance from each other (separated by more than about 10 amino acids), it is difficult to generate a single oligonucleotide that encodes all the desired variations. We can instead use one of

  two alternative methods. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then circularized simultaneously to single-stranded template DNA, and the second strand of DNA that is synthesized from the template encodes all desired amino acid substitutions. The other method involves two or more times of mutagenesis to produce the desired mutant. The first assay is as described for single mutants: wild-type DNA is used for the template, an oligonucleotide encoding the first or the first desired amino acid substitutions is circulated in that template, and the Heterogeneous double-stranded DNA is then generated. The second mutagenesis assay uses mutated DNA produced in the first mutagenesis assay as a template. Thus, this matrix already contains one or more mutations. The oligonucleotide encoding the other one or other desired amino acid substitutions is then circularized into that template, and the resulting strand of DNA now encodes mutations of the first and second mutagenesis runs. This resulting DNA can be used as a template in a third resumption of mutagenesis, and so on.

  PCR mutagenesis, a chain reaction by the DNA polymerase, is also suitable for the production of amino acid variants of the ligand polypeptide mpl. Although the following comments relate to DNA, it should be noted that the technique also covers an application with RNA. The PCR reaction technique generally refers to the following procedure (see Erlich, supra, chapter authored by R. Higuchi, pp. 61-70): When small amounts of template DNA are used as the starting material in a PCR, primers that differ slightly, by their sequence, from the corresponding region in a template DNA can be used to generate relatively large amounts of a specific DNA fragment that differs from the template only in positions where the primers are different of the matrix. For introduction of a mutation into a plasmid DNA, one of the primers is designed to cover the position of the mutation and to contain the mutation; the sequence of the other primer must be identical to a segment of the sequence of the opposite strand of the plasmid, but this sequence may be located anywhere along the plasmid DNA. However, it is

  Preferably, the sequence of the second primer is less than 200 nucleotides from that of the first primer, so that ultimately the entire amplified region of the DNA delimited by the primers can be easily sequenced. PCR amplification using a pair of primers such as the one just described produces a population of DNA fragments that differ in the position of the mutation specified by the primer, and where appropriate in other positions, since the reproduction of the matrix is a bit subject to error.

  If the ratio of the matrix to the material constituting the product is extremely low, the vast majority of the product DNA fragments contain the desired mutation or mutations. This product is used to replace the corresponding region of the plasmid that has served as a PCR template according to conventional DNA technology. Mutations at separate positions can be introduced simultaneously by using a second mutant primer or by conducting a second PCR reaction with different mutant primers and simultaneously binding the two resulting PCR fragments to the vector fragment in a third partial ligation (or higher order). In a representative example of mutagenesis by

  PCR, the template plasmid DNA (1 μg) is linearized by restriction endonuclease digestion which has a unique recognition site in the plasmid DNA outside the region to be amplified. 100 μg of this material is added to a PCR mix containing PCR buffer, which contains the four deoxynucleotide triphosphates and which is included in the GeneAmp registered trademark kits (obtained from Norwalk's Perkin-Elmer Cetus Company). , CT and Emeryville, CA), and 25 pmoles of each oligonucleotide primer, to a final volume of 50 μl. The reaction mixture is covered with 35 μl of mineral oil. It is denatured for 5 minutes at 1000C, held briefly on ice and then 11l of Thermus aquaticus (Taq) DNA polymerase (5 units / gl, marketed by the firm Perkin-Elmer Cetus), is added below. of the mineral oil layer. The reaction mixture is then introduced into a cyclic DNA heat treatment apparatus (sold by Perkin-Elmer Cetus), programmed as follows: 2 min at 55 ° C to 72 ° C, then 19 cycles established as follows. : 0 s at 94 C 30 s at 550C, and 30 s at 72 ° C.

  At the end of the program, the reaction flask is removed from the thermal cycle apparatus and the aqueous phase is transferred to a new flask, extracted with phenol / chloroform (50:50 by volume) and ethanol precipitated. , and the DNA is collected by standard operations. This material is then subjected to appropriate treatments for insertion into a vector. Another method of preparing variants, namely cassette mutagenesis, is based on the technique described by Wells et al. in Gene, 34: 315 [1985]. The starting material is the plasmid (or other vector) comprising the mpl ligand DNA to be mutated. The codon or codons existing in the mpl ligand DNA to be mutated are identified. There must be a unique restriction endonuclease site on either side of the site or identified mutation sites. If none of these restriction sites exist, they can be generated using the oligonucleotide-mediated mutagenesis method described above for introduction into appropriate locations in

  ligand DNA mpl. After the restriction sites have been introduced into the plasmid, the plasmid is sectioned at these sites in order to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation or mutations is synthesized by standard procedures. The two strands are synthesized separately and hybridized together by conventional techniques. This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 3 'and 5' ends which are compatible with the ends of the linearized plasmid, so that it can be directly soldered to the plasmid. This plasmid now contains the mutated mpl ligand DNA sequence. C. Insertion of Nucleic Acid into a Replicable Vector The nucleic acid (e.g., cDNA or genomic DNA) encoding the naturally occurring mpl ligand polypeptide or

  variant is inserted into a replicable vector for subsequent cloning (amplification of DNA) or expression. Many vectors are available, and the choice of the appropriate vector depends on (1) what it should be used for DNA amplification or for DNA expression, (2) the size of the acid nucleic acid to be inserted into the vector and (3) the host cell to be transformed with the vector. Each vector contains various components according to its function (DNA amplification or DNA expression) and the host cell with which it is compatible. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. (i) Signal sequence component

  The mpl ligand of the present invention can not only be expressed directly, but also as a fusion to a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or of the polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the mpl ligand DNA to be inserted into the vector. The selected heterologous signal sequence must be recognizable and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells which do not recognize and process the mpl ligand signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected for example from the group of alkaline phosphatase, penicillinase, lpp or enterotoxin II leaders. For the secretion of yeast, the natural signal sequence can be replaced by, for example, yeast invertase, alpha factor or acid phosphatase leaders, the C. albicans glucoamylase leader (European Patent No. 362,179). placed on public inspection on 4 April 1990) or the signal described in WO 90/13646 released to the Public Inspection on 15 November 1990. In the expression of mammalian cells, the natural signal sequence ( i.e. the mpl ligand presequence which normally

  directs the secretion of ligand mpl from its natural mammalian cells in vivo) is satisfactory, although other mammalian signal sequences may be suitable, for example signal sequences of other mpl ligand polypeptides or the same mpl ligand from a different animal species, signal sequences from an mpl ligand and signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, e.g. herpes simplex signal gD . (ii) Origin of the Replication Component The two expression and cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. In general, in cloning vectors, the sequence is capable of allowing the vector to replicate independently of the chromosomal DNA of the host, and contains replication origins or autonomous replication sequences. These sequences are well known for various kinds of bacteria, yeasts and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the origin of the plasmid

  It is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors into mammalian cells. In general, the origin of the replication component is not necessary for mammalian expression vectors (the SV40 origin may for example be used only because it contains the early promoter). Most expression vectors are "shuttle" vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression. . For example, a vector is cloned into E. coli and then the same vector is transfected into yeast cells or mammalian cells for expression, even if it is not able to replicate independently of the chromosome. the host cell. DNA can also be amplified by insertion into the genome of a host. This is readily achieved by using species of the genus Bacillus as hosts, for example by including in the vector a DNA sequence which is complementary to a sequence found in genomic Bacillus DNA. Transfection of Bacillus with this vector results in homologous recombination with the genome and insertion of mpl ligand DNA. However, the separation of

  genomic DNA encoding the mpl ligand is more complex than that of an exogenously replicated vector, because restriction enzyme digestion is required to excise mpl ligand DNA. (iii) Selection Gene Component Expression and cloning vectors should contain a selection gene, which is also considered a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells that develop in a selective culture medium. Host cells not transformed with the vector containing the selection gene do not survive in the culture medium. Representative selection genes encode proteins which, (a) confer resistance to antibiotics or other toxins, eg ampicillin, neomycin, methotrexate, or tetracycline, (b)

  or (c) provide critical nutrients unavailable in complex media, for example, the gene encoding D-alanine racemase for representatives of the genus Bacillus. An exemplary selection scheme uses a drug to stop the growth of a host cell. Cells that are successfully transformed with a heterologous gene express a protein conferring drug resistance and thus survive the selection regime. Examples of this dominant selection use neomycin drugs (Southern et al., J. Molec Appl. Genet., 1: 327 [1982]), mycophenolic acid (Mulligan et al., Science, 209: 1422 [1980]). ]) and hygromycin (Sugden et al., Mol Cell Biol., 5: 410-413 [1985]). The three examples given above use bacterial genes under eukaryotic influence to transmit resistance to the appropriate drug, G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Examples of other suitable markers that can be selected for mammalian cells include those that allow for the identification of competent cells to bind the mpl ligand nucleic acid, such as

  dihydrofolate reductase (DHFR) or thymidine kinase. Mammalian cell transformants are placed under selection pressure such that only the transformants are remarkably survivable by attaching the marker. The selection pressure is imposed by culturing the transformants under conditions in which the concentration of selection agent in the medium is successively modified so as to lead to amplification of both the selection gene and the DNA which codes for the polypeptide. ligand mpl. Amplification is the process by which genes more in demand for the production of a growth-determining protein are renewed in tandem with the chromosomes of successive generations of recombinant cells. Increased amounts of mpl ligand are synthesized from the amplified DNA. For example, cells transformed with the DHFR selection gene are first identified by culturing all the transformants in a culture medium that contains methotrexate (Mtx), which is a competition antagonist against DHFR. . A suitable host cell when using wild type DHFR is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity prepared and propagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,

  77: 4216 [1980]. The transformed cells are then exposed to increasing rates of Mtx. This leads to the synthesis of multiple copies of the DHFR gene and, at the same time, multiple copies of other DNAs including the expression vectors, such as the mpl ligand encoding DNA. This amplification technique can be used with any other suitable host, for example CHO-K1 ATCC N CCL61, despite the presence of DHFR of endogenous origin if, for example, a DHFR mutant gene that is resistant to Mtx is used (patent European N 117 060). Alternatively, host cells [particularly wild-type hosts that contain endogenous DHFR] transformed or cotransformed with DNA sequences encoding a mpl ligand, the protein

  Wild-type DHFR, and another selectable marker such as aminoglycoside-3'-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for that selectable marker, such as a aminoglycoside antibiotic, for example kanamycin, neomycin or G418. See U.S. Patent No. 4,965,199. A suitable selection gene that can be used in yeast is the trpl gene present in yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 1979). Kingsman et al., Gene, 7: 141 [1979] or Tschemper et al., Gene, 10: 157 [1980]). The trp1 gene provides a selection marker for a mutant strain of yeast that is not able to grow in tryptophan, for example ATCC N 44,076 or PEP4-1 (Jones, Genetics, 85:12 [1977]). The existence of a trp1 lesion in the yeast host cell genome then creates an efficient environment for detecting growth transformation in the absence of tryptophan. Similarly, yeast strains deficient in Leu2 (ATCC

  Nos. 622 or 38,626) are supplemented by known plasmids carrying the Leu2 gene. (iv) Promoter Component Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is actively linked to the mpl ligand nucleic acid. Promoters are untranslated sequences located upstream (5 ') of the starting codon of a structural gene (generally within about 100 to 1000 base pairs) that control the transcription and translation of a particular sequence nucleic acids, for example the nucleic acid sequence of a ligand mpl, to which they are actively linked. These promoters normally fall into two categories, namely the inducible and constitutive ones. Inducible promoters

  are promoters that initiate increased levels of transcription from DNA under their influence in response to some variation in culture conditions, for example the presence or absence of a nutrient or a change in temperature . At present, a large number of promoters recognized by a variety of potential host cells are well known. These promoters are actively linked to the DNA encoding the mpl ligand by removing the promoter from the source DNA by restriction enzyme digestion and insertion into the vector of the promoter isolated sequence. The aturel ligand mpl promoter sequence and many heterologous promoters can be used to direct the amplification and / or expression of mpl ligand DNA. However, we prefer

  heterologous promoters, because they generally provide greater transcription and higher yields of the expressed mpl ligand compared to the natural ligand promoter mpl. Advantageous promoters for use with prokaryotic hosts include β-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615 [1978] and Goeddel et al., Nature, 281: 544 [1979]) alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 [1980] and European Patent No. 36,776) and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl Acad Sci USA, 80: 21-25 [1983]). However, other known bacterial promoters are suitable. Their nucleotide sequences have been published, allowing those skilled in the art to effectively weld them to DNA encoding a mpl ligand (Siebenlist et al., Cell 20: 269 (1980)) using Binders or adapters for creating any desired restriction sites Promoters for use in bacterial systems also contain a Shine-Dalgarno (SD) sequence effectively bound to the DNA encoding the mpl ligand polypeptide. Promoter sequences are known for eukaryotes.Ekaryotic genes all have an AT-rich region located approximately 25-30 bases upstream of the site where transcription is initiated.

  Sequence observed at 70-80 bases upstream of the start of transcription of many genes is a CXCAAT region where X can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence which may be the signal for the addition of the poly A tail to the 3' end of the coding sequence. All these sequences are advantageously inserted into eukaryotic expression vectors. Examples of suitable promoter sequences that can be used with yeast type hosts include promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv Enzyme Reg., 7: 149 [1968] and Holland, Biochemistry, 17: 4900 [1978]), such as enolase, glyceraldehyde-3-phosphate

  dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Other yeast promoters, which are inducible promoters having the further advantage of transcription under the influence of growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, phosphatase acid, degradation enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for the assimilation of maltose and galactose. Advantageous vectors and promoters for use in the expression of yeasts are further described by Hitzeman et al. In European Patent No. 76,657 A. Yeast activators are also advantageously used with yeast promoters. The transcription of mpl ligands by vectors into mammalian host cells is influenced, for example, by promoters obtained from virus genomes such as polyoma virus, avian diphtheria virus (British Patent Specification No. 2,211,504 on July 5, 1989), an adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, a cytomegalovirus, a retrovirus, the hepatitis B virus and in particular the virus

  simian 40 (SV40), by mammalian heterologous promoters, e.g. actin promoter or immunoglobulin promoter, by heat shock promoters and by the promoter normally associated with the mpl ligand sequence, provided that these promoters are compatible with the cellular systems of the host. Early and late promoters of SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 origin of replication (Fiers et al., Nature, 273: 113 [1978] Mulligan and Berg, Science, 209: 1422-1427 [1980], Pavlakis et al., Proc Natl Acad Sci USA 78: 7398-7402 (19811. The immediate early promoter of human cytomegalovirus is conveniently obtained as a Hind III restriction fragment E ( Greenaway et al., Gene, 18: 355-360 [1982]) A DNA expression system in mammalian hosts using bovine papillomavirus as a vector is disclosed in US Pat. 4,419,446. A modified version of this system is described in the United States patent.

  No. 4,601,978. See also Gray et al., Nature, 295: 503-508 [1982] for the expression of cDNA encoding human interferon in monkey cells; Reyes et al., Nature, 297: 598-601 [1982] regarding the expression of human 6-interferon cDNA in mouse cells under the influence of a thymidine kinase promoter derived from herpes simplex virus; Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79: 5166-5110 [1982] on the expression of the human interferon gene 61 in cells in culture of mice and rabbits; and Gorman et al., Proc. Natl. Acad. Sci. USA, 79: 6777-6781 [1982] regarding the expression of bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells and NIH-3T3 mouse cells, with the use, as a promoter, of the long terminal repeat of Rous sarcoma virus. (v) Activator Element Component The transcription of a DNA encoding the mpl ligand of the present invention by higher eukaryotes is often enhanced by the insertion of an activator sequence into the vector. Activators are cis-acting elements of DNA, usually ranging from about 10 to

  300 base pairs, which act on a promoter to increase its transcription. Activators are relatively independent of orientation and position because they have been observed at 5 '(Laimins et al., Proc Natl Acad Sci USA 78: 993 [1981]) and 3 '(Lusky et al., Mol Cell Bio, 3: 1108 [1983]) relative to the transcription unit, in an intron (Banerji et al., Cell, 33: 729 [19831) as well as in the coding sequence itself (Osborne et al., Mol Cell Bio., 4: 1293 [1984]). Many activator sequences from mammalian genes are now known (globin, elastase, albumin, α-fetoprotein

  and insulin). However, normally an activator from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the origin of replication (100-270 base pairs), the early cytomegalovirus activator, the polyoma activator on the late side of the origin of replication, and the adenovirus activators. See also Yaniv, Nature, 297: 17-18 (1982) for activating elements for the activation of eukaryotic promoters The activator can be spliced to the vector at the 5 'or 3' position with respect to the sequence encoding the ligand mpl, but is preferably located at a 5 'site

  from the promoter. (vi) Transcription Termination Component Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) also contain sequences necessary for transcription termination and for stabilization of messenger RNA. These sequences are commonly available from the 5 'and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mpl ligand encoding mRNA. (vii) Construction and analysis of vectors

  Construction of suitable vectors containing one or more of the above listed components employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, adapted and resolded in the desired form to generate the required plasmids. For assay for confirming correct sequences in assembled plasmids, the ligation mixtures are used to transform E. coli K12 strain 294 (ATCC N 31,446) and successful transformants selected according to ampicillin or tetracycline depending on the case. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion and / or sequenced by the method of Messing et al., Nucleic Acids Res., 9: 309 [1981] or by the method of Maxam et al. , Methods in Enzymology, 65: 499 [1980]. (viii) Transient expression vectors Expression vectors that provide transient expression in mammalian cells of DNA encoding the mpl ligand polypeptide are particularly useful in the practice of the present invention. In general, transient expression involves the use of an expression vector that is able to replicate efficiently.

  in a host cell, so that the host cell accumulates many copies of the expression vector and subsequently synthesizes high levels of a desired polypeptide encoded by the expression vector. (Sambrook et al., Supra, pages 16, 17 and 16-22). Transient expression systems, including a suitable expression vector and a host cell, allow for the convenient positive identification of cloned DNA encoded polypeptides, as well as the rapid screening of such polypeptides for biological and physiological properties. desired. Thus, transient expression systems are particularly useful in the present invention for the purpose of identifying mpl ligand polypeptide analogs and variants that have biological activity as a ligand polypeptide.

  for mpl. (ix) Suitable Examples of Vertebrate Cell Vectors Other methods, vectors and host cells which are suitable for adaptation to mpl ligand synthesis in a recombinant culture of vertebrate cells are described by Gething et al. in Nature, 293: 620-625 [1981]; Mantei et al., Nature, 281: 40-46 [1979]; Levinson et al. ; European Patent Nos. 117,060 and 117,058. A plasmid particularly useful for the expression of mpl ligand in mammalian cell culture is plasmid pRK5 (European Patent No. 307,247, US Patent No. 5 258,287) or plasmid pSV16B (PCT Publication No. WO 91/08291). D. Selection and transformation of host cells

  Host cells which are suitable herein for cloning or expression of the vectors are the prokaryotic, yeast or higher eukaryotic cells described above. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example E. coli, bacilli such as B. subtilis, species of the genus Pseudomonas such as P. aeruainosa, Salmonella typhimurium or Serratia marcescens. A preferred strain of E. coli as a cloning host is E. coli 294 (ATCC N 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC N 31,537) and E. coli W3110 (ATCC No. 27,325) are suitable. These examples are illustrative rather than limiting. Preferably the host cell should secrete minimal amounts of proteolytic enzymes. Alternatively, one can advantageously use in vitro cloning methods, for example the PCR reaction

  or other nucleic acid polymerase reactions. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable hosts for mpl ligand encoding vectors. The species Saccharomyces cerevisiae, or ordinary baker's yeast, is one of the lower eukaryotic host microorganisms that is most commonly used. However, a number of other genera, species and strains are commonly available and useful in this instance, for example Schizosaccharomyces Dombe (Beach and Nurse, Nature, 290: 140 [1981], European Patent N 139 383). Public Inspection May 2, 1985), hosts of the genus Kluyveromyces (US Patent No. 4,943,529) such as, for example, L, lactis (Louvenourt et al., J. Bacteriol. 737 [19831), K. fraeilis, K. bulcaricus, K. thermotolerans and K. marxianus, Yarrowia [European Patent No. 402,226], Pichia Dastoris (European Patent No. 183,070, Sreekrishna et al., J. Basic Microbiol. 28: 265-278 [1988], Candida, Trichoderma reesia (European Patent No. 244,234), Neurosvora crassa (Case et al., Proc Natl Acad Sci USA, 76: 5259-5263 [1979]). and filamentous fungi such as, for example NeurosDora, Penicillium,

  Tolypocladium (WO 91/00357 released to the Public Inspection on Jan. 10, 1991) and hosts of the genus Aspergillus such as A. nidulans (Ballance et al., Biochem Biophys Res Commun, 112: 284). 289 [1983], Tilburn et al., Gene, 26: 205-221 [19831, Yelton et al., Proc Natl Acad Sci USA, 81: 1470-1474 [1984]) and A. niqer (Kelly and Hynes, EMBo0, J., 4: 475-479 (1985).) Host cells suitable for expression of glycosylated mpl ligand are derived from multicellular organisms.These host cells are capable of complex activities. In principle, any culture of higher eukaryotic cells can be carried out whether it is a culture derived from a vertebrate or an invertebrate. Invertebrate cells include plant and insect cells, numerous baculovirus strains and variants thereof, and corresponding permissive host cells from hosts such as e Spodoptera fruaiDerda (caterpillar), Aedes aecrDti (mosquito, Aedes albopictus (mosquito), Drosophila melanogaster (Drosophila) and Bombyx mori have been identified. See, for example, Luckow et al., Bio / Technology, 6: 47-55 [1988]; Miller et al., Genetic Engineering, edited by Setlow et al., Volume 8 (Plenum Publi-

  shing, 1986), pages 277-279; and Maeda et al., Nature, 315: 592-594 [1985]. Various viral strains for trans- fection are available to the public, for example Autocrrapha californica NPV L-1 variant and Bombvx mori NPV strain Bm-5, and these viruses can be used in this case as a virus in accordance with the present invention, particularly for the transfection of Spodoptera fruai-Perda cells.

  Cultures of cotton plant cells, but, potato, soy, petunia, tomato and tobacco can be used as hosts. Normally, plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens, which has been previously manipulated to contain mpl ligand DNA. During the incubation of the plant cell culture with A. tumefaciens, the DNA encoding the mpl ligand is transferred to the host plant cell so that it is transfected and, under appropriate conditions, expresses the DNA. ligand mpl. In addition, regulatory sequences and signal sequences compatible with plant cells are available, for example promoter sequences of nopaline synthase and polyadenylation signal sequences (Depicker et al., J. Mol Appl. Gen., 1: 561 [1982]). In addition, DNA segments isolated from the upstream region of the T-gene 780 are capable of activating or increasing the transcriptional levels of plant-expressible genes in plant tissue containing DNA. recombinant. See European Patent No. 321,196 released to the Public Inspection on June 21, 1989.

  However, the greatest interest has been attached to vertebrate cells, and the propagation of vertebrate cells in culture (tissue culture) has become a classic process in recent years (Tissue Culture, Academic Press, published under the direction Kruse and Patterson (1973)). Examples of useful mammalian host cell lines are SV40 transformed monkey kidney line CV1 (COS-7, ATCC CRL 1651); the human embryonic kidney cell line (293 or 293 cells subcloned for growth in a suspension culture, Graham et al., J. Gen. Virol., 36:59 [1977]); renal cells of young hamsters (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 77: 4216 [1980]); mouse Sertoli cells (TM4, Mather, Biol Reprod., 23: 243-251 [1980]); monkey kidney cells (CV1 ATCC CCL 70);

  kidney cells of African green monkey (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse breast tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad Sci., 383: 44-68 [1982]); MRC 5 cells; FS4 cells; and the human hepatoma cell line (Hep G2). Host cells are transfected and preferably transformed with the expression or cloning vectors of the invention described above and cultured in a conventional nutrient medium suitably modified to induce promoters, select transformants or amplify the genes encoding the desired sequences.

  Transfection refers to the attachment of an expression vector by a host cell regardless of whether or not all coding sequences are actually expressed. Many methods of transfection are known to those skilled in the art, for example CaPO4 and electroporation. The success of transfection is generally recognized when any indication of the operation of this vector appears within the host cell. Transformation means the introduction of DNA into an organism such that the DNA is replicable, either as an extrachromosomal element or by a chromosomal integrant. Depending on the host cell used, the transformation is carried out according to standard techniques appropriate for such cells. Calcium chloride treatment employing calcium chloride, as described in section 1.82 of Sambrook et al., Supra, is generally used for prokaryotes or other cells that contain substantial barriers formed by their walls. The infection with Aarobacterium tumefaciens is used for the transformation of certain plant cells, as described by Shaw et al., In Gene, 23: 315 [1983] and in WO 89/05859 placed in public inspection on June 29, 1989. In addition, plants can be transfected using sonication as described in WO 91/00358 released to the Public Inspection on January 10, 1991.

  mammalian cells lacking such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52: 456-457 [1978], is advocated. General aspects of mammalian cell host system transformation have been described by Axel in U.S. Patent No. 4,399,216 filed Aug. 16, 1983. Yeast transformations are normally performed according to the method of US Pat. Van Solingen et al., J. Bact., 130: 946 [1977] and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 [1979]. However, other methods of introducing DNA into cells, for example by nuclear injection, electroporation or protoplast fusion, can also be used. E. Host Cell Culture The prokaryotic cells used to produce the mpl ligand polypeptide of the present invention are grown in suitable media as generally described in Sambrook et al., Supra.

  The mammalian host cells used to produce the mpl ligand of the present invention can be grown in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM), Sigma are suitable for cell culture. -hôtes. In addition, any of the media described by Ham and Wallace in Meth can be used as culture media for host cells. Enz. 58:44 [1979], Barnes and Sato, Anal. Biochem., 102: 255 [1980], U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; in WO 90/03430; WO 87/00195; in U.S. Patent Re. 30,985; or in United States Patent Application Nos. 07 / 592,107 or 07 / 592,141 both filed October 3, 1990, all of which are incorporated herein by reference. Any of these media may be supplemented if necessary with hormones and / or other growth factors (eg, insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium phosphate and

  magnesium), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as the proprietary drug Gentamycin), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations which should be known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those that have been previously used with the host cell chosen for expression, and are obvious to those skilled in the art. The host cells referred to herein include in vitro cells as well as cells that are in a host animal.

  F. Amplification / gene expression detection Gene amplification and / or expression can be measured directly in a sample, for example by the standard Southern blot analysis method, Northern blot analysis method for quantitative evaluation of messenger RNA transcription (Thomas, Proc Natl Acad Sci USA, 77: 5201-5205 [1980]), fingerprint analysis (DNA analysis) or hybridization in situ using a properly labeled probe, based on the sequences provided herein. Various markers may be used, most commonly radioisotopes, especially 32p. However, other techniques can also be used, for example biotin-modified nucleotides can be used by introducing them into a polynucleotide. Biotin then serves as a binding site for avidin or for antibodies, which can be labeled with a wide variety of markers, such as radionuclides, fluorescers, enzymes, and the like. Alternatively, antibodies capable of recognizing specific duplexes, including DNA duplexes, RNA duplexes and hybrid DNA-RNA duplexes or DNA-protein duplexes can be used. The antibodies can also be labeled, and the test can be conducted

  where the duplex is bound to a surface, such that after formation of the duplex on the surface, the presence of a duplex-bound antibody can be detected. Alternatively, the expression of a gene can be measured by immunological methods such as immunohistochemical staining of tissue sections and cell culture or body fluid assay, to directly obtain quantitative evaluation of the expression. of the gene product. For immunohistochemical staining techniques, a cell sample is prepared, normally by dehydration and fixation, and then reacted with labeled antibodies specific for the coupled gene product, where the markers are usually

  visually detectable, e.g., enzymatic markers, fluorescent markers, luminescent markers, etc. A particularly sensitive staining technique that can be advantageously used in the present invention is described by Hsu et al., In Am. J. Clin. Path., 75: 734-738 [1980].

  Antibodies useful for immunohistochemical staining and / or testing of sample liquids may be monoclonal or polyclonal and may be prepared in any mammal. These antibodies may advantageously be prepared against a native mpl ligand polypeptide or against a synthetic peptide based on the DNA sequences provided herein, as described below. G. Purification of mpl ligand polypeptide The mpl ligand is preferably isolated from the culture medium as a secreted polypeptide, although it can also be isolated from host cell lysates in the case of direct expression without secretory signal. When the mpl ligand is expressed in a recombinant cell other than a cell of human origin, this ligand is completely free of proteins or polypeptides of human origin. However, it is still usually necessary to purify the mpl ligand by stripping it of other recombinant cellular proteins or polypeptides to obtain preparations which are substantially homogeneous with respect to the mpl ligand itself. In a first step, the culture medium or the lysate is centrifuged to remove cellular debris in the form of particles. The membrane and the soluble fractions of proteins are then separated. Alternatively, a commercially available protein concentration filter (e.g. ultrafiltration units) can be used.

  Amicon or Millipore Pellicon). The mpl ligand can then be stripped of the soluble protein fraction and the membrane fraction of the culture lysate, depending on whether or not the mpl ligand is bound to the membrane. This ligand is then freed from soluble protein and polypeptide impurities by release and exchange or by chromatographic methods using various gel matrices. These matrices include acrylamide, agarose, dextran, cellulose and others commonly used for the purification of proteins. Examples of suitable chromatographic procedures for the purification of proteins include: immunoaffinity (e.g., anti-hmpl Mab ligand), affinity for receptors (e.g., mpl-IgG or

  Protein A Sepharose), hydrophobic interaction chromatography (HIC) (eg, ether, butyl- or phenyl-

  Toyopearl), lectin chromatography (eg Con A-Sepharose, lens lectin-Sepharose), size exclusion (eg Sephadex G-75), cation and anion exchange columns (eg DEAE example or carboxymethyl or sulfopropyl cellulose) and reverse phase high performance liquid chromatography (RP-HPLC) (see, for example, Urdal et al., J. Chromatog., 296: 171 [1984] two successive steps of RP-HPLC are used for the purification of recombinant human IL-2). Other purification steps include, alternatively: ethanol precipitation, ammonium sulfate precipitation, chromatofocusing; preparative gel electrophoresis of sodium dodecyl sulfate and polyacrylamide, etc. Mpl ligand variants in which residues have been deleted, inserted or substituted, are collected in the same manner as a natural mpl ligand, taking into account any significant changes in properties caused by the variation. For example, the preparation of a mpl ligand fused with another protein or polypeptide, for example a bacterial or viral antigen, facilitates purification; an immunoaffinity column containing an antibody against the antigen can be used to adsorb the fused polypeptide. Immunoaffinity columns such as a polyclonal rabbit anti-mpl ligand column can be used to absorb the mpl ligand variant by binding this variant to at least one remaining immune epitope. Alternatively, mpl ligand can be purified by affinity chromatography using mpl-IgG

  purified coupled to an immobilized resin (preferably), such as Affi-Gel (Bio-Rad, Richmond, CA), etc., by means well known in the art. A protease inhibitor such as phenylmethylsulfonyl fluoride (PMSF) may also be useful for inhibiting proteolytic degradation during purification, and antibiotics may be included to prevent the growth of accidental impurities. It will be understood by those skilled in the art that purification methods suitable for a natural mpl ligand may require modification to account for changes in the character of the mpl ligand or variants thereof upon expression in a recombinant cell culture. you.

  H. Covalent Modifications of mpl ligand polypeptides The covalent modifications of mpl ligand polypeptides are within the scope of the present invention. Both a natural mpl ligand and amino acid sequence variants of the mpl ligand can be covalently modified. One type of covalent modification within the scope of the present invention is a ligand fragment mpl. Variable mpl ligand fragments having up to about 40 amino acid residues can be conveniently prepared by chemical synthesis or enzymatic or chemical cleavage of the entire or variant mpl ligand polypeptide. Other types of covalently modifying the mpl ligand or its fragments are introduced into the molecule by reacting targeted amino acid residues of the mpl ligand or fragments thereof with an organic derivatization agent that is capable of reacting with side chains. selected or N-terminal or C-terminal residues.

  Most often, cysteinyl residues are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to form carboxymethyl or carboxyamidomethyl derivatives. The cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β- (5-imidazoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl and 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues are derived by reaction with diethyl pyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Parabromophenacyl bromide is also useful; the reaction is conveniently carried out in 0.1 M sodium cacodylate at pH 6.0.

  The lysinyl and amino-terminal residues are reacted with succinic acid anhydride or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other reagents suitable for the derivatization of amino-containing residues include imido esters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal, a chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and a reaction with a glyoxylate, catalyzed by a transaminase. The arginyl residues are modified by reaction with one or more conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be conducted under alkaline conditions because of the high functional guanidine group. In addition, these reagents can react with lysine groups, as well as with the epsilon-amino group of arginine.

  The specific modification of tyrosyl residues may be carried out with particular interest by introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most often, N-acetylimidazole and tetranitromethane are used to form the O-acetyl-tyrosyl and 3-nitro derivatives, respectively. The tyrosyl residues are iodinated with 125 I or 3 I to prepare labeled proteins for use in a radioimmunoassay, the chloramine T method described above being suitable. The carboxyl side groups (aspartyl and glutamyl) are selectively modified by reaction with carbodiimides (RN = C = N-R '), where R and R' are different alkyl groups, for example 1-cyclohexyl-3- ( -morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide. In addition, the aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Derivatization with bifunctional agents is useful for crosslinking ligand mpl to a water insoluble support matrix or surface for use in the anti-ligand mpl antibody purification process, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-esters, and the like.

  hydroxysuccinimide, for example esters formed with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate) and bifunctional maleimides such as bis-N-maleimido; -l, 8-octane. Derivatizing agents such as methyl-3 - [(p-azidophenyl) dithio] propiolimidate provide photoactivatable intermediates which are capable of forming crosslinks in the presence of light. Alternatively, water-insoluble reactive matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537 and 4,330,440 are used for immobilization of proteins.

  Glutaminyl and asparaginyl residues are frequently deamidated to the respective glutamyl and aspartyl residues. These residues are deamidated under neutral or basic conditions. The deamidated form of these residues is within the scope of the present invention. Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of α-amino groups of the lysine, arginine and histidine side chains (TE Creighton , Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pages 79-86 [1983]), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group. Another type of covalent modification of the mpl ligand polypeptide within the scope of the present invention involves modification of the natural glycosylation pattern of the polypeptide. The term modification means the removal of one or more carbohydrate moieties encountered in the natural mpl ligand and / or the addition of one or more glycosylation sites that are not present in the natural mpl ligand.

  Glycosylation of polypeptides is normally linked to nitrogen or in conjunction with oxygen. Nitrogen binding refers to attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline, are the recognition sequences of the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Oxygen-linked glycosylation refers to the attachment of one of the N-acetylgalactosamine, galactose or xylose sugars to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also to be used.

  The addition of glycosylation sites to the mpl ligand polypeptide is conveniently effected by modifying the amino acid sequence to contain one or more of the tripeptide sequences described above (for glycosylation sites bound to the nitrogen). The modification may also be effected by adding one or more serine or threonine residues to the natural mpl ligand sequence or substituting one or more of these residues for the bound glycosylation sites. to oxygen). For convenience, the amino acid sequence of the ligand mpl is advantageously modified by changes in the DNA, in particular by mutation of the DNA encoding the mpl ligand polypeptide at the location of preselected bases so as to generate codons that translate to the desired amino acids. The mutation or mutations of DNA can be carried out by methods described above under the title "Ligand Amino Acid Sequence Variants".

  Another way of increasing the number of carbohydrate moieties on the mpl ligand is through the chemical or enzymatic coupling of glycosides to the polypeptide. These operations are advantageous in that they do not require the production of the polypeptide in a host cell that has abilities for nitrogen-linked or oxygen-linked glycosylation. Depending on the coupling method used, the sugar or sugars can be attached (a) to arginine and histidine, (b) to free carboxyl groups, (c) to free sulfhydryl groups such as those in the cysteine, (d) free hydroxyl groups such as serine, threonine or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan, or (f) ) to the amide group of glutamine. These processes are described in WO 87/05330 released to the Public Inspection on September 11, 1987, and by Aplin and Wriston in CRC Crit. Rev. Biochem., Pp. 259-306 [19811.

  The removal of carbohydrate moieties present on the mpl ligand polypeptide can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the polypeptide to trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all of the sugars except for the binding sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., In Arch. Biochem. Biophys., 259: 52 [1987] and Edge et al., In Anal. Biochem., 118: 131 [1981]. Enzymatic cleavage of carbohydrate moieties on polypeptides can be effected by means of various endo- and exo-glycosidases as described by Thotakura et al., In Meth. Enzymol., 138: 350 [1987]. Glycosylation at potential glycosylation sites can be prevented by use of the tunicamycin compound as described by Duskin et al., J. Biol. Chem., 257: 3105 [1982]. Tunicamycin inhibits the formation of protein-N-glycoside bonds. Another type of covalent modification of the mpl ligand comprises binding of the mpl ligand polypeptide to one of a variety of non-protein polymers, for example polyethylene glycol, polypropylene glycol or polyoxyalkylene, as taught in the US Pat. Am. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. It should be noted that some sorting of the collected mpl ligand variant will be necessary to select the optimal variant to bind to the mpl receptor and having the defined immunological and / or biological activity. above. Sorting can be carried out for stability in a recombinant cell culture or in the plasma (for example against proteolytic cleavage), high affinity for the mpl member, oxidation stability, ability to be secreted in vitro. high yields, etc. For example, a change in the immunological character of the mpl ligand polypeptide, e.g. affinity for a given antibody, is measured by a competitive immunoassay. Other potential modifications of properties of proteins or polypeptides such as oxidation-reduction stability or thermal stability, hydrophobicity or susceptibility to proteolytic degradation are analyzed by methods well known in the art. 17. General Methods for the Preparation of Antibodies to an Mpl Ligand Preparation of Antibodies (i) Polyclonal Antibodies Polyclonal antibodies against mpl ligand polypeptides or fragments thereof are generally produced in animals by subcutaneous (sc) or intra- peritoneal (ip) multiple of ligand mpl and adjuvant. It may be useful to conjugate the mpl ligand or a fragment containing the amino acid target sequence to a protein that is immunogenic to the species to be immunized, for example keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor using a bifunctional agent or a derivatizing agent, for example the maleimidobenzoyl ester of

  succinimide (conjugation with cysteine residues), Nroxysuccinimide (with lysine residues), glutaraldehyde, succinic anhydride, SOC12 or a compound RIN = C = NR wherein R and R 'are different alkyl groups . Animals are immunized against the mpl ligand polypeptide or its fragment, conjugates or mmunogenic derivatives, by combining 1 mg or 1 μg of the peptide or conjugate (respectively for rabbits or mice) with 3 volumes of complete adjuvant of Freund and injection of the solution intradermally into multiple sites. One month later, the animals received subcutaneous booster injections at multiple sites of one-fifth to one-tenth of the initial amount of peptide or conjugate in Freund's complete adjuvant. 7 to 14 days later, the animals are bled, and the serum is analyzed for ligand-mpl antibody. The animals receive booster shots until the title is level. Preferably, the animals receive booster injections of the conjugate of the same mpl ligand, but conjugated to a different protein and / or via a different cross-linking reagent. Conjugates can also be prepared in recombinant cell culture as fusion proteins. Aggregating agents such as alum are also used to promote the immune response. (ii) Monoclonal antibodies

  Monoclonal antibodies are obtained from a predominantly homogeneous antibody population, i.e., the individual antibodies constituting the population are identical, except for naturally occurring mutations, which may be present in secondary quantities. Thus, the monoclonal epithet indicates the character of the antibody, that is, it is not formed of a mixture of distinct antibodies, for example, the monoclonal antibodies ligands for mpl of the invention can be prepared by the method of obtaining hybridomas originally described by Kohler and Milstein, in Nature, 256: 495 [1975] or can be prepared by recombinant DNA methods (U.S. Patent No. 4,816,567 (Cabilly et al.) In the method of obtaining hybridomas, a mouse or other suitable host animal such as a hamster is immunized as described above to obtain lymphocytes that produce or which are capable of producing antibodies that specifically bind to the protein used for immunization Alternatively, lymphocytes can be immunized in vitro, the lymphocytes are then fused with myeloma cells via a suitable welding such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 [Academic Press, 1986]).

  The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which advantageously contains one or more substances that inhibit the growth or survival of the unfused myeloma parent cells. For example, if the parental myeloma cells do not contain the enzyme called hypoxanthine-guanine-phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas normally contains hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT deficient cells. Preferred myeloma cells are those that fuse efficiently, support high level stable expression of an antibody by the selected cells that produce the antibody and are sensitive to a medium such as HAT medium. Among these, myeloma cell lines that are preferred are murine myeloma lines, such as those derived from mouse MOPC-21 and MPC-11 tumors available from

  Salk Institute Cell Distribution Center, San Diego, Calif., United States of America, and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland, United States of America. Human myeloma and murine-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 [1984], Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987). The culture medium in which hybridoma cells are grown is titrated for the production of monoclonal antibodies against the mpl ligand. The binding specificity of monoclonal antibodies produced by hybridoma cells is advantageously determined by immunoprecipitation or by in vitro binding assay, for example by radioimmunoassay (RIA) or by assay using an enzyme-linked immunoabsorbent ( ELISA). The binding affinity of the monoclonal antibody may, for example, be determined by Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 [1980]. After identifying the hybridoma cells that produce antibodies of specificity, affinity, and / or desired activity, the clones can be subcloned by limiting the dilution processes and culturing them by conventional methods (Goding, supra ). Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium or RPMI-1640 medium. In addition, hybridoma cells can be cultured in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are conveniently separated from the culture medium, ascites fluid or serum by standard immunoglobulin purification procedures, for example protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. The DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced by standard methods (e.g. using oligonucleotide probes that are capable of specifically attaching to genes encoding the heavy and light chains). mouse antibody). The hybridoma cells of the invention serve as the preferred source of such DNA. Once isolated, the DNA can be introduced into expression vectors which are then transfected into host cells such as monkey COS cells, Chinese hamster ovary cells (CHO) or myeloma cells that produce no not otherwise immunoglobulin-like protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells. The DNA may also be modified, for example, by replacing the homologous murine sequences with the coding sequence for heavy chain and light chain constant omaines (Cabilly et al., Supra, Morisson et al., Proc Nat Acad. Sci., 81: 6851 (1984)) or by covalently binding to the coding sequence of immunoglobulin all or part of the sequence encoding a non-immunoglobulin polypeptide. immunoglobulins are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of an antigen-combining site of an antibody of the invention to create a chimeric divalent antibody. comprising an antibody combining site having specificity for an mpl ligand and another antibody combining site having specificity for a different antigen.

  Chimeric or hybrid antibodies can also be prepared in vitro by known methods of use in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be assembled using a disulfide exchange reaction or by forming a thioether linkage. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate. For diagnostic applications, the antibodies of the invention are normally provided with a detectable label. The detectable marker may be any of those capable of directly or indirectly transmitting a detectable signal. For example, the detectable label may be a radioisotope such as 3H, 14C, 32p, 3s or 125I, a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine or luciferin; a radioactive isotopic label such as, for example, 125I, 32p, 4C or 3H or an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art to separately conjugate the antibody to the detectable label, including the methods described by Hunter et al., In Nature, 144: 945 [1962]; by David et al., in Biochemistry, 13: 1014 [1974]; by Pain et al., J. Immunol. Meth., 40: 219 [1981] and by Nygren in J. Histochem. and Cytochem., 30: 407 [1982]. The antibodies of the present invention can be used in any known assay method, for example in competitive binding assays, direct and indirect sandwich assays and immunoprecipitation assays. See Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987). Competitive binding assays are based on the ability of a labeled control (which may be a mpl ligand or an immunologically reactive portion of that ligand) to compete with the analyte of the test sample (mpl ligand). to bind to a limited amount of antibodies. The amount of mpl ligand in the sample tested is inversely proportional to the amount of control that binds to the antibodies. To facilitate determination of the amount of binding control, antibodies are usually insolubilized before or after competition, so that the antibody-related control and analyte can be conveniently separated from the control and the analyte remain unrelated. Sandwich type assays involve the use of two antibodies, each capable of binding to a different immunogenic portion or epitope of the protein (mpl ligand) to be detected. In a sandwich assay, the analyte of the test sample is bound by a first antibody which is immobilized on a solid support, then a second antibody binds to the analyte thereby forming an insoluble tripartite complex. See David and Greene, U.S. Patent No. 4,376

  110. The second antibody can itself receive a detectable marker (direct sandwich assays) or can be measured using an anti-immunoglobulin antibody that carries a detectable marker (indirect sandwich assay). For example, one type of sandwich assay is an ELISA, in which case the detectable label is an enzyme (eg, horseradish peroxidase). (iii) Humanized and Humanized Antibodies Human nonhuman antibody humanization methods are well known in the art. In general, a humanized antibody contains one or more amino acid residues incorporated therein from a non-human source. These non-human amino acid residues are often referred to as "imported" residues, which are normally borrowed from a variable "import" domain. Humanization can be carried out essentially according to the method of Winter et al. (Jones et al., Nature, 321: 522-525 [1986];

  Riechmann et al., Nature, 332: 323-327 [1988]; Verhoeyen et al., Science, 239: 1534-1536 [1988]) by substituting rodent CDRs or CDR sequences for corresponding sequences of a human antibody. Accordingly, these "humanized" antibodies are chimeric antibodies (Cabilly et al., Supra) in which a variable human domain, far from being intact, has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which certain CDR residues and, where appropriate, certain FR residues are replaced by residues from analogous sites in rodent antibodies. The choice of variable human domains, both light and heavy, to be used in the preparation of humanized antibodies is very important for reducing antigenicity. In accordance with the so-called "best fit" method, the variable domain sequence of a rodent antibody is compared by screening the entire library of known variable human domain sequences. The human sequence closest to that of the rodent is then accepted as a human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 [1993], Ghothia and Lesk, J. Mol Biol., 196: 901 [1987]). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc Natl Acad Sci USA, 89: 4285 [1992], Presta et al., J. Immunol., 151: 2623 [1993] ).

It is further important that antibodies be humanized by maintaining a high affinity for the antigen and other favorable biological properties. To achieve this result, according to a preferred method, humanized antibodies are prepared by a method of parental sequence analysis and various humanized concept products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformation structures of selected candidate immunoglobulin sequences. Examination of these displays allows the analysis of the probable role of residues in the operation of the candidate immunoglobulin sequence, i.e. the analysis of residues which influence the ability of the candidate immunoglobulin to attach to its antigen. By doing so, one can choose and combine FR residues of the consensus sequence and the imported sequence so as to obtain the desired antibody characteristic, such as greater affinity for the antigen or target antigens. . In general, CDR residues are directly and significantly involved in the influence on antigen binding. For further details, see U.S. Patent Application Serial No. 07 / 934,373 filed August 21, 1992, which is a continuation in part of Application No. 07 / 715,272 filed June 14, 1991.

  Alternatively, it is now possible to produce transgenic animals (eg, mice) that are capable, after immunization, of producing a whole repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been reported that the homozygous deletion of the (J =) region gene linking the antibody heavy chains in chimeric and germ-line mutant mice resulted in complete inhibition of endogenous antibody production. . The transfer of the human germline immunoglobulin gene series into such germ-line mutant mice results in the production of human antibodies by exposure to antigens. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-255 [1993]; Jakobovits et al., Nature, 362: 255-258 [1993]; Bruggermann et al., Year in Immuno., 7:33 (1993) Human antibodies can also be produced in phage display libraries (Hoogenboom and Winter, J. Mol., Biol., 227, 381. 1991], Marks et al., J. Mol Biol 222, 581 (1991). (Iv) Bispecific antibodies

  Bispecific antibodies are monoclonal antibodies, preferably human or humanized, which possess binding specificities to at least two different antigens. Methods for producing bispecific antibodies are known in the prior art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature, 305: 537). -539 [1983]). Because of the random assortment of heavy and light chains of immunoglobulins, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific stucture. The purification of the correct molecule, which is usually carried out by affinity chromatography steps, is rather laborious, and the product yields are low. Similar processes

  are disclosed in PCT Publication No. WO 93/08829 (published May 13, 1993) and in Traunecker et al., EMBO, 10: 3655-3659 [1991].

  In accordance with a different and more preferred approach, antibody variable domains having the desired binding specificities (antibody-antigen combining sites) are fused to constant domain immunoglobulin sequences. The welding is preferably carried out with an immunoglobulin heavy chain constant domain, comprising at least a portion of the CH2 and CH3 regions forming the intermediate segment. It is preferred that the first heavy chain constant region (CH1) containing the site necessary for the light chain binding be present in at least one of the welds. DNAs encoding the immunoglobulin heavy chain fusions and, where appropriate, the immunoglobulin light chain, are inserted into separate expression vectors and cotransfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the assembly provide optimal yields. However, it is possible to insert coding sequences for two of the three peptide chains or

  for the three chains in a single expression vector when the expression of at least two polypeptide chains in equal ratios makes it possible to obtain high yields and when the ratios are not of particular importance. In a preferred form of carrying out this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid heavy chain-immunoglobulin light chain pair (offering a second specificity link) in the other arm. It has been observed that this asymmetric structure facilitates the separation of the desired bispecific compound from undesired combinations of immunoglobulin chains, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule is an easy way to separation. This approach is disclosed in the United States patent application concurrently

  N 07/931,811 filed August 17, 1992. For further details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 [1986]. (v) Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention. They are composed of two antibodies covalently bound. Such antibodies have, for example, been proposed for targeting immune system cells to undesired cells (US Patent No. 4,676,980) and for treating HIV infection (PCT documents publication numbers WO 91/00360 and WO 92/00373;

  N 03089). Heteroconjugated antibodies can be prepared by any suitable methods of cross-linking. Agents suitable for the formation of cross-links are well known in the art and are disclosed in U.S. Patent No. 4,676,980, as well as a number of techniques for forming such bonds. IV. Therapeutic use of migacaryocytopoietic mpl protein ligand and biologically active mpl ligand having hematopoietic effector function and termed megakaryocytopoietic or thrombocytopoietic protein (TPO) herein can be used in a sterile pharmaceutical preparation or formulation to stimulate megakaryocytopoietic activity or Thrombocytopenia in patients with thrombocytopenia due to impaired production, sequestration and increased platelet destruction. Hypoplasia of the spinal cord associated with thrombocytopenia (eg aplastic anemia as a result

  chemotherapy or bone marrow transplantation) can be effectively treated with the compounds of the 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. In addition, these megakaryocytopoietic proteins may be useful in the treatment of myeloproliferative thrombocytosis and thrombocytosis due to inflammatory conditions and iron deficiency.

  The megakaryocytopoetic or thrombocytopolytic protein (TPO) of the present invention can be advantageously used in connection with myelotoxic chemotherapy, chemotherapy with bone marrow removal and thrombocytopenia due to bone marrow failure. Other disorders conveniently treated with the megakaryocytopoietic proteins of the present invention include platelet-related problems or platelet alteration resulting from drug absorption, poisoning, or activation on artificial surfaces. In these cases, the compounds of the invention can be used to stimulate the budding of new, unaltered platelets. For a more complete list of useful applications, see the preamble above, including paragraphs (a) through (f) and references cited therein.

  The megakaryocytopoietic proteins of the present invention may be used alone or in combination with other cytokines, hematopoietins, interleukins, growth factors or antibodies in the treatment of disorders and conditions identified above. Thus, the compounds of the invention may be used together with other proteins or peptides endowed with thrombopoietic activity, comprising: G-CSF, GM-CSF, LIF, M-CSF, IL-1, IL-3, erythropoietin (EPO) ligand kit, IL-6 and IL-1. The megakaryocytopolytic proteins of the present invention are prepared in admixture with a pharmaceutically acceptable carrier. This therapeutic composition can be administered intravenously or nasally or pulmonally. The composition may also be administered parenterally or subcutaneously, as appropriate. When the therapeutic composition is administered systemically, it must be pyrogen-free and in parenterally acceptable solution in view of pH, isotonicity and stability. These conditions are known to those skilled in the art. In short, dosage formulations

  compounds of the present invention are prepared for preservation or administration by mixing the compound having the desired degree of purity with physiologically acceptable carriers, excipients or stabilizers. Such materials are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, acetate and other organic acid salts; anti-oxidants such as ascorbic acid; peptides of low molecular weight (less than about 10 residues) such as polyarginine, proteins such as serum albumin, 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 alcohols, such as mannitol or sorbitol; complementary ions such as sodium and / or nonionic surfactants such as Tween, Pluronics and polyethylene glycol.

  About 0.5 to 500 mg of a compound or a mixture of the megakaryocytopolytic protein in the form of the acid or of the free base or in the form of a pharmaceutically acceptable salt are formulated with a carrier, a carrier, excipient, binder, preservative, stabilizer, flavor, etc., physiologically acceptable according to accepted pharmaceutical practice. The amount of active ingredient in these compositions is chosen so as to obtain a suitable dose in the indicated range. Sterile injectable compositions may be formulated in accordance with standard pharmaceutical practice. For example, it may be desirable to dissolve or suspend the active compound in a vehicle such as water or a natural vegetable oil such as sesame oil, peanut oil or cotton seed oil. or a synthetic fatty vehicle such as ethyl oleate, etc. Buffers, preservatives, antioxidants, etc. may be incorporated according to accepted pharmaceutical practice.

  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 [e.g. poly (2-hydroxyethylmethacrylate) as described by Langer et al., J. Biomed. Mater. Res., 15: 167-277 [1981] and Langer, Chem. Tech., 12: 98-105 [1982] or a vinyl alcohol polymer], polylactides (U.S. Patent No. 3,773,919, European Patent No. 58,481), L-acid copolymers. glutamic and gammaethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 [1983]), a non-degradable ethylene-vinyl acetate copolymer (Langer et al, supra), a lactic acid-glycolic acid copolymer degradable product such as Lupron Depot branded product (injectable microspheres composed of a lactic acid-glycolic acid and leuprolide acetate copolymer) and poly-D - (-) - 3-hydroxybutyric acid (European patent No. 133,988). Although polymers of the ethylene-vinyl acetate and lactic acid-glycolic acid type allow the release of molecules for more than 100 days, some hydrogels release proteins for shorter periods. When encapsulated proteins remain in the body for an extended period of time, they may denature or agglutinate as a consequence of exposure to moisture at 37 ° C, resulting in 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 one realizes that the aggregation mechanism is a formation of intramolecular SS bonds by disulfide exchange, one can achieve a stabilization by modification of the sulfhydryl residues, by lyophilization from acid solutions, by limiting the moisture content, by the use of appropriate additives and by the development of special polymeric matrix compositions. Megakaryocytopoietic sustained release protein compositions also include a megakaryocytopoietic protein entrapped in liposomes. Liposomes containing a megakaryocytopolytic protein are prepared by known methods: German Patent No. 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985), Hwang et al., Proc Natl Acad Sci USA, 77: 4030-4034 [1980], European Patents N 52 322, 36 676, 88 046, 143 949, Japanese Patent Application No. 83-118 008, US Patent No. 4,485,045 and US Patent 4,544,545, and European Patent No. 102,294. [0008] Ordinarily, the liposomes are of the small unilamellar type (approx. 200 - 800 angstroms) according to which the content of

  lipid is greater than about 30 mole% cholesterol, the selected proportion being adjusted for optimal therapy with the megakaryocytopoietic protein. The dosage is determined by the treating physician, taking into account various factors known for their ability to modify the action of drugs, including the severity and nature of the disease, body weight, sex, diet, time and the route of administration, the absorption of other drugs, as well as other relevant clinical factors. Normally, the daily dose is in the range of 0.1 to 100 μg / kg body weight. The dose is preferably in the range of 0.1 to 50 μg / kg body weight. Preferably, it is in the range of 1 to 5 gg / kg / day. Optionally, the dosage range is the same as that established for other cytokines, including G-CSF, GM-CSF and EPO. Therapeutically effective doses can be determined by in vitro or in vivo methods. nPTLES

  Without further details, it is believed that one skilled in the art can, using the foregoing description and practical examples, realize and exploit the present invention to the fullest extent. The following practical examples therefore specifically indicate preferred embodiments of the present invention, and should not be construed as in any way limiting the other parts of this specification. EXAMPLE 1 Partial Purification of Porcine Apl Ligand Platelet-poor plasma was collected on normal pigs or with aplastic anemia. Pigs were made aplastic by a total body irradiation of 900 cGy using a 4 MeV linear accelerator. Irradiated pigs were sustained for 6 to 8 days with intramuscular injections of cefazolin. Then, their total blood volume was removed under general anesthesia, supplemented with heparin and centrifuged at 1800 x g for 30 min to form platelet-poor plasma. The megakaryocyte stimulation activity was found to be up to 6 days after irradiation.

  The aplastic porcine plasma from irradiated pigs is adjusted to 4 M NaCl and stirred for 30 min at room temperature. The resulting precipitate is separated by centrifugation at 3800 rpm in a Sorvall RC3B centrifuge and the supernatant is loaded onto a Phenyl-Toyopearl column (220 ml) equilibrated in 10 mM NaPO4 containing 4 M NaCl. The column is washed with this buffer until the A2ie value is less than 0.05 and eluted with distilled water. The eluted protein peak is diluted with distilled water to a conductivity of 15 mS and loaded onto a balanced Blue Sepharose column (240 ml) in PBS. The column is then washed with 5 times its volume of PBS and 10 mM NaPO4 (pH 7.4) containing 2 M urea. The proteins are eluted from the column with 10 mM 4 NaPO4 (pH 7.4). ), containing 2M urea and 1M NaCl. The eluted protein peak is adjusted to 0.01% octylglucoside (n-octyl-O-D-glucopyranoside) and adjusted to 1 mM EDTA and Pefabloc ( Boehringer Mannheim) and loaded directly onto CD4-IgG twin columns (DJ Capon et al., Nature, 337: 527-531 [1989]) and mpl-IgG Ultralink (Pierce) (see below). The CD4-IgG column (2 ml) is removed after

  the sample was loaded and the mpl-IgG column (4 ml) was washed with 10 times its volume of PBS and as many PBS containing 2 M NaCl and eluted with 0.1 M glycine hydrochloride at pH 2 25. The fractions are collected in 1/10 of their volume of 1M Tris hydrochloride (pH 8.0). Analysis of fractions eluted from the mpl affinity column by sodium dodecyl sulfate and polyacrylamide gel electrophoresis (Novex 4-20% gel) under reducing conditions revealed the presence of several proteins (FIG. 5). . The proteins that silver colors with the highest intensity are resolved with apparent Mr values of 66 000, 55 000, 30 000, 28 000 and

  14,000. To determine which of these proteins stimulates the proliferation of Ba / F3-mpl cell cultures, these proteins were eluted from the gel as described in Example 2 below. Ultralink Affinity Columns At 10 mg 20 mg mpl-IgG or CD4-IgG in PBS at 0.5 g Ultralink resin (Pierce) according to the manufacturer's instructions. Construction and Expression of mpl-IgG A chimeric molecule comprising the entire extracellular domain of human mpl (amino acids 1-491) and the Fc region of a human IgG1 molecule was expressed in 293 cells. A cDNA fragment encoding amino acids 1-491 of human mpl was obtained by PCR in a cDNA library of human megakaryocytic CMK cells and was sequenced. A ClaI site was inserted at the 5 'end and a BstEII site was inserted at the 3' end. This fragment was cloned upstream of the Fc coding region of IgGI in a Bluescript vector between ClaI and BstEII sites after

  partial digestion of the PCR product with BstEII because of two other BstEII sites present in the DNA which encodes the extracellular domain of mpl. The BstEII site introduced at the 3 'end of the PCR mpl product was designed so that the Fc region is in frame with the extracellular domain mpl. The construct was subcloned into the pRK5-tkneo vector between the ClaI and XbaI sites and transfected into 293 human embryonic kidney cells by the calcium phosphate method. Cells were selected in 0.4 mg / ml G418 and individual clones were isolated.

  The mpl-IgG expression of isolated clones was determined by a human Fc-specific ELISA. The best expression clone had an expression level of 1-2 mg / ml mpl-IgG. Ba / F3 mpl P Expression Cells A cDNA corresponding to the entire coding region of human mpl P was cloned into the pRK5-tkneo vector which was then linearized with NotI and transfected into the IL-dependent Ba / F3 cell line. -3 by electroporation (1 x 107 cells, 9605F, 250 V). Three days later, selection was initiated in the presence of 2 mg / ml G418. The cells were selected in groups or individual clones were obtained by limiting dilution in 96-well plates. Selected cells were maintained in RPMI containing 15% FBS, 1 mg / ml G418, 20 mM glutamine, 10 mM HEPES and 100 μg / ml Pen-Strep. Expression of mpl P in selected clones was determined by FACS analysis using rabbit anti-mpl polyclonal rabbit antibody.

  Analysis of the Ba / F3 mpl ligand The mpl ligand assay was conducted as shown in Figure 2. To detect the presence of mpl ligand from various sources, the mpl P Ba / F3 cells were deprived of IL. For 24 hours at a cell density of 5 x 105 cells / ml in a humidified incubator at 37 ° C in air containing 5% Co2. After deprivation of IL-3, the cells were plated on 96 cell culture plates at a density of 50,000 cells in 200 μl of medium with or without diluted samples and cultured for 24 hours in a culture incubator. of cells. 20 μl of serum-free RPMI medium containing 1 μCi of 3H-thymidine was added to each well for each cell for the last 6-8 hours. The cells were then harvested on 96 cell GF / C filter plates and washed five times with water. The filters were counted in the presence of 40 μl of scintillation fluid (Microscint 20) in a Packard counter of the Top Count type.

  EXAMPLE 2 Pigmented purified porcine mpl ligand Gel elution protocol Equal amounts of affinity purified mpl ligand (fraction 6 eluted from mpl-IgG column) and Laemmli 2X sampling buffer were mixed at room temperature without reducing agent and the mixture was loaded as rapidly as possible on a Novex gel with 4- 20% polyacrylamide. The sample was not heated. As a control, a ligand-free sample buffer was passed through an adjacent lane. The gel was passed through at 4-6 ° C. at 135 V for about 2 hours and 15 minutes. The buffer that was flowing was initially at room temperature. The gel was then removed from the gel bundle and the plate one side of the gel was removed. A replica of the gel was made on microcellulose as follows: A piece of nitrocellulose was

  moistened with distilled water and carefully placed on the exposed face of the gel so as to exclude air bubbles. Markings were arranged as markers on the nitrocellulose and on the gel plate so that the replica could be put back in place precisely after staining. After about 2 minutes, the nitrocellulose was carefully removed and the gel was wrapped in a plastic sheet and placed in the refrigerator. The nitrocellulose was stained with BioRad Total Protein Gold Dye, by stirring firstly in 3 times 10 ml of 0.1% Tween 20 supplemented with 0.5 M NaCl and 0.1 M NaCl. of Tris hydrochloride at pH 7.5 for about 45 minutes, then in 3 x 10 ml of purified water for 5 minutes. The gold dye was then added and allowed to grow until the bands appeared visibly in the controls. The replica was then rinsed with water, placed on the plastic envelope surrounding the gel and carefully aligned with the markers. The positions of the Novex controls were marked on the gel plate and lines were drawn to indicate the cutting positions. The nitrocellulose and the plastic wrap were then removed and the gel was cut along the lines indicated, using

  Razor blade not blunted. The sections were extended beyond the strips formed by the samples so that they could be used to determine the positions of the slices after gel staining. After removal of the slices, the remaining gel was stained with silver and the positions of the controls and the slicing marks were measured. The molecular weights corresponding to the cutting positions were determined from the Novex references. The 12 gel slices were placed in the cells of two BioRad Model 422 electroelution devices. 12-14 K molecular weight exclusion caps were used in the cells. Elution contained 50 mM ammonium bicarbonate + 0.05% SDS (pH about 7.8). 1 liter of buffer was cooled for about 1 hour in a 4-6 C refrigerator before use. The gel slices were eluted 10 mA cell (initially 40 V) in a refrigerator at 4-6 C. Elution lasted about 4 hours. The cells have

  were carefully removed and the liquid above the frit was removed with a pipette. The elution chamber was removed and any liquid above the membrane cap was aspirated with a pipette. The liquid contained in the membrane cap was removed using a Pipetman and kept. Aliquots of 50 g of purified water were then placed in the cuff, shaken, and removed until all DS crystals were dissolved. These washings were pooled with the liquid kept above. The total volume of elution samples was 300-500 μl per gel slice. The samples were placed in a 12-14 K exclusion screening Spectrapor 4 dialysis tube which had been immersed for several hours in purified water. They were dialyzed overnight at 4-6 ° C against 600 ml phosphate buffered saline (PBS is

  approximately 4 mM potassium) for 6 samples. The buffer was replaced the next morning and dialysis was continued for 2.5 hours. Samples were then taken from the dialysis bags and introduced into microcentrifuge tubes. The tubes were held on ice for 1 hour, microcentrifuged at 14 K rpm for 3 minutes and the supernatants were carefully separated from the precipitated SDS. The supernatant liquids were then held on ice for a further 1 hour and microfuged again for 4 minutes. The supernatants were diluted in phosphate buffered saline and tested for activity. The remaining samples were frozen at -700C.

  EXAMPLE 3 Porcine mpl ligand microsuction Fraction 6 (2.6 ml) from the mpl-IgG affinity column was concentrated on a Microcon-O (Amicon) apparatus. To prevent the mpl ligand from being adsorbed on the Microcon, the membrane was rinsed with 1% SDS and 5 μl of 10% SDS was added to fraction 6. A sample buffer (20 μl) of 2 × a was added to the N 6 fraction after Microcon concentration (20 μl) and the total volume (40 μl) was loaded onto a single band of a 4-20% acrylamide gel (Novex). The gel operation was conducted according to the Novex protocol. The gel was then equilibrated for 5 minutes prior to the electrical transfer assay in 10 mM 3- (cyclohexylamino) -1-propanesulfonic acid (CAPS) buffer at pH 11.0 containing 10% methanol. Electron transfer analysis on Immobilon-PSQ membranes (Millipore) was carried out for 45 minutes under a constant current of 250 mA in a Trans-Blot transfer cell (32) from BioRad. The membrane

  PVDF was stained with 0.1% Coomassie Blue R-250 in 40% methanol containing 0.1% acetic acid for one minute and decolorized for 2-3 minutes with 10% acetic acid. in 50% methanol. The only visible proteins in the 18,000-35,000 MR value regions of the Fingerprint Diagram had MR values of 30,000, 28,000 and 22,000. 30, 28 and 22 kDa bands were sequenced. of proteins. Automatic protein sequencing was performed on an Applied Biosystem model 470A sequencing apparatus equipped with an on-line PTH analyzer. The sequencing apparatus was modified for injection of 80-90% of the sample (Rodriguez, J. Chromatogr., 350: 217-225 [1985]). Acetone (about 12 # 1/1) was added to the solvent A to equilibrate the ultraviolet absorption. The proteins analyzed by electrical transfer were sequenced in the Blott cartridge. Peaks were integrated with Justice Innovation software using Nelson Analytical 970 interfaces. Sequence interpretation was performed on a VAX 5900. Henzel et al., J. Chromatogr., 404: 41-52 (1987). ]). The N-terminal sequences (use of a letter code with undetermined residues in parentheses) and the amount of material obtained (in square brackets) are shown in Table 2 '.

  TABLE 2 Amino terminal amino ligand tp 30 kDa [1; 8 pmoq (S) PAPPA (C) DPRLLNKLLRDD (H / S) VLH (G) RL (SEQ ID NO: 30) 28 kDa [0; 5 pmol] (S) PAPPAXD PR LL NK LL R DD (H) VL (GR (SEQ ID NO: 31) 18-22 kDa [Oo5 pmol] 1 XPAPPAX DP R LX (N) (K) (SEQ ID NO: 32) E ": THE 4

  Liquid Suspended Megakaryocytopoiesis Assay Human peripheral stem cells (PSCs) (from consenting patients) were diluted 5-fold with IMDM (Gibco) and centrifuged for 15 minutes at 800 x g at room temperature. The cell pellets were resuspended in IMDM medium and loaded onto 60% Percoll (density 1.077 g / ml) armacia) and centrifuged at 800 x g for 30 minutes. Low density mononuclear cells were aspirated at the interface and washed twice with IMDM, and subcultured at 1-2 x 10 7 cells / ml in IMDM medium containing 30% FBS (final volume 1). ml) per tissue culture groups in 24 cells (Costar). APAP or depleted APP ligand mpl was added in 10% portion and cultures were grown for 12-14 days in a humidified incubator at 37 ° C in air containing 5% CO 2. The cultures were also grown in the presence of 10% APP with the addition of 0.5 g of mpl-IgG initially and the 2nd and 4th days. The APP had been depleted in mpl ligand by passage through an mpl-IgG affinity column. To quantitatively evaluate megakaryocytopolesis in these liquid suspension cultures, a modified version of the Solberg et al. and a radioactively labeled mouse IgG monoclonal antibody (HP1-1D) against GPIIbIIIa (procured by Dr. Nichols, Mayo Clinic) was used. 100 Mg of HII-1D (see B.

  Grant et al., Blood 69: 1334-1339 [1987]) were radioactively labeled with 1 mCi Na2sI using the product Enzymobeads (Biorad, Richmond, CA) according to the instructions given by the manufacturer. Radioactively labeled HP1-1D was stored at -70 ° C in PBS containing 0.01% octyl glucoside. Representative specific activities were 1-2 x 10G cpm / μg (more than 95% precipitation with 12.5% trichloroacetic acid). Liquid suspension cultures were established in triplicate for each experimental point. After 12 to 14 days of culture, the 1 ml cultures were transferred to 1.5 ml Eppendorf tubes and centrifuged at 800 xg for 10 minutes at room temperature, and the resulting cell pellets were resuspended at room temperature. 100 μl of PBS containing 0.02% EDTA and 20% calf serum. To the resuspended cultures was added 10 μg of 12 S-HPI-1 D in 50 μl of assay buffer and incubated for 60 minutes at room temperature (RT) with occasional shaking. Then, the cells were collected by centrifugation at 800 x g for 10 minutes at RT and washed twice with the assay buffer. Counting was done on the caps

  during i minute in a gamma counter (Packard). Nonspecific binding was determined by addition of 1 μg of unlabeled HP1-1D for 60 minutes prior to addition of labeled HP1-1D. Specific binding was determined from the bound total of 25I-HPI-1D minus the bound amount in the presence of excess unlabeled HP1-1D. EIEPLE 5 Oligonucleotide Proteins for PCR Based on the amino-terminal amino acid sequence obtained from 30 kDa, 28 kDa and 18-22 kDa proteins, degenerate oligonucleotides were designed for use as reaction primers. Polymerase chain reaction (PCR) (see Table 4). Two groups of primers were synthesized, namely a 20-member positive-sense group encoding amino acid residues 2-8 (mpl 1) and an antisense group of 21 complementary elements of sequences encoding amino acids 18-24 (mpl). 2).

  TABLE 4 Primer oligonucleotide primer groups mpl 1: 5 'CCN GCN CCN CCN GCN TGY GA 3' (factor of (SEQ ID N: degeneration 2048) 35) mpl 2: 5 'NCC RTO AR MAC RTG RTC RTC 3' ( factor (SUQ ID N: degeneration 2048) 36) Porcine genome DNA, isolated from porcine peripheral blood lymphocytes, was used as a template for the PCR reaction. The 50 μl of reaction mixture contained 0.8 μg of porcine genome DNA in 10 mM Tris hydrochloride (pH 8.3), 50 mM KCl, 3 mM MgCl 2, 100 μg / ml beef serum albumin. 400 μM dNTPs, 1 μM of each primer group and 2.5 units of Taq polymerase. The initial denaturation of the matrix was carried out at 940C for 8 minutes, followed by 35 cycles of 45 seconds at 940C, 174 2714670 for one minute at 550C and for one minute at 72 C. The final cycle was allowed to continue for 10 minutes at 72oC. The PCR reaction products were separated by 12 'polyacrylamide gel electrophoresis and visualized by staining with ethidium bromide. It was concluded that if the amino-terminal amino acid sequence was encoded by a single exon, then the correct PCR reaction product would have to be 69 base pairs. A DNA fragment of this size was eluted from the gel and subcloned into the plasmid pGEMT (Promega). The sequences of three clones are shown below in Table 5.

  TABLE 5 69 base pair gemT3 porcine genome fragments 5'CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA 3'GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATITAMTG ACGGAGCACT TGACCACGTT CAGCACGGC [69 bp] (SEQ ID NO: 37) ACTGG TGCAA GTC (SEO ID NO 38) gemT7 5'CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 3'GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATMAMTG ACGAAGCACI CGACCACGTC CATCACGGC [69 pbl (SEQ ID NO: 39) GCTGG TGCAGQ GTAGTG CCG (SEQ ID NO: 40) gemT9 - PRLLNKLLR (SEQ ID NO: 32) 5 'CCAGCACCGCCGGCATGTGACCCCCGACTCCTAAATAAACTGCTTCGTGACG 3' GGTCGTGGCGGCCGTACACTGGGGGCTGAGGATTTGAIrCGCGAAGCACTGCQ ATCATGTCTATCACGGT 3 '(SEQ ID NO: 41) TAGTACAGATAGTGCCA 5' (SEQ ID NO: 42) The position of the PCR primers is indicated by the underlined bases. These results verify the N-terminal sequence obtained for amino acids 9-17 for the 30 kDa, 28 kDa and 18-22 kDa proteins and indicate that this sequence is encoded by a single exon of pork DNA. wrEPLE 6 Human mpl ligand gene

  Based on the results of Example 5, a 45-mer deoxyoligonucleotide, called pR45, was designed and synthesized for sorting a genomic library. The 45-mer deoxy oligonucleotide had the following sequence: 5 'GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TFr-ATT-TAG-GAG-TCG 3' (SEO ID NO: 28) This oligonucleotide was 32 P-phosphorous labeled with (T32P) -ATP and T4 kinase and used for sorting a human genomic DNA library into Xgeml2 under weak hybridization and hybridization conditions. washing (see Example 7). Positive clones were picked, plaque purified and analyzed by restriction mapping and Southern blot analysis. Clone NO 4 was selected for another analysis. A 2.8 kb BamHI-XbaI fragment that hybridized to the 45-mer oligonucleotide was subcloned into pBluescript SK-. Partial DNA sequencing of this clone was performed using as primers oligonucleotides specific for the porcine ligand mpl DNA sequence. The resulting sequence confirmed that DNA encoding the human homolog of porcine ligand mpl was isolated. An EcoRI restriction site was detected in the sequence, which allowed the Applicant to isolate an EcoRI-XbaI fragment of

  390 base pairs of the 2.8 kilobase BamHI-XbaI fragment and subclone it to pBluescript SK-. Both strands of this fragment were sequenced. The human DNA sequence and the deduced amino acid sequence are shown in Figure 9 (SEQ ID N: 3 and 4). The predicted positions of introns in the genomic sequence are also indicated by arrows and define a presumed exon ("exon 3"). Examination of the predicted amino acid sequence confirms that a serine residue is the first amino acid of the mature mpl ligand, as determined from direct amino acid sequence analysis. Immediately upstream of this codon, the predicted amino acid sequence suggests to a high degree a signal sequence involved in the secretion of the mature mpl ligand. This region encoding the signal sequence is presumably interrupted at the position of nucleotide 68 by an intron. In the 3 'direction, it appears that the exon terminates with nucleotide 196. This exon therefore encodes a sequence of 42 amino acids of which 16 are likely part of a signal sequence and 26 are part of the mature human mpl ligand.

  EAMPLE 7 cDNA of the whole human apl ligand Based on the human "exon 3" sequence (Example 6), two non-degenerate oligonucleotides corresponding to the 3 'and 5' ends of the 3 "texon sequence were synthesized (Table 6) TABLE 6 Oligonucleate PM primers not derived from human cDNA 5 'GCT Primer AGC TCT AG AAT TGC TCC TCG TGG TCA TGC TTC T 3' SEQ ID NO: Direct: 4 43) 5 'CAG Primer TOT GCC GTG AAG GAC ATG G 3 '(SEQ ID NO: inverse 44 44)

  These two primers were used in PCR reactions using as template DNA from various human cDNA libraries or 1 ng of Quick Clone cDNA (Clonetech) from various tissues under the conditions described in Example 5 The expected length of the correct PCR product was 140 base pairs. After analysis of the PCR products on a 12% polyacrylamide gel, a DNA fragment of the expected length was detected in the cDNA libraries prepared from adult renal cells, 293 fetal kidney cells and CDNA prepared from human fetal liver (Clonetech Catalog No. 7171-1). A fetal DR2 (Clonetech HL1151x) fetal liver cDNA library was screened with the same 45-mer oligonucleotide as that used for sorting the human genomic library. The oligonucleotide was labeled with (γ32P) -ATP using T4 polynucleotide kinase. The bank was sorted under low stringency hybridization conditions. The filters were prehybridized for 2 hours and then hybridized with the probe overnight at 42 ° C in 20% formamide containing 5x SSC, Denhardt lox medium, 0.05 M sodium phosphate (pH 6.5). 0.1% sodium pyrophosphate, 50 μg / ml ultrasound-treated salmon sperm DNA, for 16 hours. The filters were then rinsed in 2x SSC and washed once in 0.1 SSC, 0.1% SDS at 42 ° C. The filters were exposed overnight to Kodak X-ray film. Positive clones were picked, plaque purified and the length of the insertion segment determined by PCR using oligonucleotides flanking the BamHI segment.

  XbaI Cloning in DR2 (Catalog No. Clonetech 6475-1). 5 μl of bacteriophage strain was used as the source constituting the matrix. The initial denaturation was carried out for 7 minutes at 94 ° C. followed by 30 amplification cycles (1 minute at 940 ° C., 1 minute at 520 ° C. and 1.5 minutes at 72 ° C.). The final extension was 15 minutes at 72 C. Clone N FL2b had a 1.8 kilobase insert and was chosen for subsequent analysis. The plasmid pDR2 (Clonetech, cloning of XDR2 and pDR2 and Expression System Library Protocol Handbook, page 42) contained in the end segments of bacteriophage XDR2 was released according to the instructions given by the manufacturer (Clonetech, cloning of XDR2 and pDR2 and Expression System Library Protocol Handbook, pages 29-30). Restriction analysis of plasmid pDR2-FL2b with BamHI and XbaI revealed the presence of an internal BamHI restriction site in the insertion segment occupying approximately the position

  650. Digestion of the plasmid with BamHI-XbaI cut the insertion segment into two fragments, one of 0.65 kilobase and the other of 1.15 kilobase. The DNA sequence was determined with three different classes of template derived from plasmid pDR2-FL2b. Sequencing of the double helix plasmid DNA was performed with the AB1373 automated fluorescent DNA sequencing apparatus (Applied Biosystems, Foster City, Calif.) According to standard protocols for dideoxynucleoside triphosphate terminators labeled with a dye (color terminators) and step initiators synthesized according to use (Sanger et al., Proc Natl Acad Sci USA, 74: 5463-5467 [1977], Smith et al., Nature, 321: 674-679 (1986)) Direct sequencing of plasmid fragments amplified by a polymerase chain reaction was performed with the AB1373 sequencing apparatus using reactions involving conventional initiators and dye-labeled terminators. single-stranded template was generated with the Janus vector

  M13 (DNASTAR, Inc. Madison, Wisconsin) (Burland et al., Nucl Acids Res., 21: 3385-3390 [1993]). BamHI-XbaI (1.15 kilobase) and BamHI (0.65 kilobase) fragments were isolated from plasmid pDR2-FL2b, the ends were filled with T4 DNA polymerase in the presence of deoxynucleotides, and then 2714670 179 under -cloned into the SmaI site of Janus M13. Sequencing was performed according to standard protocols for dye-labeled universal and reverse M13 initiators, or step initiators and dye-dyed terminators. Manual sequencing reactions were performed on single-stranded M13 DNA using step initiators and classical dideoxy terminator chemistry (Sanger et al., Proc Natl Acad.

  Sci. USA, 74: 5463-5467 [1977]), 33P phosphorus-labeled α-dATP and Sequenase (United States Biochemical Corp., Cleveland, Ohio). DNA sequence assembly was performed with the Sequencher V2.1b12 apparatus (Gene Codes Corporation, Ann Arbor, Michigan). The nucleotide sequence and deduced hML sequence are shown in Figure 1 (SEQ ID N: 1).

  EXAMPLE 8 Isolation of the gene (TPO) from the human mpl ligand Human genomic DNA clones of the TPO gene were isolated by sorting a human genomic library into X-GemI2 with pR45, the oligonucleotide probe already described, under conditions of low rigor (see Example 7) or under rigorous conditions with a fragment corresponding to the 3 'half of human cDNA encoding the mpl ligand (BamHI site at the 3' end). Two overlapping lambda clones covering 35 kilobases were isolated. Two overlapping (BamHI and EcoRI) fragments containing the entire TPO gene were subcloned and sequenced. The structure of the human gene is composed of 6 exons with 7 kilobases of genomic DNA (Figure 14A, B and C). The delineations of all exon / intron junctions are consistent with the consensus motif established for mammalian genes (M. B. Shapiro et al., Nucl Acids Res 15: 7155 [1987]). Exon 1 and exon 2 contain a 5 '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 in exon 3. The entire carboxyl domain and the untranslated 3'-half as well as about 50 amino acids of the erythropoietin-like domain are The four amino acids involved in the deletion observed in hML-2 (hTPO-2) are encoded at the 5 'end of exon 6.

  EXAMPLE 9 Transient expression of mpl-human ligand (hXL) In order to subclone the entire insert segment contained in pDR2-FL2b, the plasmid was digested with XbaI to completion and then partially digested with BamHI. A DNA fragment corresponding to the 1.8 kilobase insert was gel purified and subcloned into pRKS (pRKS-hmpl I) (see U.S. Patent No. 5,258,287 for US Pat. construction of pRK5) under the influence of the immediate early promoter of cytomegalovirus. The DNA of the pRKS-hmpl I assembly was prepared by the polyethylene glycol method and transfected into 293 human embryonic kidney cells maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with F-12 nutrient mixture. 20 mM HEPES (pH 7.4) and 10% fetal calf serum. The cells were transfected by the calcium phosphate method as described by C. Gorman [1985] in DNA Cloning: A Practical Approach (published under the direction of DM Glover), Volume II, pages 143-190, IRL Press, Washington. , DC). 36 hours after the transfection, the supernatant fluid of the transfected cells was

  subjected to activity titration in the proliferation assay (see Example 1). The supernatant fluid of 293 cells transfected with the pRK vector simply showed that there was no stimulation of Ba / F3 or Ba / F3-mpl cells (FIG. 12A). The supernatant fluid of cells transfected with prK5-hmpl I had no effect on Ba / F3 cells but dramatically stimulated proliferation of Ba / F3-mpl cells (Figure 12A), indicating that this cDNA encodes a human mpl ligand endowed with functional activity.

  EaPLE 10 human tp ligand h] L2, hIL3 and hL4 ligands In order to identify alternatively spliced hML forms, the initiators corresponding to each end of the hML coding sequence were synthesized. These initiators were used in the polymerase chain reaction at room temperature to amplify human adult liver RNA. In addition, internal initiators flanking selected regions of interest (see below) were constructed and used in the same way. Direct sequencing of the ends of the PCR reaction product revealed the existence of a single sequence exactly corresponding to the sequence of the cDNA isolated from the human fetal liver library (see FIG. 1 [SEQ ID NO: 1] ). However, a region near the C-terminus of the EPO domain (in the middle of the PCR reaction product) exhibited a complex sequence motif suggesting the existence of possible splice variants in this region. To isolate these splice variants, the initiators shown in Table 7 flanking the region of interest were used in a PCR reaction as templates for human adult liver cDNA.

  TABLE 7 Phropi.odna.3e1 human XL isoform PCR initiators: 5 "TGTGGACTTTAGCTTGGGAGAATG3 '(SEQ ID NO: 45) pbx4.f2: 5'GGTCCAGGGACCTGGAGGTTrG3' SEQ ID NO: 46) The PCR reaction products were subdivided into cloned with M13 blunt ends Sequencing of individual subclones revealed the existence of at least 3 LM isoforms.One of them, hML (also called hML332), is the most long and corresponds exactly to the isolated sequence of the fetal liver library.The sequences of the four human mpl ligand isoforms listed from the longest (hML) to the shortest (hML-4) are shown in Figure 11 [SEQ ID N: 6, 8, 9 and 10].

  EXAMPLE 11 Construction and transient expression of human mpl ligand isoforms and hML2, hML3 and hML substitution variants (R153A, R154A) The hML2 and hML3 isoforms and the hML substitution variant (R153A, R154A) were reconstituted from hML using the recombinant PCR technique described by Russell Higuchi in PCR Protocols, A Guide to Methods and Applications, Acad. Press, edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White. In all assemblies, the "external" initiators used are shown in Table 8 and the "overlapping" initiators are shown in Table 9.

  TABLE 8 External Initiators Cla.FL.F2: 5'ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G3 '(SEQ ID NO: 47) HMPLL-R: 5GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA3' (SEQ ID NO: 47) ID NO: 48) TO ~~~~~~~ -

  TABLE 9 Initiatora overlapping hML-2: MLa4.F: 5'CTC CTT GGA ACC AGC GGC AGG ACC 3 '(SEQ ID NO: 49) ML.4.R 5'GGT CCT GCCG CTG GGT TCC AAG GAG 3' (SEO ID NO 50) hML-3: hML 116+: 5'CTG CTC CGA GGA AAG GAC TTC TGG ATT 3 '(SEQ ID NO: 51) hMLA1 16-: 5'AAT CCA GMAA GTC CTT TCC TCG GAG CAG 3' ( SEO ID NO: 52) hML (R153A.R154A): RR-KO-F: 5'CCC TCT GCG TCG CGG CGG CCC CAC CCA C 3 '(SEO ID NO: 53) RR-KO-R: 5'GTG GGT GGG GCC GCC GCG ACG CAG AGG G3 '(SEQ ID NO: 54) All PCR amplifications were performed with cloned Pfu DNA polymerase (Stratagen) using the following conditions: the initial denaturation of the matrix was carried out at 94 ° C. for 7 minutes, followed by 30 cycles of 1 minute at 94 ° C., 1 minute at 55 ° C. and 1.5 minutes at 72 ° C. The last cycle was allowed to continue for 10 minutes at 72 ° C. C. The final PCR product was digested with ClaI-XbaI, gel purified and cloned into pRKStkneo. 293 cells were transfected with the various assemblies described above and the supernatant was titrated using the Ba / F3-mpl proliferation assay. The hML-2 and hML-3 isoforms did not show detectable activity in this assay, however the hML (R153A, R154A) activity was similar to that of hML, indicating that the treatment at this dibasic site is not necessary for the activity (see figure 13).

  EXAMPLE 12 Mouse mpl ligand cDNA mML, iML-2 and mUL-3 mML cDNA isolation A DNA fragment corresponding to the entire coding region of the human mpl ligand was obtained by PCR, purified on gel and random primed in the presence of 32P-dATP and 32P-dCTP This probe was used to screen 106 clones of a mouse liver cDNA library in XGT10 (Clontech catalog number ML3001a). of filters were hybridized in 35% formamide containing 5x SSC, 10x Denhardt's medium, 0.1% SDS, 0.05M sodium phosphate (pH 6.5), 0.1% pyrophosphate of sodium, 100 μg / ml sonicated ultrasound sperm DNA overnight in the presence of the probe The filters were rinsed in 2x SSC and washed once in 0.5x SSC 0.1 % of SDS at 42 ° C. The hybridization bacteriophages were plaque purified and the cDNA inserts were subcloned into the EcoRI site of plasmid B1. uescript SK The "LD" clone with a 1.5 kilobase insert was chosen for further analysis and both strands were sequenced as described above for human ML cDNA. . The nucleotide sequence and deduced amino acid sequence from the LD clone are shown in Figure 14 (SEQ ID N: 1 and 11). The deduced mature ML sequence derived from this clone had a length of 331 amino acid residues and was identified as mML33X (OR mML-2 for reasons described below). Considerable identity for the nucleotide sequence and the deduced amino acid sequence was observed in the EPO-like domains of these isoforms

  ML. However, when the deduced amino acid sequences of the human and murine ML isoforms were aligned, it was found that the murine sequence exhibited a tetrapeptide deletion between the 111-114 human residues corresponding to the 12 nucleotide deletion following the position of nucleotide 618 appearing in the human (see above) and porcine cDNAs (see below). As a result, other clones were examined for possible murine ML isoforms. One of the clones, L7 ", had a 1.4 kilobase insertion segment with a deduced sequence of 335 amino acids containing the missing" missing "tetrapeptide LPLQ.This form is believed to be the entire murine ML isoform and is called mML or mML33s The nucleotide sequence S and the deduced amino acid sequence for mML are shown in Figure 16 (SEQ ID NO: 12 and 13) Finally, the "L2" clone was isolated and sequenced. deletion of 116 nucleotides corresponding to

  hML3 and is therefore called mML-3. A comparison of the deduced amino acid sequences of these two isoforms is shown in Figure 16. Expression of recombinant mML. Expression vectors for the murine ML isoform were prepared essentially as described in Example 8. Clones encoding mML and mML-2 were subcloned into the plasmid pRKStkneo, a mammalian expression vector that performs the following functions: expression under the influence of the CMV promoter and an SV40 polyadenylation signal. The resulting expression vectors, mMLpRKtkneo and mML2pRKtkneo were exposed to transient transfection in 293 cells by the calcium phosphate method. After the transient transfection, the medium was conditioned for five days. Cells were maintained in high glucose DMEM medium, supplemented with 10% fetal calf serum. Expression of murine mpl (mmpl) in Ba / F3 cells. Stable cell lines expressing c-mpl were obtained by transfection of mmpl-pRKtkneo, essentially as described for human mpl in Example 1. Briefly, an expression vector (20 Mg linearized), containing the coding sequence whole murine mpl (RC Skoda et al.

  EMBO, J. 12: 2645-2653 [1993]) was transfected into Ba / F3 cells by electroporation (5 x 10 'cells, 250 volts, 960 pF), followed by selection vis-à-vis -vis resistance to neomycin with 2 mg / ml of G418. The mpl expression was assessed by flow cytometric analysis using mouse anti-mpl-IgG rabbit antisera. Ba / F3 cells were maintained in RPMI 1640 medium from WEHI-3B cells as the source of IL-3. Supernatant fluids from 293 transiently transfected cells with mML and mML-2 were analyzed in BaF3 cells transfected with mmpl and hmpl as described in Example 1. Inp ~ 13 pML ligand tLP ligand pML and pML- 2

  Porcine ML (pML) cDNA was isolated by RACE PCR reaction. Briefly, an oligo dT initiator and two specific initiators were designed based on the exon sequence of the porcine ML gene encoding the amino terminus of purified ML from aplastic pig serum. CDNA prepared from various aplastic pig tissues was obtained and amplified. A 1,342 base pair PCR reaction product cDNA was found in the kidney and subcloned. Several clones were sequenced and found to encode the mature pork mpl ligand (not containing complete secretion signal). CDNA was found to encode a mature protein of 332 amino acids (pML332) having the sequence shown in Figure 18 (SEQ ID NO: 9 and 16).

  Procedure: isolation of the pML gene and cDNA. Genomic clones of the porcine ML gene were isolated by screening an EMBL3 porcine genomic library (Clontech Inc.) with plasmid pR45. The library was sorted essentially as described in Example 7. Several clones were isolated and the exon encoding the amino acid sequence identical to that obtained from purified ML was sequenced. The porcine ML cDNA was obtained from a modified version of the RACE PCR reaction protocol. Two specific ML initiators were designed based on the porcine ML gene sequence. Polyadenylated messenger RNA was isolated from the kidney of aplastic pigs essentially as described above. The cDNA was prepared by reverse transcription with the BamDT ~~~ 5 ~ initiator (BamdT: 5'GACTCGAGGATCCATCGAI I), (SEQ ID NO: 55) directed against the mRNA tail-forming polyadenosine. An initial PCR amplification run (28 cycles of 60 seconds at 950C, 60 seconds at 580C and 90 seconds at 72 C) was conducted using the ML-specific direct h-fwd-1 primer or initiator (h -fwd-1: 5'GCTAGCTCTAGAAATTGCTCCTCGTGGTCATGCTTCT 3) (SEQ ID NO: 43) and the BAMAD primer or initiator (BAMAD: 5 'GACTCGAGGATCCATCG 3) (SEQ ID NO: 56) in a reaction volume of 100 ml (50 mM KCl, 1.5 mM MgCl 2, 10 mM Tris pH 8.0, 0.2 mM dNTP, with 0.05 U / ml Amplitaq polymerase [Perkin Elmer Inc.]). The PCR reaction product was then digested with Call, extracted with phenol-chloroform (1: 1), ethanol precipitated and ligated to 0.1 mg of Bluescript SK- vector (Stratagene Inc.) which had been cut with Clal and Kpn 1. After incubation for 2 hours at room temperature, a quarter of the ligation mixture was added directly at a second resumption of the PCR reaction (22 cycles as described above) using a second direct primer

  ML-specific (fwd-1: 5 'GCTAGCTCTAGAAGCCCGGCTCCTCCTGCCTG 3) (SEQ ID NO: 57) and T3-21 (oligonucleotide which binds to a sequence adjacent to the multiple cloning region in the Bluescript SK- vector): (5 CGAAATTAACCCTCACTAAAG 3 ') (SEQ ID NO: 58). The resulting PCR reaction product was digested with XbaI and ClaI and subcloned into Bluescript SK-. Several clones from independent PCR reactions were sequenced. Again, a second form, called pML-2, encoding a protein with a deletion of 4 amino acid residues (328 amino acid residues), was identified (see Figure 21 [SEQ ID NO: 21]). A comparison of the amino acid sequences of pML and pML-2 shows that the second form is identical, except that the QLPP tetrapeptide corresponding to residues 111-114 inclusive has been deleted (see FIG. 22 [SEQ ID NOS: 18 and 21]). . The deletions of four amino acids observed in murine, human and porcine ML cDNA appear precisely in the same position in the predicted proteins.

  E: = OPLE 14 Thrombopoltin CM Spruce (TPO), induction of GPIbIII platelet antigen expression, CMK cells are maintained in RMPI 1640 medium (Sigma) additionally containing 10% fetal calf serum and 10 mM glutamine. In the test preparation, the cells were harvested, washed and resuspended at a density of 5 x 10 5 cells / ml in serum free GIF medium supplemented with 5 mg / l of blinding. beef line, 10 mg / l apo-transferrin, and trace elements (1 x). Using a 96-well flat bottom plate, the TPO control or experimental samples are added to each cell at appropriate 100-fold dilutions. 100 ~ 1 of the CMK cell suspension is added to each well and the plates are incubated at 37 ° C in a 5% CO2 incubator for 48 hours. After incubation, the plates are centrifuged at 1000 rpm at 40C for 5 minutes. The supernatant liquids are discarded and 100 μl of the anti-GPIIbIII monoclonal antibody 2D2 conjugated to a FITC is added to each well. After incubation 40C for 1 hour, the plates are again centrifuged at 1000 rpm for five minutes. Supernatant liquids containing the unbound antibody are discarded and 200 μl of 0.1% serum albumin wash solution in PBS are added to each well. The washing step with 0.1% BSA serum albumin is repeated three times. The cells are then analyzed on a FASCAN device using the standard single-parameter analysis measuring the relative fluorescence intensity.

  EXAMPLE 15 Thrombopolin (TPO) DAMI Assay by Measurement of the Endomitotic Activity of DAMI Cells on 96-Cell Microtiter Plates DAMI cells were maintained in IMDM medium supplemented with 10% horse serum (Gibco) with in addition 10 mM glutamine, 100 ng / ml penicillin G and 50 μg / ml streptomycin. For the assay, the cells were harvested, washed and resuspended at a density of 1 × 10 7 cells / ml in IMDM medium supplemented with 1% horse serum. In a 96-well round bottom plate, 100 μl of the TPO control or experimental samples is added to the DAMI cell suspension. The cells are then incubated for 48 hours at 37 ° C. in an incubator containing 5% CO 2. After the incubation, the plates are centrifuged in a Sorvall 6000B centrifuge

  at 1000 rpm for five minutes at 4 ° C. The supernatant liquids are discarded and the washing step is repeated with 200 g of PBS 0.1% beef serum albumin. The cells were fixed by adding 200 M1 of 70% PBS ice-cold ethanol and resuspended by aspiration. After incubation at 40 ° C. for 15 minutes, the plates are centrifuged at 2000 rpm for 5 minutes and 150 μl of RNAse at 1 mg / ml containing 0.1 mg / ml of propidium iodide and 0, are added to each well. 05% Tween 20. After incubation for one hour at 37 ° C., the variations in the DNA content are measured by flow cytometry. Polyploidy is measured and quantitatively evaluated as follows: (T cells in G2 + N / cells% in <G2 + M) with TPO Normalized ratio of polyplo [of (RNPI - (cells% dars> G2 + N / cells % in <G2 * N) in the control

  EXAMPLE 16 Thrombopoletin In Vivo (TPO) Assay (Mouse Platelet Bounce Assay) In Vivo Assay for 3% S Determination of Platelet Production C57BL6 (from Charles River) mice were injected intraperitoneally ( IP) 1 ml goat anti-platelet mouse serum (6 amps) on the first day to induce thrombocytopenia. On the 5th and 6th days, the mice are administered in two intraperitoneal injections the factor or PBS as a control. On the 7th day, 30 μCi of Na23SSO4 was injected intravenously into 0.1 ml of physiological saline and the percentage of 35S incorporation of the injected dose into the circulating platelets was measured in blood samples taken from treated mice. and control mice. Platelet counts and leukocyte counts were performed at the same time on blood taken from the retro-orbital sinus.

  EXAMPLE 17 Thrombopoline (TPO) KIRA ELISA Assay by Measurement of Phosphorylation in the Chimeric Receptor mpl-Rue.gD The human mpl receptor was described by Vigon et al., PNAS, USA 89: 5640-5644 (1992). A chimeric receptor comprising the extracellular domain (ECD) of the mpl receptor and the transmembrane (TM) and intracellular (ICD) domain of Rse (Mark et al., J. of Biol Chem 269 (14): 10720-10728 [1994] ]) with a carboxyl-terminated polypeptide (ie Rse.gD) was prepared for use in the KIRA ELISA assay described herein. See Figures 30 and 31 for a schematic description of the assay. (a) Preparation of a capture agent A monoclonal anti-gD (clone 5B6) was produced against a peptide derived from Herpes simplex virus glycoprotein D (Paborsky et al., Protein Engineering 3 (6): 547-553 (1990)) The purified mother preparation was adjusted to 3.0 mg / ml phosphate buffered saline (PBS) at pH 7.4 and 1.0 ml aliquots were stored at - 200C. (B) Preparation of an anti-phosphor tyrosine antibody

  Monoclonal anti-phosphotyrosine, clone 4G10, was purchased from UBI (Lake Placid, NY) and combined with biotin using a long end segment biotin-N-hydroxysuccinimide (Biotin-X-NHS). , Research Organics, Cleveland, OH). (c) Ligand The mpl ligand was prepared by the recombinant techniques described herein. The purified mpl ligand was stored at 40C as stock solution. (d) Preparation of nucleic acid Rse.gD double stranded synthetic oligonucleotides were used to reconstitute the coding sequence for the 10 C-terminal amino acids (880 - 890) of human Rse and to add 21 other amino acids containing an epitope for antibody 5B6 and a stop codon. Table 10 shows the final sequence of the synthetic portion of the fused gene.

  TABLE 10 Synthetic double-stranded portion of the gene fused Rae human coding strand: 5'-TGCAGCAAGGGCTACTGCCACACTCGAGCTGCGCAGATGCTAGCCTCAAGA TGGCTGATCCAAATCGATTCCGCGGCAAAGATCTTCCGGTCCTGTAGAAGCT-3 '(SEQ ID NO: 59) non-coding strand (antisense): 5'.AGCTTCTACAGGACCGGAAGATCImTGCCGCGGAATCGAMTGGATCAGCCA TCTTG AGGCTAGCATCTGCGCAGCTCGAGTGTGGCAGTAGCCCTTGCTGCA-3' ( SEQ ID NO: 60) Synthetic DNA was ligated to the cDNA encoding human Rse 1-880 amino acids at the PstI site beginning at nucleotide 2644 of the published human Rse cDNA sequence (Mark et al. Journal of Biological Chemistry 269 (14): 10720-10728 [1994]) and at the HindIII sites of the multiple linker of the expression vector pSV17.ID.LL (see Fig. 32 AL, SEQ ID N: 22) for create

  the expression plasmid pSV.ID.Rse.gD. Briefly, the expression plasmid comprises the dicistronic primary transcript which contains the DHFR coding sequence delimited by the intron splice sites, 5 'donor and 3' acceptor, followed by a sequence which encodes the Rse.gD. . The entire (unspliced) message contains DHFR as the first open reading frame and therefore generates the DHFR protein to allow the selection of stable transformants. (e) Preparation of nucleic acid mpl-Rse.gD The expression plasmid pSV.ID.Rse.gD produced in the manner described above was modified to produce the plasmid pSV.ID.M.tmRd6 which contained the coding sequences of human mpl ECD (amino acids 1-491) fused to the transmembrane domain and to the intracellular domain of Rse.gD (amino acids 429-911). Synthetic oligonucleotides were used to bind the coding sequence of a portion of the extracellular domain of human mpl to a portion of the Rse coding sequence in a two-step PCR cloning reaction as described by Mark et al. in J. Biol. Chem. 67: 26166-26171 (1992). The primers or initiators used for the first PCR reaction were as follows: M1 (5'-TCTCGCTACCGTTTACAG-3 ') (SEO ID NO: 61) and M2 (5'-CAGGTACCCACCAGGCGGTCTCGGT-3') (SEQ ID NO: 62) with a cDNA template of mpl and R1 (5'-GGGCCATGACACTGTCAA-3 ') (SEQ ID NO: 63) and R2 (5'GACCGCCACCGAGACCGCCTGGTGGGTACCTGTGGTCCTT-3') (SEQ ID NO: 64) with a cDNA template of cSR. The PvuII-SmaI portion of

  this fused junction was used for assembly of the entire chimeric counter. (f) Transformation of cells CP12.CHO cells (European Patent No. 307,247 published March 15, 1989) were electroporated with the plasmid pSV.ID.M.tmRd6 which had been linearized at a unique NotI site of its structure. The DNA was ethanol precipitated after phenol / chloroform extraction and resuspended in 20 μl of 1/10 Tris EDTA. Next, 10 g of DNA was incubated with 107 Chinese hamster ovary DP12 cells in 1 ml of PBS on ice for 10 minutes before electroporation at 400 volts and 330 gf. The cells were reintegrated for a period of 10 minutes before being seeded in a non-selective medium. After 24 hours, the cells were supplemented with nucleoside-free medium for the selection of stable DHFR + clones. g) Selection of Transformed Cells for Use in the KIRA ELISA Assay The MPL / Rse.gD expression clones were identified by western-blot analysis of whole cell lysates after fractionation by sodium dodecyl sulfate and sodium dodecyl sulfate gel electrophoresis. polyacrylamide using the 5B6 antibody which detects the identity of the gD epitope. (h) Medium The cells were cultured in F12 / DMEM 50:50 (Gibco / BRL, Life Technologies, Grand Island, NY). The medium additionally contained 10% diafiltered FBS (HyClone, Logan, Utah), 25 mM HEPES and 2 mM L-glutamine. (i) KIRA ELISA

  DP12.CHO cells transformed with mpl-Rse.gD were seeded (3 × 10 4 per well) into the cells of a flat-bottomed culture dish 96 cells in 100 g medium and cultured overnight. at 370C in the presence of 5% CO2. The following morning, supernatant liquids from the cells were decanted and the plates were gently pressed onto paper towels. Fifty grams of medium containing experimental samples or 200, 50, 12.5, 3.12, 0.78, 0.19, 0.048 or 0 ng / ml of ligand mpl were then added to each well. The cells were stimulated at 37 ° C. for 30 minutes, the supernatants of the cells were decanted and the plates were again gently pressed onto paper towels. To lyse the cells and solubilize the chimeric receptors, 100 g of lysis buffer was added to each well. The lysis buffer consisted of 150 mM NaCl containing 50 mM HEPES (Gibco), 0.5% Triton-X 100 (Gibco), 0.01% thimerosal, 30 kIU / ml aprotin (ICN Biochemicals , Aurora, OH), 1 mM 4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride (AEBSF; ICN Biochemicals), 50 μM leupeptin (ICN Biochemicals) and 2 mM sodium orthovanadate (Na3VO4; Sigma Chemical Co., St. Louis, MO), pH 7.5. The plate was then shaken gently on a plate shaker (Bellco Instruments, Vineland, NJ) for 60 minutes at room temperature. As the cells solubilized, an ELISA microtiter plate (Nunc Maxisorp, Inter Med, Denmark) coated overnight at 4 ° C anti-gD 5B6 monoclonal antibody (5.0 μg / ml in 50 mM carbonate, pH 9.6, 100 μl / cell) was decanted, pressed onto a paper towel pad and blocked with 150 μl / well of buffer buffer [PBS containing 0.5% BSA (Intergen Company,

  Purchase, NY) and 0.01% thimerosal] for 60 minutes at room temperature with gentle shaking. After 60 minutes, the anti-gD 5B6 coated plate was washed 6 times with wash buffer (PBS containing 0.05% Tween 20 and 0.01% thimerosal) using an automatic washing apparatus. of plates (ScanWasher 300, Skatron Instruments, Inc., Sterling, VA). The lysate containing the solubilized MPL / Rse.gD from the cell culture microtiter wells was transferred (85 l / well) into an anti-gD 5B6 coated ELISA cell and blocked and incubated for 2 hours at room temperature. the ambient temperature with moderate agitation. Unbound mpl-Rse.gD was washed out with buffer and 100 μl of biotin-bound 4G10 (anti-phosphotyrosine) diluted 1:18,000 in diluent buffer (PBS containing 0.5%). of beef serum albumin, 0.05% Tween-20, 5 mM EDTA and 0.01% thimerosal), i.e. 56 ng / ml were added to each well. After incubation for 2 hours at room temperature, the plate was washed and 100 # 1 of horseradish peroxidase-conjugated streptanidin (HRPO) (Zymed Laboratories, San Francisco, CA) diluted 1:60 000. in a diluent buffer were added to each well. The plate was incubated for 30 minutes at room temperature while moderately shaking. The avidin-free conjugate was removed by washing and 100 μl of the positively prepared substrate solution (tetramethylbenzidine [TMB]; 2-component substrate composition; Kirkegaard and

  Perry, Gaithersburg, MD) were added to each cell. The reaction was allowed to continue for 10 minutes, after which the color development was stopped by the addition of 100 μl / well of 1.0M H3PO4. Absorption at 450 nm was read with reference wavelength of 650 nm (ABS4s, / 6s0) using a vmax plate reader (Molecular Devices, Palo Alto, CA) under the control of a Macintosh Centris 650 computer (Apple Computers, Cupertino, CA) and DeltaSoft software (BioMetallics, Inc., Princeton, NJ). The reference curve was constructed by stimulation of dpl2.trkA, B or C.gD cells with 200, 50, 12.5, 3.12, 0.78, 0.19, 0.048 or 0 ng / ml of ligand for mpl and this curve represents the variation in the number of ng / ml of TPO as a function of the average value ABS4s0, / s0 standard deviation, using DeltaSoft software. The concentrations of the samples were obtained by interpolation of their absorbance on the standard curve and are expressed in terms of TPO activity, in ng / ml (Figure 43). It has been found that the mpl ligand is capable of activating the chimeric mpl-Rse.gD receptor in a concentration-dependent and ligand-specific manner. In

  furthermore, it has been found that the mpl-Rse.gD KIRA-ELISA assay has a tolerance of up to 100% human serum (shown) or 100% plasma (not shown), which allows use this test to easily sort patient samples and pK samples. Table of EC values of TPO _ EC50 EC 50 TPO form (cells) (weight / volume) (molarity) Hu TPO 332 (293) 2 56 nglml 67.4 PM Mu TPO 332 (293) 3.69 na / ml 9711 PM Hu TPO 153 (293) -41 ng / ml -108 nM Hu TPO 155 (E.coli) 0.44 n / ml 11.6 DM Hu TPO 153met (E.co / i), 829 naml 21.8 gM

  EXAMPLE 18 Receptor Based ELISA Test for Thrombopoietin (TPO) Osage ELISA assay plates were coated with rabbit F (ab ') 2 anti-human IgG (Fc) in pH 9.6 carbonate buffer at 4 C overnight. Plates were blocked with 0.5% bovine serum albumin in PBS at room temperature for 1 hour. The harvest of the chimeric receptor-containing fermentation apparatus, mpl-IgG, was added to the plates which were incubated for 2 hours. Double serial dilutions (0.39-25 ng / ml) of the reference substance (TPO332 produced in 293 cells with concentration determination by quantitative amino acid analysis) and serially diluted samples in 0.5% serum albumin of beef and 0.05% Tween 20 were added to the plates which were incubated for 2 hours. Tethered TPO was detected with protein A-freed biotin-bound rabbit antibodies against TPOss5 that had been produced in E. coli (incubation for 1 hour) followed by streptavidin-peroxidase (incubation for 30 min) with 3,3 ', 5,5'-tetramethylbenzidine as substrate. The absorption was read at 450 nm. The plates were washed between the steps. For data analysis, the reference curve is adapted using a 4-parameter curve fitting program using a Kaleidagraph device. The concentrations of the samples were calculated from the calibration curve.

  EXAMPLE 19 Xpression and purification of TPO from 293 cells 1. Preparation of 293 cell expression vectors A cDNA corresponding to the entire open reading frame of TPO was obtained by PCR reaction using the following oligonucleotides as initiators

  TABLE 1i 293 Cla.FL.F Polymerase Chain Reaction Initators: 5 'ATC GAT ATC GAT CAG ACC GAC ACC CCG GCC AG 3 (SEQ ID NO: hmplI-R: 5' GCT AGC TCT AGA AGC GGA AGG GAG CTG TAC ATG AGA 3 '(SEQ ID NO: 48) The plasmid pRKS-hmplI (described in Example 9) was used as template for the reaction in the presence of pfu DNA polymerase (Stratagene). was carried out for 7 minutes at 94 ° C. followed by 25 amplification cycles (1 min at 940 ° C., 1 min at 55 ° C. and 1 min at 720 ° C.) The final prolongation was 15 minutes at 720 ° C. The reaction product PCR was was purified and cloned between ClaI and XbaI restriction sites of plasmid pRK5tkneo, pRK5 derived vector modified to express a neomycin resistance gene under the influence of the thymidine kinase promoter, in order to obtain the vector pRKStkneo.ORF ( open reading frame) A second assembly corresponding to the epo peer domain was generated in the same way but in a using Cla.FL.F as the forward primer and the reverse reverse primer Arg.STOPXb & S TCT AGA TCT AGA TCA CCT GAC GCA GAG GGT GGA CC 3 '(SEQ ID NO: 66)

  The final assembly is called pRK5-tkneoEPO-D. The sequence of the two constructs was verified as described in Example 7. 2. Transfection of Human Embryo Kidney Cells The constructs were transfected into human embryo kidney cells by the CaPO 4 method. As described in Example 9, 24 hours after transfection, the selection of neomycin-resistant clones was undertaken in the presence of 0.4 mg / ml G418. Ten to fifteen days later, individual colonies were transferred to 96-well plates and allowed to grow to confluence. The expression of Ms3 or ML332 in the conditioned medium from these clones was estimated using the Ba / F3-mpl proliferation assay method (described in Example 1). 3. Purification of rhML332

  The conditioned medium of 293-rhML332 was loaded onto a Blue-Sepharose column (Pharmacia) which had been equilibrated with 10 mM sodium phosphate pH 7.4 (buffer A). The column was then washed with 10 times its volume of buffer A and the same amount of buffer A containing 2 M urea. The column was then eluted with buffer A containing 2 M urea and 1 M NaCl. . The pooled eluate from the Blue-Sepharose column was then loaded directly onto a WGA-Sepharose column equilibrated in buffer A. The WGA-Sepharose column was then washed with 10 times its volume of buffer A containing 2 M urea and 1 M NaCl and eluted with the same buffer containing 0.5 M N-acetyl-D-glucosamine. The WGA-Sepharose eluate was loaded on an HPLC-C4 column (Synchrom, Inc.) equilibrated with 0.1% TFA. The HPLC-C4 column was eluted with a discontinuous gradient of propanol (0-25%, 25-35%, 35-70%). It was found that rhML332 was eluted in the 28-30% propanol region of the gradient. In gel electrophoresis of sodium dodecyl sulfate and polyacrylamide, the purified rhML332 migrates as a broad band in the 68-80 kDa region of the gel (see Figure 15).

  4. Purification of rhML15s3 The 293-rhMLIs3 conditioned medium was resolved on Blue-Sepharose as described for rhML332. The Blue Sepharose eluate was loaded directly onto an affinity column for mpl as described above. The rhML1s3 eluted from the affinity column for mpl was purified to homogeneous consistency using an HPLC-C4 column operating under the same conditions as for rhML332. In gel electrophoresis of sodium dodecyl sulfate and polyacrylamide, the purified rhMLs53 resolves into two dominant bands and two secondary bands with a Mr value of about 18,000-21,000 (see Figure 15).

  EXAMPLE 20 Expression and purification of TPO from Chinese hamster ovary (CHO) cells 1. Description of CHO expression vectors The expression vectors used in the electroporation protocols described below were designated as follows: pSV15 .ID.LL.MLORF (integer or hTPO332), and pSV15.ID.LL..MLEPO-D (truncated or hTPO53) '. The relevant characteristics of these plasmids are shown in Figures 23 and 24. 2. Preparation of CHO expression vectors A cDNA corresponding to the full open reading frame of hTPO was obtained by polymerase chain reaction using oligonucleotide primers. of Table 12.

  TABLE 12 PCR primers of CHO Cla.FLF2 expression vectors 5 'ATC GAT ATC GAT AGC AGC ACA CCC CGG CCA G (SE ID NO: 47) ORF. Sal 5 'AGT CGA CGT CGA CGT CGG CAG TGT CTG AGA ACC 3 (SEO ID NO: 67) pRKS-hmplI (described in xamples 7 and 9) was used as a template for the reaction in the presence of DNA polymerase pfu (Stratagene). The initial denaturation was carried out for 7 minutes at 94 ° C., followed by 25 amplification cycles (1 min at 94 ° C., 1 min at 55 ° C. and 1 min at 72 ° C.). The final extension was 15 minutes at 72 degrees C). The PCR reaction product was purified and cloned between ClaI and SalI restriction sites of plasmid pSV15.ID.LL to obtain the vector pSV15.ID.LL.MLORF. A second construct corresponding to the homologous domain EPO was generated in the same way but using Cla.FL.F2 as a forward primer and the following inverse primer:

  EPOO.Sai 5 'AGT CGA CGT CGA CTC ACC TGA CGC AGA GGG TGG ACC 3 (SEQ ID NO: 68) The final construct is named pSV15.ID.LL.MLEPO-D. The sequence of the two constructs was verified as described in Examples 7 and 9. For the most part, the coding sequences for the whole ligand and the truncated ligand were introduced into the multiple cloning site of the vector PSV15. .LL expression of CHO. This vector contains the SV40 promoter / early promoter region, a modified splice unit containing mouse DHFR cDNA, a multiple cloning site for the introduction of the gene of interest (in this case the described TPO sequences), the polyadenylation signal of SV40 and the origin of replication and the beta-lactamase gene for the selection and amplification of the plasmid in bacteria. 3. Methodology for establishment of stable CHO cell lines, expressing recombinant human TPO332 and recombinant TPO1s3 ~ 5 ~ a. Description of the CHO parental cell line The Chinese hamster ovary (CHO) cell line used for expression of the TPO molecules described herein is designated CHO-DP12 (see European Patent No. 307,247). at the Public Inspectorate on March 15, 1989). This mammalian cell line was cloned from parental (CHO-K1 DUX-B11 (DHFR-) line transfection from Dr. Franck 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 cells and clones can be selected for the presence of DHFR cDNA vector sequences by growth on a medium lacking nucleoside supplements (glycine, hypoxanthine and thymidine). This selection system of CHO cell lines giving stable expression is commonly used. b. Transfection method (electroporation) TPO332 and TPOIs3 expression cell lines were generated by transfection of DP12 cells by electroporation (see, for example, G. L. Andreason, J. Tiss.

  Cult. Meth., 15, 56 (1993)) with, respectively, the linear pSVI5.ID.LL.MLORF or pSV15.ID.LL.MLEPO-D plasmids. Three (3) restriction enzyme reaction mixtures were established for each plasmid cutoff: 10 μg, 25 μg, and 50 μg of the vector with the NOTI enzyme by standard molecular biology methods. This restriction site is found only once in the vector in the 3 'linearization region and outside the TPO ligand transcription units (see Fig. 23). The 100 M1 reaction mixtures were incubated overnight at 37 ° C. The following day the mixtures were extracted once with phenol-chloroform-isoamyl alcohol (50: 49: 1) and ethanol precipitated. on carbon snow for about 1 hour. The precipitate was then collected by microcentrifugation for 15 minutes, and dried. The linearized DNA was resuspended in 50 μl of 1: 1 Ham DMEM-F12 medium supplemented with conventional antibiotics and 2 mM glutamine.

  The DP12 cells growing in the suspension were collected, washed once in the medium described for resuspension of the DNA and finally resuspended in the same medium at a concentration of 107 cells per 750 μl. Aliquots of cells (750 μl) and each linearized DNA mixture were incubated together at room temperature for 1 hour and then transferred to a BRL electroporation chamber. Each reaction mixture was then exposed to electroporation in a conventional 350 volt BRL electroporation apparatus set at 350 gF and low capacitance. After electroporation, the cells were allowed to settle in the apparatus for 5 minutes and then on ice for another 10 minute incubation period. The electroporated cells were transferred to 60 mm cell culture dishes, containing 5 ml of standard CHO cell culture medium (DMEM-F12 50:50 high glucose, no glycine, additionally containing 1X GHT, 2mM glutamine and 5% fetal calf serum) and were grown overnight in a cell culture incubator in the presence of 5% CO. c. Selection and Sorting Method The following day, the cells were trypsinized off the plates by standard methods and transferred to 150 mm tissue culture dishes containing DHFR selective medium (1: 1 Ham DMEM-F12 medium). , described above, additionally containing 2% or 5% dialyzed calf fetus serum but lacking glycine, hypoxanthine and thymidine, constituting the normal DHFR selection medium that the Applicant has used). Cells from each 60 mm dish were then subcultured into 5 x 150 mm dishes. Then, we incubated

  cells for 10 to 15 days (with change of medium) 10 to 37 ° C / 5% CO2 until clones begin to mature and reach sizes allowing transfer to the 96-well plates. at 4 to 5 days, the cell lines were transferred to 96-well plates using yellow sterile tips adapted to a 50 ml pipettman The cells were allowed to grow to confluence (usually 3-5 days), then the plates were trypsinized and two copies of the original plate were repro- duced Two of these copies were stored in the freezer for a short time, with the cells of each cell diluting in 50 g of fetal serum. of 10% calf in DMSO Serum-free medium samples conditioned for 5 days were titrated from the confluent cells in the third tray for TPO expression by the activity test method. The maximal expression clones on the basis of this assay were resuscitated after storage and amplified into two 150 mm confluent T-plates for transfer to the cell culture group for treatment. adaptation of the suspension, a new titration and the establishment of a bank.

  d. Amplification Protocol Several higher titre cell lines from the selection described above were then subjected to a standard metho-trexate amplification regimen to generate high titer clones. CHO cell clones are grown and seeded in 10 cm dishes at 4 methotrexate concentrations (ie, 50 nM, 100 nM, 200 nM, and 400 nM), at two or three cell numbers (10s, 5x 10s). and 10'6 cells per bunch). These cultures are then incubated at 37 degrees C / 5% CO2 until the clones have established themselves and are ready to be transferred to 96 cell dishes for the rest of the test. Several high-titre clones from this selection were again subjected to higher concentrations of methotrexate (ie, 600 nM, 800 nM, 1000 nM and 1200 nM), and, as previously, resistant clones were allowed to occur. establish and then transferred to 96-cell bundles and titrated.

  4. Culture of stable CHO cell lines expressing TPO332 and recombinant human TPO1s3 The cells in the library are thawed and the cell population is amplified by conventional cell growth methods in serum-free medium or in serum-containing medium . After amplification to sufficient cell density, the cells are washed to remove the spent cell culture medium. The cells are then cultured by any conventional method comprising batch, semicontinuous or continuous culture at 25-40 C, at neutral pH, with a dissolved O 2 content of at least 5% until the TPO constituted by secretion has accumulated. The liquid from the cell culture is then separated from the cells by mechanical means such as centrifugation. 5. Purification of Recombinant Human TPO from CHO Cell Culture Fluids The harvested cell culture fluid (HCCF) is directly loaded onto a fast-flowing Blue Sepharose 6 column (Pharmacia) equilibrated in sodium phosphate 0, 0.1 M at pH 7.4 containing 0.15 M NaCl in a ratio of about 100 ml of HCCF per liter of resin and at a linear flow rate of about 300 ml / hr / cm 2. The column is then washed with 3 to 5 times its volume of equilibration buffer and then 3 to 5 times its volume of 0.01 M sodium phosphate pH 7.4 containing 2.0 M urea. The TPO is then eluted with 3 to 5 column volumes of 0.01 M sodium phosphate pH 7.4 containing 2.0 M urea and 1.0 M NaCl.

  The pooled liquid from the TPO-containing Blue Sepharose column is then loaded onto a 6 MB Sepharose column containing wheat germ lectin (Pharmacia), equilibrated in 0.01 M sodium phosphate pH 7.4 containing 2.0 M urea and 1.0 M NaCl in a ratio of 8 to 16 ml eluted Blue Sepharose liquid per ml resin at a flow rate of about 50 ml / hr / cm 2. The column is then washed with 2 to 3 times its volume of equilibration buffer. The TPO is then eluted with 2 to 5 column volumes of 0.01 M sodium phosphate pH 7.4 containing 2.0 M urea and 0.5 M N-acetyl-D-glucosamine. The pooled liquid from the wheat germ lectin column is then adjusted to a final concentration of 0.04% C12E, and 0.1% trifluoroacetic acid (TFA). The resulting liquid is loaded onto a reversed phase C4 column (Vydac 214TP1022) equilibrated in 0.1% TFA and 0.04% C12 at a loading of about 0.2 to 0.5 mg of protein 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 and 0.04% C12E *. The first phase is composed of a linear gradient ranging from 0 to 30% acetonitrile in 15 minutes. The

  second phase is composed of a linear gradient ranging from 30 to 60% acetonitrile in 60 minutes. TPO is eluted with about 50% acetonitrile. The liquid is collected on the basis of sodium dodecyl sulfate and polyacrylamide gel electrophoresis. The liquid collected on C4 is then diluted with 2 volumes of 0.01 M sodium phosphate at pH 7.4 supplemented with 0.15 M NaCl and diafiltered against approximately 6 volumes of 0.01 M sodium phosphate at pH 7. 4 plus 0.15 M NaCl on an Amicon YM ultrafiltration membrane or the like having molecular weight exclusion limits of 10,000 to 30,000 daltons. The resulting diafiltrate can then be processed directly or further concentrated by ultrafiltration. The diafiltrate / concentrate is adjusted to a final concentration of 0.01% Tween-80. All or a portion of the diafiltrate / concentrate equivalent to 2-5% of the calculated column volume is then loaded onto a Sephacryl S-300 HR (Pharmacia) column equilibrated in 0.01 M sodium phosphate at pH 7.4 containing 0.15 M NaCl and 0.01% Tween-80 and chromatographed at a flow rate of about 17 ml / hr / cm 2. The TPO-containing fractions which are free of aggregates and proteolytic degradation products are pooled on the basis of sodium dodecyl sulfate and polyacrylamide gel electrophoresis. The resulting pooled liquid is filtered through a Millex-GV filter or the like of 0.22 μm and stored at 2-8oC.

  EXWMPLE 21 Transformation and induction of TPO protein synthesis in. coli 1. Construction of E. coli TPO expression vectors Plasmids pMP21, pMP151, pMP41, pMP57 and pMP202 are all designed to express the first 125 amino acids of TPO downstream of a small leader that varies between different assembly products. Leaders offer mainly a high level of translation initiation and rapid purification. Plasmids pMP210-1, -T8, -21, -22, -24, -25 are designed to express the first 153 amino acids of TPO downstream of an initiating methionine and differ only in the use of the codons for the first 6 amino acids of the TPO, while the plasmid

  pMP251 is a derivative of plasmid pMP210-1 in which the carboxy-terminus of TPO is extended by two amino acids. All the plasmids indicated above produce high levels of intracellular TPO expression in E. coli upon induction of the tryptophan promoter (DG Yansura et al., Methods in Enzymology (edited by DV Goeddel). 185: 54-60, Academic Press, San Diego [1990]). Plasmids pMP1 and pMP172 are intermediates in the construction of the above TPO intracellular expression plasmids. (a) Plasmid mMP1 Plasmid pMP1 is a secretory vector for the first 155 amino acids of TPO, and was constructed by binding DNA fragments together as shown in Fig. 33. The first of these fragments was the vector pPho21 in which the small Mlul-BamHI fragment had been removed. The vector pPho21 is a derivative of phGH1 (CN Chang et al., Gene 55: 189-196 [1987]) in which the human growth hormone gene has been replaced by the phoA gene of E. coli, and the MluI restriction site was incorporated by manipulation into the coding sequence for the STII signal sequence at amino acids 20-21. The following two fragments, a 258 base pair piece of Hinfl-PstI DNA from pRKS-hmplI (Example 9) encoding TPO amino acids 19-103, and the following synthetic DNA encoding amino acids 1-18 5-CGCGTATGCCAGCCCAGGCTCCTCCTGCTGTGACCTCCGAGTCCTCAGTAAACTGCT'CG TG ATACGGTCGGGCGA GAACACTGGAGGCTCAGGAGTCA GAGAAGC ACTGA-5 '(SEQ ID NO: 69) (SEQ ID NO: 70) were previously ligated with the T4 DNA ligase and the

  second was cut with PstI. The fourth was a 152 base pair PstI-HaeIII fragment from pRK5hmpII encoding amino acids 104-155 of TPO. The latter was a 412 base pair StuI-BamHI fragment from pdh108, containing the transcription terminator lambda as previously described (Scholtissek, S. et al., NAR 15: 3185 [1987]) (b) pMP21 plasmid. Plasmid pMP21 is designed to express the first 115 amino acids of TPO using a 13 amino acid leader sequence comprising part of the STII signal sequence. Three (3) DNA fragments were ligated together as shown in Figure 34, the first of these fragments being the pVEG31 vector in which the short XbaI-SphI fragment had been removed. The vector pVEG31 is a derivative of pHGH207-1 (H.A., de Boer et al., In Promoter Structure and Function (published under the direction of R.L.

  Rodriguez and MJ Chamberlain), 462, Praeger, New York [1982]), in which the human growth hormone gene had been replaced by the vascular endothelial growth factor gene annoyance (this identical vector fragment can be obtained from this plasmid). The second part of the ligation was a synthetic DNA duplex with the following sequence: 5-CTAGAATTATGAAAAAGAATATCGCATI ,, 'CTTCTTAA TTAATACTTT'CTTATAGGAAAGAAGAATTGCGC-5 (SEQ ID NO: 71) (SEO ID NO: 72) The last piece was a 1072 base pair MluI-SphI fragment derived from pMP1 encoding 155 amino acids of TPO. (c) Plasmid pMP151 Plasmid pMP151 is designed to express the first 155 amino acids of TPO downstream of a leader sequence comprising 7 amino acids of the STII signal sequence, 8 histidines, and a factor Xa cleavage site. As shown in Fig. 35, plasmid pMP151 was constructed by ligating three DNA fragments together, the first of these fragments being the already described pVEG31 vector from which the small XbaI-SphI fragment had been removed. The second was a synthetic DNA duplex having the following sequence:

  SCTAGAMTTATGAAAAAGAATATCGCAM-CATCACCATCACCATCACCATCACATCGAAG GTCGTAGCC TrAATATIiTiiCTTATAGCGTAAAGTAGTGGTAGTGTAGTGTAGTGTAGCTTC CAGCAT-5 '(SEO ID NO: 73) (SEQ ID NO: 74) The last was a 1064 base pair BgII-SphI fragment derived from PMP11 encoding 154 amino acids of TPO. Plasmid pMP11 is identical to plasmid pMP1 with the exception of a few codon modifications in the STII signal sequence (this fragment can be obtained from pMP1). d) Plasmid pMP202 Plasmid pMP202 is very similar to the pMP151 expression vector except that the factor Xa cleavage site in the leader sequence has been replaced by a thrombin cleavage site. As shown in Figure 36, plasmid pMP202 was constructed by ligating three DNA fragments together. The first of these fragments was pVEG31 already described in which the short fragment XbaI-SphI had been removed. The second was a synthetic DNA duplex

having the following sequence: 5.-CTAGAATTATGAAAMAAGAATATCGCATTCATCACCATCACCATCACCATCACATCGAA CCACGTAGCC TrAATACr ICIATrAGCGTAAAGTAGTGGTAGTGGTA "TGGTAGTGTAGCTT GGTGCAT-5 '(SEQ ID NO: 75) (SEQ ID NO: 76) The last piece was a 1064 base pair BgII-SphI fragment derived from plasmid pMP11 previously described (e) Plasmid pMP172 Plasmid pMP172 is a secretory vector for the first 153 amino acids of TPO, and is an intermediate for the construction of pMP210 As shown in Figure 37, plasmid pMP172 was prepared by ligation of three DNA fragments together, the first of which was the vector pLS32IamB whose small section EcoRIHindIII had been removed The second was a 946 base pair EcoRI-HgaI fragment derived from plasmid pMP11 previously described. chunk was a synthetic DNA duplex having the following sequence: 5'-TCCACCCTCTGCGTCAGGT (SEQ ID NO: 77) GGAGACGCAGTCCATCGA-5 '(SEQ ID NO : 78) (f) Plasmid pMP210 Plasmid pMP210 is designed to express the first 153 amino acids of TPO after a translation initiation methionine. This plasmid was actually designed as a plasmid library in which the first six TPO codons were randomized in the third position of each codon, and it was constructed as shown in Figure 38 by ligation of three fragments of DNA. The first of these fragments was the already described PVEG31 vector in which the short XbaI-SphI fragment had been removed. The second was a synthetic DNA duplex shown below, first treated with DNA polymerase I (Klenow) and then digested with the enzymes XbaI and HinfI, and encoding the initiating methionine and the first 6 codons

  randomized TPO. 5 'GCAGCAGT-CTAGAA1TATGTCNCCNGCNCCNCCNGCNTGTGACCTCCGA GTTCTCAGTAAA (SEQ ID NO: 79) CAAGAGTCATITGACGAAGCACTGAGGGTACAGGmG-S (SEQ ID NO: 80) The third was an 890 base pair HinfI-SphI fragment derived from pMP172 encoding TPO amino acids 19-153. The pMP210 plasmid library of about 3700 clones was retransformed on high tetracycline LB plates (50 μg / ml) to select high translation initiation clones (DG Yansura et al., Methods: A Companion to Methods in Enzymology 4: 151-158 [1992]). Of the 8 colonies that appeared on the high tetracycline plates, five of the best in terms of TPO expression were subjected to DNA sequencing and the results are shown in Fig. 39 (SEQ ID N 23 , 24, 25, 26, 27 and 28). (g) Plasmid pMP41 Plasmid pMP41 is designed to express the first 155 amino acids of TPO fused to a leader sequence of 7 amino acids of the STII signal sequence followed by a factor Xa cleavage site. The plasmid was constructed as shown in Figure 40 by ligation of three pieces of DNA together, the first of which was the previously described pVEG31 vector in which the short XbaI-SphI fragment had been removed. The second was the following synthetic DNA duplex: 5 '- CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (SEOID NO: 81) TTAATACTmCTTATAGCGTAAATAGCTTCCAGCAT-' (SEO IDNO: 82) The last piece of the ligation was the 1064 base pair BgII-SphI fragment derived from plasmid pMP11 described above. (h) Plasmid pMP57

  Plasmid pMP57 expresses the first 155 amino acids of TPO downstream of a leader sequence of 9 amino acids of the STII signal sequence and the Lys-Arg dibasic site. This dibasic site provides a means of removing the leader sequence with the ArgC protease. This plasmid was constructed as shown in Figure 41 by ligation of three pieces of DNA together. The first of these pieces was the previously described pVEG31 vector in which the small XbaI-SphI fragment had been removed. The second was the following synthetic DNA duplex: 5'-CTAGAATTATGAAAAAGAATATCGCAIICTTCTTAAACGTAGCC (SEQ ID NO: 83) TTAATAC1TrTCTTATAGCGTAAAGAAGAATTTGCAT-5 (SEO ID NO: 84) The last part of the ligation was the derived 1064 base pair BgII-SphI fragment. of the plasmid pMPl1 described above:

  (i) Plasmid pMP251 Plasmid pMP251 is a derivative of pMP210-1 in which two other amino acids of TPO are included at the carboxy terminus. As shown in Figure 42, this plasmid was constructed by ligating two pieces of DNA together, the first of which was the previously described pMP21 in which the small XbaI-ApaI fragment had been removed. The second part of the ligation was a 316 base pair XbaI-ApaI fragment derived from pMP210-1. 2. Transformation and Induction of E. coli with TPO Expression Vectors The above TPO expression plasmids were used to transform the E. coli strain 44C6 (w3110 tonAl rpoHt, long clpP, GalE) to CaCl2 heat shock method (Mandel et al., J. Mol Biol 53: 159-162 [1970]). Transformed cells were first grown at 37 ° C in LB medium containing 50 μg / ml carbenicillin until the optical density (600 nm) of the culture reached about 2-3. The LB culture was then diluted 20-fold in M9 medium containing 0.49% casamino acids (w / v) and 50 μg / ml carbenicillin. After growth with aeration at 30 ° C. for 1 hour, indole-3-acrylic acid was added to a final concentration of 50 μg / ml. The culture was then allowed to proceed at 30 ° C. with aeration for a further hour and at this point the cells were harvested by centrifugation.

  EIMP> 22 Production of biologically active TPO (Met'1-153) in B. coli The procedures given below for the production of biologically active folded TPO (met-1-153) can be applied by analogy to isolation of other variants of TPO comprising the N-terminal and C-terminal forms (see Example 23). A. Isolation of Insoluble TPO (Met '1-153) E. coli cells expressing TPO (Met-1-153) encoded by plasmid pMP210-1 are subjected to fermentation as described above. Normally, about 100 g of cells are resuspended in 1 liter (10 S volumes) of cell disruption buffer (10 mM Tris, 5 mM EDTA, pH 8) with a Polytron homogenizer and the cells are centrifuged at room temperature. 5000 xg for 30 minutes. The washed cell pellet is resuspended in 1 liter of cell disruption buffer with the Polytron homogenizer, and the cell suspension is passed through an LH cell disruptor (LH Inceltech, Inc.) or through a microfluidizer (Microfluidics International) according to the manufacturers instructions. The suspension is centrifuged at 5000 x g for 30 min and the cells are resuspended, and the suspension is centrifuged a second time to obtain a washed refractile body pellet. The washed pellet is used immediately or stored frozen at -700C.

  B. Solubilization and purification of monomeric TPO (Met '1-153) The pellet obtained above is resuspended in 5 volumes by weight of 20 mM Tris buffer at pH 8 supplemented with 6-8 M guanidine and 25 mM DTT (dithiothreitol) and the suspension is stirred for 1-3 h or overnight at 40C to solubilize the TPO protein. High concentrations of urea (6-8 M) are also useful but generally give lower yields compared to guanidine. After solubilization, the solution is centrifuged at 30,000 x g for 30 min producing a clear supernatant liquid containing the protein.

  Denatured monomeric TPO. The supernatant liquid is then chromatographed on a Superdex 200 gel filtration column (Pharmacia, 2.6 x 60 cm) at a flow rate of 2 ml / min and the protein is eluted with 20 mM sodium phosphate at pH 6 , 0, with 10 mM DTT. The fractions containing the denatured monomeric TPO protein eluted between 160 and 200 ml are pooled. The TPO protein is further purified on a semi-preparative C4 reverse phase column (VYDAC 2 x 20 cm). The sample is loaded at a rate of 5 ml / min on 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 min). The purified reduced protein is eluted at about 50% acetonitrile. This material is used for refolding to obtain a biologically active TPO variant. C. Production of biologically active TPO (Met-1-153) About 20 mg of reduced, denatured, monomeric TPO protein is diluted in 40 ml of 0.1% TFA in 50% acetonitrile in 360 ml of refolding buffer optimally containing the following reagents: 50 mM Tris 0.3 M NaCl 5 mM EDTA 2 g CHAPS detergent 25% glycerol 5 mM oxidized glutathione 1 mM Glutathione reduced pH adjusted to 8 3

  After mixing, the refolding buffer is gently stirred at 4 ° C for 12-48 hours to obtain the maximum yields of refolding of the correct form of disulfide-bound TPO (see below). The solution is then acidified with trifluoroacetic acid to a final concentration of 0.2%, filtered through a filter of 0.45 or 0.22 Am and 1/10 of the volume of acetonitrile is added. This solution is then pumped directly onto an inverted phase C4 column and the purified folded TPO (Met '1-153) is eluted with the same gradient program as above. The biologically active folded TPO is eluted at about 45% acetonitrile under these conditions. Unsuitable versions of TPO bound to the disulfide are eluted earlier. The final purified TPO (Met-1-153) has a purity of greater than 95% as determined by sodium dodecyl sulfate gel chromatography and reverse-chained C4 analytical chromatography. For animal studies, the purified C4 column material was dialyzed into physiologically compatible buffers. Isotonic buffers (10 mM sodium acetate, pH 5.5, 10 mM sodium succinate, pH 5.5 or 10 mM sodium phosphate, pH 7.4) containing 150 mM NaCl and 0.01 Tween 80's have been used. Because of this high activity of TPO in the Ba / F3 assay (a semi-maximal stimulation is reached at about 3 μg / ml), it is possible to obtain the biologically active material using many different conditions regarding buffer, detergent and redox. However, under most conditions only a small amount of suitably folded material (<10%) is obtained. For industrial production processes, it is desirable to obtain refolding efficiencies of at least 10%, more preferably 30-50% and especially more than 50%. Many different detergents (Triton X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, sarkosyl, Tween 20 and Tween 80, Zxittergent 3-14, etc.) were subjected to an efficacy evaluation for maintenance of high folding yields. Of these detergents, only the family of

  CHAPS (CHAPS and CHAPSO) has been shown to be generally useful in the refolding reaction to limit protein aggregation and incorrect disulfide formation. CHAPS proportions above 1% have been very useful. Sodium chloride was required to obtain the best yields, with optimal concentrations between 0.1 and 0.5 M. The presence of EDTA (1-5 mM) limited the degree of oxidation (and aggregation) catalyzed by a metal that has been observed with certain preparations. Glycerol concentrations of more than 15% have produced the optimum conditions for refolding. For maximum yields, it was essential to have oxidized and reduced glutathione or oxidized and reduced cysteine at the same time as a pair of oxidation-reduction reagents. Generally, higher endings were observed when the molar ratio of the oxidized reagent was equal to or greater than the reduced reactive member of the oxidation-reduction pair. PH values between 7.5 and about 9 were optimal for folding of these TPO variants. Organic solvents (e.g. ethanol, acetonitrile, methanol) have been tolerated at concentrations of 10-15% or less. Higher concentrations of organic solvents have increased the amount of improperly folded shapes. Tris buffer and phosphate buffer were generally used. A 4 C incubation also produced higher proportions of correctly folded TPO. Folding yields of 40-60% (based on the amount of reduced and denatured TPO used in the refolding reaction) are representative of TPO preparations that were purified by the first C4 step. Active material can be obtained with less pure preparations (eg immediately after the Superdex 200 column or after the initial extraction of refractive bodies), although yields are lower due to precipitation and interference. large-scale non-TPO proteins during the TPO refolding process.

  Since TPO (Met '1-153) contains 4 cysteine residues, it is possible to generate three different disulfide versions of this protein: version 1: disulfide between cysteine residues 1-4 and 2-3 version 2: disulfide between cysteine residues 1-2 and 3-4 version 3: disulfides between cysteine residues 1-3 and 2-4. During the initial scanning in the determination of refolding conditions, several different peaks containing the TPO protein were separated by reverse phase C4 chromatography. Only one of these peaks had significant iological activity as determined using the Ba / F3 assay. Then, the folding conditions were optimized in order to obtain this version preferentially. Under these conditions, poorly folded conversions account for less than 10-20% of the total monomeric TPO obtained. The disulfide unit for biologically active TPO was located by mass spectrometry and protein sequencing at 1-4 and 2-3 (ie version 1). Aliquots of the various C4 resolved peaks (5-10 nmol) were subjected to trypsin digestion (mole ratio of trypsin to protein, 1:25). The mixture after digestion was analyzed by matrix-assisted laser desorption mass spectroscopy before and after reduction with dithiothreitol (DTT). After reduction, the masses corresponding to most of the larger TPO tryptic peptides were detected. In the unreduced samples, some of these masses were missing and new masses were observed. The mass of the new peaks basically corresponded to the sum of the individual tryptic peptides involved in the disulfide pair. It was thus possible to unequivocally assign the disulfide unit of recombinant biologically active TPO folded to form 1-4 and 2-3. This is consistent with the known disulfide pattern of erythropoietin, a related molecule.

  D. Biological Activity of Recombinant Recombinant TPO (Met 1-153) Refolded and purified TPO (Met '1-153) is endowed with activity in both in vitro and in vivo assays. In the Ba / F3 assay, semi-maximal stimulation of thymidine incorporation into Ba / F3 cells was achieved at a concentration of 3.3 μg / ml (0.3 μM). In normal and myelosuppressed animals, produced by sublethal X irradiation, TPO (Met '1-153) stimulated with high potency (the activity was observed at doses as low as 30 ng / mouse). production of new platelets. EXAMPLE 23 Production of other biologically active TPO variants in E. coli Three different TPO variants produced in E. coli, purified and refolded into biologically active forms, are shown below. (1) MLF - 13 residues of STII signal sequence of bacterial origin are fused to the N-terminal domain of the

  TPO (residues 1-155). The resulting sequence is MKKNIAFLLNAYASPAPPAC --- CVRRA (SEQ ID NO: 85) where the leader sequence is underlined and C .... C represents the sequence Cys' to Cysls51. This variant was constructed to create a tyrosine for radio-iodination of TPO for receptor studies and biological studies. (2) H8MLF - 7 residues from the STII sequence, 8 histidine residues and the IEGR sequence of Factor Xa enzymatic cleavage are fused to the N-terminal domain (residues 1-155) of TP0. The sequence is MKKNIAFHHHHHHHIEGRSPAPPAC ..... CVRRA (SEQ ID NO: 86) where the leader sequence is underlined and C .... C represents the sequence of Cys 'to Cys'. This variant, once purified and refolded, can be treated with Enzyme Factor Xa which cleaves after the arginine residue of the IEGR sequence giving a TPO variant of 155 residues with serine as amino acid N -terminal natural. (3) T-H8MLF - this form is prepared as described above for variant (2) except that a thrombin-sensitive IEPR sequence is fused to the N-terminal domain of TPO. The resulting sequence is MKKNIAFHHHHHHHHIEPRSPAPPAC .-- CVRRA (SEQ ID NO: 87) where the leader sequence is underlined and C .... C represents the Cys sequence. at Cys'sl. This variant, after purification and refolding, can be enzymatically treated with thrombin to generate an N-terminal natural TPO variant of 155 residues in length.

  A. Isolation, solubilization and purification of biologically active monomeric variants (1), (2) and (3) of TPO. All variants were expressed in E. coli. Most variants were found in refractile bodies, as observed in Example 22 for TPO 1-153). Identical methods of isolation, solubilization and purification of monomeric variants of TPO were carried out as described in Example 22. The same folding conditions were used as those used for TPO (Met-1). 153) with overall returns of 30-50%. After refolding, the TPO variants were purified by C4 reverse phase chromatography in 0.1% TFA using an acetonitrile gradient as previously described. All TPO variants (in the unproteolysed form) were endowed with biological activity as determined in the Ba / F3 assay, with semi-maximal activities of 2-5 μM.

  B. Proteolytic processing of variants (2) and (3) to generate authentic N-terminal TPO (1-155). The TPO (2) and (3) variants indicated above were designed with a leader peptide capable of enzymatic cleavage before the normal N-terminal amino acid residue of TPO. After refolding and purification of variants (2) and (3) as described above, each was digested with the appropriate enzyme. For each variant, acetonitrile from the C4 reverse phase step was removed by passing a moderate stream of nitrogen over the solution. Then, both variants were treated with Factor Xa or thrombin as described below. For the TPO variant (2), 1 M Tris buffer at pH 8 was added to the acetonitrile free solution to a final concentration of 50 mM and the pH was adjusted to 8 if necessary. NaCl and CaCl 2 were added in respective proportions of 0.1 M and 2 mM. Factor Xa (New England Biolabs) was added to achieve an approximate molar ratio of enzyme to variant of 1:25 to 1: 100. The sample was incubated at room temperature for 1-2 hours to obtain maximum cleavage determined by a change in sodium dodecyl sulphate gel migration representing the loss of

  leader sequence. Then, the reaction mixture was purified by C4 reverse phase chromatography using the same gradient and conditions as noted above for the purification of properly folded variants. The uncleaved variant B was separated under these conditions from the cleaved variant (2). The N-terminal amino acids were shown to be SPAPP, indicating that the elimination of the N-terminal leader sequence was successful. Factor Xa also generated varying degrees of internal cleavage in the TPO domain; cleavage was observed after the arginine residue in the NO 118 position, generating another N-terminal sequence of TTAHKDP (SEQ ID NO: 88). On non-reducing sodium dodecyl sulphate gels, a single band at about 17,000 daltons was observed for the Factor Xa cleaved variant; on reducing gels, two molecular weight bands approximately equal to 12,000 and 5,000 daltons appeared, in accordance with the cleavage at arginine 118. This observation also confirmed that both parts of the molecule had been held together by a disulfide bond between the first and fourth residues

  of cysteine, as deduced from the trypsin digestion experiments described above. In the Ba / F3 bioassay, the purified TPO variant (1-155), after removal of the N-terminal leader sequence and with internal cleavage, had a peak activity of 0.2 to 0.3 picomole. The intact variant with the leader sequence had a half maximal activity of 2-4 picomoles. For variant (3), the digestion buffer consisted of 50 mM Tris pH 8 containing 2% CHAPS, 0.3 M NaCl, 5 mM EDTA and human or bovine thrombin (Calbiochem) in a weight ratio of enzyme to TPO variant protein of 1:25 to 1:50. Digestion was conducted at room temperature for 2-6 hours. The progress of digestion was determined on sodium dodecyl sulfate gels as described above for the Factor Xa cleavage reaction. In general, more than 90% cleavage of the leader sequence was obtained at this time. The resulting TPO was purified on reverse phase C4 columns as described above and found to have the desired N-terminus by amino acid sequencing. Only very small amounts (less than 5%) of internal cleavage at the same arginine-threonine bond as observed with Factor Xa were obtained. The resulting TPO protein had a high biological activity with semi-maximal responses in the assay

  Ba / F3 at a protein concentration of 0.2-0.4 picomole. In the mpl receptor-based ELISA, this protein had a semi-maximal response for a purified protein concentration of 2-4 ng / ml (120-240 picomolar concentration) while the intact variant containing the leader sequence was less potent by a factor of 5 to 10 in both trials. In animal studies, the HPLC purified cleaved protein was dialyzed into physiologically acceptable buffers with 150 mM NaCl, 0.01% Tween 80, and 10 mM sodium succinate at pH 5. , 5, or 10 mM sodium acetate, pH 5.5, or 10 mM sodium phosphate pH 7.4. Using HPLC or sodium dodecyl sulfate gels, the purified protein was stable for several weeks when stored at 4 ° C. In normal and myilosuppressed mice, this purified TPO with the authentic N-terminal sequence has been very active, stimulating platelet production at doses as low as 30 ng / mouse.

  EXAMPLE 2A Synthetic mpl ligand Although human mpl ligand (hML) is usually prepared by recombinant methods, it can also be synthesized by enzymatic ligation of synthetic peptide fragments using the methods described above. The synthetic production of hML allows the incorporation of unnatural amino acids or synthetic functionalities such as polyethylene glycol. Previously, a mutant of the serine protease subtilisin BPN, namely the substiligase (S221C / P225A), was manipulated to obtain efficient ligation of peptide esters in aqueous solution (Abrahmsen et al., Biochem., 30: 4151). -4159 [1991]). It has now been demonstrated that synthetic peptides can be enzymatically sequentially fused to produce enzymatically active long chain peptides and proteins such as ribonuclease A (Jackson et al., Science, [1994]). This technology, described in more detail below, has allowed the Applicant to synthesize chemically long proteins that could previously only be obtained by recombinant DNA technology.

  A general strategy for the synthesis of hML1s3 with the use of subtiligase is shown in Scheme 1. Starting from a peptide completely freed of its protection, corresponding to the C-terminal fragment of the protein, a protected peptide ester is added to N-terminus and activated at the C-terminus at the same time as the subtiligase. When the reaction is complete, the product is isolated by reverse phase HPLC and the protecting group is removed from the N-terminus. The next peptide fragment is fused, deprotected, and the method is repeated using successive peptides until the full length protein has been obtained. The method is analogous to solid phase methodology in which an N-terminally protected and C-terminally activated peptide is fused to the N-terminus of the foregoing peptide and the protein is synthesized in one direction.

  C-N. However, since each coupling results in an addition of up to 50 residues and the products are isolated after each ligation, longer proteins of high purity can be synthesized in reasonable yields. Scheme 1. Synthesis strategy of hML using subtiligase R-NH-Peptide2-CO-R '+ H2N-Peptide1-CO2 1) Subtiligase R-NH-Peptide2-CO-NH-Peptide1-CO2; 2) Zn / CH3CO2H H2N-Peptide2-CO-NH-Peptidel-C02 1 3) Repeat 1 + 2 H2N-Peptide3-CO-NH-Peptide2-CO-NH-Peptide1 -CO2 0 0 ~~ R- R = ko0 = NH NH2 ~~~~ N0 ~ O (CH2) 4 NH2 On the basis of the knowledge acquired concerning sequence specificity of the subtiligase as well as the amino acid sequence of the biologically active "epo domain" of hML, the Applicant has divided hMLs53 into seven fragments with a length of 18 to 25 residues. Experimental ligation tetrapeptides were synthesized to determine the proper junctions for ligation for the 18-25 parent moiety. Table 13 shows the results of these experimental ligations.

  TABLE 13 Experimental Ligations of hML. Donor and nucleophilic peptides were dissolved in a proportion of 10 mM in 100 mM tricine (pH 7.8) at 22 ° C. Ligase was added to a final concentration of 10 μM from a stock solution. 1.6 mg / ml (about 70 MM) and allowed to ligation overnight. The yields are based on the percentage of ligation relative to the hydrolysis of the donor peptides. Site 1Donor (glc-K-NH2) Nucelophil-NH2 | lldrolMe,.% Uzatior4z 1 (23/24) HVLH SRLS 92 08 (SEO ID NO: 89) (SEO ID NO: 90). (22123) SHVL HSRL 48 52 (SEQ ID NO: 91) (SEQ ID NO: 92) 2 (46/47) AVDF SLGE 22 78 (SEO ID NO 93) (SEO ID NO: 94) 3 (69/70) AVTL LLEG 53 47 (SEO .D _ NO: 95) (SEQ ID NO: 96)

  4 (89/90) LSSL LGQL 95 0 5 (SEO ID NO: 97) (SEQ ID NO: 98), (88/89) C (acm) LSS LLGQ 0 0 0 0 (SEC ID NO: 99) lSEQ ID NO: 100) (90/91) SSLL GQLS 45 (SEQ ID NO: 101) (SEQ ID NO: 102) (8 8/8 9) CLSS LLGQ 90 10 (SEQ ID NO: 103) (SEQ ID NO. : 100). (107/108) LQSL LGTQ 99 01 (SEQ ID NO: 104) (SEQ ID NO: 105) (106/107) ALOS LLGT 70 (SEQ ID NO: 106) (SEQ ID NO: 107) ) 6 (128/129) NAIF LSFQ 60 40 (SEQ ID NO: 108) (SEO ID NO: 109)

  Based on these experiments, the ligation peptides shown in Table 14 must be efficiently bound by the subtiligase. A suitable protecting group for the N-terminus of each donor ester peptide was necessary to prevent autocligation. We have selected an isonicotinyl protecting group (iNOC) (Veber et al., J. Org Chem., 42: 3286-3289 [1977]) because of its solubility in water. It can be incorporated in the last step of solid phase peptide synthesis and is stable to anhydrous HF used for deprotection and peptide cleavage from a solid phase resin. In addition, it can be removed from the peptide after each ligation under moderately reducing conditions (Zn / CH3CO2H) to obtain a free N-terminus for subsequent ligations. A glycolate-lysyl-amide ester (glc-K-NH2) was used for C-terminal activation based on previous experiments which showed that this ester was effectively acylated by the subtiligase ( Abrahmsen et al., Biochem., 30: 4151-4159 (1991).) Glc-K-amide activated peptides protected by an iNOC group can be synthesized by standard solid phase methods as shown (Scheme 2). The peptides are then sequentially fused until the complete protein has been produced, and the final product is refolded in vitro.On the basis of homology with EPO, it is assumed that pairs of disulfides are formed between cysteine residues 7 and 151 and between 28 and

  85. Oxidation of disulfides can be accomplished by simply stirring the reduced material under an oxygen atmosphere for several hours. The folded material can then be purified by HPLC and the active protein-containing fractions can be pooled and lyophilized. Alternatively, disulfides can be differentially protected to influence sequential oxidation between specific pairs of disulfides. Protection of cysteines 7 and 151 with acetamidomethyl (acm) groups would provide oxidation of 28 and 85. The acm groups could then be removed and residues 7 and 151 would be oxidized. Conversely, residues 28 and 85 could be protected by acm and oxidized groups in case sequential oxidation would be required for correct folding. Optionally, cysteines 28 and 85 may be replaced with another natural or non-natural residue other than Cys to ensure proper oxidation of cysteines 7 and 151.

  TABLE 14. Peptide fragments used for the total synthesis of h-ML in the presence of subtiligase Fraciment Sicuence 1 (SEQ ID NO: 110) iNOC-HN-SPAPPACDLRVLSKLLRDSHVL-glc-K-NH2 (1-22) 2 (SEQ ID NO: 111) iNOC-HN-HSRLSQCPEVHPLPTPVLLPAVDF-glc-K-NH2 (23-46) 3 (SEQ ID NO: 112) iNOC-HN-SLGEWKTQMEETKAODILG AVTL-glc-K-NH2 (47-69) 4 (SEQ ID NO: 113) ## STR1 ## -HN-LLGTOLPPQGRTTAHKDPNAIF-glc-K-NH 2 (107-128) 7 (SEQ ID NO: 116)

  H2N.LSFQHLLRGKVRFLMLVGGSTLCVR-CO2 (129-153) Peptide ligation is performed at 250C in 100mM tricine at pH 8 (freshly prepared and degassed by vacuum filtration on a 5nm filter). Normally, the C-terminal fragment is dissolved in a buffer (2-5 mM peptide) and a 10 × subtilisase stock solution (1 mg / ml in 100 mM tricine, pH 8) is added to bring the final enzyme concentration at about 5 μM. A 3-5 mol excess of the glc-K-NH 2 activated donor peptide is then added in solid form, dissolved, and the mixture is allowed to stand at 25 ° C. Ligations are monitored by inverted C18-phase analytical HPLC (gradient CH3CN / H2O with 0.1% TFA). The ligation products are purified by preparative HPLC and lyophilized. Removal of the protective isonicotinyl (iNOC) group was carried out by stirring HC1-activated powdered zinc with the protected peptide in acetic acid. The powdered zinc is removed by filtration and the acetic acid is evaporated under vacuum. The resulting peptide can be used directly in the next ligation and the process can be repeated. Of

  Synthetic hMLs3 can be fused by methods analogous to those described above for synthetic or recombinant hMIs4.332 to produce full-length synthetic or semi-synthetic hML. Synthetic hML offers many advantages over the recombinant. Unnatural side chains can be introduced to improve power or specificity. Polymeric functionalities such as polyethylene glycol may be incorporated to improve the duration of action. For example, polyethylene glycol can be attached to lysine residues of individual fragments (Table 14) before or after one or more ligation steps have been performed. Protease-sensitive peptide bonds can be removed or modified to improve stability in vivo. In addition, heavy atom derivatives can be synthesized to facilitate the determination of structure.

  Scheme 2. Solid Phase Synthesis of Peptide Fragments for Ligation of Segments 0 0 H ~ 14Nb H2N XQ ~ a Br O 1 2 B0C-NH <, o NHY-1 c, d (automatic synthesis) RO 3 A (P eptide ## STR1 ## wherein R (cleavage) ## STR2 ## ) 4 ii NH2!

  Isonicotinyl (iNOC) glycolate-lysylamine (glc-K-NH2) a) Lysyl-paramethylbenzhydrylamine (MBHA) resin 1 (0.63 meq / g, Advanced ChemTech) is stirred with bromoacetic acid (5). equivalents) and diisopropyl carbodiimide (5 equivalents) for 1 hour at 25 ° C in dimethylacetamide (DMA) to form the bromoacetyl derivative 2 b). The resin is washed extensively with DMA, and individual Boc-protected amino acids (3 equivalents, Bachem) are esterified by stirring with sodium bicarbonate (6 equivalents) in dimethylformamide (DMF) for 24 hr. C to produce the corresponding glycolate-phenylalanyl-amide resin 3. The mino-acetylated resin 3 is washed with DMF (3 times) and dichloromethane (CH 2 Cl 2) (3 times) and can be stored at room temperature for several months. The resin 3 can then be loaded into an automatic peptide synthesizer (Applied Biosystems 430A) and the peptides can be extended using conventional solid phase methods (5). c) The N-a-Boc group is removed with a 45% solution of trifluoroacetic acid in CH2Cl2. d)

  The following Boc protectin amino acids (5 equivalents) are preactivated with benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate (BOP, 4 equivalents) and N-methylmorpholine (NMM, 10 equivalents) in DMA and coupled for 1 to 2 hours. e) The final Na-Boc group is removed (TFA / CH2Cl2) to give the resin 4 and the isonicotinyl (iNOC) protecting group is introduced as previously described (4) with stirring using 4isonicotinyl-2,4-carbonate -dinitrophenyl (3 equivalents) and NMM (6 equivalents) in DMA at 25 C for 24 h. f) Cleavage and deprotection of the peptide by treatment with anhydrous HF (5% anisole / 5% ethylmethylsulfide) at 0 ° C. for 1 hour gives the peptide protected by the iNOC group and glycolate-lys-amide activated, which is purified by reverse-phase C18 HPLC (gradient CH3CN / H2O, 0.1% TFA). The identity of all substrates is confirmed by mass spectrometry. It should be noted that the present invention can be implemented in accordance with the foregoing description in accordance with the references and with readily available raw materials. Nevertheless, the Claimant has filed with the American Type Culture Collection,

  Rockville, Md., United States of America (ATCC) the cell line shown below: Escherichia coli, DH10B-pBSK-hmplI 1.8, ATCC Reference No. CRL 69575, filed Feb. 24, 1994. Plasmid, pSV15.ID.LL.MLORF, ATCC Reference No. CRL, filed December 1994; and CHO DP-12 cells of Chinese hamster ovary, ML 1/50 MCB (registration N 1594), reference NO. ATCC CRL 11 770, filed December 6, 1994. This deposit was made in accordance with the provisions of the Treaty of Budapest concerning the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedures and Related Regulations (Budapest Treaty). These provisions ensure the maintenance of a viable crop for 30 years from the filing date. The agencies are available from ATCC under the conditions established by the Budapest Treaty, and their obtaining is subject to an agreement between the applicants and the ATCC which ensures availability subject to the issuance of the corresponding United States patent. The availability of the deposited strain should not be construed as an authorization to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. It goes without saying that the present invention has been described for explanatory purposes but not limiting, and that many modifications can be made without departing from its scope. All references cited herein are expressly incorporated herein by reference.

Claims (5)

  1. Polypeptide ligand mpl isolated substantially homogeneous.   The ligand ligand polypeptide according to claim 1, characterized in that it is selected from the group consisting of (a) a fragment polypeptide; (b) a variant polypeptide; and (c) a chimeric polypeptide.   3. The ligand ligand polypeptide according to claim 1, characterized in that it is selected from the group consisting of (a) the polypeptide which is isolated from a mammal; b) the polypeptide which is prepared by recombinant means; and (c) the polypeptide which is prepared by synthetic means.   4. The ligand ligand polypeptide according to claim 1, characterized in that it is selected from the group consisting of (a) a polypeptide which is human; and (b) a polypeptide that is non-imminogenic in a human.   6. A fragmentary polypeptide according to Claim 2, characterized in that it is represented by the formula X-hTPO (7-151) -Y in which hTPO (7-151) represents the amino acid sequence of thrombopoietin ( TPO) human (hML) from Cys7 to Cys15 inclusive; ~ S ~ X represents an amino group of Cys7 or the amino-terminal amino acid residue (s) selected from the group M, MA, MPA, MPPA, (SEQ ID NO: 117) MAPPA, (SEQ ID NO: 118) MPAPPA (SEO ID NO: 119) MSPAPPA, (SEQ ID NO: 120) A, PA, APP, APPA, (SEQ ID NO: 121) PAPPA, (SEQ ID NO: 122) SPAPPA, (SEQ ID NO: 123) Y represents the carboxy terminal group of Cys1 'or the carboxy-terminal amino acid residue (s) selected from the group V, VR, VRR, VRRA. (SEO ID NO: 124) VRRAP, (SEO ID NO: 125) VRRAPP ,. (SEQ ID NO: 126) VRRAPPT ,. (SEO ID NO: 127) VRRAPPrT, (SEQ ID NO: 128) VRRAPPTTA, (SEO ID NO: 129) VRRAPP1TAV. (SEQ ID NO: 130) VRRAPP1TAVP, (SEQ ID NO: 131) VRRAPP1TAVPS, (SEQ ID NO: 132) VRRAPPTTAVPSR, (SEQ ID NO: 133) VRRAPPTTAVPSRT, (SEQ ID NO: 134) VRRAPPTTAVPSRTS, (SEQ ID NO: 135) VRRAPPTrAVPSRTSL, (SEQ ID NO: 136) VRRAPPTrAVPSRTSLV, (SEQ ID NO: 137) VRRAPPTFAVPSRTSLVL. (SEQ ID NO: 138) VRRAPPTIAVPSRTSLVLT, (SEO ID NO: 139) VRRAPPTFAVPSRTSLVLTL, (SEQ ID NO: 140) VRRAPPTTAVPSRTSLVLTLN, (SEQ ID NO: 141) VRRAPPTTAVPSRTSLVLTLNE, (SEQ ID NO: 142) VRRAPPTTAVPSRTSLVLTLNEL, (SEQ ID NO: 143) VRRAPPTTAVPSRTSLVLTLNELP, (SEO ID NO: 144) and extensions of one or more amino-terminal amino acid residues comprising one or more of residues 176-332 of human ML as shown in Figure 1 (SEQ ID N: 1).   7. A fragmentary polypeptide according to claim 6, characterized in that it is selected from the group TPO (1-153) and TPO (1-245).   A fragmentary polypeptide according to claim 2, characterized in that its amino acid sequence comprises SPAPPACDLRVLSKLLRDSHVL, (SEQ ID NO: 110) HSRLSQCPEVHPLPTPVLLPAVDF, (SEQ ID NO: 111) SLGEWKTQMEETKAQDILGAVTL, (SEQ ID NO: 112) LLEGVMAARGQLGPTCLSSLL ( SEQ ID NO: 113) GQLSGQVRLLLGALOS. (SEQ ID NO: 114) LLGTQLPPQGRTTAHKDPNAIF. (SEQ ID NO: 115) LSFQHLLRGKVRFLMLVGGSTLCVR., And (SEQ ID NO: 116) and combined forms.   Polypeptide according to Claim 6, characterized in that it is unglycosylated.   An isolated polypeptide, characterized in that it is encoded by a nucleic acid having a sequence that hybridizes under moderately stringent conditions to nucleic acid molecules having a nucleic acid sequence shown in Figure 1 (SEQ ID No. 2).   Polypeptide according to Claim 10, characterized in that it is biologically active.   12. Polypeptide according to claim 1, characterized in that it is selected from the group hML, hMLls3, hML (R153A, R154A), hML2, hML3, hML4, mML, mML2, mML3, pML and pML2.   Polypeptide according to claim 2, characterized in that its amino acid sequence comprises amino acid residues 1 to X of Figure 1 (SEQ ID N: 1), where X is selected from the group of numbers 153, 155 , 164, 174, 191, 205, 207, 217, 229, 245 and 332.   14. Isolated mainly homogeneous isolated mpl ligand polypeptide, characterized in that it shares at least 80% sequence identity with the polypeptide of claim 13.   Polypeptide according to claim 13, characterized in that X is equal to 153.   16. Chimera, characterized in that it comprises the ligand mpl according to claim 13 fused to a heterologous polypeptide.   Chimera according to claim 16, characterized in that the heterologous polypeptide is an immunoglobulin-like polypeptide.   Chimer according to claim 16, characterized in that the heterologous polypeptide is an interleukin polypeptide.   19. Chimera characterized in that it comprises N-terminal residues 1 to about 153-157 of hML substituted with one or more, but not all, of added or substituted human EPO residues in N-terminal residues of hML in positions corresponding to the alignment shown in Figure 10.   An antibody characterized in that it is capable of binding to the ligand polypeptide mpl according to claim 13.   21. Hybridoma cell line, characterized in that it is capable of producing the antibody of claim 20.   22. Isolated nucleic acid molecule, characterized in that it encodes the mpl ligand polypeptide of claim 1.   23. Isolated nucleic acid molecule, characterized in that it encodes the mpl ligand polypeptide of claim 13.   24. Isolated nucleic acid molecule, characterized in that it comprises the open-reading frame nucleic acid sequence shown in Figure 1 (SEQ ID N: 2).   25. An isolated nucleic acid molecule according to claim 24, characterized in that it encodes a ligand mpl polypeptide selected from the group consisting of hML, hMLS3, hML (R153A, R154A), hML2, hML3, hML4, mML, mML2, mML3, pML and pML2.   26. An isolated nucleic acid molecule, characterized in that it is selected from the group consisting of (a) a cDNA clone comprising the sequence of leotides of the coding region of the ligand mpl gene; (b) a DNA sequence capable of hybridizing under stringent conditions to a clone of (a); and (c) a genetic variant of any of the DNA sequences of (a) and (b) which encodes a polypeptide having a biological property of a natural mpl ligand polypeptide.   27. isolated DNA molecule, characterized in that it has a sequence capable of hybridizing to a DNA sequence according to FIG. 1 (SEQ ID No. 2) under moderately rigorous conditions, and in that it encodes a biologically active ligand ligand polypeptide.   28. A nucleic acid molecule according to claim 25, characterized in that it further comprises a promoter operably linked to the nucleic acid molecule.   29. Expression vector, characterized in that it comprises the nucleic acid sequence according to the claim operably linked to condition sequences recognized by a host cell transformed with the vector.   30. Host cell, characterized in that it is transformed with the vector according to claim 29.   31. A method of using a nucleic acid molecule encoding the ligand polypeptide mpl to effect the production of this polypeptide, characterized in that it consists in culturing the host cell according to claim 30.   32. The method of claim 31, characterized in that the ligand mp1 polypeptide is isolated from the host cell.   33. A process according to claim 31, characterized in that the mpl ligand polypeptide is isolated from the culture medium of the host cell.   34. A method for detecting the presence of mpl ligand polypeptide, characterized in that it consists in hybridizing the DNA encoding the mpl ligand polypeptide to a nucleic acid of a sample to be analyzed and detecting the presence of polypeptide DNA. ligand mpl.   35. Process for amplifying a tested sample of nucleic acid, characterized in that it consists in initiating a nucleic acid polymerase reaction with a nucleic acid encoding a ligand mpl polypeptide.   36. A composition characterized in that it comprises the ligand ligand polypeptide according to claim 1 and a pharmaceutically acceptable carrier.   37. The composition of claim 36, comprising a therapeutically effective amount of the mpl ligand polypeptide of claim 1 for the treatment of a mammal afflicted with or at risk for thrombocytopenia.   38. The composition of claim 36 further comprising a therapeutically effective amount of an agent selected from the group consisting of cytotoxin, colony stimulating factor or interleukin.   39. A composition according to claim 38, characterized in that the agent is selected from KL, LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-2, IL-3, IL-2, 5, IL-6, IL-7, IL-8, IL-9 and IL-11.   5. A substantially homogeneous isolated mpl agonist, characterized in that: (a) it stimulates the incorporation of labeled nucleotides (3H-thymidine) into the DNA of Ba / F3 cells under the control of IL-3 transfected with help from human mpl; or (b) it stimulates the incorporation of 35S into circulating platelets in the platelet rebound test.