FI121573B - A method for preparing an isolated mpl ligand polypeptide - Google Patents

Description

METHOD FOR PREPARING AN INSULATED MPL LIGAND POLYPEPTIDE

Field of the Invention

The present invention also relates to the isolation, purification, and reconstitution or chemical synthesis of proteins that affect the survival, proliferation, differentiation or maturation of hematopoietic cells, in particular platelet progenitor cells. In particular, the present invention relates to the cloning and expression of nucleic acids encoding and activating a protein ligand capable of binding to mpl, a member of the cytokine receptor superfamily. This invention further relates to the use of these proteins either alone or in combination with other cytokines for the treatment of immune or hematopoietic disorders including thrombocytopenia.

BACKGROUND OF THE INVENTION I. Hematopoietic System

The hematopoietic system produces fully developed, highly specialized blood cells that are known to be essential for the survival of all mammals. These mature cells include: red blood cells specialized in oxygen and carbon dioxide transport, T and B lymphocytes responsible for the cell and antibody-selected immune responses, platelets or platelets specialized in forming blood clots, and granulocytes and macrophages. that specialize in cancer cells and extra cells to fight infection. Granulocytes are further subdivided into neutrophils, eosinophils, basophils, and cytocytes, which are specialized cell types with distinct functions. It is noteworthy that all of these specialized, fully developed blood cells are derived from one common primitive cell type called the pluripotent (or totipotent) stem cell, which is primarily found in the bone marrow [Dexter et al., Ann. Rev. Cell Biol., 3: 2423-441 (1987)].

Fully developed, highly specialized blood cells must be produced in large quantities continuously throughout the life of the mammal. A very large majority of these specialized blood cells are intended to remain functionally active for only a few hours to weeks (Cronkite et al., Blood Cells, 2: 263-284 (1976)). Thus, continuous regeneration of fully developed blood cells, primitive stem cells themselves, and any intermediate or lineage-bound progenitor cell lines between primitive and fully developed cells is necessary to maintain the mammal's normal steady-state blood cell requirement.

At the core of the hematopoietic system is a pluripotent stem cell (s). These cells are relatively small in number and undergo increased self-renewal to produce germline cells or are increasingly transformed into mature lineage-restricted progenitors, which eventually form a highly specialized, fully developed blood cell (blood cells).

.... For example, certain multipotent progenitors of which • · · \ *. referred to as CFC-Mix and derived from stem cells, undergo proliferation (self-renewal) and • · i * 'production, producing colonies containing all types of myeloid cells: red cells, neutrophils, megakaryocytes (platelet parent cells), macrophages, basophils, eosinophils, and mast cells. Other progenitor cells of the lymphoid lineage undergo T- proliferation and development. . *. cells or B cells.

• · · • · ·. ·; ·. In addition, there is another intermediate class of CFC-Mix progenitors and myeloid cells that are in- • · V * termediate commitment to the function of their offspring. These pedigree - restricted progenitor cells are classified as the progeny they produce. Based on age. Thus, known immediate myeloid cells are: • erythroid colony forming monofilament (CFU-E) for red blood cell production, granulocyte / macrophage colony forming cells (GM-CFC) for neutrophil and macrophage production, megakaryotic cells (Meg-CFC) for the production of megakaryocytes, eo-cinophilic colony forming cells (Eos-CFC) for the production of eosinophils and basophilic colony forming cells (Bas-CFC) for the production of mast cells. Other intermediate progenitor cells of varying lineage or degree of self-renewal between pluripotent stem cells and fully developed blood cells are known (or see below) or are likely to be detected.

The basic principle of a normal hematopoietic cellular system appears to be reduced ability to self-regenerate as multipotency has been lost and lineage restriction and maturity have been acquired. Thus, at the other end of the hematopoietic cell spectrum, there is a pluripotent stem cell that has the ability to self-renew and differentiate into all sorts of lineage-specific progenitor cells. This ability is the basis of bone marrow transplantation, where primitive stem cells re-occupy the entire hematopoietic cell system. At the other end of the spectrum are greatly descended progenitor cells and their offspring, which have lost their ability to self-replicate but have acquired fully developed functional activity.

· ♦: ** Various hematopoietic growth factors or cytokines carefully control proliferation and development of stem cells and genes.

: * Γ: The contribution of these growth factors in vivo is complex and is not fully known. Some growth factors, such as ine. . ·. terleukin-3 (IL-3), able to stimulate, and Multiport *. exams stem cells and progenitor cells of different generations, including megakaryocytes, for example. Other factors, such as granulocyte / macrophage colony-stimulating factor (GM-CSF), were initially thought to be functional. restricted to GM-CFC. Later, however, • - • ·. ···. tnn that GM-CSF also influenced e.g. • proliferation and development of megakaryocytes. Thus, it was found that IL-3 and GM-CSF had overlapping biological activities, although the efficacy was different. It was later found that both interleukin-6 (IL-6) and interleukin-11 (IL-11), while having no apparent effect on the formation of meg colonies alone, act synergistically with IL-3 to stimulate the maturation of megakaryocytes. Yonemura et al., Exp. Hematol., 20: 1011-1016 (1992)].

Thus, hematopoietic growth factors may affect the growth and differentiation of one or more lineages, may partially overlap with other growth factors in a single progenitor line, or may act synergistically with other factors.

It also appears that hematopoietic growth factors may exert their effects at different stages of cell development from a totipotent stem cell through a variety of bound germ-restricted progenitors to a fully developed blood cell. For example, erythropoietin (epo) appears to promote the proliferation of only mature erythroid progenitor cells. IL-3 appears to act in the past, affecting primitive .... dense stem cells and germ-restricted intermediate progenitor cells. Other growth factors, such as stem cell factor (SCF), may even contribute more to the development of a primitive cell.

• ·

From the foregoing, it will be appreciated that novel φ *. · / Hematopoietic growth factors that affect the survival, proliferation, differentiation, or maturation of any blood cells or their parent cells would be beneficial. . *. especially to help rebuild the impaired haematopoietic system caused by the disease, or after radiation or chemotherapy.

• · · *. *: II. Megakaryocytopoiesis - Platelet Production • · ·

The regulation of megakaryocytopoiesis and platelet production is reviewed in Mazur, Exp.

'·· *. Hematol., 15: 248 (1987) and Hoffman, Blood, 74: 1196-1121212 (1989). Briefly, bone marrow pluripotent stem cells differentiate into megakaryocytic, erythrocytic and myelocytic cell lines. It is believed that there is a hierarchy of megakaryocytic progenitors committed to their future role between stem cells and megakaryocytes. At least three classes of megakaryocytic progenitor cells have been identified, namely burst forming unit megakaryocytes (BFU-MK), colony forming unit megakaryocytes (CFU-MK), and low density megakaryocyte precursor cells (LD-CFU-MK). Megakaryocytic maturation itself is a continuous developmental event, separated into stages based on standard morphological criteria. The earliest identified member of the megakaryocyte family (MK or xneg family) is the megakaryotes. These cells are initially 20 to 30 μπ in diameter and have a basophilic cytoplasm and a slightly irregular nucleus with a loose, somewhat networked chromatin and several nuclei. Later, megakaryoblasts may contain up to 32 nuclei (polyploid), but the cytoplasm remains scarce and immature. As the process of maturation progresses, the nucleus becomes stripped and compacted, the amount of cytoplasm increases, and it becomes more acididophilic and granular. For this family *. the most mature cells can give the appearance of liberated blood platelets • in the periphery. Normally less than 10% of the megaka: * 'ryocytes are in the blast phase and more than 50% are fully developed. The arbitrary morphological classifications commonly used for the megakaryocyte series are the megakaryoblast of the earliest ::: form; intermediate promegakaryocyte or basophilic megakaryocyte and mature forms of the last forms .. '. (acididophilic, granular, or platelet-producing) mega- • · ·. ·: ·. megakaryocytic. The mature megakaryocyte extends cytoplasmic fibers to the capillaries where they detach and disintegrate into individual platelets (Williams et al., Hematol ...: logY / 1972).

Megakaryocytopoiesis is believed to involve a number of regulatory • ·, ···. common factors [Williams et al., Br. J. Haematol., 52: 173-6-6 (1982) and Williams et al., J. Cell Physiol., 110: 101 (1982)]. Early levels of megakaryocytopoiesis are believed to be mitotic with respect to cell proliferation and colony initiation from CFU-MK, but are not affected by platelet count [Burstein et al., J. Cell Physiol., 109: 333 (1981) and Kimura et al., Exp. . Hematol., 13: 1048 (1985)]. The subsequent stage of maturation is non-mitotic and involves nuclear polyploidization and cytoplasm maturation and is probably regulated by the number of peripheral platelets in the feedback mechanism [Odell et al., Blood, 48: 765 (1976) and Ebbe et al., Blood, 32: 787 (1966). )].

The existence of a distinct and specific megakaryocyte-colony stimulating factor (MK-CSF) is controversial [Mazur, Exp. Hematol., 15: 340-350 (1987)]. However, many authors believe that both the vital process for survival and platelet production is likely to be regulated by the cytokine (s), who are solely responsible for this process. The assumption of the existence of a megakaryocyte / platelet specific cytokine (s) has provided the basis for research for over 30 years, but to date such cytokine has not been purified, sequenced, or identified by assay as unique MK-CSF '. I (TPO).

• · \ Although MK-CSFs have been reported to be partially purified from experimentally produced thrombocytopenia [Hill • · · .1. * Et al., Exp. Hematol., 14: 752 (1986)], and conditioned medium (CM) of human embryonic kidney (McDonald et al., J. Lab. Clin. Med. , 85: 59 (1975)] and: from human urinary extracts of plastic anemia and idiopathic thrombocytopenic purpura [Kawakita et al., Blood, 6: 556 (1983)] and plasma [Hoffman et al., J.

• * ♦ * · * / Clin. Invest., 75: 1174 (1985)], their physiological function is still unknown in most cases.

: * · *: Use of Pokeweed Mitogen Activated Spleen Cells • ·. ***; medium (PWM-SpCM) and the used medium for rat myelomonocyte • · · 7 distal cell line WEHI-3 (WEHI-3CM) have been used as megakaryocyte potentiators. PWM-SpCM contains factors that increase CFU-MK growth [Metcalf et al., Pro. Natl. Acad. Sci., USA, 72: 1744-1748 (1975); Quesenberry et al., Blood, 65: 214 (1985); and Iscove, N.N., in Hematopoietic Cell Differentiation, ICN-UCLA Symposia on Molecular and Cellular Biology, Vol 10, edited by Golde et al. (New York, Academy Press), pages 37-52 (1978)], and one of these is interleukin-3 (IL-3), a multi-genital colony stimulating factor (multi-CSF) [Burstein, Blood Cells, 11: 469 (1986)]. Other factors for this medium have not yet been identified or isolated. WEHI-3 is a rat 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 wide variety of hematopoietic cells (Ihle et al., J. Immunol., 13: 282 (1983)). IL-3 has also been found to act synergistically with many 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)], including erythropoietin; ni (EPO) and interleukin-1 (IL-1), very early multi- ·] .t; • induction of potent precursors and formation of very large hematopoietic colonies.

• · *. . Other sources of megakaryocyte potentiators have been found in nutrient media used in rat, lung, bone and macrophage cell lines, peritoneal inflammatory cells, and human embryonic mammalian cells. Despite some contradictory data [Mazur, Exp. Hematol., 15:: 340-350 (1987)], there is some evidence [Geissler, et al., Br. J. Haematol., 60: 233-238 (1985)] that φ -activated T lymphocytes play a proactive role in megakaryl · · * * · '/ osytopoiesis as opposed to monocytes. These disruptions suggest that secretions of activated T lymphocytes, such as interleukins, may be regulators of MK development [Geissler et al., Exp. Hematol., ··· 8 15: 845-853 (1987)]. A few megakaryocyte poeses using purified erythropoietin, EPO, [Vainchenker et al., Blood, 54: 940 (1979); McLeod et al., Nature, 261: 492-494 (1976); and Williams et al., Exp. Hematol., 12: 734 (1984), show that this hormone has a potentiating effect on MK colonization. This has also been demonstrated in both serum-free and serum-containing cultures and in the absence of additional cells [Williams et al., Exp. Hematol., 12: 734 (1984)].

EPO was expected to be more involved in the single-cell and double-cell stages of megakaryocytosis, as opposed to the action of PWM-SpCM in the four-cell stage of mega-karyocyte development. The interaction of all these factors in both early and late stages of megakaryocyte development remains to be elucidated.

Data from several laboratories suggest that the only multigene factors with independent MK-colony stimulating activity are GM-CSF and IL-3 and, to a lesser extent, B-cell stimulating factor IL-6 [Ikebuchi et al., Proc. Natl. Acad. Sci. USA, 84: 9035 (1987)]. Later, many authors have reported that IL-11 and the Leukemia Inhibitor (LIF) act synergistically with IL-3 to increase megakaryocyte size and ploidy [Yonemura et al., British Journal of Hematolo - * · \ ** gy, 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); Bru · ♦ · 'no et al., Exp. Hematol., 19: 378-381 (1991); and Yonemura et al., Exp. Hematol., 20: 1011-1016 (1992)].

ί Other interesting documents include: ···

Eppstein et al., U.S. Patent 4,962,091; Chong, U.S. Patent 4,879,111; Fernandes et al., U.S. Patent 4,604,377; Wissler et al., U.S. Patent 4,512,971; Gottlieb, U.S. Patent 4,468,379; * .. · * Bennet et al., U.S. Patent 5,215,895; Kogan et al., U.S. Pat. exam 5,250,732; Kimura et al., Eur. J. Immunol., 20 (9): • ·. · * ·. 1927-1931 (1990); Secor et al., J. of Immunol., 144 (4): 91484-1489 (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); All 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 crucial elements of the blood's coagulation mechanism. Circulatory deficiency of platelets, known as thrombocytopenia, occurs in a variety of clinical settings and conditions. Thrombocytopenia is generally defined as the amount of platelets less than 150 x 10 9 / liter. The major causes of thrombocytopenia can be broadly divided into three classes based on the platelet life cycle, namely (:.) Impaired platelet production, (2) sequestration of the platelets in the spleen (enlarged spleen), or (3) increased auto-thrombosis of the platelets. chemotherapy and radiation). In addition, patients with • * '· /; rapid administration of large volumes of scarce platelet-containing blood products, · thrombocytopenia can develop ill · *. . because of his passing.

• · · ** / Clinical manifestations of thrombocytopenia depend on the severity of thrombocytopenia, its cause, and • · · · possible coagulation deficiencies.

Generally, patients with platelet counts between j 20 and 100 x 109/1 are at risk of excessive bleeding after injury, while patients with platelet counts. below 20 x 109/1, may bleed spontaneously for no reason.

m · · These latter patients are candidates for platelet transplants with immune system risk and · * · *: viral risk. For any given degree of thrombocytopenia, · »*. ***; in a pause, bleeding tends to be more severe when the cause is ··· 10 reduced production rather than increased platelet degradation. In the latter situation, the accelerated platelet turnover rate results in the circulation of younger, larger, and haemostatically more effective platelets. Thrombocytopenia may be due to a variety of conditions which are briefly described below. A more detailed description can be found in Schafner, A.I., "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 impaired platelet production

Causes of congenital thrombocytopenia include structural aplastic anemia (Fancor's syndrome) and congenital amegacaryocytic thrombocytopenia, which may be associated with skeletal malformations. The acquired diseases of platelet production may be caused by either mega-karyocyte hypo-plasma or ineffective thrombopoiesis. Megakaryocytic hypoplasia can be caused by a variety of diseases including bone marrow immature (including idiopathic forms or bone marrow depression caused by chemotherapeutic agents or r: *. Radiation), myelofibrosis, leukemia and metastatic penetration of the bone marrow. In some situations, k »· *. . toxins, infectious agents, or drugs may interfere with thrombopoiesis relatively selectively; transient thrombocytopenias caused by alcohol and certain viral infections and mild thrombocytopenia associated with the use of thiazide diuretics are included in the pre-I * · • L * signs. Lo-s; ': boxing ineffective thrombopoiesis following megaloblastic processes (deficiency of folate or vitamin B12) can also cause thrombocytopenia with commonly occurring anemia and * · · leukopenia.

j · ·; · * Current treatment of thrombocytopenia due to reduced platelet production depends on the bone marrow »* '*; »· 11 identification of the underlying cause of the failure and improvement. Platelet transplants are usually reserved for patients with severe bleeding complications, or for protection during surgical procedures, since isoimmunization can lead to inactivation of new platelet transplants. Mucosal haemorrhage resulting from severe thrombocytopenia can be ameliorated by oral or intravenous administration of antifibrinolytic agents to the patient. However, thrombotic complications may develop if anti-fibrinolytic agents are used in patients with disseminated intravascular coagulation (DIC).

(b) Thrombocytopenia due to the isolation of platelets in the spleen

Splenic enlargement of any cause may be associated with mild to moderate thrombocytopenia. This is a largely passive process of platelet isolation in the spleen (hypersplenism), in contrast to the active destruction of platelets by immune system-mediated thrombocytopenia, which will be described below. Although the most common cause of hypersplenism is splenic hyperplasia due to increased blood pressure in the portal vein of S * · * · due to alcohol ψ * •; Cirrhosis caused by thrombocytopenia are also associated with other infiltration-related infiltration •. other forms of hyperplasia or lymphoproliferative splenic enlargement. Platelet counts generally do not fall below 50 I · * · * «'x 109/1 as a result of hypersplenic activity alone.

9 · · V * (c) Thrombocytopenia due to non-immune mediated platelet destruction • tjci Thrombocytopenia may be due to accelerated platelet destruction by a variety of non-Immunological processes. Disorders of this type include disseminated intravascular coagulation, s: “· intravenous prosthetic devices, extracorporeal circulation, and thrombotic small blood vessel diseases such as thrombotic thrombocytic purpura. In all these,

• H

In 12 cases, circulating platelets that are exposed to either artificial surfaces or abnormal intravascular membrane are either consumed at these sites or damaged and then prematurely cleaned by the reticuloendothelial system. Disease conditions or diseases in which disseminated intravascular coagulation (DIC) can originate are described in more detail in Braunwald et al. (editors), Harrison's Principles of Internal Medicine, 11th Edition, page 1478, McGraw Hill (1987). Intravascular prosthetic devices, including cardiac valves and intra-aortic spheres, may cause mild-to-moderately destructive thrombocytopenia and transient thrombocytopenia in patients undergoing cardiopulmonary bypass, or hemodialysis may result in extravascular or peripheral hemorrhage.

(d) Drug-induced immune thrombocytopenia

More than 100 drugs have produced immunologically mediated thrombocytopenia. However, only quinidine, quinine, gold, sulfonamides, cephalothin and heparin are well characterized. Drug-induced thrombocytopenia is often very severe and typically occurs abruptly within a few days of the patient receiving a sensitizing medication • · · • · ...

·. *.

• (e) Immune (autoimmune) thrombocytopenic purpura (itp) j In adults, ITP is a chronic disease characterized by autoimmune platelet destruction. The autoantibody is usually IgG, although other immunoglobulins. ·. lines have been reported. Although ITP autoantibody has been found to be associated with the platelet membrane GPIIbIIIa «· · *. , platelet antigen specificity is not identified · · · V: in most cases. Extravascular destruction of sensitized platelets occurs in the spleen and liver reticuloendothelial system. Although more than half of all ITP cases are idiopathic, many patients have underlying rheumatic or autoimmune diseases (eg systemic lupus erythematosus) or lymphoproliferative diseases (eg chronic lymphocyte leukemia). .

(f) HIV-induced ITP

ITP is an increasingly common complication of HIV infection [Morris et al., Ann. Intern. Med., 96: 714-717 (1982)] and may occur at any stage of the disease in both patients with acquired immune deficiency (AIDS), patients with AIDS-related complex and patients with HIV. infection, but without the symptoms of AIDS. HIV infection is a person-to-person transmission disease that is ultimately characterized by a profound loss of cellular immune function and the appearance of opportunistic infection and cellular malignancy. The primary immunological abnormality resulting from HIV infection is the progressive reduction and functional impairment of CD4 cell surface glycoprotein-expressing T-lym phosphocytes [Lane et al., Ann. Rev. Immunol., 3: 477 (1985)]. The disappearance of CD4 helper / inducer T cell function is probably at the root of cell and fluid deficiencies in immunity, leading to opportunistic infections and the malignancy characteristic of AIDS (H. Lane, supra).

t · · ί · 1 Although unknown, the mechanism of HIV-related ITP is believed to be different from that of a non-HIV-infected ITP [Walsh et al. al., N. Eng.

: 1: J. Med., 311: 635-639 (1984) and Ratner, Am. J. Med. , 86: 194-189 (1989)].

• · · IV. Current treatment of thrombocytopenia

The severity and urgency of the clinical situation dictate a therapeutic solution for patients with thrombocytopenia. to manage the premises. The treatment is the same in HIV-related and • · · • · · ·. • non-HIV-related thrombocytopenia, and although there are several treatment options available, treatment remains controversial.

Platelet counts in patients with diagnosed thrombocytopenia have been successfully increased with Glu corticosteroid therapy (eg prednisolone), however most patients respond incomplete or relapsed when the glucocorticoid dose is reduced or discontinued. Based on studies in patients with HIV-associated ITP, some researchers have suggested that glucocorticoid treatment may lead to exposure to AIDS. Glucocorticoids are usually given to the patient if the platelet count falls below 20 x 10 9/1 or if there is spontaneous bleeding.

For patients who do not respond well to glucocorticoids, 4- (2-chlorophenyl) -9-methyl-2- [3- (4-morpholinyl) -3-propanon-1-yl] 6H-thieno [3, 2, f] [1,2,4] triazolo [4,3, a] [1,4] diazepine compound (WEB 2096) for the treatment of a non-HIV-related severe ITP case. A patient with platelet counts of 37,000 to 58,000 / μΐ was treated with WEB 2086 and after 1 to 2 weeks, platelet counts increased to 140,000 to 190,000 / μl [EP 361077 and Lohman et al., Lancet, 1147 (1988)].

Although the optimal treatment for the acquired amegakaryocytic thrombocytopenic purpura (AATP) is uncertain, it has been shown that anti-thymocyte globulin (ATG), • an · serum for human thymus tissue, produces long-lasting. 1 2 31 its complete relief [Trimble et al., Am. J. Hema., 1: 3, 37: 126-127 (1991)]. However, a recent report demonstrates that the hematopoietic effects of ATG are due to: Thimerosal, whereby the protein is probably active in mercury; as a carrier (Panella et al., Cancer Research, 50: 4429-4435 (1990)).

Good results have been reported for spleen removal. The removal of the spleen removes the platelet destruction • · · *. its most important point and the most important source of autoantibody production in many patients. This procedure results in a long-term untreated spread of symptoms 1 in a large number of patients. However, since surgery should generally be avoided in immunologically compromised patients, splenectomy is recommended only in severe cases of thrombocytopenia (eg, severe HIV-associated ITP) in patients who do not produce response to 2-3 weeks of glucocorticoid therapy or do not achieve sustained response after discontinuation of glucocorticoid therapy. Based on current scientific experience, it is unclear whether patients with AIDS are exposed to splenectomy.

In addition to treatment with prednisolone and splenectomy, certain cytotoxic agents, e.g., vincristine and azidothymidine (AZT, zidovudine), also show promise in the treatment of HIV-induced ITP; however, the results are preliminary.

From the foregoing, it is to be understood that one way of treating thrombocytopenia would be to obtain an agent capable of accelerating the differentiation and maturation of megakaryocytes or their precursors into a platelet-producing form. Considerable effort has been made to identify such an agent, commonly referred to as "thrombopoietin" (TPO). Other TPO names commonly found in the literature include thrombocytopoiesis stimulating factor (TSF), megakaryocyte colony stimulating factor (MK-CSF), megakaryocyte stimulating factor, and megakaryocyte potentiator. TPO activity was observed as early as 1959 (Rak et al., Med. Exp., 1: 125) • ·: 1 ·· and attempts to characterize and purify this substance have continued to this day. . Although there have been reports of partial purification of TPO-active polypeptides [see, e.g., Tayrien et al., J. Biol. Chem., 262: 3262 (1987) and Hoffman et al., J. Olin. Invest. 75:. . ·. 1174 (1985)], others have argued that TPO is not a distinct independent entity with its own aids, but rather. it is simply a polyfunctional · · · V · form of the known hormone [IL-3, Sparrow et al., Prog. body.

Biol. Res., 215: 123 (1986)]. Thrombopoietic activity; · 1 ·. a molecule having a significant therapeutic value of 16. Although no protein has been unequivocally identified as TPOs, considerable interest surrounds the recent finding that mpl, a putative cytokine receptor, can transduce a thrombopoietic signal.

V. Mpl is a Megakaryocytopoietic Cytokine Receptor It is believed that the proliferation and maturation of hematopoietic cells are strictly regulated by factors that positively or negatively alter pluripotent stem cell proliferation and multigenerational differentiation. These effects are mediated by the high affinity binding of extracellular protein factors to specific cell surface receptors. These cell surface receptors share a significant homology and are generally classified as members of the cytokine receptor family. Members of the Su family include receptors for the following factors: IL-2 (β 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)); 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)); grananyl '*: lymphocyte macrophage colony stimulating factor (GM-CSF) [Gearing et al., EMBO J., 8: 3667-3676 (1991); Hayashida et al., Proc. Natl. Acad. Sci. USA, 244: 9655-9659]. Glycocyte Colony Stimulating Factor (G-CSF) i · · * [Fukunaga et al., Cell, 61: 341-350 (1990a); Fukunaga (1990)]; 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., 17

Blood, 76: 31-35 (1990)]; leukemia inhibiting 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)] and also prolactin receptors [Boutin et al., Proc. Natl. Acad. Sci. USA, 88: 7744-7748 (1988)]; Edery et al., Proc. Natl. Acad. Sci. USA, 86: 2112-2166 (1989)), growth hormone (GH) [Leung et al., Nature, 330: 537-543 (1987)] and the ciliary neurotrophic factor (CNTF) [Davis et al., Science, 253: 59-63 (1991).

Members of the cytokine receptor family can be grouped into three functional classes (for a review, see Nicola et al., Cell, 67: 1-4 (1991)). The first class includes single chain receptors, such as the erythropoietin receptor (EPO-R) or the granylocyte colony stimulating factor receptor (G-CSF-R), which bind the ligand with high affinity through the extracellular domain and also produce an intracellular signal. Another class of receptors, called α-subunits, include the interleukin-6 receptor (IL-6-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 family. These α-subunits bind the ligand with low affinity, but cannot · · · Ί mediate intracellular sigma. With a high affinity, a binding receptor capable of producing; 1 signal, produces a heterodimer between the α-subunit and the third class of cyto- • · *. * ·· kinetic receptors, the so-called β-subunit: units, e.g., Bc, three α-subunits, IL-3- Ra and GM-CSF-R, a common β-subunit.

•

Evidence that mpl is a cytokine receptor. . ·. member of the genus family, derived from sequence homology [Gea- · · · ·; * ·. ring, EMBO J., 8: 3667-3676 (1988); Bazan, Proc. Natl.

2 · ·

Acad. Sci. USA, 87: 6834-6938 (1990); Davis et al., V · Science, 253: 59-63 (1991) and Vigon et al., Proc. Natl.

* ...: Acad. Sci. USA, 89: 5640-5644 (1992)] and its ability; transmits proliferative signals.

• · • · • 18 •

The protein sequence derived from molecular cloning of rat c-mpl reveals that this protein is homologous to other cytokine receptors. The extracellular domain contains 4,665 amino acid residues and consists of two subdomains, each with four highly conserved cysteines, and a specific motif in the N-terminated subdomain and the C-terminated subdomain. Li-gand binding extracellular domains are predicted to have similar double B-barrel fold (β-Barrel fold) structural geometries. This double extracellular domain is highly homologous to the signal transducing chain common to the IL-3, IL-5 and GM-CSF receptors and to the low affinity binding LIF domain [Vigon et al., Oncogene, 8: 2607-2615 (1993)]. Thus, mpl may belong to the class of cytokine receptors that bind the ligand with low affinity.

Comparison of rat mpl and human mature mpl P reveals that these two proteins show 81% sequence identity. In particular, the N-terminal and C-terminal extracellular subdomains share a sequence identity of 75% and 80%, respectively. The best conserved mpl region is the cell glue domain, which shows 91% amino acid identity, whereby the sequence of 37 residues • · ·: · 1 near the transmembrane domain is identical in both lαs. Therefore, mpl is reported to be a cytokine receptor; One of the best-preserved members of the thoracic superfamily (Vi- •: / ·· gon, supra) The evidence that mpl is a functional receptor capable of transducing a proliferative signal is derived from the construction of such chimeric receptors. . ·. of tea containing the extracellular domain cyto- • · · '.V.' a high affinity receptor for known <S · *. cytokine, and which have a mpl cell membrane domain. Since no known mpl ligand has been reported, it was necessary to construct a chimeric, high-affinity ligand. din binding extracellular domain of the first 19 classes of cytokine receptors, such as IL-4R or G-CSFR. Vi-gon et al., Supra, fused c-mpl of the extracellular domain of G-CSFR with both the transmembrane domain and the cytoplasmic domain. The IL-3 dependent cell line, BAF / B03 (Ba / F3), was transfected with the G-CSFR / mpl chimera along the entire length of the G-CSFR control. Cells transfected with the chimera grew equally well in the presence of cytokine IL-3 or G-CSF. Similarly, cells transfected with G-CSFR also grew well in the presence of either IL-3 or G-CSF. All cells died in the absence of growth factors. A similar experiment was performed by Skoda et al., EMBO J., 12 (7): 2645-2653 (1993), in which both the extracellular domain and the transmembrane domain of the human IL-4 receptor (hIL-4-R) were fused to rat mplr1 the fusion was transfected into a murine IL-3 dependent Ba / F3 cell line. Ba / F3 cells transfected with wild-type hIL-4-R normally proliferated in the presence of either IL-4 or IL-3 specific species. Ba / F3 cells transfected with hIL-4R / mpl normally proliferated in the presence (in the presence or absence of hIL-4), indicating that in Ba / F3 cells, the mpl cellular domain contains all elements , which are necessary for the transmission of a proliferative signal.

These chimeric experiments demonstrate the ability of mpl to produce a cell proliferative signal, but do not reveal whether the extracellular domain of mpl can bind the ligand. These results are consistent with at least two possibilities, namely, mpl is a single-chain receptor (class one), such as EPO-R or G-CSFR, or is a β-subunit of signal transduction (class three) that requires α-subunit ,. ·. units such as IL-3 (Skoda et al., supra).

. ·; ·. VI. The Mpl ligand is thrombopoietin (TPO).

As described above, it has been proposed that serum • ♦ · V * contains a unique factor, sometimes called throm • · ♦ * ...: bopoietin (TPO), which acts synergistically differently. with other cytokines to promote megakaryocyte growth and maturation. No such natural factor has ever been isolated from serum or any other source, even though countless groups have persistently tried it. Although it is not known whether mpl is capable of directly binding a megakaryocyte stimulating factor, recent experiments indicate that mpl is involved in the transduction of the proliferative signal from the factor or factors found in the serum of patients with aplastic bone marrow [Methia et al., Blood, 82 (82). : 1395-1401 (1993)].

Evidence for a unique serum colony forming factor that is clearly distinguishable from IL-1ar, IL-3, IL-4, IL-6, IL-11, SCF, EPO, G -CSF and GM-CSF, which transduces a proliferative signal via mpl, has been obtained from the study of the distribution of c-mpl expression in primitive and task-bound hematopoietic cell lines and mpl-antisense studies in one of these cell lines.

Using reverse transcriptase PCR [(RT) -PCR] in immunologically purified human hematopoietic cells, Methia et al., Supra, showed that strong mpl mRNA messages were only found in purified CD34 + cells, megakaryocytes, and platelets. Bone .... Nuclear CD (BM) purified CD34 + cells represent approximately 1% of all bone marrow cells and are enriched in all genotypes (such as erythroid, granulomacrophage and megakary- • 1 ϊ · 1 oscotic) in primitive and task-bound pre-V1 • cells.

♦ t · / · Mpl antisense oligodeoxynucleotides were shown to: attenuate the formation of megakaryocytic colonies from pluripotent CD34 + cells cultured in serum from patients with aplastic bone marrow · «» * ··, [megakaryocyte colony stimulating MK-CSA]). These same antisense oligodeoxynucleotides had no effect on erythroid or granulomacrophage colony formation.

♦

It is still unknown whether the mpl directly bound the ligand and whether the serum factor, which was shown to be caused by megakaaryocytopoiesis, mpl. However, it has been suggested that if mpl directly binds the ligand, its amino acid sequence is likely to be highly conserved and has cross-species reactivity due to the remarkable sequence identity between the extracellular domains of human and rat mpl [Vigon et al., Supra, ( 1993)]. VII. Destinations

In view of the foregoing, it is understood that there is a general and continuing need in the art for the isolation and identification of molecules capable of stimulating the proliferation, differentiation and maturation of hematopoietic cells, particularly megakaryocytes or their mast cells, for therapeutic use in the treatment of thrombocytopenia. It is believed that such a molecule is an mpl ligand and thus there is a further need to isolate such ligand (s) in order to evaluate their function (s) in cell growth and differentiation.

Accordingly, it is an object of this invention to provide a pharmaceutically pure molecule capable of stimulating the proliferation, differentiation and / or maturation of megakaryocytes into a mature platelet producing form.

Another object is to produce a molecule in a form for therapeutic use in the treatment of a haematopoietic disorder, particularly thrombocytopenia.

Another object of the present invention is to isolate, purify, and in particular identify protein binding gands capable of binding and mediating in vivo the cytokine superfamily receptor known as mpl. . *. proliferative signal.

m · ·

Still another object is the production of nucleic acid molecules encoding such protein ligands and the use of these nucleic acid molecules to produce mpl binding ligands in recombinant cell culture. for diagnostic and therapeutic use.

»* ··» »· #« «22

Still another object is the production of derivatives and modified forms of protein ligands, including amino acid sequence variants, glycoprotein variants, and covalent derivatives thereof.

Another object is to produce fusion polypeptide forms that combine the mpl ligand and the heterologous protein and its covalent derivatives.

Still another object is to produce variants of polypeptide forms by combining mpl ligand with amino acid additions and substitutions from the EPO sequence to produce a protein capable of regulating proliferation and growth of both platelets and red blood cell progenitors.

Still another object is the production of an immunogenic Len to generate antibodies against mpl ligands or fusion forms thereof, and to obtain antibodies capable of binding such ligands.

These and other objects of the invention will be apparent to those skilled in the art upon reading the patent specification as a whole.

Summary of the Invention

The objects of the invention are achieved by the production of an isolated protein that promotes mammalian megakaryocytopoietic proliferation and maturation, termed "mpl ligand" (ML) or "thrombopoietin" (TPO) and is capable of stimulating. . # Loima megakaryocyte proliferation, maturation and / or. : differentiation into a mature platelet producing form.

This substantially homogeneous protein can be purified from a natural source using the method of (1) contacting a source plasmid containing the purified mpl ligand molecules, an immobilized receptor, • · · · toripolypeptide, particularly npl or mpl fusion polypeptide immobilized on a substrate, under conditions where the mpl ligand molecules to be purified adsorb to the immobilized receptor polypeptide. 23 (2) washing the immobilized receptor polypeptide and its substrate to remove non-adsorbent material, and (3) eluting the mpl ligand molecules from the immobilized receptor polypeptide on which they are absorbed. , elution buffer.

Preferably, the natural source is mammalian plasma or urine containing the mpl ligand. Optionally, the mammal is aplastic and the immobilized receptor is a mpl-IgG fusion.

A potentially preferred megakaryocytopoietic proliferation and maturation promoting protein is an isolated, substantially homogeneous mpl ligand polypeptide prepared by synthetic or recombinant means.

Preferably, the "mpl ligand.L" polypeptide or "TPO" of the present invention has at least 70% overall sequence identity with the highly purified substantially homogeneous porcine mpl ligand polypeptide amino acid sequence and at least 80% sequence identity with porcine. mpl ligand polypeptide "EPO domain". Optionally, the mpl ligand of the present invention is a mature human mpl ligand (hML) having the fully developed amino acid sequence shown in Figure 1 (SEQ ID NO: 1), or a variant thereof, or modified after transcription. form, or a protein having: * ·. · has about 80% sequence identity with the mature human mpl-li · · · * · .. gand. The mpl ligand variant is possibly human. The mature mpl ligand (hML) fragment, especially the amino terminus. . ·. the other, or "EPO domain" fragment. Preferably, the amino terminus fragment contains substantially all of the human ML sequence between the first and fourth cysteine residues, but may contain significant additions, deletions, or substitutions upstream of that region. . According to this embodiment, the fragment poly- 'j'; the peptide may be represented by the formula:

. * ··. X - hML (7-151) - Y

Wherein hML (7-151) represents the human TPO (hML) amino acid sequence from Cys7 to Cys151, including Cys7 and Cys151; X represents the amino group residue of Cys7 or one or more amino acid residues of leader sequences containing mature hML or related amino acid residue extensions such as Met, Tyr, or, for example, proteolytic (e.g., factor Xa or thrombin) cleavage sites; and Y represents the carboxy terminus group of Cys151 or one or more amino acid residues of the carboxy terminus of mature hML or related extensions.

Optionally, the mpl ligand polypeptide or fragment thereof may be fused to a heterologous polypeptide (chimeric). A preferred heterologous polypeptide is a cytokine, a colony stimulating factor or an interleukin or fragment thereof, in particular kit ligand (KL), IL-1, IL-3, IL-6, IL-11, EPO, GM-CSF or LIF. A possible preferred heterologous polypeptide is an immunoglobulin chain, especially human IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgD, IgM, or a fragment thereof, containing in particular the heavy chain constant domain of IgG.

Another aspect of the present invention provides a composition comprising an isolated mpl agonist which is biologically active and is capable of preferentially stimulating the incorporation of labeled nucleotides (e.g., 3H-thymidine.n) with human mpl. transfected IL-3 dependent Ba / F3 cells into DNA. Optionally, the mpl agonist is a biologically active mpl ligand and is preferably capable of stimulating? ·; Attachment of 35S to circulating platelets ··· in mouse platelet rebound assays. 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 insulating T antibody capable of binding to mpl ligand.

s ***: An isolated antibody capable of binding to the mpl ligand may optionally be fused to another polypeptide, and the antibody or its fusion may be used above. • · · 25 to isolate and purify the mpl ligand from the source described with the immobilized mpl. In another aspect of this embodiment, the invention provides a method of detecting an mpl ligand in vitro or in vivo, the method comprising contacting an antibody sample, particularly a serum sample, suspected of containing the ligand, and detecting the ligand if binding has occurred.

In still other embodiments, the invention provides an isolated nucleic acid molecule encoding an mpl ligand or fragment thereof, wherein said nucleic acid molecule may optionally be labeled with a detectable moiety, and the nucleic acid molecule has a sequence complementary to a m under highly stringent conditions with a nucleic acid having the coding sequence for mpl ligand. Preferred nucleic acid molecules are molecules encoding the mpl ligand of human, porcine and rat, and include RNA and DNA, both genomic and cDNA. In another aspect of this embodiment,. ; at the nucleic acid molecule is a DNIA that encodes the mp1 · · · 'ligand and additionally contains a replication-capable vector. . wherein the DNA is operably linked to control sequences recognized by a vector transformed host. Optionally, the DNA is a cDNA having the sequence 5'-3 '(SEQ ID NO: 2), 3'-5' or a fragment thereof shown in Figure 1 * · *. * *. This aspect further includes host cells, preferably CHO cells transformed with a vector; ***, a method for using DNA to generate mpl ligand production, wherein the method preferably comprises: · · · · · · expression of mpl ligand-encoding cDNA in culture of transformed host cells and recovery of mpl ligand from host cells or host cell culture. The mpl ligand prepared in this manner is preferably a human mpl ligand.

The invention further includes a method for isolating and purifying TPO (ML) from a TPO producing microorganism, the method comprising the steps of: (1) lysing or leasing cells containing TPO; (2) separating the potentially soluble material from the insoluble material containing TPO; (3) dissolving the TPO in the insoluble material with a dissolution buffer; (4) separating the dissolved TPO from other soluble and insoluble materials; (5) causing the TPO to rotate in the redox oxidation-reduction buffer; and (6) separating the correctly cured TPO from the incorrectly rotated TPO.

The process allows the insoluble material containing TPO to be dissolved by a chaotropic agent, wherein the chaotropic agent is selected from the guanidine salt, sodium thiocyanate or urea.

In addition, the process requires that the dissolved TPO be separated. other soluble and insoluble materials • ♦. using one or more steps selected from • · · .1 'centrifugation, gel filtration, and reverse phase • · · · · *. . chromatography. The process bypass step • · · * · 1 · requires an oxidation-reduction buffer that contains both an oxidant and a reducing agent. Generally, the oxidant is oxygen or a compound containing at least one disulfide bond and the reducing agent is a compound containing at least one: T: free sulfhydryl. Preferably, the oxidant is selected from oxidized glutathione (GSSG) and cystine, and is selected from the group consisting of oxidized glutathione (GSSG) and cystine. the reducing agent is selected from reduced glutathione (GSH) and cysteine. Most preferably, the oxidant is oxidized glutathione (GSSG) and the reducing agent is reduced glutathione (GSH). It is also preferred that the molar ratio of the oxidant is equal to or greater than the molar ratio of the reducing agent. The oxidation-reduction buffer further comprises a detergent, preferably selected from CHAPS and CHAPSO detergents in an amount of at least 1%. The oxidation-reduction buffer further contains NaCl, preferably in a concentration of about 0.1 to 0.5 M, and glycerol, preferably in a concentration of greater than 15%. The pH of the oxidation-reduction buffer is preferably from about 7.5 to about 9.0, and the recirculation step is carried out at 4 ° C for 12 to 48 hours. The rewiring step produces a biologically active TPO in which a disulfide bond is formed between the Cys residue closest to the amino terminus and the Cys residue closest to the carboxy terminus of the EPO domain.

The invention further includes a method for purifying a biologically active TPO from a microorganism, comprising the steps of: (1) disrupting at least the extracellular membrane of the microorganism; • · · ·: (2) treating the resulting lysis product containing • ♦ V *: TPO with a chaotropic agent; : (3) recycle the TPO and

(4) separating the impurities and improperly twisted TPO

• ♦ l of correctly twisted TPO.

Brief description of the images «» ·

Figure 1 shows the cDNA for human mpl ligand (hML). a derived amino acid sequence (SEQ IID NO: 1) and a coding nucleotide sequence (SEQ ID NO: 2). Nucleotides are numbered at the beginning of each line. Untranslated 5 'and: T: 3' regions are shown in lower case. The amino acid residues {*** are numbered above the sequence starting at Ser 1 of the mature mpl- · · · ligand (ML) protein sequence Ser 1. The boundaries of the putative exon 3 are indicated by arrows and the possible N-. · 28 glycosylation sites are boxed. Cysteine residues are indicated by a dot above the sequence. The underlined sequence corresponds to the N-terminal sequence determined from porcine plasma purified mpl ligand.

Figure 2 shows the method used to determine the binding of 3H-thymidine to mpl ligand. To determine the presence of mpl ligand from various sources, mpl P Ba / F3 cells were maintained in the absence of IL-3 for 24 hours in a humidified incubator at 37 ° C in an atmosphere of 5% CO 2 and air. After being deprived of IL-3, cells were cultured in 96-well culture plates with or without diluted samples and cultured for 24 hours in a cell culture incubator. To each well was added 20 μΐ serum-free RPMI medium containing 1 μΟ 3H-thymidine for the last 6-8 hours. The cells were then harvested into 96-well filter plates and washed with water. Filters were then counted.

Figure 3 shows the effect of protonase, DTT and heat on APP's ability to stimulate Ba / F3 mpl cell proliferation. For APP protonase digestion, proncLasi (Boehringer Mannheim) or bovine serum albumin was coupled to Affi · · · * * Gell0 (Biorad) and incubated separately with APP · · · · /. C temperature. The resins were then removed by centrifugation and the supernatants analyzed.

APP was also heated at 80 ° C for 4 minutes. or was placed on 100 μΜ DTTrhem and then to- * * * »*. dialysis against PBS.

• <·

Figure 4 shows elution of mpl ligand activity. Phenyl-Toyopearl, Blue-Sepharose and Ultralink mpl columns * * *. Fractions from the 4-8 mpl affinity column were fractions with peak «♦ * activity eluted from the column.

: * · *: Figure 5 shows the m l "" of the eluted Ultralink mpl fractions; SDS-PAGE. 200 μl of 2-η 2 to 81 »ml ml of acetone containing 1 mM / 1 HCl was added at 20 ° C at 10 · ·. After three hours at -20 ° C, the samples were centrifuged to 4 · 2 · 29 and the resulting pellets were washed 2x with acetone at -20 ° C. The acetone pellets were then dissolved in 30 Mira SDS solubilization buffer containing 100 μιηο / 1 DTT and heated at 90 ° C. temperature for 5 minutes. The samples were then separated on a 4-20% SDS polyacrylamide gel and the proteins were visualized by silver staining.

Figure 6 shows the elution of mpl ligand activity from SDS-PAGE. The fraction from the 6 mpl affinity column was separated on a 4-20% SDS polyacrylamide gel under non-reducing conditions. After electrophoresis, the gel was sliced into 12 equal regions and eluted as described in the examples. Electro-eluted samples were dialyzed into PBS and analyzed at 1/20 dilution. The Mr standards used to calibrate the gel were the Novex Mark 12 standards.

Figure 7 shows the effect of mpl ligand-free APPr on human megakaryocytopoiesis. The mpl ligand-depleted APP was prepared by passing 1 ml through a 1 ml affinity column (700 Mg mpl-IgG / ml NHS supercoose, Pharmacia). Human peripheral stem cell cultures were prepared using 10% APP or 10% APP deprived of mpl-f · · * * ligand, and cultures were cultured for 12 days.

• · V *: Your megakaryocytopoiesis were quantified as described in the prefixes.

Figure 8 shows the effect of mpl-IgG on the stimulation of megakaryocytopoiesis induced by human APPr. Preparations— M »j * J *. cultures of human peripheral stem cells using 10% APP and cultures were cultured for 12 days. On days 0, 2 and> 4 mpl-IgG (0.5 Mg) or ΛΝΡ-R-IgG (0.5 β9) was added.

• t ·

Then, after 12 days, your megakaryocytopoiesis were quantified as * * *, 'as described in the examples. Dual Samples The average of the roads is represented graphically and the actual data of the dual samples in parentheses.

Figure 9 shows both strands of a 390 bp fragment of human genomic DNA encoding the mpl ligand. The deduced amino acid sequence of "exon 3" (SEQ ID NO: 3), the coding sequence (SEQ ID NO: 4) and its complement (SEQ ID NO: 5) are shown.

Figure 10 shows the deduced amino acid sequence of the mature human mpl ligand (hML) (SEQ ID NO: 6) and the deduced amino acid sequence of the fully human erythropoietin (hEPO) (SEQ ID NO: 7). The predicted amino acid sequence of the human mpl ligand is aligned for alignment with the human erythropoietin sequence. Identical amino acids are boxed and spacing produced for optimal alignment is indicated by dashed lines. Possible N-glycosylation sites are underlined by a single line for hML and a dashed line for hEPO. The two cysteines important for erythropoietin activity have been shown with a high score.

Figure 11 shows the amino acid sequences of the fully mature human mpl ligand isoforms hML (SEQ ID: 6), hML2 (SEQ ID NO: 8), hML3 (SEQ ID NO: 9) and hML4 (SEQ ID NO: 10).

Identical amino acids are boxed 3a For optimal alignment, the added openings were not indicated by dashed lines.

Figures 12A, 12B, and 12C show the effect of human mpl ligand on Ba / F3 mpl cell proliferation (A), in vitro human megakaryocytopoiesis determined using rat radiolabeled rat. monoclonal list • ·,. ·. An IgG antibody specific for megakaryocyte glycoprotein GPIIbIIIa (B) and a rat thrombopoietic assay as measured in the platelet elastic response assay (C).

293 cells were transfected using the CaPO method · [Gorman, C. in DNA Cloning: A New Approach 2: 143 -: * j *; 190 (1985)] with pRK5 vector alone, pRK5 hML or. ···. pRK5-ML153 overnight (pRK5-ML153 was generated by the addition of the terminal codon 153 residual hML by PCR).

The medium was then conditioned for 36 hours and the stimulation of Ba / F3 mpl cell proliferation as described in Example 1 (A) or in vitro meqarocytopoiesis was determined. (B). Megakaryocytopoiesis was quantified using a murine 12II-radiolabeled monoclonal antibody (HP1-1D) to megakaryocyte-specific glycoprotein GPIIbIIIa, as described in Grant et al., Blood 69: 1334-1339 (1987). The effect of partially purified recombinant ML (rML) on platelet production (C) in vivo was determined using the Kimmo rebound thrombocytosis assay described in McDonald, T. P., Proc. Soc. Exp. Biol. Med., 144: 1006-10012 (1973). Partially purified rML was prepared from 200 ml of spent medium containing recombinant ML. The medium was passed through a 2 ml Blue-Sepharose column equilibrated in PBS and the column was washed with PBS and eluted with a 2 mol / L PBS solution containing both urea and NaCl. The active fraction was dialyzed into PBS and endotoxin-free BSA was added to give a concentration of 1 mg / ml. The sample contained less than one unit of endotoxin / ml. Mice were injected with either 64,000, 32,000, or 16,000 units of rML or filler alone.

• · · • *. · Each group consisted of six mice. Mean and standard deviation of each group are reported, p values were determined by a 2 tailed (2 tailed) T-test. comparing medians.

Figure 13 compares the isoforms of human mpl ligand • ·,. ·. and the effect of variants in the Ba / F3 mpl cell proliferation assay. hML, mock, hML2, hML3, hML (R153A, R154A) and hML15 were determined using various dilutions as in Example 1. has been described.

Figures 14A, 14B and 14C show the deduced amino acid sequence of human mpl ligand J · · ·] (hML) or human TPO (hTPO) (SEQ ID NO: 1) and human ***** coding sequence for genomic DNA (SEQ ID NO: 11). Nucleotides and amino acid residues are numbered at the beginning of each line.

Figure 15 shows SDS-PAGE of purified 293-rhML332 and purified 293-rhML153.

Figure 16 shows the nucleotide sequence: the coding sequence (SEQ ID NO: 12) and the deduced amino acid sequence (SEQ ID NO: 13) of the rat ML isoform open reading frame.

This rat mature mpl ligand isoform contains 331 amino acid residues, four less than the expected full-length mML, and is therefore referred to as mML2. Nucleotides are numbered at the beginning of each line. The amino acid residues have been numbered above the sequence starting at Ser 1. The possible 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 the ML isoform (mML) of this rat. Nucleotides are numbered at the beginning of each line. The amino acid residues are numbered above the sequence starting at Ser 1. This fully prepared rat mpl ligand isoform contains 335 amino acid residues and is believed to be a full-length mpl ligand, designated mML. The signal sequence is indicated by a dashed underline and the probable cleavage point is indicated by an arrow. The untranslated 5 'and 3' • · · • V regions are indicated by lower case letters. The two deletions observed as a result of alternative splicing (mML2 and · '· .. mML3) are underlined. Four cysteine residues are represented by •: * ·. · Points. Seven possible N-glycosylation sites • ·,. *. is boxed.

m · · • · ·

Figure 18 compares the deduced amino acid sequence (SEQ ID NO: 9) of the human ML isoform hML3 with a rat. men's ML isoform known as mML3 (DEQ ID NO: 16). The predicted amino acid sequence of human mpl ligand is? · · • j 'aligned with the murine mpl ligand sequence; * · *. Identical amino acids are boxed and, for optimal alignment, the spacing added is indicated by dotted lines. The amino acids are numbered at the beginning of each line.

Figure 19 compares murine ML (SEQ ID NO: 17), 33 porcine ML (SEQ ID NO: 18), and human ML (SEQ ID NO: 18). SEQ ID NO: 6) predicted amino acid sequences of mature ML isoforms. The amino acids are arranged in rows, with dashed spaces added for optimal alignment. The amino acids are numbered at the beginning of each line and identical residues are boxed. Possible N-Gly cosylation sites are indicated by a shaded box and cysteine residues are indicated by a dot. The conserved double-stranded amino acid motif representing a potential protease cleavage site is underlined. The deletion of four amino acids in all three species (ML2) is indicated in bold box.

Figure 20 shows the porcine ML isoform (pML) cDNA sequence (SEQ ID NO: 19) and the predicted mature protein sequence (SEQ ID NO: 18). This porcine mpl ligand iso-form contains 332 amino acid residues and is believed to be a full-length porcine mpl ligand, designated pML. Nucleotides are numbered at the beginning of each line. The amino acid residues are numbered above the sequence starting at Ser 1.

Figure 21 shows the sequence of the porcine ML isoform (pML2) cDNA (SEQ ID NO: 20) and the predicted mature protein sequence (SEQ ID NO: 21). This porcine mpl ligand iso- • · ί / · | The form contains 328 amino acid residues and is a deletion of four residues of the full-length porcine: * · .. second mpl ligand: * ·. · The form, designated pML2. Nucleotides are numbered * · *. at the beginning of each line. The amino acid residues are numbered above the sequence, starting with Ser 1.

• · ·

Figure 22 compares the full-length ML isoform of a pig. pML deduced amino acid sequence (SEQ ID NO: 18) and the deduced amino acid sequence of the ML isoform designated as swine • φ · pML2 | · · · [* (SEQ ID NO: 21). The predicted amino acid sequence of pML is aligned with the pML2 sequence. Identical, * "· amino acids are boxed and, for optimal alignment, · · · the inserted spaces are indicated by dashed lines.

• · · • · * Amino acids are numbered at the beginning of each line.

• · • · • · · 34

Figure 23 shows plasmid pSVI5 used for transfection of CH0-rhTP0332 into host CH0-DP12 cells. ID. LL. MLORF ("full length" or TP0332) essential features.

Figure 24 shows the essential features of plasmid pSVI5.ID.LL.MLEP0-D ("truncated" or TP0153) used for transfection of CH0-rhTP0153 into host CH0-DP12 cells.

Figures 25A, 25B and 25C show the effect of E. coli-rhTPO (Het-153) on platelets (A), red blood cells (B) and white blood cells (C) in normal mice. Two groups, each consisting of 6 female C57 B6 mice, were injected daily with either PBS buffer or 0.3 μg E. coli-rhTPO (Met-1 153) (100 μΐ subcutaneously). On day 0 and on days 3 to 7, 40 μΐ of blood was drawn from the fundus of the eye. This blood was immediately diluted in 10 ml commercial diluent and complete blood counts were obtained on a Serrono Baker Hematology Analyzer 9018. Data are presented as means ± standard error of the mean.

Figures 26A, 26B, and 26C show the effect of E. coli-rhTPO (Met-1 153) on platelets (A), red blood cells (B) • V, and white blood cells (C) at an almost lethal dose of radiation. mice. Two groups, each consisting of: * .. 10 female C57 B6 mice, were irradiated with almost * c.a. 750 cGy gamma radiation from a source of 137Cs and were injected daily with either PBS buffer or • · · 3.0 μg E. coli-rhTPO (Het_., 153) (100 μΐ subcutaneously). On day 0 and subsequent intervals, 40 μΐ of blood was drawn. from the bottom of the eye socket. This blood was immediately diluted with any commercial diluent and complete blood counts were obtained on a Serrono Baker Hematology Analyzer 9018: *** :. Data are presented as means ± mean j ***: mean error.

• · ·

Figures 27A, 27B, and 27C show the effect of CH0-rhTP0332 on (A) platelets (platelets), (B) blood red blood cells (erythrocytes), and (C) white blood cells (leukocytes) in normal mice. Two groups each comprising 6 female C57 B6 mice were injected daily with either PBS buffer or 0.3 μ <3 CH0-rhTP0332 (100 μΐ subcutaneously). On day 0 and on days 3 to 7, 40 μΐ of blood was drawn from the fundus of the eye. This blood was immediately diluted in 10 ml commercial diluent and complete blood counts were obtained on a Serrono Baker Hematology Analyzer 9018. Data are presented as means ± standard error of the mean.

Figure 28 shows dose-response curves for different forms of rhTPO from different cell lines. Dose-response curves were generated using rhTPO obtained from the following cell lines: hTP0332 from CHO (full-length Chinese hamster ovary cells); hTPOMet'1153 (truncated form of N-terminal methionine from E. coli); hTP0332 (full-length TPO from human 293 cells); Met-less 166 E-Coli (truncated form [rhTP0155] without terminal methionine from E. coli). Groups of six C57B6 mice were injected daily for 7 days with rhTPO, depending on the group. Each day, 40 μΐ of blood was drawn from the fundus for complete blood counts.

• · · i. * The above data are the maximal effects observed with the various treatments, and except for j (met 153 E-Coli), the effects occurred on the day of treatment 7.

In the aforementioned "met 153 E-Coli" group, the maximal effect • ·, was observed on day 5. The data are presented as means ± mean · · ·, ·; mean error of average.

• · ·

Figure 29 shows dose-response curves for comparison. Activity of full-length and "truncated" form of rhTPO produced in CHO cells by truncated form of E. coli | · · 'Activity. Groups of 6 female: T: C57B6 mice were injected daily with 0.3 μg of different types of rhTPO. On days 2 to 7, 40 μΐ of blood was drawn from the • • · fundus of the eye for complete blood counts. The treatment groups were TP0153, a truncated form of TPO from E. coli; TP0332 (mixed fraction), a full length TPO containing approximately 80 to 90% full length and 10 to 20% truncated form; TP0332 (30K fraction) = refined truncated fraction from the original "sekci" preparation; TP0332 (70K fraction) = purified fraction of full-length TPO from the original "mixed" formulation. Data are presented as means ± standard error of the mean.

Figure 30 is a sketch showing KIRA ELISA assay for determination of TPO. The figure shows the MPL / Rse.gD chimera and the essential parts of the original receptors as well as the final construction (right part of the picture) and the method diagram (left part of the picture) showing the essential steps of the analysis.

Figure 31 is a method diagram for performing KIRA ELISA analysis showing each step in the method.

Figures 32A-32L produce pSVI17 used to express Rse.gD in Example 17. ID. LL-ekE; nucleotide sequence of the press vector (SEQ ID NO: 22).

Figure 33 is a schematic representation of the construction of plasmid pMP1.

Figure 34 is a schematic representation of the construction of plasmid pMP21.

• · · i *. * Figure 35 is a schematic representation of the construction of plasmid pMP151.

Figure 36 is a schematic representation of the construction of plasmid pMP202: * ·. ·.

Figure 37 is a schematic representation of the construction of plasmid pMP172.

• · ·

Figure 38 is a schematic representation of the plasmid pMP210 val-. making.

Figure 39 is a table showing the plasmid pMP210

The five best expressing TPO clones obtained from j '(SEQ ID NO

NOs: 23, 24, 25, 26, 27 and 28).

• t ***: Figure 40 is a schematic representation of the construction of plasmid pMP41.

Figure 41 is a schematic representation of the construction of plasmid pMP57 ♦ · • · ··· 37.

Figure 42 is a schematic representation of the construction of plasmid pMP251.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Generally, the following words are used to indicate a definition when used in the specification, examples, and claims.

"Chaotropic agent" refers to a compound which, in aqueous solutions and at appropriate concentrations, can cause a change in the space configuration or conformation of a protein by at least partially disrupting the forces responsible for maintaining the protein's normal secondary and tertiary structure. Such compounds include, for example, urea, guanidine 'HCl and sodium thiocyanate. High concentrations of these compounds, usually 4 to 9 M, are normally required to produce a conformational effect on proteins.

"Cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokiin.lt and traditional • · ·: polypeptide hormones. Cytokines include growth hormone, • ·:. * ·· insulin-like growth factors, human growth hormone, human: N-methionyl growth hormone, bovine growth hormone,: · · · parathyroid hormone, thyroxine, insulin, proinsulin, , glycoprotein hormones such as • · · follicle stimulating hormone (FSH), thyroid stimulator

* »I

mild hormone (TSH) and luteinising hormone (LH), hematopoiesis. ethical growth factor, liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor (TNF-α and TNF-β), anti-mullerian (mullerian): T: Substance, murine gonadotropin-related peptide, human * ': bin, activin, vascular endothelial growth factor, integrin, nerve growth factors such as NGF-β, platelet growth factor, • transforming growth factors (TGF: t), such as TGF-? 38 and TGF-β, insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factors, interferons such as interferon-α, -β and -γ, colonies. stimulating factors (CSFs) such as macrophage-CSF (M-CEF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF), interleukins (ILs) such as IL-1, IL-Ια, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 and other polypeptides factors including LIF, SCF and kit ligand as used in the foregoing terms is intended to include proteins derived from natural sources or from recombinant cell culture. Similarly, the terms are intended to include biologically active equivalents that differ in, for example, one or more amino acids or in the type and extent of glycosylation.

The terms "mpl ligand", "mpl ligand polypeptide", "ML", "thrombopoietin" or "TPO" are used interchangeably herein and include any polypeptide having the ability to bind to the mpl, a member of the cytokine receptor superfamily and having the biological property of ML as defined below. An exemplary biological property is the ability to stimulate the incorporation of labeled nucleotides (e.g., 3H-thymidine) into the DNA of human mpl P transfected IL-1: 1: 3-dependent Ba / F3 cells. Another exemplary biological property is the ability to stimulate the incorporation of 35S into circulating platelets in mouse water; flakes elastic response assay. This definition includes a polypeptide isolated from a mpl ligand source, tl # *** as in porcine plaque plasma described herein, or from another source such as another animal species. human, or prepared using recombinant or synthetic methods, and includes variant forms, including functional derivatives, fragments, alleles, isoforms, and analogs thereof.

The "mpl ligand fragment" or the "TPO fragment" is part of the sequence of I · Ί1 naturally occurring, full-length mpl ligand or one of the TPOs deleted. 39 more amino acid residues or carbohydrate units. The deleted amino acid residue (s) can be present anywhere on the peptide, including at or within the N-terminal or C-terminal end. The fragment has at least one biological property that is common to the mpl ligand. Mpl ligand fragments typically have a consecutive sequence of at least 10, 15, 20, 25, 30, or 40 amino acid residues identical to the mpl ligand sequences isolated from a mammal, including a ligand isolated from porcine aplastic plasma, or a human or rat ligand, in particular its EPO clomain. Representative examples of N-terminal fragments are hML153 or TPO (Met'11-153).

As used herein, "mpl ligand variants" or "mpl ligand sequence variants" mean a biologically active mpl ligand, as defined below, having less than 100% sequence identity with mpl ligand isolated from recombinant cell culture or porcine plaque plasma. or a human ligand having the deduced sequence shown in Figure 1 (SEQ ID NO: 1). Usually, the biologically active mpl ligand variant has a amino acid sequence having at least about 70% • ♦: * ·· amino acid sequence identity with the mpl ligand isolated from porcine aplastic plasma, or rat · * · ,; or with a mature human ligand or fragments thereof (see Figure 1 (SEQ ID NO: 1)), preferably at least about 75 µg / l *. %, more preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 85%. at least about 90%; and most preferably at least about 95% · · · * *% amino acid sequence identity.

• · · * · | * "Chimeric mpl ligand" is a pclypeptide containing the full-length mpl ligand or one or more i *** fragments thereof fused or bound to another heterologous polypeptide or one or more fragments thereof. • · * yes. The chimera has at least one biological property ♦ · ··· 40 in common with the mpl ligand. The second polypeptide is typically a cytokine, an immunoglobulin, or a fragment thereof.

The terms "isolated mpl ligand", "highly purified mpl ligand" and "substantially homogeneous mpl ligand" are used interchangeably and refer to mpl ligand purified from mpl ligand source or prepared by recombinant or synthetic methods and containing other peptides or proteins to (1) obtain at least 15 and preferably 20 amino acid residues of the internal amino acid sequence or N-terminal using a rotary cup sequencer or the best commercially available amino acid sequencer sold or modified by the methods disclosed in this patent application, or (2) homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver. Here, homogeneity means that the contamination by other source proteins is less than about 5%.

"Biological property" when used in conjunction with either "mpl ligand" or "isolated mpl ligand" refers to thrombopoietic activity or in vivo effector activity or activity or antigenic activity or activity. or a fragment thereof, either directly or indirectly induced or effected by the mpl ligand (either in its native or denatured · * ·. conformation). Effector functions include ♦ · I include mpl binding and any mpl carrier ··· binding activity, agonism or antagonism, particularly ♦ · · including proliferative signal transduction. replication, DNA regulatory action, regulation of other cytokines • · · · * · biological activity, activation (deactivation) of the receptor (particularly} »♦ · * 'cytokine), regulation of acceleration or silencing, ** *. corresponding. Antigenic activity refers to having an epitope ·· · or an antigenic site capable of cross-reacting with antibodies raised against • · 9 9 ··· 41 native mpl ligands. The major antigenic function of the mpl ligand polypeptide is that it binds with an affinity of at least about 10 6 L / mole to an antibody raised against mpl ligand isolated from porcine aplastic plasma. Typically, the polypeptide binds with an affinity of at least about 107 L / mole. Most preferably, the antigenic cyclic mpl ligand polypeptide is a polypeptide that binds to an antibody raised against the mpl ligand and has one of the effector functions described above. Antibodies used to determine "biological activity" are rabbit polyclonal antibodies formed by formulating mpl-ligand isolated from recombinant cell culture or porcine plaque plasma into complete Freund's adjuvant, injecting subcutaneously, and boosting the immune response to the intraperitoneal injection. antibody titer of the ligand.

"Biologically active" when used in conjunction with either "mpl ligand" or "isolated mpl ligand" means an mpl ligand or polypeptide that exhibits thrombolytic activity or is not shared by porcine aplastic plasma. effector function of the mpl ligand isolated or expressed in recombinant cell culture described herein. Herein, the major known effector function of the mpl ligand or polypeptide is binding to mpl and stimulating the incorporation of labeled nucleotides (3H-thymidine) ···: * j '; human mpl P-transfected, IL-3 dependent

Ba / F3 cell DNA. Another known effector function of the mpl ligand or polypeptide herein is the ability to stimulate 35S · · · VV. attachment to circulating platelets hi-f · · • · · *. ren platelet elastic response assay. Yet another known effector function of the mpl- «·· * V '· ligand is the ability to stimulate human megakaryocytopoiesis in vitro, which can be quantified using * ·· a radiolabeled monoclonal antibody to the glycoprotein of the megakaryocyte. ··· 42 for GPIIbIIIa.

"Amino acid sequence identity%" with respect to 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 of the mpl ligand isolated from porcine aplastic plasma or in the amino acid residue of 1 (SEQ ID NO: 1), after the sequences are arranged in rows and the necessary spacing is added to obtain a maximum percent identity of the sequence without considering conservative substitutions as part of the sequence identity. Any N-terminal, C-terminal, or intrinsic deletions, deletions, or insertions related to the mpl ligand sequence should not be construed as affecting sequence identity or homology. Thus, exemplary biologically active mpl ligand polypeptides that are considered to have identical sequences include prepro-mpl ligand, pro-mpl ligand, and mature mpl ligand.

"Mpl ligand microsequencing" can be carried out using any suitable standard method, provided that the method is sufficiently sensitive. In one such method, the efficiently purified polypeptide obtained from SDS gels or the final HPLC step is directly sequenced by automated Edman digestion (phenyliso- * ·, · thiocyanate digestion). a 470A Applied Biosystems Gas Phase Sequencer equipped with a? 41 ··· 120A Phenylthiohydantione (PTH) amino acid analyzer. In addition, the same can be sequenced. mpl ligand fragments prepared using? 14 ** j * 'chemical digestion (e.g., CnBrra, hydroxylamine, 2-? · V * nitro-5-thiocyanobenzoate) or enzymatic digestion ** (e.g. trypsin, clostripainine, Staphylococcal protease) followed by purification of the fragment (e.g., HPLC).

* · * PTH amino acids are analyzed using ChromPerfect data ♦ ·? system (Justice Innovations, Palo Alto, CA). The interpretation of the sequence · · r «* 44 43 is carried out in VAX 11/785 Digital Equiment CO. computer as described in Henzel et al., J. Chromatography, 404: 41-52 (1987). Optionally, samples of HPLC fractions can be electrophoresed on 5-20% SDS-PAGE, electrophoresed on a PVDF mem membrane (ProBlott, AIB, Foster City, CA) and stained with Coo-massie in Brilliant Blue [Matsurdiara , J. Biol. Chem., 262: 10035-10038 (1987)]. The specific protein identified by staining is removed from the blotting paper and the N-terminal sequencing is performed with the gas phase sequencing apparatus described above. For internal protein sequences, the HPLC fractions are dried in vacuo (SpeedVac), resuspended in appropriate buffers, and resolved using cyanogen bromide, Lys-specific enzyme Lys-C (Wako Chemicals, Richmond, VA) or Asp-N (Boehringer Mannheim, Indianapolis). IN). After digestion, the resulting peptides are sequenced as a mixture or separated by HPLC; with C4 on P1 developing with a propanol gradient in 0.1% TFA prior to gas phase sequencing.

"Thrombocytopenia" is defined as the amount of platelets less than 150 x 109/1 blood.

"Thrombopoietic activity" is defined as biological activity. «* As a biological activity consisting of accelerating the proliferation, differentiation and / or maturation of megakaryocytes or precursors of megakaryocytes into the blood-forming form of these cells. This activity can be measured by various assays, including in vivo charcoal. renal platelet elastic response synthesis, determination of platelet cell surface antigen induction as measured by human Leu chemistry megakaryoblastic cell in i'CMK) anti-platelet immunoassay (anti-GPIIbIII) and polyploidisation in D · · · · · · · · · · · · · · ·

"Thrombopoietin" (TPO) is defined as a compound, jol-j ** '; la has thrombopoietic activity or is capable of increasing serum platelet counts in a mammal. TPO is able to * 9 i * # <* advantageously increase the amount of endogenous platelets.

• M

44 is at least 10%, more preferably 50%, and most preferably is capable of raising platelet levels in a human being above 150 x 109/1 blood.

"Isolated mpl ligand nucleic acid" is RNA or DNA containing more than 16 and preferably 20 or more than 20 consecutive nucleotide bases encoding a biologically active mpl ligand or fragment thereof, complementary to RNA or DNA, or hybridizes to RNA or DNA and remains stable bound under moderate to severe conditions. This RNA or DNA is free of at least one contaminating source nucleic acid with which it is normally linked in a natural source, and preferably is free of any other mammalian RNA or DNA. The expression "free from at least one contaminant source nucleic acid with which it is normally associated" includes a case where the nucleic acid is present in the source or in a natural cell but has a different chromosomal location or is otherwise flanked by nucleic acid sequences not normally found in the source cell. An example of an isolated mpl ligand nucleic acid is RNA or DNA encoding a biologically active mpl ligand having at least 75% sequence identity, more preferably at least 80%, more preferably at least 85% : inen, vie- • · · • · ·.! more preferably 90% and most preferably 95% sequence identity to human, rat or porcine mpl ligand.

When referring to expression, the term "control sequences" refers to DNA sequences necessary for expression of a functionally linked coding sequence in a particular host organism. Control sequences which are suitable for prokaryotes include, for example, a promoter, possibly an operator sequence, a ribosome binding site, and possibly other further elicited known sequences. Science in Eukaryotic Cells · * · *; utilizing promoters, polyadenylation signals, and enhancers.

With reference to nucleic acids, the term "operably linked" means that the nucleic acids are placed in a functional relationship with another nucleic acid sequence. For example, the DNA of the secretory leader of the pre-sequence te.i is operably linked to the DNA of the polypeptide if it is expressed as a pre-protein involved in secretion of the polypeptide; the promoter or enhancer is operably linked to a coding sequence where it affects transcription of the sequence; or a riboson binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are consecutive and, in the case of a secretory leader, contiguous and in reading. However, boosters do not need to be adjacent. The coupling is accomplished by attachment at appropriate restriction sites. In the absence of such sites, synthetic oligonucleotide adapters or linkers are used according to standard practice.

When referring to an element, "exogenous" refers to a nucleic acid sequence which is foreign to a cell or homologous to a cell, but in a position in the host cell's nucleic acid where the element is not normally found.

• ·

The terms "cell," "cell line," and "cell culture" are used interchangeably herein and such designations include all progeny of a cell or cell line. Thus, for example, terms such as "trans formant it" and "transformed cells" include the primary target gene. Lun and derived • · ·: cultures regardless of the number of passages. It is also to be understood that not all offspring may be identical: in their DNA content, due to intentional or unintentional mutations. Includes mutant offspring with the same function or biological activity as originally detected by screening the · · · * · '* transformed cell. Where distinctive denominations are used, they appear in the context. mode connection.

46 "Plasmids" are autonomously replicating circular DNA molecules that have independent replication origins and are designated by the lower case letter "p", preceded and / or followed by capital letters and / or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or may be constructed from such available plasmids according to published methods. In addition, other similar plasmids are known in the art and will be apparent to those skilled in the art.

By reference to DNA, the term "restriction enzyme digestion" refers to the catalytic cleavage of internal phosphodiester bonds within a DNA by an enzyme that functions only at specific locations and sites within the DNA sequence.

Such enzymes are called "restriction endonucleases". Each restriction endonuclease recognizes a specific DNA sequence called a restriction site that shows double symmetry. The various restriction enzymes used herein are commercially available and their reaction conditions, co-factors, and other requirements are used as reported by the enzyme suppliers.

In general, restriction enzymes are named using the abbreviations: ·. *. uppercase letters followed by others • ·. ·. : letters representing the micro-orcianism from which each Λ 'restriction enzyme was originally derived, and then followed by • · · *. . mero, which indicates the specific enzyme. Generally, about 1 Mg of the plasmid or DNA fragment is used with about 1-2 units of enzyme in about 20 ml / well of buffer solution. Changes · · *. · * Detail the appropriate buffers and amounts of substrates for specific restriction enzymes! :: pause. An incubation period of about 1 hour at 37 ° C is usually used, but this may vary depending on the supplier. according to instructions. After incubation, the protein or poly · peptide is removed by extraction with phenol or chloroform and

| J

· '* The digested nucleic acid is recovered from the aqueous: * · *: fraction by ethanol precipitation. Digestion with restriction enzymes can be followed by bacterial alkaline phosphatase hydrolysis of terminal 5'-phosphates to prevent the two restriction fragments of the DNA fragment from forming a circle or closed loop, restriction site. Unless otherwise noted, plasmid digestion is not followed by 51-terminal dephosphorylation. Methods and reagents for carrying out dephosphorylation are conventional as described in Sections 1.56 to 1.61 in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

"Recovery" or "isolation" of a particular DNA fragment from a restriction digestion product means separation of the digestion product on a polyacrylamide or agarose gel by electrophoresis, identification of the fragment of interest by comparing its mobility with the removal of glazed DNA fragments of known molecular weight, separation from 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 (1930).

"Southern analysis" or "Southern blotting" is a success. A method for verifying the presence of DNA sequences in a restriction endonuclease di- · · * of a DNA or a composition containing DNA. in a gestational product by hybridizing to a known labeled oligonucleotide or DNA fragment. Southern analysis typically involves electrophoretic separation of DNA digestion products on agarose gels, denaturation of DNA after electrophoretic separation, and transfer of DNA to nitrocellulose membrane, nylonmembracin or other suitable membrane medium, or by means of an enzyme-labeled probe as described in Sambrook · et al., paragraphs 9.37 to 9.52. in the book, above.

: * · *: "Northern analysis" or "Northern blotting" is a method used to identify RNA sequences that hybridize to a known probe such as gonucleotide, DNA fragment, cDNA or fragment thereof or RNA fragment. The probe is labeled with a radioisotope such as 32P or biotinylation or enzyme. The RNA to be analyzed is usually electrophoretically separated on an agarose or polyacrylamide gel, transferred to a nitrocellulose membrane, nylon membrane or other suitable membrane, and hybridized with a probe using standard techniques well known in the art, such as those described in Sections 7.39-7.52 et al. in the book, above.

"Coupling" is the process of forming phosphodiester bonds between two nucleic acid fragments. To connect two fragments, the ends of the fragments must be compatible with each other. In some cases, the ends are directly compatible after endonuclease digestion. However, it may be necessary to first reconcile to endonuclease digestion For equalization, the DNA is treated with 10 units of the Klerow fragment of DNA polymerase I or T4 • · · ·: DNA polymerase in the presence of four deoxyribonucleotide triphosphates • · · · 1 → * for at least 15 minutes at 15 ° C. The DNA is then purified by extraction with phenol-chloroform and ethanol precipitated. The DNA * * / fragments to be introduced are brought into solution in approximately equimolar ft · · * ·: · * solution. also contains ATP, ligase buffer, and ligase, such as T4 DNA ligase in an amount of about 10 units / 0.5 Mg DNA. If the DNA is to be inserted into a vector, the vector is first linearized by digestion with the appropriate restriction endonuclease (s). Then additional • linearized fragment was treated with bacterial alkaline phosphate • · · phosphatase or calf intestinal phosphatase itself related i. ........

"· To prevent frying in the breeding fence.

: * · *: "Production" of DNA from cells refers to the isolation of plasmid DNA from the culture of host cells. Commonly used methods for preparing DNA are the large-scale and small-scale plasmid preparations described in Sambrook et al., Paragraphs 1.25-1.33. in the book, above. Once prepared, the DNA can be purified using methods well known in the art, such as those described in Sambrook et al., Paragraph 1.40. in the book, above.

&Quot; Oligonucleotides " 1988, or via deoxynucleoside H-phosphonate intermediates as described in Froehler et al., Nucl. Acids Res., 14: 5399-5407 (1986)]. Other methods include the polymerase chain reaction as defined below, and other auto-primer methods and oligonucleotide syntheses on solid supports. All of these methods are described in Engels et al., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989).

These methods are used if the entire nucleic acid sequence of the germ is known or available for nucleic acid • · | .t *. a sequence that is complementary to the coding strand • sequence. Alternatively, if the target amino acid sequence • · • »· *. . As known, possible 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 method or technique in which small amounts of a specific copy of a nucleic acid, RNA and / or · · · DNA are amplified as described in U.S. Patent 4,683,195, published 28. • · · ** / view, 1987. In general, sequence information from or to the ends of the region of interest must be available, such that the oligonucleotide • 50 dialects can be designed; these primers are identical or similar to the opposite strands of the template of sequence amplification. The 5 'terminal nucleotides of the two primers may match with the ends of the amplified material. The polymerase chain reaction (PCR) can be used to amplify specific RNA sequences, specific DNA sequences derived from total genomic DNA and sequences of cDNA, bacterophage or plasmid, etc., reproduced from total cellular RNA. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263 (1987); edited by Erlich, PCR Technology (Stockton Press, NY, 1989). As used herein, the polymerase chain reaction is considered to be one, but not the only, exemplary nucleic acid polymerase reaction method for amplifying a test sample of a nucleic acid, comprising using a known nucleic acid as a primer and nucleic acid polymerase to form a specific fragment of a nucleic acid.

"Harsh conditions" refers to conditions in which (1) low ionic strength and high temperature washing are used, e.g., 0.015 M NaCl / 0.0015 M sodium citrate / 0.1% NaDodSO 4 (SDS) at 50 ° C temperature, or (2) · * · *. during hybridization, a denaturing wave such as. ; formamide, for example 50% (v / v) of formamide. mead containing 0.1% bovine serum albumin / 0.1% • · ·. Ficoll / 0.1% polyvinylpyrrolidone / 50 mmol / l sodium phosphate buffer, pH 6.5, containing 750 mmol / l NaCl and 75 mmol / l sodium citrate. ° C · · · · * * temperature. In another example, 50% formamide, 5 x SSC (0.75 mol / l NaCl, 0.075 mol / l sodium citrate), 50 mmol / l sodium phosphate (pH 6.8) is used, 0.1% Sodium Pyrophosphate, 5 xDenhardt Solution, Sound Wave · *. ·. la salmon sperm DNA (50 μq / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C, i; *; * washes at 42 ° C in 0.2 x SSC and ϊ *: 0.1% SDS.

51 "Moderate conditions" are described in Sambroo et al. in the work, supra, and include the use of lesser washing liquid and hygridization conditions (e.g., temperature, ionic strength, and% SDS) as described above. An example of moderately severe conditions includes, for example, conditions such as overnight incubation at 37 ° C in a solution containing 20% formamide, 5 x SSC (150 mmol / l NaCl, 15 mmol / l trisodium citrate), 50 mmol / l 1 sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate and 20 μΐ / ml denatured salmon sperm DNA, after incubation of the filters in 1 x SSC at approximately 37-50 ° C temperature. One skilled in the art knows how to adjust temperature, ionic strength, etc., as is necessary to adjust factors such as probe length and the like.

"Antibodies" (ABs) and "immunoglobulins" (Igs) are glycoproteins that share the same structural features. Although antibodies showed binding specificity for a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules lacking antigen specificity. For example, the lymphatic system produces small amounts of myeloma. produce large quantities of the latter quality polypeptic. : tides.

• · ·; ·, “Natural antibodies and immunoglobulins” are • · · *. . usually heterotetrameric glycoproteins with a weight of about 150,000 daltons and two · | · · ··· 'from an identical light chain (L chain) and two identical heavy chains (H chain). Each light chain is linked to the heavy chain by a single covalent disulfide-ß # · # ß disulfide, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has · · · regular disulfide bridges at regular intervals.

i: "· Each heavy chain has at its other end a variable domain (VH) followed by a set of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end; the light chain constant domain is aligned with the heavy chain first constant do-Main, and the light chain variable domain is aligned with the heavy chain variable domain. Specific amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains [Clot-hia et al.f. 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 certain portions of the variable domains differ widely in their sequence between antibodies and are used in the binding and specificity of each specific antibody to its particular antigen. However, the variability is not evenly distributed along the variable domains of the antibodies. It is enriched in three segments called complementarity determining regions (CDRs) or hypervariable regions in both light chain and heavy chain variable domains. The more conserved portions of the variable domains are called the framework (FR). The variable dooms of natural heavy chains and light chains each contain four FR regions that have a large β-plate configuration and are joined by three • ·; complementarity determining domains that form • · loops connecting the β-sheet structure or, in some cases, • · ♦ \. in it, forming part of it. In each chain, the complementarity determining regions are held in close proximity to the J * · · '' FR regions and the other chain complementarity determining regions J · · · '* contribute to the antigen binding sites of the antibodies. (see: Kabat et al., Sequences of Proteins of Immunological Interests, National Institute of Health, Bethesda, MD (1987)). Constant domains are not directly involved in the binding of the antibody to the antigen. , but they exhibit various t: *; * effector functions, such as the involvement of antibodies in antibody-dependent cell toxicity.

• · · • · • · · · · 53

Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each having one antigen binding site, and the remaining "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F (ab ') 2 fragment which has two antigen binding sites and yet is capable of crosslinking the antigen.

"Fv" is the smallest antibody fragment containing the complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in a tight, non-covalent context. It is in this configuration that the three complementarity determining regions of each variable domain interact to determine the antigen binding site on the surface of the VH-VL dimer. Collectively, these six complementarity determining regions produce antigen binding specificity for the antibody. However, any variable domain (or Fv half containing only three antigen-specific complementarity determining regions) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

· * · *; The Fab fragment also contains a light chain constant • ·. ·. : domain and first heavy domain of heavy chain • ·; ·. (CH1). Fab 'fragments differ from Fab fragments by a few · · · *. . addition of residues at the carboxy- • · * * end of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the I · · · “* antibody. The Fab'-SH name is used herein to refer to * * Fab ', where the cysteine residue (s) of the constant domains carry (s) a free, thiol group. F (ab ') 2 - *, · .ϊ antibody fragments were originally produced as Fab1 fragment pairs between hinge cysteines. Other chemical couplings of antibody fragments are also known.

• · ·

The "light chains" of any vertebrate antibody (imt: ** · munoglobulins) can be divided into two distinct types, called kappa and lamb ··· ♦ · · · ··· ♦ 54 daxis (λ) based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be divided into different classes. There are five important classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called a, delta, epsilon, γ and μ, respectively. The structures and three-dimensional configurations of various classes of immunoglobulins are well known.

The term "antibody" is used in the broadest sense and includes in particular single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions having polyepitopic specificity, and antibody fragments (e.g., Fab, F (ab) 2) and Fv) if they exhibit the desired biological activity.

As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., *. that the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present to a minor extent. Monoclonal antibodies are highly specific.

.Ϊ. * Fis and are targeted to one antigenic site • · · *. *. In addition, as opposed to conventional (polyclonal) antibody preparations, which typically contain t different anti-determinants (• epitopes) and different antibodies, each monochlorine antibody is targeted against one determinant on the surface of the antigen. .

• · ·

In addition to the specificity of monoclonal antibodies, they are | · ··· 'are advantageous in the sense that they are synthesized: * · *; hybridoma culture that has not been contaminated by other immunoglobulins. The term "monochlorine" indicates the nature of the antibody in the sense that it is derived from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies intended to be used in accordance with the present invention may be prepared using the hybridoma method first described by Kohler & Milstein, Nature 256: 495 (1975), or may be made using recombinant DNA techniques [see, e.g., U.S. Pat. U.S. Patent No. 4,816,567 to Cabilly et al.].

In particular, monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequences in antibodies of a particular species or class of antibodies, or - while the remainder of the chain (s) is identical or homologous to the corresponding sequences in antibodies derived from another species or to another class or subclass of antibodies, as well as fragments of such antibodies if they exhibit the desired biological function. • V; giant activity [U.S. Patent 4,816,567 (Cabilly et al.); • ·, ·. : and Morrison et al., Proc. Natl. Acad. Sci., USA, 81: 6851 - * · ·; ·. 6868 (1984)].

«· · *. . The "humanized forms" of non-human (e.g., rat) antibodies are chimeric Immunoglobulins, | * · · '·' Immunoglobulin chains or fragments thereof (such as Fv, * · · .......

'Fab, Fab', F (ab ') 2 or other antigen-binding antibody sub sequences) which contain a minimal sequence derived from non-human immunoglobulin. Most of the humanized antibodies are human immunoglobulins (recipient vcista), in which residues from the • complementarity determining region (CDR) i: ·; * of the recipient are replaced by residues M ·; are derived from a non-human species (donor antibody), 56 CDRs, such as the mouse, rat, or rabbit CDR, which have the desired specificity, affinity, and capacity. In some cases, residues of the human immunoglobulin Fv framework have been replaced by corresponding non-human residues. In addition, the humanized antibody may contain residues that are not found in the recipient antibody or in the sequences of the imported CDR region or frame. These variants are made to further improve and optimize antibody performance. Generally, the humanized antibody will contain substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDRs correspond to the non-human immunoglobulin CDRs and all or substantially all of the FRs are FR regions of the human immunoglobulin consensus sequence. . The humanized antibody optimally also contains at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin FC region. For further details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

· * · *. "Non-immunogenic in humans" means that when contacted with a pharmaceutically acceptable polypeptide. in an inflammatory carrier and in a therapeutically effective amount ♦ ·· * »e. with appropriate human tissue, has no detectable state of sensitivity or resistance to the polypeptide, | · · ·· ** after the second administration of the polypeptide after an appropriate latency period of 2 ♦ ** (e.g., 8-14 days).

II. Preferred Embodiments of the Invention The preferred polypeptide (s) of the present invention (s) are (are) substantially homogeneous poly-, ··· peptide (s). called mpl- * * * ligand (mpl-ligands) or trorabopoietin (TPO), t: ***, which has the property of binding to mpl, a member of the * * cytokine receptor family and has the biological property ·· «» * Stimulate the incorporation of labeled nucleotides (3H-thymidine) into the DNA of human mpl P transfected IL-3 dependent Ba / F3 cells. The more preferred mpl ligand (s) are isolated mammalian protein (s) having hematopoietic, in particular megakaryocytopoietic or thrombocytopoietic activity - namely, capable of stimulating or pro-liferation, maturation and / or differentiation into a mature platelet producing form. The most preferred polypeptide of this invention is (is) the human mpl ligand (s), including fragments thereof having a haematopoietic, megakaryocytopoietic or thrombopoietic activity. Possibly, this human mpl ligand (these human mpl ligands) lacks glycosylation. Other preferred human mpl ligands are the "EPO domain" of hML, designated hML153 or hTP0153, a truncated form of hML called hML245 or hTP0245, and a mature full-length polypeptide having the amino acid sequence shown in Figure 1 ( SEQ ID NO: 1) and is designated as hML, hML1 or hTP0332, and the biologically active substitution variant hML (R153A, R154A).

Possible preferred polypeptides of this invention are biologically or immunologically active variants of mpl ligands selected from hML2, hML3, hML4, aa *, mML, mML2, mML3, pML and pML2.

A possible preferred polypeptide of the present invention is the biologically active * (*) * mpl ligand variant (s) having at least 70% amino acid sequence identity to the human. with mpl ligand [see Fig. 1 (SEQ ID NO: 1)], with rat mpl ligand .j. # [see Fig. 16 (SEQ ID NO: 12 & 13)], pig recombinant * · · mpl ligand [see Fig. 19 (SEQ ID NO: 18)] or with porcine rapl ligand isolated from porcine plaque plasma, preferably at least 75%, The sequence identity is at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%.

The mpl ligand isolated from porcine aplastic plasma has the following characteristics: (1) The partially purified ligand elutes from the gel filtration column run to either PBS solution, PBS solution containing 0.1% SDS or PBS solution containing contains 4 mol / L MgCl 2 with a Mr value of 60,000 to 70,000.

(2) Ligand activity is destroyed by proton.

(3) The ligand is stable against low pH (2.5) with SDS up to 0.1% and urea concentration of 2 mol / L.

(4) The ligand is a glycoprotein based on its binding to various lectin columns.

(5) An efficiently purified ligand elutes from plain SDS-PAGE with a Mr value of 25,000-35,000. Smaller amounts of activity are also eluted with a Mr value of ~ 18,000-22,000 and 60,000.

(6) Highly purified lic; Andi is resolved by reduced SDS-PAGE at doublet with a Mr value of 28,000-31,000.

♦ V. (7) The amino terminal sequence having bands • ·: 18,000, 28,000 and 31,000 bands is the same - SPAP- •, · * PACDPRLLNKLLRDDHVLHGR (SEQ ID NO: 29).

• * ·· * ». (8) The ligand binds and elutes from the following affinity columns: t * · ♦ ·· * Blue-Sepharose, Ϊ * Φ * · * * CM Blue-Sepharose, MONO-Q, s: ': mono -s, s *: * i Lentil lectin-Sepharose, * · * · WGA-Sepharose, • t »I .. Con A-Sepharose,:: ** · Ether 650m Toyopearl,] '\ Butyl 650m Toyopearl, ·« · * K • 9

»M

59

Phenyl 650m Toyopearl, and

Phenyl-Sepharose.

More preferred mpl ligand polypeptides are those polypeptides encoded by human genomic DNA or cDNA having the amino acid sequence shown in Figure 1 (SEQ ID NO: 1).

Other preferred, naturally occurring, biologically active mpl ligand polypeptides of this invention include prepro-mpl ligand, pro-mpl ligand, fully prepared mpl ligand, mpl ligand fragments, and glycolylation variants of the above.

Still other preferred polypeptides of the invention include sequence variants and chimeras of the mpl ligand. Usually preferred mpl ligand sequence variants and chimeras are biologically active mpl ligand variants having at least 70% amino acid sequence identity to human mpl ligand or mpl ligand isolated from porcine plaque plasma, preferably at least 75%: 80% f, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% sequence identity. An exemplary preferred mpl ligand variant is the hML-domain of the N-terminal.se domain.

Il ·: variant (called the "EPC domain" because of its sequence homology to erythropoietin). The preferred hML-EPO domain contains about 153 first amino acid residues of the mature hML and is called hML15. A possible preferred variant of the hML sequence comprises a variant wherein one or more of the basic or dibasic amino acid residues in the C-terminal domain is sub-. titered with a non-basic amino acid residue (e.g., hydro-, ·, ·, phobic, neutral, acidic, aromatic residue, ·, Gly, Pro, and the like). A preferred sequence variant of the C-terminal domain of hML comprises the sequence 1'1, in which Arg residues 153 and 154 are replaced by Ala residues. This variant is called hML ,,? (R153A, • · · • t • · · · • • • • 1 · 60 R154A). An alternative preferred hML variant comprises either hML332 or hML153 in which amino acid residues 111-114 (QLPP or LPPQ) have been deleted or replaced by a different tetra peptide sequence (e.g., AGAG or the like). The deletion mutants described above are referred to as A4hML332 or A4hML153.

A preferred chimera is a fusion between the mpl ligand or fragment thereof (defined below) and the heterologous polypeptide or fragment thereof. For example, hML153 can be fused to an IgG fragment to increase serum half-life or to IL-3, G-CSF or EPO to produce a molecule with increased thrombopoietic or chimeric hematopoietic activity.

An alternative preferred human mpl ligand chimera is the "ML-EPO domain chimer" consisting of N-terminal 153-157 hML residues substituted by one or more, but not all, human EPO residues in approximately the same order, as shown in Figure 10 (SEQ ID NO: 7). In this embodiment, the hML chimera is likely to be about 153 to 166 residues in length, with single residues or segments of residues from the human EPO sequence being inserted or substituted into the hML sequence at positions corresponding to the sequence sequence that is precursor to the sequence. 10 (SEQ ID NO: 6). Exemplary sequence inserts • · * .1 ·· N-terminal portion of hML could include: · .. one or more N-glycosylation sites (EPO): 1 · · · 24-27, 38-40 and 83 - 85; one or more of the four predetermined bipolar (bundle) amphipathic α-helix bundles at positions 9-22, 59-76, 90-107 and 132-152; and • · · other very well preserved areas, including N-. terminal and C-terminal regions and residual positions (epo) 44-52 • · · [see, e.g., Wen et al., Blood, 82: 1507-1516 lit * · | (1993) and Boissel et al., J. Biol. Chem. , 268 (21): 15983-15993 (1993)]. It is possible that this "ML-EPO domain chimeric" has mixed thrombopoietic-erythropoietic • · · (TEPO) biological activity.

Other preferred polypeptides of this invention include mpl ligand fragments having at least 10, 15, 20, 25, 30, or 40 consecutive amino acid residue sequences identical to porcine aplastic. with the mpl ligand sequence isolated from plasma or with the human mpl ligand sequence described herein (see, e.g., Table 14, Example 24). A preferred mpl ligand fragment is human ML [1-X], where X is 153, 164, 191, 205, 207, 217, 229 or 245 [see Figure 1 (SEQ ID NO: 1) for the sequence of residues 1-X]. Other preferred mpl ligand fragments include fragments produced by chemical or enzymatic hydrolysis or digestion of the purified ligand.

Another preferred aspect of the invention is a method for purifying mpl ligand molecules, comprising contacting a mpl ligand source containing mpl ligand molecules with an immobilized receptor polypeptide, in particular mpl or mpl fusion polypeptide, under conditions where , washing the immobilized medium to remove non-adsorbed material, and eluting the molecules to be purified from the immobilized receptor polypeptide with elution buffer. The source containing the mpl ligand may be plasma, where the immobilized receptor is preferably mpl-IgG fusion.

Alternatively, the mpl ligand-containing source is a recombinant cell culture with a concentration of mpl ligand. either in culture medium or in cell lysis products are generally higher than in plasma or other natural sources. In this case, the mpl-IgG-im- described above. The egg-affinity method, although still useful, is not usually necessary and may use more conventional protein purification methods known in the art. In short, a preferred purification method for producing a substantially homogeneous mpl ligand Comprises the removal of finely divided cellular debris, either host cells or disrupted fragments, for example by centrifugation or ultrafiltration, optionally the protein may be concentrated by a commercially available protein concentration filter. followed by separation of the ligand from the other impurities using one or more steps selected from immunoaffinity, ion exchange (e.g. DEAE or matrices containing carboxymethyl or sulfopropyl groups), affinity columns which are Blue-Sepharose CM Blue-Sepharose, MONO-; Q, MONO-S, Lentil lectin-Sepharose, WGA-Sepharose, Con A-Sepharose, Ether Toylpearl, Butyl Toypearl, Phenyl Toypearl, Protein A Sepharose, SDS-PAGE, Reverse Phase HPLC1 (e.g. . silica gel with added aliphatic groups) or Sephadex molecular sieve or size exclusion chromatography and ethanol or ammonium sulfate precipitation. To prevent proteolysis, a protease inhibitor such as methylsulfonyl fluoride (PMSF) can be coupled to any of the above steps.

In another preferred embodiment, the present invention provides an isolated antibody capable of binding to mpl ligand. The preferred mpl ligand isolated 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, Vol. 13, Elsevier Science Publishers, Amsterdam (1985); and Huse, et al., Science, 246: 1275-1281 (198S)). A preferred isolated antibody to mpl-1 · · · · gand is an antibody that binds to · · · · · · · · · · · · mpl ligand with an affinity of at least about 10 6 · · · 1 / mol. More preferably, the antibody binds with an affinity of at least about 10 7 l / mol. Most preferably, an antibody. is formed against an mpl ligand having any of the effector functions described above. An isolated antibody capable of binding to the mpl ligand may be possibly fused to another polypeptide and the antibody or its i1; fusions can be used to isolate such mpl ligand · · ./. sex and purification from the source described for the mpl 63 polypeptide immobilized above. In another preferred aspect of this embodiment, the invention provides a method of detecting an mpl ligand in vitro or in vivo, comprising contacting the antibody with a sample, particularly a serum sample suspected of containing the ligand, and detecting the ligand if binding has occurred.

In still other preferred embodiments, the invention provides an isolated nucleic acid molecule encoding an mpl ligand or fragments thereof, wherein the nucleic acid may be labeled with a detectable moiety or may be unlabeled and the nucleic acid molecule will have a sequence complementary to or in moderate conditions with it. The preferred mpl ligand nucleic acid is RNA or DNA encoding a biologically active mpl ligand having at least 75% sequence identity, more preferably at least 80%, even more preferably at least 85%, even more preferably 90% % and most preferably 95% sequence identity with human mpl ligand. More preferred isolated nucleic acid molecules are DNA sequences that encode a biologically active mpl ligand and are intermediate.

: tu: • ·! / ·· (a) DNA based on the coding region of a mammalian mpl ligand gene [e.g. DNA comprising the nucleotide sequence shown in Figure 1 (SEQ ID NO: 2) or fragments thereof]; A DNA capable of hybridizing to the DNA of • · ♦ (a) under at least moderately stringent conditions and. (c) DNA that is degenerate with respect to DNA as defined in (a) or • · · (b) as a result of degeneracy of the genetic code · · · · · ·

It is possible that the novel mpl ligands described herein are i1 "; members of a family of ligands or cytokines which have a suitable sequence identity such that their DNA can hybridize with the DNA of Figure 1 (SEQ ID NO: 2). (or its complement or fragments) under mild to moderate conditions. Thus, another aspect of the present invention includes DNA that hybridizes: under mild to moderate stringency with DNA encoding mpl ligand polypeptides.

In another preferred embodiment of the present invention, the nucleic acid molecule is a cDNA encoding an mpl ligand and further comprising a replication-capable vector wherein the cDNA is operably linked to control sequences recognized by the host transformed with the vector. This aspect further includes host cells transformed with a vector and a method for using cDNA to induce mpl ligand production, the method comprising expressing cDNA encoding mpl ligand in a culture of transformed host cells and recovering the mpl ligand from the culture of the host cell. The mpl ligand prepared in this manner is preferably a substantially homogeneous human mpl ligand. Preferred host cells for producing mpl ligand are Chinese Hamster Ovary (CHO) cells.

• · • · • · III. Production Methods • · · • · .1 * Some writers have long assumed that • · · *. . multi-origin specific humoral factors regulate • · · * · [* platelet production. It has been hypothesized that two distinct cytokine activities utilized to denote megakaryocyte colony stimulating factor (meg- • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · megakaryocytopoiesis and thrombopoiesis [Williams et al., J. Ce: 11 Physiol., 110: 101-104 (1982); Williams et al., Blood Cells, 15: 123-133 (1989); and Gordon et al., Blood, 80: 302-307 (1992). Under this assumption, meg-CSF stimulates the proliferation of megakaryocyte progenitor cells, whereas thrombopoietin primarily affects the maturation of more differentiated cells and ultimately the release of platelets. Since the 1960s, the induction and occurrence of both meg-CSF and thrombopoietin activity in animal and human plasma, serum and urine following thrombocytopenic episodes has been well documented [Odell et al., Proc. Soc. Exp. Biol. Med., 108: 428-431 (1961); Nakeff et al., Acta Haematol., 54: 340-344 (1975); Specter, Proc. Soc. Exp. Biol., 108: 146-149 (1961); Schreiner et al., J. Clln. Invest., 49: 1709-1713 (1970); Ebbe, Blood, 44: 605-60 (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); Hille et al., Exp. Hematol., 20: 354-360 (1992); and Hegyi et al., Int. J. Cell Cloning, 8: 236-244 (1990)]. These activities are reported to be gender specific and distinct from known cytokine actin *: ** densities [Hill, RJ et al., Blood, 80: 346 (1992); Erickson-Miller, CL, et al., Brit. J. Haematol., 84: 197-203 (1993); Straneva, JE, et al., Exp. Hematol., 20: 4750 (1992), and Tsukada, J. et al., Blood 81: 866-867 · ·· (1993)] To date, attempts to purify meg-CSF or thrombopoietin from thrombocytopenic plasma or urine have been unsuccessful.

• · · • · · IV. Consistent with the above observations • ♦ ♦ *. In those depicting thrombocytopenic plasma, we have observed that porcine aplastic plasma (APP) derived from <1-irradiated pigs stimulates human megakaryocytopoiesis in vitro. We have found that this stimulatory activity of 66 is abrogated by the soluble extracellular domain of c-mpl, confirming that APP is a potential source of putative mpl ligand (ML). We have now successfully purified the mpl ligand from APP and amino acid sequence information was used to isolate rat, porcine and human ML cDNA. These mpl ligands have sequence homology to erythropoietin and exhibit both meg-CSF and thrombopoietin-like activities.

1. Purification and identification of the plasmid mpl ligand

As stated above, aplastic plasma from various species has been reported to contain activities that stimulate hematopoiesis in vitro, but no hematopoietic stimulating factor isolated from plasma has been reported previously. One source of aplastic plasma is plasma from irradiated pigs. This porcine aplastic plasma (APP) stimulates human hematopoiesis in vitro. To determine if APP mpl ligand was present, its effect was determined by measuring the binding of 3 H-thymidine to Ba / F3 cells transfected with human mpl P (Ba / F3 mpl) using the method shown in Figure 2. APP stimulates 3H-thymidine incorporation into Ba / F3 mpl cells but not in Ba / F3 control cells (namely, they were not transfected with human mpl P). In addition, such activity 9 9 s.1 ··· was not observed in normal porcine plasma. These results indicated that APP contained a factor (s) that expressed a proliferative signal via the mpl receptor and therefore could be (or may be) present at this receptor. nature- • · · ·; ·. a null ligand. This was further supported by the observation that treatment of APP 9 9 9 with soluble mpl-IgG prevented APP from stimulating. effect on Ba / F3 mpl cells.

Activity in APPr appeared to be a protein because 1 hour * ·] 2 proton, DTT or heat destroyed activity in APP (Figure 3). The activity was also non-dialyzable. Activity was 9! 3: however, low pH resistant (pH 2.5 for 2 hours) • 1 · and was shown to bind to a number of lectin affinity 2 · 9 3 99 9 67 columns and eluted from them, indicating that it was a Gly-coprotein. To further elucidate the structure and identity of this active agent, it was purified by affinity purification from APPr using the mpl-IgG chimera.

APP was treated according to the method described in Examples 1 and 2. Briefly, the mpl ligand was purified using hydrophobic interaction chromatography (HIC), immobilized color chromatography, and mpl affinity chromatography. The activity recovery from each step is shown in Figure 4 and fold purification is shown in Table 1. The total activity recovery value using the mpl affinity column was approximately 10%. The peak specific activity fraction (F6) obtained from the Mpl affinity column was estimated to be 9.8 x 10 6 units / ml. The total purification from 5 liters of APP was approximately 4 x 10 6 times (0.8 units / ml to 3.3 x 10 6 units / mg) with a reduction of proteins 83 x 10 6 (from 250 g to 3 mg). We estimated that the specific activity of the ligand eluted from the mpl affinity column was <3 x 10 6 units / mg.

Table 1. Purification of the Mpl ligand ... Specific Purification Supplement V Protein - Single Activated - Sample Volume Ininitol / ml Units Organs l * /: mg / ml Units / mg _% __ • ♦ · '' "I · nv- ".-... i" * ·.: APP 5000 50 40 200,000 0.8 __1

Phenyl 4700 0.8 40 200.000 50 94 62 i:: --------- ::: Blue-SeD. 640 0.93 400 256.000 430 128 538 lii—— 1 - - - - —— mpl (μΙ)

Iffrakt. 5-7) 1 12 5x10'4 | 1666 20,000 | 3.300 · 000 | 10: 4.100,000: Protein was determined by the Bradford assay.

i “*: Protein concentrations · of Mpl eluted fractions 5-7 are estimates based on the color intensity of the silver stained SDS gel. One unit is determined to cause 50% of maximal stimulation of Ba / F3 cell proliferation.

Analysis of the fractions eluted from the Mpl affinity column using SDS-PAGE run (4-20%, Novex gel) under reducing conditions revealed the presence of several proteins (Figure 5). Proteins that stained with silver at the highest intensity differed 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 of Ba / F3 mpl cell cultures, proteins 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 gel slice containing proteins having a Mr value of 28,000 to 32,000, with less activity eluted from the gel region of 18,000 to 22,000 (Figure 6). In these regions, the only visible proteins had Mr values of 30,000, 28,000, and 18,000-22,000. To identify and obtain the sequence of the proteins from the distinct proteins in this region of the gel (i.e., bands 30, 28, and 18-22 kDa), these three the protein was electrically transferred to PVDF and sequenced as described in Example 3. The resulting amino terminal sequences are shown in Table 2.

• ·

Table 2. Amplified amino acid sequences of the Mpl ligand ······························································································ ((S) PAPPA (C) D PRLLNKLLRPD (H / S) VLH (G) RL ID NO: 30) 28 kDa. 1 5 10 15 20 25 • ·

.1! 1. (S) PAPPAXDPRLLNKLLRDD (H) VL (H) GR (SEQ ID NO: 3tL

* 18-22 kDa Y; 1 5 io XPAPPAXDPRLX (N) (K) __ | (SEQ ID NO: 32j_ • · · · · · · · · · · 69

Computer-assisted analysis revealed that these amino acid sequences are novel. Because all three sequences were the same, it was believed that the 30 kDa, 28 kDa and 18-22 kDa proteins were closely related and may have different forms of the same novel protein. In addition, this protein (s) was (are) a likely candidate for natural mpl ligand because activity was resolved by SDS-PAGE over the same 4-20% gel range (28,000-32,000). In addition, the partially purified ligand migrated at a Mr value of 17,000 to 30,000 when subjected to gel filtration chromatography using a Superose 12 column (Pharmacia). It is believed that the different Mr forms of the ligand are the result of differences in proteolysis or glycosylation, or other post-translational or pre-translational variants.

As previously described, human antisense mpl RNA inhibited megakaryocytopoiesis in human bone marrow cultures enriched with CD 34 + precursor cells without affecting the lineage differentiation of other hematopoietic cells (Methia et al., Supra). This result suggests that the mpl receptor might play a role in megakaryocyte differentiation and proliferation in vitro. To further elucidate the role of mpl ligand in megakaryocytopoiesis, the effects of APP and non-mpl ligand-free APP on in vitro human megakaryocytopoiesis were compared. The effect of APP on human megakaryocytopoiesis was determined using a variation of the megakaryotic * *. · Osytopoiesis assay described in Example 4 in a liquid suspension. In this assay, human peripheral stem cells (PSCs) were treated with APP before and after mpl-IgG affinity chromatography. GPIIbIIIa stimulation of megi-karyocytopoiesis was quantified. with 125 I-anti-IIbIII antibody (Figure 7). As * ··· * in Figure 7, 10% of APP induced approximately 3- ♦ · ♦ * · 'fold stimulation, whereas the mpl-ligand: * · *: APP had no effect at all. Significant ,··*. was that the mpl ligand-depleted APP did not induce the proliferation of Ba / F3 mpl cells.

: ·: «· ··« • * • ♦ «« · 70

In another experiment, soluble human mpl-IgG added on days 0, 2, and 4 to 10% APP-containing cultures counteracted the stimulatory effect of APP on human mega-karyocytopoiesis (Figure 8). These results indicate that the mpl ligand has a role in the regulation of human megakaryocytopoiesis and is thus useful in the treatment of thrombocytopenia.

2. Molecular cloning of the Mpl ligand

Based on the amino terminal amino acid sequence derived from 30 kDa, 28 kDa and 18-22 kDa proteins (see Table 2 above), two de-generating oligonucleotide primer halves were designed and used to amplify porcine genomic DNA by PCR. It was concluded that if one exon encodes an amino terminal amino acid sequence, then the correct PCR product was expected to be 69 base pairs in length. A DNA fragment of this size was detected and subcloned into pGEMT. The sequences of the oligonucleotide PCR primers and the three resulting clones are shown in Example 5. The amino acid sequence [PRLLNKLLR (SEQ ID NO: 33)] of the peptide encoded between the PCR primers was identical to that obtained by sequencing the amino acid protein of porcine ligand 28 (see above) and 30 kDa protein sequences residues 9 - • · · 17).

A synthetic oligonucleotide based on the sequence of the PCR fragment was used to screen for the human genomic DNA sequence. A 45 &apos; oligonucleotide, designated pR45, was designed and synthesized based on the sequence of the PCR fragment. This oligonucleotide i · · had the following sequence:. 5'GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT

• · I

* · “1 TAG-GAG-TCG 3 '(SEQ ID NO: 34) 1 · * 2 1 This deoxyoligonucleotide was used to screen the human genomic DNA library in Igem12 for less stringency; in hybridization and washing conditions as in Example 6. Positive clones were picked, plaque purified f · · • · • ·

M

0 · 2 * · 3 • · 71 and analyzed by restriction mapping and Southern blotting. The 390 bp EcoRI-XbaI fragment, which hybridized to the 45-mer, was subcloned into pBluescript SK-. DNA sequencing of this clone confirmed the presence of isolated DNA encoding the human homolog of the porcine mpl ligand. The human DNA sequence and the deduced amino acid sequence are shown in Figure 9 (SE ID NOs: 3 & 4). Predicted positions of introns in the genomic sequence are also indicated by arrows and limit the putative exon ("exon 3").

Based on the human "exon 3" sequence (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 template cDNA prepared from various human tissues. The expected size of the correct PCR product was 140 base pairs. After analysis of the PCR products, a 12% polyacrylamide gel detected the expected size of a DNA fragment from cDNA libraries prepared from fully developed human kidney, 293 fetal kidney cells, and cDNA prepared from human fetal liver.

The fetal liver cDNA library (7 x 10 6 clones) in lambda DR2 was next screened with the same 45-mer oligonucleotide used to screen the human genomic library and the fetal liver cDNA library at a less stringent level. · Under conditions of hydration. Positive clones were picked, plaque purified and insert size determined by PCR 9 /·.·. For other analyzes, one clone with a? T insert of 1.8 kb was selected. Using the me- * »« ·· * described in Example 7. Methods were obtained from the nucleotide sequence of human mpl ligand (hML).

I · I and the deduced amino acid sequence. These sequences are. shown in Figure 1 (SEQ ID NOs: 1 & 2).

I * * • j · * 3. Structure of human mpl ligand (hML) II · ** 'cDNA sequence of human mpl ligand (hML) [Figure 1; *; * · (SEQ ID NO: 2)] contains 1774 nucleotides followed by the son **. ly (A) tail. It contains 215 AP λf nucleotides of the 51 untranslated sequence and the 318 untranslated region of 498 nucleotides.

»· · • · t« M) 9 • · 72

The putative start codon at the nucleotide position (216-218) is in the consensus sequence, which favors the initiation of eukaryotic translation. The open reading frame is 1059 nucleotides in length and encodes a polypeptide comprising 353 amino acid residues starting at nucleotide position 220. The N-terminus of the predicted amino acid sequence is highly hydrophobic and probably corresponds to the signal peptide. Computer analysis of the predicted amino acid sequence [von Heijne et al., Eur. J. Biochem., 133: 17-21 (1983)] indicates a possible signal peptidase cleavage site between residues 21 and 22. Cleavage at this position would produce a mature polypeptide having 332 amino acid residues, starting from the amino terminal sequence obtained from porcine plasma purified mpl ligand. The ligand comprising 332 amino acid residues has a predicted non-glycosylated molecular weight of about 38 kDa. It has 6 potential N-glycosylation sites and 4 cysteine residues.

Comparison of the mpl ligand sequence to the gene bank sequence database revealed 23% identity between the amino terminal 153 residues of mature human mpl ligand and human erythropoietin [Figure 10 (SEQ ID NOs: 6 & 7). Given conservative substitutions, this region of hML exhibits 50% co-morbidity with human erythropoietin (hEPO). Both hEPO and hML contain- w «·,,. ,.

*. four cystennes. Three of these four cysteines have 44 i * '· retained in hML, including the first and last 4 · </ ·· cysteine. Studies on site-directed mutagenesis have shown that the first and last cysteine of erythropoietin form a disulfide bond which is required for function (Wang, FF et al., Endocrinology 116: 2286-2229 (1983)). Similarly, the first and last cysteines in hML may also form a crucial disulfide bond.

«· I ** 4 · ·

None of the glycosylation sites has been retained in hML.

* 44V * All possible NML-linked glycosylation sites of hML are located in the carboxy-terminal half of the hML polypeptide.

• · 4 ^ 4 4 4 v * 4 4 4 4 • 4 · 73

Like hEPO, the hML mRNA does not contain the consensus polyadenylation sequence AAUAAA or the regulatory element AUU-UA, which is present in the 3 'untranslated regions of many cytokines and is believed to affect the stability of mRNA [Cell et al. , 46: 659-667 (1986)]. Northern blotting technology reveals small amounts of one 1.8 kb hML RNA copy product in both fetal and adult liver. After prolonged exposure, a weaker zone of the same size could be detected in the adult kidney. By comparison, human erythropoietin is expressed in the fetal liver and in response to hypoxia 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 significance of the C-terminal region of hML remains to be determined. Based on the presence of six potential sites for N-linked glycosylation and the ability of the ligand to bind to lectin affinity columns, this region of the hML is likely to be glycosylated. In some gel elution experiments, we detected activity that resolved at a Mr value of about 60,000, which may represent a full-length glycosylated molecule. The C-terminal region can thus function as a blood. to stabilize the circulating hML and half- • · · *. ·. to increase darkness. In the case of erythropoietin, the glycosylated form has complete biological activity in vitro but significantly reduced plasma half-life relative to glycosylated erythropoietin [Takeuchi et al. ., J. Biol. chem., 265: 12127 -: T: 12130 (1990)? Narhi et al., J. Biol. Chem., 266: 23022-23026 (1991) and Spivack et al., Blood, 7: 90-99 (1989)].

. The C-terminal domain of hML contains two bivalent base amino acid sequences [Arg-Arg motifs at positions 153-154 and • φ · 245-246] which could serve as possible processing sites. . Cleavage at these sites may be the reason for the formation of 30, 20 and 18-22 kDa forms of ML isolated from APP. Significantly, the Arg153 - Arg154 sequence is expressed in terms of. illegally following the erythropoietin-like domain of ML.

These observations indicate that full-length ML can represent a precursor protein that undergoes limited proteolysis to produce a fully mature ligand.

4. Human mpl ligand isoforms and variants Human mpl ligand isoforms: or alternatively, the stranded forms were detected by PCR in fully developed human liver. Briefly, primers were synthesized corresponding to both ends of the hML coding sequence as well as selected internal regions. These primers were used in the RT-PCR reaction to amplify mature human liver RNA as described in Example 10. In addition to the full-length form designated hML, three other forms called hML2, hML3 and hML4 were observed or derived. The fully prepared deduced amino acid sequences of all four isoforms are shown in Figure 11 (SEQ ID NOs: 6, 8, 9 & 10). hML3 has a 116 nucleotide deletion at position 700, which results in both amino acid deletion and a reading frame change. The cDNA now encodes a fully mature polypeptide of 265 amino acids that differs from the hML sequence at amino acid residue 139. Finally, hML4 has both a 12 nucleotide deletion after nucleotide position 618 (also found in murine and porcine sequences (see *). (i)) and the 116 bp deletion observed in hML3.

Although no clone has been isolated from human with a · · - * 'only 12 base pair deletion (after nucleotide 619) (hML2), this form is unlikely to exist because such J.:.: isoform has been identified in both mouse and pig (see • · · ::: below) and because it has been identified along with a 116 nucleotide deletion in hML4.

. Both substitution variants of hML, in which the · ··· seeded Arg153 to Arg154 sequence was replaced by two alanine residues and a truncated form of the · EPO domain of hML, were constructed to determine whether the · · * ... · ML long necessary for biological activity.

♦

A substitutional variant of the duplex Arg153 - Arg154 sequence. ti, designated hML (R153A, R154A), was constructed using the PCR method as described in Example 10. The truncated form of the "EPO domain", hML53, was also prepared using the PCR method by adding the stop codon Argl53: after.

5. Expression of recombinant human mpl ligand (rhML) in transiently transfected human embryonic kidney cells (293 cells)

To confirm that the cloned human cDNA encoded the ligand for the mpl receptor, the ligand was expressed in mammalian 293 cells under the control of the immediate early promoter of the cytomegalovirus using expression vectors pRK5-hML or pRK5-hML153. Transiently transfected human embryonic kidney 293 cell supernatants were found to stimulate 3H-thymidine incorporation into Ba / F3 mpl cells but not in parental Ba / F3 cells (Figure 12A). The media of 293 cells transfected with the pRK vector alone did not contain this activity. Addition of Mpl-IgG to the medium removed the stimulation (data not shown). These results indicate that the cloned cDNA encodes human functional ML (hML).

The purpose is to determine whether the "EPO domain" alone I · '·, bind to mpl and activate it, the truncated form of hML, · · *. rhML153, was expressed in 293 cells. It was found that the supernatants of the transfected • · ·. * * Cells had the same activity as the supernatants of the full-length hML (Fig. 12 &) expressing the C-terminus of ML. the domain is not required for binding to the c-mpl receptor and for its activation • ·· ι.ί '.

6. Mpl ligand stimulates megakaryocytopoiesis and. thrombopoiesis • · ·. • f. Both the full-length form of recombinant hML and the truncated form of rhML153 stimulate human megakaryocytic topoeia in vitro (Figure 12B). This effect was observed in the absence of other exogenously added hematopoietic growth factors. With the exception of IL-3, ML was the only · · hematopoietic growth factor investigated that demonstrated this · · · · 76 activity. IL-11, IL-6, IL-1, erythropoietin, G-CSF, IL-9, LIF, kit ligand (KL), M-CSF, OSM and GM-CSF had no effect on megakaryocytopoiesis when examined; separately in our specification (data not shown). This result indicates that ML has megakaryocyte stimulating activity and indicates that ML has a role in the regulation of megakaryocytopoiesis.

It has been shown that thrombopoietic activity in the plasma of thrombocytopenic animals stimulates platelet production in the mouse elastic response thrombocytosis assay [McDonald, Proc. Soc. Exp. Biol. Med., 14: 1006-1001 (1973) and McDonald et al., Scan !. J. Haematol., 16: 326-334 (1976)]. In this model, mice are suddenly induced with thrombocytopenia using specific anti-platelet serum, resulting in predictable elastic response thrombo-bocytosis. Such immunothrombocytemic mice are more responsive to exogenous thrombopoietin-like activities than normal mice [McDonald, Proc. Soc. Exp. Biol. Med., 14: 1006-1001 (1973)] as well as exhypoxic mice are more sensitive to erythropoietin. than normal mice [McDonald, et al., J. Lab. Clin. Med., *. *. 77: 134-143 (1971)]. To determine whether platelet production in vivo stimulated platelet production in mice with elastic response thrombocytosis, a partial injection of pure *: * was given. * 1 dedicated rhML. Platelet counts and platelet incorporation of 35S were then quantified. In mice injected with 64,000 or 32,000 units of rML, platelet production was significantly increased; as evidenced by the <20% increase in platelet count • · · (p = 0.0005 and 0.0001, respectively) and ~ 40% increase in 35S incorporation into platelets (p = 0.003) in treated • · · * · * * mice compared to mice injected with filler alone (Fig. 12C). This level of stimulation is comparable to: ·. *. to the level we have observed with EL-6 in this model • ·. ···. (data not shown). Treatment with 16,000 units • · · 77 rML did not significantly stimulate platelet production. These results indicate that ML stimulates platelet production in a dose-dependent manner and thus has thrombopoietin-like activity.

293 cells were also transfected with the other hML isoform constructs described above and the supernatants were analyzed using the Ba / F3 mpl proliferation assay (see Figure 13). hML2 and hML3 did not show detectable activity in this assay, however, the activity of hML (R153A, R154A) was similar to that of hML and hML153, indicating that processing at the dibasic Arg153-Arg154 site is not necessary but not detrimental to activity.

7. Megakaryocytopoiesis and mpl ligand

It has been suggested that megakaryocytopoiesis is regulated at multiple cell levels (Williams et al., J. Cell Physiol., 110: 101-104 (1982) and Williams et al., Blood Cells, 15: 123-133 (1989)). This is largely based on the finding that certain hematopoietic growth factors stimulate the proliferation of megakaryocyte progenitors, while others appear to primarily affect maturation. The results presented here suggest that ML functions both proliferatively. *. as a factor and as a maturation factor. Several lines of evidence point to the fact that ML stimulates the proliferation of megakaryocyte progenitor cells. Firstly, APP (swine aplastic plasma). *: stimulates both proliferation and maturation of human megakaryocytes in vitro, and this stimulation is inhibited by complete mpl-IgG (Figs. 7 and 8). In addition, the formation of megakaryocyte colonies by c-mpl antisense oligonucleotides. blocking it [Methia et al., Blood, 82: 1395-1401 ···. ·; ·. (1993)] and the finding that c-mpl can transfer the proliferative signal in the cells to which it has been transfected [Skoda et al., EMBO, 12: 2645-2653 (1993) and Vigon et al., Oncogene, 8: 2607-2615 (1993)] also show that ML stimulates proliferation. clear expression of c-mpl during all stages of megakary-,. ···, oocyte differentiation [Methia ·, · 78 et al., Blood, 82: 1395-1401 (1993)] and ability of recombinant ML. The rapid stimulation of platelet production in vivo indicates that ML also affects maturation. The availability of recombinant ML makes it possible to carefully evaluate its role in the regulation of megakaryocytopoiesis and thrombosis and to assess its ability to influence other hematopoietic lineages.

8. Isolation of the human mpl ligand (TPO) gene Human genomic DNA clones of the TPO gene were isolated by screening the human genomic library in A-Gem12 with pR45 under less stringent conditions or under very stringent conditions using a fragment corresponding to human mpl. 31 of the cDNA encoding the ligand. Two partially overlapping 35 kb lambda clones were isolated. Two partially 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 consists of 6 exons within 7 kb of genomic DNA. The boundaries of all exon / intron junctions are consistent with the consensus motif created for human genes [Shapiro, M.B. et al., Nucl. Acids ... Res., 15: 7155 (1987)]. Exon 1 and exon 2 contain 5'- • * · '* • · *. *. the first four amino acids of the untranslated sequence and 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 31-untranslated domain of the erythropoietin-like domain and <50 • l * t: amino acids are encoded in exon 6. involved in the deletion of hML-2 (hTP0-2) is encoded at the 5 'end of exon 6.

• · ·

Southern blot analysis of human genomic DNA showed that the TPO gene is present in a single copy. The chromosomal location of the · · · *. * * Gene was determined using Fluorescent In situ Hybridization (FISH) mapping to chromosome 3q27-28.

«·. · * ·. 9. Expression and Purification of TPO from 293 Cells • · ·· · Preparation and purification of 79 ML or TPO from 293 cells is described in detail in Example 19. In short, the cDNA corresponding to the entire open reading frame of TPO , was obtained by PCR using pRK5-hmpl I. The PCR product was purified and cloned between the restriction sites Clal and Xba I of the plasmid pRK5tkneo (plasmid pRK5tkneo), between the restriction sites Clal and XbaI of the plasmid pRKStkneo (a vector derived from pRK5, engineered to express the neomycin resistance gene).

Another vector encoding the EPO homologous domain was similarly constructed, except using different PCR primers, to obtain a final construct called pRK5-tkneoEP0-D.

These two constructs were transfected into human embryonic kidney cells using the CaPO 4 method and neo-mycine resistant clones were selected and allowed to grow into a solid plate. The expression of ML153 or ML14 in the medium used to cultivate these clones was determined using the Ba / F3 mpl proliferation assay.

The purification of rhML was performed as described in Example 19. In short, the 293-rhML332 contains *. *. the conditioned medium was added to Blue-Sepharose (Pharmacia), which was then washed with a buffer containing 2 µl mol / L urea. The column was eluted with buffer containing 2 µmol / l urea and 1 mol / l NaCl. The combined eluate from the Blue-Sephrarose column was then directly transferred to the WGA- • · · »#! : Sepharose column, washed with 10 column volumes of buffer containing 2 mol / L urea and 1 mol / L NaCl, and eluted. was added with the same buffer containing 0.5 mol / L N-acetyl- · · · D-glucosamine. The WGA-Sepharose eluate was loaded on a C4 HPLC column (Synchrom, Inc.) and eluted with a periodic propanol gradient. According to SDS-PAGE, the purified 293-rhML> 332 migrates as a broad band within the 68-80 kDa gel (see Figure 15).

• ·. ···. Purification of rhML153 was also performed as described in Examples ··· 80 in 19. Briefly, the spent medium containing 293-rhML153 was separated on Blue-Sepharose column as described for rhML. The Blue-Sepharose eluate was directly applied to the mpl affinity column as described above. RhML153 eluted from the Mpl affinity column was purified to homogeneity using a C4 HPLC column run under the same conditions as for rhML1. According to SDS-PAGE, purified rhML153 is distinguished into two major and two minor bands with a Mr value of &lt; 18,000-22,000 (see Figure 15).

10. The murine mpl ligand DNA fragment corresponding to the coding region for 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 screen 106 clones of mouse liver cDNA library in IgT10. The rat clone [Figure 16 (SEQ ID NOs: 12 & 13)] containing an insert of 1443 bp was isolated and sequenced. The putative start codon at nucleotide position 138-141 was in the consensus sequence favoring the onset of eukaryotic translation (Kozak, M., J. Cell Biol., 108: 229-241 (1989)). This sequence defines an open reading frame of 1056 nucleotides which predicts • · *. 352 amino acid primary translation products. This open reading frame is flanked by 5 'end 137 nucleotides and · 247 nucleotides of the 3' untranslated sequence. There is no poly (A) tail after the 3'- • · untranslated region, indicating that the clone is unlikely to be complete. Predicted: The N-terminus of the known amino acid sequence is highly hydrophobic and probably represents a signal peptide. Computer Analysis [von. Heijne, G., Eur. J. Biochem. , 133: 17-21 (1983)] indicated ··· a possible signal peptidase cleavage site between residues • · · 21 and 22. Cleavage at that position would yield a 331 ··· • · · · * * amino acid mature polypeptide (35 kDA), identified · · · * ... · as mML331 (or mML2 for the reasons explained below). The sequence contains 4 cysteines, all of · ·, ···. ki have been conserved in the human sequence, and seven possible 81 · N · glycosylation sites, 5 of which have been conserved in the human sequence. Again, as in the case of hML, all seven potential N-glycosylation sites are located in the C-terminal half of the protein.

Compared to the sequence of the human mpl ligand, significant identity was found in the "EPO domains" of these mpl ligands with respect to both nucleotide sequences and derived amino acid sequences. However, when the deduced amino acid sequences of human and mouse mpl ligands were aligned, the mouse sequence appeared to have a tetrapeptide deletion between residues 111-114, corresponding to a deletion of 12 nucleotides after nucleotide position 618, which was detected in both human (see above) and porcine (see below) in. Thus, additional clones were examined for possible ML isoforms of the rat. One clone encoded a 335 amino acid derived polypeptide containing the "missing" tetrapeptide LPLQ. This shape is believed to be full-length rat ML and is called mML or mML ^. The nucleotide sequence and derived amino acid sequence of mML are shown in Figure 17 (SEQ ID NOs: 14 & 15). This cDNA clone consists of 1443 base pairs followed by a poly (A) tail.

• ·

It has an open reading frame of 1068 bp, flanked by 5'-S * ·· * untranslated sequence 134 and 3-untranslated sequence 241 bases. The putative start codon is at nucleotide position 138-140. The open reading frame encodes a predicted protein consisting of 356 amino acids, the first 21 of which are highly hydrophobic and are likely to act as a secretory signal.

Finally, a third rat clone was isolated, sequenced, and found to contain a 116 nucleotide deletion corresponding to hML3. Therefore, this isoform of the rat was named mML3. A comparison of the deduced amino acid sequences of these two isoforms is shown in Figure 18 (SEQ ID NOs: 9 & 16).

• ·. ·· *. The overall amino acid sequence identity between human and ML · rat · Figure · 82 (Figure 19 (SEQ ID NOs: 6 & 17)) is 72%, but this homology is not uniformly distributed. The region defined as the "EPO domain" (human sequence amino acids 1 to 153 and mouse sequence amino acids 1 to 149) is better preserved (homology 86%) than the protein carboxy-terminal region (62% homology). This may further indicate that only the "EPO domain" is important for the biological activity of the protein. Interestingly, of the two dibasic amino acid motifs observed in hML, only the double base motif immediately following the "EPO domain" (residual 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 form a fully mature ligand. Alternatively, proteolysis between Arg153 and Arg154 may facilitate hML clearance.

An expression vector containing the entire coding sequence for mML was transiently transfected into 293 cells as described in Example 1. The medium used from these cells stimulates 3H-thymidine incorporation into Ba / F3 cells, which expressed either murine or human mpl but m *. 1. It had no effect on the parental (no mpl) cell • · ·

'· Line. This indicates that cloned rat ML cDNA

• · • 2 encodes a functional ligand capable of activation *. 1t of both rat and human ML receptor (mpl).

11. Porcine mpl ligand ·· ♦ · Porcine ML (pML) cDNA was isolated by RACE PCR,

as described in Example 13. 13 4 2 bp PCR cDNA

• · 1 · was detected in the kidney and subcloned. A number of clones ··· were sequenced and found to encode a porcine mpl-1 · · 1 gand with 332 amino acid residues and designated pML (or pMI ja) with a nucleotide sequence and a deduced amino acid sequence. is shown in Figure 20 (SEQ ID NOs: 18 & 19).

• ·. ···. Again, another form was identified, designated 2 x 83 pML2, which encoded a protein with a deletion of 4 amino acid residues (228 amino acid residues) [see Figure 21 (SEQ ID NO: 21)]. Comparison of the pML and pML2 amino acid sequences shows that the latter form is identical except that the tetrapeptide QLPP corresponding to residues 111-114 has been deleted [see Figure 22 (SEQ ID NOs: 18 & 21)]. The deletions of four amino acids found in both rat and porcine ML cDNA occur at exactly the same position in the predicted proteins.

Comparison of the predicted amino acid sequences of fully mature ML from human, mouse and porcine [Figure 19 (SEQ ID NOs: 6, 17 & 18)] shows that the overall sequence identity is 72% between mouse and human, 68% between mouse and porcine. and 73% between pig and human. The homology is substantially higher in the amino terminal half of ML (EPO homologous domain). The identity of this domain is between 80% and 84% between any two species, while the identity of the carboxy terminated half (carbohydrate domain) is only 57% to 67%. A double-base amino acid motif that could represent a protease cleavage site is present at the carboxyl terminus of the erythropoietin homology domain. This: *. *. the motif has remained between the three species in this position [ku-, ·. ; va 19 (SEQ ID NOs: 6, 17 & 18)]. Another bipartite target that is present at positions 245 and 246 in the human sequence, "· · *". is not present in mouse or porcine sequences. The Mouse and Pig • · · '/ ML sequence contains 4 cysteines, all of which are conserved | »*« · * In the human sequence. The murine ligand has seven possible · · ·. * * N-glycosylation sites and six in porcine ML, 5 of which are conserved in the human sequence. Again, all 54; #J potential N-glycosylation sites are located in the C-terminal half of the protein.

, .J. ' 12. Expression and Purification of TPO from Chinese Hamster Ovary (CHO) *: ...

Γ Expression vectors used in CHO cells; transfection, is named as: pSVIS.ID.-

LL.MLORF (full length or TPO ,,,) and pSVI5. ID. LL.MLEPO D

84 (truncated, i.e. TP0153). The essential properties of these plasmids are shown in Figures 23 and 24.

Transfection methods are described in Example 20. Briefly, cDNA corresponding to the entire open reading frame of TP0 was obtained by PCR. The PCR product was purified and cloned between the two restriction sites (Clal and SalI) of plasmid pSVI5.ID.LL to obtain the vector pSVI5.ID.LL.MLORF. Another construct corresponding to the EPO homologous domain was constructed in the same manner except using a different inverse region (EPOD.Sal). The final construct of the vector encoding the TPO EPO homologous domain is called pSVIS.ID.-LL.MLEPO-D.

These two constructs were linearized with NotI and transfected into Chinese hamster ovary cells (CHO-DP12 cells, EP 307 247, published March 15, 1989) by electroporation. 107 cells were electroporated into a BRL electroporation apparatus (350 V, 330 mF, low capacitance) in the presence of 10, 25 or 50 mg of DNA as described [Andreason, G.L., J. Tissue Cult. Meth., 15.56 (1993)]. On the day following transfection, cells were partitioned with DHFR-selective medium (50: 50 glucose-rich DMEM-F12 in the absence of glycine, 2 · ·] ./ · mmol / l glutamine, 2-5% dialyzed fetal calf serum. 'mia). Then, 10-15 days later, individual colonies • 4 »*. . were transferred to 96-well plates and allowed to grow as a single plate. The expression of ML153 or ML332 in the medium used from these clones was determined using the Ba / F3 mpl proliferation assay (described in Example 1).

The method for purifying and isolating TPO from CHO cell culture fluid is described in Example 20.

Briefly, the recovered cell culture fluid (HCCF) « is added to the Blue Sepharose column (Pharmacia) in a ratio of J · ··· * about 100 L HCCF / 1 L resin. The column is then washed with: '*' and 3-5 column volumes of buffer followed by 3-5 * · · ** '; column volume of buffer containing 2.0 mol / l urea * · 85 aa. The TPO is then eluted with 3-5 volumes of buffer containing both 2.0 mol / l urea and 1.0 mol / l NaCl.

The combined Blue Sepharose eluate containing TPO is then added to a Wheat Germ Lectin Sepharose column (Pharmacia) equilibrated in Blue Sepharose elution buffer at a ratio of 8-16 ml Blue Sepharose eluate / 1 ml resin. The column is then washed with 2-3 column volumes of equilibration buffer. TPO is then eluted with 2 to 5 column volumes of buffer containing 2.0 mol / L urea and 0.5 mol / L N-acetyl-D-glucosamine.

The Wheat Germ Lectin eluate containing TPO is then acidified and C12E8 is added to give a final concentration of 0.04%. The resulting combination is added to a C4 reverse phase column equilibrated in 0.1% TFA, 0.04% C12E8 with a loading of approximately 0.2-0.5 mg protein / 1 ml resin.

The protein elutes in a biphasic linear gradient consisting of acetonitrile containing 0.1% TFA and 0.04% C12E8, and the products are pooled by SDS-PAGE.

The combined C4 product is then diluted and diafilter-JV. is added against about 6 volumes of buffer with an Amicon YM-mem-* · • «» · bra or equivalent ultrafiltration membrane with a Dalton molecular weight cut-off of 10,000 to 30,000 daltons. . , push. The resulting diafiltrate can then be directly processed or may be further concentrated by ultrafiltration.

t t # »L * The final Tween-80 concentration of the diafiltrate / concentrate is usually adjusted to 0.01%.

All or part of the diafiltrate / concentrate corresponding to 2-5% of the calculated column volume is then added to a ··· /: *: Sephacryl S-300 HR column (Pharmacia) equilibrated.1. in 0.01% Tween-80, and chromatographed. Fractions containing TPO that do not contain aggregates or proteolytic degradation products are pooled by SDS-PAGE. The resulting combined product is filtered and stored at 2-8 ° C.

13. Methods for Transforming and Inducing TPO Synthesis in an Microorganism and for Isolation, Purification and Recirculation of TPO Prepared in It The construction of E. coli TPO expression vectors is described in detail in Example 21. Briefly, plasmids pMP21, pMP151, pMP41, pMP57 were all designed to express the first 155 amino acids of TPO under a variable downstream sequence between different constructs. The lead sequences primarily allow large-scale translation to be started and rapid purification. Plasmids pMP210-1, -T8, -21, -22, -24, -25 are designed to express the first 153 amino acids of TPO below the start methionine and differ only in codon usage for the first 6 amino acids of TPO, whereas plasmid pMP251 has a derivative of pMP210-1, in which the carboxy terminated end of TPO is extended by two amino acids. All of the above plasmids produce high levels of intracellular expression of TPO in E. coli after induction of the tryptophan promoter [Yansura, DG et al., Methods in Enzymology (Ed. Goeddel, DV) 185: 54-60, Academic Press , San Diego (1990)]. Plasmids pMP1 and pMP172 are intermediates in the formation of the above-mentioned TPO intracellular expression vectors.

• · · • m

The above TPO expression vectors were used to transform E. coli using the CaCl2 heat shock method [Mandel, M. et al., J. Mol. Biol., 53: • · · *. 1: 159-162 (1970)] and other methods described in Example 21. Briefly, transformed cells were first grown at 37 ° C until the optical density (600 nm) of the culture reached approximately 2-3. Then: the culture was diluted and the culture was grown under aeration, then acid was added. The culture was then allowed to continue growing for a further 15 hours with aeration, after which the cells were harvested by centrifugation.

• • • • • • • • • • • • • • • 87 · 87

The isolation, purification, and rewrapping methods set forth below to produce biologically active, recombinant human TPO or fragments thereof are described in Examples 22 and 23 and can be used for any TPO variant, including N- and C-. to recover the terminal extended forms. Other methods suitable for recycling recombinant or synthetic TPO can be found in the following patents: Builder et al., U.S. Patent 4,511,502; Jones et al., U.S. Patent 4,512,922; Olson, U.S. Patent 4,518,526; and Builder et al., U.S. Patent 4,620,948; for a general description of a method for the recovery and recycling of various recombinant proteins expressed in Liu intact form in E. coli.

Ά. Recovery of insoluble TP0

A microorganism such as E. coli that expresses TPO encoded by any suitable plasmid is fermented under conditions where TPO precipitates in insoluble "refractile bodies". Optionally, the cells are first washed with cell lysis buffer. Typically, about 100 g of cells are resuspended in about 10 volumes of cell lysis buffer (e.g., 10 mM Tris, 5 mM EDTA, pH 8), for example, using a Polytron homogenizer and cells ... centrifuge at 5,000 x g for 30 minutes • · *. 1st period. The cells are then lysed using any conventional technique such as sonic shock, sonic vibrator, pressure oscillation, chemical or enzymatic methods. For example, the washed cell pellet obtained above may be resuspended in an additional 10 volumes of cell lysis buffer homogenizer and the cell suspension transported through an LH Cell Disrupter (LH Inceltech, Inc.). or Microfluidizer (Microfluidics International) • · ·. ···. through the manufacturer's instructions. The TP0-containing fine particle is then separated from the liquid phase and optionally washed with any suitable liquid. For example, a suspension of 88 cell lysis product can be centrifuged at 5,000 xg for 30 minutes, resuspended, and possibly centrifuged. a second time to wash the refractive body to form a pellet. The washed pellet may be used immediately or may be stored frozen (e.g. at -70 ° C).

B. Dissolution and purification of monomeric TP0

The insoluble TPO in the pellet of the refractive body is dissolved in the dissolving 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. Representative chaotropic agents include urea, guanidine1HCl and sodium thiocyanate. A preferred chaotropic agent is guanidine1HCl. The concentration of the chaotropic agent is usually 4 to 9 M, preferably 6 to 8 M. The pH of the soluble buffer is maintained by any suitable buffer in the pH range of about 7.5 to 9.5, preferably 8.0 to 9.0 and most preferably 8.0. Preferably, the dissolving buffer also contains a reducing agent to facilitate formation of the monomeric form of TPO. Suitable reducing agents include organic compounds containing free thiol (RSH). Representative reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), mercapto. support ethanol, glutathione (GSH), cysteamine and cysteine. Edul- • · · *. The first reductant is dithiothreitol (DTT). Optionally, the solubilizing buffer may contain a mild oxidant (e.g., molecular molecular oxygen) and a sulphite salt to form monomeric TPO by sulfitolysis. In this embodiment, the resulting TPO-S sulfonate is subsequently recycled in the presence of an oxidation reducing buffer (e.g., GSH / GSSG) to form a properly rotated TPO.

. . ·. Typically, the TPO protein is further purified using, for example, centrifugation, gel filtration chromatography, and reverse phase column chromatography.

1 By way of example, the following method has yielded a yield of monomeric TPOs of 2 to 89 pivotal monomeric TPO. . The pellet formed by the refractive body is resuspended in about 5 volumes of weight-soluble buffer (20 mM Tris, pH 8, contains 6-8 mol / l guanidine and 25 mmol / l DTT) and the resulting suspension is stirred for 1-3 hours. or overnight at 4 ° C to effect dissolution of the TPO protein. High concentrations of urea (6-8 M) are also useful, but generally give somewhat lower yields compared to guanidine. After dissolution, the solution is centrifuged at 30,000 x g for 30 minutes to produce a clear supernatant containing denatured monomeric TPO protein. The supernatant is then chromatographed using a Superdex 200 gel filtration column (Pharmacia, 2.6 x 60 cm) at a flow rate of 2 mL / min and the protein eluted with 20 mM Na phosphate pH 6.0 containing 10 mM DTT: a. Fractions containing the monomeric denatured TPO protein and eluted between 160 and 200 ml are pooled. The TPO protein is further purified using a semi-preparative C4 reverse phase column (2 x 20 cm, VYDAC). The sample is added at a rate of 5 ml / min to a column equilibrated in 0.1% TFA (trifluoroacetic acid) containing 30% acetonitrile. The protein eluted with a linear gradient of acetonitrile (30-60%, 60 min). The purified, ... reduced protein elutes at about • · · * acetonitrile. \ 50%. This material is used to re-circulate TP0 to obtain a biologically active TPO variant.

♦ ♦ i ** C. Recirculation of TPO to produce the form of biologically active • · ♦. ** after dissolution of TPO and further purification, the biologically active form is obtained by recirculation of the denaturated monomeric TPO in the oxidation-reduction buffer. TPO. due to its high potency (half of the maximum stimulation ···. ··· in the Ba / F3 assay is obtained at approximately 3 pg / ml) it is possible to obtain a biologically active substance using a variety of buffer. , detergent and oxidation-reduction conditions. However, under most conditions, only a small amount of correctly twisted material (<10%) will be obtained. For commercial production processes, it is desirable that the yields of the recycled material be at least 10%, more preferably 30-50% and most preferably> 50%. A variety of detergents, including Triton X-100, dodecyl beta-maltoside, CHAPS, CHAPSO, SDS, sarcosyl, Tween 20 and Tween 80, Zwittergent 3-14 and others, were found to be suitable for producing at least some degree of twisted material. Of these, however, the most preferred detergents were the CHAPS detergents (CHAPS and CHAPSO), which were found to work best in the reuptake reaction and to limit protein aggregation and inappropriate disulfide formation. Most preferred were amounts of CHAPS greater than about 1%. Sodium chloride was required for the best yields, with an optimum amount of 0.1 to 0.5 mol / L. The presence of EDTA (1-5 mmol / L) in the oxidation-reduction buffer was preferred to limit the amount of metal-catalyzed oxidation (and accumulation) observed in some preparations. Glycerol concentrations above 15% provided optimum conditions for re-rotation. For maximum yields, it was essential that the oxidation-reduction buffer contained an oxidation-reduction pair consisting of both oxidized and reduced organic thiol (RSH). Suitable oxidation-reduction pairs include mercaptoethanol, glutathione (GSH), · · *. *. cysteamine, cysteine and their corresponding oxidized forms.

• · ·

Preferred oxidation-reduction pairs were glutathione (GSH): • · * 'oxidized glutathione (GSSG) or cysteine: cystine. Edul- • · · *. *: The additional oxidation-reduction pair was glutathione (GSH): oxidized glutathione (GSSG). Generally, higher yields were observed when the molar ratio of oxidized member of the oxidation-reduction pair was equal to or greater than the reduction ratio of the oxidation-reduction pair. · '; Member. To circumvent these TPO variants ···. *: ·. pH values of 7.5 to about 9 were optimal. Organic solvents (e.g., ethanol, acetonitrile, methanol) could be used in acceptable concentrations of 10 to 15% or more ♦ · · t: ♦ · · • · 91 lower concentrations. Large amounts of organic solvents increased the amount of incorrectly twisted forms. Tris and phosphate buffers were generally useful. Incubation at 4 ° C also produced higher amounts of correctly twisted TPO.

Typical yields of the re-twisted material are 40-60% (based on the amount of reduced and denatured TP0 used in the re-twisting reaction) for TPO preparations purified by the first C4 step. The active agent can be obtained using less pure formulations (e.g. directly after the Superdex 200 column or after extraction of the first refractive unit), although the yields are lower due to extensive precipitation of non-TPO proteins and interference during the TPO refolding process.

Because TPO contains four cysteine residues, it is possible to produce three different disulfide variants of this protein: Variant 1: Disulfides between cysteine residues 1-4 and 2-3; Variation 2: Disulfides between cysteine residues 1-2 and 3-4; Variation 3: Disulfides between cysteine residues 1-3 and 2-4.

During the initial investigation, when configuring • · '. First, a number of different peaks containing <♦ »• · 1 * containing TPO were separated by C4 reverse phase chromatography *. Only one of these peaks had significant biological activity as determined by the Ba / F3 assay. Then: .ί .: The rewind conditions were selectively optimized to produce that * ·· · variation. Under these conditions, the mis-twisted variants were less than 10-20% in the dissolution step; of the total amount of monomeric TPO obtained.

• · ·

The disulfide model of biologically active TP0 has been determined to have 1-4 and 2-3 mass spectrometry and

• · I

* · '' Through the sequencing of ain, whereby the cysteines are numbered Γ2: ♦ · ·

»1 S

• 2 · 92 sequentially starting from the amino terminus. This crosslinking pattern of cysteine is consistent with the known disulfide binding pattern of a nearby erythropoietin molecule.

D. Biological Activity of Recombinant Recycled TPO

Re-twisted and purified TPO has activity in both in vitro and in vivo assays. For example, in the Ba / F3 assay, TPO (Met 11-1153) stimulated thymidine incorporation into Ba / F3 cells such that half-maximal stimulation was achieved at a concentration of 3.3 pg / ml (0.3 µM). In the Mpl receptor based ELISA assay, half-maximal activity occurred at a concentration of 1.9 ng / ml (120 pM). In normal animals and in animals in which bone marrow function is blocked by near lethal X-rays, re-twisted TPO (Met'111-153) was highly effective (activity was observed at doses as low as 30 ng / mouse) to stimulate the production of new platelets. Similar biological activity was observed with other forms of TPO recycled according to the methods described above (see Figures 25, 26 and 28).

14. Methods for measuring thrombopoietic activity

Thrombopoietic activity can be measured in different ways. in these assays, including Ba / F3 mpl ligand counts. *. the in vivo determination of the platelet elastic response synthesis, the platelet cell surface antigen induction assay as measured by the anti-platelet • · ·, '* · le immunoassay (anti-platelet). -GPIIbIIIa) for human leukemia megakaryoblastic cell line (CMK) [see Sato et al., Brit. J. Haematol., 72: 184-190 (1989)] (see also liquid suspension megakaryocytopoiesis assay described in Example 4) and induction of polyploidization in megakaryoblastic cellular (DAMI) [see Ogura et al., Blood, 72 (1): 49-60 (1988)]. The maturation of megakaryocytes · · · · * * from immature, extensively synthesized DNA

»M

t: • »·

• · I

From 9,493 cells to morphologically identifiable megakaryocytes, the process involves the expression of cytoplasmic cells, acquisition of membrane antigens (GPIIbIIIa), platelet endor replication and release as described in the background. The genus-specific promoter of megakaryocyte maturation (namely, mpl ligand) would be expected to induce at least some of these changes in immature megakaryocytes, leading to platelet release and thrombocytopenia 1 spread. Thus, assays were designed to measure the presence of these parameters in immature megakaryocyte cell lines, namely CMK and DAMI cells. The CMK assay (Example 4) measures the incidence of specific platelet marker, GPIIbIIIa, and platelet leakage. The DAMI assay (Example 15) measures endo replication because ploidy inserts are the hallmarks of mature megakaryocytes. In detectable megakaryocytes, ploidy values are 2N, 4N, 8N, 16N, 32N, etc. Finally, in vivo mouse platelet elastic response assay (Example 16) is useful in demonstrating that administration of a test compound (herein mpl ligand) to a patient results in platelet counts.

Two other in vitro assays have been developed to measure TPO activity. The first is the kinase receptor activation (KIRA) ELISA assay, in which CHO cells are transfected with the mpl-Rse chimera and Rse's tyrosine phospho-≤ <* * · ration is measured by ELISA after vii *> 1ϊ mpl portion is exposed to mpl ligand (see Example 17).

«1: 1 ·· The other is a receptor based ELISA with an ELISA plate,

I I

·, 1 ··, coated with rabbit anti-human IgG, captures? it! the mpl-IgG human chimeric receptor that binds the mpl ligand to be determined. A biotinylated rabbit polyclonal antibody against mpl ligand (TP0155) is used to detect a recombinant mpl ligand measured using streptavidin peroxidase as described in Example 18 e1.

# · Ι V '15. In vivo Biological Response of Normal and Nearly Killed Mice Treated with TPO • 4 * 9 · # · 94 Highly Irradiated Mice

Both normal and near-lethal irradiated mice were treated with truncated and full-length TPO isolated from Chinese Hamster Ovary (CHO) cells, E. coli and human embryonic kidney cells (293 cells). Both forms of TPO produced in these three hosts stimulate platelet production in mice, however, full-length TPO isolated from CHO appeared to produce the greatest response in vivo. These results indicate that appropriate glycosylation of the carboxy terminal domain may be necessary for optimal in vivo activity.

(a) The "Met" form of the E. coli EPO domain produced in E. coli rhTPO (Het-153) (at the Met-1 position plus the first 153 residues of human TPO) (see Example 23) was injected daily for normal female C57 B6 mice as described in the descriptions of Figures 25A, 25B and 25C. These figures show that the non-glycosylated truncated form of TPO produced in E. coli and recirculated as described above is capable of stimulating approximately double the increase in platelet production in normal mice without affecting the red or white blood cell population.

• «·! · 'This is the same molecule that was injected daily into almost lethal irradiated (137Cs) female • ·

• · · · C57 B6 mice as described in Figures 26A, 26B and 26C

• · in explanations, stimulates platelet recovery and reduced: Nadir (Nadir) but did not affect red blood cells or • J1. Leukocyte.

• · · (b) CH0-rhTP0332 Full length TPO form produced in CHO and · · · injected daily into normal female • · ·

*. additional C57 B6 mice as described in Figures 27A, 27B

And · 27C, produced approximately a 5-fold increase in platelet production in normal mice unaffected by the red blood cell or white blood cell population.

• · • · · · 95 • (c) CHO-rhTPO ^? E. coli-rhTPO (Met-153); 293 rhTP0332; and E. coli-rhTP0155

Dose-response curves were generated from the treatment of normal mice with rhTPO from different cell lines (CH0-rhTP0332; E. coli-rhTPO (Met-153); 293-rhTP0332; and E. coli-rhTP0155), as described in Figure 28. described. This figure shows that all tested forms of the molecule stimulated platelet production, however, the full-length form produced in CHO had the highest in vivo activity.

(d) Dose-response curves for CHO-rhTP0153 / CHO-rhTPO? and CHO-rhTPO? were generated from the treatment of normal mice with various forms of CHTP-produced rhTPO (CHO-rhTP0153, CH0-rhTP03), as described in the description of Figure 29. This figure shows that all CHO forms of the molecule tested stimulated platelet production but that the full length 70 kDa form had the highest in vivo activity.

16. General recombinant production of Mpl ligand and variants

Preferably, the mpl ligand is prepared using standard recombinant methods involving the production of the mpl ligand polypeptide by culturing cells transfected to express the mpl ligand nucleic acid ·· ·: * (typically by transforming cells with an expression vector), • · *. * ·· and the polypeptide. cells. However, it is possible to imagine that the mpl ligand can be produced by homologous recombination, or by recombinant preparation. methods using control elements inserted into cells that already contain DNA encoding the mpl ligand. For example, a strong promoter / enhancer element, supp. a promoter or an exogenous transcription modifying element may be inserted into the genome of the intended host cell sufficiently close and sufficiently directed to effect transcription of the DNA encoding the desired mpl; T: ligand polypeptide. The control element does not encode the mpl ligand, but DNA is native to the genome of the host cell. Next, cells that make the receptor polypeptide of the present invention or increase or decrease its expression, if desired, are screened.

Thus, the invention relates to a method of producing an mpl ligand, the method comprising introducing a transcription modifying element into the genome of a cell containing a mpl ligand nucleic acid molecule sufficiently close to the nucleic acid molecule and thereby targeting said nucleic acid molecule to effect its transcription, culturing a cell containing the transcription modifying element and the nucleic acid molecule. The invention also refers to a host cell comprising a native mpl ligand nucleic acid molecule operably linked to exogenous control sequences recognized by the host cell.

A. Isolation of DNA Encoding the Mpl Ligand Polypeptide The DNA encoding the mpl ligand polypeptide can be obtained from any cDNA library prepared from tissue believed to have mpl ligand mRNA and express it at a detectable level. The Mpl ligand gene can also be obtained from a genomic DNA library or, by in vitro oligonucleotide synthesis, from a complete nucleotide • · ·: · 'or amino acid sequence.

• ·

Libraries are screened by a probe designed to identify the gene of interest or the protein encoded by it. In the case of cDNA expression libraries, suitable probes include monoclonal or · or polyclonal antibodies that recognize and specifically bind to the mpl ligand. In the case of cDNA libraries, suitable probes include oligonucleotides of about 20 to about 80 bases in length encoding mpl- · · · *. known or believed portions of the ligand cDNA of the same or different species; and / or complementary or homologous cDNAs or fragments thereof encoding their gene or a corresponding gene. Suitable probes for screening genomic DNA libraries include, but are not limited to, oligonucleotides, cDNAs, or fragments thereof that encode their gene or equivalent gene, and / or homologous genomic DNA. or fragments thereof. Screening of the cDNA or genomic library by a selected probe can be accomplished using standard methods as described in Sambrook et al. in the book, above.

An alternative means of isolating the gene encoding mpl ligand is to use PCR methodology as described in Sambrook et al. in the book, above. This method requires the use of oligonucleotide probes that hybridize to DNA encoding the mpl ligand. Procedures for selecting oligonucleotides are described below.

A preferred method for practicing this invention is the use of carefully selected oligonucleotide sequences for screening cDNA libraries from various tissues, preferably from human or porcine (adult or fetal) kidney or liver cell lines. For example, human fetal liver cellular cDNA libraries are screened using oligonucleotide probes. Alternatively, human genomic libraries can be screened using oligonucleotide probes.

The oligonucleotide sequences selected as probes • · · *. 1: should be long enough and unambiguous enough to minimize false positives. The actual (s) • ·! .1 ·· nucleotide sequence (s) is usually designed based on the mpl ligand regions with the smallest codon excess.

* · · · 1 · 1; Oligonucleotides may be degenerate at one or more positions. The use of degenerate oligonucleotides is. especially important when screening a library for a species where • · ♦ IV. the use of a selective codon is unknown.

• · «*. The oligonucleotide must be labeled so that it can be detected ♦ ♦ · V: when it hybridizes to its DNA in the screening library. The preferred labeling method is ATP (e.g. γ32Ρ: η); · [·. and the use of polynucleotide kinase to radiolabel the 5 'end of the oligonucleotide. However, other methods, including but not limited to biotinylation or enzyme labeling, may be used to label the oligonucleotide.

Of particular interest is the mpl ligand nucleic acid encoding the full-length mpl ligand polypeptide. In some preferred embodiments, the nucleic acid sequence contains a native mpl ligand signal sequence. Nucleic acid having the entire protein coding sequence is obtained by screening a selected cDNA library or genomic libraries using the deduced amino acid sequence.

B. Amino acid sequence variants of native mpl ligand

The amino acid sequence variants of the mpl ligand are prepared by introducing appropriate nucleotide changes into the mpl ligand DNA, or by in vitro synthesis of the desired mpl ligand polypeptide. Such variants include, for example, deletions, insertions, or substitutions of residues in the porcine mpl ligand amino acid sequence. For example, the carboxy terminated portions of a fully mature full length mpl ligand can be removed by proteolytic cleavage either in vivo or in vitro, or by cloning and expression of a fragment or DNA encoding a full length mpl ligand to produce a biologically active variant. Whatever the deletion is, the combination of insertion and substitution may be prepared to provide the final construct, provided that the final construct has the desired biological activity. modifies post-translational processes of the mpl ligand, such as the number or position of glycosylation site: 1 · 1: To design the amino acid sequence variants of the mpl ligand, the location of the mutation and the nature of the mutation depend on the · · # «I. mpl- The mutation sites may be modified individually or in series, e.g. by (1) substitution, first by conservative amino acid choices and then by · · · more radical choices, depending on the result achieved. , (2) removing the target residue, or (3) adding the same or a different category of residue near the antibody, or using combinations of 1-3.

A useful method for identifying certain residues or regions of the mpl ligand polypeptide which are preferred sites for mutagenesis is called 'alanine scanning mutagenesis' as described in Cunningham and Wells, Science, 244: 1081-1085 (1989). e.g., charged residues such as arg, asp, his, lys, and Glu) and replaced by any, preferably neutral or negatively charged, amino acid (most preferably alanine or polyalanine) for the purpose of interaction of amino acids with the surrounding aqueous environment within or outside the cell. which show functional susceptibility to substitutions is then enhanced by further addition of other variants to the substitution sites. Thus, although the site for introducing an amino acid sequence modification is predetermined, the nature of the mutation For example, to optimize the performance of a mutation at a particular site, sub-sweep or random mutagenesis is performed in the target codon or target region and ... expressed mpl ligand variants are screened for an optimal combination of the desired activity · · · • · *.

• · * *. “Forming amino acid sequence variants, there is a · ·; '·· two principal variables: the location of the mutation site and • · *. * ·: The nature of the mutation. For example, variants of mpl ligand polypeptide variants: * · * include variants of the mpl ligand sequence: * · *: and may represent naturally occurring alleles (which do not require mpl ligand DNA manipulation) or pre-. . ·. certain mutant forms made from mutated • · · IV. ' club DNA to either enter the allele or variant that ♦ · · *. not meet in nature. In general, the location and the nature of the selected mutation depend on the specificity of the mpl ligand to be transformed.

• «·; · [·. Deletions in the amino acid sequence generally comprise from about 1 to about 30 residues, more preferably from about 1 to about 10 residues, and are typically consecutive. Alternatively, deletions of the mpl ligand amino acid sequence may include some or all of the carboxy-terminated glycoprotein domain. Deletions of the amino acid sequence may also include one or more of the first six amino terminus residues of the mature protein. Possible deletions of the amino acid sequence comprise one or more residues in one or more loop regions located between the "helical bundles". Successive deletions are generally made with an even number of residues, but one or an odd number of deletions are within the scope of this invention. Deletion can be generated in regions of low homology among mpl ligands sharing the highest sequence identity to modify mpl ligand activity. Deletion can be generated in regions of low homology between human mpl ligand and other mammalian mpl ligand polypeptides that share most of the sequence identity with human mpl ligand. Deletions of mammalian mpl ligand polypeptide in regions of substantial homology with other mammalian mpl ligands are more likely to significantly modify the biological activity of the mpl ligand. Number of consecutive deletions • · • ♦ · *. The number of 1i is chosen to maintain the tpl · · ·: 1 · 1 -AR structure of the mpl ligands in the target domain, e.g., beta-· pleated (beta-pleated) plate or alpha helix.

Additions of the amino acid sequence include amino: 1: 1: and / or carboxyl-terminal fusions, which vary in length from one residue to the polypeptides containing. . ·. one hundred or more residues, and internal additions of one or more amino acid residues. Intrinsic inserts (i.e., insertions in the complete mature mpl ligand ···: sequence) can generally vary in length from about · · · * to about 1 to about 10 residues, more preferably from about 1 to about 5 residues; ·] ·. and most preferably between 1-3 residues. An exemplary fusion of exemplary fusion is a fusion of mpl ligand or fragment thereof and another cytokine or fragment thereof. Examples of terminal insertions include the fully prepared mpl ligand having the N-terminal methionyl residue, the product of direct expression of the fully mature ligand in recombinant cell culture, and the fusion of the heterologous N-terminal signal sequence to the N-terminus of the mature mpl ligand molecule. to facilitate secretion from recombinant hosts. Such signal sequences are generally obtained from the intended host cell species and are thus homologous to it. Suitable sequences include STII or Ipp in E. coli, alpha factor in yeast, and viral signals such as herpes gD in mammalian cells.

Other insert variants of the mpl ligand molecule include fusion to the N or C terminus of the mpl ligand of immunogenic polypeptides (i.e., not endogenous to the host to which the fusion is administered), such as bacterial polypeptides encoded by beta-lactamase or enzyme E. coli trp locus, or yeast protein, and C-terminated fusions with proteins with long half-lives, such as immunoglobulin constant regions (or ... other immunoglobulin regions), albumin or ferritin, as described in WO 89/02922, published April 6, 1989.

• · • '·· The third group of variants is formed by the amino acid «· ί / · | posubstituutiovariantit. With these variants, at least one t amino acid residue from the mpl ligand molecule has been removed and t1-1 different residue has been added. The sites most interesting for substitution mutagenesis include the sites identified as the active · · · I ”site (s) of the mpl ligand, and the sites where the other • · · *. the amino acids found in the analogs are substantially different in terms of side chain size, charge, or hydrophobicity, but also have a high degree of sequence identity. at a selected site between different mpl ligand species. 102 and / or one mpl ligand member in various animal analogs.

Other places of interest are sites where the specific residues of mpl ligand from different family members and / or species in one member are similar. These sites, especially those in the sequence of at least three other identical conserved sites, have been substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 3 under the preferred title substitution below. If such substitutions result in a change in biological activity, then more significant changes, named exemplary substitutions in Table 3 or described below with reference to amino acid classes, are produced and the products screened.

• • • • • • • • • • • • • • • • • · · · · · · · · · · · · · · · · · · · · · · · · · · · 1 1 1 1 1 1 1 1 1 t t t t t t t t t::::::::::::::::::::::: · · · · · · · ·

Table 3.

103

Original Exemplary Preferred residue substitutions substitutions

Ala (A) Val; Leu; Ile Vai

Arg (R) Lys; Gin; Asn Lys

Asn (N) Gln; His; Lys; Arg Gin

Asp (D) Glu Glu

Cys (C) Ser Ser

Gin (Q) Asn Asn

Glu (E) Asp Asp

Gly (G) Pro Pro

His (H) Asn; Gin; Lys; Arg Arg

Ile (I) Leu; Or; Met; Area; Phe; norleucine Leu

Leu (L) norleucine, Ile; Or;

Met; Area; Phe Ile

Lys (K) Arg; Gin; Asn Arg

Met (M) Leu; Phe; Ile Leu

Phe (F) Leu; Or; Ile; Area Leu

Pro (P) Gly Gly

Ser (S) Thr Thr

Thr (T) Ser Ser ... Trp (W) Tyr Tyr • ♦ ♦ · [Tyr (Y) Trp; Phe; Thr; Ser Phe 5 '** Val (V) Ile; Leu; Met; Phe; • ♦; *·* Area; norleucine Leu • ♦

Significant modifications in the function of the mpl ligand or in t ;: immunological identity are accomplished by selecting t *: *: substitutions that differ significantly in their effect on (a) maintaining the structure of the polypeptide backbone. . ·. in the region, for example as a sheet or helical structure, (b) the molecular charge or hydrophobicity of the target site, or (c) the size of the side chain. Naturally occurring residues are divided into ··· V * groups based on common side-chain properties: »·» (1) hydrophobic: norleucine, Met, Ala, Vai, Leu, · · · Ile; • (1) · 104 (2) neutral hydrophilic: Cys, Ser, Thr; (3) acidic: Asp, Glu; (4) basic: Asn, Gln, His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions require the replacement of one member of these classes. Such substituted residues may also be introduced into conservative substitution sites or, more preferably, into residual (non-conservative) sites.

In one embodiment of the invention, it is desirable to inactivate one or more protease cleavage sites present in the molecule. These sites are identified by looking at the encoded amino acid sequence, for trypsin, e.g., for arginyl or lysinyl residues. Once the protease cleavage sites have been identified, they are rendered inactive with respect to proteolytic cleavage by substituting the targeted residue with another residue, preferably a basic residue such as glutamine, or a hydrophobic residue such as serine; removing the residue; or by adding the residual prolyl immediately to the ice. after the session.

• 1 *. 1, In another embodiment, any methionyl »1«] · 1 'residue other than the methionyl residue which initiates the signal sequence, or any residue located about three residues from the N- or C-terminal to each such methionyl residue, »ϊ! is substituted by another residue (preferably according to Table 3) or removed. Alternatively, about 1-3. the remainder is added next to such places.

«T»

Any cysteine residues that are not excessively involved in maintaining the proper conformation of the mpl ligand can also be substituted, usually by serine, for molecular

To improve oxidation stability of the iin and to prevent abnormal crosslinking. It has been found that the first and fourth cysteines in the epo domain, numbered from the amino terminus, are necessary to maintain a suitable conformation, but that the second and third are not. Thus, the second and third cysteines in the epo domain can be substituted.

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

Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution, deletion, and insertion variants of mpl ligand DNA. This technique is well known in the art as described in Adelman et al., DNA, 2: 183 (1983). Briefly, DNA for the mpl ligand is converted by hybridizing the oligonucleotide encoding the desired mutation to a DNA template, the template being a single-stranded form of a plasmid or bacteriophage which encodes a. contains the unmodified or native DNA sequence of the mpl ligand. After hybridization, DNA polymerase ♦ * · is used to synthesize the entire second complementary strand of the template, thus containing the oligonucleotide primer and coding for the selected change in the mpl ligand DNA.

Generally, oligonucleotides of sT length of at least 25 nucleotides are used. An optimal oligonucleotide has 12 to 15 nucleotides which are perfectly complementary. tary to the template on either side of the nucleotide (s) encoding the mutation. This ensures that the oligonucleotide hybridizes properly to the single-stranded 11 V DNA template molecule. Oligonucleotides are readily synthesized using techniques known in the art, such as those described in Crea et al., Proc. Natl. Acad.

• 4 r · «44 • · •« 4 · 4 106 sei. USA, 75: 5765 (1978).

The DNA template may be constructed using vectors either derived from bacteriophage M13 vectors (commercially available M13mpl8 and M13mpl9 vectors are suitable) or vectors containing a single-stranded phage origin of replication as described in Viera et al. et al., Meth. Enzy-Mol., 153: 3 (1987). The DNA thus mutated can be inserted into one of these vectors to form a single-stranded template. The production of a single-stranded template is described in Sections 4.21 to 4.41 of Sambrook et al. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY 1989).

Alternatively, the single-stranded DNA template may be generated by denaturing the DNA of the double-stranded plasmid (or other) using standard techniques.

To alter the native DNA sequence (for example, to form amino acid sequence variants), the oligonucleotide is hybridized to a single-stranded template under suitable hybridization conditions. The DNA polymerizing enzyme, usually the K1enow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template. using an oligonucleotide as a primer for synthesis. Here's how • ♦ \ *. a heteroduplex molecule is formed such that one strand of DNA * * # ·. * * encodes a mutated form of mpl ligand and the other strand * * (original template) encodes an innate, * * · * · * S unmodified sequence of mpl ligand. This heteroduplex mole * * *. ·. · Kyl is then transformed into a suitable host cell, usually an f * ·· * · * prokaryote, such as E. coli JM101. After the cells have grown, they are transferred to agarose plates and screened using an oligonucleotide primer, which is a transfected. diol-labeled with 32-phosphate, containing <«· m, ·. bacterial colonies. The mutated region is then removed and introduced into a suitable vector to produce the protein, usually an expression vector of the type typically used to transform a suitable host.

t · «« · 4 · »9 107

It is precisely the method described above that can be modified to create a homoduplex molecule, whereby both strands of the plasmid contain a mutation (s). The variants are as follows: The single-stranded oligonucleotide is ligated to the single-stranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP) and deoxyribotomidine (dTTP), is combined with a modified thiodeoxyribibosocytosine called dCTP (aS) (available from Amersham Corporation). This mixture is added to the template oligonucleotide complex. To this mixture is added DNA polymerase, which then forms a DNA strand identical to the template except for the mutated bases. In addition, this new DNA strand contains dCTP (aS) instead of dCTP, which serves to protect the strand against digestion by a recombinant enuclease.

After the template strand of the double-stranded heteroduplex is nicked by a suitable restriction enzyme, the template strand can be cleaved using ExoIII nuclease or other suitable nuclease outside the region containing the mutagenic site (s). The reaction is then stopped to give a molecule that is only partially mono-. stranded. Then a complete double-strand • · · * · is formed. : DNA homoduplex using DNA polymerase in the presence of all 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 formed;; ·. in many ways. If the amino acids are located near · · · t ···. mutually in the polypeptide chain, they can be mutated simultaneously using a single oligonucleotide encoding all the desired amino acid substitutions. However, if the amino acids are located at some distance (separated by more than 108 about 10 amino acids), it is more difficult to form a single oligonucleotide that encodes all the desired changes. Instead, one of the two alternative methods may be used.

In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then ligated to the single-stranded template DNA simultaneously, and another DNA strand synthesized from the template encodes all the desired amino acid substitutions.

An alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the individual mutants; wild-type DNA is used to obtain the template, the oligonucleotide encoding the first desired amino acid substitution (s) is inserted into this template and then a heteroduplex DNA molecule is formed. The second round of mutagenesis uses as template the mutated DNA produced in the first round of mutagenesis. Thus, this template already contains one or more mutations. The oligonucleotide encoding the other desired amino acid substitution (s) is then inserted into this template, and the resulting DNA strand now encodes the mutations as well as the first mutation in mutagenesis. . for that second round. This resulting # · · '* DNA can be used as a template in the third round of · · · ***** mutagenesis, etc.

PCR mutagenesis is also suitable for the preparation of amino acid variants of the mpl ligand polypeptide. Although the next! #; T; description refers to DNA, it is to be understood that the technique: **: ': can also be used in the case of RNA. The PCR technique generally refers to the following method (see Erlich, supra, · · · * .. Chapter written by R. Higuch, pp. 61-70). When small amounts of template DNA are used as a starting material in PCR, • · ·! Primers differing slightly in their sequence: *** may be used to generate relatively large amounts of a specific DNA fragment that differs from the template sequence in positions where the primers differ from the template. To generate a mutation in plasmid DNA, one of the primers is designed to extend beyond the mutation position and include the mutation; the sequence of the second primer must be identical to the sequence of the opposite strand of the plasmid, but this sequence may be located anywhere along the plasmid DNA. However, it is preferred that the sequence of the second primer be located 200 nucleotides away from the sequence of the first primer so that at the end the entire amplified region of the DNA bound by the primers can be easily sequenced. PCR amplification using a primer pair as described above results in a population of DNA fragments that differ in the position of the mutation defined by the primer and possibly in other positions, since template duplication is somewhat prone to error.

If the ratio of template to product material is extremely low, the great majority of the product DNA fragments will contain the desired mutation (s). This product material is used to replace the corresponding region in the plasmid, which served as a PCR template, using the standard · · • · ·. : DNA technology. Mutations at different positions can be produced concurrently by either using a mutant at another region. . or by carrying out a second PCR using different mu · · · * · / tant primers and inserting the two resulting PCR · ♦ · * fragments simultaneously into a vector fragment in a three-part • ··· · ♦,, ...

*. * * in the pasting event (or more).

In a specific example of PCR mutagenesis, DNA from the template plasmid (1 µg) is linearized by digestion with a restriction endonuclease having a unique recognition site on the outside of the amplified region of the plasmid DNA. 100 ng of this material is added to a PCR buffer containing · · * ··· * containing four deoxynucleic: * ·: otide triphosphate contained in GeneAmp ® kits (obtained from Perkin-Elmer Cetus, Norwalk, CT and Emeryville, CA) ··· 110 and 25 pxnol of each oligonucleotide primer, to a final volume of 50 μΐ. The reaction mixture is covered with 35 silicon mineral oils. The reaction mixture is denatured for five minutes at 100 ° C, placed on ice for a short time, and then added with 1 μus of Thermus aquaticus (Taq) DNA polymerase (5 units / μΐ, purchased from Perkin-Elmer Cetus) below the mineral oil layer. The reaction mixture is then placed on a DNA Thermal Cycler heat exchange apparatus (purchased from Perkin-Elmer Cetus), which is programmed as follows: 2 min. 55 ° C; 30 sec at 72 ° C, then 19 turns as follows: 30 sec at 94 ° C; 30 sec at 55 ° C and 30 sec at 72 ° C.

At the end of the program, the reaction flask is removed from said heat exchange apparatus and the aqueous phase is transferred to a new flask, extracted with phenol / chloroform (50:50 by volume) and precipitated with ethanol and DNA.

is recovered using standard techniques. This material is then subjected to suitable treatments for insertion into the · * ": vector.

• ·: Another method for making variants, cassettes • ·· 9 • ·; thymagenesis, based on the technique described in Jul • • · *. #; in Wells et al., Gene, 34: 315 (1985). The starting material • · · * li is a plasmid (or other vector) containing mutant! ::; · * Mpl ligand DNA. The codon (s) is identified in the mutant mpl ligand DNA to be mutated. There must be a unique restriction endonuclease site on each side of the mutation site (s) to be identified. If there are no complete · · · 'restriction sites, they must be generated using, ···. the oligonucleotide-mediated mutagenesis method described above to produce them at appropriate sites in the mpl-S · * 1 * ligand DNA. Once the restriction sites are generated into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding a DNA sequence

Ill between restriction sites but containing the desired mutation (s) is synthesized using standard methods. The two strands are synthesized separately and then hybridized together using standard technology. This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 3 'and 5' ends that are compatible with the ends of the linearized plasmid so that it can be directly inserted into the plasmid. This plasmid now contains the mutated mpl ligand di-DNA sequence.

C. Insertion of nucleic acid into a replicable vector

Nucleic acid (e.g., cDNA or genomic DNA) encoding the native or variant mpl ligand polypeptide is added to a replicable vector for further cloning (DNA amplification) or expression. Many vectors are available and the choice of a suitable vector will depend on (1) whether it is used for DNA amplification or expression, (2) the size of the nucleic acid to be added to the vector and (3) the host cell to be transformed with the vector. Each vector contains different components depending on its function (amplification of DNA or expression of DNA) and the host cell with which it is compatible. Vector * · ·; ·. components generally include one or more of the following; including, but not limited to: signal sequence, origin of replication, · ··, one or more marker genes, enhancer gene::: promoter, and transcription termination sequence.

i ·; · * (I) Signal sequence component The mpl ligand of the present invention can be expressed not only directly but also by fusion with a heterologous polypeptide, preferably a signal sequence polypeptide or other polypeptide having a specific cleavage site. at the N-terminus of the protein or polypeptide. Generally, the 2 · *! * Signal sequence may be a component of the vector or it may be a · · ·: *. · Part of the mpl ligand DNA inserted into the vector. The selected · · · heterologous signal sequence should be 112 such that it is recognized and processed by the host cell (i.e., the signal lipeptide cleaves). In the case of prokaryotic host cells that do not recognize and process the signal sequence of the native mpl-ligand, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of alkaline phosphatase, penicillinase II, or heat. For yeast secretion, the native signal sequence may be substituted, for example, by yeast comparator, alpha factor or acid phosphatase leader sequences, the C. albicans glucoamylase leader sequence (EP 362179, published April 4, 1990), or by the signal described in WO 90/13646. published November 15, 1990. In expression in a mammalian cell, the native signal sequence (i.e., the mpl ligand precursor, which normally directs mpl ligand secretion from its native mammalian cells in vivo) is satisfactory, although other mammalian signal sequences may be suitable, additional sequences from other mpl ligand polypeptides or from the same mpl ligand derived from different animal species, signal sequences from mpl ligand and signal sequences from secreted polypeptides of the same la- * * *: Jin or related species, and viral secretory sequences, e.g. ·; ·. ^ gD signal.

'·. ; (ii) The origin of replication component · · · * / Both expression and cloning vectors contain a t · · nucleic acid sequence that allows the vector to replicate.

j · J

· * Occurrence in one or more selected host cells.

Generally, in cloning vectors, this sequence is a sequence that allows the vector to replicate independently of the host chromosomal DNA and contains the replication ali ·; ·. or autonomously replicating sequences. Such sequences are known by a wide variety of bacteria, j: yeast and viruses. Plasmid origin of replication • · ·: *. PBR322 is suitable for most Gram-negative bacteria, · · · · ..., 2 μ: η plasmid origin is suitable for yeast and 113 different viral origin (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not required for mammalian expression vectors (SV 40 origin can typically be used only because it contains an early promoter).

Most expression vectors are "shuttle" vectors, i.e.. they are capable of replication in at least one class of organism, but can be transfected into another organism for expression. For example, a vector is cloned in an E. coli cell and then the same vector is transfected into yeast cells or mammalian cells for expression, although it is unable to replicate independently of the host cell chromosome.

DNA can also be amplified by adding it to the host genome. This is readily accomplished using Bacillus species as hosts, for example by including in the vector a DNA sequence complementary to the sequence found in Bacillus genomic DNA. Transfection of Bacillus with this vector results in homologous recombination with the genome and insertion of mpl ligand DNA. However, recovery of genomic DNA encoding mpl ligand is multiplicable. : more complex than the exogenously replicated vector • ·· • * ...

recovery because restriction enzyme digestion is required • · »*. . mpl ligand DNA.

• ·· *. * (Iii) Selection gene component • j · * Expression and cloning vectors should contain the · · · · * * selection gene, also called the strain marker. This gene encodes a protein essential for the survival or growth of transformed::: host cells grown in a selective medium. Isantasolut, ^ .j ·. which have not been transformed with a vector containing the select gene will not survive in the medium. Typical val: genes encode proteins that (a) confer resistance to antibiotics and other toxins, e.g.

ampicillin, neomycin, methotrexate or tetracycline 114? (b) complement auxotrophic deficiencies; or (c) provide critical nutrients that are not available from the complex medium, e.g., a gene encoding D-alanine racemase for Bacillus.

In one example of a selection scheme, a drug is used to stop growth of a host cell. Cells that have been successfully transformed with a heterologous gene will express a protein that produces drug resistance and thus maintains the selection system. Examples of such a dominant selection include neomycin [Southern et al., J. Molec. Appl. Genet., 1: 327 (1982)], mycophenolic acid (Mulligan et al., Science, 209: 1422 (1980)) or hygromycin (Sugden et al., Mol. Cell. Biol., 5: 410-413 (1985)]. In these three examples given above, bacterial genes are used under eukaryotic control to mediate resistance against the drug G418 or neomycin (geneticin), xgpt (mycophenolic acid po) or hygromycin.

Examples of other suitable selectable markers in the case of mammalian cells include markers that allow the identification of cells capable of uptake of the mpl ligand nucleic acid, such as di-hydrofolate reductase (DHFR) or thymidine kinase. Ni- • ·; ·. the transformed cells in the sack are subjected to a selection pressure that only the transformed cells are uniquely modified to withstand because of their I:: ** uptake by the marker. The selection pressure is determined by culturing the transformed cells under conditions where the concentration of the selection agent in the medium is sequentially altered, resulting in amplification of both the selection gene and the DNA encoding the mpl ligand polypeptide. Reproduction is a process by which. ···. genes that have a greater need for growth in the production of critical protein • · · · * in successive generations of recombinant cells. Larger amounts of mpl ligand are synthesized from the amplified DNA.

115

For example, cells transformed with the DHFR-selectant gene are first identified by culturing all transformed cells in medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When using wild-type DHFR, a suitable host is the Chinese Hamster Ovary (CHO) cell line, which lacks DHFR activity and is prepared and propagated as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). The transformed cells are then exposed to increasing amounts of Mtx. This results in the synthesis of multiple copies of the DHFR gene and simultaneous multiple copies of other DNA containing Express expression vectors, such as DNA encoding the mpl ligand. This amplification technique can be used in the case of any other suitable host, e.g. ATCC No. CCL61 in the case of a CHO-K1 host, despite the presence of endogenous DHFR, for example, when using a mutant DHFR gene which is highly resistant to Mtx (EP Patent 117,060). Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) that have been transformed or co-transformed with mpl · · *: ligand, wild-type DHFR protein or other selectable marker, such as aminoglycoside-3. -phosphotransfera- • ·; ·. sin (APH), encoding DNA sequences, can be selected • ·· *. · According to cell growth in culture medium containing selective

«I * f J

♦ ·· a viable marker selectant such as aminoglycates:; ** a cosidic antibiotic, e.g., kanamycin, neomycin or

I · J

· '* G418: n. See U.S. Patent 4,965,199.

A suitable selection gene for use in yeast is the trp1-1 gene present in the 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 produces a selection marker for a yeast mutant strain that lacks the ability to grow in tryptophan, as exemplified by strain: * · / ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85: 12 (1977)].

The presence of the Trp1 lesion in the yeast host cell genome then provides 116 an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC No. 20,622 or 38,626) have been amplified using known plasmids carrying the Leu2 gene.

(iv) The promoter component

Expression and cloning vectors usually contain a promoter recognized by the host organism and operably linked to the mpl ligand nucleic acid. The promoters are untranslated sequences located above (5 ') the start codon of the structural gene (usually about 100-1000 bp) and control a particular nucleic acid sequence such as the mpl ligand nucleic acid sequence, is operationally connected. Such promoters are typically divided into two classes, inducible and constitutive. Inducible promoters are promoter pathways that initiate increasing transcription of DNA under their control in response to a change in culture conditions, e.g., a change in the presence or absence of a nutrient, or a change in temperature. At present, hy-: * i '; a large number of promoters of different possible • · /. : Host cells recognize. These promoters are operatively linked to the mpl ligand-encoding DNA by deleting * ·· *. . promoter from source DNA by restriction enzyme digestion * · ♦ *. * and insertion of the isolated promoter sequence into the vector · * · *. Both the native mpl ligand promoter sequence / 5 * · '* and many heterologous promoters can be used to direct the amplification and / or expression of mpl ligand DNA. However, heterologous promoters are preferred because they generally allow higher transcription and higher yields of the expressed mpl ligand compared to the native mpl ligand promoter i: *; * promoters which are suitable for use in the case of * * *; * / prokaryotic hosts, the β-lactase? '**! land and lactose promoter systems are included [Chang et al., 117

Nature, 275: 615 (1978); and Goeddel et al., Nature, 281: 544 (1979)], a basic phosphatase promoter system, a tryptophan promoter system (trp-proto-motor system) [Goeddel, Nucleic Acids Res., 8: 4057 (1989), and 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. allows those skilled in the art to operably link them to DNA encoding the mpl ligand (Siebenlist et al., Cell, 20: 269 (1980)) using linking fragments and adapters to produce any necessary restriction sites, and promoters for use in bacterial systems also include Shine-Dalgarno (SD sequence) operably linked to DNA encoding the mpl ligand polypeptide.

For eukaryotes, promoter sequences are known. Virtually all eukaryotic genes have an AT-rich region located about 25-30 bases upstream from where transcription begins. Another sequence observed at 70 and 80 bases upstream of many

j'J *. gene transcription start site, is the CXCAAT region where X

* ·, ·. : can be any nucleotide. For most eukaryotic • ·· «· '. there is an AATAAA sequence at the 3 'end of the genes, which may be a signal signal. . 3 'end of the coding sequence to insert a li poly A tail ♦ * «* he. All of these sequences are suitably inserted into eukaryotic expression vectors.

• **! * - * Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. ? * i t Chem. , 255: 2073 (1989)] 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 dehydro-1M-genease, hexochemase, pyruvate decarboxylase , phosphofruk-} ***! toxinase, glucose-6-phosphate isomerase, 3-phospho- * 118 glycerate mutase, pyruvate kinase, triosephosphate isoxerase, phosphoglucose isomerase and glucokinase.

Other yeast promoters, which are inducible promoters and have the additional advantage of growth-controlled transcription, include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, nitrogen metabolite-degrading enzymes, and promoter regions of the enzymes responsible for the utilization of maltose and galactose. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al., European Patent 73,675. Yeast enhancers are also preferably used with yeast promoters.

For example, transcription of the Mpl ligand from vectors in mammalian host cells is controlled by promoters derived from viral genomes such as polyomavirus, fowlpox virus (UK Patent 2 211 504, published July 5, 1989), adenovirus (such as Adenovirus 2), bovine animal. papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferably

Simian Virus 40 (SV40) genomes, heterologous ni *. sack promoters, e.g., actin promoter or * ·, ·,: immunoglobulin promoter, heat shock promoter, \ ·: ..... .

and a promoter normally associated with the mpl ligand * ·· *,. sequence, provided that such promoters are f ·.: *, * compatible with the host cell system.

g * ·; · * 'The early and late promoters of SV40 virus t J · / * are conveniently obtained as a restriction fragment of SV40 which also contains the SV40 virus replication initiation site i. Fiers et al., Nature, 273: 113 (1978); Mulligan and Berg, Science, 209: 1422-1427 (1980)? Pavlakis et al., .1. Proc. Natl. Acad. Sci. USA, 78: 7398-7402 (1981). Human «» t., The immediate early promoter of the cytomegalovirus, is provided by: *; * suitably HindIII E as a restriction fragment.

Greenaway et al., Gene, 18: 355-360 (1982). A system for expressing DNA in mammalian hosts using bovine 119 papillomavirus as a vector is disclosed in U.S. Patent 4,419,446. A variant of this system is described in U.S. Patent 4,601,978. See also Gray et al., Nature, 295: 503-508 (1982). regarding the expression of immune interferon-encoding cDNA in monkey cells; Reyes et al., Nature, 297; 598-601 (1982) for expression of human interferon beta cDNA under control of a thymidine kinase promoter derived from the herpes simplex virus; Canaani and Berg, Proc. Natl. Acad. Sci. US, 79: 5166-51570 (1982) regarding expression of the human interferon B1 gene in cultured mouse and rabbit cells; 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 murine NIH-3T3 mouse using Rous's sarcoma repeat to a promoter.

(v) Enhancer Element Component Transcription of the mpl ligand encoding DNA of this invention by higher eukaryotes is often increased by introducing the enhancer sequence into a vector. Enhancers are elements in the cis-position of DNA that are • ·. ·. : usually about 10-300 base pairs and acting on the · · ·. • t * promoter to increase its transcription. Boosters • · · *. . are relatively independent of orinination and position • · · * ·. * and have been found to be 5 '[Laimins et * ·: ·' et al., Proc. Natl. Acad. Sci. USA, 78: 993 (1981)] and 3 '· * * * [Lusky et al., Mol. Cell Bio., 3: 1108 (1983)] with respect to a transcription unit in an intron [Banerji et al., Cell, 33: 729 (1983)] and in the coding sequence itself [Osborne: * · *: et al., Mol. Cell Bio., 4: 1293 (1984)]. Many enhancers are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).

However, an enhancer from a eukaryotic cell virus is typically used. Examples include SV40-: ***: enhancer on the late side of the replication origin (bp 100-270), late promoter of the cytomegalovirus, late promoter of the polyomatehost replication origin, and adenovirus enhancers. See also Yaniv, Nature, 297: 17-18 (1982) for enhancer elements for activating eukaryotic promoters. The enhancer may be inserted into the vector at position 5 'or 3' to the coding sequence for the mpl ligand, but preferably is located 5 'to the promoter.

(vi) Transcription Termination Component Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nuclear cells from other multicellular organisms) also contain the sequences necessary for transcription termination and mRNA. Such sequences are commonly available from the 5 'and randomly 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions include nucleotide segments that are copied as polyadenylated fragments in the non-translated portion of the mpl ligand encoding mRNA.

(vii) Construction and Analysis of Vectors

Suitable vectors containing one or more of the above-listed components are: · · standard ligation technique. Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated to form the plasmids required in the desired form.

• t · • · ·

For the purpose of displaying the correct sequences for analysis in the plasmids formed, the linkers are used to transform E. coli K12 strain 294 (ATCC No. 31,446) and successful transformants are selected for ampicillin resistance or tetracycline resistance, where appropriate. ; ·. compound. Transformants are prepared from plasmids, ani / i ···. lysing them by restriction endonuclease digestion and / or sequencing using the method described in Messing et al., Nucleic Acids Res., 9: 309 (1981), 9 9, or is described in Maxam et al., Met 121 hods in Enzymology, 65: 499 (1980).

(viii) Temporary Expression Vectors Especially useful in the practice of this invention are expression vectors that allow the temporary expression of DNA encoding the mpl ligand polypeptide in mammalian cells. Generally, transient expression involves the use of an expression vector which, when replicated, is capable of efficiently replicating in a host cell such that the host cell collects multiple copies of the expression vector and in turn synthesizes large amounts of the desired polypeptide encoded by the expression vector. Sambrook et al., Supra, pages 16.17-16.22. Temporary expression systems comprising a suitable expression vector and a host cell allow for the appropriate positive identification of polypeptides encoded by the cloned DNA molecules and for the rapid screening of such polypeptides for the desired biological or physiological properties. Thus, transient expression systems are particularly useful in the invention for identifying analogs and variants of the mpl ligand polypeptide having the biological activity of the mpl ligand polypeptide.

• (ix) Suitable exemplary vertebrate cell vectors Other methods, vectors, and host cells that are: suitable for adaptation to mpl ligand synthesis by recombinant. . ·. in vertebrate cell culture, are described in • · ·

Vy sa Gething et al., Nature, 293: 620-625 (1981); Mantei et al., ♦ ♦ * et al., Nature, 281: 40-46 (1979); Levinson et al., EP Patent 117,060 and EP Patent 117,058. A particularly useful plasmid for expressing mpl ligand in mammalian cell culture is pRK5 (EP patent). 307,247, U.S. Patent 5,258,287) or pSVI6B (PCT Publication WO 91/08291).

, ···. D. Host Cell Selection and Transformation * ·

Suitable host cells for cloning and expression of vectors here include prokaryotic cells, yeast cells, and higher · eukaryotic cells described above.

122

Suitable prokaryotes include eubacteria such as Gram-negative or Gram-positive organisms, for example E. coli, Bacillus bacteria such as B. subtilis, Pseudomonas species such as P. aeruginosa, Salmonella typhimurium or Serratia marcescans. One preferred E. coli clone host is E. coli 294 (ATCC No. 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC No. 31,537) and E. coli W3110 (ATCC No. 31,437) are preferred. 27,325) are suitable. These examples are illustrative rather than limiting. Preferably, the host cell should secrete minimal amounts of proteolytic enzymes. Alternatively, in vitro cloning methods, e.g., PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for mpl-ligand-encoding vectors. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used among the lower eukaryotic host microorganisms. However, several other genera, species, and strains are generally available and useful herein, such as Schizosaccharomyces pombe [Beach and Nurse, Nature, 290: 140 (1981); EP Patent 139! '/ 383, published May 2, 1985), Kluyveromyces-h. (U.S. Patent 4,943,529), such as K. lactis [Lou- vencourt et al. ., J. Bacteriol., 737 (1983)], K. fragilis, K.

. ·. : bulgaricus, K. thermotolerans and K. marxianus, Yarrowia (EP 402 226), Pichia pastoris (EP 183 070; ♦ · ♦

Sreekrishna et al., J. Basic Microbiol., 28: 265-278 t: * (1988)], Candida, Trichoderma reese (EP 244 234), in a batch of Neurospora [Case et al., Proc. Natl. Acad. Sci. USA, 76; 5259-5263 (1979)] and filamentous fungi, such as, for example, the Neurospores, Penicillium, Tolypocladium (WO 91/00357, published 10).

January, 1991) and Aspergillus hosts such as A. nidulans ···. [Ballance et al., Biochem. Biophys. Res. Commun. , 112: 284-2-29 (1983); Tilburn et al., Gene, 26: 205-221 (1983); · · ·: V Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 (1984) and A. niger (Kelly and Hynes, EMBO J., 4: 475-479 123 (1985) 3).

Suitable host cells for expression of the glycosylated mpl ligand are derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activity. In principle, any culture of a k9r-more eukaryotic cell is useful, whether it is a vertebrate or an invertebrate cell culture. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants and corresponding optional insect host cells from hosts have been identified, such as Spodoptera frugiperda (butterfly larvae), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogas-ter (fruit fly). See, e.g., Luckow et al., Bio / Technology, 6: 47-55 (1988); Miller et al., Genetic Engineering, editors Setlow et al., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature, 315: 592-594 (1985). A variety of virus strains for transfection are publicly available, e.g., Ll variant of Auto-grapha californica NPV and Bm-5 strain of Bombyx Mori NPV, and such viruses may be used herein as a virus in accordance with the present invention, particularly Spodoptera; \ j for transfection of frugiperda cells.

• · ^ Cotton, corn, potato,, · can be used as hosts. ; plant cell cultures of soybean, petunia, tomato and tobacco. Typically, plant cells are transfected by incubation with certain strains of Agrobacterium tumefaciens 2 · · · 'previously manipulated to contain mpl-ligand DNA. During incubation of the plant cell «

s.: ..: DNA encoding mpl ligand with A. tumefaciens

V: transferred to the plant cell host for transfection. and expressing mpl ligand DNA under appropriate conditions. Li- • · · ···. In addition, regulatory and signal sequences are available that are capable of activating and increasing transcriptional levels of genes expressed in plant tissue containing recombinant DNA, which are isolated from the region of the T-124 T-DNA 780 gene. European Patent 321,196, issued June 21, 1989.

However, interest has been most focused on vertebrate cells and the addition of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, Academic Press, edited by Kruse and Patterson, (1973)). Examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol., 3: 6: 59 (1977); hamster chick kidney cells (BHK, ATCC CCL 10); ovarian cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)); mouse Sertolin cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)). ]; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human target: ** [: 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; sasolut (Hep G2, HB 8065); mouse mammary tumor (MTT 060562, · · · ATCC CCL51); TRI cells [Mather et al., Annals N. Y. Acad.

t i ·

Sci., 383: 44-68 (1982)]; MRC 5 cells; FS4 cells and s: • Human liver tumor line (Hep G2).

The host cells are transfected, and preferably transformed, with the expression or cloning vectors described above of this invention and cultured in standard nutrient medium modified if appropriate. ·. such as to induce promoters, select the transformants, or select the gene to encode the desired sequences • · · ♦ · · ♦. .

:. * to duplicate.

··· s ...: Transfection means that the host cell takes up 125 expression vectors whether or not the coding sequences are actually expressed. Numerous methods of transfection are known to those skilled in the art, such as the CaPO 4 method and electroporation. Successful transfection is generally recognized when any indication of the activity of this vector occurs in the host cell.

Transformation refers to the introduction of DNA into an organism such that the DNA is capable of replication, either as an extrachromosomal element or as a chromosomal entity. Depending on the host cell used, the transformation is performed using standard techniques suitable for the cells. Calcium treatment using calcium chloride as described in Sambroo et al 1.82. in the above work is generally used in the case of prokaryotes or other cells containing significant cell wall barriers. Agrobacterium tumefaciens infection is used to transform certain plant cells as described in Shaw et al., Gene, 2 3: 315 (1983) and WO 89/05859, published June 29, 1989. In addition, the plants can be transfected using ultrasound treatment such as is described in WO 91/00358, published Jan. 10, 1991. In the case of mammalian cells lacking such cell walls, the · phosphate precipitation method described in

Graham and van der Eb, Virology, 52: 456-457 (1978), are preferred. General considerations regarding transformations of mammalian host cell systems are disclosed in Axel, U.S. Pat. No. 4,399,216, issued August 16, 1983.

Transformations into yeast are typically carried out according to the method J · · ··· * described by Van Solingen et al., J.

• · · * Bact. , 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad.

Sci. (USA), 76: 3829 (1979). However, other, ·· * can be used. coat methods for introducing DNA into cells such as injection, electroporation, or protoplast fusion.

E. Culture of Host Cells ··· 8 ... · Prokaryotic cells used to produce the mpl ligand polypeptide of the present invention are cultured in appropriate media as described generally by Sambrook et al. in the book, above.

The mammalian host cells used to produce the mpl ligand of this invention can be cultured in various 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 culturing host cells. In addition, any of the media described in Ham and Wallace, Meth, may be used as culture media for host cells. Enz., 58: 44 (1979), Barnes and Sato, Anal. Biochem., 102: 255 (1980), U.S. Patents 4,767,704; 4,657,866; 4,927,762 or 4,560,655; WO 90/03430; WO 87/00195; U.S. Patent 30,985; or U.S. Serial No. 07 / 562,107 or 07 / 562,141, 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 (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, · · · · •, calcium, magnesium and phosphate), buffers. (such as HEPES), nucleosides (such as adenosine- and thymidine), antibiotics (such as Gentamycin ™ -. * tj drug), trace elements (defined as inorganic compounds which are usually present in final concentrations in the micromolar range) and glucose or s.

• the corresponding energy source. Any other necessary supplements may also be included in suitable concentrations known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those used above for expression of the host cell selected for expression and will be apparent to those skilled in the art.

The host cells referred to in this publication include cells in in vitro culture and cells in · · · 5 ... in the host animal.

127 F. Detection of gene amplification / expression

Amplification and / or expression of a gene can be directly measured from a sample, for example, using a conventional Southern blotting method or a Northern blotting method to determine the amount of mRNA transcription [Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)], dot blotting (DNA analysis) or in situ hybridization using a suitably labeled probe based on the sequences provided herein. Various labels may be used, most commonly radioisotopes, especially 32P. However, other technical methods can be used, such as biotin-modified nucleotides for insertion into a polynucleotide. Biotin then acts as a binding site for avidin or antibodies which may be labeled with a wide variety of labels such as radionuclides, fluorescent agents, enzymes or the like. Alternatively, antibodies that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes, may be used. In turn, the antibodies may be labeled and an assay may be performed in which the duplex is bound to the surface such that upon the formation of the duplex on the surface, the presence of antibody bound to the duplex can be detected.

• · · * ·,. Alternatively, gene expression may be measured using immunological methods such as tissue sections. immunohistochemical staining and analysis of cell culture or body fluids to directly quantify the expression of the gene product. Using an immunohistochemical staining technique, a cell sample is prepared by drying and fixing the cell specimen and then reacting the product with labeled antibodies φ * »* · · · · · · ·. with specificity for the linked gene product * x * ··. whereby the labels are usually visually detectable, such as enzymatic labels, fluorescent labels, · »: luminescent labels and the like. A particularly sensitive dyeing ankle suitable for use in the present invention is described in Hsu et al., Am. J. Clin. Path., 75: 734-738 (1980).

Antibodies useful for immunohistochemical staining and / or analysis of sample fluids may be either monoclonal or polyclonal and may be prepared in any mammal. Conveniently, antibodies can be made against the native mpl ligand polypeptide or against a synthetic peptide based on the DNA sequences produced herein as described below.

6. Purification of the Mpl ligand polypeptide

The Mpl ligand is preferably recovered as a polypeptide secreted from the culture medium, although it can also be recovered from the lysis products of the host cell when directly expressed in the absence of a secretion signal.

When the mpl ligand is expressed in a recombinant cell other than a human cell, the mpl ligand is completely free of human-derived proteins or polypeptides. However, it is usually still necessary to purify the mpl ligand from other recombinant cellular proteins or polypeptides in order to obtain preparations which are substantially homogeneous with respect to the mpl-ligand itself. As a first step, the culture medium or cell lysis product is centrifuged to remove finely divided cell debris. Then, the membrane fraction and the soluble protein fraction are separated. Alternatively, a commercially available t I i * protein enrichment filter (e.g., Amicon or Millipore Pellicon ultrafiltration units) may be used. The mpl ligand is then purified from the *** · V · fraction and the membrane fraction of the soluble protein of the culture lysis product, depending on whether mpl- * t *. ligand membrane bound. The mpl ligand is then purified from the contaminating soluble proteins and poly1 * '* peptides by salting and chromatography or chromatography using various gel matrices. These matrices include acrylamide, agarose, dextran, cellulose, and others commonly used in protein purification. Exemplary chromatographic methods suitable for protein purification include immunoaffinity (e.g., anti-hmpl ligand Mab), receptor affinity (e.g. mpl-IgG or protein A-Sepharose), hydrophobic interaction chromatography (HIO) (e.g. ether), e.g. or phenyl-Toyopearl), lectin chromatography (e.g. Con A-Sepharose, lentil-lectin-Sepharose), size exclusion (e.g. Sephadex G-75), cation and anion exchange columns (e.g. DEAE or carboxymethyl or sulfopropyl cellulose), and reverse phase high performance liquid chromatography (RP-HPLC) [see, e.g., Urdal et al., J. Chromatog., 296: 171 (1984), using two consecutive RP-HPLC steps to purify recombinant human IL-2]. Other purification steps may optionally include ethanol precipitation; ammonium sulfate precipitation; chromatofocusing; preparative SDS-PAGE and the like.

Mpl ligand variants, which have been removed or added or substituted with residues, are recovered in the same manner as the native mpl ligand, taking into account any essential variations caused by variation; 1 · 1; set properties changes. For example, the preparation of a fusion of mpl ligand with another protein or polypeptide, e.g.

* · With bacterial or viral antigen, to facilitate purification; an immuno-affinity column containing an antibody to the antigen may be used to adsorb the fusion polypeptide. Immunoaffinity columns, such as rabbit dust from a clonal anti-mpl ligand column, can be used to absorb the mpl ligand variant by binding to at least one of the remaining immune epitope. Alternatively, «Il V! The mpl ligand can be purified by affinity chromatography. using purified mpl-IgG coupled to 4 <· Λ. , »· '. (preferably) to an immobilized resin such as Affi-Gel • 1010 (Bio-Rad, Richmond, CA) or the like, well within the art. by known means. A protease inhibitor such as phenylmethyl sulphate ························································ING Prot Prot 130 kuten kuten kuten kuten kuten kuten 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 35 35 30 30 30 45 45 One skilled in the art will appreciate that purification methods suitable for purifying native mpl ligand may require modification to account for changes in the nature of the mpl ligand or variants thereof upon expression in recombinant cell culture.

H. Covalent Modifications of Mpl Ligand Polypeptide Covalent variants of Mpl ligand polypeptides are encompassed within the scope of this invention. Both native mpl-li-Gand and mpl-ligand amino acid sequence variants can be covalently modified. One type of covalent modification which is included within the scope of this invention is the mpl ligand diffraction. Various mpl ligand fragments having up to about 40 amino acid residues may be conveniently prepared by enzymatic or chemical synthesis or by chemical cleavage of the full or variant mpl ligand polypeptide. Other types of covalent conversion of the mpl ligand or fragments thereof are made by reacting the targeted amino acid residues of the mpl ligand or fragments thereof with an organic derivative which is capable of reacting with selected side chains of N * or C terminus residues. ·. : with.

• · · • ·; ·. Most commonly, cysteine residues are reacted with · · · *. . earth-α-haloacetates (and corresponding amines) such as chloro · · · *. * acetic acid or chloroacetamide to provide carboxymethyl • · · * · · · * or carboxyamidomethyl derivatives. Derivatives of cysteinyl residues are also formed by reacting them with bromotrifluoroacetone, α-bromo-β- (5-imidazoyl-β) -propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl-2-pyridyl ···. disulfide, p-chloro-mercury (2) benzoate, 2-chloro-mercapto (2) -4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3 * x * diazole.

Derivative of Histidyl Residues • · · • *.

• M

131 by reacting with diethyl pyrocarbonate at pH 5.50-7.0 because this substance is relatively specific for the histidyl side chain. Parabromophenacyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

The lysinyl and amino terminated residues are reacted with succinic anhydride or other carboxylic acid anhydrides. The formation of derivatives with these agents has the effect of reversing the charge of lysinyl derivatives. Other suitable reagents for forming derivatives of amino-containing residues include imidoesters, such as methyl picolinimidate; doksaalifosfaatti-view; pyridoxal; chloroborohydride; Triniti robentseenisulfonihappo; O-methylisourea; 2,4-pentanedione-ni; and trans-aminase catalyzed reaction with glyoxylate.

The arginyl residues are converted by reaction with one or more conventional reagents such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. The formation of derivatives of arginine residues requires the reaction to be carried out under basic conditions due to the high pKa value of the functional group of the Gaucidine. In addition, these reagents may react with lysine. and the epsilon amino group of arginine.

• · · ··, specific conversion of tyrosyl residues can be · · · *. . is particularly advantageous when adding spectral labels with • · · 132 imides (RN = C = N-R ′) wherein R and R ′ are different alkyl groups such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or with 1-ethyl-3- (4-azonia-4,4-dimethyl-lipentyl) -carbodiimide. In addition, the aspartyl and glutamyl residues are converted to the asparaginyl and glutaminyl residues by reaction with ammonium ions.

Formation of derivatives with bifunctional agents is useful for crosslinking the mpl ligand to a water-soluble support matrix or surface for use in a method for purifying anti-mpl ligand antibodies and vice versa. Commonly used cross-linking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g. 31-dithiobis (succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents, such as methyl 3 - [(p-azidophenyl) dithio] propioimidate, produce light-activated intermediates that are capable of forming bridges in the presence of light. Alternatively .. . • · · • · * reactive with water are used to immobilize proteins. *. insoluble matrices such as cyanogen bromide activated carbohydrates and reactive substrates, which are described in U.S. Patent 3,969,287; 3,691,016; 4,195,128; 4 • · 247,642; 4,229,537 and 4,330,440.

♦ Glutaminyl and asparaginyl residues are deamidated: often to the corresponding glutamyl and aspartyl residues.

These residues are deamidated in neutral or basic. conditions. The deamidated form of these residues is included in the · · ·. ·; ·. within the scope of the present invention.

• · ·

Other variations include hydroxylation of proline and lysine • · · · · · *, phosphorylation of hydroxyl groups of the seryl and threonyl moieties, methylation of the α-amino groups of the lysine, arginine and histidine side chains [T. E. Creigh- • · · ♦ · • • • · ··· 133 ton, Proteins: Structure and Molecular Properties, W. H..

Freeman & Co., San Francisco, pp. 79-86 (1983)] acetylation of an N-terminal amine and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the mpl ligand poly peptide within the scope of this invention involves altering the native glycosylation pattern of the polypeptide. Modification means removing one or more carbohydrate moieties found in native mpl ligand and / or adding one or more glycosylation sites not present in native mpl ligand.

Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of the asparagine residue. The tripeptide sequences asparagine-X-serine and Asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences by which the carbohydrate moiety is enzymatically attached to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylate refers to the attachment of one N-acetylgalactosamine, galactose or xylose sugar · '· *: to a hydroxyamino acid, most commonly serine • ·. * ·., Or threonine, although 5-hydroxy-· proline or 5-hydroxylysine may also be used .

• · · /. : Addition of glycosylation sites to the mpl ligand polypeptide is conveniently accomplished by altering the amino acid sequence of • · · · · *** to include one or more of the tripeptide sequences described above (for the N-linked glycosylation sites). ). The conversion can also be accomplished by adding one or more serine or threo [beta]: V residues to the native mpl ligand sequence or by substitution (O-linked glycosylation sites). To facilitate this, the amino acid sequence t · '! * Of the mpl ligand is preferably altered through changes in the DNA level, in particular by mutating the DNA encoding the mpl ligand polypeptide for · · · · · · 134 selected bases. DNA coding mutation (s) can be made using the methods described above under the heading "Amplic acid sequence variants of the Mpl ligand".

Another way to increase the number of carbohydrate moieties in the mpl ligand is by chemical or enzymatic coupling of glycosides to the polypeptide. These methods are advantageous in the sense that they do not require the production of a polypeptide in a host cell having glycosylation capacity to effect N- or O-linked glycosylation. Depending on the coupling pattern used, the sugar (s) may be linked to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, (c) e) aromatic residues such as aromatic residues of phenylalanine, tyrosine or tryptophan; or (f) an amide group of glutamine. These methods are described in WO 87/05330, published September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981).

Removal of the carbohydrate moieties present in the Mpl ligand polypeptide can be accomplished chemically or • ·. ·. : enzymatically. Chemical deglycosylation requires • · ·. exposing the polypeptide to trifluoromethane sulfate. . phonic acid compound or the like. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), whereby the polypeptide remains intact. Chemical deglycosylation is described in Hakimuddin et al., Arch. Biochem.

: Biophys., 259: 52 (1987) and Edge et al., Anal. Biochem., # ···. 118: 131 (1981). Enzymatic cleavage of the carbohydrate moieties of the polypeptides can be accomplished using a variety of methods. Endo and exo-glycosidases as described in Thotakura et al., Meth. Enzymol., 138: 350 (1987).

• · · • · 135 •

Glycosylation at potential glycosylation sites can be prevented using a tunicamycin compound as described in Duskin et al., J. Biol. Chem., 257: 3105 (1982). Tunicamycin prevents the formation of protein N-glycosidic bonds.

Another type of covalent modification of the mpl ligand comprises coupling the mpl ligand polypeptide to a variety of non-proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, as disclosed in U.S. Patents 4,640,835; 4,496,689; 4,310,144; 4,670,417; 4,791,192; or 4,179,337. Mpl ligand polypeptides covalently linked to the above polymers are referred to herein as pegylated mpl ligand polypeptides.

It will be appreciated that some sort of screening for the recovered mpl ligand variant is required to select the optimal variant which binds to the mpl and has the immunological and / or biological activity as defined above. Stability in recombinant cell culture or plasma (e.g., proteolytic cleavage), high affinity for the mpl member, oxidative stability, ability to excrete in high yields and the like can be screened. For example, a change in the immunological nature of the mpl ligand polypeptide, such as affinity for a particular antibody. is measured in a competition type specification. Other possible variations in the properties of the protein or polypeptide, such as oxidation-reduction resistance or heat resistance, hydro-: phobia or susceptibility to proteolytic degradation, are determined using methods well known in the art. methods.

17. General Methods for the Preparation of Antibodies to mpl Ligand *:: · ♦ · JJ · Preparation of Antibody (i) Polyclonal Antibodies · * · · * ... φ Polyclonal Antibodies mpl Ligand Polypeptides · T's or fragments thereof are usually formed in animal * ♦ · ♦ V by multiple injections of mpl ligand and adjuvant by multiple subcutaneous (se) or ··· ♦ · • · ··· 136 intraperitoneal (ip) injections. It may be advantageous for the mpl ligand or fragment containing the target amino acid sequence to be conjugated to a protein which is immunogenic in the species to be immunized, e.g., key hole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, a derivative forming agent, for example maleimidobenzoylsulphosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), Glu-taraldehyde, succinic anhydride, SOCl2 or R1N = C1-NR1.

Animals are immunized against mpl ligand polypeptide or fragment, immunogenic conjugates or derivatives by combining 1 mg or 1 μg peptide or conjugate (for administration to rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution through multiple sites. One month later, the animals are boosted with 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant through multiple subcutaneous injections. Then 7-14 days later the animals are bled and the antibody titer of mpl ligand is determined from the serum.

Animals are given a booster vaccine until the titer is equal. ·:; ·. Tuu. Preferably the animals are boosted with the same mpl- «* · * ·. j ligand conjugate, however, conjugated via a different protein 'and / or a different crosslinking reagent. Conjugates * · · can also be prepared in recombinant cell culture as protein fusions. Aggregating agents such as alum are also used to increase the immune response.

(ii) Monoclonal Antibodies «* · * Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, namely, the individual antibodies which make up the population of« »· * .. t are identical in nature except for * 1 *. mutations that may be present in minor amounts. Thus, the quantum monoclonal indicates the nature of the antibody so that it is not a mixture of separate antibodies.

For example, monoclonal antibodies to the mpl ligand of this invention may be prepared using the hybridoma method first described by Kohler & Milstein, Nature 256: 495 (1975), or may be made using recombinant DNA techniques (U.S. Patent 4,816). 567 (Cabilly et al.).

In the hybridoma method, a mouse or other suitable host animal such as a hamster is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used to produce the immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma swabs so prepared are inoculated and grown in a suitable medium, preferably containing one or more agents that inhibit the growth and survival of non-fused, parenteral myeloma cells. For example, if parental myeloma cells "i · lack the enzyme hypoxanthine guanine phosphoribosyl transferase, · rase (HGPRT or HPRT), the hybridoma medium contains * * · typically hypoxanthine, aminopterin and thymidine *" * * '* (HAT-inhibiting medium) growth of defective cells »# · * '* *.

Preferred myeloma cells are cells that fuse efficiently, support stable, efficient expression of the antibody of selected antibody producing cells. levels and are sensitive to media such as HAT media. Among these, preferred myeloma cell lines are i · V rat myeloma lines, such as lines derived from • i *; * / sin MOPC-21 and MPC-II mouse tumors and are available • • 9 «* • * n * 138

Salk Institute Cell Distribution Center, San Diego, California, USA; and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland, USA. A human myeloma cell line and a mouse-human heteromyeloma cell line 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, pages 51-63, Marcel Dekker, Inc., New York, 1987].

From the medium in which the hybridoma cells are grown, the production of monoclonal antibodies directed against the mpl ligand is determined. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can be determined, for example, using Munson & Pollard's Scatchard analysis, Anal. Biochem., 107: 220 (1980).

After hybridoma cells have been identified that produce antibodies with the desired specificity, · · *. 1. affinity and / or activity, clones can be subcloned • · · *; '1 using restrictive dilution methods and culturing using the standard methods (Goding, supra). This is what • ·:. 1: Mediums suitable for mixing include, for example, Dulbecco's Modified Eagles Medium or • · · 'RPMI-160 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in an animal.

; Monoclonal antibodies secreted by subclones • · ·. • f. is conveniently separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxylation, gelatin electrophoresis, dialysis or affinity. purity chromatography.

• ·

. ···. DNA encoding the monoclonal antibodies of the invention

139 are readily isolated and sequenced using conventional techniques (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies. The hybridoma cells of the invention serve as a preferred source of such DNA. is isolated, it can be inserted into expression vectors which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells which otherwise do not produce immunoglobulin protein in the production of recombinant monoclonal antibodies. can also be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains for rat homologous sequences (Cabilly et al., Morrison et al., Proc. Nat. Acad. Sci., 81: 6851 (1984)) or by covalently linking. i an immunoglobulin coding sequence, all or part of the non-immunoglobulin polypeptide coding sequence.

Typically, such non-immunoglobulin polypeptides are substituted for the standard dosages of the antibody of the invention or are substituted for the antibody of the invention. *. one antigen junction site for variable domains • · · * · to generate a chimeric divalent antibody, · · · ** containing one antigen junction site with • · mpl ligand specificity and another antigen junction site with different specificity antigen.

t #:: Chimeric antibodies or hybrid antibodies can also be prepared in vitro using synthetic pro. ; *: methods known in the field of teen chemistry, including suc- •. methods for crosslinking. For example, immunotoxins may be formed using a disulfide exchange reaction or by forming a thioether bond. Examples of reagents suitable for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate.

• ·. ···. For diagnostic applications, the antibodies of the invention are typically labeled with a detectable moiety. The detectable portion may be any portion which is capable of producing, directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope such as 3H, UC, 32P, 35S or 125I, a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, Rhodamine or luciferin; radioactive isotopic labels such as 125 I, 32 P, UC or 3 H, or an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for conjugating the antibody to a detectable moiety may be used, including those described in Hunter et al., Nature 144: 945 (1962); David et al., Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40: 219 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407 (1982).

The antibodies of this invention can be used in any assay method such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies; A Manual of Techniques, pages 147-158 (CRC Press, Inc., .... 1987).

• · · '• · *. Competitive binding assays are based on the ability of the labeled standard (which may be the mpl ligand or its immunologically reactive moiety) to compete with the test sample. *: binding of a limited amount of antibody to the product to be analyzed (mpl ligand). The amount of mpl ligand in the test sample is inversely proportional to the amount of standard that binds to the antibodies. Commit. • · ·. ·; ·. substances are generally rendered insoluble before or after competition so that the antibody-bound standard and the product to be analyzed can be suitably distinguished from the unbound standard and the product to be analyzed.

;. · ·. The sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic moiety or epitope of a detectable protein (mpl ligand). In the sandwich assay, the product to be analyzed is bound by a first antibody immobilized on a solid support, and then a second antibody bound to the product to be analyzed to form an insoluble tripartite complex. David & Greene, U.S. Patent 4,376,101. The second antibody may itself be labeled with a detectable portion (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody labeled with a detectable portion (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay in which the detectable portion is an enzyme (e.g. horseradish peroxidase).

(iii) Humanized and Human Antibodies Methods for humanizing non-human antibodies are well known in the art. Generally, the humanized antibody will have one or more amino acid residues introduced into it from a non-human source. These nonhuman amino acid residues are often referred to as "import residues, typically taken from a variable" import "domain. Humanization can be accomplished essentially following the method of Winter and co-workers [Jones et al., Nature, 321 : 522-525 (1986); 1 'Riechmann et al., Nature, 332, 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)]. «♦ * · corresponding sequences of human antibody in rodent CDR-: i .: residues or CDR sequences. Thus, such" hu- ♦ ··: humanized "antibodies are chimeric antibodies (Ca-Billy et al. ., supra), in which less than an intact human variable domain is substituted by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies, with some CDR residues and possibly substantially * .. · * some FR residues are substituted by residues which are * * · ': analogous to rodent antibodies.

• ·. · * ·. The selection of human variable domains for the production of humanized antibodies, both light and heavy, is very important in reducing antigenicity. According to the so-called "best fit" method, the variable domain sequence of a rodent antibody is screened against a complete library of known human variable domain sequences. The human sequence closest to the rodent sequence is then adopted as a human reading frame (FR) for the humanized antibody [Sims et al., J. Immunol., 151: 2296 (1993); Clothia and Lesk, J. Mol. Biol., 196: 901 (1987)]. The second method employs a specific reading frame derived from the consensus sequence of all human antibodies having a particular subset of light and heavy chains. The same reading frame 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)].

Furthermore, it is important that the antibodies be humanized so that high affinity for the antigen and other favorable biological properties are maintained. In order to achieve this goal, according to a preferred method, humanized antibodies are prepared using parenteral sequences. '. analysis process and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are: .i. * Familiar to those skilled in the art. Computer programs are available that describe and represent probable three-dimensional: conformational structures of selected candidate immunoglobulin sequences. Examination of these representations enables the analysis of the likely role of residues in the function of the candidate immunoglobulin sequence, namely, the analysis of residues that affect the ability of the immunoglobulin present to bind to antigen. . In this way, FR residues can be selected and combined • ·. * ··. consensus and import sequence to achieve the desired antibody characteristic, such as increased affinity for the target antigen (s). In general, CDR residues have a direct and most significant effect on antigen binding. For more information, see U.S. Patent Application Serial No. 07 / 934,373, filed August 21, 1992, which is a continuation of U.S. Patent Application Serial No. 07 / 715,272, filed June 14, 1991.

Alternatively, it is now possible to produce transgenic animals (e.g., mice) which, upon immunization, are capable of producing a complete range of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain binding region (Jh) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of a group of human germline immunoglobulin genes into such germline mutant mice results in the production of human antibodies after antigen sensitization. See, e.g., 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 by phage display • • [. *. in sequences [Hoogenboom and Winter, J. Mol. Biol., 227, 381 (1991); Marks et al., J. Mol. Biol., 222, 581 (1991)].

t · '* (iv) Bispecific Antibodies • · ·' · Bispecific antibodies are monoclonal, preferably human or humanized antibodies, which have binding specificity for at least two different antigens. Methods for preparing bispecific antibodies are known in the art.

• · ·

Conventionally, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy-chain-light chain pairs, whereby these two heavy chains have different specificities [Millstein · * · ': and Cuello, Nature, 305: 537-539 (1983)]. Immunoglobulii- • ·. * · *; Because of the random sorting of their heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, which is usually accomplished by affinity chromatography steps, is quite laborious and the product yields are low. Similar methods are disclosed in PCT Patent Publication No. WO 93/08829 (published May 13, 1993) and in Traunecker et al., EMBO, 10: 3655-3659 (1991).

In another and more preferred embodiment, antibody variable domains having the desired binding specificities (antibody-antigen binding sites) are fused to immunoglobulin constant domain sequences. The fusion is preferably with a constant domain of an immunoglobulin heavy chain comprising at least a portion of the hinge region and the CH2 and CH3 regions. It is preferred that the constant region (CH1) of the first heavy chain contains the site necessary for light chain binding and is present in at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected with a suitable host. täorganismiin. This allows for great flexibility in adjusting the relative proportions of the three polypeptide fragments in the embodiments, whereby different ratios of the three polypeptide chains used to construct the vector produce optimal yields. However, it is possible to add

IM

t / '· The coding sequences of two or all three polypeptide chains in a single expression vector when expression of at least two polypeptide chains in the same ratio produces • · ·. ·; resulting in high yields or when relationships are of little importance. In a preferred embodiment of this approach, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with binding specificity in one arm and a hybrid immunoglobulin heavy chain chain. . of the zipper pair (which produces one binding specificity • · · 145) in one arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations because the presence of the immunoglobulin light chain on only one half of the bispecific molecule allows for an easy method of separation. This solution is disclosed in pending patent application 07/931 811, filed August 17, 1992.

Further details on the generation of bispecific antibodies can be found, for example, in Suresh et al., Methods in Enzymology, 121: 210 (1986).

(v) Heteroconjugate antibodies

Heteroconjugate antibodies are also included within the scope of this invention. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Patent 4,676,980) and to treat HIV infection (PCT Publication Nos. WO 91/00360 and WO 92/00373; EP Publication 03089). Heteroconjugate antibodies may be prepared using any suitable cross-linking method. Suitable crosslinkers are well known in the art and have been published; In U.S. Patent No. 4,676,980, in addition to a few crosslinking methods, • · * * IV. Of the megakaryocytopoietic protein, mpl ligand *. . din, therapeutic use • · * * · * · A biologically active mpl ligand with a haematopoietic effector function, referred to herein as * * · V · megakaryocytopoietic or thrombocytopoietic protein (TPO), can be used in sterile pharmaceuticals. megakaryocyto- · * · * in the preparation or formulated product; stimulation of poetic or thromobopoietic activity. ' sex in patients with thrombocytopenia due to impaired platelet production, retention, or increased destruction. Bone marrow hypoplasia associated with thrombocytopenia (e.g., aplastic anemia after chemotherapy or bone marrow transplantation) can be effectively treated with the compounds of this invention as well as diseases such as disseminated intravascular coagulation (DIC), immune thrombocytopenia (including ITP and non-HIV-associated ITP), chronic idiopathic thrombocytopenia, congenital thrombocytopenia, myelodysplasma and thrombotic thrombocytopenia. In addition, these mesakaryocytopoietic proteins may be useful in the treatment of myeloproliferative thrombocytotic diseases and in the treatment of thrombocytosis due to inflammatory diseases and iron deficiency.

Preferred uses of the megakaryocytopoietic or thrombo-cytopoietic protein (TPO) of this invention are: myelotoxic chemotherapy for the treatment of leukemia or solid tumors, myeloablative chemotherapy in the context of autologous or autogenous bone marrow transplantation, myelodysplastic aplastic, myelodysplastic, myelodysplastic,

Still other diseases usefully treated with the megakaryocytopoietic proteins of this invention include deficiency or damage to platelets due to drugs, poisoning, or activation on artificial surfaces. In these cases, these «« · *; The compounds may be used to stimulate the "leakage" of new "intact" blood platelets. For a more complete list of usable · * · .. applications, see the "Background" section above, especially sections (a) to (f) and. references cited therein.

«· · Rt» ···. The megakaryocytopoietic proteins I * · of this invention may be used alone or in combination with other cytokines. hematopoietins, interleukins, growth factors or the like for the treatment of the aforementioned diseases and conditions. Thus, these compounds may be used *: * · * in combination with thrombopoietic activity, ***. with another protein or peptide including G-CSF, GM-CSF, LIF, M-CSF, IL-1, IL-3, erythropoietin (EPO),! · 'Kit ligand, IL-6 and IL-11.

The megakaryocytopoietic proteins of the present invention are prepared in admixture with a pharmaceutically acceptable carrier. This therapeutic composition can be administered to a patient intravenously or through the nose or lungs. The composition may also be administered parenterally or subcutaneously if desired. When administered systemically, the therapeutic composition should be pyrogen-free and in a parenterally acceptable solution having appropriate pH, isotonicity and stability. Those skilled in the art are aware of these circumstances. Briefly, dosage formulations of the compounds of this invention are prepared for storage or use by mixing a compound of the desired purity with physiologically acceptable carriers, excipients, and stabilizers. Such agents are non-toxic to the recipient at the dosages and concentrations employed and include buffers such as phosphate, citrate, acetate, and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) peptides 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, and. dextrins; chelating agents such as EDTA; sugar alcohols, * 9,, ·, such as mannitol or sorbitol; counter-ions such as sodium, · · · ·, and / or non-ionic surfactants such as t ·

Tween, Pluronics or polyethylene glycol.

About 0.5 to 500 mg of the compound or mixture of megakaryocytopoietic proteins in the form of free acid or base V * or a pharmaceutically acceptable salt thereof are mixed with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, with a stabilizing agent, a flavoring agent, etc. according to accepted pharmaceutical practice. The amount of active agent in these compositions is such that a suitable dosage will be obtained within a given range.

Sterile injectable compositions may be formulated in accordance with conventional pharmaceutical practice. For example, it may be desirable to dissolve or suspend the active compound in a vehicle such as water or in a naturally occurring vegetable oil such as sesame, peanut or cottonseed oil or in a synthetic fatty vehicle such as ethyl oleate or the like. Buffers, preservatives, antioxidants and the like may be added in accordance with accepted pharmaceutical practice.

Suitable examples of sustained release formulations include semipermeable matrices of polypeptide-containing solid hydrophobic polymers in the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels [e.g. poly-2-hydroxyethyl methacrylate as described in Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982), or ···, *, polyvinyl alcohol], polylactides (U.S. Pat. No. 3,773,919, U.S. Patent No. 58,481), L-glutamic acid and gamma-ethyl. L-, * * glutamate copolymers (Sidman et al., Biopolymers, 22: * * * 547-556 (1983)), non-degradable ethylene vinyl acetate (Langer® * * * ♦ et al., Supra), decompose lactic acid-glycolic acid copolymer - M. * rituals such as Lupron Depot ™ (microspheres for injection, consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid (EP-A-133 988). ).

· «· J * l '. Polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow the release of molecules over * * · * · * * for 100 days, while certain hydrogels release · proteins for a shorter period. When encapsulated proteins remain in the body for a prolonged period, they may be denatured or removed. * ··. accumulate as a result of being exposed • «

»M

149 at 37 ° C resulting in a loss of their biological activity and possible changes in their immunogenicity. Rational policies can be designed to stabilize the protein depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation through disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling moisture content, using appropriate additives and developing specific polymer matrix compositions.

Long-acting megakaryocytopoietic protein compositions also include megakaryocytopoietic protein trapped within liposomes. Liposomes containing megakaryocytopoietic protein are prepared using methods known per se: DE 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); EP 52 322; EP 36,676; EP 88 046; EP 143,949; EP 142,641; Japanese Patent Application 83-118008; U.S. Patents 4,485,045 and 4,544,545, and European Patent 102,324. Typically, liposomes are small (about 200-800 Å), a monolayer type with a lipid content greater than about 30 moles % cholesterol, the selected proportion being adjusted for optimal treatment with the megakaryocytopoietic protein • · · 1 ·.

. ·. : The dosage will be determined by the attending physician taking into consideration the • · · • ·. . ·. various factors known to alter the effect of drugs, including severity and type of disease, patient's weight, sex, diet, time and route of administration, other medications, and other relevant clinical factors.

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Preferably, the dosage is 0.1 to 50 µg / kg body weight. &Quot; 1 kg body weight. More preferably, the initial dose is 1 to 5 / µg / kg / day. In most cases, the dosage range is the same as for other cytokines, Dosage Range for G-CSF, GM-CSF, and EPO In particular, $ 150 effective dosages can be determined using either in in vitro or in vivo methods.

eXAMPLES

Without further explanation, it is believed that one skilled in the art, utilizing the foregoing patent specification and illustrative examples, will make and utilize the present invention to the fullest extent. Thus, the following working examples are particularly directed to preferred embodiments of the present invention and are not to be construed as limiting the remainder of the disclosure in any way.

Example 1

Partial purification of porcine mpl ligand Low platelet-rich plasma was collected from normal or aplastic anemic pigs. Pigs were aplastic by irradiation with 900 cGy of whole body using a 4mEV linear accelerator. Irradiated pigs were given cefazolin by intramuscular injection for 6-8 days. Subsequently, their entire blood volume was removed under general anesthesia, the blood was treated with heparin and centrifuged at 1800 x g for 30 minutes to produce low platelet-containing plasma. Megakaryocyte-stimulating activity was found to peak at 6 days after irradiation • · ♦: 1.

• · Swine aplastic plasma obtained from irradiated pigs ♦ · ϊ1 · is made up to 4 molar with NaCl and mixed at 30% .1. : for one minute at room temperature. The resulting precipitate • ·. is removed by centrifugation at 3800 rpm in a Sor-1 · 1 L interval in an RC3B centrifuge and the supernatant is applied to a Phenyl-To-· · * yopearl column (220 mL) equilibrated in 10 mM NaPO 4 containing mol / l NaCl. The column is washed • · ♦ * ··· 1 with this buffer until A280 is <0.05 and eluted with dH2O.

V 'The peak of the eluted protein is diluted with dH 2 O to the leading number · 1 · 1; 15 mS and applied to a Blue-Sepharose column equilibrated1 ···. (240 mL) in PBS. The column is then washed with a volume of * 5 columns each of PBS and 10 mM NaOH (pH 7.4) containing 2 mol / L of PBS (pH 7.4). 1 urea. Proteins are eluted from the column with 10 mM NaPO 4 (pH 7.4) containing 2 mol / L urea and 1 mol / L NaCl. The peak of the eluted protein is made up to 0.01% for octyl glucoside (n-octyl-BD-glucopyranoside) and 1 mM for both ED-TA and Pefabloc (Boehringer Mannheim) and applied directly to a series of connected CD4-IgG columns. (Capon, DJ et al., Nature, 337: 525-531 (1989)) and mpl-IgG-Ultralink column (Pierce) (see below). The CD4 IgG column (2 mL) is removed after the sample has been added and the mpl IgG column (4 mL) is washed with 10 column volumes of both PBS and 2 M NaCl in each case. , and eluted with 0.1 M glycine-HCl, pH 2.25. Fractions are collected in a tenth volume of 1 M Tris-HCl (pH 8.0).

Analysis of the fractions eluted from the Mpl affinity column using SDS-PAGE run (4-20%, Novex gel) under reducing conditions revealed the presence of several proteins (Figure 5). Proteins which are stained with silver at the highest intensity differ at an apparent Mr of 66,000, 55,000, 30,000, 28,000, and 14,000. In order to determine which of these proteins stimulate the proliferation of Ba / F3 mpl cell cultures, these proteins were eluted from the gel as described in Example 2 below.

• · · • ·· 'Ultralink affinity columns *. . Add 10-20 mg mpl-IgG or CD4-IgG in PBS Addition to 0.5 g Ultralink Resin (Pierce) according to manufacturer's • · · * ··· in accordance with.

* Mpl-IgG formation and expression

A chimeric molecule containing the entire extracellular domain of human mpl (amino acids 1-491) and the Fc region of the human IgG1 molecule was expressed in 293 cells. The cDNA fragment coding for amino acids 1-491 of human mpl was obtained by PCR from a cDNA library of a human megakaryocytic β-1 CMK cell and sequenced. Clal-152 was added at the 5 'end and BstEII at the 3' end. This fragment was cloned over the Fc coding region of IgG in the Bluescript vector between the ClaI and BstEII sites after partial digestion of the PCR product using BstEII due to two other BstEII sites present in the DNA encoding the extracellular mpl. domain. The BstEII site produced at the 3 'end of the mpl PCR product was designed to have an Fc region in reading frame with the extracellular domain of mpl. The construct was subcloned into the pRK5 tkneo vector between the ClaI and XbaI sites and transfected into human embryonic 293 kidney cells using the calcium phosphate method. Cells were selected at 0.4 mg / ml G418 and individual clones were isolated. Mpl-IgG expression from isolated clones was determined using a human Fc specific ELISA. The clone that produced the best expression had an expression level of 1-2 mg / ml mpl-IgG.

Ba / F3 mpl P expressing cells

The cDNA corresponding to the entire coding region of human mpl P was cloned into the pRK5 tkneo vector and then linearized with NotI and transfected into an 11-3 dependent cell line by Ba / F3 electroporation (1 x 107 cells, 9605 F, 250V). Three days later, the launch of · '· 1. choice in the presence of 2 mg / ml G418. Cells Intermediate • ·. ·. : pooled or individual clones were obtained with limited · · · • ·; dilution in 96-well plates. Selected • · 1. cells were maintained in RPMI medium containing 15% FBS, 1 mg / ml G418, 20 µg / ml glutamine, 10 mM HEPES and 100 mg / ml Pen-

j · J

· 1 Strep. Mpl P expression in selected clones was determined using FACS analysis and polyclonal anti-mpl P rabbit antibody.

Assay for Ba / F3 mpl Ligand ··· The Mpl ligand assay was performed as shown in Figure 2. Libraries of mpl Ligand from Different Sources: To determine the presence of mpl P-Ba / F3 cells were maintained in the absence of 153 IL-3 for 24 hours at a cell density 5 x 105 cells / ml in a humidified incubator at 37 ° C in an atmosphere of 5% CO 2 and air. After the cells were maintained in the absence of IL-3, they were cultured in 96-well culture plates at a density of 50,000 cells in or without 200 mL of medium diluted and cells were cultured for 24 hours in a cell culture incubator. To each well was added 20 μΐ serum-free RPMI medium containing 1 μ ΐ 2 ΐ 3H-thymidine for the last 6-8 hours. The cells were then harvested into 96-well GF / C filter plates and washed 5 times with water. Counting from filters in the presence of 40 μΐ scintillation fluid (Microscint 20) was performed on a Packard Top Count.

Example 2

Highly purified porcine mpl ligand

Geelieluointimenetelmä

Equal amounts of affinity purified mpl ligand (fraction 6 eluted from the mpl IgG column) and 2X Laemmli sample buffer were mixed at room temperature without reducing agent and applied as quickly as possible to the 4-20% Novex polyacrylamide gel. The sample was not heated. As a control, the sample buffer was run without the ligand · · · i slide on an adjacent line. The gel was run at 4-6 ° C in a patient at 135 V for approximately 2 1/4 hours.

The running buffer was initially at room temperature. The gel was then removed from the gel box and the plate on the other side of the gel · ·,; 1; was removed.

• · ·

The gel lump was prepared for nitrocellulose as follows: A piece of nitrocellulose was moistened with distilled water and carefully placed on the exposed gel surface, by blowing off air bubbles. Basic markings were made on the · · · * ·] 1 nitrocellulose and gel plate so that the folds could be accurately inserted after staining. Approximately i ***: After 2 minutes, the nitrocellulose was carefully removed and the gel was wrapped in a plastic wrap and placed in a refrigerator.

• · · • • • 154

Nitrocellulose was stained with Biorad's Total Golden Protein Dye by first mixing it in 3 x 10 ml of 0.1% Tween 20 + 0.5 M NaCl + 0.1 M Tris-HCl, pH 7.5 , for about 45 minutes and then with 3 x 10 ml purified water for 5 minutes. The gold color was then added and allowed to develop until zones in the standards were visible. The repeats were then rinsed with water, placed on top of a plastic wrap on a gel, and carefully aligned with the base marks. The positions of the Novex standards were marked on a gel plate and lines were drawn to indicate the intersections. The nitrocellulose and plastic wrap were then removed and the gel was cut along the indicated lines using sharp razors. The sections extended over the sample lines so that they could be used to determine the position of the slices when the gel was stained. After removing the slices, the remaining gel was stained with silver and the positions and shear marks of the standards were measured. The molecular weights corresponding to the breakpoints were determined from the Novex standards.

12 slices of gel were then placed in cells on two Biorad Model 422 electroelution devices. The cells used membrane lids (caps) of 12 to 14K molecular weight cut-off. The elution buffer is 50 mM ammonium bicarbonate + 0.05% SDS (pH about 7.8). One liter of buffer was cooled for approximately one hour in a 4-6 ° C cold store before use. The gel slices were eluted at a current of 10 mA / cell (initially 40 V) in a 4 to 6 ° C cold room. Elution took about 4 hours.

• · ·

The cells were then carefully removed with a pipette to remove the supernatant. The elution chamber was removed and the supernatant liquid was removed by pipette. The liquid in the mem-»· · lid was removed with a Pipetman. and saved. 50 µl of purified water was then placed on the lid, mixed and removed until all Γ **: SDS crystals had dissolved. These washes were combined with the · · · above saved fluid. The total eluted sample volume was 300-500 μΐ / gel slice. Samples were placed in a 10 mm Spectrapor 4 12-14K cut-off dialysis tube soaked for several hours in purified water. They were dialyzed overnight at 4-6 ° C against 600 ml of phosphate-buffered saline (PBS about 4 mM potassium) per six samples. The buffer was changed the next morning and dialysis was continued for 2.5 hours. Samples were then removed from dialysis bags and placed in microfuge tubes. The tubes were placed on ice for 1 hour, microfuged at 14K rpm for 3 minutes, and the supernatants carefully removed from the precipitated SDS. The supernatants were then placed on ice for about 1 hour or more and microfuged again for 4 minutes. Supernatants were diluted with phosphate buffered saline and subjected to activity assay. The remaining samples were frozen at -70 ° C.

Example 3

Micro-sequencing of porcine mpl ligand

Fraction 6 (2.6 mL) from the mpl-IgG affinity column was concentrated on a Microcon-10 (Amicon). To prevent the Mpl ligand from being absorbed into the Microcon, the membrane was rinsed with 1% SDS and 5 µl of 10% SDS. was adjusted to fraction 6. 2X sample buffer (20 μΐ) was added to * · .1 ·: after fractionation of Microcon (20 μΐ) and fractional volume (40 μΐ) was added to 4-20% one line of gradient acrylamide gel (Novex). The gel was run according to Novex instructions. The gel was then equilibrated for 5 min before electroblotting in 10 mM 3- (cyclohexylamino) -1-propanesulfonic acid buffer (CAPS buffer), pH 11.0 containing 10% methanol. Electric blotting * · · ***. Immobilon-PSQ membranes (Millipore) were subjected to a sustained current of 250: mA in a BioRad Trans-Blot transfer cell (32) within 45 minutes. PVDF membrane t2 ': stained with 0.1% Coomassie Blue R-250 40- · · · ..1. in methanol, 0.1% acetic acid for 1 minute and decolorization with 10% acetic acid in 50% for 2 to 3 minutes. methanol. The only proteins visible in the Mr region of the spot were 18,000-35,000 with Mr values of 30,000, 28,000 and 22,000.

Bands at 30, 28 and 22 kDars were subjected to protein sequencing. Automated protein sequencing was performed using a Model 470A Applied Biosystem sequencer equipped with an on-line PTH analyzer. The sequencer had been modified to inject 80 to 90% of the sample (Rodriguez, J. Chromaticog., 350: 217-225 (1985)). Acetone (12 μΐ / ΐ) was added to solvent A to balance UV absorbance. The electroblotted proteins were sequenced in a Blott cassette. The peaks were integrated with Justice Innovation 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 (using one letter code, uncertain residues in parentheses) and the amount of material obtained (in parentheses) are shown in Table 2 '.

Table 2 '. Amino terminal sequences of Mpl ligand.

30 kDa [1.8'pmol] V * 1 5 10 15 20 25 • · (S) PAPPA (C) D PRLLNKLLRDD (H / S) VLH (G) RL (SEQ ID NO: 30) »·: * ·· 28 kDa [0.5 pmol] 1 5 10 15 20 25 ti: (S) PAPPAXDPRLLNKLLRDP (H) VL (H) GR_ (SEQ ID NO: 31) ΓΓ: 18-22 kDa [0.5 pmol] 1 5 10:. ·. XPAPPAXDPRLX (N) (K) _ (SEQ ID NO: 32) ··· ···•••••••••••••••••••••••••• · · • · • · 1 · · · · · · 157

Example 4

Liquid suspension assay for megakaryocytopoiesis

Human peripheral stem cells (PSC) (obtained from consenting patients) were diluted 5-fold with IMDM medium (Gibco) and centrifuged at 800 x g for 15 minutes at room temperature. Cell pellets were resuspended in IMDM medium and layered on 60% Percoll (density 1.077 g / ml) (Pharmacia) and centrifuged at 800 x g for 30 minutes. The low density mononuclear cells were aspirated from the dividing surface and washed twice with IMDM medium and cultured at 1-2 x 10 6 cells / ml in IMDM medium containing 30% fetal calf serum (FBS) (final volume 1 ml) in 24-well tissue culture. (Costar). APP or depleted mpl ligated APP was added to 10% and cultures were grown for 12-14 days in a humidified incubator at 37 ° C in 5% CO 2 and air. Cultures were also grown in the presence of 10% APP, and 0.5 μg mpl-IgG was added on days 0, 2, and 4. From APP, the mpl ligand was removed by passing APP through a mpl-IgG-affinity column.

To determine the amount of megakaryocytopoiesis from these liquid suspension cultures, · · · ϊ * was used according to Solberg et al. a variant of the method using a radiolabeled rat monoclonal IgG antibody (HP1-1D) against GPIIb s * · (obtained by Dr. Nichols, Mayo Clinic). Ra · · *: diol was labeled with 100 µg HPl-1D [see Grant, B. et. al., Blood, 69: 1334-1339 (1987)] with 1 mCi of Na125I using Enzymobeads (Biorad, Richmond, CA) as described in the manufacturer's instructions. The radiolabeled HP1-1D was stored at -70 ° C in PBS containing 0.01% ox- * * *. tyyliglukosidia. Typical specific activity was 1 x 2 x 10 6 cpm / µg (> 95% precipitated with 12.5% trichloroacetic acid).

♦ · *

Liquid suspension cultures were arranged in triplicate for 158 wrists for each experimental site. After 12-14 days, 1 ml cultures in culture 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 in 100 μΙζΆΆΠ of PBS containing 0, 02% EDTA and 20% fetal bovine serum. The resuspended cultures were supplemented with 10 ng of 125I-HP1-ID in 50 silicon assay buffer and incubated for 60 minutes at room temperature (RT) with occasional shaking. The cells were then harvested by centrifugation at 800 x g for 10 minutes at room temperature and washed twice with assay buffer. The pellets were counted within 1 minute on a gamma counter (Packard). Non-specific binding was determined by the addition of 1 µg of unlabeled HPI-1D 60 minutes before the addition of labeled HPI-1D. Specific binding was determined as the total amount of 125 I-HP1-1D minus the amount bound in the presence of excess unlabeled HP1-1D.

Example 5

PCR primers for oligonucleotide

Based on the amino terminal amino acid sequence obtained from the 30 kDa, 28 kDa and 18-22 kDa proto · · ·: * scaffolds, degenerate oligonucleotides were designed for use as primers for polymerase chain reaction (PCR) (see Table j). 4). Pools of the two primers were synthesized, with the positive sense-20-mer pool encoding amino acid residues 2-8 (mpl 1) and the anti-sense-21 mer pool being »I · t *; ·. complementary to sequences encoding amino acids 18-24 (mpl 2).

Table 4. Pools of degenerate oligonucleotide primers c · * t I «. * .. -r ~ -! - Γ v * · mpl 1: 5 'CCN GCN CCN CCN GCN TGY GA 3' (2.048-fold degenerate (SEQ ID NO: 35) * ... · mpl 2: 5 'NCC RTG NAR NAC RTG RTcj RTC 3' (2.048-tert.degenerate) (SEQ ID NO: 36) ·· * i · »k · • · ··· • · «· 1,159

Porcine genomic DNA isolated from porcine peripheral blood lymphocytes was used as template for the polymerase chain reaction (PCR). The reaction mixture, 50 μΐ, contained: 0.8 Mg porcine genomic DNA in a mixture of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 100 Mg / ml BSA, 400 Mmol / L dNTP, 1 Mmol / L each primer pool and 2.5 units Taq polymerase. The template was first denatured at 94 ° C for 8 minutes and then subjected to 35 45 second cycles at 94 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C. The final cycle was allowed to last for 10 min at 72 ° C. The PCR products were separated using electrophoresis on a 12% polyacrylamide gel and visualized by staining with ethidinium bromide. It was concluded that if the amino-terminal amino acid sequence was encoded by a single exon, then the correct length of the PCR product was assumed to be 69 base pairs. This complete DNA fragment was eluted from the gel and subcloned into pGEMT (Promega). The sequences of the three clones are shown in Table 5.

«·« • · i «i«:: «• 1% · • · 1« * 1 1 • · · • I1 · • · • If • «» f · ·.

• 1 I

* * · · • · 1 • If ·· · f ♦ · t · · ··· 4 ♦ · I I · • • · · i:

Mf * »· · • · 1» »f · I1 • · • ·« 1 «160

Table 5. Porcine genomic DNA fragments of 69 base pairs

f H

gemT3

5'CCAGCGCCGC CAhcCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA 3'GGTCGCGGCG GT3GGACACT GGGGGCTGAG GATTTATTTG ACGGAGCAQI

TGACCACGTT CAGCACGGC [69 bp] (SEQ ID NO: 37) ACTGGTGCAA GTCGTGCCG_ (SEQ ID NO: 38) _ gemT7

5'CCAGCACCTC CG3CATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 3'GGTCGTGGAG GcbGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCACT

CGACCACGTC CATCACGGC [69 bp] (SEQ ID NO: 39) GCTGGTGCAG GTAGTGCCG_ (SEQ ID NO: 40) __ gemT9

PRLLNKLLR {SEQ ID

NO: 32) i 5 'CCAGCACCGCCGGCATGTGAQCCCCGACTCCTAAATAAACTGCTTCGTGACG 3' GGTCGTGGCGGCCGTACACTGGGGGCTGAGGATTTATTTGACGAAGCA £ T ££ i \ ».. .. ATCATGTCTATCACGGTGTGTGT · • * - * · \. Underlined * · · * · '· bases indicate the location of the PCR primers. These results confirm the N-terminal sequence obtained for amino acids 9-17 of the 30 kDa, 28 kDa, and 18-22 kDa proteins. , and show that this sequence is encoded by a single exon of porcine DNA. x Example 6 f # ·. V. Human mpl ligand gene%

Based on the results from Example 5, the 45-mer 4 · · '· * / deoxyoligonucleotide, called pR45, • ·. * · "Was designed and synthesized for screening the genomic library Γ *'. The 45-mer had the following sequence : • · M »f · t · •« · 161 5 'G CC-GTG -AAG-G AC-GTG-GTCrGT C-ACG-AAG-CAG-TTT-ATT-TAG-G AG-T CG 3' ( äEQ ID NO: 28) This oligonucleotide was 32P-labeled with (γ32Ρ) -ATP and T4 kinase and was used to screen the human genomic DNA library in vgem12 under less stringent hybridization and wash conditions (see Example 7). positive clones, plaque purified and analyzed using restriction mapping and Southern blotting. Clone 4 was selected for further analysis.

A 2.8 kb BamHI-XbaI fragment that hybridized to 45-mer, pBluescript SK- was subcloned. Partial DNA sequencing of this clone was performed using oligonucleotide primers specific for the porcine mpl-ligand DNA sequence. The resulting sequence confirmed the presence of isolated DNA encoding the human homolog of porcine mpl ligand. We observed the EcoRI restriction site in the sequence, which allowed us to isolate the 390 bp Eco RI-Xba I fragment from the 2.8 kb Bam HI-Xba I and subclone it into 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 NOs: 3 & 4). The predicted • ·% · • - * positions of the introns in the genomic sequence are also indicated by arrows and the 4 · * · · · »* ϊ defines a putative exon (" exon 3 ").

• »; * ·· Examination of the predicted amino acid sequence confirms * ·? / * · That the serine residue is the first amino acid of the fully prepared mpl ligand as determined by the direct amino acid sequence *; sianalyysistä. The amino acid sequence predicted just above this codon gives strong indications of the signal sequence involved in secretion of the fully mature mpl ligand * · · d. Probably the coding region of this signal sequence is interrupted by the intron at nucleotide position 68.

In the V '31 direction, the exon appears to terminate at nucleotide # ♦ * * ϊ 196. Thus, this exon encodes a sequence of 42 amino acids, 16 of which are likely to be part of the signal. * · 162 additional sequences and 26 are part of a fully mature human mpl ligand.

Example 7

Human full-length mpl ligand cDNA

Based on the human "exon 3" sequence (Example 6), two non-degenerate oligonucleotides corresponding to the 3 'and 5' ends of the "exon 3" sequence were synthesized (Table 6).

Table 6. Non-degenerate PCR oligonucleotide primers for human cDNA

Fwd primer: 5 'GCT AGC TCT AGA AAT TGC TCC TCG TGG TCA TGC TTC T 3' (SEQ ID NO: __43J__

Rvs primer: 5'CAG TCT GCC GTG AAG GAC ATG G 3 'm (SEQIDNO: -44) These two primers were used in PCR reactions using DNA from various human cDNA libraries as template, or ng Quick Clone cDNA (Clonetech) from various tissues using the conditions described in Example 5. The expected size of the correct PCR product was 140 base pairs. After analysis of the PCR products on a 12% polyacrylamide gel, the expected size DNA fragment was detected in cDNA libraries prepared from adult kidney, 293 fetal kidney cells and. *. CDNA prepared from human fetal liver (Clone-tech cat. # 7171-1).

• · · ί.ϊ: Fetal liver cDNA library in λ DR2 (Clonetexh cat.

# HL1151X) was screened with the same 45-mer oligonucleotide; which was used to screen the human genomic library.

• · ·. ·; ·. The oligonucleotide was labeled with (γ32Ρ) -ATP using T4 polynucleotide kinase. The library was screened under less stringent hybridization conditions. The filters were pre-hybridized for 2 hours, then hybridized with the probe overnight at 42 ° C in 20% formamide with. was 5 x SSC, 10 x Denhardt, 0.05 mol / L sodium phosphate 163 (pH 6.5), 0.1% sodium pyrophosphate, 50 μg / ml sonicated salmon sperm DNA for 16 hours. The filters were then rinsed in 2 x SSC and then washed once with 0.5 x SSC, 0.1% SDS at 42 ° C. The filters were exposed to a Kodak X-ray film overnight. Positive clones were picked, plaque purified and insert size determined using PCR and oligonucleotides flanking the BamHI-Xba I cloning in λ DR2 (Clonetech cat. # 6475-1). 5 μΐ of the phage stock product was used as the Temp source. The first denaturation was carried out for 7 minutes at 94 ° C, followed by 30 cycles of amplification (1 minute at 94 ° C, 1 minute at 52 ° C, and 1.5 minutes at 72 ° C). The final extension took place within 15 minutes at 72 ° C. Clone # FL2b had a 1.8 kb insert and was selected for further analysis.

Plasmid pDR2 (Clonetech, ADR2 & pDR2 Cloning and Expression System Library Protocol Handbook, page 42), contained in the lDR2 phage arms, was recovered as described in the manufacturer's instructions (Clonetech, 1DR2 & pDR2 Cloning and Expression System Library Protocol Handbook, pages · * · *; 29 - 30). Restriction analysis of pDR2-FL2b plasmid with BamHI and · · · ·: XbaI indicated the presence of an internal BamHI restriction site. in the insert at approximately 650. Plasmid disruptor · · ·. BamHI-XbaI cut the insert into two fragments, one 0.65 kb and one 1.15 kb. The DNA sequence was determined by:: “· using three different classes of * · * 'template from plasmid pDR2-FL2b. DNA sequencing of double-stranded plasmid DNA was performed on an automated, ·. · · Fluorescent ABI373 (Applied Biosystems, Foster City, Ka. • · ·: lifornia) DNA sequencer using standard · ··· methods for color-labeled dideoxynucleoside triphosphate terminators (dye terminators) and tailored 2 · * ”synthesized walking primers [Sanger et al., Proc.

j'v Natl. Acad. Sci. USA, 74: 5463-5467 (1977); Smith et al.,

Nature, 321: 674-679 (1986)]. Direct sequencing of the plasmid polymerase chain-amplified fragments was performed on an ABI373 sequencer using customized primers and dye terminator reactions. The single-stranded template was constructed using the M13 Janus vector [DNASTAR, Inc., Madison, Wisconsin] [Burland et al., Nucl. Acids Res., 21: 3385-3390 (1993)]. Fragments Bam-HI-Xbal (1.15 kb) and BamHI (0.65 kb) were isolated from plasmid pDR2-FL2b, amplified with T4 DNA polymerase in the presence of deoxynucleotides and then subcloned into the Smal site of the M13 Janus vector. Sequencing was performed using standard methods for dye-labeled M13 universal primers and inverse primers or walk primers and dye terminators. Manual sequencing reactions were performed with single-stranded M13 DNA using walking primers and standard dideoxy-terminator chemistry [Sanger et al., Proc. Natl. Acad. Sci., USA, 74-5463-5467 (1977)], 33β-labeled α-dATP and Sequenase (United States Biochemical Corp., Cleveland, Ohio). DNA sequence assembly was performed on a Sequencher V2.1bl2 (Gene Codes Corporation, Ann Arbor, Michican). The nucleotide sequence and derived sequence j of the hML are shown in Figure 1 (SEQ ID NO: 1).

Example 8

Isolation of the human mpl ligand (TPO) gene. : Human genomic DNA clones of the TPO gene were isolated by · · · screening the human genomic library in A-Gem12 I · · * !! using pR45, the oligonucleotide probe previously described, under less stringent conditions (see Example 7) or under very stringent conditions with the aid of a fragment corresponding to the 3 'end of the human cDNA encoding mpl ligand. (From Bam HI to 3 'end). Two were partially isolated. *: ·. an overlapping lambda clone 35 kb in length. Two ···. the partially overlapping fragment (BamHI and EcoRI) containing the entire TPO gene was subcloned and sequenced. The structure of the human gene consisted of 6 exons within 7 kb of genomic DNA (Figures 14 A, B and C). The boundaries of all the exon 165 excon / intron junctions were consistent with the consensus motif formed on mammalian genes [Shapiro, M.B., et al., Nucl. Acids Res., 15: 7155 (1987)]. Exon 1 and exon 2 contain the 5 'untranslated sequence and the first 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 of the erythropoietin-like domain and 3-1 untranslated and <50 amino acids are encoded in exon 6. The four amino acids involved in hML-2 ( hTPO-2), encoded at the 5 'end of exon 6.

Example 9

Temporary expression of the human mpl ligand (hML) to subclone the full-length insert contained in pDR2-FL2b was completely digested with XbaI, then partially with Bam HI. The DNA fragment corresponding to the 1.8 kb insert was gel purified and subcloned into pRK5 (pRK5-hmpl1) (see U.S. Patent 5,258,287 for pRK5 formation) under the control of the immediate early promoter of the cytomegalovirus. DNA from construct pRK5-hmpl1 was prepared by the PEG method. and transfected into human embryonic kidney 293 cells, · · · • ·. which were maintained in Dulbecco's modified Eagle Maintenance • · · *. . in DMEM supplemented with F-12 nutrient mixture, 166 ta Ba / F3 cells but significantly stimulated Ba / F3 mpl cell proliferation (Figure 12A), indicating that this cDNA encodes a functionally active human mpl ligand.

Example 10

To identify alternatively spliced forms of human mpl ligand hML2, hML3, and hML4, primers corresponding to either end of the hML coding sequence were synthesized. These primers were used in RT-PCR to amplify mature human liver RNA. In addition, the internal primers flanking the selected regions of interest (see below) were constructed and used in the same manner. Direct sequencing of the ends of the PCR product revealed one sequence that exactly matched the sequence of cDNA isolated from the human fetal liver library [see Figure 1 (SEQ ID NO: 1)]. However, the region near the C-terminus of the EPO domain (in the middle of the PCR product) showed a complex sequence model, suggesting the existence of possible stranding variants in that region. To isolate these filament variants, the primers shown in Table 7 flanking the region of interest were used as templates in the PCR method for fully human liver cDNA.

. *. : Table 7. PCR primers for human ML isoform.

• · · ♦ · _ ___ KO phmpllcdna.3e1: 5, TGTGGACTTTAGCTTGGGAGAATG3, (SEQ ID NO: 45) 0 'pbx4.f2: 5'GGTCCAGGGACCTGGAGGTTTG3' (SEQ ID NO: 46) PCR products subclone M. Sequencing of single subclones revealed at least 3 ML- *. * * Isoforms. One of them, hML (also called: * · *; also called hML ^), is the longest form and exactly matches the fetus, · “. liver isolated sequence from the 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 NOs: 6, 8, • · · 9 & 10).

Example 11 167

Formation and transient expression of human mpl ligand isoforms and substitution variants, hML2, hML3 and hML (R153A, R154A)

The isoforms hML2 and hML3 and the substitution variant hML (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, editors M.A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White.

The "outside" primers used in all constructs are shown in Table 8 and the "overlapping" primers are shown in Table 9.

Table 8. External primers

Cla.FL.F2: 5'ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G3 '(SEQ ID NO: __47) HMPLL-R: 5'GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA3' (SEQ ID NO: : _._ 48) _ _ • «* • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • ••. . Table 9. Partially overlapping primers

| · · "_ I

hm l-2:: MLA4.F: 5'CTC CTT GGA ACC CAG GGC AGG ACC 3 '(SEQ ID NO: 49) MLA4.R 5'GGT CCT GCC CTG GGT TCC AAG G AG 3' __ (SEQ ID NO: 49) : 50) h M L-3:: j: hMLAl 16+: 5'CTG CTC CGA GGA AAG GAC TTC TGG ATT 3 '(SEQ ID NO: 51) h M LA 116-: 5'AAT CCA GAA GTC CTT TCC TCG GAG CAG 3 '(SEQ ID NO: 52), -. hML (R153A. R154A): ** 'RR-KO-F: 5'CCC TCT GCG TCG CGG CGG CCC CAC CCA C 3' (SEQ ID NO: 53) ·· *: RR-KO-R: 5'GTG GGT GGG GCC GCC GCG ACG CAG AGG G 3 '(SEQ ID NO: 54) • «• ·« ·· 168

All PCR amplifications were performed using cloned Pfu DNA polymerase (Stratagene) using the following conditions: First template denaturation was performed at 94 ° C for 7 minutes, then 30 cycles of 1 minute at 94 ° C, 1 minute at 55 ° C, and 1.5 minutes at 72 ° C. The final cycle was allowed to last for 10 minutes at 72 ° C. The final PCR product was digested with Clal-Xbal, gel purified and cloned in pRKStkneo. 293 cells were transfected with various constructs as described above and the supernatant was analyzed using the Ba / F3 mpl proliferation assay. In this assay, hML-2 and hML-3 did not show detectable activity, however, the activity of hML (R153A, R154A) was similar to that of hML, indicating that processing at this diacid site was not required for activity (see Figure 13).

Example 12

Isolation of murine mpl ligand cDNA mML, mML-2 and mML-3 mML cDNA A DNA fragment corresponding to the entire coding region of the human mpl ligand was obtained by PCR, purified by :. * ·· was gelated and randomly labeled by sensitization in the presence of 32P-dATP and 32P-dCTP. This probe was used to screen for 106 clones of mouse liver cDNA library in IgT10 (Clontech cat # ML3001a). The dual · · · filters were hybridized in 35% formamide containing 5 x SSC, 10 x Denhardtra, 0.1% SDS, 0.05 mol / L. sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 100 mg / ml sonicated salmon sperm DNA: overnight in the presence of a probe. Filters were rinsed in T x 2 x SSC and then washed once in 0.5 x Γ * ': SSC, 0.1% SDS at 42 ° C. The hybridisation phage was plaque purified and the cDNA inserts were subcloned into the Eco RI site of Bluescript SK. Clone • · * ··· '' LD 'with a 1.5 kb insert was selected for further analysis 169 and both strands were sequenced as described above for human ML cDNA. The nucleotide sequence and deduced amino acid sequence obtained from clone LD are shown in Figure 14 (SEQ ID NOs: 1 & 11). The fully prepared ML sequence derived from this clone had 331 amino acid residues in length and was identified as mML1 (or mML-2 for the reasons described below). Significant identity of both the nucleotide sequence and the deduced amino acid sequence was observed in EPO-like domains of these mpl ligands. However, when the deduced amino acid sequences of human and mouse mpl ligands were aligned, the mouse sequence appeared to have a tetrapeptide deletion between residues 111-114 of the human sequence, corresponding to a 12 nucleotide deletion after nucleotide position 618 observed in both human (see below) and cDNA. Thus, additional clones were examined for possible ML isoforms of the rat. One clone, "L7", had a 1.4 kb insert and a 335 amino acid derived sequence containing the "missing" tetrapeptide N LPLQ. This shape is believed to be a full-length rat ML and is referred to as mML or mML ^. The nucleotide sequence and derivative of mML • · ·

The amino acid sequence shown in Fig. 16 (SEQ ID NO

NOs: 12 & 13). Finally, clone "L2" was isolated and sequenced. soitiin. This clone had a 116 nucleotide deletion which: corresponded to hML3 and was therefore designated mML-3. Comparison of the 1 · · derived amino acid sequences of these two isoforms is shown in Figure 16.

i:: * Expression of recombinant mML

Expression vectors for the rat mpl ligand • · · * .i. · Were prepared essentially as described in Example 8.

• · · V * Clones encoding mML and mML-2 were subcloned into the pRK5Tkneo vector, a mammalian expression vector which, ···. produces expression under the control of the CMV promoter and SV40 polyadenylate signal. The resulting express- • «·! . * sieve vectors, mMLpRKtkneo and mML2pRKtkneo, were transiently transfected ··· into 293 cells using the calcium phosphate method. After transient transfection, the medium was conditioned for five days. Cells were maintained in glucose-rich DMEM medium supplemented with 10% fetal bovine serum.

Expression of rat mpl (mmpl) in Ba / F3 cells

Stable cell lines expressing c-mpl were obtained by transfecting mmpl pRKtkneo as described essentially with human mpl in Example 1. Briefly, an expression vector (20 μq ^ linearized) containing the entire coding sequence for rat mpl [Skoda, RC et al., EMBO J., 12: 2645-2653 (1993)], were transfected into Ba / F3 cells by electroporation (5 x 10 6 cells, 250 V, 960 μΈ) and subsequently selected for neo-mycine resistance 2 mg / ml of G418. Mpl expression was determined by flow cytometric analysis using rabbit anti-mouse mpl IgG antiserum. Ba / F3 cells were maintained in RPMI medium obtained from WEHI -3B cells and served as a source of IL-3. Supernatants of 293 cells transiently transfected with both mML and mML-2 were analyzed in BaF3 cells transfected with both mmpl and hmpl as described in Example 1.

• · · * · .. Example 13

Sian mpl-ligand cDNA

• ·, pML and pML-2 • j *. Porcine ML (pML) cDNA was isolated by the RACE PCR method t · ·. Briefly, the oligo-dT primer and 2 specific primers were designed based on the exon sequence of the porcine ML gene encoding the amino terminus of ML * purified from porcine plaque serum. A cDNA prepared from various aplastic pig tissues was obtained and amplified.

, ···. The PCR cDNA product of 1342 bp was detected in the kidney and subcloned. Several clones were sequenced and • · ·; · * Were found to encode a fully mature porcine mpl ligand • · · (which did not contain complete secretion signal). It was found that the cDNA encoded a 332 amino acid mature protein (pML332) having the sequence shown in Figure 18 (SEQ ID NOs: 9 & 16).

Method Isolation of the pML gene and cDNA. Genomic clones of the porcine ML gene were isolated by screening the porcine genomic library in EMBL3 (Clontech Inc.) with pR45. The library was substantially screened as described in Example 7. Several clones were isolated and an exon encoding an amino acid sequence similar to that obtained from purified ML was sequenced. Porcine ML cDNA was obtained using a modification of the RACE PCR method. Two specific ML primers were designed based on the sequence of the porcine ML gene. The polyadenylated mRNA was isolated from the kidney of aplastic pigs, essentially as previously described. CDNA was prepared using reverse transcription and BamdT primer (BamdT: 5 'GACTCGAGGATCCATCGAI IIIII VI IIIIIII TT 3j (SEQ ID NO: 55)) targeted to the polyadenosine tail of mRNA. PCR amplification (28 rounds at 95 ° C for 60 seconds, At 60 ° C for 60 seconds and at 72 ° C for 90 seconds) The first round was performed using the ML-specific h- • · t * .ί forward-1 primer. • · · (h-forward-1: 5 ’G CT AG CT CT AG AA ATT GCTCCTCGTGGTCATG CTT CT 3j (SEQ ID NO: 43) • j ·. And BAMAD primer • · · (BAMAD: 5’ GACTCGAGGATCCATCG 3j. (SEQ ID NO: 56) in 100 ml reaction mixture (50 mM KCl, 1.5 mM MgCl, 10 mM Tris buffer, pH 8.0, 0.2 mmol / l dNTPs and 0.05 U / ml Amplitaq polymerase (Perkin Elmer I * ··. Inc.) The PCR product was then digested with Clal, extracted • · Phenol-chloroform (1: 1), precipitated with ethanol and ·: · * Was added to 0.1 mg of Bluescript SK vector (Stratagene • · * ... * Inc.) truncated with Clal and Kpn I. The ligand 172 thiocyte mixture was incubated for two hours at room temperature, then one quarter of it was directly added to the second round of PCR (22 rounds as described above) using another ML-specific forward-1 primer (forward-1: 5 'GCTAGCTCTAGAAGCCCGGCTCCTCCTGCCTG ) (SEQ ID NO: 57) and T3-21 (an oligonucleotide that binds to a sequence adjacent to a multiple cloning site in a Bluescript SK vector): (5 'CGAAATTAACCCTCACTAAAG 3') (SEQ ID NO: 58).

The resulting PCR product was digested with XbaI and ClaI and subcloned into Bluescript SK- vector. Several clones from independent PCR reactions were sequenced.

Again, another form, designated pML-2, which encodes a protein with 4 amino acid residue deletions (328 amino acid residues) was identified [see Figure 21 (SEQ ID NO: 21)]. Comparison of the pML and pML-2 amino acid sequences shows that the latter form is identical except that the tetrapeptide QLPP corresponding to residues 111-114 is deleted [see Figure 22 (SEQ ID NOs: 18 & 21)].

• * *:. * The four amino acid deletions observed in rat, • human, and porcine ML cDNAs occur in exactly the same position in the predicted proteins.

Example 14 • · i CMK assay for platelet antigen GPU.

··· bIIIa expression induced thrombopoietin (TPO) induction. CMK cells are maintained in RMPI 1640 medium • · · **; (Sigma) supplemented with 10% fetal calf serum 1 * * · * and 10 mmol / l glutamine. For assay, cells were harvested, washed and resuspended at a cell number I ** 'of 5 x 10 5 cells / ml in serum-free GIF medium supplemented with 5 mg / l bovine insulin, 10 µg / l apo-transferrin, 1x with trace elements. Then, in a 96- * · * ··· * well flat-bottomed dish, 173 TPO standard or test samples are added to each well at appropriate dilutions in 100 μ1: η volumes. Add 100 μΐ CMK cell suspension to each well and incubate the plates at 37 ° C in a 5% CO2 incubator for 48 hours. After incubation, the plates are centrifuged at 1000 rpm at 4 ° C for five minutes. The supernatants are discarded and 100 μΐ FITC-conjugated GPIIbIIIa 2D2 monoclonal antibody is added to each well. The plates are incubated at 4 ° C for one hour and then centrifuged again at 1000 rpm for five minutes. The supernatants containing unbound antibody are discarded and 200 μΐ of 0.1% BSA-PBS is added to each well. The 0.1% BSA-PBS washing step is repeated three times. The cells are then analyzed on a FASCAN using a standard one-parameter assay that measures fluorescence intensity.

Example 15

DAMI Assay for Thrombopoietin (TPO) by Measuring Endomytotic Activity of DA-MI Cells in 96-well Microtiter Plates «» ·: *. * DAMI cells are maintained in IMDM medium with 10% equine serum (Gibco). ) supplemented with 10 mmol / l glutamine, 100 ng / ml penicillin G, and: 50 μg / ml streptomycin. For assay, cells are harvested, washed, and resuspended at a volume of 1 x 10 ° cells / ml in IMDM medium containing 1% horse serum. Then with a 96-well round bottom. 100 μΐ TPO standard or test samples are added to t: * · * DAMI cell suspension. Cells are then incubated for 48 hours at 37 ° C in an incubator with: ***; is 5% CO 2. After incubation, the plates are centrifuged in a SorI *** · Vail 6000B centrifuge at 1000 rpm for 5 • · ♦ minutes at 4 ° C. The supernatants are poured out and the washing step repeated with 200 μΐ of PBS-♦ ♦ * ··· * 0.1% BSA. Cells are fixed by adding 200 μΐ ice-cold 174 cold 70% ethanol-PBS and resuspended by aspiration. The plates are incubated at 4 ° C for 15 minutes, then centrifuged at 2000 rpm for five minutes and 150 μΐ RNAase at a concentration of 1 mg / ml in propidium iodide is added to each well. and 0.05% Tween 20. After incubation for one hour at 37 ° C, changes in the DNA concentration are measured using a flow cytometer. The polyploid is measured and quantified as follows:

Normalized Polyploid Ratio (NPR) = (Cells,%> G2 + M / Cells,% <G2 + M) with TP0 (Cells,%> G2 + M / Cells,% <G2 + M): s) in control Example 16

Thrombopoietin (TPO) In Vivo Assay (Mouse Platelet Elastic Response Assay) In vivo Assay For 35S Platelet Production Assay C57BL6 mice (obtained from Charles River) were injected intraperitoneally (IP) with 1 ml of goat anti-mouse serum (IP) ampoules) on day 1 to produce thrombo · #:. * bocytopenia. On days 5 and 6, mice are given two intraperitoneal injections of factor or PBS as a control. On day 7, 30 µl of Na? 35SO, in 0.1 ml saline and injected I. * are administered intravenously. addition of 35S dose to circulating blood »·« /: *. platelets are determined as a percentage of blood samples obtained from treated mice and control mice. Blood-. platelet count and white blood cell count are simultaneously counted f ♦ * * · * * '* from blood obtained from the fundus • 4 * \ ta *: T: Example 17 * I *** · KIRA ELISA for Thrombopoietin (TPO) measures - <4 «phosphorylation of the club chimeric mpl-Rse.gD receptor ♦ · ·

The human mpl receptor is disclosed in Vigon et al., PNAS, USA, 89: 5640-5644 (1992). A chimeric receptor containing the mpl receptor extracellular domain (ECD) and the Rse transmembrane domain (TM domain) and the intracellular domain (ICD) was prepared for use in the 175 KIRA ELISA described herein [Mark et al., J. of Biol. Chem., 269 (14): 10720-10728 (1994)], and having a carboxyl-terminated flag polypeptide (i.e., Rse.gD). See Figures 30 and 31 for a schematic description of the assay.

(a) Manufacture of a captive agent

Monoclonal anti-gD (clone 5B6) was raised against a peptide derived from Herpes simplex virus glycoprotein D (Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)). The concentration of the purified stock solution was adjusted to 3.0 mg / ml in phosphate buffered saline (PBS), pH 7.4, and 1.0 ml samples were stored at -20 ° C.

(b) Preparation of Anti-Phosphotyrosine Antibody Monoclonal anti-phosphotyrosine, clone 4G10, was purchased from UBI (Lake Placid, NY) and biotinylated using long-arm biotin-N-hydroxysuccinamide (Biotin-X-NHS), Research Organics, Cl. ).

(c) Ligand ·· ·; ', · Mpl ligand was prepared using the one described herein! recombinant technology. The purified mpl ligand was stored at 4 ° C as a stock solution.

Preparation of Rse.gD Nucleic Acid. /. Synthetic double-stranded oligonucleotides were used to rearrange the coding sequence of the human Rse C-terminal 10 amino acids (880 - t · 890) and to add an additional 21 amino acids containing epi-% · * ··· for substance 5B6 and a stop codon. Table 10 · 1 · · V1 shows the final sequence of the synthetic portion of the fusion gene.

i «j · x: 44 · 9 ** I • 1 · • · • s • ·» 4 · 176

Table 10. Synthetic double-stranded portion of the human Rse fusion gene

Tape coding: 5'-TGCAGCAAGGGCTACTGCCACACTCGAGCTGCGCAGATGCTAGCCTCAAGA TGGCTG ATCCAAATCGATTCCGCGGCAAAGATCTTCCGGTCCTGTAGAAGCT-3 '(SEQ ID __NO: 59)

Non-sense coding strand: 5'-AGCTTCTACAGGACCGGAAGATCTTTGCCGCGGAATCGATTTGGATCAGCCA TCTTG AGGCTAGCATCTGCGCAGCTCGAGTGTGGCAGTAGCCCTTGCTGCA-3 ': (60)

Synthetic DNA was inserted into cDNA encoding amino acids 1 to 880 of human Rse starting from nucleotide 2644 of the human Rse cDNA sequence published at the PstI site [Mark et al., Journal of Biological Chemistry 269 (14): 10720-10728 (1994)] and the HindIII sites in the multi-link fragment of the expression vector pSVI7.ID.LL (see Figure 32 A-L; SEQ ID NO: 22) to generate the expression plasmid pSV.ID.Rse.gD. Briefly, the expression plasmid contains two primary copies containing two · · · · sistrons containing a sequence encoding DHFR, which is a 5′-stranded donor. and bound by the intron insertion sites of the 3 'stranding acceptor, followed by the sequence encoding Rse.gDin. Full length. . ·. The (non-threaded) messenger contains DHFR as the first open ♦ ♦ · reading frame and therefore produces DHFR to allow the selection of stable transformed products.

Preparation of Mpl-Rse.gD Nucleic Acid The expression plasmid pSV.ID.RSE.gD, produced as described above, was modified to yield the · · ·. ··· . plasmid pSV. ID.M.tmRd6 containing the human mpl ECD • ♦ * · 'coding sequences (amino acids 1-491) fused to the · · ·: *. * Rse.gD transmembrane domain and intracellular • ♦ · domain (amino acids) 429-911). Synthetic 177 oligonucleotides were used to insert the coding sequence of the extracellular domain portion of the human mpl into the portion of the Rse coding sequence in a two-step PCR cloning reaction as described by Mark et al., J. Biol. Chem., 267: 26166-26171 (1992). The primers used in the first PCR reaction were M1 (5'-TCTCGCTACCGTTTACAG-3 ') (SEQ ID NO: 61) and M2 (5'-CAGGTACCCACCAGGCGCaTCTCGGT-3j (SEQ ID NO: 62j mpl with cDNA template and R1). (5'-GGGCCATGACACTGTCAA-3 ') (SEQ ID NO: 63) and R2 (5'-GACCGCCACCGAGACCGCCTGGTGg? 3TACCTGTGGTCCTT-3') (SEQ ID NO: 64)

Rse with cDNA template. The PvuII-SmaI portion of this fusion junction was used to form the full-length chimeric receptor.

(f) Cell transformation DP12. CHO cells (EP Patent 307,247, published 15th June).

March 1989) was subjected to electroporation pSV.ID.-: ·. With M.tmRd learmed from a unique • · · * ·. ; Notl site in the plasmid backbone. The DNA was precipitated with ethanol after the m ··· / phenol / chloroform extraction and resuspended in 20 · · · l of 1/10 Tris-EDTA. Thus, 10 µg of DNA were incubated with • · · * · '* 107 CHO DP12 cells in 1 ml of PBS on ice for 10 minutes before electroporation at 400 V and 330 pF. Cells were transferred back onto ice for 10 minutes before being cultured in non-selective medium. Then, 24 hours later, · · · I .. cells were given nucleoside-free medium • · · · ** to select stable DHFR + clones.

V (g) Selection of transformed cells for use in: KIRA ELISA »· · 178

Clones expressing MPL / Rse.gD were identified by fractionating the whole cell lysis products by SDS-PAGE followed by Western blotting using antibody 5B6 which detects the gD epitope tag (tag).

(h) Culture media

Cells were grown in F12 / DMEM 50: 50 (Gibco / BRL, Life Technologies, Grand Island, NY).

The medium was supplemented with 10% diafiltered FBS (HyClone, Logan, Utah), 25 mmol / L HEPES and 2 mmol / L L-glutamine.

(X) KIRA ELISA

DP12.CHO cells transformed with Mpl-Rse.gD were cultured (3 x 10 4 cells / well) in flat-bottom 96-well dishes at 100 µl of medium and cultured overnight at 37 ° C in an atmosphere of 5% CO 2. The next morning, the wells of the wells were decanted and the plates lightly pressed with a paper towel. Then, 50 μΐ of medium containing either test samples or 200, 50, 12.5, 3.12, 0.78, 0.19, 0.048 or 0 ng / ml mpl ligand was added to each well. Cells were stimulated at 37 ° C for 30 minutes, wells were decanted, and plates were pressed again. lightly with a paper towel. For lysis of cells and chime • · ·. ·. : 100 μΐ lysis buffer was added to each well to dissolve the receptors. The lysis buffer contained 150 mM 1 NaCl containing 50 mM HEPES (Gibco), 0.5% Trit · · * · * ton-X 100 (Gibco), 0.01% thimerosal, KIU / ml of aprotinin (ICN Biochemicals, Aurora, OH), 1 mmol / 14 of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF; · · · · * ICN Biochemicals), 50 Mmol / L of leupeptin (ICN Biochemicals) and 2 mmol / L sodium orthovanadate (Na, VO; Sigma Chemical Co., St. Louis, MO), pH 7.5. The plate was then mixed with a gentle agitator (Bellco Instruments, Vineland, V NJ) for 60 minutes at room temperature.

• · * • mmm · As cells were lysed, an ELISA microtiter plate 179 (Nunc Maxisorp, Inter Med, Denmark) overnight at 4 ° C coated with 5B6 anti-gD monoclonal antibody (5 , 0 μg / ml in 50 mM carbonate buffer, pH 9.6, 100 μl / well), decanted and pressed with a paper towel and blocked with 150 μΐ / well Block Puffer Buffer [PBS solution, containing 0% BSA (Intergen Company, Purchase, NY) and 0.01% thimerosal] for 60 minutes at room temperature with gentle stirring. After 60 minutes, the dish coated with anti-gD 5B6 was washed 6 times with wash buffer (PBS solution containing 0.05% Tween-20 and 0.01% thimerosal) using an automated plate washer (ScanWasher 300, Skatron Instuments, Inc. ., Sterling, VA).

The lysed cell suspension containing the dissolved MPL / Rse.gD from the cell culture microtiter well was transferred (85 μΐ / well) to an anti-gD 5B6-coated and sealed ELISA well and incubated for 2 hours at room temperature with gentle agitation. Unbound mpl-Rse.gD was removed by washing with wash buffer and 100 μΐ biotinylated 4G10 (anti-phosphotyrosine) diluted 1: 18,000 in dilution buffer (PBS solution containing 0.5% BSA) was added to each well. 0.05% • ·

Tween-20, 5 mM EDTA and 0.01% thimerosal), i.e. 56 µg / ml. Incubated for 2 hours at room temperature, which was · · ♦; afterwards the plate was washed and 100 μΐ • · ♦ strepta-* * ♦ conjugated with horseradish peroxidase (HRPOtn) (Zymed Laboratories, S. San Francisco, CA) was diluted * * · * 'diluted 1: 60,000 in dilution buffer. . The plate was incubated for 30 minutes at room temperature with gentle agitation. The free avidin conjugate was washed and 100 μΐ of a freshly prepared substrate solution [tetramethylbenzidine (TMB) was added to each well. 2-pack • · · '···' substrate pack; Kirkegaard and Perry, Gaithersburg, MD).

The reaction was allowed to proceed for 10 minutes, after which the development of the · · ·: V color was stopped by the addition of 100 μΐ / well of 1.0 · · · M H3PO4. The absorbance at 450 nm was read at a reference wavelength of 650 nm (ABS / 450/650) using a vmax plate reader (Molecular Devices, Palo Alto, CA) controlled by Macintosh Centris 650 (Apple Computers, Cupertino, CA) and DeltaSoft (BioMetallics, Inc., Princeton, NJ).

A standard curve was generated by stimulating dpl2.trkA, B, or C.gD cells with 200, 50, 12.5, 3.12, 0.78, 0.19, 0.048 or 0 ng / ml mpl ligand, and the curve was plotted against ng / ml TP0 relative to the mean ABS450 / 650 ± standard deviation using DeltaSoft software. The concentrations of the samples were obtained by interpolation of their absorbance to a standard curve and expressed as TP0 activity in ng / ml.

It was found that the mpl ligand was able to activate the chimeric mpl-Rse.gD receptor in a concentration-dependent and ligand-specific manner. In addition, it was found that the mpl-Rse.gD KIRA ELISA tolerated up to 100% human serum (shown) or 100% plasma (not shown), allowing the assay to be used readily to screen patient and pK samples.

Standard curve of TP0332 produced by 293 cells 0.9-T-—; 0.8-j ': B Medium: * ·· 0.7-f: J 100% f * ·] · 0.6-human /: o: serum / ··· to 0.5- /

i:: δ: J

• LT £

/ ° -4i J

<0.3-f / • · · "Iff v: 0.2 it ™ \ = *** r% ·« · _ '** 0--1 i 1111ii | —Γττττπη — τ-ττττπη — r — τπτπη — i mun: * · *: P.01 0.1 1 10 100 1000 * ..! "> Tpn Concentration '··· *' (ng / ml) 181

Summary of TPO EC50s EC50 EC50 TP Form O (Cells) (w / v), (molarity) f

Hu TPO 332 (293) __ 2.56 no / ml__67.4 pM

Mu TPO 332 (293) __ 3.69 na / ml__97.1 pM

Hu TPO 153 (293) __- 41 na / ml __- 1.08 nM

Hu TPO 155 (E. coli) __ 0.44 na / ml__11.6 pM

Hu TPO 153met (E. coli) 0.829 na / mi__21.8 pM

Example 18

Thrombopoietin (TPO) receptor based ELISA

ELISA plates were coated with rabbit F (ab ') 2 anti-human IgG (Fc) in carbonate buffer at pH 9.6 at 4 ° C overnight. Plates were blocked with 0.5% bovine serum albumin in PBS • · · · • * at room temperature for one hour. Fermentation Product,: /.! containing the chimeric receptor, mpl-IgG, was added to the plates and incubated for 2 hours. Double serial dilutions (0.39-25 ng / ml) were added to the plates. standard (TP0332 produced in 293 cells and concentration ·; ·, determined by quantitative amino acid analysis) and serially diluted samples in 0.5% bovine serum albumin, 0 was added to the plates, 05% tt · * ··· * Tween 20 and incubated for 2 hours. Binding • ♦ · 'TPO was detected using protein A-purified, biotinylated rabbit antibodies against TP0155 produced in E. coli (1 hour incubation) followed by • streptavidin peroxidase (30 minutes). incubation) and • · ·: · '3,35,5' -tetramethylbenzidine as substrate. The absorbance was read at 450 nm. The plates were washed between steps. For data analysis, the standard curve was fitted using the Kaleidagraph four-parameter curve fitting program. Concentrations of samples were calculated from a standard curve.

Example 19 Expression and Purification of TPO from 293 Cells 1. Preparation of 293 Cell Expression Vectors By PCR, a cDNA corresponding to the entire open reading frame of TPO was obtained using the following oligonucleotides as primers.

Table 11. PCR primers for 293 cells

Cla.FL.F: 5 'ATC GAT ATC GAT CAG CCA GAC ACC CCG GCC AG 3' (SEQ ID NO: __65) hmpll-R: 5 'GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA 3' (SEQ. ID NO: _ 48)

The reaction was performed using PRK5-hmpl I (described in Example 9) as a template in the presence of pfu DNA polymerase (Stratagene). The first denaturation took place within 7 min at 94 ° C and was followed by 25 cycles of amplification (1 min at 94 ° C, 1 min at 55 ° C and 1 min at 72 ° C). The bulging extension occurred within 15 minutes at 72 ° C. The PCR product was purified and cloned into a plasmid

! ** .. between the restriction sites Clal and Xbal the vector pRK5tkneo.ORF

Whereby said plasmid pRKStkneo was from pRK5. derived vector that was modified to express neo · · · ·; ·. under the control of the thymidine kinase promoter of the mycine resistance gene. Another structure corresponding to the homolig Epo domain. nelma was constructed in the same way except using Cla.- • · · * ··· * FL.F as a forward primer and the following inverse primer: * · * 'Arg.STOP.Xba: 5'TCT AGA TCT AGA TCA CCT GAC GCA GAG GGT GGA CC 3 'm: T: (SEQ ID NO: 66) t' *. The final construct is called pRK5-tkneoEP0-D.

• · ·

The sequence of both constructs was found to be correct as described in * · ·: · * in Example 7.

These two constructs were transfected into human embryonic kidney cells using the CaPO4 method as described in Example 9. Then, 24 hours after transfection, the selection of neomycin resistant clones was started in the presence of 0.4 mg. / ml G418.10, then 15 days later individual colonies were transferred to 96 well plates and allowed to grow until confluence. The expression of ML153 or ML4 in the medium used for these clones was determined using the Ba / F3 mpl proliferation assay (described in Example 1).

3. Purification of rhML332

The spent medium of 293-32 → 32 was added to a Blue-Sepharose column (Pharmacia) equilibrated in 10 mM sodium phosphate, pH 7.4 (buffer A). The column was then washed with 10 column volumes of both buffer A and buffer A containing 2 mol / L urea. The column was then eluted with buffer A containing 2 mol / l urea and 1 mol / l NaCl. The Blue-Sepharose elution pool was then applied directly to the WGA-Sepharose column equilibrated in buffer A. The WGA-Sepharose column was then washed with 10 column volumes of buffer A containing 2 mol / L urea and 1 mol / L NaCl, and » · · * Eluted with the same buffer containing 0.5 mol / L N -acetyl · D -glucosamine. The WGA-Sepharose eluate was placed in C4. HPLC column (Synchrom, Inc.) equilibrated in · * · · · 0.1% TFA. The C4 HPLC column was eluted with a periodic gradient of propanol (0-25%, 25-35%, 35-70%).

• · · /; ·. It was found that rhML 4 was eluted in a gradient range of 28-30% propanol. Purified by SDS-PAGE. rhMLjjj is shifted as a broad band over the range of 68-80 kDa of the gel (see Figure 15).

Purification of rhML153: * · *: The medium used for 293-rhML153 was separated on Blue-X ** '; Sepharose column as described for rhML ^.

· · ·

The Blue-Sepharose eluate was directly applied to the mpl affinity column as described above. RhML153 eluted from the Mpl affinity column • · * · ♦ · * was purified to homogeneity using a C4 HPLC column under the same conditions as described for purification of rhML1. According to SDS-PAGE, purified rhML153 is distinguished into 2 major zones and 2 minor zones with Mr-Avon <18,000-21,000 (see Figure 15).

Example 20 Expression and Purification of TPO from CHO Cells 1. Expression of CHO Expression Vectors The expression vectors used in the electroporation methods described below are designated as: pSVI5. ID. LL.MLORF (full length or hTP0332) and pSVI5.ID.LL.MLEPO-D (truncated or hTP0153).

The relevant properties of these plasmids are shown in Figures 23 and 24.

2. Preparation of CHO Expression Vectors By PCR, a cDNA corresponding to the entire open reading frame of hTPO was obtained using the oligonucleotide dialogues of Table 12.

Table 12. PCR primers for the CHO expression vector

Cla.FL.F2 5 'ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G 3' (SEQ ID NO: __47) «· '. * · ORF. Sai 5 'AGT CGA CGT CGA CGT CGG CAG TGT CTG AGA ACC 3' (SEQ ID NO: f · · '• · · * 67) e · ·

PRK5-hmpl I was used as a template in the reaction. (described in Examples 7 and 9) in the presence of pfu DNA polymerase. lessa (Stratagene). The first denaturation took place within 7 minutes at 94 ° C and was followed by 25 cycles of multiple (1 min at 94 ° C, 1 min at 55 ° C and 1 min) · · · * ··· * 72 ° C). The final extension took place within 15 minutes at 72 ° C. The PCR product was purified and cloned between the plasmid: * · * · din pSVI5.ID.LL restriction site vectors * ··. rin pS VI5. ID. LL.MLORF to obtain. Another construct which · · · corresponded to a homologous EPO domain was constructed in the same manner except using Cla.FL.F2 as a forward primer and a · · · reverse primer: 185 EPOD.Sal 5 'AGT CGA CGT CGA CTC ACC TGA CGC AGA GGG TGG ACC 3' (SEQ ID NO: 68)

The final construct is called pSVI5.ID.LL.MLE-PO-D. The sequence of both constructs was found to be correct as described in Examples 7 and 9.

In essence, the coding sequences for the full-length and truncated ligand were inserted into the multiple cloning site of the CHO expression vector pSVI5.ID.LL. This vector contains the SV40 early promoter / enhancer region, a modified fusion unit containing the murine DHFR cDNA, a multiple cloning site to introduce the gene of interest (TPO sequences described herein), SV40 polyadenylation signaling and replication. for the selection and amplification of the plasmid of the beta-lactamase gene in bacteria.

3. Methodology for Creating Stable CHO Cell Lines Expressing Recombinant Human TP0332 and TP0153 a. Parent CHO Cell Line CHO Host Cell Line (Chinese Hamster Ovary Cell * * * * • line) used for the TPO molecules described herein. is known as CH0-DP12 (see EP 307 247, published March 15, 1989). This mammalian hand cell line was clonally selected from the parental line [CHO- «,. ·, K1 DUX-B11 (DHFR-) -, obtained from Dr. Frank Lee, Stanley, 'Y.' ford University, courtesy of Dr. L. Chasin] from transfection with the t · «* vector expressing preproinsulin to obtain clones with reduced insulin requirements. These cells are also DHFR-negative (DHFR-) and clones can be selected for the presence of 9 9 f '. * * DHFR cDNA vector sequences on the basis of growth in the absence of medium. nucleoside complete- «j ** ·. (glycine, hypoxanthine and thymidine). This selection system is commonly used for the stable expression of CHO cell lines.

Transfection Method (Electroporation) 186 TP0332 and ΤΡ0153 expressing cell lines were generated by transfecting DP12 cells with electroporation [see, e.g., Andreason, G.L., J. Tiss. Cult. Meth., 15, 56 (1993)] using respectively linearized pSVI5.ID.LL.MLORF or pSVI5.ID.LL.MLEPO-D plasmids. Three (3) restriction enzyme reaction mixtures were arranged per plasmid section; 10 pq, 25 μφ and 50 μq of vector with NOTI using standard molecular biology techniques. This restriction site is only detected once in the vector in the linearization region 3 · and outside the transcription units of the TPO ligand (see Figure 23). The 100 μ1: η reaction mixtures were then incubated overnight at 37 ° C. The following day, the mixtures were extracted once with phenol-chloroform-isoamyl alcohol (50: 49: 1) once and precipitated with ethanol on dry ice for approximately one hour. The precipitate was then collected by microcentrifugation for 15 minutes and dried. The linearized DNA was resuspended in 50 Miraan Ham's DMEM-F12 medium 1: 1 supplemented with standard antibiotics and 2 mmol / L glutamine.

The DP12 cells growing in suspension were harvested, washed • f * 9 kerran once in the medium described by resuspension of the DNA, and finally resuspended in the same medium at a cell concentration of 107: 750 cells / 750 μΐ. Cell samples (750 μΐ) and each mixture of unirradiated DNA were incubated at room temperature for one hour and then transferred to a BRL electroporation chamber. Each mixture was then held electro-. pairing in a standard BRL electroporation device at 350 V at 330 MF and low capacitance • · · * · | * there. After electroporation, the cells were allowed to equip. in tea for 5 minutes and then on ice for another 10 minutes * '*; during the incubation period. Electroporated cells were transferred to 60 mmm cell culture plates containing 4 ml of 5 ml of standard complete medium for CHO cell culture (glucose-rich DMEM-F12, 50 187: 50, glycine supplemented with 1 x GHT, 2 mmol / l glutamine, and 5% fetal calf serum) and grown overnight in a 5% CO 2 cell culture incubator.

c. Selection and screening method

The next day, cells were removed from the plates by trypsin using standard methods and transferred to 150 mm tissue culture plates containing DHFR selective medium (Ham's DMEM-F12, 1: 1 medium supplemented with either 2% or 5%). % dialyzed fetal calf serum but lacking glycine, hypoxanthine, and thymidine (this is the standard DHFR selection medium we use). Cells from each 60 mm dish were re-cultured in 5/150 mm dishes. The cells were then incubated for 10-15 days (one culture change) at 37 ° C / 5% CO 2 until the clones began to appear and reached a size suitable for transfer to 96-well plates. Then, after a period of 4-5 days, the cell lines were transferred to 96-well plates using sterile yellow tips. *. find in a pipettman set at 50 ml.

The cells were allowed to grow into a single plate (usually • · 3-5 days) and then the cells were removed with trypsin • · V ”and 2 copies of the original tray were re-produced in the delta. Two of these copies were briefly stored in a freezer, whereby cells in each well were diluted in 50 µl of 10% fetal calf serum in DMS0. Five days from serum-free culture medium used • · ·. ·: *. TP0 expression from Ba / F cells based on Ba / F cells was determined in third series from wells containing cells that had grown up to fusion.

• · · Based on this assay, the best expressing clones were recovered from the stock and expanded to two 150 mm. to a pooled T-flask for transfer to a cell culture group for suspension adjustment, reconfiguration and storage.

d. Amplification method

Several of the highest-titer cell lines from the selection described above were then passed through a standard methotrexic replication system to form higher-titer clones. CHO cell clones are expanded and cultured in 10-cm plates at four concentrations of methotrexate (namely 50 nM, 100 nM, 200 nM and 400 nM) and two or three (105, 5 x 105 and 106 cells / plate) cells. ). These cultures are then incubated at 37 ° C / 5% CO 2 until the clones are formed and are suitable for transfer to 96-well plates for further assays. From this selection, several higher titer clones were again exposed to higher concentrations of methotrexate (namely 600 nM, 800 nM, 1000 nM and 1200 nM), and as above, resistant clones are allowed to form and then transferred to 96-well plates and analyzed.

4. Cultivation of stable CHO cell lines expressing recombinant human TP0332 and TP0153

The deposited cells are thawed and the cell population is expanded using standard cell culture methods in either serum-free or serum-containing medium. After expanding to a sufficient cell density, the cells are washed to remove media used for cell culture.

• · ·. **: The cells are then cultured using any standard *. ·.! including the method: batch cultivation, feed batch cultivation: T: or continuous culture at 25-40 ° C, neutral pH, with a dissolved O 2 content of at least 5%. . *. because constitutively secreted TPO has accumulated.

· · ·

The cell culture fluid is then separated from the cells by mechanical means, such as centrifugation.

• Purification of recombinant human TPO from culture fluids of CHO • · · cells

The harvested cell culture fluid (HCCF) is directly applied • ·, ···. Blue-Sepharose 6 Fast Flow column (Pharmacia) equilibrated in 0.01 M Na phosphate, pH 7.4, containing 0.15 M NaCl at approximately 100-1189 cell culture medium (HCCF) / l of resin and at a linear flow rate of about 300 ml / h / cm2. The column is then washed with 3-5 column volumes of equilibration buffer followed by 3-5 column volumes of 0.01 M Na phosphate, pH 7.4 with 2.0 mol / L urea. The TPO is then eluted with 3 to 5 column volumes of 0.01 M Na phosphate, pH 7.4 with 2 mol / L urea, 1.0 mol / NaCl.

The Blue-Sepharose pool containing TPO is then added to a Wheat Germ Lectin Sepharose 6MB column (Pharmacia) equilibrated in 0.01 M Na phosphate, pH 7.4 containing 2.0 mol / L 1 urea and 1.0 mol / L NaCl, 8 to 16 ml Blue-Sepharose pool / ml resin at a flow rate of about 50 ml / h / cm 2. The column is then washed with about 2-3 column volumes of equilibration buffer. The TPO is then eluted with 2-5 column volumes of 0.01 M Na phosphate, pH 7.4, containing 2.0 mol / L urea, 0.5 mol / L N -acetyl-D-glucosamine. ·

The Wheat Germ Lectin Pool is then adjusted to a final concentration of 0.04% C12E8 and 0.1% trifluoroacetic acid (TFA). The resulting pool is applied to a C4 reverse phase column • ·] · /; (Vydac 214TP1022) equilibrated in 0.1% TFA and 0.04% C12E8 with a load of · · · *. . approximately 0.2 to 0.5 mg protein / ml resin at a flow rate of 157 ml / h / cm 2.

The protein is eluted in a two-phase linear gradient of · · · *. · * Consisting of acetonitrile containing 0.1% TFA and 0.04% C12E8. The first phase consists of: a linear gradient of 0 to 30% acetonitrile over 15 minutes. The second phase consists of a linear gradient of 30 to 60% acetonitrile in 60 minutes. TPO elutes at about 50% acetonitrile.

The pool is manufactured by SDS-PAGE.

: * · *: The C4 pool is then diluted with 2 volumes of 0.01 M

: ***; Na phosphate, pH 7.4, containing 0.15 mol / L NaCl, and diafiltered against approximately 6 volumes of 0.01 M Na phosphate, pH 7.4, 0.15 mol / L NaCl, Amicon YM 190 or equivalent ultrafiltration membrane with a molecular weight cutoff of 10,000 to 30,000 Daltons. The resulting diafiltrate can then be directly processed or further concentrated by ultrafiltration. The final Tween-80 concentration of the diafiltrate / concentrate is adjusted to 0.01%.

The diafiltrate / concentrate, in whole or in part, corresponding to 2 to 5% of the calculated column volume, is loaded on a Sephacryl S-300 HR column (Pharmacia) equilibrated in 0.01 M Na-phosphate, pH 7.4 containing 0.16 mol / L NaCl, 0.01% Tween-80, and chromatographed at a flow rate of approximately 17 ml / h / cm 2. Fractions containing TP0 that do not contain clumps or proteolytic degradation products are pooled by SDS-PAGE. The resulting combined product is filtered as a 0.22 μ η filter, with a Millex-GV filter or equivalent, and stored at 2-8 ° C.

Example 21 Transformation and Induction of TPO Protein Synthesis in E. coli Construction of the E. coli TPO Expression Vector Plasmid pMP21, pMP151, pMP41, pMP57 and pMP202 is • * ·· all designed to express the first 155: amino acids of TP0 between different constructs below a variable leader sequence. Leading sequences allow for a high degree of translational initiation and rapid purification. Plasmids pMP210-1, -T8, -21, -22, -24, -25 are designed to express the first 153 amino acids of TP0 below the start methionine and differ only in codon function. work for the first 6 amino acids of TP0, whereas plasmid pMP251 is a derivative of pMP210-1, in which the carboxy-terminated end of TPO is extended by two amino acids. All of the above plasmids produce to a large extent the intracellular expression of TP0 * .. * in E. coli after induction of the tryptone · · · ··· * fan promoter [Yansura, D.G. et al.,

Methods in Enzymology (ed. Goeddel, D.V.), 185: 54191-60, Academic Press, San Diego (1990)]. Plasmids pMP1 and pMP172 are intermediates in the construction of the above-mentioned intracellular expression plasmids of TPO.

(a) Plasmid pMP1

Plasmid pMP1 is the secretion vector of the first 155 amino acids of TPO and was constructed by inserting the 5 fragments of DNA as shown in Figure 33. The first was the vector pPho21, which had a small MluI-BamHI fragment deleted. pPho21 is a derivative of phGH1 [Chang, CN et al., Gene 55: 189-196 (1987)], in which the human growth hormone gene is replaced by the E. coli phoA gene and the MluI restriction site is engineered to encode the STII signal sequence at amino acids 20- 21.

The following two fragments, a 258 bp Hinf1-Pst1 DNA fragment from pRK5-hmpl I (Example 9) encoding amino acids 19-103 of TP0, and the following synthetic DNA encoding amino acids 1-18

5'-CGCGTATGCCAGCCCGGCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCG

TG

: ** · *: ATACGGTCGGGCCGAGGAGGACGAACACTGGAGGCTCAGGAGTCATTTGACGAAGC

ACTGA-5 '(SEQ ID NO: 69): (SEQ ID NO: 70) was primed with T4 DNA ligase, and the second site of surgery. · *. · With Pstl. The fourth was a 152 bp Pstl-Haell fragment derived from pRK5hmpl1 encoding amino acids 104-155 of TPO. The last was a 412 bp Stul-BamHI fragment derived from pdh108 containing lambda linked to a transcriptional terminator. , as previously described (Scholtissek, S. et al., NAR 15: * 3185 (1987)).

(b) Plasmid pMP21 • ·

Plasmid pMP21 was designed to express the first 155 amino acids of TPO by a 13 amino acid leader sequence containing part of the STII signal sequence. It was constructed by linking three (3) DNA fragments as shown in Figure 34, the first of which was the vector pVEG31, from which the small Xba I-Sph I fragment has been deleted. The vector pVEG31 is a derivative of pHGH207-1 (de Boer, HA et al., In Promoter Structure and Function (edited by Rodriguez, RL and Chamberlain, MJ), 462, Praeger, New York (1982)), with the human growth hormone has been replaced by the vascular endothelial growth factor gene (this identical vector fragment can be obtained from this latter vector).

The second part of the ligation was a synthetic DNA duplex having the following sequence:

5'-CTAG AATTAT G A AA AAG AATAT CGCATTTCTTCTTAA

TTAATACTTTTTCTTATAGCGTAAAGAAGAATTGCGC-5 '(SEQ ID NO: 71) \ (SEQ ID NO: 72)

The last song was 1072 from pMP! *: *. the Mlul-Sphl fragment encoding the TPO · · · · base. : 155 amino acids.

• ^ '(c) Plasmid pMP151 • · · *. . Plasmid pMP151 was designed to express the pre-• · · * / first 155 amino acids of TP0 below the leader sequence, whereby | The · · ·: · * leader sequence comprises 7 amino acids of the STII signal sequence, 8 · · · *. * 'Histidine and a factor Xa cleavage site. As shown in Figure 35, pMP151 was constructed by linking a chromosomal DNA fragment, the first of which was the previously described vector pVEG31, in which the small Xba I-Sph I fragment had been deleted. The second was a synthetic DNA duplex having • · · t .. the following sequence: i: · · ·

5'-CT AG AATT ATG AAAAAG AAT ATCGCATTTC AT C ACC ATCACCATCACCATCAC ATCG AAG

* · ·

GTCGTAGCC

• *

'· ♦ · * TTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTTC

CAGCAT-5 '(SEQ ID NO: 73) (SEQ ID NO: 74) 193

The last was a 1064 bp BglII-Sph1 fragment derived from pMP11 encoding the 154 amino acids of TPO. Plasmid pMP11 is identical to pMP1 except that it has a few codon changes in the STII signal sequence (this fragment can be obtained from pMP1).

(d) Plasmid pMP202

Plasmid pMP202 is very similar to the Express vector pMP151 except that the factor Xa cleavage site in the leader is replaced by the thrombin cleavage site. As shown in Figure 36, pMP202 was formed by fusion of three DNA fragments. The first was pVEG31, previously described, from which a small Xba I-Sph I fragment had been deleted. The second was a synthetic DNA duplex having the following sequence:

5'-CT AG AAH ATG AAAAAG AAT ATCGCATTT CATCACCAT CACCATCACCATCACATCG AA CCACGTAGCC

TTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTT

GGTGCAT-5 '.... (SEQ ID NO: 75) \ · [(SEQ ID NO: 76) • · · • · *. . The last fragment was a 1064 bp Bgll-SphI fragment from the previously described plasmid pMP11.

Plasmid pMP172 is a secretory vector of the first 153 amino acids of TP0 and is an intermediate for the formation of pMP210.

: As shown in Fig. 37, pMP172 was prepared by inserting ···: * · *; three DNA fragments, the first of which was the vector pLS321amB, from which a small Eco RI-HindIII moiety was removed. The second was a 946 bp EcoRI-Hgal fragment | · ··· * from the plasmid pMP11 described above. The last fragment was: ***: synthetic DNA duplex having the following sequence: 5'-TCCACCCTCTGCGTCAGGT (SEQ ID NO: 77) GGAGACGCAGTCCATCGA-5 '(SEQ ID NO: 77) 78) 194 (£) Plasmid pMP210

Plasmid pMP210 was designed to express the first 153 amino acids of TPO after translation initiation methionine. In fact, this plasmid was prepared as a library of plasmids in which the first 6 codons of TPO were randomized to the third position of each codon, and was constructed, as shown in Figure 38, by inserting three DNA fragments. The first was the previously described vector pVEG31, in which the small Xba I-Sph I fragment had been deleted. The second was a synthetic DNA duplex, shown below, which was first treated with DNA polymerase i (Klenow) and then digested with XbaI and Hinfl and encodes the TPO start methionine and the first 6 randomized codons.

5'-GCAGCAGTTCTAGAATTATGTCNCCNGCNCCNCCNGCNTGTGACCTCCGA

ACACTGGAGGCT

GTTCTCAGTAAA (SEQ ID NO: 79) CAAGAGTCATTTGACGA; W3CACTGAGGGTACAGGAAG-5 '(SEQ ID NO: 80) The third was an 890 base pair Hinfl-derived from pMP172.

Sph1 fragment encoding amino acids 19-153 of TP0.

The plasmid pMP210 library, containing approximately 3,700 clones, was re-transformed into LB plates containing tetracycline-rich ♦ ·· *. · · (50 μg / ml) to select large translational initiation clones [Yansura, DG et al. ., Methods: Companion to Methods in Enzymology 4: 151-1998 (1992)]. Of the eight colonies that appeared on plates containing tetracycline-rich five, · · · * · * "for TP0 expression were subjected to DNA sequencing and the results are shown in Figure 39 (SEQ. ID NOs:: *. ·, 23, 24, 25, 26, 27 and 28).

• ·. ···. (g) Plasmid pMP41 • · ·

Plasmid pMP41 was designed to express the first 155 amino acids of TPO fused to a leader sequence consisting 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 DNA fragments, the first of which was the previously described vector pVEG31, in which the small Xba I-Sph I fragment had been deleted. The second was the following synthetic DNA duplex: 5'-CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (SEQ ID NO: 81) TTAATACn I ICTTATAGCGTAAATAGCTTCCAGCAT-5 '(SEQ ID NO: 82)

The last fragment of the ligation was a 1064 bp BglII-SphI fragment from the previously described plasmid pMP11.

(h) Plasmid pMP57

Plasmid pMP57 expresses the first 155 amino acids of the TPO below the leader sequence, the leader sequence consisting of 9 amino acids of the STII signal sequence and the Lys-Arg base. This dibasic site allows the leader to be removed by ArgC. This plasmid was constructed as shown in Figure 41 by ligation of three pieces of DNA. The first of these was the previously described vector pVEG31, of which a small Xbal- • ·

The Sph1 fragment had been deleted. The second was the following synthetic duplex: * · • · · 5'-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAAACGTAGCC (SEQ ID NO: 83) «, TT AAT ACTTTTT CTT AT AGCGT AAAG AAG AATTTGCAT * 5 '(SEQ ID NO: 84 ) · · · • ··: The last part of the ligation was a 1064 bp Bgll-Sphl fragment from the previously described plasmid pMP11.

: (i) Plasmid pMP25l • · ·

Plasmid pMP251 is a derivative of pMP210-1 in which two additional amino acids of TPO are inserted at the carboxy terminus. As shown in Figure 42, this * ··· * plasmid was constructed by linking two DNA capsules; palettes, the first of which was previously described in pMP21, • ·. ·· *. from which the small Xbal-Apal fragment had been deleted. Another part of the ligation was the 316 bp Xba I-Apal fragment from pMP210-1.

196 2. Transformation and Induction of E. coli by TPO Expression Vectors

The above TPO expression plasmids were used to transform E. coli strain 44C6 (w3110 tonAA rpoHts ΙοηΔ clpPA GalE) using the CaCl2 thermal shock method [Mandel, M. et al., J. Mol. Biol., 53: 159-162 (1970)]. The transformed cells were first grown at 37 ° C in LB medium containing 50 mg / ml carbenicillin until the culture had an optical density (600 nm) of about 2-3. The LB culture was then diluted 20-fold in M9 medium containing 0.49% (w / v) casino acids and 50 mg / ml carbenicillin. The culture was grown under aeration at 30 ° C for 1 hour, after which indole-3-acrylic acid was added to a final concentration of 50 µg / ml. The culture was then allowed to continue growing at 30 ° C with aeration for a further 15 hours, after which the cells were harvested by centrifugation.

Example 22

Production of Biologically Active TPO (Mef'1-lSS) E.

• · ·: · 'in coli cell • ♦ *. *: The methods given below for the production of biologically active recombinant TPO (met-11-153) can be used uniformly to recover other TPO variants, including N and C. -terminal Pi * j *: denoted forms (see Example 23).

A. Recovery of Insoluble TPO (Met'11-153) E. coli Cells Expressing Plasmid pMP210-1-1 · ♦ II '.' encoded by TP0 (Met'111-153) is fermented as above. described. Typically, about 100 g of cells are resuspended in 1 µl (10 volumes) of cell lysis buffer (10 »'*: mmol / L Tris buffer, 5 mmol / L EDTA, pH 8). using a homogenizer and centrifuging the cells at 5,000 xg for 30 minutes. The washed cell pellet «· ** · is resuspended in 1 L of cell lysis buffer using a Polytron homogenizer and the cell suspension is transported with LH Cell. Disrupter (LH Inceltech, Inc.) or Microfluidizer (Microfluidics International) according to the manufacturer's instructions Centrifuge the suspension at 5,000 g for 30 minutes and resuspend and centrifuge a second washed refractile body. The washed pellet is used immediately or stored frozen at -70 ° C.

B. Dissolution and purification of monomeric TPO (Met'11-153)

The pellet obtained above is resuspended in 5 volumes of 20 mM Tris buffer, pH 8 with 6-8 mol / L guanidine and 25 mmol / L DTT (dithiothreitol), and the suspension is stirred for 1-3 hours. or overnight at 4 ° C to effect solubilization of the TPO protein. High concentrations of urea (6-8 M) are also useful but generally result in lower yields compared to guanidine. After solubilization, the solution is centrifuged at 30,000 x g for 30 minutes to obtain a clear supernatant containing • · *. denatured monomeric TPO protein. The supernatant liquid is then chromatographed on a Superdex 200 gel filtration column at a flow rate of 2 ml / min (Pharmacia, 2.6 x 60 cm) and the protein eluted with 20 mM Na phosphate, pH 6 The fractions containing denatured ··· * / · monomeric TPO protein eluted between 160 ml and 200 ml are then pooled. on a semiprep C4 reverse phase column (2 x ··· 20 cm VYDAC) The sample is added at a rate of 5 mL / min to column equilibrated in 0.1% TFA (trif • · · * ... * is eluted with a linear gradient of acetonitrile (20-60% in 60 minutes). Purified reduced protein is eluted when acetonitrile is used. is about 50% This material was reused to circulate the biologically active TPO to get the ant.

198 C. Formation of Biologically Active TPO (Met'11-153)

About 20 mg of monomeric, reduced and denatured TPO protein in 40 ml of 0.1% TFA / 50% acetonitrile is diluted in 360 ml of a circulating buffer, which contains optimally the following reagents: mmol / l Tris buffer 0.3 mol / l NaCl 5 mmol / l EDTA 2% CHAPS detergent 25% glycerol 5 mmol / l oxidized glutathione 1 mmol / l reduced glutathione adjusted to pH 8.3 .

After mixing, the refold buffer is gently stirred at 4 ° C for 12 to 48 hours to obtain maximum refold yields of the correct disulfide-bonded form of TPO (see below). The solution is then acidified with TFA to a final concentration of 0.2%, filtered with 0.45 or 0.22 micron e · *. *, through a filter and add 1/10 volume of acetonitrile.

This solution is pumped directly onto a C4 reversed phase column and * ** purified, re-twisted TPO (Met 1-153) is eluted. *: Using the same gradient program as above. Recirculation *. ·. * Now the active TPO is eluted with acetonitrile at about 45% under these conditions. False disulfide-linked variants of TPO elute earlier. The purity of high purity purified TPO (Met "11-153) is greater than 95% ··· as determined by SDS gels and analytical C4 reverse phase * · · • C4 purification for animal experiments. * the substance was dialyzed into physiologically compatible buffers.

• · · T ...: Isotonic buffers (10 mM Na acetate, pH: *. 5.5., 10 mM Na succinate, pH 55, or 10 mM Na. Phosphate pH 7.4) containing 150 mM NaCl and 0.01% Tween 80.

Due to the high potency of TPO in the Ba / F3 assay 199 (half maximal stimulation is achieved at a concentration of about 3 pg / ml), it is possible to obtain a biologically active agent using a variety of buffer, detergent, and oxidation-reduction conditions. However, under most conditions, only a small amount of correctly twisted material (<10%) is obtained. For commercial production processes, it is desirable that the yields of recycled TPO be at least 10%, more preferably 30-50%, and most preferably> 50%. The efficacy of many different detergents (Triton X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, sarcosyl, Tween 20 and Tween 80, Zwittergent 3-14, etc.) was reassessed to support high yields of fused TP0. Of these detergents, it was found that only the CHAPS group (CHAPS and CHAPSO) were generally useful in the TP0 recirculation reaction to limit protein accumulation and mis-disulfide formation. Concentration of CHAPS above 1% was most beneficial. Sodium chloride was needed to obtain the best yields at an optimum concentration of 0.1 mol / l to 0.5 mol / l.

· * "; The presence of EDTA (1-5 mol / l) limited some of the preparation. metal - catalyzed oxidation detected by these metals; (and accrual). Glycerol concentrations greater than 15% • · · *. . produced optimal re-twisting conditions.

• · 4 *. * To obtain maximum yields, both the oxidized i: ...

* · * That reduced glutathione or oxidized or reduced | · * * Cysteine as an oxidation-reduction reagent pair. Generally, higher yields were found when the molar ratio of oxidized reagent was equal to or greater than the reduced reagent member of the oxidation-reduction-paper. For circulating these TPO variants, the pH of 7.5 to about 9 · · · "... was optimal. The concentration of organic solvents (e.g., ethanol, *:“ · acetonitrile, methanol) may have been • · · · * Probably 10-15% or less Higher concentrations of organic solvents increased the amount of incorrectly twisted forms · · «Tris and phosphate buffers were generally useful Incubation at 4 ° C also correctly produced larger amounts of 200 twisted TPO .

Yields of re-twisted TPO of 40 to 60% (based on the amount of reduced and denatured TPO used in the re-twisting reaction) are typical in the case of TPO preparations purified through the first C4 step. The active agent can be obtained by using impure preparations (e.g. directly after the Superdex 200 column or after extraction of the first refractive unit), although lower yields are due to extensive precipitation of non-TPO proteins and interference during the TPO re-cycling process.

Since TPO (Mef111-153) contains 4 cysteine residues, it is possible to form three different disulfide variants of this protein: Variant 1: Disulfides between cysteine residues 1-4 and 2-3; Variation 2: Disulfides between cysteine residues 1-2 and 3-4; Variation 3: Disulfides between cysteine residues 1-3 and 2-4.

• ·: During initial investigations to set up a rewind. conditions were separated by C4 reverse phase chromatography. a number of different peaks containing TPO.

«« F

Only one of these peaks had significant biological s:: · activity as determined by Ba / F3 assay. Then, the * f · * · * 'rewind conditions were optimized to selectively produce that variation. Under these conditions, the misfolded variants represent less than 10-20% of the total amount of mono-esteric TPO obtained.

, ···, The disulfide model of biologically active TPO is determined to be 1 to 4 and 2 to 3 using mass spectrometry and protein sequencing (i.e., variant 1). Samples of various C4-separated peaks (5-10 nmol) were lysed with Tim Trypsin (1: 25 molar ratio of trypsin to protein). The degradation mixture was analyzed using matrix-assisted laser desorption mass spectrometry before and after reduction with 201 DTT. After reduction, masses corresponding to most of the larger tryptic peptides of TPO were observed. In unreduced samples, some of these masses were missing and new masses were observed. The masses of the new peaks basically corresponded to the sum of the individual tryptic peptides involved in the disulfide pair. Thus, it was possible to unequivocally demonstrate that the disulfide model of the recombinant, recombinant, biologically active TPO is 1 to 4 and 2 to 3. This is consistent with the closely related disulfide model of the molecule, erythropoietin.

D. Biological Activity of Recombinant Recirculated TPO (Met I-153)

Re-twisted and purified TPO (Met'111-153) has activity in both in vitro and in vivo assays. In the Ba / F3 assay, half-maximal stimulation was achieved by incorporation of thymidine into Ba / F3 cells at a concentration of 3.3 µg / ml (0.3 µM) of said TP0. In the Mpl receptor ELISA assay, half-maximal activity was observed at a concentration of 1.9 ng / ml (120 pM). In normal animals and animals with bone marrow i '. activity is attenuated by an almost lethal X-ray • 1 ... ...

; in that, TPO (Met 11-153) was very effective (activity * * was observed at doses as low as 30 t x ng / mouse) to stimulate the production of new platelets.

X * * ** * Example 23

Production of other biologically active TPO variants in E. coli

• M

A: Three different TPO variants produced by E.

* coli, purified and recycled into * · · * biologically active forms, are shown below.

# · *! '(1) MLF - 13 residues from the bacterial signal sequence STII are fused to the N-terminal do *' * 'of TPO (residues 1 to 155). . The resulting sequence is MKKNIAFLLNAYASPAPPAC ..... CVRRA (SEQ ID NO: 85)

wherein the synthetic leader sequence is underlined and C ..... C

202 represents Cys7 to Cys151. This variant was constructed to produce tyrosine for radioiodination of TPO for receptor and biological studies.

(2) H8MLF - 7 residues from the STII sequence, 8 histidine residues, and the Factor Xa enzymatic cleavage IEGR are fused to the N-terminal domain of TPO (residues 1 - 155). The sequence is MKKNIAFHHHHHHHHIEGRSPAPPAC ..... CVRRA (SEQ ID NO: 86) wherein the leader sequence is underlined and C ..... C represents residues Cys7-Cys151. This variant, purified and refolded, can be treated with Factor Xa, which cleaves the IEGR sequence after the arginine residue to produce a TPO variant with a length of 155 residues having a natural N-terminal serine amino acid.

(3) T-H8MLF - is prepared as described above for Variant (2) except that the thrombin sensitive sequence IEPR 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 3a C ..... C represents the residues Cys7-Cys151. This variant, after purification and recirculation, can be treated with thrombin: to produce a natural N-terminal variant of TPO having a length of 155 residues.

i 1!

Recovery, solubilization and purification of monomeric biologically active TPO variants * (1) / (2) and (3) All variants were expressed in E. coli. Most of the variants were found in the refractive bodies, as observed in Example 22 for TPO (Met 1-153). Sessa. Similar procedures were employed to recover, dissolve, and purify the M · Ί * monomeric TPO variants as described in Example 22. Similar re-circulation conditions were used as in the case of TPO (Met'11-1-153) with total yields of 30-50%. After recirculation, the TPO variants were purified by C4-203 reverse phase chromatography in 0.1% TFA using an acetonitrile gradient as previously described. All TPO variants (in their non-proteolysed forms) had biological activity as determined by the Ba / F3 assay, with half-maximal activities of 2 to 5 pM.

B. Proteolytic Processing of Variants (2) and (3) to Produce Right N-terminal TPO (1-155)

The TPO variants (2) and (3) described above were designed to have an enzymatically cleaved leader peptide before the normal N-terminal amino acid residue of TPO. Variants (2) and (3) were recirculated and purified as described above, followed by digestion with a suitable enzyme. For each variant, the acetonitrile from the C4 reverse phase step was removed by carefully blowing nitrogen onto the solution. The two variants were then treated with either factor Xar or thrombin as described below.

In the case of TPO variant (2), 1 M Tris buffer, pH: *** value 8 was added to the acetonitrile-free solution so that a final concentration of 50 mM was obtained and the pH adjusted to 8, if necessary. . NaCl was added to a concentration. : 0.1 M and CaCl 2 were added to a concentration of 2 mM. Factor Xa (New England Biolabs) was added such that the enzyme and • · · .V. mole ratio of variant was about 1: 25 to 1: · · · · 100 · The sample was incubated at room temperature for 1 to 2 hours to achieve maximal cleavage, which was determined as the change in migration on SDS gels. · · Representing the loss of leader sequence. Thereafter, the reaction mixture. ·: *. was purified by C4 reverse phase chromatography using •. ···. the same gradient and the same conditions as described above in connection with the purification of the circulating variants • · ·: The non-cleavable variant B was separated from the cleaved variant (2) by these conditions.

204 N-terminal amino acids were found to be SPAPP, indicating successful removal of the N-terminal leader sequence. Factor Xa also produced varying amounts of internal cleavage within the TPO domain; cleavage was observed after the arginine residue at position 118, yielding an additional N-terminal sequence TTAHKDP · (SEQ ID NO: 88). With unreduced SDS gels, a single band was detected in the case of the factor Xa cleaved variant at about 17,000 daltons; two bands were observed on the reducing gels with molecular weights of about 12,000 and 5,000 daltons, which is consistent with cleavage at arginine 118. This observation confirmed that the two moieties were held together by a disulfide bond between the 1st and 4th cysteine residues, as in derived from the tryptic digestion experiments described above. In the biological Ba / F3 assay, the purified TPO (1-155) variant had a half-maximal activity of 0.2-0.3 picomolar after removal of the N-terminal leader sequence and in the presence of internal cleavage. In the intact variant having the leader sequence, the half-maximal activity was 2-4 picomolar.

In the case of variant (3), the digestion buffer consisted of 50: * · · mmol / L Tris buffer, pH 8, 2% CHAPS detergent, · 0.3 mol / L NaCl: a, 5 mmol / l EDTA and human ·. : or bovine thrombin (Calbiochem), whereby the ratio of ent · · · / ··, enzyme to TPO variant protein was 1: 25 to 1: 50 • · · by weight. Digestion was performed at room temperature for 2 to 6 hours. Digestion progression was determined by SDS gels as described above for the factor Xa cleavage reaction. In general, over this time, more than · · ·: · · · 90% of the leader sequence was cleaved. The resulting TPO was purified on C4 reverse phase columns as described above and ···. was found to be the desired N-terminal according to S · * · * amino acid sequencing. Only very small (<5%) amounts of internal • · · • · ·: * cleavage were obtained for the same arginine-threonine bond, · ·· as noted above for factor Xa. The resulting TPO protein 205 had high biological activity with half maximal responses occurring at a concentration of 0.2-0.4 picomolar in the Ba / F3 assay. In the Mpl receptor-based ELISA assay, this protein had a half-maximal response with 2-4 ng / ml purified protein (120-240 picomolar), whereas the intact variant containing the leader sequence was 5-10 times less potent in each assay. For animal experiments, HPLC-purified digested protein was dialyzed into physiologically acceptable buffers containing 150 mM NaCl, 0.01% Tween 80 and 10 mM sodium succinate, pH 5.5, or 10 mM 1 sodium acetate, pH 5.5, or 10 mmol / l sodium phosphate, pH 7.4. Purified protein by HPLC and SDS gels was stable for several weeks when stored at 4 ° C. In normal mice and in mice with impaired bone marrow function, this purified TPO with the correct N-terminal sequence was very active in stimulating platelet production at doses as low as 30 ng / mouse.

Example 24! * · *: Synthetic mpl ligand · * · · Although human mpl ligand (hML) is usually prepared by recombinant methods, it can also be. : to synthesize synthetic peptide fragments by enzymatic ligation using the methods described below. Synthetic production of hML permits the incorporation of non-natural amino acids or synthetic functionalities such as polyethylene glycol. Previously, serine pro:, i .: subtilisin BPN mutant of theasase subtiligase V · (S221C / P225A) was engineered to efficiently incorporate peptide esters φ. ·; ·. aqueous solution [Abrahmsen et al., Biochem. , 30: 4151-4159 (1991)]. It has now been shown that synthetic peptides can 2.

*! * enzymatically linked enzymatically • · ·: V active long peptides and proteins such as ribonuc

»M

leaase A (Jackson et al., Science (1994)).

206 This technology, which is described in more detail below, has enabled us to synthesize chemically long proteins that previously could only be made by recombinant DNA technology.

A general procedure for synthesizing hML153 using subtiligase is shown (Scheme 1). Starting with a completely unprotected peptide corresponding to the C-terminal fragment of the protein, the N-terminal protected, C-terminal activated ester peptide is added together with the subtilase. After the reaction is complete, the product is isolated by reverse phase HPLC and the protecting group is removed from the N-terminus. The following peptide fragment is annealed, deprotected and the process repeated using successive peptides until a full-length protein is obtained. The process is similar to the solid phase methodology where the N-terminally protected, C-terminally activated peptide is linked to the N-terminus of the previous peptide and the protein is synthesized in the C-to-N direction. However, since each coupling results in up to 50 residues and products are isolated Afterwards, much longer, highly purified proteins can be synthesized with yields in the spring.

• · • • • ♦ ♦ ♦ ♦ ♦ ♦ ♦ t t t t t t t t t t t t t • 1 2 3 · • *: 2: ·: • • • • • • • • • • • • • • • • • • • • •: · · 207

Scheme 1. Procedure for synthesizing hML using subtiligase R-NH-peptide '2-CO-R' + H2N-peptide -j-CO2

| 1) SUBTILIGAASI

R-NH-peptide 2-CO-NH-peptide ^ -CO2

J, 2) Zn / CH 3 CO 2 H

H2N Peptide 2-CO-NH Peptide ^ 002, r 3) Repeat '1 + 2 H 2 N Peptide 3rCO-NH Peptide 2 "CO-NH Peptide - | -CO 2

0 o

R = R, = ^ ο-νΝΗγ ^ ΝΗ2 · 'O (CH2) 4 • · · ·: V NH2 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • ·: •: ·: ·:

Based on our knowledge of the sequence specificity of subtiligase: and biologically active hML. "epo-domain" amino acid sequence, we split the hML153 into seven fragments of 18-25 residues. Test ligation tetrapeptides were synthesized to determine appropriate ligation junctions at 18-25 µm. Table 13 shows the results of these experimental ligations.

208

Table 13. Test ligations for hML. The donor peptides and nucleophilic peptides were dissolved at a concentration of 10 mM in 100 mM tricine (pH 7.8) at 22 ° C. The ligase was added to give a final concentration of 10 μΜ from a stock solution of 1.6 mg / ml (7070 μΜ) and the ligation was allowed to proceed overnight. Yields are based on% ligation of donor peptides compared to hydrolysis.

Location Luavatus- (glc-K-NH2) Nucleophile · νη2 Hydrolysis% __% Ligation and. - - --- - = 1 (23/24) HVLH] SRLS 9 2 0 8 _j (SEQ ID NO: 89) __ (SEQ ID NO: 90) ___ (22/23) SHVL HSRL 48 52 _ (SEQ ID NO: 91) __ (SEQ ID NO: 92) ___ 2 (46/47) AVDF SLGE 2 2 7 8 __ (SEQ ID NO: 93) __ (SEQ ID NO: 94) ___ 3 (69/70) A VTL LLEG 5 3 4 7 _i (SEQ ID NO: 95) __ (SEQ ID NO: 96) ___ 4 (89 / 9I0) LSSL [_GQL 9 5 0 5 _! (SEQ ID NO: 97) (SEQ ID NO: 98} _ _ • · ·!: · '(88/89) C (acm) LSS LLGQ 0 0 0 0 * · __ (SEQ ID NO: 991 __ (SEQ ID NO: 100) ___: '·· (90/91) SSLL GQLS 4 5 5 5 V ·: __ (SEQ ID NO: 101) __ (SEQ ID NO: 102) ____ (88/89) CLSS LLGQ 90 10 | T: __ (SEQ ID NO: 103) (SEQ ID NO: 100) ___ 5 (107/1 08) LQSL .LGTQ 9 9 .01. __ (SEQ ID NO: 104) V> (SEQ ID NO: 105) ___ • · · (106/107) ALQS \ LLGT 7 0 3 0 __ (SEQ ID NO: 1061 __ (SEQ ID NO: 107) ___ * * · 6 (128/129) NAIF LSFQ 60 40 ··· '* · ; · _I (SEQ ID NO: 108) (SEQ ID NO: 109) __ • · 1 1 9 9 9 9 209

Based on these experiments, the subtiligase should efficiently couple the ligation peptides shown in Table 14. To prevent self-attachment, a suitable protecting group was required to protect the N-terminus of each donor ester peptide. We chose the isonicotinyl protecting group (iNOC) [Veber et al., J. Org. Chem., 42: 3286-3289 (1977) because of its water solubility, it can be added in the final step of solid phase peptide synthesis and it is stable to the anhydrous HF used to deprotect and cleave the peptides from the solid phase resin. In addition, it can be removed from the peptide after each ligation under mildly reducing conditions (Zn / CH 3 CO 2 H) to produce the free N-terminus for subsequent ligations. The glycoate-lysyl amide ester (glc-K-NH 2 ester) was used to activate the C-terminus based on previous experiments which demonstrated that subtiligase acylated it effectively [Abrahmsen et al., Biochem., 30: 4151-4159 (1991). ]. Glc-K-amide-activated peptides protected by an iNOC protecting group can be synthesized using standard solid phase methods as outlined (Scheme 2). The peptides are then sequentially coupled until a complete protein is obtained and the final product is recycled in. : in vitro. Based on homology to EP0, the disulfide pairs • · · ... 1 are believed to be formed between cysteine residues 7 and 151 and • · ·. between cysteine residues 28 and 85. The oxidation of the disulfides can be accomplished simply by stirring the reduced material in an oxygen atmosphere for several hours.

f 2 3 1 V 2 The recycled material can then be purified by HPLC and the fractions containing the active protein are combined and freeze-dried. Alternatively, disulfides may be protected in various ways to prevent sequential oxidation between specific pairs of disulfides. Removal of the cysteines 7 and 151 by acetamidomethyl groups (acm groups) would ensure oxidation of residues 28 and 85 of i: 3. The acetamidomethyl groups • could then be removed and residues 7 and 151 oxidized.

: 4: Otherwise, residues 28 and 85 could be protected by acetamidomethyl groups and oxidized if sequential oxidation is required due to correct rotation. Optionally, cysteines 28 and 85 may be substituted by another natural or non-natural residue other than Cys to assure proper oxidation of cysteines 7 and 151.

Table 14. Peptide fragments used for total synthesis of h-ML using Fragment Sequence 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-SLGEWKTQMEETKAQDILGAVTL-glc-K-NH2 (47-69) • · · · · · · 4 ( SEQ ID NO: 113) • · • · • · \. iNOC-HN-LLEGVMAARGQLGPTCLSSLL-glc-K-NH2 (70-90) • · · «· · •: t: 5 (SEQ ID NO: 114) 2 · · iNOC-HN-GQLSGQVRLLLGALQS-glc-K-NH2 ( 90-106) • · · · · · · 6 (SEQ ID NO: 115) »··· • · · iNOC-HN-LLGTQLPPQGRTTAHKDPNAIF-glc-K-NH2 (107-128) t: • · · 7 (SEQ ID NO: 116) • · • · ♦ ♦ ♦ • · '**** H2N-LSFQHLLRGKVRFLMLVGGSTLCVR-CO2 (129-153) 211

Peptide ligations are performed at 25 ° C in 100 mM Tricine, pH 8 (freshly prepared and degassed by vacuum filtration through a 5 μΜ filter). Typically, the C-terminal fragment is dissolved in buffer (2 to 5 mM peptide) and 10-fold subtiligase stock solution (1 mg / ml in 100 mM Tricine, pH 8) is added to give a final enzyme concentration of 55 μΜ. 3-5 molar excess of glc-K-NH2-activated donor peptide as a solid is then added, dissolved and allowed to stand at 25 ° C. The ligations are monitored by reverse phase C18 HPLC (CH3CN / H2O gradient with 0.1% TFA). The ligation products are purified by preparative HPLC and freeze-dried. Removal of the isonicotinyl protecting group (iNOC protecting group) was accomplished by mixing HCl-activated zinc dust with a protected peptide in acetic acid. The zinc dust was removed by filtration and the acetic acid was evaporated in vacuo. The resulting peptide can be used directly in the subsequent ligation and the process is repeated. Synthetic hML153 may be ligated using methods consistent with those described above for synthetic or recombinant hML15, 32; 5 to produce synthetic or semisynthetic full-length • · *. * ·· hML.

Synthetic hML has many advantages over recombinant. Unnatural side chains can be added to it for improved power or specificity. Can be added ··· t *: ·. polymer functionalities such as polyethylene glycol to improve the duration of action. For example, polyethylene glycol. li may be attached to the lysine residues of individual fragments (Table 14) before or after one or more ligation steps. Protease-sensitive: *! *: Peptide bonds may be removed or altered to improve t '' stability in vivo. In addition, heavy metal derivatives may be synthesized to facilitate the determination of the structure.

• · • · • · 212

Scheme 2. Solid phase synthesis of peptide fragments to effect segmentation ligation

0:] O

h2jN '\ ^^ \ / mbha \ aj b I \ HART SI / -1 - * - ΰΓ Π T VHARTSI / -► R | O R V_y 1 2 o o c ^ ^ (automated synthesis) R OR ^ 3

O O

NH C, f (cleavage) H2N li I r T] f | \ resin) —-- ►

OR OR V_S

4 i ^ o \ H * PEPTIDI \ ™ r ^ o ^ mr & m2 5 OR O (CHj) 4 I l I ** »i

: *: I. . I

• · j ·. : Isonicotinyl (iNOC) glycolate-lys ^ over-amide. (glc-K-NHo) • (a) Lysyl paramethylbenzhydrylamine resin 1 h: • (MBHA resin) (0.63) meq / g, Advanced ChemTech) is mixed with bromoacetic acid (5 eq) and diisopropylcarbodiimide (5 V: eq) for 1 hour at 25 ° C in dimethyl ·· * V: Acetamide (DMA), the bromoacetyl derivative 2 b) Thoroughly wash the resin with DMA and esterify the single Boc protected amino acids (3 equiv., Bachem) with 1 · T by mixing them with sodium bicarbonate (6 equiv.). Methylformamide (DMF) at 50 ° C for 24 hours to afford the corresponding glycolate-phenylalanyl-amide resin 3. The aminoacetylated resin 3 is washed with DMF (3x) and dichloromethane (CH 2 Cl 2) (3x) and can be stored at room temperature for several months. The resin 3 can then be loaded onto an automated peptide synthesizer (Applied Biosystems 430A) and the peptides are eluted using standard solid phase methods (5). The N-α-Boc group is removed with a 45% trifluoroacetic acid solution in CH 2 Cl 2. d) The following Boc-protected amino acids (5 eq.) are preactivated using benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP, 4 eq.) and N-methylmorpholine (NMM. 10 eq.) in DMA and coupled for 1-2 hours. e) Removal of the final α-α-Boc group (TFA / CH 2 Cl 2) to give compound 4 and addition of the isonicotinyl protecting group (iNOC protecting group) as described above (4) by stirring 4-isonicotinyl-2-4-dinitrophenyl -carbonate (3eq) and NMM (6eq) in DMA at 25 ° C for 24 hours. f) Peptide cleavage and deprotection by treatment with anhydrous HF (5% anisole / 5% ethylmethylsulfide) at 0 ° C for 1 hour gives an iNOC-protected glycolate-lysamide-activated peptide 5 which is purified by reverse phase C18. By HPLC (CH 3 CN / H 2 O gradient, 0.1% TFA). The identity · ** · * of all substrates is confirmed by a mass spectrometer.

• *! ** Supplemental Power of Attorney The invention, as claimed, is warranted by the foregoing patent specification and readily available: T: references and starting materials. Nonetheless, the Applicants have deposited with the American Type Culture, Collection, Rockville, Md., USA (ATCC) a cell line ··· shown below: »» ·

Escherichia coli, DH10B-pBSK-hmpl 1 1.8, ATCC acces- · »· V: sion no. CRL 69575, deposited February 24, 1994.

• · ·

Plasmid, pSVI5. ID. LL.MLORF, ATCC Accession no. CRL 75958, deposited December 2, 1994; and «fr

CHO DP-12 cells, ML 1/50 MCB (labeled # 1594), ATCC

• · «« · 214 accession no. CRL 11770, deposited December 6, 1994.

This deposit was made under the terms and conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of the Patent Procedure (Budapest Treaty). This ensures that the live culture is maintained for 30 years from the deposit date. The organisms are made available by the ATCC under the Budapest Treaty and subject to a contract between the patent applicants and the ATCC, which guarantees unrestricted availability after the publication of the relevant US patent. The availability of the deposited stock shall not be construed as a license to practice the invention, contrary to the rights granted to any government under its patent laws.

Although the invention has necessarily been described in connection with preferred embodiments and specific working examples, one skilled in the art, having read the patent specification above, will be able to make various modifications, substitutions of equivalents, and modifications of the subject matter disclosed herein without departing from its spirit and scope. Thus, the invention may be practiced in ways other than those specifically described herein. It is therefore intended that the protection afforded by this patent is limited only by the appended claims and their equivalents.

«·, All references cited herein are appended to this name-

* · V

· * ♦ /; ·. as a reference.

* · · * * I · · • · ♦ 4 ** * ♦ · * · · rf »» ·· «* I t • · * * * * * ** · * * ♦ • ·» »> f * • · · «t 4 9« «« ·

Claims (34)

A process for the preparation of an isolated mpl ligand polypeptide having the amino acid sequence SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO: 29) or SPAPPACDLRVLSKLLRDSHVLHSRL, characterized in that the mpl-ligand polypeptide is Method according to claim 1, characterized in that the isolated mpl ligand polypeptide is encoded by a nucleic acid having a sequence which hybridizes under moderate stringency to a nucleic acid having the sequence consisting of nucleotides 119-196 of Figure 9 (SEQ ID NO: 4 or SEQ ID NO: 2). 5). Method according to claim 1 or 2, characterized in that the portion of the human mpl ligand has the sequence hML (1-153), hML (7-151), hML (1-191), hML (1-205) or hML ( 1 -217). The method according to claim 3, characterized in that the portion of the human mpl ligand contains an N-terminated methionine. • · · • · · · · Method according to claim 1, characterized in that the mpl ligand polypeptide is modified at the amino acid sites R153-R154 and / or R245-R246 of the mpl ligand according to Figure 14. The method according to claim 5, characterized in that the cleavage site at the amino acid site R153-R154 of the mp1- · · * ligand polypeptide is ... inactivated by substitution. The method of claim 6, wherein the mpl-: T: at position R153-R154 of the ligand polypeptide is substituted by an Ala residue. The method according to any one of claims 1 to 7, characterized in that the mpl ligand polypeptide binds to a non-protein based polymer. • ♦ ♦ · • ♦ ♦ Process according to claim 8, characterized in that said polymer is polyethylene glycol, polypropylene glycol or polyoxyalkylene. The method according to any one of claims 1 to 9, characterized in that the mpl ligand polypeptide is fused to a heterologous polypeptide. The method of claim 10, wherein said heterologous polypeptide is EPO, G-CSF, IgG or IL-3. A method according to claim 10, characterized in that said amino acid sequence is SEQ ID NO: 85, 86 or 87. The method according to claim 10, characterized in that it comprises a portion of the mpl ligand (SEQ ID NO: 1) or mpl ligand (SEQ ID NO: 9) of Figure 1. The method of claim 10, characterized in that it comprises a portion of the mpl ligand of Figure 1 (SEQ ID NO: 1) and that said portion of the mpl ligand of Figure 1 (SEQ ID NO: 1) has the structure: MPPA (SEQ ID NO: 1). NO: 117) MAPPA, (SEQ ID NO: 118): * · · M PAPPA, (SEQ ID NO: 119) y / MS PAPPA, (SEQ ID NO: 120): APPA, (SEQ ID NO: 121) PAPPA, (SEQ ID NO: 122) spappa, (seq id no: 123) VRRA, (SEQ ID NO: 124) VRRAP, (SEQ ID NO: 125); VRRAPP, (SEQ ID NO: 126) *: ***: VRRAPPT, (SEQ ID NO: 127) VRRAPPTT, (SEQ ID NO: 128) VRRAPPTTA, (SEQ ID NO: 129) VRRAPPTTAV, (SEQ ID NO: 130) ) i 'VRRAPPTTAVP, (SEQ ID NO: 131) VRRAPPTTAVPSR, (SEQ ID NO: 132) VRRAPPTTAVPSRT, (SEQ ID NO: 134) VRRAPPTTAVPSRTS, (SEQ ID NO: 135) VRRAPPTTAVPSL, (SEQ ID NO: 136) VRRAPPTTAVPSRTSLV, (SEQ ID NO: 137) VRRAPPTTAVPSRTSLVL, (SEQ ID NO: 138) VRRAPPTTAVPSRTSLVLT, (SEQ ID NO: 139) VRRAPPTTAVPSRTSLVLTL, (SEQ ID NO: 140) VRRAPPTTAVN 141) VRRAPPTTAVPSRTSLVLTLNE, (SEQ ID NO: 142) VRRAPPTTAVPSRTSLVLTLNEL, (SEQ ID NO: 143) VRRAPPTTAVPSRTSLVLTLNELP, (SEQ ID NO: 144), one or more of residues 176-332 of the human ML or SEQ ID NO: 1. : 1). The method of claim 11, characterized in that it comprises a portion of the mpl ligand of Figure 1 (SEQ ID NO: 1) and that said portion of the mpl ligand of Figure 1 (SEQ ID NO: 1) has the structure: SPAPPACDLRVLSKLLRDSHVL, (SEQ ID NO: 1). NO: 110) HSRLSQCPEVHPLPTPVLLPAVDF, (SEQ ID NO: 111) SLGEWKTQMEETKAQDILGAVTL, (SEQ ID NO: 112) LLEGVMAARGQLGPTCLSSLL, (SEQ ID NO: 113) GQLSGQVRLLLGALQS, (SEQ ID NO: 114T) (SEQ ID NO: 114)! : 115) LSFQHLLRGKVRFLMLVGGSTLCVR, (SEQ ID NO: 116) • · ·. and a combination thereof. The method according to claim 10, characterized in that it contains * · * a portion of the mpl ligand of Figure 1 (SEQ ID NO: 1) and said portion of the mpl of Figure 1. - the ligand (SEQ ID NO: 1) contains at least one of the following amino acids of SEQ ID NO: 1: 1-3, 6-6, 9, 13-15, 17-28, 30, 32-34, 36-41, ·: **: 43-50, 53-59, 61-68, 71, 73, 75-80, 82-88, 90-96, 98-99, 102-103, 105-107,. ·! ·. 110, 112-113, 115-119, 121-130, 132-133, 135, 137-140, 143-150, and 152-156 • · ·. ···. and combinations thereof. The method according to claim 10, characterized in that it comprises a portion of the mpl ligand of Figure 1 (SEQ ID NO: 1) and that said portion of the mpl ligand of Figure 1 (SEQ ID NO: 1). : 1) contains in its structure at least one of the amino acids of SEQ ID NO: 1: 7-8,10-12, 16, 29, 31, 35, 42, 51-52, 60, 69, 72, 74, 81, 89, 97, 100-101, 104, 108-109, 111, 114, 120, 131, 134, 136, 141-142 and 151, and combinations thereof. The method according to claim 10, characterized in that it comprises a portion of the mpl ligand of Figure 1 (SEQ ID NO: 1) and that said portion of the mpl ligand of Figure 1 (SEQ ID NO: 1) contains the sequence of SEQ ID NO: 1. at least one of these amino acids 111-114, 139-140, 142-144 and 147-150, and combinations thereof. The method of claim 10, characterized in that it comprises a portion of the mpl ligand of Figure 1 (SEQ ID NO: 1) and that said portion of the mpl ligand of Figure 1 (SEQ ID NO: 1) contains the sequence of SEQ ID NO: 1. amino acids encoded by at least one of these: exon 1, exon 2, exon 3, exon 4, exon 5 and exon 6 according to Figures 14A, 14B and 14C (SEQ ID NO: 1), and combinations thereof. A method according to claim 10, characterized in that it comprises a portion of the mpl ligand of Figure 1 (SEQ ID NO: 9) and that said portion of the mpl ligand of SEQ ID NO: 9 (SEQ ID NO: 1) contains the sequence SEQ ID NO: ID NO: 9 of at least one of the amino acids 1-2, 5-8, 10, 12, 13-20, 22-30, 32, 34-35, 38-54, 56, 59-67, 69-81, 83 , 85-105, 107-113, 115-126, 128-130, 132, 134-145, 147, 149, and 151-153, and these. : combinations. • ·· • · • 9 • · • · ·. 21. The method of claim 10, wherein said? · · * · / Heterologous polypeptide comprises an immunoglobulin chain or portion thereof. The method of claim 21, wherein said heterologous polypeptide comprises IgG, IgM, IgA, IgD, IgE, or a portion thereof. • 9. ···. The method of claim 22, wherein said heterologous polypeptide comprises IgG1, IgG2, IgG3, IgG4, or a portion thereof. • 9 9 9 9 99 9 An isolated nucleic acid molecule, characterized in that it encodes a polypeptide produced by the method of any one of claims 1-23. The isolated nucleic acid of claim 24, wherein the nucleic acid is operably linked to a promoter. The isolated nucleic acid of claim 24 or 25, wherein the isolated nucleic acid has a sequence which hybridizes under moderately stringent conditions to a nucleic acid having the sequence of nucleotides 119-196 of Figure 9 (SEQ ID NO: 4 or SEQ ID NO: 5) and encodes an mpl ligand or portion thereof having at least one biological property of said mpl ligand. The expression vector 27, characterized in that it contains the nucleic acid of any one of claims 24 to 26. A host cell, characterized in that it is modified by an expression vector according to claim 27. A method for producing an mpl ligand, which comprises expressing the nucleic acid of any one of claims 24 to 26 in a suitable host. · *. *; The method of claim 29, wherein the mpl ligand polypeptide is derived from a host cell or a culture of a host cell:. * media. The method according to claim 30, characterized in that the host cell is derived from a CHO cell line. «· · • · · A method of producing an antibody capable of binding to an mpl ligand polypeptide, characterized in that the antibody is prepared to interact with the mpl ligand polypeptide of claim 1. • · · • · · · The method of claim 32, wherein the · ·: * · * method produces a monoclonal antibody. • · · · · · · · · · A process for the preparation of a pharmaceutical composition for the treatment or prophylaxis of thrombocytopenia, characterized by the addition of the mpl ligand polypeptide prepared according to claim 1 and a pharmaceutically acceptable carrier. ·· · • · .1 2 3 • • • • • • • • • · · · · · · · · · · · · · · · · · · · · · · · · · · · ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··· 2 • · 3