SK87596A3 - Thrombopoietin - Google Patents

Abstract

Disclosed is a mpl ligand polypeptide prepared in the process comprising: (i) screening of the human genome library with oligonucleotide based on the genome sequence imaged in the picture 9 for isolation of genome DNA, comprising mpl ligand exon encoding the sequence imaged in the picture 9 together with remaining gene exons, (ii) inserting DNA to the express vector, (iii) transfection of the mammal cell by gene express vector and (iv) isolation of the mpl ligand polypeptide from the cell cultivation medium, as well as nucleic acid, express vector and host cell and the method for preparation of polypeptide and its use.

Description

Technical field

The invention relates to the isolation, purification, recombinant or chemical synthesis of proteins which affect the survival, proliferation, differentiation or maturation of hematopoietic cells, in particular platelet progenitic cells. In particular, this invention relates to the cloning and expression of nucleic acids encoding a protein hgand that is capable of linking and activating mpl, a member of the cytokine receptor superfamily. The invention further relates to the use of these proteins alone or in combination with other cytokines for the treatment of immune system diseases or haematopoietic disorders, including thrombocytopenia.

BACKGROUND OF THE INVENTION

I. Blood system

The hematopoietic system produces mature highly specialized blood cells that are necessary for the survival of all mammals. These mature cells include erythrocytes specialized in the transport of oxygen and carbon dioxide, T- and B-lymphocytes responsible for the transmission of cellular and antibody responses of the immune system, platelets or thrombocytes, specialized in the formation of blood clots, granulocytes and macrophages that they are specialized both as feeders and as cells to help fight infection. Granulocytes are further subdivided into neutrophils, e2nophils, basophils and mast cells, specialized cell types with individual functions. Remarkably, all of these specialized mature blood cells are derived from one type of undifferentiated cell, referred to as a pluripotent (or totipotent) cell strain found in the bone marrow (Dexter et al., Ann. Rev. Cell Biol., 3 : 423-441 [1987]).

Mature highly specialized blood cells must be produced in large quantities continuously throughout the life of the mammal. The vast majority of these specialized blood cells maintain their functional activity for only a few hours to weeks (Cronkite et al., Blood Cells, 2: 263-284 [1976]). Thus, to maintain the normal steady state of mammalian blood cell needs is necessary

the continual recovery of mature blood cells, undifferentiated stem cells, as well as a whole series of transient cells or progenitoma cells, imparting ancestral properties that lie between undifferentiated and mature cells.

Stem pluripotent cells are located at the center of gravity of the blood system. There are relatively few of these cells. They are renewed by proliferation, producing daughter stem cells or transforming in a series of different steps into more and more mature, gender-restricted progenitic cells, until finally, highly specialized mature blood cells are formed.

For example, some multipotent progenitoma cells, referred to as stem cell-derived CFCMix, proliferate (self-renewal) and develop over time to whole cell groups containing all the different myeloid cells such as erythrocytes, neutrophils, megakaryocytes (precursors of platelets), macrophages , eosinophils and mast cells. Additional progenitoma cells of the lymphoid lineage proliferate and develop into T-cells and B-cells.

Between CFC-Mix progenitoma cells and myeloid cells there is another series of progenitoma cells mediating association with their progeny. These gender-restricted pregenitoma cells are classified based on the progene they produce. Thus, known immediate precursors to myeloid cells are: erythroid cell group forming units (CFU-E) for erythrocytes, cells forming granulocyte / macrophage cell groups (GM-CFC) for neutrophils and macrophages, cells forming megakaryocyte cell groups (Meg-CFC) for megakaryocytes, cells forming eosinophil cell groups (Eos-CFC) for eosinophils, and cells forming basophil cell groups (Bas-CFC) for mast cells. Other mediating cells are known to exist between pluripotent stem cells and mature blood cells (see below), or are likely to be discovered, having interdependent degrees of gender limitation and self-renewal.

An essential principle for the normal functioning of the blood-cell system is to suppress the diminution of multipotent self-renewal and to develop gender constraints and maturity. Thus, at one end of the haematopoietic cell spectrum lies a stem pluripotent cell having the ability to self-renew and differentiate to all of the interface-specific progenitoma cells of the cell. This ability is the basis of bone marrow transplant treatment when undifferentiated stem cells re-enter

it will populate the whole hematopoietic cell system. At the opposite end of this spectrum are highly gender-restricted predecessors and their progeny who have lost self-renewal ability but have acquired mature functional activity.

Proliferation and development of stem cells and gender-restricted progenitoma cells can be safely regulated by various hematopoietic growth factors or cytokines. At present, we do not know the completely complex function of these growth factors in vivo. Some growth factors, such as interleukin-3 (IL-3), are able to stimulate multipotent stem cells as well as the progenitoma of cells with multiple genes inherited, including, for example, megakaryocytes. Initially, it was thought that the granulocyte / macrophage cell growth factor (GM-CSF) was only limited to GM-CFC in its effect. Later it was found that GM-CSF also affects the proliferation and development of, inter alia, megakaryocytes. Thus, IL-3 and GM-CSF were found to have broader biological activities, varying in intensity. More recently, interleukin-6 (IL-6) and interleukin-11 (IL-11) have been found to have synergistic effects with IL-3 and stimulate megakaryocyte maturation (Yonemura), although they do not have a significant effect on meg-colony group formation alone. et al., Exp. Hematol., 20: 1011-1016 [1992]).

Thus, hematopoietic growth factors may affect the growth and differentiation of one or more genera, their effect may overlap with other growth factors in influencing a simple progeny cell development line, or may act synergistically with other factors.

It also appears that hematopoietic growth factors can act at different stages of cell development, ranging from totipotent stem cells, through various gender-restricted hereditary cell progenithomas to mature blood cells. For example, erythropoietin (epo) has been found to accelerate the proliferation of only mature erythroid progenitoma cells. IL-3 acts earlier, affecting already undifferentiated stem cells and transient gender-restricted cell progenitoma.

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Other growth factors, such as stem cell factor (SCF), can affect even more differentiated cell development.

This suggests that such new hematopoietic growth factors that affect the survival, multiplication, division or maturation of any blood cells or their predecessors from which they originate will be useful, especially if they assist in

- 4 re-establishment of a reduced haematopoietic system due to disease or radio- or chemotherapy.

Π. Megakaryocytopoiesis - platelet formation

Analysis of published data on the regulation of megakaiyocytopoiesis (megakaryocyte formation) and platelet formation is reported by: Mazur, Exp. Hematol., 15: 248 [1987] and Hoffinan, Blood, 74: 1196-1212 [1989]. In short, phiripotent bone marrow stem cells are divided into megakaryocyte, erythrocyte and myelocyte cell lines. It is believed that there is a staggered arrangement of megakaryocyte progenitoma cells with hereditary information between stem cells and megakaryocytes. At least three classes of megakaryocyte progenitoma cells have been identified: the budding megakaryocyte unit (BFU-MK), the colony-forming megakaryocyte unit (CFU-MK) and the low-density megakaryocyte progenitoma cell (LD-CFU-MK). Megakaryocyte maturation itself is a continuation of development that can be divided into several stages according to standard morphological criteria. The first distinguishable element of the megakaryocyte (MK or meg) family are megakaryoblasts. These cells, with an initial diameter of 20 to 30 μιη, have basophilic cytoplasm and a slightly irregular free nucleus, partially chromatin reticulates and multiple nuclei. Megakaryoblasts may contain up to 32 nuclei (ployploid), but the cytoplasm remains thin and immature. By maturation, the nucleus becomes more lobed and pycnotic, the amount of cytoplasm increases, and this becomes more acidophilic and granular. At the periphery of the most mature cells of this family, platelets may appear and release. Typically, 10% of megakaryocytes are in the embryonic stage and more than 50% are mature. In any morphological classifications commonly applied to a series of megakaryocytes, the earliest form is megakaryoblasts, the intermediate form is promegakaryocytes or basophilic megakaryocytes, and the latest developmental form is mature (acidophilic, granular or platelet producing) megakaryocytes. Mature megakaryocytes extend the fibers of the cytoplasm into curved spaces where they separate and fragment into individual platelets (Wilhams et al., Hematology, 1972).

Megakaryocyte formation is thought to involve several regulatory factors (Wilhams et al., Br. J. Haematol., 52: 173 [1982] and Wilhams et al., J. Cell.

Physiol., 110: 101 [1982]). The first stage of megakaryocyte formation is postulated to be mitotic, it relates to cell proliferation and the initiation of a colony (group) of cells with CFU-MK, but the process is not affected by the platelet count (Burstein et al., J. Cell Physiol., 109: 333 [1981] ] and Kimura et al., Exp. Hematol., 13: 1048 [1985]). The second stage of maturation is non-mitotic, involves nuclear polyploidization and maturation of the cytoplasm, and is likely driven by a mechanism of feedback by the amount of peripheral platelets formed (Odeli et al., Blood, 48: 765 [1976] and Ebbe et al., Blood, 32: 787 [1968]).

The existence of a different and specific factor stimulating megakaryocyte cell colonies (MK-CSF) has been discussed by Mazur, Exp. Hematol., 15: 340-350 [1987]. However, most authors assume that as vital a process as platelet production will be driven by cytokine (s) solely by response to this process. Hypotheses about the existence of specific cytokine (s) for the megakaryocyte / platelet system have been the basis of research work for over 30 years - but to date no such citokine has been cleared, its sequence defined and experimentally prepared as specific MK-CSF (TPO) ).

Although it has been reported in the literature that MK-CSF was partially purified from experimentally induced thrombocytopenia (Hill et al., Exp. Hematol., 14: 752 [1986]) and from modified human embryonic kidney [CM] medium (McDonald et al. , J. Lab. Clin. Med., 85:59 [1975]) and male urinary extracts in a patient with aplastic anemia (anemia) and idiopathic thrombocytopenic purpura (Kawakita et al., Blood, 6: 556 [1983]) and plasma (Hoffman et al., J. Clin. Invest., 75: 1174 [1985]), yet their physiological function was in most cases unknown.

Modified media from mitogen-activated spleen cells from Phytolacca americana L. (PWM-SpCM) and the murine myelomonocyte cell line WEHI-3 (WEHI-3CM) were used as megakaryocyte potentiators. PWM-SpCM contains growth-enhancing factors CFU-MK (Metcalf et al., Pro. Natl. Acad. Sci., USA, 72.T744-1748 [1975]; Quesenberry et al., Blood, 65: 214 [1985]; and Iscove, NN, in Hematopoietic Cell Differentiation, ICN-UCLA Symposium on Molecular and Cellular Biology, Vol. 10, Golde et al., eds. [New York, Academy Press] pp. 37-52 [1978]), one of these are interleukin-3 (IL-3), a colony stimulating factor of cells of several genera (multi-CSF [Burstein, Blood Cells, 11: 469 [1986]). Other factors in this medium

were not identified and isolated. WEH1-3 is a murine myelomonocyte cell line secreting a relatively large amount of IL-3 and less GM-CSF. IL-3 has been found to enhance the growth of a wide variety of blood cells (Ihle et al., J. Immunol., 13: 282 [1983]). It has also been found that IL-3 has a synergistic effect with a number of known blood-inducing 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 (EPO) and interleukin-1 (IL-1), by introducing initial multipotent precursors and forming very large mixed groups of hematopoietic cells.

Additional sources of megakaryocyte potentiators have been found in modified media of mouse lung, bone, macrophage cell lines, peritoneal effusion cells, and human (human) embryo kidney cells. Despite some contradictory data (Mazur, Exp. Hematol., 15: 340-350 [1987]), there is evidence (Geissler et al., Br. J. Haematol., 60: 233-238 [1985]) that a greater impact on activated T-lymphocytes than monocytes have megakaryocyte production. This finding suggests that activated T-cell secretions such as interleukins may be regulatory factors in the development of MK (Geissler et al., Exp. Hematol., 15: 845-853 [1987]). A number of works on the preparation of purified erythropoietin (EPO) megakaryocytes (Vainchenker et al., Blood, 54: 940 [1979]; McLeod et al., Nature, 261: 492-4 [1976]; and Williams et al., Exp. Hematol., 12: 734 [1984]) indicates that this hormone has an enhancing effect on the formation of a group of MK cells. This has been demonstrated in cultures both with and without serum and in the absence of accompanying cells (Williams et al., Exp. Hematol., 12: 734 [1984]). EPO is more commonly used in connection with a one or two cell stage of megakaryocyte formation, while PWM-SpCM is used in a four cell stage of megakaryocyte development. The effect of all these factors on both the first and last stages of megakaryocyte development remains to be elucidated.

According to data from several laboratories, it is believed that the only multirodial factors that individually have stimulating activity against a group of MK cells are GM-CSF and IL-3 and, to a lesser extent, IL-6 as a B-cell stimulating factor (Ikebuchi et al. , Proc. Natl. Acad. Sci. USA, 84: 9035 [1987]). More recently, several authors have reported that IL-11 and leukemia inhibitory factor (LIF) act synergistically with IL-3, increasing megakaryocyte size and ploidy (Yonemura et al., British Journal of Hematology, 84: 16-23 [ 1993]; Burstein et al., J.

- 7 Cell. Physiol., 153: 305-312 [1992]; Metcalf et al., Blood, 76: 50-56 [1990]; Metcalf et al., Blood, 77: 2150-2153 [1991]; Bruno et al., Exp. Hematol., 19: 378-381 [1991]; and Yonemura et al., Exp. Hematol., 20: 1011-1016 [1992]).

Further work on this subject is: Eppstein et al., U.S. Pat. U.S. Patent 4,962,091; Chong, U.S. Pat. U.S. Patent 4,879,111; Femandes et al. U.S. Patent 4,604,377; Wissler et al. U.S. Patent 4,512,971; Gottlieb, U.S. Pat. U.S. Patent 4,468,379; Bennett et al. U.S. Patent 5,215,895; Kogan et al. U.S. Patent 5,250,732; Kimura et al., Eur. J. Immunol., 20 (9): 1927-1931 [1990]; Secor et al., J. of Immunol., 144 (4): 1484-1489 [1990]; Warren et al., J. of Immunol., 140 (1) 94-99 [1988]; Warren et al., Exp. Hematol., 17 (11): 1095-1099 [1989]; Bruno et al., Exp. Hematol., 17 (10): 1038-1043 [1989]; Tanikawa et al., Exp. Hematol., 17 (8): 883-888 [1989]; Koike et al., Blood, 75 (12): 2286-2291 [1990]; Lotem, Blood, 75 (5): 1545-1551 [1989]; Rennick et al., Blood, 73 (7): 1828-1835 [1989]; and Clutterbuck et al., Blood, 73 (6): 1504-1512 [1989],

ΙΠ. thrombocytopenia

Platelets are critical elements of the blood coagulation mechanism. In thrombocytopenia, which occurs under different clinical conditions and diseases, platelet levels are reduced below 150 x 10 ⁹ per liter. The main causes of thrombocytopenia can be divided into three groups based on the platelet lifetime range: (1) impaired platelet formation in the bone marrow, (2) platelet secretion in the spleen (splenomegaly) or (3) increased platelet breakdown in the peripheral circuit (i.e., autoimmune thrombocytopenia or chemo- and radiotherapy). Patients who have received large volumes of platelet-poor blood products with rapid help may also develop thrombocytopenia as a result of dilution.

Clinical haemorrhage as a manifestation of thrombocytopenia results from the severity of thrombocytopenia, its causes and from the likely concomitant coagulation disorders. In general, patients who have platelets between 20 and 100 x 10 ⁹ per liter are at risk of significant post-traumatic bleeding, while patients with platelets less than 20 x 10 ⁹ per liter may bleed spontaneously. These latter patients are candidates for platelet transfusion, which may be accompanied by immunological and viral risks. At any stage of thrombocytopenia, bleeding may become more severe if:

- 8 the cause is a decrease in platelet production rather than an increase in their breakdown. In the latter case, the accelerated platelet transformation results from the circulation of younger, larger and more hemostatically effective platelets. Thrombocytopenia can be caused by a number of diseases, which are briefly described below. A more detailed description can be found in Schafner, AI, "Throbocytopenia and Disorders of Platelet Function," College of Medicine, 3rd Ed., John J. Hutton et al., Eds., Little Brown and Co., Boston / Toronto / London [1990] ]

(a) Thrombocytopenia caused by the formation of damaged platelets

Causes of congenital thrombocytopenia include congenital aplastic anemia (Fanconi syndrome) and congenital amegakaryocyte thrombocytopenia, which may be associated with skeletal deformities. Secondary diseases of platelet production are caused by either megakaryocyte hypoplasia or ineffective platelet production. Megakaryocyte hypoplasia can be caused by a variety of conditions, including bone marrow aplasia (including congenital forms or myelosuppression by chemotherapeutic agents or radiotherapy), myelofibrosis, leukemia, and bone marrow attack by metastatic tumor or granulomas. In some cases, the toxins, infectious agents, or drugs may selectively interfere with thrombopoiesis; such examples include transient thrombocytopenia caused by alcohol and some viral infections and moderate thrombocytopenia associated with the administration of thiazide diuretics. Finally, ineffective thrombopoiesis may also be the cause of thrombocytopenia as a result of megaloblast processes (lack of B12 or folic acid derivatives), the course being accompanied by anemia and leucopenia.

Concomitant treatment of thrombocytopenias due to decreased platelet production depends on the identification and reversibility of the underlying cause that caused the bone marrow disorder. Platelet transfusions are usually given to patients with severe bleeding complications or during surgical procedures, as isoimmunization may lead to hypersensitivity to further platelet transfusions. Mucosal bleeding resulting from intense thrombocytopenia may be improved following oral or intravenous administration of antifibrinolytic agents. When antifibrinolytic agents are administered to patients with multiple intravascular coagulation (DIC), thrombotic complications may occur.

(b) Thrombocytopenia due to spleen separation

Spleen enlargement due to any cause may be associated with mild to moderate thrombocytopenia. This is a predominantly passive process (hypersplenism) of platelet secretion by the spleen, whereas the active breakdown of platelets by the spleen in cases of immuno-mediated thrombocytopenia will be discussed below. Although the most common cause of hypersplenism is the accumulation of blood in the enlarged spleen, caused by increased porterial pressure due to alcoholic cirrhosis, other forms of bloodied, infiltrating or lymphoproliferative enlarged spleen are also associated with thrombocytopenia. As a result of hypersplenism alone, the platelet count is not less than 50 x 10 ⁹ per liter.

(c) Thrombocytopenia caused by mediated non-immune platelet breakdown

Thrombocytopenia may be due to the accelerated breakdown of platelets by various non-immunological processes. Disorders of this type include multiple intravascular coagulation, prosthetic intravascular devices, extracorporeal blood circulation, and thrombotic microvascular therapy techniques such as thrombotic platelet purpura. In all these cases, circulating platelets are stressed, either by contact with artificial surfaces or abnormal intravenous conditions, or consumed at these sites, or damaged and then prematurely removed by the reticuloendothelial system. Disease or disorder conditions from which multiple intravascular coagulation (DIC) may develop are described in detail in Braunwald et al. (eds), Harrison's Principles of College of Medicine, llth Ed., p. 1478, McGraw Hill [1987]. Intravascular prosthetic devices, including heart valves and intra-aortic balloons, may cause mild to moderate destructive thrombocytopenia and, on the other hand, transient thrombocytopenia may occur in those patients who are connected to a cardiac lung bypass or bypass or damage to platelets in the extracorporeal circuit.

(d) Drug-induced immune thrombocytopenia

More than 100 kinds of drugs cause immunologically mediated thrombocytopenia. However, only the effects of quinidine, quinine, gold, sulfonamides, cephalotin

10 and heparin were well characterized. Drug-induced thrombocytopenia is often very severe and typically occurs abruptly on those days when the patient is taking the appropriate medication.

(e) Immune (autoimmune) thrombocytopenic purpura (ITP)

ITP is a chronic disease in adults characterized by autoimmune platelet breakdown. The antibody itself is usually IgG, although other immunoglobulins have also been reported. Although ITP self-antibody has been found to be associated with the GPHblUa platelet membrane, in many cases the specificity of the platelet antigen has not been determined. The extracorporeal decomposition of sensitized platelets occurs in the spleen-endothelial system of the spleen and liver. Although more than half of all ITP cases are congenital, many patients suffer from rheumatic or autoimmune diseases (that is, systemic lupus erythematosus) or lymphoproliferative disorders (that is, chronic lymphocytic leukemia).

(f) HIV-induced ITP

ITP is a common major complication of HIV infection (Monis et al., Ann. Intern. Med., 96: 714-717 [1982]), and may occur at any stage of the disease, even in patients diagnosed with AIDS, who have AIDS symptoms, as well as those who are infected with HIV but without AIDS symptoms. HIV infection is a communicable disease characterized by severe loss of cellular immunity as well as the occurrence of opportunistic infections and malignancy. The primary immunological abnormality resulting from HIV infection is the progressive depletion and impairment of T cell function by displacing CD4 glycoprotein from the cell surface (Lane et al., Ann. Rev. Immunol., 3: 477 [1985]). By losing the CD4 glycoprotein, which aids respectively resp. induces the function of T cells, presumably severe cellular and humoral immunity disorders leading to opportunistic infections and malignancies that are characteristic of AIDS (H. Lane, supra).

Although the mechanism of HIV-related ITP is unknown, it is believed to be different from that of ITP that is not associated with HIV infection (Walsh et al., N. Eng. J. Med, 311: 635-639 [1984]; and Ratner, Am. J. Med., 86: 194-198 [1989]).

IV. Overview of thrombocytopenia therapy

The therapeutic approach to the treatment of patients with thrombocytopenia determines the intensity and warmth of the clinical situation. The treatment is similar when HTV is associated with thrombocytopenia than when it is not associated with HIV, and although various treatments have been used, therapy remains controversial.

Platelet counts in patients diagnosed with thrombocytopenia increased significantly by treatment with glucocorticoids (eg prednisolone), but in most patients the response was incomplete or relapsed when the dose of glucocorticoid was reduced or discontinued. Based on a study of patients who have mt ITPs associated with HTV, some authors have suggested that glucocorticoid therapy may lead to increased susceptibility to AIDS. Glucocorticoids are usually administered when the platelet count falls below 20 x 10 ⁹ / bter or when spontaneous bleeding occurs.

Patients who do not take glucocorticoids have been successfully administered the HIV compound in severe cases of ITP associated with HIV:

4- (2-chlorophenyl) -9-methyl-2- [3- (4-morfolniyl) -3-propanone-yl] 6H-thieno [3,2, f | [1,2,4] triazolo [4,3, a] [1,4] diazepine (WEB 2086).

Patients who have platelet counts of 37000-58000 / μΙ with HEBIB WEB 2086 and after 1-2 weeks of Healing, platelet counts increased to 140000-190000 / μ1 (EP 361 077 and Lohman et al., Lancet, 1147 [ 1988]).

Although we do not know exactly the optimal method of Healing the obtained amegakaryocyte thrombocytopenic purpura (AATP), it has been found (Trimble et al., Am. J. Hematol., 37: 126-127 [1991]) that administration of antitymocyte globulin (ATG), which is equine antisera against human thymus tissue, prolonged the time of complete resolution of disease symptoms. According to other data, the blood-induced effects of ATG are due to the thimerosal, since the protein is believed to act as a mercury carrier (Panella et al., Cancer Research, 50: 4429-4435 [1990]).

Good results after spleen surgery (splenectomy) are reported in the literature. Splenectomy will reduce the likelihood of platelet breakdown in many patients and remove the major source of autoantibody production. Using this procedure will extend the remission interval in many patients even without medication. However, since surgical procedures are generally not used in patients with immune problems, splenectomy is recommended only in severe

cases of thrombocytopenia (i.e., severe ITP associated with HIV) in patients who have not responded to 2-3 weeks of glucocorticoid therapy or have not sustained a response after discontinuing glucocorticoid administration. Based on a review of scientific knowledge, it is not confirmed whether splenectomy increases the susceptibility of AIDS patients.

The promising results of HIV-induced ITP treatment with some cytotoxic agents such as vincristine and azidotimidine (AZT, zidovudine) can be attributed to the splenectomy and prednisolone treatment, but the results can only be considered preliminary.

As is apparent from the foregoing, one method of treating thrombocytopenia is to obtain a substance capable of accelerating the differentiation and maturation of megakaryocytes or their precursors to the platelet-producing stage. In the past, considerable efforts have been made to identify such a substance, commonly referred to as "thrombopoietin" (TPO). TPO is also known in the literature under other names such as: thrombocytopoiesis stimulating factor (TSF), megakaryocyte group 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) and attempts to characterize and purify this substance have continued to date. On the one hand, there is information on partial purification of TPO based on active polypeptides (see, for example, Tayrien et al., J. Biol. Chem., 262: 3262 [1987], but on the other hand, it is reported that TPO is not an individual individual in terms of its action, but rather resembles the polyfunctional manifestations of the known hormone (IL-3, Sparrow et al., Prog. Clin. Biol. Res., 215: 123 [1986]). Although no protein has been unequivocally identified as TPO, the recent discovery that mpl, an apparent cytokine receptor, can transmit a thrombopoietic signal, is of great interest.

V. Mpl is a megakaryocytopoietin cytokine receptor

It is believed that the proliferation and maturation of hematopoietic cells are totally controlled by factors that positively or negatively modulate the proliferation of cells of the pluripotent strain and multi-natal differentiation. These effects are mediated through the high-efficiency coupling of factors of the monocell proteins to specific cell surface receptors. These cell surface receptors are extensively

homologous and are generally classified as superfamily of cytokine receptors. Elements of this superfamily include receptors for: IL-2 (β and γ chains) (Hatakeyama et al., Science, 244: 551-556 [1989]; Taheshita et al., Science, 257: 379382 [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 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]; Tavemier et al., Cell, 66: 1175-1184 [1991], BL-6 (Yamasaki et al., Science, 241: 825-828 [1988]; Hihi 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: 56905694 [1992]), granulocyte and macrophage colony stimulating factor (GM-CSF) (Fukunaga et al., Cell, 61: 341-350 [1990a]; Fukunaga et al. 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. , Cel 1, 57: 277-285 [1989]; Jones et al., Blood, 76: 31-35 [1990], leukemia inhibitory factor (LIF) (Gearing et al., EMBO J., 10: 2839-2848 [1991], oncostatin M (OSM) (Rose et al. , Proc. Natl. Acad. Sci. USA, 88: 8641-8645 [1991]) as well as receptors for prolactin (Boutin et al., Proc. Natl. Acad. Sci. USA, 88: 77447748 [1988]; Edery; et al., Proc. Natl. Acad. Sci. USA, 86: 2112-2116 [1989]), growth hormone (GH) (Leung et al., Nature, 330: 537-543 [1987]) and ciliary (cilia) neurotrophic factor (CNTF) (Davis et al., Science, 253: 59-63 [1991]).

Members of the superfamily of cytokine receptors are divided into three functional categories (see review by Nicola et al., Cell, 67: 1-4 [1991]). The first group comprises single chain receptors, such as the erythropoietin receptor (EPO-R) or granulocyte colony stimulating factor receptor (G-CSF-R), which binds a high affinity ligand via the estracellular domain (extracellular region) and also generates an intracellular signal. The second group of receptors, the so-called α-subunits, includes the interleukin-6 receptor (IL6-R), the granulocyte and macrophage colony stimulating factor receptor (GM-CSF-R), the interleukin-3 receptor (IL3-Ra) and other superfamily cytokine receptors. These α-subunits bind a low affinity ligand but cannot transmit an intercellular signal. The high affinity receptor capable of signal transduction is generated by a heterodimer between the α-subunit and a member of the third family of cytokine receptors, designated

as β-subunits, for example β _ς , the common β-subunit for the three α-subunits 1L3-Ra and GM-CSF-R

Evidence that mpl is a member of the superfamily of cytokine receptors is provided by the homologous sequence (Gearing, EMBO, J., 8: 3667-3676 [1988]; Bazan, Proc. Natl. Acad. Sci. USA, 87: 6834-6938 [1990] Davis et al., Science, 253: 59-63 [1991] and Vigon et al., Proc. Natl. Acad. Sci. USA, 89: 5640-5644 [1992]) and its ability to transmit proliferation signals.

The deduced sequence of proteins from the molecular cloning of murine c-mpl shows that this protein is a homologue of other cytokine receptors. The extracellular domain contains 465 amino acid residues and consists of two subdomains, each containing four strongly protected cysteines and a specific motif in the N-terminal subdomain and C-terminal subdomain. The extracellular regions of the binding ligands are believed to have similar structural geometry to the double closed β-cylindrical shape. This double extracellular region is strongly homologous to the signal transduction chain common to the IL-3, IL-5 and GM-CSF receptors, as well as the LJF low activity binding region (Vigon et al., Oncogene, 8: 2607-2615 [1993]). Thus, mpl may belong to the family of cytokine receptors with low activity bound ligand.

A comparison of murine mpl and mature human mpl P shows that the two proteins are 81% identical in sequence. More specifically, the N-terminal and Cterminal extracellular subdomains are 75% identical in sequence, respectively. to 80%. The most protected mpl sphere is a region of the cytoplasm having 91% amino acid sequence identity, with a sequence of 37 residues near the transmembrane domain, which is identical in both agents. Therefore, mpl is reported to be one of the most protected elements of the superfamily of cytokine receptors (Vigon, supra).

Evidence that mpl is a functional receptor capable of transmitting a proliferation signal is provided by the construction of chimeric receptors containing an extracellular domain from a cytokine receptor that has a high affinity for the known cytokine with the mpl domain of the cytoplasm. Since no mpl ligand was reported in the literature, it was necessary to construct a chimeric high affinity ligand linking the extracellular region of a first cytokine receptor family, such as IL-4R or G-CSFR Vigon et al. (See above) linked the extracellular region of G-CSFR with both transmembrane and c-mpl domains

cytoplasm. The IL-3 dependent cell line, BAF / B03 (Ba / F3) was infected with the G-CSFSJmpl chimera simultaneously with full-length G-CSFR regulation. Cells infected with the chimera grew equally well in the presence of the cytokine IL-3 or G-CSF. Similarly, cells infected with G-CSFR also grew well in either IL-3 or G-CSF. All cells died in the absence of growth factors. Similar experiments were performed by Škoda et al., EMBO J., 12 (7): 2645-2653 [1993], linking both the extracellular and transmembrane regions of the human IL-4 receptor (hIL-4-R) with the mouse cytoplasmic mpl domain and Infected into mouse IL-3 susceptible Ba / F3 cell line Ba / F3 cells infected with wild-type hIL-4-R were normally propagated in the presence of either IL-4 or IL-3. Ba / F3 cells infected with hIL-4R (/?) Were normally propagated in the presence of bTL-4 (in the presence or absence of IL-3), demonstrating that in the Ba / F3 cells the mpl cytoplasmic domain contains all the elements necessary for proliferation signal transmission.

These chimeric experiments suggest that the mpl cytoplasmic domain has a signaling ability to proliferate, but does not indicate whether the mpl cytoplasmic domain can bind a ligand. These results are consistent with at least two likely hypotheses, namely, mpl is a single chain receptor (first group), such as EPO-R or G-CSFR, or is a β-subunit that transmits a signal (third group), requiring α- a subunit such as IL-3 (Skoda et al., supra).

VI. Mpl ligand is thrombopoietin (TPO)

As indicated above, serum was thought to contain a unique factor, sometimes referred to as thrombopoietin (TPO), which synergistically interacts with various second cytokines and accelerates the growth and maturation of megakaryocytes. No such natural factor has so far been isolated from serum or other sources, despite the fact that many scientific teams have made considerable efforts to do so. Even though it is not known whether mpl is capable of directly binding a megakaryocyte stimulation factor, recent experiments have shown that mpl is contained in a factor or factors found in the serum of aplastic bone marrow patients that transmit a multiplication signal (Methia et al. , Blood, 82 (5): 1395-1401.

Evidence that a unique colony-forming factor of serum cells and distinct from IL-1α, IL-3, IL-4, IL-6, IL-11, SCF, EPO, G-CSF, and GM-CSF transmits a proliferation signal using mpl, provides an assay for distribution of c-mpl expression in undifferentiated α

general hematopoietic cell lines, and the results of "an.isense" mpl work in one of these cell lines.

Using reverse transcriptase (RT) -PCR in immuno-purified human blood cells, Methia et al. (see above) showed that strong mpl mRNA information was found only in CD34 ⁺ purified cells, megakaryocytes and platelets. Bone marrow (BM) purified CD34 ⁺ cells represent about 1% of all BM cells and are spread in undifferentiated and common progenitors of all genera (i.e., erythroid, granulomacrophage and megakaryocyte).

Antisense mpl oligodeoxynucleotides suppress megakaryocyte colony formation from pluripotent CD34 ⁺ cells cultured in sera of patients with aplastic bone marrow (a rich source of megakaryocyte group-stimulating active elements [MK-CSA]). The same "antisense" oligodeoxynucleotides had no effect on the formation of a group of erythroids or granulomacrophages.

It is not yet known whether mpl is directly bound to a ligand and whether serum factor indicates that the cause of megakaryocytopoiesis acts through mpl. It was believed that if mpl is directly ligand-bound, its amino acid sequence was likely to be strongly protected and has a species cross-reactivity due to its significant sequence identity between human and murine mpl extracellular domains (Vigon et al., Supra [ 1993]).

VII. OBJECTS OF THE INVENTION

As is apparent from the foregoing, there is a continuing need to isolate and identify molecules capable of stimulating the proliferation, differentiation and maturation of blood cells, particularly megakaryocytes or their precursors, for therapeutic use in the treatment of thrombocytopenia. It is believed that such a molecule is an mpl ligand, and then there is a further need to isolate such a ligand (s) and to evaluate its role (s) in cell growth and differentiation.

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

Another object is to obtain a molecule of suitable shape for therapeutic use in the treatment of blood-bearing diseases, in particular thrombocytopenia.

It is a further object of the present invention to isolate, purify and identify protein ligands capable of binding in vivo a receptor from the superfamily of cytokinins, known as mpl, and transmit a proliferation signal.

It is a further object of the present invention to provide nucleic acid molecules encoding such protein ligands and the use of these nucleic acid molecules for the preparation of mpl binding ligands in a recombinant cell culture for diagnosis and therapeutic use.

It is a further object of the present invention to provide derivatives and modified forms of protein ligands, including amino acid sequence variants, glycoprotein variant forms, and covalent derivatives thereof.

It is another object of the present invention to provide a mixture of polypeptide forms by coupling the mpl ligand and the heterologous protein and covalent derivatives thereof.

It is another object of the present invention to provide a variant of the polypeptide forms by linking the mpl ligand to amino acid additions and substitutions from the EPO sequence, thereby producing a protein capable of regulating both the proliferation and growth of both platelets and red blood cell progenitors.

It is a further object of the present invention to prepare immunogens for obtaining antibodies against mpl ligands or associated forms thereof, as well as obtaining antibodies capable of linking such ligands.

These and other objects of the present invention will be apparent to those of ordinary skill in the art, taking into account the overall specification.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention is accomplished by providing an isolated mammalian protein that accelerates megakaryocytopoietic proliferation and maturation, referred to as " ^mplII ligand (ML) or thrombopoietin (TPO) "

This truly homogeneous protein can be purified from natural sources by a method consisting of; (1) contacting the source plasma containing the mpl ligand molecules to be purified with the immobilized polypeptide receptor, in particular the mpl or mpl linked (fused) polypeptide immobilized on a carrier,

under conditions wherein the purified mpl ligand molecules are selectively adsorbed on the immobilized polypeptide receptor, (2) washing the immobilized polypeptide receptor and its carrier and removing the unadsorbed substance, and (3) eluting the mpl ligand molecules from the immobilized polypeptide receptor on , using an elution buffer. Preferably, the natural source is mammalian plasma or urine containing the mpl ligand. The mammal may be aplastic and the immobilized receptor may be a 7β / β-IgG mixture.

A preferred protein for accelerating megakaryocytopoietic proliferation and maturation may be an isolated homogeneous polypeptide mpl ligand, prepared by a synthetic or recombinant route.

The mp1 or TPO polypeptide polypeptide of the present invention has at least a total 70% sequence identity to the amino acid sequence of a highly purified homogeneous porcine polypeptide mpl Ugand and at least 80% sequence identity to a "EPO region" of a porcine polypeptide Ugand mpl. The mpl ligand of the invention is a mature human mpl ligand (hML) having the final amino acid sequence shown in FIG. 1 (SEQ ID NO: 1), or a variant of a post-transcriptionally modified form thereof, or is a protein having about 80% sequence identity to the mature human mpl ligand. Optionally, the Ugandan variant may be an mpl fragment, particularly an amino-terminal or "EPO-region" fragment, of mature human Ugandan mpl (hML). It is preferred that the amino-terminal fragment retains substantially all of the human ML sequence between the first and fourth cysteine residues, but may contain substantial added, removed or replaced portions, but outside of this delimited region. Accordingly, the polypeptide fragment has the following formula:

X-hML (7-151) -Y, wherein hML (7-151) represents the human TPO (hML) amino acid sequence from Cys ⁷ to Cys ^151, inclusive; X represents the amino group of Cys ⁷ or one amino-terminal or more amino-terminal amino acid residues of mature hML or an extension of the amino acid residues in addition to Met, Tyr or a major sequence containing, for example, proteolytically cleaved positions (i.e., Factor Xa or thrombin); and Y represents a carboxy-terminal group of Cys ¹⁵¹ or one or more carboxy-terminal amino acid residues of mature hML or extra extension.

The mpl polypeptide ligand or fragment thereof may be linked to a heterologous polypeptide (chimera). A preferred heterologous polypeptide is a cytokine, cell group stimulating factor, or interleukin, or a fragment thereof, particularly Utligand (KL), IL-1, IL-3, IL-6, IL-11, EPO, GM-CSF, or LIF. Optionally, the heterologous polypeptide is an immunoglobin chain, particularly human IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgD, IgM, or fragments thereof, particularly comprising an IgG heavy chain constant region.

Another aspect of the present invention provides a compound comprising an isolated mpl agonist that is biologically active and is preferably capable of stimulating the introduction of labeled nucleotides (e.g., ³ H-thymidine) into Ba / F3 DNA of IL-3 dependent cells infected with human mpl. The mpl agonist may be a biologically active mpl ligand, and is preferably capable of stimulating the introduction of ³⁵ S into circulating platelets in a mouse platelet reaction assay. Suitable mpl agonists may be ILML153, hML (R153A, R154A), hML2, hML3, hML4, mML, mML2, mML3, pML, and pML2, or fragments thereof.

Another object of the present invention is to provide an isolated antibody capable of binding to the mpl ligand. An isolated antibody capable of binding to an mpl ligand may optionally be coupled to a second polypeptide, and the antibody or a mixture thereof may be used to isolate and purify the mpl ligand from the starting materials, as previously described for immobilized mpl. In another aspect, the present invention provides a method for detecting mpl ligand in vitro or in vivo comprising contacting the antibody with a sample, particularly a serum sample, which is believed to contain the ligand and determining if binding with the ligand occurs.

In other embodiments, the invention provides an isolated nucleic acid molecule encoding the mpl ligand or fragments thereof, wherein the nucleic acid molecule may optionally be labeled such that a portion thereof is detectable and the nucleic acid molecule has a sequence that is complementary to the nucleic acid molecule sequence encoding the ligand mpl, or hybridizes under mild to intense conditions with the same nucleic acid molecule. Preferred nucleic acid molecules are those that encode the human, porcine and murine mpl ligand and include RNA and DNA, both genomic and cDNA. In other aspects of this aspect, the nucleic acid molecule is a DNA encoding an mpl ligand and further comprises a reproducible vector in which the DNA is linked useful for the control of the resolved sequences

vector using a transformed host. The DNA may be a cDNA having the sequence shown in FIG. 15-3 '(SEQ ID NO: 2), 3'-5' or a fragment thereof. This aspect further includes host cells, particularly CHO cells transformed with the vector, and a method of using DNA to efficiently produce mpl ligand, preferably comprising extending the cplNA encoding the mpl ligand in a culture of transformed host cells and recovering mpl ligand from the host cells or host cell culture. . It is preferred that the mpl ligand thus prepared is a human Hgand mpl.

The invention further encompasses a method of treating a mammal having a hematopoietic disorder, in particular thrombocytopenia, by administering a therapeutically effective amount of Uganda mpl to the mammal. Optionally, Ugpl mpl can be administered with a cytokine, particularly a colony stimulating factor or interleukin. Preferred colony stimulating factors or interleukins are: kit-Ugand (KL), LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-3, IL-6 and IL-11.

The invention further includes a method for isolating and purifying TPO (ML) from TPO-producing microorganisms, comprising:

(1) rupture or release of TPO containing cells, (2) inseparable separation of soluble material from insoluble material containing TPO, (3) dissolution of TPO from insoluble material using dissolution buffer, (4) separation of dissolved TPO from other dissolved and undissolved material, (5) dissolving (masking) TPO in redox buffer, and (6) separating suitably masked TPO from unmasked TPO.

The process comprises, in order to dissolve the insoluble TPO-containing material, the use of a chaotropic substance, wherein the chaotropic substance is selected from guanidine, sodium thiocyanate or urea. The process further comprises separating the dissolved TPO from other soluble and insoluble materials, which may take place in one or more stages and may be centrifugation, gel filtration, or reverse phase chromatography. In the step involving masking the TPO, it is

uses a redox solution containing an oxidizing and reducing agent. Generally, the oxidizing agent is oxygen or a compound containing at least a disulfide bond and the reducing agent is a compound containing at least one free sulfhydryl. Preferably, the oxidizing agent is selected from oxidized glutathione (GSSG) and cystine and the reducing agent is selected from reduced glutathione (GSH) and cysteine. The best oxidizing agent is oxidized glutathione (GSSG) and the reducing agent is reduced glutathione (GSH). Preferably, the molar ratio of oxidizing agent to reducing agent is equal to or greater than 1. The redox buffer also contains a detergent, preferably selected from CHAPS and CHAPSO, at a concentration of at most 1%. The redox buffer also contains NaCl, preferably in a concentration range of about 0.1-0.5 M, and glycerin, preferably at a concentration greater than 15%. The pH of the redox buffer is about 7.5-9.0 and the masking step is performed at 4 degrees for 12-48 hours. In the masking step, a biologically active TPO is produced in which a disulfide bond is formed between Cys, which is closest to the amino terminal, and Cys, which is closest to the carboxy terminal of the EPO region.

Further, the invention includes a method of purifying biologically active TPO from microorganisms, comprising:

(1) lysing at least the extracellular membrane of microorganisms, (2) treating the lysate with TPO with a chaotropic agent, (3) masking TPO, and (4) separating impurities and unmasked TPO from suitably masked TPO.

Brief description of the figures

Fig. 1 represents the deduced amino acid sequence (SEQ ID NO: 1) of the human Uganda mpl (hML) cDNA and the coding nucleotide sequence (SEQ ID NO: 2). The nucleotides are numbered at the beginning of each row. The untranslated 5 'and 3' regions are indicated by a lower case. The amino acid residues are numbered above the sequence beginning at Ser 1 of the mature mpl ligand (ML) protein sequence. The boundaries of the putative exon 3 are indicated by arrows and the potential N-glycosylation positions are boxed. Cysteine residues are indicated by a dot above the sequence. The underlined sequences correspond to the N-terminal sequence determined from Uganda mpl, purified from porcine plasma.

Fig. 2 shows the procedure for introducing mpl ³ H-thymidine Uganda. To determine the presence of mpl ligand from various sources, mpl P Ba / F3 cells were maintained in a fasted state (without IL-3) for 24 hours in a humidified incubator at 37 ° C in 5% CO ₂ and air. Starved cells were plated in 96 well culture plates with or without diluted samples and cultured in a cell culture incubator for 18 hours. 20 μΐ serum-free RPMI media containing 1 pCi of ³ H-thymidine was added to each well and the samples left in the incubator for 6-8 hours. Cells were then harvested and collected on 96 microfiltration plates and washed with water. The samples were then evaluated.

Fig. 3 shows the effect of pronase, DTT and heat on the ability of APP to stimulate proliferation of Ba / F3- / npZ cells. For APP pronase digestion, pronase (Boehringer Mannheim) or cow's serum albumin was combined with AffigellO (Biorad) and incubated individually with APP for 18 hours at 37 ° C. They were then separated by centrifugation of the resin and the supernatants were tested. APP was also heated to 80 ° C for 4 min, or 100 μΜ DTT was prepared and dialyzed against PBS.

Figure 4 shows the elution of Uganda mpl activity from Phenyl-Toyopearl, Blue-Sepharose and Ultralink-wpZ columns. Fractions 4-8 from the mpl affinity column are rich fractions of activity peaks eluted from the column.

Figure 5 shows the SDS-PAGE of the eluted fraction from the Ultralink-nzpZ column. To 200 μΐ of each of Fractions 2-8, 1 ml of acetone containing 1 mM HCl was added at -20 ° C. After three hours at -20 ° C, the samples were centrifuged and the resulting sediment was washed twice with acetone at -20 ° C. The acetone sediment was then dissolved in 30 μΐ SDS-dissolution buffer, 100 μΜ DTT was obtained and heated at 90 ° C for 5 minutes. The samples were then dissolved on a 4-20% SDS-polyacrylamide gel and the proteins were visualized by silver staining.

Fig. 6 shows elution of mpl ligand activity from SDS-PAGE. Fraction 6 from the mplafinity column was dissolved on a 4-20% SDS-polyacrylamide gel under non-reducing conditions. After electrophoresis, the gel was cut into 12 equal portions and electroeluted,

- 23 as shown in the examples. Electroheated samples were dialyzed into PBS and examined at a 1/20 dilution. 12 Novex Mark standards were used to calibrate the gel.

Fig. 7 shows the effect of APP with ligandorn mpl consumed on human megakaryocytopoiesis. APP with consumed ligandorn mpl was prepared by passing 1 ml through 1 ml mpl affinity columns (700 gg / w / R / - IgG / ml NHS-superose, Pharmacia). Human peripheral stem cell cultures were prepared with 10% APP or 10% APP with ligandorn mpl consumed and cultured for 12 days. Megakaryocytopoiesis was determined as described in the Examples.

Fig. 8 shows the effect of mp / -IgG on APP stimulation of human megakaryocytopoiesis. Human peripheral stem cell cultures were prepared at 10% dilution with APP and cultured for 12 days. On day 0, day 2, and day 4, mpl-IgG (0.5 µg) or ANP-R-IgG (0.5 µg) was added. After 12 days, megakaryocytopoiesis was evaluated as described in the Examples. The graph shows the mean values from a pair of samples (measured values are given in brackets).

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

Fig. 10 shows the deduced amino acid sequence of mature human Uganda mpl (hML) (SEQ ID NO: 6) and mature human erythropoietin (hEPO) (SEQ ID NO: 7). The predicted amino acid sequence for human Ugand mpl is in line with the human erythropoietin sequence. Identical amino acids are framed and dashes indicate gaps introduced for optimal alignment. Potential positions for N-glycosylation are underlined with a solid line for hML and a dashed line for hEPO. Two cysteines, which are important for erythropoietin activity, are marked with a large dot.

Fig. 11 shows the deduced amino acid sequences of mature human isoforms

Ugpl mpl hML (SEQ ID NO: 6), hML2 (SEQ ID NO: 8), hML3 (SEQ ID NO: 9) and

24 hML4 (SEQ ID NO: 10). Identical amino acids are framed and dashes indicate the introduced gaps for optimal alignment.

Fig. 12A, 12B and 12C show the effect of human Ugpl mpl on the proliferation of Ba / F3-z /? / (A) cells, in vitro human megakaryocytopoiesis measured using the radiolabeled mouse IgG monoclonal antibody specific for the megakaryocyte glycoprotein GPIIbIII _and (B), and mouse thrombopoiesis measured in platelets by reaction assay (C).

293 cells were infected overnight _CaPO4 method (Gorman, C in DNA Cloning: A New Approach 2: 143-190 [1985]) with pRK5 vector alone, pRK5 pRK5hML or the MLI ₅₃ (pRK5-MLi5 ₃ was generated by introducing a stop codon after residue 153 of hML by PCR). The cultures were conditioned for 36 hours and assayed to stimulate Ba (F3-w / β) cell proliferation as described in Example 1 (A) or in vitro for human megakaryocytopoiesis (B). Megakaryocytopoiesis was determined using ¹²⁵ L of radiolabeled mouse IgG monoclonal antibody (HP1-1D) to megakaryocyte-specific glycoprotein GPIIbIIIa as described (Grant et al., Blood 69: 1334-1339 [1987]). The effect of partially purified recombinant ML (rML) on in vivo platelet production (C) was determined using the reaction platelet assay reported in McDonald, TP Proc. Soc. Exp. Biol. Med. 144: 1006-1012 (1973). Partially purified rML was prepared from a 200 ml conditioned culture containing recombinant ML. The culture was passed through a 2 ml Blue-Separose column equilibrated in PBS and the column was washed with PBS and eluted with PBS containing 2 M urea and 2 M NaCl. The active fractions were dialyzed into PBS and 1 mg / ml was prepared with B SA-free endotoxin. The sample contained less than one endotoxin unit / ml. Mice were injected with 64000, 32000 and 32000 respectively. 16000 rML units or excipient alone. Each group consisted of 6 mice. The mean and standard deviation for each group is shown. The p-values were determined by comparing the medians of the 2 tail T-test.

Fig. 13 compares the effect of isoforms and variants of human Uganda mpl in the Ba / F3-wp / cell proliferation assay. HML, mock, hML2, hML3, hML (R153A, R154A) and hMLi _{53 were} tested at various dilutions as shown in Example 1.

Figures 14A, 14B and 14C show the deduced amino acid sequence (SEQ ID NO: 1) of human mpl ligand (hML) or human TPO (hTPO) and the sequence encoding human genomic DNA (SEQ ID NO: 11). Nuldeotides and amino acid residues are numbered at the beginning of each row.

Fig. 15 shows purified 293-rhML ₃₃₂ and purified 293-rhMLi ₅₃ by SDS-PAGE.

Fig. 16 shows the nucleotide sequence of the cDNA coding (SEQ ID NO: 12) and the deduced amino acid sequence (SEQ ID NO: 13) in the uncovered murine ML isoform. This mature mouse isoform of mpl ligand contains 331 amino acid residues, four of which are smaller than the apparent full length of mML and is therefore designated mML2. Nucleotides are numbered at the beginning of each row. Amino acid residues are numbered above the sequence beginning with Ser 1. Potentially suitable N-glycosylation positions are underlined. Cysteine residues are indicated by a dot above the sequence.

Fig. 17 shows the cDNA sequence (SEQ ID NO: 14) and the predicted protein sequence (SEQ ID NO: 15) of the isoform of this murine ML (mML). Nucleotides are numbered at the beginning of each row. The amino acid residues are numbered above the sequence beginning with Ser 1. The mature murine mpl ligand isoform contains 335 amino acid residues and is believed to have the full length mpl ligand, designated mML. The signal sequence is indicated by broken underlines and the likely cleavage point is indicated by an arrow. 5 'and 3' untranslated areas are marked with a lower case. The two nucleotide loss sites due to alternative splicing (mML2 and mML3) are underlined. Four cysteine residues are indicated by a dot. The seven potential N-glycosylation positions are bounded by a box.

Fig. 18 compares the deduced amino acid sequence of the human ML isoform of hML3 (SEQ ID NO: 9) and the murine ML isoform designated mML3 (SEQ ID NO: 16). The predicted amino acid sequence of the human mpl ligand is aligned with the murine mpl ligand sequence. Identical amino acids are framed and dashes are indicated by gaps introduced for optimal alignment. Amino acids are numbered at the beginning of each series.

Fig. 19 compares the predicted amino acid sequences of the mature ML isoforms of murine ML (SEQ ID NO: 17), porcine ML (SEQ ID NO: 18) and human ML (SEQ ID NO: 6). Amino acid sequences are aligned with the gaps indicated by dashes introduced for optimal alignment. Amino acids are numbered at the beginning of each row, identical residues are delimited by a line. Potential N-glycosylation positions are indicated by shading and cysteine residues are indicated by a dot. The protected di-basic amino acid motif, which represents a potential protease cleavage position, is underlined. The omission of the four amino acids occurring in all three species (ML2) is indicated by a thick box.

Figure 20 shows the cDNA sequence (SEQ ID NO: 19) and predicted mature protein sequence (SEQ ID NO: 18) of the porcine ML isoform (pTvTL). This porcine mpl ligand isoform, which contains 332 amino acid residues and is believed to have the full length porcine mpl ligand, is designated pML. Nucleotides are numbered at the beginning of each row. Amino acid residues are numbered above the sequence beginning with Ser 1.

Figure 21 shows the cDNA sequence (SEQ ID NO: 20) and predicted mature protein sequence (SEQ ID NO: 21) of the porcine ML isoform (pML2). This porcine mpl ligand isoform, which contains 328 amino acid residues and is a form with four residues of the full length porcine mpl ligand, is referred to as pML2. Nucleotides are numbered at the beginning of each row. Amino acid residues are numbered above the sequence beginning with Ser 1.

Fig. 22 compares the deduced amino acid sequence of the full-length porcine ML isoform designated pML (SEQ ID NO: 18) and the porcine ML isoform designated pML2 (SEQ ID NO: 21). The predicted amino acid sequence for pML is aligned with the pML2 sequence. Identical amino acids are framed and dashes indicate gaps introduced for optimal alignment. Amino acids are numbered at the beginning of each series.

Figure 27 shows the respective characteristics of plasmid pSVI5.ID.LL.ML0RF ("full length" or TPO332) used to infect CHO-DP12 host cells in the preparation of CHO-rhTPO 332 ·

Figure 24 shows the respective characteristics of plasmid pSVI5.ID.LL.MLEP0-D ("cut off" or TPO153) used to infect CHO-DP12 host cells in the preparation of CHO-rhTPO153.

25A, 25B and 25C show the effect of E. coli-rhTPO (Met ¹ , 153) on platelets (A), red blood cells (B) and white blood cells (C) of ordinary mice. Two groups of 6 female C57 B6 mice were injected daily with either PBS buffer or 0.3 µg E. co / z-rhTPO (Me ¹ , 153) (100 µΐ sc.). On day zero and then on 3.-7. 40 μ of blood from the orbital cavity was collected on days. This blood was immediately diluted to 10 ml with a commercial diluent and complete blood analysis was obtained on a Serrono Baker Hematology Analyzer 9018. Data are presented as mean ± standard deviation from mean.

26A, 26B and 26C show the effect of E. coli z-rhTPO (Me ¹ , 153) on platelets (A), red blood cells (B) and white blood cells (C) of nearly lethally irradiated mice. Two groups of 10 female C57 B6 females, which were nearly 750 fatally irradiated with 750 cGy gamma radiation from a ¹³⁷ Cs source, were injected daily with either PBS buffer or 0.3 µg E. coli-rhlVQ (Me ¹ , 153) (100 µl sc) .). On day 0 and the following days, 40 µl of blood from the orbital cavity was collected. This blood was immediately diluted to 10 ml with a commercial diluent and a complete blood analysis was obtained on a Serrono Baker Hematology Analyzer 9018. Data are presented as means ± standard deviation of means.

Figure 27A, 27B and 27C show the effect of CHO-rhTPO ₃ to 32 (A) platelets (thrombocytes), (B) red blood cells (erythrocytes) and (C) white blood cells (leukocytes) in normal mice. Two groups of 6 female C57 B6 female mice were injected daily with either PBS buffer or 0.3 µg CHO-rhTPC 332 332 (100 µl sc). At zero and 3.-7. On 40 days, 40 µl of orbital kidney blood was collected. This blood was immediately diluted to 10 ml with a commercial diluent and a Serrono Baker

- 28 Hematology Analyzer 9018 a complete blood analysis was obtained. Data are presented as means ± standard deviation of means.

Figure 28 shows dose response curves for interface forms of rhTPO obtained from different cell lines. Dose response curves were generated against rhTPO from the following cell lines: I1TPO332 from CHO (full length Chinese hamster ovary cells); hTPOMet-1153 (truncated form derived from E. coli with N-terminal methionine); ITPO332 (full length TPO from 293 human cells); 155 E. coli without methionine (truncated form [rhTPO 155] without terminal methionine from E. coli). Groups of 6 female C57 B6 female mice were injected daily for 7 days with rhTPO depending on the group. 40 μΐ of blood was collected from the orbit every day and a complete analysis was performed. The data presented represents the maximum observable effects of the various treatments except for (met 153 E.coli) observed on day 7 of treatment. For the above "met 153 E.Col" group, the maximal effect was observed on day 5. Data are presented as means ± standard deviation of means.

Fig. 29 shows dose response curves comparing whole-chain activity and "truncated" forms of rhTPO produced in CHO cells with the truncated form of E. coli. Groups of 6 female C57B6 female mice were injected daily with 0.3 gg of rhTPO of various types. At 2.-7. On day 40, 40 g of blood from the orbital cavities were collected and a complete analysis was performed. Individual groups were treated with TPO153, which is a truncated form of TPO from E. Col., TPO332 (mixed fraction), which is a full length TPO, containing approximately 80-90% full length and 10-20% truncated forms; TPO332 (30K fraction), which is a purified truncated fraction from the original mixed fraction; TPO332 (70K fraction), which is a purified full-length fraction of TPO from the original mixed fraction. Data are presented as means ± standard deviation of means.

Ohr.30 is a figure showing the progress of the KIRA ELISA TPO measurement assay. The figure shows the MPL / Rse.gD chimera and substantial portions of the parental receptors, as well as the final shape (right side of the figure) and the flow diagram (left side of the figure) showing the substantial steps of the experiment.

Fig. 31 is a stream record of KIRA ELIŠA experiment showing each step of the process.

Figures 32A-32L show the nucleotide sequences (SEQ ID NO: 22) of the expression vector pSVI17.ID.LL used to express Rse.gD in Example 17.

33 is a schematic representation of the preparation of plasmid pMP 1.

Fig. 34 is a schematic representation of the preparation of plasmid pMP21.

Fig. 35 is a schematic representation of the preparation of plasmid p.MP151.

Figure 36 is a schematic representation of the preparation of plasmid pMP202.

Fig. 37 is a schematic representation of the preparation of plasmid pMP172.

Fig. 38 is a schematic representation of the preparation of plasmid pMP210.

Figure 39 is a table of the five best formed TPO clones from the pMP210 plasmid bank (SEQ ID NO: 23, 24, 25, 26, 27 and 28).

Fig. 40 is a schematic representation of the preparation of plasmid pMP41.

Fig. 41 is a schematic representation of the preparation of plasmid pMP57.

Fig. 42 is a schematic representation of the preparation of plasmid pMP251.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The following words and phrases refer to the definitions used in the description, examples, and claims.

- 30 'Chaotropic substance' refers to a compound which, in aqueous solutions, at appropriate concentrations, may cause a change in the spatial configuration or shape of the protein by at least partial disruption of the forces required to maintain the normal secondary and tertiary structure of the protein. Such compounds are, for example, urea, guanidine HCl and sodium thiocyanate. In order to exert the desired effect of a chaotropic agent on a protein, high concentrations, usually 4-9M, of these compounds are required.

"Cytokine" is a generic term for proteins derived from a population of one cell that act on the other cell as intracellular mediators. Examples of such cytokines are lymphokines, monokines, and known polypeptide hormones. Cytokines include growth hormone, insulin-like growth factors, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle-stimulating hormone (FS), thyroid stimulating hormone (TSH), leutinizing hormone (LH), hematopoietic growth factor, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α (TNF-α) inhibiting agent (TNF-α) TNF-β), mousegonadotropin-linked peptide, inhibin, active, vascular endothelial growth factor, integrin, nerve growth factors such as NGF-β, platelet growth factor, transforming growth factors (TGFs) such as TGF-α and TGF- β, growth factor-I and growth factor-Π, insulin-like, erythropoietin (EPO), osteoinductive factors, interferons such as s interferon-α, -β and -γ, colony growth factors (CSFs) such as macrophages-CSF (M-CSF), granulocytes / macrophages-CSF (GM-CSF) and granulocytes CSF (G-CSF), interleukins such as IL-1, IL-2, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 and other polypeptide factors including LIF, SCF and kit ligand. For the purposes of the present invention, the foregoing compounds from natural sources or from recombinant cell cultures can be used. Likewise, their biological equivalents, i.e., differing in amino acid sequence by one or more amino acids or in the type or extent of glycosylation, are also included.

"MpZ ligand", "mp1 polypeptide ligand" "ML", "thrombopoietin" or "TPO" are used interchangeably herein and include any polypeptide,

which has the ability to associate with mpl, a member of the superfamily of cytokine receptors, and has biological properties of ML as defined below. An important biological property is the ability to stimulate the introduction of labeled nucleotides (e.g., ³ H-thymidine) into IL-3 dependent DNA of Ba / F3 cells infected with human mpl P. Another important biological property is the ability to stimulate ³⁵ S introduction into circulating blood platelets mouse platelet reaction assay. This definition includes a polypeptide isolated from a mpl Uganda source, such as the aplastic porcine plasma described herein or from another source, such as other animal species, including humans or prepared by recombinant or synthetic means, and including variant forms including functional derivatives, fragments, alleloinorphs. , isoforms and analogues thereof.

An "mpl ligand fragment" or "TPO fragment" is a portion of a naturally occurring mature full length mpl ligand or TPO sequence that has omitted one or more amino acid residues or carbohydrate units. The omitted amino acid residues (residues) can occur in the peptide at any site, including N-terminal or C-terminal termini, or within. The fragment must assume at least one biological property from the Uganda mpl. · Uganda mpl fragments will typically have a sequential sequence of at least 10, 15, 20, 25, 30, or 40 amino acid residues that are identical to the mammalian Uganda mpl sequences including Uganda isolated aplastic porcine plasma or human or murine ligand, especially their EPO region. Typical examples of N-terminal fragments are hMLi ₅₃ or TPO (Met'1-153).

"Ugpl mpl variants" or "Ugpl mpl sequence variants" are defined herein to represent a biologically active mpl ligand, defined below, that has less than 100% sequence identity to a mpl hgand isolated from recombinant cell culture or from aplastic porcine porcine. plasma or human hgandom having the derived sequence shown in FIG. 1 (SEQ ID NO: 1). Typically, the biologically active variant of Uganda will have an mpl amino acid sequence having at least about 70% amino acid sequence identity with Ugandal mpl isolated from aplastic porcine plasma or mature or human mouse Ugand or fragment thereof (see Fig. 1 [SEQ ID NO: 1] ), preferably at least about 75%, more preferably at least about 80%, even more preferably at least about 85%, even more preferably a value of at least about 85%, and most preferably at least about 95%.

- 32 "Chimeric ligands ηψΓ" is a polypeptide containing the full length ligand of mpl or one or more fragments thereof, linked to or linked to a second heterologous polypeptide or to one or more fragments thereof. The chimera transfers at least one biological property from the mpl ligand. The second polypeptide will typically be a cytokine, an immunoglobin, or fragments thereof.

The terms "isolated mp1 ligand", "high purity mpl ligand" and "substantially homogeneous mpl ligand" are used interchangeably to mean mpl ligand that has been purified from an mpl ligand source or has been prepared by recombinant or synthetic means and is free of other peptides or proteins (1) obtaining at least 15 and preferably 20 amino acid residues of the N-terminal or internal amino acid sequence using a linker sequencer or best purchased commercially available amino acid sequencer or modified according to published methods at the date of the present invention, or (2) homogenizing by SDS-PAGE to reduce or reduce conditions using Coomassie blue or, preferably, a silver color. Here, homogeneity means less than about 5% contamination by other starting proteins.

"Biological property" when used in conjunction with either an "mpl ligand" or an "isolated mpl ligand" means either a thrombopoietic activity or an in vivo effector or antigen function or an activity that is directly or indirectly caused or caused by an mpl ligand (whether whether natural or denatured) or a fragment thereof. Effector functions include coupling of mpl and coupling of any carrier that binds to activity, agonism or antagonism of mpl, particularly the transmission of a proliferation signal, including replication, DNA regulatory function, modulation of other cytokine biological activity, receptor (especially cytokine) activation, deactivation, up and down regulation cell growth or differentiation and the like. Antigenic function means the existence of an epitopic or antigenic position that is capable of reacting with an antibody raised against the native Uganda mpl. The major antigenic function of the mpl polypeptide ligand is that it is associated with an affinity of at least about 10 ⁶ l / mol with the mpl Uganda raised antibody isolated from aplastic porcine plasma. Typically, the polypeptides associate with an affinity of at least about 10 ⁷ L / mol. The antigenically active mpl polypeptide ligand is preferably

A polypeptide that associates with an antibody raised against an mpl ligand having one of the above effector functions. Antibodies used herein to define "biological activity" are rabbit polyclonal antibodies, formed by formulating an mpl ligand isolated from recombinant cell culture or from aplastic porcine plasma in complete Freund's adjuvant, by subcutaneous administration of the dosage form by injection and boosting immuno-response by intraperitoneal injection. forms until the antibody titer of mpl ligand has stabilized.

"Biologically active" when used in conjunction with either "mpl ligand" or "isolated mpl ligand" means an mpl ligand or a polypeptide having

thrombopoietic activity or involved in the effector fimigation of mpl ligand isolated from aplastic porcine plasma or expressed ("expressed") in recombinant cell culture described herein. A fundamental known effector function of the mpl ligand or polypeptide of the present invention is to attach to mpl and stimulate the introduction of labeled nucleotides ( ³ H-thymidine) into IL-3 dependent IL / 3-dependent DNA cells infected with human mpl P. Another known effector function The mpl ligand or polypeptide of the present invention is the ability to stimulate the introduction of ³⁵ S into circulating blood platelets in a mouse platelet reaction assay. Yet another known effector function of the mpl ligand is the ability to stimulate in vitro human megakaryocytopoiesis, which can be quantitated using radiolabeled monoclonal antibodies specific for the megakaryocyte glycoprotein GPIIbII4I _a .

"Percent amino acid sequence identity", taking into account 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 mpl ligand sequence isolated from aplastic porcine plasma or mouse or human Ugand having a derived sequence of the amino acids shown in FIG. 1 (SEQ ID NO: 1) by sequencing the sequences and introducing gaps, if necessary, to achieve maximum sequence identity and without non-violent substitution as part of the sequence identity. There must be no N-terminal or C-terminal or internal extensions, deletions, or insertions into the mpl ligand sequence that would affect sequence identity or homology. Thus, exemplary biologically active polypeptide mpl ligands will be

34 have identical sequences that include the prepro- / np / ligand, the γτο-mpl ligand, and the mature mpl ligand.

'Micro-sequencing of mpl ligand' can be carried out by any suitable standard procedure which ensures sufficient sensitivity of the method. In one such method, highly purified polypeptides, obtained from SDS gels or from HPLC end stage, were directly sequenced by automated Edman (phenylisothiocyanate) degradation using a 470A Applied Biosystems gas sequencing model equipped with a 120A phenylthiohydantoin (PTH) amino acid analyzer. . Uganda mpl fragments prepared by chemical (e.g. CNBr, hydroxylamine, 2-nitro-5-thiocyanobenzoate) or enzymatic (e.g. trypsin, clostripaine, staphylococcal protease) leaching and then purification (HPLC) can be sequenced in a similar manner. PTH amino acids were analyzed using a ChromPerfect data system (Justice Innovations, Palo Alto, CA). Sequence interpretation was performed on a VAX 11/785 Digital Equipment Co., according to the procedure described by Henzel et al., J. Chromatography, 404: 41-52 [1987]. Freely aliquots of HPLC fractions were electrophoresed on 5-20% SDS. -PAGE, an electrotransfer on a PVDF membrane (ProBlott, AIB, Foster City, CA) and stained with Coomassie Brilliant Blue (Matsurdiara, J. Biol. Chem., 262: 10035-10038 [1987], The specific protein identified in color was removed For internal protein sequences, the HPLC fractions were vacuum dried (SpeedVac), resuspended in appropriate buffer, and cyanogen bromide, Lys-specific enzyme Lys-C (Wako Chemicals, Richmond). , VA) or Asp-N (Boehringer Mannheim, Indianapolis, IN.) After digestion, the resulting peptides were sequenced sequentially or after HPLC dissolution on a C4 column elicited with a propanol gradient in 0.1% TFA, prior to sequencing. gas phase.

'Thrombocytopenia' is defined by a platelet count of less than 150 x 10 ⁹ per Hter of blood.

"Thrombopoietic activity" is defined as a biological activity that includes the acceleration of proliferation, differentiation and / or maturation of megakaryocytes or their

- 35 precursors to a form of these cells that produces platelets. This activity can be measured by various assays, including an in vivo murine platelet reaction synthesis assay, a platelet cell surface antigen induction assay, as measured by an anti-platelet immunoassay (anti-GPHblIIa) for human leukemia megakaryoblast (CMK.) Cell line. and induction of polyploidization in the megakaryoblast cell line (DAMI).

"Thrombopoietin" (TPO) is defined as a compound that has thrombopoietic activity or is capable of increasing serum platelet counts in mammals. Preferably, TPO is able to increase the number of endogenous platelets by at least 10%, even more preferably by 50%, and most preferably if it is able to increase the platelet count in a human to a value greater than 150 x 10 ⁹ per liter of blood.

"Isolated mpl ligand nucleic acid" is RNA or DN A, containing greater than 16, preferably 20 or more, sequenced nucleic bases that encode a biologically active mpl ligand or fragment thereof, is complementary to RNA or DNA, or hybridizes to RNA or DNA, and remains stably bound under mild to severe conditions. The RNA or DNA is free of at least one contaminating nucleic acid source with which it normally occurs under natural conditions and preferably contains no mammalian RNA or DNA. The expression is free of at least one contaminating nucleic acid source that normally occurs under natural conditions' includes the case where the nucleic acid is present in the source material or in natural cells but is at a different chromosome position or otherwise flanked by nucleic acid sequences, not as commonly found in source material cells. An example of an isolated mpl nucleic acid ligand is RNA or DNA that encodes a biologically active mpl ligand and which has at least 75% sequence identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably 95% sequence identity with human, mouse or porcine mpl ligand.

"Regulatory sequences", when referring to expression, means the DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Regulatory sequences suitable for prokaryotes include,

For example, supportive, selectable and efficient sequences, a ribosome binding site, and probably other sequences not yet known. Eukaryotic cells are known to process promoters, polyadenylation signals, and enhancers.

"Operatively linked" when referring to nucleic acids means that the nucleic acids are functionally aligned with the sequence of other nucleic acids. For example, DNA presequence, or as a secretory, is operably linked to DNA of a polypeptide when it is in the function of a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is usefully associated with a coding sequence when positioned so as to aid translation. In general, the term "operably (usable) spliced" means that the spliced DNA sequences are immediately adjacent and, in the case of secretion directing, are immediately adjacent and are in the interpretation phase. However, the magnifiers need not be immediately adjacent. The bonding is carried out by tying in suitably defined places. In the absence of such sites, synthetic oligonucleotide adapters or binders are used in accordance with conventional practice.

"Exogenous" when referring to an element means a nucleic acid sequence that is foreign to or homologous to the cell but at a position in the host cell of the nucleic acid in which the element is not normally found.

"Cell", "cell line" and "cell culture" are used interchangeably in the present invention and such designations include all descendants of cells or cell lines. Thus, for example, terms such as "transformants" and "transformed cells" include the subject primary cells and cultures derived therefrom regardless of the number of transfers. It is also understood that not all progeny cells may be exactly identical in DNA content due to deliberate or ill-considered mutations. Also included are mutant progeny that have the same function or biological activity as the original transformed cell. In the case of different designations, this will be the context.

"Plasmids" are autonomously reproducible circular DNA molecules with an independent source of reproducibility and are indicated in the present invention by the lowercase letter "p" preceding and / or followed by capital letters and / or numbers. According to this patent, the starting plasmids are either commercially available or publicly unrestrictedly available or can be obtained from such available plasmids by published procedures. It can also be other plasmid equivalents known to those of ordinary skill in the art.

"Restriction enzyme digestion", when referring to DNA, means the catalytic cleavage of internal phosphodiester DNA bonds with an enzyme that

acts only at certain positions or locations of the DNA sequence. Such enzymes are termed "restriction endonucleases". Each restriction endonuclease distinguishes a specific DNA sequence called a "restriction site" that has a double symmetry. The various restriction enzymes used in this invention are commercially available and their reaction conditions, co-factors and other requirements have been applied as determined by the suppliers of these enzymes. Restriction enzymes are commonly indicated by abbreviations consisting of a capital letter, followed by additional letters representing the microorganism from which each restriction enzyme was originally derived, followed by a numeral denoting a specific enzyme. Generally, about 1 µg of plasmid or

The DNA fragment was used with about 1-2 units of enzyme in about 20 μΐ of buffer. Suitable buffers and substrate amounts for specific restriction enzymes are specified by the manufacturer. Usually, incubation for 1 hour at ° C was used, but can be adapted to the supplier's instructions. After incubation, the protein or polypeptide is removed by phenol or chloroform extraction and the leached nucleic acid is separated from the aqueous phase by ethanol precipitation. The restriction enzyme digestion may be followed by hydrolysis of the terminal 5 'phosphates with bacterial alkaline phosphatase to prevent the two restriction digested ends of the DNA fragment from joining and thereby cyclization or the formation of a closed loop that might interfere with the introduction of another DNA fragment at the restriction site. Unless otherwise defined, plasmid digestion is not followed by 5 'terminal dephosphorylation. Methods and reagents for dephosphorylation are conventional, as described in sections 1.56-1.61 of Sambrook et al., Molecular Cloning: A Laboratory Manual [New York: Cold Spring Harbor Laboratory Press, 1989].

- 38 'Recovering' or 'isolating' a given DNA fragment from a restriction leach means separating the leach by electrophoresis on polyacrylamide or agar, identifying the desired fragment by comparing its mobility to the mobility of labeled fragments of known molecular weight, separating the gel portion containing the desired fragment and separating the gel from DNA. This procedure is generally known. It is reported, for example, by Lawn et al., Nucleic Acids Res., 9: 6103-6114 [1981] and by Goeddel et al., Nucleic Acids Res., 8: 4057 [1980],

"Southern analysis" or "Southern blotting" is a method to confirm the presence of DNA in a restriction endonuclease extract or DNA-containing mixture by hybridization with a known labeled oligonucleotide or DNA fragment. Typical Southern analysis involves separating DNA leaches on an agar gel followed by denaturation of the DNA and transfer of the DNA to nitrocellulose, nylon, or other suitable membrane carrier for analysis by radiolabeled, biotinylated, or enzymatically labeled probe (sample) as set forth in Sections 9.37-9.52 of Sambrook et al., supra.

"Northern analysis" or "Northern blotting" is a method used to identify RNA sequences that hybridize to a known sample, such as an oligonucleotide, DNA fragment, cDNA, or fragment thereof, or RNA fragment. The sample is labeled with a radioisotope such as ³² P, or biotinylation or enzymatically. The procedure for RNA analysis typically consists of its electrophoretic separation on an agar or polyacrylamide gel, transfer to nitrocellulose, nylon or other suitable membrane, and hybridization to a sample using standard procedures well known in the art, such as those disclosed in Sections 7.39-7.52 of the above-cited publication by Sambrook et al.

"Binding" ("ligation") is the process of forming phosphodiester bonds between two fragmentary nucleic acids. In order to bind two fragments, the fragment ends must be compatible with each other. In some cases, the termini will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the frayed ends that normally occur after endonuclease digestion into blunt ends that are suitable for binding. Dulling process

the termination consists in treating the DNA in a suitable buffer for at least 15 minutes at 15 ° C with about 10 units of a Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of four deoxyribonucleotide triphosphates. The DNA is then purified by phenol-chloroform extraction and precipitation with ethanol. The DNA fragments that are linked together are placed in solution in approximately equimolar amounts. The solution further comprises ATP, a ligase buffer, and a ligase such as T4 DNA ligase, of about 10 units per 0.5 µg of DNA. If the DNA is to be ligated into the carrier, the carrier must first be linearized by digestion with the appropriate restriction endonuclease (s). Then the linearized fragment is treated with bacterial alkaline phosphatase or intestinal calf phosphatase to prevent self-binding during this process.

"Preparation" of DNA from cells means isolation of plasmid DNA from a host cell culture. Commonly used methods of DNA preparation are the broad and narrow-scale plasmid preparations described in sections 1.25-1.33 of Sambrook et al., Supra. After preparation, the DNA may be purified by methods well known in the art, for example as set forth in section 1.40 of Sambrook et al., Supra.

"Oligonucleotides" are short-chain, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (such as those known from phosphotriester, phosphonate or phosphoramidite chemistry, using solid-phase procedures as disclosed in EP 266 032 published May 4 1988, or the procedure using deoxynucleoside H-phosphonate intermediates as described by Froehler et al., Nucl. Acids Res., 14: 5399-5407 [1986]). Other methods include the polymerase chain reaction as defined below and other self-initiation procedures and oligonucleotide synthesis on solid supports. All of these methods are disclosed in Engels et al., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989). These methods are used when the entire nucleic acid sequence of the gene is known or a complementary nucleic acid sequence to the coding strand is available. Thus, if the target amino acid sequence is known, it is also believed that the potential nucleic acid sequences and preferred coding residues for each amino acid residue are known. The oligonucleotides are then purified on polyacrylamide gels.

"Polymerase Chain Reaction" or "PCR" refers to a process or method in which the minimum amounts of specific portions of nucleic acid, RNA and / or DNA are increased as set forth in US Patent No. 5,768,549. No. 4,683,195, issued July 28, 1987. In general, it is desirable to have sequence information about the terminations (or even further) of the region of interest, so that oligonucleotide primers can be determined by this; these primers may be identical or similar in sequence to the opposite strands of the template being enlarged. The 5 'terminal nucleotides of the two primers can blend in with the terminations of the enlarged species. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, and the like. (see: Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263 [1987]; Erlich, ed., PCR Technology, Stockton Press, NY, 1989). In the present invention, PCR is reported as one, but need not be the only example of a nucleic acid polymerase reaction method for enlarging a nucleic acid test sample which involves using a known nucleic acid as a primer and nucleic acid polymerase enlarging or generating a specific portion of the nucleic acid.

"Stringent conditions" are those in which (1) low ionic strength and high temperature are used for scrubbing, for example, 0,015 M NaCl / 0,0015 M sodium citrate / 0,1% NaDodSO ₄ (SDS) at 50 ° C or (2) during the hybridization, a denaturing agent such as formamide is used, for example 50% (v / v) formamide with 0.1% cow serum albumin / 0.1% Ficoll / 0.1% polyvinalpyrohdone / 50 mM sodium phosphate as buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 ° C. Another example is the use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, ultrasonic DNA of salmon sperm obtained (50 µg / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C, washing at 42 ° C in 0.2 x SSC and 0.1% SDS.

"Moderately stringent conditions" are disclosed in Sambrook et al., (Cited above) and include the use of a wash solution and hybridization conditions (temperature, ionic strength, and% SDS) less stringent than above. Examples of moderately stringent conditions are conditions such as overnight incubation at 37 ° C in a solution containing:

- 41 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate and 20 μΐ / πύ DNA from denatured carved salmon sperm , followed by washing the filters with 1 x SSC at a temperature of about 37-50 ° C. Trained experts know how to adjust temperature, ionic strength, etc. and when other factors such as experiment time and the like need to be adjusted.

"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies have binding specificity to specific antigens, immunoglobulins also include antibodies and other antibody-like molecules that are not specific for antigens. For example, polypeptides of the latter species are produced at low concentrations by the lymphatic system and at increased concentrations by myelomas.

"Natural antibodies and immunoglobulins" are usually heterotetrameric glycoproteins having about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a single covalent disulfide bond, while there are different numbers of disulfide linkages between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable region (V _H ) at one end and a plurality of constant regions beside it. Each light chain has a variable region at one end (V1) and a constant region at the other end; the light chain constant region is aligned with the first heavy chain constant region, and the light chain variable region is aligned with the heavy chain variable region. The interface between the light and heavy chain variable regions is believed to be specific amino acid residues (Clothia et al., J. Mol. Biol., 186: 651-663 [1985]; Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82: 4592-4596.

The term "variable" refers to the fact that certain portions of the variable regions vary greatly in sequence between antibodies and are used in linking and specificity of each individual antibody to its specific antigen. However, variability is not

also divided into antibody variable regions. It is concentrated in three segments, called complementarity determining spheres (CDRs) or hypervariable spheres, both in the light and heavy chain variable regions. The most protected portions of the variable regions are called framework (FR). All natural heavy and light chain variable regions include four FRs, predominantly receiving the β-sheet configuration, joined by three CDRs that form a loop link and in some cases form part of the β-sheet structure. CDRs are held together in each strand, in close proximity to the FR portions, and CDRs from the second strand contribute to the generation of antigen binding sites of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institue of Health, Bethesda, MD [1987]). ). The constant domains do not directly participate in the binding of the antibody to the antigen, but have different effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity.

Papain leaching of antibodies provides two identical antigen binding fragments, called "Fab" fragments, each with one antigen binding site and a residual "Fc" fragment, whose name reflects its ability to crystallize immediately. Treatment with pepsin gives an F (ab ') 2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

"Fv" is the smallest fragment of an antibody that includes the overall ability to distinguish an antigen and has a binding site. This region consists of a dimer of the variable region of one heavy and one light chain in tight non-covalent association. This configuration is characterized in that the three CDRs of each variable region interact by providing an antigen binding site on the surface of the V _H -V _L dimer. Six CDRs together form a binding antigen specific antibody. However, even one variable region (or half of an Fv that contains only three antigen-specific CDRs) is able to recognize and bind antigen, albeit with lower affinity than the full binding site.

The Fab fragment also contains the light chain constant region and the first heavy chain constant region (CH1). Fab 'fragments differ from Fab fragments by adding several residues to the carboxyl terminus of the heavy chain CH1 region that contain one or more cysteines from the antibody hinge region. Fab'SH is a designation used herein for Fab 'in which the cysteine residue (s)

- 43 constant regions carry a free thiol group. F (ab ') 2 antibody fragments were originally prepared as paired Fab' fragments having hinge cysteines between them. Chemical coupling of antibody fragments are also known.

The term "light chains" of antibodies (immunoglobulins) from any vertebrate species may refer to one or two distinctly different types, called kappa (χ) and lambda (λ), which are based on amino acid sequences and their constant domains.

Depending on the amino acid sequences of the constant domain of their heavy chains, we divide immunoglobulins into five groups. The five main groups of immunoglobulins are: IgA, IgD, IgE, IgG and IgM, and some of them can be subdivided into subgroups (isotypes), e.g. IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains that correspond to different immunoglobulin groups are termed α, delta, epsilon, γ, and μ. Subunit structures and three-dimensional configurations of different immunoglobulin classes are well known.

The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compounds with polyepitopic specific as well as antibody fragments (e.g. Fab, F (ab ') 2 and Fv) if they have the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies having an identical population, except for possibly naturally occurring mutations that may be present in minimal amounts. Monoclonal antibodies are highly specific, acting directly against a single antigenic site. Further, contrary to the preparation of conventional (polyclonal) antibodies, which typically include different antibodies directed directly against different determinants (epitopes), each monoclonal antibody acts directly against one determinant on the antigen. In terms of their specificity, it can be added that monoclonal antibodies are advantageous when synthesized using a hybrid culture not contaminated by other immunoglobulins. epithet

- 44 "monoclonal" indicates the nature of such an antibody, obtained from a substantially homogeneous population of antibodies, and not one that has been prepared as the desired antibody production by another particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by the hybrid method first described by Kohler & Milstein, Nature, 256: 495 (1975) or may be prepared by recombinant DNA methods (see, e.g., US Patent 4,816,567). [Cabilly et al.]).

Monoclonal antibodies of the invention include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to (or homologous to) the corresponding sequences in antibodies derived from a particular species or belonging to a group or subgroup of particular antibodies whereas the remainder of the chain (s) is identical to (or homologous to) the corresponding sequences in antibodies derived from other species or belonging to a group or subgroup of other antibodies, also to fragments of such antibodies as long as they have the desired biological activity (US patent 4,816,567 (Cabilly et al.) And Morrison et al, Proc. Natl. Acad. Sci. USA, 81: 6851-6855 [1984]).

The "humanized" forms of non-human (non-human) (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other antigen-binding antibody sub-sequences) that contain minimal a sequence derived from a non-human immunoglobulin. Humanized antibodies are mostly human immunoglobulins (recipient antibodies) in which residues from the complementary determining regions (CDRs) of the recipient are replaced by residues from non-human CDRs (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, the human immunoglobulin Fv framework residues corresponding to w may be replaced

In addition, humanized antibodies may contain residues that are found neither in the antibody recipient nor in the imported CDRs or in the main (carrier) sequences. These modifications are made to further refine and optimize antibody preparation. Thus, the humanized antibodies will comprise substantially all of at least one or preferably two variable domains in which all or substantially all of the CDRs correspond to the CDRs of a non-human immunoglobulin and all or substantially all of the JFRs are FRs from

45 identical human immunoglobulin sequences. The humanized antibody will optimally also comprise at least a portion of an immunoglobulin constant sphere (Fc), preferably a human immunoglobulin. Additional information is provided by 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 treatment of a human tissue with a therapeutically effective dose of the polypeptide in a pharmaceutically acceptable carrier will not result in any state of sensitivity or resistance to the polypeptide after the second administration of the polypeptide after the appropriate latency period (i.e. 8-14 days).

Π. Preferred embodiments of the invention

Particularly preferred polypeptides of the invention are homogeneous polypeptide (s) related to mpl ligand (s) or thrombopoietin (TPO), which can bind to mpl, a superfamily of cytoldine receptors, and which have the biological ability to stimulate the introduction of radionucleotides ( ³ H-thymidine) into the DNA of the Ba / F3 cells dependent on IL-3 and with human mpl P. More preferred mpl ligand isolated proteins to a mammal having krvotvomú activity, in particular the megakaryocytopoietic and platelets, being capable of stimulating the proliferation, maturation and / or differentiation of immature megakaryocytes or their precursors into a mature platelet-producing form. The most preferred polypeptides of the invention are human mpl ligand (s), including fragments thereof having haematopoietic, megakaryocytopoietic or thrombopoietic activity. This human mpl ligand may lack glycosylation. Other preferred human mpl ligands are the "EPO-domain" of hML, referred to as hMLi ₅₃ or ITPO153, the truncated shape of hML, referred to as I1ML245 or ITPTP245, and the mature full-length polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO: 1), referred to as hML, I1ML332 or ITPTP332, and a biologically active surrogate variant of hML (R153A, R154A).

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

Preferred polypeptides of the invention are biologically active variants of mpl ligands having an amino acid sequence comprising at least 70% of the same amino acid sequence with a human mpl ligand (see Fig. 1 [SEQ ID NO: 1]) with a mouse mpl ligand (see Fig. 16). [SEQ ID NOS: 12 & 13]), with recombinant porcine mpl ligand (see Figure 19 [SEQ ID NO: 18]) or porcine mpl ligand isolated from aplastic porcine plasma, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 95%.

The Mpl ligand isolated from aplastic porcine plasma has the following characteristics:

(1) The partially purified ligand elutes from a gel filtration column packed with either PBS, PBS containing 0.1% SDS or PBS containing 4M MgCl ₂ with Mr 60000

70000;

(2) Ligand activity is abolished by pronase;

(3) The ligand is stable at low pH (2.5), SDS up to 0.1% and 2M urea;

(4) The ligand is a glycoprotein, bound to different lectin column columns;

(5) The high purity ligand elutes from the non-reduced SDS-PAGE with a Mr 25000 35000. Smaller amounts also elute with a Mr of about 18000-22000 and 60000;

(6) The high purity ligand is redissolved as a doublet with Mr 28000 and 31000;

(7) The amino-terminal sequence in the bands 18000-22000, 28000 and 31000 is the same as - SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO: 29); and (8) The ligand binds and elutes from the following affinity column columns

Blue-Sepharose

CM Blue-Sepharose

MONO-Q, MONO-S, Lentil Lectin-Sepharose, WGA-Sepharose,

Con A-Sepharose

Ether 650m Toyopearl,

Butyl 650 m Toyopearl,

Phenyl 650m Toyopearl a

Phenyl-Sepharose

The most preferred polypeptides (mpl ligand) are those encoded by human genes or cDNA having the amino acid sequence shown in FIG. 1 (SEQ ID NO: 1).

Other preferred naturally occurring biologically active polypeptides (mpl ligand) of the invention are prepro-mp / ligand, gamma-mpl ligand, mature mpl ligand, mpl ligand fragments, and glycosylation variants thereof.

Other preferred polypeptides of the invention will play variants and chimeras of the mpl ligand sequences. Preferred variants and chimeras of mpl ligand sequences are biologically active variants of mpl ligand, wherein the amino acid sequence is at least 70% identical to the amino acid sequence of human mpl ligand or mpl ligand isolated from aplastic porcine plasma, preferably at least 75%, more preferably at least 80% more preferably at least 85%, more preferably at least 90% and most preferably at least 95%. A particularly preferred variant of mpl Uganda is a variant of the N-terminal domain of hML (referred to as the "EPO-domain" because of its erythropoietm homology sequence). The preferred hML EPO-domain contains about the first 153 amino acid residues of mature hML and is referred to as IL11515. An arbitrarily preferred variant of the hML sequence comprises a sequence in which one or more of the mono- or di-amino acid residues in the C-terminal domain is substituted with non-basic amino acid residues (e.g., hydrophobic, neutral, acidic, aromatic, Gly, Pro, and the like). A preferred variant of the hML sequence of the C-terminal domain comprises a sequence in which the Arg residues 153 and 154 are replaced by Ala residues. This variant is referred to as hML ₃ 32 (R153A, R154A). Alternatively, a preferred hML variant comprises either I1ML332 or I1ML153 in which the amino residues 111-114 (QLPP or LPPQ) are deleted or replaced in different tetrapeptide sequences (e.g., AGAG and the like). The further reduction of the mutants is designated as ₃₃ 2 or A4hML A4hMLi5 _third

A preferred chimera is formed by coupling the mpl ligand or fragment thereof (defined below) to a heterologous polypeptide or fragment thereof. For example, hMLi ₅₃ may attach to an IgG fragment, thereby improving its serum half-lives or IL-3, G-CSF or EPO, to produce a molecule with higher thromboprost or chimeric hematopoietic activity.

Alternatively, a preferred human mpl Uganda chimera is a "MLEPO domain chimera" that contains N-terminal 153 to 157 hML residues substituted with one or more (but not all) residues of human EPO, which are approximately aligned in FIG. 10 (SEQ ID NO: 7). A particular block sequence that is inserted into the N-terminal portion of hML may contain one or more N-glycosylation sites at (EPO) positions 24-27, 38-40, and 83-85; one or more of the four putative amphipathic α-spiral bundles at (EPO) positions 9-22, 59-76, 90-107 and 132-152; and other highly protected regions, including N-teminal and C-terminal regions and residue positions (epo) 44-52 (see, e.g., Wen et al., Blood, 82: 1507-1516 [1993] and Boissel et al., J Biol. Chem., 268 (21): 15983-15993 [1993]). It is expected that the "ML-EPO domain chimera" will have a mixed thrombopoietic erythropoietic (TEPO) biological activity.

Other preferred polypeptides of the invention include mpl ligand fragments having a consecutive sequence of at least 10, 15, 20, 25, 30 or 40 amino acid residues that are identical to the mpl ligand sequences isolated from aplastic porcine plasma or the human mpl ligand described herein (see Table 14, Example 24). A preferred fragment of the mpl ligand is human ML [1-X], wherein X is 153, 164, 191, 205, 207, 217, 229 or 245 (see Fig. 1 [SEQ ID NO: 1] v

for residue sequences 1-X). Other preferred mpl ligand fragments include fragments that result from chemical or enzymatic hydrolysis or leaching of the purified ligand.

Another preferred aspect of the invention is a method of purifying mpl ligand molecules comprising contacting a mpl ligand source containing mpl ligand molecules with a static polypeptide receptor, particularly an mpl or mpl linked polypeptide, under conditions wherein the purified mpl ligand molecules are selectively adsorbed on a static a polypeptide receptor; washing the static carrier to remove unadsorbed substances and eluting the purified molecules from the static polypeptide receptor with the high-boiling timing solution. The source containing mpl hgand may be plasma, with the static receptor preferably being the mpl-IgG junction.

Alternatively, the source containing mpl Ugand may be a recombinant cell culture wherein the concentration of mpl Ugand in either the culture medium or cell lysates is higher than in plasma or other natural sources. In this case,

the useful / npZ-IgG immunoaffinity method described above usually does not need to be used, but commonly known protein purification methods are used. A preferred purification method that ensures a substantially homogeneous mpl ligand consists of: removing damaged residues of either host cells or digested fragments by, for example, centrifugation or ultrafiltration; concentrating the protein using a commercially available protein concentration filter; separating the Uganda from other impurities in one or more stages by any of the following methods: immunoaffinity, ion exchange (e.g. DEAE or carboxymethyl or sulfopropyl group containing beds), Bhie-Sepharose, CM Blue-Sepharose, MONO-Q, MONO- S, Lentil lectin-Sepharose, WGA-Sepharose, Con A-Sepharose, Etlier Toypearl, Butyl Toypearl, Phenyl Toypearl, Protein A Sepharose, SDS-PAGE, Reversed Phase HPLC (e.g., aliphatic-linked silica) or molecular sieves Sephadex or Separation Chromatography based on molecular sizes; washing with ethanol or ammonium sulfate solution. In any of the foregoing steps, a protease inhibitor such as methylsulfonylphioride (PMSF) may be used to inhibit proteolysis.

Another preferred embodiment of the invention is the isolation of an antibody capable of binding to mpl Ugand. A preferred isolated mpl Uganda antibody is a monoclonal antibody (Kohler and Molstein, Nature, 256: 495-497 [1975]; Campbell, Laboratory Techniques in Biochemistry and Molecular Biology, Burdon et al., Eds, Volume 13, Elsevier Science Publishers, Amsterdam [ And Huse et al., Science, 246: 12751281 [1989]). The preferred isolated mpl ligand antibody is an antibody that binds to the mpl ligand with an affinity of at least about 10 ⁶ l / moL More preferred antibody associates with an affinity of at least 10 ⁷ l / moL The most preferred antibody is an antibody that acts against the mpl Ugand that it has one of the effector functions described above. An isolated antibody capable of binding to the mpl ligand may optionally be attached to a second polypeptide, and the antibody itself, or such mixture thereof, may be used to isolate and purify the mpl ligand from its source, as described above for a static mpl polypeptide. In further preferred aspects of this aspect of the invention, the present invention provides a method for detecting mpl Ugand in vitro or in vivo, comprising contacting the antibody with a sample, particularly a serum sample suspected of containing Ugand and detecting when the ligand is combined with the antibody .

In further preferred examples of the invention, the invention provides for isolation of a nucleic acid molecule that encodes an mpl ligand or fragments thereof, wherein the nucleic acid molecule can be labeled to be detectable or unlabelled and the nucleic acid molecule has a sequence that is complementary to the molecule or hybridizes under strong or milder conditions with a nucleic acid molecule having a sequence encoding an mpl ligand. The preferred mpl ligand nucleic acid is RNA or DNA that encodes a biologically active mpl ligand with a 75% sequence identity, preferably at least 80%, more preferably at least 85%, even more preferably 90%, and most preferably 95%. sequence identity with human mpl Hgandom. Preferred isolated nucleic acid molecules are DNA sequences encoding a biologically active mpl Hgand, selected from: (a) DNA based on the coding region of the mammalian mpl ligand genes (e.g., DNA comprising the non-cleotide sequence shown in Figure 1 (SEQ ID NO: 2)) fragments thereof); (b) DNA capable of hybridizing to the DNA of (a) under, at least, milder conditions; and (c) degenerate DNA to DNA according to (a) or (b), wherein degeneration refers to the genetic code. It is to be expected that the novel mpl ligands described herein may be members of the ligand family or cytokines having the appropriate sequence identity so that their DNA can hybridize to the DNA of FIG. 1 (SEQ ID NO: 2) (or complement or fragments thereof) under mild to moderate conditions. Thus, another aspect of the invention includes DNA that hybridizes under mild to moderate conditions to DNA that encodes mpl ligand polypeptides.

In another preferred embodiment of the invention, the nucleic acid molecule is a cDNA encoding mpl Ugand and further comprising a replicable vector, in which the cDNA is operably linked to regulate sequences distinguished by the host transformed vector. This aspect further comprises vector-transformed host cells and a method of using cDNA to influence the preparation of mpl Ugand, which comprises expressing a cDNA encoding mpl Ugand in a culture of transformed host cells and separating mpl Ugand from a host cell culture. The mpl Ugand prepared in this way is preferably a substantially homogeneous human mpl Ugand. Preferred host cells for the preparation of mpl Uganda are Chinese Hamster Ovary (CHO) cells.

The present invention further comprises a preferred method of treating a mammal having an immunological or blood-borne disease, particularly thrombocytopenia; who includes

Administering a therapeutically effective amount of the mpl ligand to the mammal. The Mpl ligand may also be administered in combination with a cytokine, particularly a cell group stimulating factor or interleukin. Preferred cell group stimulatory factors or interleukes include: kit-ligand, LEF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-2, IL-3, IL-5, IL-6, IL -7, IL-8, IL-9, or IL-11.

ΙΠ. Methods of preparation

For a long time, several authors have suggested that platelet production is regulated by various hereditary specific humoral (body) factors. Two different cytokine functions, termed megakaryocyte cell group stimulating factor (meg-CSF) and thrombopoietin, have been found to regulate megakaryocyte and platelet production (Williams et al., J. Cell Physiol., 110: 101-104 [1982]; Williams et al., Blood Cells, 15: 123-133 [1989] and Gordon et al., Blood, 80: 302-307 [1992]). According to this hypothesis, meg-CSF stimulates the proliferation of progenitic megakaryocytes, while thrombopoietin primarily affects the maturation of more differentiated cells and the ultimate platelet release. The induction and occurrence of meg-CSF and thrombopoietin in plasma, serum and urine of animals and humans, with ongoing thrombocytopenic events, have been well documented in the literature since 1960 (Odeli et al., Proc. Soc. Exp. Biol. Med., 108 : 428431 [1961]; Nakeff et al., Acta Haematol., 54: 340-344 [1975]; Specter, Proc. Soc. Exp. Biol., 108: 146-149 [1961]; Schreiner et al., J. Clin. Invest., 49: 1709-1713 [1970]; Ebbe, Blood, 44: 605-608 [1974]; Hoffinan et al., N. Engl. J. Med., 305: 533 [1981]; al., Exp. Hematol., 17: 1122-1127 [1988]; Mazur et al., Exp. Hematol., 13: 1164 [1985]; Mazur et al., J. Clin Invest., 68: 733-741 [1981]; Sheiner et al., Blood, 56: 183-188 [1980]; Hill et al., Exp. Hematol., 20: 354-360 [1992]; and Hegyi et al., Int. J. Cell Cloning , 8: 236-244 [1990]). These actions are referred to as specifically hereditary and distinct from known cytokines (Hill RJ et al., Blood, 80: 346 [1992]; Erickson-Miller CL et al., British J. Haematol., 84: 197-203 [1993] Straneva et al., Exp. Hematol. 20: 4750 [1992] and Tsukada J. et al., Blood, 81: 866867 [1993]). So far, attempts to purify meg-CSF or thrombopoietin from thrombocytopenic plasma or urine have not been successful.

In accordance with the above observations describing thrombocytopenic plasma, it was found that aplastic porcine plasma (APP) obtained from irradiated pigs,

- 52 stimulates the production of human megakaryocytes in vitro. It was further found that this stimulatory activity can be abolished by the soluble extracellular c, -mpl domain, confirming

APP as a potential source of putative mpl hgand (ML). Further, mpl Ugand was successfully purified from APP and amino acid sequence information was used to isolate murine, porcine and human ML cDNAs that are sequence homologous to erythropoietin and act similar to meg-CSF and thrombopoietin.

1. Purification and identification of plasma mpl Uganda

As indicated above, aplastic plasma from various species has been reported to contain components that stimulate in vitro hematopoiesis, but it has not been reported in the literature that some blood-inducing blood-stimulating factor has been isolated. One source of aplastic plasma comes from irradiated pigs. This aplastic porcine plasma stimulates human hematopoiesis in vitro. To determine if APP contained mpl Ugand, its effect was examined by measuring the introduction of ³ H-thymidine into Ba / F3 cells infected with human mpl P (Ba / F3-z »pZ) by the procedure shown in Figs. 2. APP stimulates the introduction of ³ H-thymidine into Ba / F3-wp / cells but not into control Ba / F3 cells (i.e., uninfected with human mpl P). Consequently, no effect was observed in normal porcine plasma. These results indicate that APP contained a factor or factors that transmit a proliferative signal via the mpl receptor and thus must be a natural ligand. This conclusion was further supported by the finding that the action of soluble mpl-IgG on APP blocks the stimulatory effects of APP on Ba / F3-zzipZ cells.

This effect of APP appears to be proteinaceous because pronase, DTT and / or heat act to degrade this effect (Fig. 3). The effect of APP was also non-dialysable. The effect was also stable at low pH values (pH 2.5 for 2 hours) and was shown to bind and elute from lectin affinity column columns indicating that it was a glycoprotein. To further elucidate the structure and identity of this effect, it was necessary to separate it from APP using the wp / -IgG chimera.

APP was processed as described in Examples 1 and 2. Briefly, mpl Ugand was purified using hydrophobic interaction chromotography (HIC), immobilized color chromatography and zwpZ-affinity chromatography. The recovery of effect after each step is shown in FIG. 4 and the fold purification is shown in Table 1. The overall recovery of effect with the zzzpZ-affinity column column was approximately 10%. faction

- 53 with a maximum effect (F6) from the z / / / - affinity column column had an estimated specific effect of 9.8 x 10 ⁶ units / mg. Total purification from the original 5 liters of APP was approximately 4 x 10 ^6- fold (from 0.8 units / mg to 3.3 x 10 ⁶ units / mg) with 83 x 10 ^6- fold protein reduction (from 250 g to 3 gg) ). The specific effect of the Ugand eluted from the zzZ / affinity column is estimated to be 33 x 10 ⁶ units / mg.

Table 1

Purification of mpl ligand

sample volume dainty Prateín ma / ml Units per ml units Specif. unit / mg activity yield % Multiple cleaning APP 5000 50 40 200,000 0.8 1 Phenyl 4700 0.8 40 200,000 5 0 94 62 Blue-Aug. 640 0.93 400 256,000 430 128 538 znp / (μΙ) (Fxns 5-7) 1 2 5x1 O ' ⁴ 1666 20,000 3,300,000 1 0 4,100,000

Protein was determined by Bradford assay. The protein concentration of zzzpZ-eluted fractions 5-7 was estimated based on the silver staining intensity of the SDS-gel. One unit is defined in such a way that it causes 50% of the maximal stimulation of Ba / F3 cell proliferation.

Analysis of the recovered fractions from the mpl affinity column column by SDSPAGE (4-20%, Novex gel) was performed under reducing conditions, revealing the presence of cf several proteins. Proteins that cause the most intense silver staining dissolve with Mr 66000, 55000, 30000, 28000 and 18000-22000. In order to determine which of these proteins stimulates proliferation of BaZF3mpl cell cultures, the proteins were gelled as described in Example 2.

The results of this experiment showed that the higher effect fraction eluted from the gel layer containing the proteins with Mr 28000-32000 while the lower effect fraction eluted from the gel layer with Mr value 18000-22000 (Fig. 6). The only visible proteins in these regions had Mr 30000, 28000 and 18000-22000. To identify and obtain a protein sequence for dissolved proteins in this region

- 54 gel (bands 30, 28 and 18-22 kDA), the three proteins were electroblotted by PVDF and sequenced as described in Example 3. The amino terminal sequences obtained are shown in Table 2.

Table 2

Aminothermal sequences of mpl ligand

30 kDa 1 2 3 4 5 6 7 8 9 10 11 12 (S) P A P P A (C) D P R L L N K L L D D (H / S) V L H (G) R L (SEQ ID NO: 29) 28 kDa 1 2 3 4 5 6 7 8 9 10 11 12 (S) P A P P A X D P L L N K L L D D (H) V L (H) G R (SEQ ID NO: .31) 18-22 kDa 1 5 10 X P A P P A D D L L (N) (K) (SEQ ID NO: 33)

Computer analysis revealed that these amino acid sequences were new. Since all sequences were the same, it can be assumed that proteins corresponding to 30 kDa, 28 kDa, and 18-22 kDa are only different forms of the same novel protein. Furthermore, this protein could be considered as a natural mpl ligand because it had activity in the same region after dissolution (28000-32000, SDS-PAGE, 4-20% gel). In addition, the partially purified ligand migrated with a Mr value of 17000-30000 when subjected to gel filtration chromatography using a Superose 12 column (Phannacia). Different Mr ligand forms are believed to occur due to proteolysis or glycosylation differences or other post- or pre-translational modifications.

As previously reported in the literature, antisense human mpl RNA suppressed megakaryocyte production in human bone marrow cultures enriched with CD 34 ⁺ progenitoma cells without affecting the differentiation of other hematopoietic lineage lineages (Methia et al., Supra). This result suggests that the mpl receptor may play a role in influencing the in vitro differentiation and proliferation of megakaryocytes. In order to further elucidate the role of mpl Uganda in megakaryocyte production, the effect of APP and non-mpl Uganda APP in vitro on human megakaryocyte production was compared. Effect of APP on production of human megakaryocytes

was determined using a modification of the megakaryocyte formation assay in the liquid suspension described in Example 4. In this assay, human peripheral cell strains (PSC) were treated with APP before and after wpZ-IgG affinity chromatography. GPIIbIIa stimulation of megakaryocyte formation was quantified with ¹²⁵ I-antiII _b III _and antibody (Fig. 7). As seen in FIG. 7, 10% of APP caused approximately 3-fold stimulation, while APP without mpl ligand had no effect. Clearly, APP without mpl ligand does not induce proliferation of Ba / F3- / npZ cells.

In another experiment, soluble human mpl-IgG, when added at zero, second and fourth days to cultures containing 10% APP, neutralized the stimulatory effects of APP on the production of human megakaryocytes (Fig. 8). These results suggest that mpl ligand plays a role in regulating the production of human megakaryocytes and thus may be useful in the treatment of thrombocytopenia.

2. Molecular cloning of mpl ligand

Starting from the amino-terminal amino acid sequence obtained from the 30 kDa, 28 kDa, and 18-22 kDa protons (see Table 2, above), pools of two degenerate oligonucleotide primers were used that were used to amplify porcine genomic DNA by PCR. the amino acid sequence was encoded by a single exon, then the correct PCR product length of 69 bp could be expected. A DNA fragment of this size was found and cloned into pGEMT. Figure 5 shows the sequences of oligonucleotide PCR primers and three clones. The amino acid sequence (PRLLNKLLR [SEQ ID NO: 33]) of the peptide, encoded between the PCR primers, was the same as that obtained by protein amino-terminal sequencing of the porcine ligand (see residues 9-17 for the 28 and 30 kDa porcine sequence sequences above).

A synthetic oligonucleotide, based on the PCR fragment sequence, was used for screening searches in a human genomic DNA library. Based on the sequence of the PCR fragment, a 45-membered oligonucleotide, designated pR45, was designed and synthesized. This oligonucleotide has the following sequence: 5 'GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG 3' (SEQIDNO: 34)

This deoxyoligonucleotide was used for screening in the human genomic DNA library in Xgem12 under mild hybridization conditions and

washes according to Example 6. Positive clones were selected, spots purified and analyzed by restriction mapping and Southem transfer. The 390 bp EcoRI-XbaI fragment, which hybridized to a 45-membered oligonucleotide, was cloned into pBhiescript SK-. DNA sequencing of this clone confirmed that DNA encoding a human porcine mpl ligand homolog was isolated. The human DNA and deduced amino acid sequences are shown in FIG. 9 (SEQ ID NOS: 3 & 4). Predicted intron positions in the genomic sequence are indicated by arrows and define 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 a chemical pattern of cDNA prepared from various human tissues. The expected size of the correct PCR product was 140 bp. After analysis of PCR products on a 12% polyacrylamide gel, the expected DNA fragment was detected in cDNA libraries prepared from human adult kidneys, 293 fetal kidney cells and cDNA prepared from human fetal liver.

Next, screening was performed by sorting in a fetal liver cDNA library (7x10 ⁶ clones) in lambda DR2 using the same 45-member oligonucleotide that was used for the same activity in the human genomic library and the fetal liver cDNA library under mild hybridization conditions. Positive clones were selected, plaques were cleaned, and the insert size was determined by PCR. One 1.8 kb insert size was selected for further analysis. Using the procedures described in Example 7, nucleotides and deduced amino acid sequences of human mpl Uganda (hML) were obtained. These sequences are shown in FIG. 1 (SEQ ID NOS: 1 & 2).

in

3. Structure of human mpl Uganda (hML)

The human mpl Uganda (hML) cDNA sequence (Fig. 1 [SEQ ID NO: 2]) contains 1774 nucleotides terminated by a poly (A) tail (tail). It contains 215 nucleotides from the 5 'untranslated sequence and from the 3' untranslated region 498 nucleotides. The putative initiation codon at nucleotide position (216-218), with a consensus sequence, is preferred for eukaryotic translation initiation. The framework has a length of 1059 nucleotides encoded by 353 polypeptide residues

The N-terminal of the predicted amino acid sequence is very hydrophobic and probably corresponds to a signal peptide. Computer analysis of the predicted amino acid sequence (von Heijne et al., Eur. J. Biochem., 133: 17-21 [1983]) indicates the potential cleavage position for the signal peptidase between residues 21 and 22. Cleavage at this position could release the mature polypeptide from 332 amino acid residues beginning with the amino terminal sequence, which was obtained from the mpl ligand purified from porcine plasma. The predicted non-glycosylated molar mass of the ligand from 332 amino acid additions is about 38 kDa. It has 6 potential N-glycosylation positions and 4 cysteine residues.

Comparison of the mpl ligand sequence with the Genbank sequence database showed a 23% identity between the 153 amino terminal residues of the mature human mpl ligand and human erythropoietin (Fig. 10 [SEQ ID NOS: 6 & 7]). Taking into account conservative substitutions, this region of hML has 50% similarity to human erythropoietin (hEPO). Both hEPO and hML contain four cysiteins. Three of the four cysteines are conserved in hML, including the first and last cysteine. Mutagenic positioning experiments have shown that the first and last cysteine of erythropoietin forms the disulfide bond required for proper functionality (Wang,

F. F. et al., Endocrinology 116: 2286-2292 [1983]). Analogously, the first and last cysteine of hML may also form a critical disulfide bond. No glycosylation position is conserved in hML. All potential N-linked glycosylation positions in hML are located in the carboxyterminal portion of the hML polypeptide.

As in hEPO, hML mRNA does not contain the same polyadenylation sequence AAUAAA, nor the regulatory element AUUUA, which is present in the 3 'non-transferable regions of many cytokines and is believed to affect mRNA stability (Shawet et al., Cell, 46: 659-667 [1986]). Northern blot analysis showed low levels of 1.8 kb hML RNA transcription alone in fetal and adult liver. After prolonged exposure, weaker binding of the same size can be detected in adult kidneys. By comparison, human erythropoietin is found in the fetal liver and in response to hypoxia also in adult kidney and liver (Jacobs et al., Nature, 313: 804.809 [1985] and Bondurant et al., Afo / ec. Cell. Biol., 6: 2731-2733 [1986]).

The importance of the C-terminal region of hML needs to be clarified. Based on the presence of six potential positions for N-linked glycosylation and the ability of the ligand to bind in the lectin affinity column, this region of hML is likely to

glycosylated. In some goal elution experiments, effective dissolution was observed with a Mr of about 60,000, which may represent the full length of the glycosylated molecule. The C-terminal region can then act to stabilize and increase the half-life of hML. In the case of erythropoietin, the non-glycosylated form has complete biological activity in vitro but has a significantly reduced plasma half-life relative to glycosylated erythropoietin (Takeuchi et al., J. Biol. Chem., 265: 12127-12130 [1990]; Narhi et al., J. Biol. Chem., 266: 23022-23026 [1991] and Spivack et al., Blood, 7: 90-99 [1989]). The C-terminal domain of hML contains two di-amino acid sequences [Arg-Arg junction at positions 153-154 and 245-246], which can serve as potential treatment positions. Cleavage at these positions may be suitable to release the forms 30, 28 and 18-22 kDa from ML isolated from APP. Significantly, the Arg153-Arg154 sequences follow the erythropoietin-like ML domains. These observations indicate that the full length of ML may be a protein precursor that is subject to limited proteolysis to release the mature ligand.

4. Isoforms and variants of human mpl Uganda

Isoforms or alternatively spliced forms of human mpl Uganda are detected by PCR in human adult liver. Briefly, primers were synthesized correspondingly to each termination for the selected internal regions of the ML coding sequence. These primers are used in RT-PCR to expand RNA from human adult liver as shown in Example 10. In addition to the full-length form, designated hML, three other forms, designated hML2, hML3 and hML4, are observed or derived. . The mature deduced amino acid sequences of all four isoforms are shown in Figure 11 (SEQ ID NOS: 6, 8, 9 & 10). At position 700, 116 nucleotides are missing in hML3, which results from the missing amino acids and the shift. The cDNA now encodes a mature polypeptide that is 265 amino acids in length and deviates from the hML sequence at amino acid residue 139. Finally, hML4 has deleted nucleotide 12 after nucleotide position 618 (also found in mouse and porcine sequences [see below]) as well as removal 116 bp, which was also found in hML3. Although no clones, except for the removal of 12 bp (after nucleotide 619), or U isolated in humans (designated as hML2), this form is likely to exist as such

The 59 isoforms were identified in both mouse and pig (see below) and because they were identified in conjunction with the removal of 116 nucleotides in hML4.

And replacement options hML in which dibasic Argi5 ₃ -Argi54 sequence was replaced with two alanine residues and the "EPO-domain" truncated form hML f is formed to determine whether the biological activity was needed full length ML. A divalent sequence replacement variant Arg153-Arg154, referred to as hML (R153A, R154A), was generated using PCR as set forth in Example 10. The "EPO-domain" truncated form, hML 153, was also generated using PCR using stopcodon Argl53.

5. Expression of recombinant human mpl Uganda (rliML) into transiently infected human embryonic kidney (293) cells

To confirm that the cloned human cDNA encoded Hgand for mpl, Ugand was extruded into 293 mammalian cells under regulation with the immediate cytomegalovirus start promoter, using pRK5hML or pRK5-hMLi53 expression vectors. Transient human embryonic kidney supernatants 293 were found to be cells stimulate the introduction of ³ H-thymidine into Ba / F3-wp / cells but not into parental Ba / F3 cells (Fig. 12A). Medium from 293 cells infected with the pRK vector alone did not have this effect. Addition of mp / -IgG to the medium abolishes this stimulation (results not shown). These results indicate that the cloned cDNA encodes functional human ML (hML).

To determine whether the "EPO-domain" alone could bind and activate mpl, defined in 293 cells, the truncated form of hML, rhMLi5 _third Supernatants from infected cells were found to have activity similar to supematants from cells expressing full length hML (Fig. 12A), indicating that the C-terminal domain of ML is not required for binding and activation of c-mpl.

6. Mpl Ugand stimulates megakaiyocyte and platelet production

Like the full-length form of recombinant hML rhML, the cut-off form of rhML153 stimulated the production of human megakaryocytes in vitro (Fig. 12B). This effect was observed in the absence of other exogenously added hematopoietic growth factors. In addition to IL-3, of the hematopoietic growth factors tested, only ML had this effect. 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 megakaryocyte formation when tested separately by these assays (results not shown). This result demonstrates that ML has megakaryocyte stimulatory activity and determines the role of ML in regulating megakaryocyte production.

Those effects that lead to platelet formation and found in the plasma of thrombocytopenic animals have been shown to stimulate platelet production in a mouse reaction in a thrombocytosis assay (McDonald, Proc. Soc. Exp. Biol. Med., 14: 1006-1010 [ 1973] and McDonald et al., Scand. J. Haematol., 16: 326-334 [1976]). In this experiment, mice have acute thrombocytopenia induced by the use of specific serum that counteracts platelets, resulting in predictable induced thrombocytosis. Such immunothrombocyte mice are more resistant to thrombopoietin-like external effects than normal mice (McDonald, Proc. Soc. Exp. Biol. Med., 14: 1006-1010 [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 rML stimulates platelet production in vivo, thrombocytosis-induced mice were partially injected with partially purified rhML. Platelet count and quantitation of ³⁵ S into platelets were then quantified. Injection of 64000 or 32000 rML units in mice significantly increased platelet counts, namely, ~ 20% increased platelet counts (p = 0.0005 and 0.0001, respectively), and ~ 40% increased the incorporation of ³⁵ S into platelets ( p = 0.003) in treated mice versus control mice that were injected with excipient alone (Fig. 12C). This level of stimulation is comparable to that observed with IL-6 in the same experiment (results not shown). Treatment with 16,000 rML units did not show significant stimulation of platelet production. These results show that ML stimulates platelet production in a dose-dependent manner and thus has a similar effect to thrombopoietin.

In addition, 293 cells were also infected with other prepared hML isoforms, which were already described above, and supernatants were tested using the Ba / F3-mp / proliferation assay (see Fig. 13). hML2 and hML3 had no detectable effect in this assay, but the effect of hML (R153A, R154A) was similar to that of hML and I1ML153, indicating that treatment at the Argi53-Arg154 divalent positions is neither necessary nor detrimental to the effect observed.

7. Megakaryocyte formation and mpl ligand

Based on the literature, megakaryocyte production is thought to be regulated at a variety of cellular levels (Williams et al., J. Cell. Physiol., 110: 101-104 [1982] and Williams et al., Blood Cells, 15: 123-133 [1989]). This finding is based on the observation that certain hematopoietic growth factors stimulate megakaryocyte progenitor proliferation, while other factors appear to affect mainly maturation. The results presented in this invention show that ML affects both proliferative and maturation factor. Different evidence supports the fact that ML stimulates megakaryocyte progenitor proliferation. First, APP stimulates both proliferation and maturation of human megakaryocytes in vitro, and this stimulation is completely inhibited by wp / -IgG (Figs. 7 and 8). Furthermore, inhibition of megakaryocyte colony formation by c-mpl antisense oligonucleotides (Methia et spoL, Blood, 82: 1395-1401 [1993]) and the finding that c-mpl can transduce a proliferative signal in cells into which it has been infected (Skoda et al. , EMBO, 12: 2645-2653 [1993] and Vigon et al., Oncogene, 8: 2607-2615 [1993]) also indicate that ML stimulates proliferation. The apparent expression of c-mpl during all degrees of megakaryocyte differentiation (Methia et al., Blood, 82: 1395-1401 [1993]) and the ability of recombinant ML to rapidly stimulate platelet production in vivo shows that ML also affects maturation. The availability of recombinant ML makes it possible to reliably evaluate its role in regulating megakaryocyte and platelet production, as well as its potential effect on other hematopoietic lineages.

8. Isolation of the human mpl ligand (TPO) gene

Human TPO genomic DNA clones were isolated by screening the human genomic library in X-Gem12 with pR45 under relatively strong conditions or under very strong conditions with a fragment corresponding to the 3 'half of the human cDNA encoding the mpl ligand. Two overlapping lambda clones measuring 35 kb were isolated. Two overlapping fragments (BamH1 and EcoRI) containing the full TPO gene were subcloned and sequenced (see Figures 14A, 14B and 14C).

in

The structure of the human gene consists of 6 exons of 7 kb genomic DNA. The interfaces of all exon / intron junctions are consistent with the identical motif that has been determined in mammalian genes (Shapiro, M. B., et al., Nucl. Acids Res. 15: 7155 [1987]). Exon 1 and exon 2 contain a 5 'untranslated sequence and an initial four

- 62 amino acids of the signal peptide. The remainder of the secretory signal and the first 26 amino acids of the mature protein are encoded in exon 3. The entire carboxyl domain and 3 'untranslated, as well as 5050 amino acids of the erythropoietin-like domain are encoded in exon 6. The four amino acids contained in the deletion observed in hML-2 -2), are encoded at the 5 'end of exon 6.

Analysis of human genomic DNA by Southem blot shows that the TPO gene is present in a single copy. Chromosome localization in the gene was determined by fluorescent in situ hybridization (FISH), which mapped chromosomes 3q27-28.

9. Expression and purification of TPO from 293 cells

Preparation and purification of ML or TPO from 293 cells is described in detail in Example 19. Briefly, cDNA corresponding to the fully open reading frame of TPO was obtained by PCR using pRK5-hmp / 1. The PCR product was purified and cloned between the Cla I and Xhal restriction positions of plasmid pRK5tkneo (a pRK5tkneo derived vector modified to express a neomycin-resistant gene by regulation with a thymidine kinase) to obtain the pRK5tkneo.ORF vector (encoding the fully open vector) reading frame).

A second vector encoding the homologous domain of EPO was generated in the same manner, but different PCR primers were used to obtain the final shape designated pRK5-tkneoEPO-D.

These two vectors were infected with human embryonic kidney cells by the CaPC > 4 method and resistant neomycin clones were selected and allowed to grow until confluence. Using the Ba / F3-mpl proliferation assay, ML153 or ML332 clones were expressed in conditioned media.

In Example 19, the purification of rhML ₃ 32 is described. Briefly, 293-rhML ₃ 3 ₂ conditioned medium was used in a Blue-Sepharose (Pharmacia) column, where it was then washed with a buffer containing 2 M urea. The column was eluted with a buffer containing 2 M urea and 1 M NaCl. The entire eluate collected from the Blue-Sepharose column was then directly applied to the WGA-Sepharose column, washed with a 10-fold volume of buffer before the column volume containing 2 M urea and 1 M NaCl and eluting with the same buffer containing 0 5 M N-acetyl-D-glucosamine. The eluate from the WGA-Sepharose column was applied to C4-HPLC

63 columns (Synchrom, Inc.) and eluted discontinuously with propanol. SDS-PAGE the purified 293-rhML33 ₂ migrated as a broad band in the 68-80 kDa gel (see FIG. 15).

In Example 19 also discloses cleaning rhMLi53 · Briefly, 293-rhMLi53 conditioned media was resolved on Blue-Sepharose column in the same way as described for rhML ₃ 3 _2nd The Blue-Sepharose eluate was directly applied to the m 2 Z-affinity column as described above. RI1ML153, which eluted from the znpZ-affinity column, was purified and homogenized using a C4-HPLC column under the same conditions as used for rhML ₃ 3 ₂ . SDI-PAGE purified RI1ML153 is divided into two main and two small bands with Mr -18000-22000 values (see Fig. 15).

10. Mouse mpl ligand

By means of PCR, gel purification and radiolabelling in the presence of ³² P-dATP and ³² P-dCTP, a DNA fragment corresponding to the coding region of human mpl Uganda was obtained. This sample was used for screening by 10 ⁶ clones of the mouse liver cDNA library in XGTIO. A mouse clone (Fig. 16 [SEQ ID NOS: 12 & 13]) containing 1443 base pair inserts was isolated and sequenced. The putative initiation codon at nucleotide position 138-141 had a consensus sequence with a sequence that is favorable for eukaryotic translation initiation (Kozak, MJ Cell Biol., 108: 229-241 [1989]). This sequence defines an open reading frame of 1056 nucleotides, predicting a primary translation product of 352 amino acids. This open reading frame is flanked by 137 nucleotides at the 5 'end and 247 nucleotides at the 3' end of the untranslated sequence. There is no poly (A) tail (tail) over the untranslated region 3 ', indicating that the clone is probably not complete. The terminal of the predicted amino acid sequence is very hydrophobic and probably represents a signal peptide. Computer analysis (von Heijne, G., Eur. J. Biochem., 133: 17-21 [1983]) indicates the potential cleavage position for signal peptidase in

between residues 21 and 22. Cleavage at that position would generate a mature polypeptide of 331 amino acids (35 kDa) identified as ₃ mML 3R (or mML2 the need, resulting from the following text). The sequence contains 4 cysteines, all retained in the human sequence and seven potential N-glycosylation sites, of which 5 are conserved in the human sequence Similar to hML, all seven potential N-glycosylation sites are located in the C-terminal portion of the protein.

In comparison to human ML, both nucleotides and deduced amino acid sequences were observed in the "EPO-domains" of these MLs. However, when the deduced amino acid sequences of human and murine ML were aligned, the murine sequence was found to have removed the tetrapeptide between residues 111-114, corresponding to the removal of 12 nucleotides after nucleotide 618, which can be encountered both in human (see above) and in pig (see below) cDNA. Additional clones were therefore tested to detect probable isoforms of murine ML. One clone encoded a polypeptide consisting of a deduced 335 amino acid sequence that contained the "missing" tetrapaptide LPLQ. This form is believed to be the full length of murine ML and is referred to as mML or 111ML335. The nucleotide and deduced amino acid sequence for mML are shown in FIG. 17 (SEQ ID NOS: 14 & 15). This cDNA clone consists of 1443 base pairs followed by a poly (A) tail. It includes a 1068 bp open reading frame flanked by 134 '5' end bases and 241 '3' untranslated sequence bases. The expected initiation codon lies at nucleotide position 138-140. The open reading frame encodes a predicted protein of 356 amino acids, of which the first 21 are very hydrophobic and probably useful as a secretory signal.

Finally, a third mouse clone was isolated, sequenced and was found to contain a 116 nucleotide deletion, corresponding to hML3. This mouse isoform was then renamed to mML3. A comparison of the deduced amino acid sequences of the two isoforms is shown in FIG. 18 (SEQ ID NOS: 9 & 16).

The overall amino acid sequence identity between human and murine ML (Fig. 19 [SEQ ID NOS: 6 & 17]) is 72%, but this homology is not uniform. The region defined as the "EPO-domain" (amino acids 1-153 of the human sequence and 1-149 of the mouse sequence) is better protected (86% homology) than the carboxyterminal region of the protein (62% homology). This may further imply that only the "EPO-domain" is important for the biological activity of the protein. Remarkably, of the two di-amino acid motifs found in hML, only the di-motif found immediately after the "EPO-domain" (residue position 153-154) in the human sequence is also present in the murine sequence. This is consistent with the possibility that the full length of ML may represent a protein precursor that is subject to limited proteolysis to release the mature ligand. Similarly, proteolysis between Arg 15 ₃ -Argi ₅ 4 can facilitate purification of hML.

An expression vector containing the entire mML coding sequence was transiently infected with 293 cells as described in Example 1. Conditioned medium from these cells stimulated ³ H-thymidine incorporation into Ba / F3 cells expressing either mouse or human mpl but had no effect. to the parent cell line (without mpl). Accordingly, the cloned murine ML cDNA encodes a functional Ugand capable of activating both the murine and human ML receptors (mpl).

11. Pig mpl Ugand

Porcine ML (pML) cDNA was isolated by RAČE PCR as described in Example 13. A 1342 bp PCR cDNA product was found in the hips and subcloned. Some clones were sequenced and a porcine mpl Ugand 332 amino acid residue, designated pML (or pML ₃₃₂ ), whose nucleotide and deduced amino acid sequence is shown in FIG. 20 (SEQ ID NOS: 18 & 19).

The second form, designated pML2, encoding a protein with a 4 amino acid residue deletion (228 amino acid residues) was also identified (see Fig. 21 [SEQ ID NO: 21]). A comparison of the amino acid sequences for pML and pML2 shows that the last shape is identical except that it has the tetrapeptide QLPP corresponding to residues 111-114 included (see Fig. 22 [SEQ ID NOS: 18 & 21]) removed. The four amino acid deletion, observed in both murine and porcine ML cDNA, was observed at exactly the same position as the predicted proteases.

Comparison of the predicted amino acid sequences of mature ML from human, mouse and pig (Fig. 19 [SEQ ID NOS: 6, 17 & 18]) shows that the sequence identity between mouse and human is 72%, between mouse and pig is 68%, and between pig and man is 73%. The homology is substantially greater in the amino terminal portion of ML (EPO homology domain). This domain has 80 to 84% identity between any two species, while the carboxyterminal portion (carbohydrate domain) is only 57 to 67% identical. A dibasic amino acid motif that may represent a protease cleavage site is found at the carboxyl terminus of the erythropoietin homology domain. This motif is conserved for the species at the positions shown in FIG. 19 (SEQ ID NOS: 6,17 & 18). The second di-position present at positions 245 and 246 of the human sequence is not found in the mouse and pig sequences. The murine and porcine ML sequence contains 4 cysteines,

All are conserved in the human sequence. Furthermore, there are seven potential N-glycosylation positions in mouse Uganda and six such positions in porcine ML, of which 5 are conserved in the human sequence. All potential Nglycosylation positions are located in the C-terminal portion of the protein.

12. Expression and purification of TPO from Chinese hamster ovary cells (CHO cells)

Expression vectors used to infect CHO cells are designated as follows: pSVI5.ID.LL.MLORF (full length TPO ₃₃₂ ), and pSVI5.ID.LL.MLEPO-D (truncated form or TPOis ₃ ). The properties of these plasmids are shown in FIG. 23 and 24.

Infection procedures are described in Example 20. Briefly, by PCR, cDNA was obtained which corresponded to the fully open TPO reading frame. The PCR product was purified and cloned between two restriction sites (ClaI and SalI) of plasmid pSVI5.ID.LL to obtain the vector pSVI5.ID.LL.MLORF. The second procedure, corresponding to the EPO homology domain, was performed in the same manner, but using a different reverse primer (EPOD.Sal). The final composition of the vector encoding the EPO homology TPO domain is designated pSVI5.ID.LL.MLEPO-D.

These two constructs were linearized with NotI and infected with Chinese hamster ovary cells (CHO-DP12 cells, EP 307 247, published March 15, 1989) by electroporation. 10 ⁷ cells (350 Volt, 330 mF, low capacitance) were electroporated in a BRL electroporation apparatus in the presence of 10, 25 or 50 mg of DNA as described in the literature (Andreason, GLJ Tissue Cult. Meth. 15, 56 [1993]) . The day after infection, cells were digested in DHFR selective medium (high glucose DMEM-F12 50:50 without glycine, 2 mM glutamine, 2-5% dialyzed calf serum). Ten to 15 days later, individual colonies were transferred to 96-well plates (microplates) and allowed to grow until confluence. Expression MLi ₅₃ or ML ₃₃₂ in the conditioned media from these clones was assessed using the Ba / F3-ILC proliferation assay (described in Example 1).

The process of purifying and isolating TPO from the pooled CHO cell culture fluid is described in Example 20. Briefly, the pooled cell culture fluid (HCCF) is applied to a Blue Sepharose column (Pharmacia) at a rate of approximately 100 L HCCF per liter resin. The column is then washed with a buffer solution having a volume of 3 to 5 times the volume of the column and then with the same amount of buffer.

of a solution containing 2.0 M urea. Then TPO was digested with a 3 to 5-fold amount of buffer against column volume containing 2.0 M urea and 1.0 M NaCL

The entire amount of Blue Sepharose column containing TPO containing was then applied to a Wheat Germ Lectin Sepharose column (Pharmacia) which was equilibrated with Blue Sepharose elution buffer at a ratio of 8 to 16 ml Blue Sepharose Eiate per ml resin. The column was then flushed with a 2 to 3-fold column volume of equilibration buffer. TPO was then eluted with a 2- to 5-fold column volume buffer containing 2.0 M urea and 0.5 M N-acetyl-D-glucosamine.

The eluate from the Wheat Germ Lectin column containing TPO was then acidified and C ₁₂ E _{8 was added} up to a final concentration of 0.04%. The resulting stock solution was applied to a C4 reverse phase column that was equilibrated with 0.1% TFA; 0.04% Ci2E ₈ in the feed was a solution containing about 0.2 to 0.5 mg protein per ml of resin.

The protein was eluted in a two-phase linear gradient of acetonitrile containing 0.1% TFA and 0.04% C ₁₂ E ₈ and the resulting solution was obtained by SDS-PAGE.

The accumulated C4 reversed phase column solution was then diluted and diafiltered against approximately six volumes of buffer on Amicone YM or using an ultrafiltration membrane equivalent that allowed to cut 10,000 to 30,000 Da molecular weight. The resulting diafiltrate can then be directly processed or further concentrated by ultrafiltration. The diafiltrate / concentrate is usually adjusted to a final concentration of 0.01% Tween-80.

All or just a portion of the diafiltrate / concentrate corresponding to 2-5% of the calculated column volume is then applied to a Sephacryl S-300 HR column (Pharmacia) equilibrated with a buffer containing 0.01% Tween-80 and subjected to chromatography. Fractions containing TPO that do not have aggregates and proteolytic degradation products are then processed and pooled by SDS-PAGE. The resulting solution was filtered and stored at 2-8 ° C.

13. Methods of transformation and induction of TPO synthesis in microorganism and its isolation, purification and refolding

The construction of E. coli TPO expression vectors is described in detail in Example 21. Briefly, all plasmids pMP21, pMP151, pMP41, pMP57 and pMP202 are constructed to express the first 155 amino acids of TPO in the expression direction of the short fragment, which may vary according to of different constructions. In particular, the short fragments ensure a high level of translation initiation and a rapid purification process. Plasmids pMP210-1, -T8, -21, -22, -24, -25 are engineered to express the first 153 amino acids of TPO in the expression direction of methionine initiation and differ only in the codon used for the first 6 amino acids of TPO, while plasmid pMP251 is a derivative pMP210-1, wherein the carboxyterminal termination of TPO is extended by two amino acids. All of the above plasmids produce high levels of intracellular expression of TPO in E. coli using inducing tryptophan as a promoter (Yansura, DG et al., Methods in Enzymology (Goeddel, DV, Ed.) 185: 54-60, Academic Press, San. Diego [1990]). Plasmids pMP1 and pMP172 are intermediates in the construction of the above-mentioned intracellular expression plasmids TPO.

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

The isolation, purification and refolding procedures (multiple procedure) set forth below for the preparation of biologically active, multiple human TPO or fragments thereof are described in Examples 22 and 23 and can be used to recover any variant of TPO, including N and C terminally extended forms. . Other procedures suitable for refolding recombinant or synthetic TPO are disclosed in the patents: Builder et al., U.S. Pat. U.S. Patent 4,511,502; Jones et al. U.S. Patent 4,512,922; Olson U.S. Pat. U.S. Patent 4,518,526 and Builder et al. U.S. Patent 4,620,948; for a general description of recovery and a multiple process for various recombinant proteins expressed in insoluble form into E. coli.

A. Recovery of insoluble TPO

A microorganism, such as E. coli, expressing TPO encoded by any suitable plasmid is fermented under conditions where the TPO is stored in

insoluble 'refractile bodies'. The cells are first washed with a buffer which decomposes them. Specifically, about 100 g of cells are resuspended in about 10 volumes of disintegrating cell buffer (e.g., 10 mM Tris, 5 mM EDTA, pH 8) with a homogenizer (e.g. Polytron) and the cells are centrifuged at 5000 x g for 30 minutes. The cells are then lysed using any conventional method, such as tonic shock, ultrasonic vibrations, pressure cycling, chemical or enzymatic methods. For example, the above washed cell sediment may be resuspended in an additional 10 volumes of disintegrating cell buffer with a homogenizer and the cell suspension passed through LH Cell Disrupter (LH Inceltech, Inc.) or through a Microfluidizer (Microfluidics Intemational) according to the manufacturing instructions. The particulate mass containing TPO is then separated from the liquid phase and washed with any suitable liquid. For example, the cell lysate suspension may be centrifuged at 5000 x g for 30 minutes, resuspended, and centrifuged arbitrarily for a second time to yield a washed refractile body sediment. The washed pellets can be used immediately or stored at low temperature (e.g. -70 ° C) at will.

B. Solubilization and purification of monomeric TPO

The insoluble TPO in the refractile body pellets is then solubilized with a solubilizing buffer. The solubilizing buffer comprises a chaotropic agent and is usually buffered at basic pH values and further comprises a reducing agent to improve the yield of monomeric TPO. Typical chaotropic agents may be urea, guanidine hydrochloride, and sodium thiocyanate. A preferred chaotropic agent is guanidine hydrochloride. The concentration of the chaotropic agent is usually 4-9 M, preferably 6-8 M. The pH of the solubilizing buffer is maintained with any suitable buffer in the pH range of about 7.5-9.5, preferably 8.0-9.0 and preferably 8.0. Preferably, the solubilizing buffer also contains a reducing agent to form the monomeric form of TPO. Suitable reducing agents include organic compounds containing free thiol (RSH). Typical reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), mercaptoethanol, glutathione (GSH), cysteamine and cysteine. A preferred reducing agent is dithiothreitol (DTT). The solubilizing buffer may contain a moderately oxidizing agent (e.g., molecular oxygen) and sulfites, and may be formed by sulfitolysis of TPO monomers. In this embodiment of the invention, the resulting TPO-S-sulfonate

later refolded in the presence of a buffered redox solution (e.g., GSH / GSSG), resulting in its own multiple TPO.

The TPO protein is typically further purified using, for example, centrifugation, goal filtration chromatography, and reverse phase column chromatography.

By way of illustration, the following procedure is given which yields suitable yields of monomeric TPO. The refractory body sediment is resuspended in about 5 volumes (by weight) of solubilizing buffer (20 mM Tris, pH 8, with 6-8 M guanidine and 25 mM DTT) and stirred for 1-3 hours or overnight at 4 ° C. to affect the solubilization of the TPO protein. High urea concentrations (6-8 M) are also used, but generally the yields using guanidine are higher. After solubilization, the solution is centrifuged at 30,000 x g for 30 min. to obtain a clear supernatant containing denatured TPO monomers. The supernatant is then chromatographed on a Superdex 200 goal filter column (Pharmacia, 2.6 x 60 cm) at a flow rate of 2 ml / min and the protein eluted with 20 mM sodium phosphate, pH 6.0, with 10 mM DTT. Fractions containing denatured TPO protein monomers, eluting between 160 and 200 ml, were collected. The TPO protein is further purified on a semi-preparative C4 reverse phase column (2 x 20 cm VYDAC). The sample is fed at a rate of 5 ml / min to a column which is equilibrated with 0.1% TFA (trifluoroacetic acid) with 30% acetonitrile. The protein was eluted with a linear gradient of acetonitrile (30-60% in 60 min). The purified and reduced protein elutes in approximately 50% acetonitrile. Such a substance is used for refolding to obtain a biologically active variant of TPO.

C. Refolding TPO to form a biologically active form

After solubilization and purification of TPO, the biologically active form is obtained by refolding denatured monomeric TPO in a redox buffer. Due to the high potency of TPO (half of the maximum stimulation in the Ba / 1-3 assay is achieved at approximately 3 µg / ml), it is likely that the biologically active material can be obtained by using various buffering, detergent and redox conditions. However, under most conditions used, only a small amount (<10%) of a suitable multiple material is obtained. For commercial production processes, it is preferred that these yields be at least 10%, more preferably when they are 30-50% and most preferably when they are> 50%. It has been shown that a variety of detergent compositions, including Triton X-100, dodecyl-71-

beta-maltoside, CHAPS, CHAPSO, SDS, sarcosyl, Tween 20 and Tween 80, Zwittergent 3-14 and others. Of these, however, the most preferred detergents were those belonging to the CHAPS detergent family (CHAPS and CHAPSO) which are best suited for a multiple reaction and limit protein aggregation and inappropriate disulfide formation. Preference is given to concentrations of CHAPS that are greater than 1%. The best yields are obtained when sodium chloride is used at optimal concentrations of 0.1 M and 0.5 M. Addition of EDTA (1-5 mM) to the thix redox solution is also preferred, thereby limiting the metal catalyzed oxidation (and aggregation), which is observed in some preparations. The presence of glycerine at a concentration of more than 15% also ensures optimum multiple conditions. Maximum yields are obtained when oxidized and reduced organic thiol (RSH) is used as a redox pair in a redox buffer. Suitable redox pairs include mercaptoethanol, glutathione (GSH), cysteamine, cystine with their corresponding oxidized forms. Preferred redox pairs are glutathione (GSH): oxidized glutathione (GSSG) or cysteine: cystine. The most preferred redox pair is glutathione (GSH): oxidized glutathione (GSSG). Generally, higher yields are observed when the number of moles of the oxidized redox pair is equal to or greater than the number of moles of the reduced redox pair. Optimal pH values for the refolding of these TPO variants are between 7.5 and 9. Organic solvents (e.g., ethanol, acetonitrile, methanol) at concentrations of 10-15% or less may also be used. Higher concentrations of organic solvents increase the amount of unsuitable multiple forms. Generally, tris and phosphate buffers are used. Incubation at 4 ° C also contributes to high concentrations of suitably fold TPO product.

Typical multiplicity yields are between 40% and 60% TPO (calculated on the amount of reduced and denatured TPO used for the multiple reaction) that was purified in the first C4 stage. The active material can also be obtained by less efficient techniques (e.g., directly from a Superdex 200 column or by initial extraction of refractory bodies), but yields are lower due to significant precipitation and overlap of non-TPO proteins during the TPO multiplication process.

Since TPO contains 4 cysteine residues, three different versions of the disulfide of this protein can be distinguished:

version 1: disulfides between cysteine residues 1-4 and 2-3 version 2: disulfides between cysteine residues 1-2 and 3-4 version 3: disulfides between cysteine residues 1-3 and 2-4.

During the initial investigation of conditions that affect multiplication, several different peaks containing TPO protein were separated by C4 reverse phase chromatography. Only one of these peaks had significant biological activity, as confirmed by the Ba / F3 assay. Subsequently, the multiplication conditions were optimized to increase the yield of the substance. Under these conditions, inappropriate versions of the desired product were formed in an amount of less than 10-20% of the total amount of monomeric TPO that was obtained in the solubilization step.

By means of mass spectrometry and protein sequencing, a suitable disulfide model for the biological activity of TPO has been found to have a model of disulfides between cysteine residues 1-4 and 2-3, with the cysteines being calculated sequentially from the amino terminal portion. This cysteine cross-linked model is consistent with the known model of disulfide bonds in the corresponding erythropoietin molecule.

D. Biological effect of recombinant, multiply TPO

Multiply multiplied and purified TPO has activity in both in vitro and in vivo assays. For example, in the Ba / F3 assay, half of the maximal stimulation of thymidine introduction into Ba / F3 cells was achieved for TPO (Me ¹ 1-153) at 3.3 pg / ml (0.3 pM). In the wp / -receptor-based ELISA, half of the maximal effect was achieved at 1.9 ng / ml (120 pM). In normal animals and myelosupresných to prepare X-radiation near the lethal dose, was refolding TPO (Met ¹ 1-153) highly potent (activity was seen at doses of 30 ng / mouse), when stimulated to form new blood vessels. A similar biological effect was observed for other forms of TPO, obtained by multiplying according to the procedures described above (see Figures 25, 26 and 28).

14. Methods of measuring thrombopoietic activity

Thrombopoietic activity can be measured by a variety of assays, including the Ba / F3 mpl ligand assay described in Example 1, in vivo mouse platelet synthesis response assay, platelet surface antigen induction assay, as measured by anti-platelet immunoassay (anti-GPIIblIIa). leukemic

megakaryoblast cell lines (CMK) (see Sato et al., Brit. J. Heamatol., 72: 184-190 [1989]) (see also megakaryocyte formation assay in the liquid suspension described in Example 4) and induction of polyploidization in the megakaryoblast cell line (DAMI) (see Ogura et al., Blood, 72 (1): 49-60 [1988]). The maturation of megakaryocytes from immature cells, predominantly those that do not synthesize DNA, to morphologically identifiable megakaiyocytes is a process consisting of the formation of cytoplasmic organelles, the acquisition of membrane antigens (GPHblIIa), endoreplication and the release of platelets as described above. It is believed that the gender specific promoter (i.e., mpl ligand) of megakaryocyte maturation induces at least some of these changes in immature megakaryocytes, leading to platelet release and amelioration of thrombocytopenia. Assays have been designed to detect the presence of these parameters in immature megakaryocyte cell lines, i.e. CMK and DAMI cells. The CMK assay (Example 4) detects the presence of a specific platelet signaling marker, GPHblIIa, and platelet shedding. The DAMI assay (Example 15) measures endoreplication because an increase in chromosome count is a sign of mature megakaryocytes. Distinguishable megakaryocytes have chromosome values of 2N, 4N, 8N, 16N, 32N, etc. Finally, an in vivo murine platelet reaction assay (Example 16) is used to demonstrate an increase in platelet counts when a test compound (in this case the mpl ligand) is administered.

Two additional in vitro assays were developed to measure TPO activity. The first is the activation of the kinase receptor (KIRA) ELISA, in which CHO cells are infected with the mplRse chimera and the Rse tyrosine phosphorylation is measured by ELISA after treatment of a portion of the mpl chimera with the mpl ligand (see Example 17). The second is a receptor-based ELISA in which an ELISA plate coated with rabbit non-human IgG captures the human mp / -IgG receptor binding mpl ligand as determined by this assay. A biotinylated rabbit polyclonal antibody to mpl ligand (TPOi ₅₅₎ is used to detect bound mpl ligand which is measured using streptavidin-peroxidase as described in Example eighteenth

In vivo biological response of normal and sublethally irradiated mice treated with s

TPO

Normal and sublethal irradiated mice were treated with TPO (chopped and full length) isolated from Chinese hamster ovary (CHO), E. coli, and human kidney embryonic (293) cells. Both forms of TPO produced in these three hosts stimulated platelet formation in mice, but full length TPO isolated from CHO had the highest effect in vivo. These results indicate that glycosylation of the carboxyterminal domain alone is required for optimal in vivo activity.

(a) E. co / z-rhTPO (Met-1,153) The "Met" form of the EPO domain (Met at position -1 plus the first 153 residues of human TPO) produced in E. coli (see Example 23) was administered injected daily to female C57 B6 mice as described in the legends of FIG. 25A, 25B and 25C. These figures show that the non-glycolyzed truncated form of TPO, produced in E. coli and multiplied by the method described above, is capable of stimulating an approximately 2-fold increase in platelet formation in normal mice without affecting the population of red and white blood cells.

The same molecule administered daily by injection of sublethally irradiated ( ¹³⁷ Cs) female C57 B6 mice, as described in the legends of FIG. 26A, 26B, and 26C, stimulated platelet recovery and decreased nadir, but had no effect on erythrocytes and leukocytes.

(b) CHO-rhTPO ₃₃ 2

Full-length form of TPO produced in CHO and administered daily to normal female C57 B6 mice as described in the legends of FIG. 27A, 27B and 27C elicited about a 5-fold increase in platelet production in normal mice without affecting the erythrocyte or leukocyte population.

(c) CHO-rhTPO ₃ 32; E. colTPTPO (M _e tl 153); 293 32 _3-rhTPO; and E. co / z-rhTPOi ₅ 5

Single dose response curves were constructed based on treatment of normal rhTPO mice with different cell lines (CHO-rhTPO ₃₃₂ ; E. coli-z-rhTPO (Met-1, 153-293-rhTPO ₃₃ 2; and E. Co / z-rhTPOus) as described in the legend to Fig. 28. This figure shows that all tested forms of the molecule stimulate platelet production, but the full-length form produced in CHO had the highest in vivo activity.

(d) CHO-rhTPO153, CHO-rhTPO wicked and CHO-rhTPO ₃₃ 2

Single dose response curves were constructed based on treatment of normal mice with various forms of rhTPO produced in CHO (CHOrhTPO153, CHO-rhTPO “cut off” and CHO-rhTPC1) as described in the legend to FIG. This figure shows that all tested CHO forms of the molecule stimulate platelet production, with the full in vivo form having a full length of 70 Kda.

General recomhinant preparation of mpl ligand and variants

Preferably, the Mpl ligand is prepared by standard recombinant procedures, which include preparing the mpl ligand polypeptide by culturing infected cells for expression of the mpl ligand nucleic acid (typically by transforming the cells with an expression vector) and separating the polypeptide from the cells. However, it is contemplated that the mpl ligand may be produced by homologous recombination or recombinant production methods utilizing regulatory elements introduced into cells that already contain DNA encoding the mpl ligand. For example, a dynamics enhancer / enhancer element, a suppressor, or an exogenous transcriptional modulation element can be introduced into the genome of a given host cell at such a distance and in a direction that is appropriate to affect the transcription of the DNA encoding the desired mpl ligand polypeptide. The regulatory element does not encode mpl Ugand, rather the DNA is native to the genome of the host cell. Further, as desired, cells can be screened to prepare the receptor polypeptide of the invention, or screened for increased or decreased expression levels.

Thus, the invention also relates to a method of preparing mpl Uganda, comprising inserting a transcriptional modulating element into the genome of a cell containing the mpl Uganda nucleic acid molecule, such that the element is at a suitable distance and position relative to the nucleic acid molecule to affect its transcription, wherein the next step comprises culturing a cell comprising a transcriptional modulating element and a nucleic acid molecule. The invention also relates to a host cell comprising the original mpl ligand nucleic acid molecule capable of attachment to exogenous regulatory sequences that are distinguishable by the host cell.

A. Isolation of DNA encoding the mpl ligand polypeptide

DNA encoding the mpl ligand polypeptide can be obtained from any cDNA library prepared from tissues expected to possess the mpl ligand mRNA, and

that they express it in a detectable manner. The mpl ligand gene can also be obtained from a genomic DNA library or by in vitro oligonucleotide synthesis from a full nucleotide or amino acid sequence.

Libraries are screened with labeled samples to identify the gene or protein encoded by it. For cDNA expression libraries, suitable samples include monoclonal or polyclonal antibodies that discriminate and specifically bind to the mpl ligand. For cDNA libraries, suitable samples include oligonucleotides of about 20-80 bases in the long chain that encode known or expected portions of the mpl ligand cDNA from the same or different species; and / or complementary or homologous cDNAs or fragments thereof that encode the same or a similar gene. Suitable samples for screening genomic DNA libraries include oligonucleotides, cDNA or fragments thereof that encode the same or a similar gene and / or homologous genomic DNA or fragments thereof. Screening of the cDNA or genomic library with the selected sample is performed by standard procedures as described in Chapters 10-12 of Sambrook et al. (see above).

An alternative to isolating the gene encoding the mpl ligand is to use PCR methodology as described in section 14 of Sambrook et al. (see above). This method requires the use of oligonucleotide probes that will hybridize to the DNA encoding the mpl ligand. Methods for selecting oligonucleotides are set forth below.

A preferred embodiment of the invention is the use of reliably selected oligonucleotide sequences for screening cDNA libraries from a variety of tissues, preferably human or porcine kidney (adult or germline) cell lines or liver. For example, cDNA libraries of a human germline cell line are screened using oligonucleotide samples.

The oligonucleotide sequences selected as samples should be suitably long and unambiguous so as to minimize the effect of false positives. The actual nucleotide sequence is usually constructed based on regions of mpl Uganda having at least a coding redundancy. The oligonucleotides may be degenerate into one or more positions. The use of degenerate oligonucleotides is of particular importance where the library is screened from species in which the use of the preferred code is not known.

Oligonucleotides must be labeled so that they can be detected after hybridization to DNA during screening in the library. The preferred marking method is the use of ATP

(e.g., γ ³² P) and a polynucleotide kinase to radiolabel the 5 'end of the oligonucleotide. The use of other labeling methods is not limited, for example biotinylation or enzyme labeling may be used.

Of particular importance is the mpl ligand nucleic acid which encodes the entire mpl ligand polypeptide chain. In some preferred embodiments, the nucleic acid sequence comprises the native signal sequence of mpl Uganda. A nucleic acid having the entire coding sequence of a protein is obtained by screening selected cDNAs or genomic libraries using a deduced amino acid sequence.

B. Amino acid sequence variants of the native mpl ligand

The amino acid sequence variants of 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, deletion or insertion or substitution of residues in the amino acid sequence in porcine mpl Uganda. For example, portions of the carboxy terminus of mature full mpl ligand may be removed by proteolytic cleavage, either in vivo or in vitro, or by cloning and f expression of a fragment or DNA encoding the full length of mpl. Ugand to form a biologically active variant. Any combination of removal, insertion, and substitution can be made to achieve a final shape having the desired biological activity. Amino acid changes may also alter the posttranslational processes of mpl Uganda, such as changing the number or position of glycosylation sites. In constructing amino acid sequence variants of mpl Uganda, the location of the mutation position and the nature of the mutation will depend on the modified characteristic (s) of mpl Uganda. The mutation positions may be modified individually or in series, for example, (1) by substituting the first one with the conserved amino acid selection and then more radical selection until the result is obtained, (2) removing (deletion) target residues or (3) inserting (inserting) residues of the same or different class adjacent to the position being positioned or a combination of points 1-3.

A method useful for identifying certain residues or regions of the mpl ligand polypeptide in order to find preferred mutagenesis sites is termed "alanine scanning mutagenesis" and is described by Cunningham and Wells, Science, 244: 10811085 (1989). In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are first identified and replaced with any residue, but preferably neutral or negatively charged

an amino acid (preferably alanine or polyalanine) to affect the interaction of amino acids with the surrounding aqueous environment within or outside the cell. These domains, demonstrating functional sensitivity to substitutions, are then further developed by introducing additional or other variants into (or for) the substitution positions. Thus, while the position for introducing the amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize a mutation at a given position, ala sense or random mutagenesis was introduced into the final code or region, and the expressed variants of mpl Uganda were screened to obtain the optimal combination of the desired activity.

There are two main variables in the construction of amino acid sequence variants: the location of the mutation position and the nature of the mutation. For example, variants of the mpl ligand polypeptide include variants from the mpl ligand sequence, and may represent naturally occurring alleles (which do not require manipulation of the mpl ligand DNA), either predetermined mutant forms prepared by DNA mutation or achieved in alleles or in a non-natural variant. In general, the location and nature of the mutation selected will depend upon modification of the mpl Ugandan characteristics.

Deletions of the amino acid sequence generally range from 1 to 30 residues, preferably 1 to 10 residues, and are typically contiguous residues. The removal of the amino acid sequence for mpl Ugand may also be part or all of the carboxy-terminal of the glycoprotein domain. Deletions of the amino acid sequence may also include one or more of the first 6 amino-terminal residues of the mature protein. Any deletion of the amino acid sequence includes one or more residues in one or more loop regions that exist between "spiral bundles". Deletions of adjacent residues as well as single or irregularly located residues are within the scope of the invention. Removals can be performed in regions of low homology between mpl ligands that contribute the highest sequence identity to mpl ligand modification activities. Alternatively, removals can be performed in regions of low homology between human mpl Ugande and other mpl polypeptides of mammalian mammals that share the highest sequence identity to human mpl Ugande. Removals of mammalian mpl ligand polypeptides in regions of substantial homology with the other mammalian mpl ligands will be more suitable for modifying the biological activity of mpl Uganda. The number of consecutive deletions is determined to be

protected the tertiary structure of the mpl ligand in the affected domain, e.g. "Beta-pleated sheet" or "alpha-helix".

Insertion of an amino acid sequence includes amino- and / or carboxy-terminal fusion ranges, which may be a single residue or up to polypeptides containing one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Intra-sequential insertions (e.g., insertion into the mature sequence of mpl Uganda) may be insertion of 1 to 10 residues, preferably 1 to 5 residues, even more preferably 1 to 3 residues. An example of a preferred fusion is the fusion of an mpl ligand or fragment thereof with a second cytokine or fragment thereof. Examples of terminal insertions include coupling of mature mpl Ugand to an N-terminal methionyl residue, the artifact of direct expression of mature mpl Ugand in a recombinant cell culture, and fusion of a heterogeneous N-terminal signal sequence to the N-terminal of mature mpl Ugand molecule to facilitate recombination . Such signal sequences can generally be obtained from and intended to be homologous to the intended host cell species. Suitable sequences include STK or Ipp for E. coli, alpha factor for yeast, and viral signals such as herpes gD for mammalian cells.

in

Other variants of insertion into the mpl ligand molecule include coupling to the N or C-terminal of the mpl ligand of immunogenic polypeptides (e.g., non-endogenous to the host to which the coupling is made), e.g. bacterial polypeptides such as betalactamase or an enzyme encoded by E. coli at the trp or yeast protein position and C-terminal linkages to proteins having a long half-life, such as immunoglobulin constant regions (or other immunoglobulin regions), albumin or ferritin as described in WO 89/02922 published April 6, 1989.

A third group of variants are amino acid substitution variants. These variants have at least one amino acid residue in the mpl ligand molecule removed and a different residue introduced in its place. The most important positions for substitutional mutagenesis include those identified as active positions of mpl Uganda and positions where amino acids found in other analogs differ substantially in properties such as side chain size, charge, hydrophobicity, but where there is also a high degree of sequence identity in the selected position among different mpl Uganda species and / or in different animal analogs of one mpl Uganda representative.

Other positions of interest are those in which the individual mpl ligand residues obtained from different representatives of the family and / or animal species are identical in one representative. These positions, particularly those in accordance with the sequence of the last three otherwise identically conserved positions, are substituted. in a rather conservative way. Such conservative substitutions are listed in Table 3 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes will be introduced, which are indicated by the exemplary substitutions in Table 3 or, as discussed below, in reference to amino acid classes, and the products are screened.

Table 3

original sample Preferred Rest substitutions substitutions Ala (A) Val; Leu; Ile wall Arg (R) Lys; gin; same time Lys Asn (N) gin; His; Lys; Arg gin Asp (D) Glu 'Glu Cys Ser Ser Gin (Q) ' same time same time Glu (E) Asp Asp Gly for for His (H) Asn; gin; Lys; Arg Arg lle (2) Leu; Val; Met; Ala; Phe; norleucine Leu Leu (L) norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; gin; same time Arg Met (M) . Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (A) Ile; Leu; Met; Phe; Ala; norleucine Leu

Substantial modifications in the functionality or immunological identity of the mpl ligand are accompanied by selective substitutions that differ significantly in their effect on maintaining (a) the backbone structure of the polypeptide in the region of substitution, such as leaf or spiral alignment, (b) charge or hydrophobicity of the molecule. or (c) a side chain size. Naturally occurring residues are divided into groups based on the general side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Be;

(2) neutral hydrophilic: Cys, Ser, Thr;

(3) acidic: Asp, Glu;

(4) basic: Asn, Gln, His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will result in the replacement of one of these groups for another. Such substituted residues may also be introduced into conserved (conserved) substitution positions, preferably into remaining (non-conserved) positions.

In one embodiment of the invention, it is desirable to inactivate one or more protease cleavage sites found in the molecule. These positions are identified by the coding sequence of the encoded amino acid sequence, in the case of trypsin, for example, for an arginyl or lysinyl residue. When the protease cleavage positions are identified, they become inactive against proteolytic cleavage by substitution of the selected residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine; deletion of the residue; or by inserting a prolyl residue immediately after the selected residue.

According to another embodiment, each one methionyl residue other than the initial methionyl residue of the signal sequence, or each one located within about three N- or C-terminal residues of each such methionyl residue, is substituted with a different residue (preferably in accordance with Table 3) or deleted. Alternatively, approximately 1-3 residues can be inserted close to such positions.

Also, any cysteine residues not included in maintaining the intrinsic conformation of the mpl ligand can be substituted, generally with serine, to improve

the oxidative stability of the molecule and avoiding abnormal crosslinking. It has been found that the first and fourth cysteine in the epo domain, counted from the amino terminal, is needed to maintain self conformation, but the second and third cysteine is not required for this purpose. As a result, the second and third cysteine in the epo domain may be substituted.

Nucleic acid molecules encoding amino acid sequence variants of the mpl ligand can be prepared by a variety of methods known in the art. These methods include: isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonuldeotide (or site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis of pre-engineered variants, or a non-variant version of the mpl ligand polypeptide. The scope of the methods used is not limited by these methods

Oligonucleotide-mediated mutagenesis is the preferred method for preparing, substituting, deleting, and inserting variants of mpl Uganda. This technique is well known in the art and has been described by Adelman et al., DNA, 2: 183 (1983). Briefly, mpl Uganda DNA is altered by hybridization with an oligonucleotide encoding the desired mutation into a DNA template, wherein the template (the matrix) is a single-stranded form of a plasmid or bacteriophage containing unchanged or natural sequence mpl Uganda DNA. After hybridization, DNA polymerase is used to synthesize the entire second complementary strand of the template, which will thus include the oligonucleotide primer and encode a selection change in the mpl Uganda DNA.

r

Generally, oligonucleotides having a length of at least 25 nucleotides are used. The optimal oligonucleotide will have 12 to 15 such nucleotides that are completely complementary to the template on both sides of the nucleotide (s) encoding the mutation. This ensures that the oligonucleotide will effectively hybridize to the single-stranded DNA template molecule. Ogonucleotides can be synthesized using techniques known in the art as described by Crea et al., Proc. Natl. Acad. Sci. USA, 75: 5765 (1978).

The DNA matrix may be generated using those vectors that are either derived from bacteriophage M13 vectors (commercially available M13mp18 and M13mp18 vectors are suitable) or those containing a single chain phage replication source as described by Viera et al., Meth. Enzymol., 153: 3 (1987).

Then, the DNA to be mutated can be inserted into one of these vectors to generate a single-stranded template. The preparation of the single chain template is described in Sections 4.21-4.41 of Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratories JPress, NY 1989).

Alternatively, the single-stranded DNA matrix may be generated by denaturating the double-stranded plasmid (or other) DNA using standard procedures.

For an alternative to the natural DNA sequence (for example, to generate amino acid sequence variants), the single-stranded oligonucleotide can be hybridized under appropriate hybridization conditions. The DNA polymerization enzyme, usually a Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template, using the oligonucleotide as a primer for synthesis. A heteroduplex molecule is formed in such a way that one DNA strand encodes a mutated form of the mpl ligand and the other strand (the original template) encodes a natural, unaltered sequence of the mpl ligand. This heteroduplex molecule is then transformed into suitable host cells, usually using a prokaryote such as E. coli JM101. When cells are grown, they are plated on agar plates and screened using an oligonucleotide primer that is radiolabeled with ³² P to identify bacterial colonies containing the mutated DNA. The mutated region is then removed and placed in a suitable vector for the production of the protein, generally an expression vector of the type typically used to transform a suitable host.

The above method may be modified in such a way that a homoduplex molecule is formed in which both plasmid chains contain the mutation (s). The modifications are as follows: the single-stranded oligonucleotide is reinforced to the single-stranded matrix by the procedure described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP) and deoxyribotymidine (dTTP), is combined with a modified thio-deoxyribocytosine, also called dCTP- (αS) (obtainable from Amersham Corporation). This mixture is then added to the template-oligonucleotide complex. Upon addition of the DNA polymerase to this mixture, a DNA strand identical to the template, except for the mutated base, is generated. This new DNA strand will already contain dCTP- (αS) instead of dCTP, which will serve to protect it against restriction digestion of the endonuclease.

After the double stranded heteroduplex matrix strand has been refined with a suitable restriction enzyme, the strand strand may be digested with a β χοΙΠ nuclease or other suitable nuclease next to the region containing the position (s) to be mutagenized. Then the reaction stops and a molecule that is only partially single-stranded remains. Full double stranded DNA homoduplex is then generated using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP and DNA ligase. This homoduplex molecule can then be transformed into suitable host cells such as E. coli JM101, as mentioned above.

DNA encoding mpl ligand mutants with more than one substitutable amino acid can be generated by one of several methods. If the amino acids are located closer to each other in the polypeptide chain, they can be mutated simultaneously using a single oligonucleotide that encodes all of the desired amino acid substitutions. On the other hand, if the amino acids are located at some distance from each other (separated by more than ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. In this case, one of the following two alternative methods shall be used.

In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. These oligonucleotides are then simultaneously stiffened on a single stranded DNA matrix and the second DNA strand, which is synthesized from the template (matrix), will encode all of the desired amino acid substitutions.

An alternative method involves two or more mutagenesis steps to prepare the desired mutant. The first step is as described for single mutants: wild-type DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution (s) is added to the template, and then a heteroduplex DNA molecule is generated. In the second mutagenesis step, the mutated DNA prepared in the first mutagenesis step is used as template. Thus, this template already contains one or more mutations. An oligonucleotide encoding another desired amino acid substitution (s) is then added to this template, and the resulting DNA strand now encodes mutations from both the first two steps of mutagenesis, and so on.

PCR mutagenesis is also suitable for preparing amino acid variants of the mpl ligand polypeptide. While the following procedure is for DNA, it is understood that it can

used for RNA. In the PCR method, reference is generally made to the following procedure (see above, Erlich, chapter by R. Higuchi, pp. 61-70): However, in the PCR procedure, a small amount of DNA template is used as the starting material, then such primers that only slightly different from the corresponding region in the DNA template can be used to generate relatively large amounts of a specific DNA fragment that differs from the matrix sequence only at positions where the primers differ from the template. To introduce a mutation into a plasmid DNA, one of the primers is constructed in such a way that it overlaps the mutation position and contains the mutation; the sequence of the second primer must be identical to that of the opposite strand sequence of the plasmid, but this sequence can be located anywhere along the plasmid DNA. Preferably, the sequence of the second primer is located at a distance of 200 nucleotides from the first, so that the entire extended DNA region flanked by the primers can easily be sequenced. A PCR extension in which a pair of primers is used in a similar manner instead of one primer results in a population of DNA fragments that differs at the mutation position specified by the primer and possibly at other positions because the copying template is somewhat inaccurate.

If the ratio of template to produced material is extremely low, the overwhelming predominance of the production DNA fragments involves the desired mutation (s). This produced material is used to replace the corresponding region in the plasmid, which serves as a PCR template using standard DNA technology. Mutations at separate positions can be introduced simultaneously by either a mutant second primer or by creating a second PCR with different mutant primers and ligating the two resulting PCR fragments simultaneously to the vector fragment in three (or more) portions of the ligation.

In a specific example of mutagenesis, a PCR plasmid DNA matrix (1 gg) is linearized by digestion with a restriction endonuclease having a specific recognition position in the plasmid DNA outside the magnification region. From this material, 100 ng is added to a PCR mixture containing PCR buffer containing four deoxynucleotide triphosphates and contained in the GeneAmp® kits (obtained from Perkin-Elmer Cetus, Norwalk, CT and Emeryville, CA) and 25 pmoles of each oligonucleotide primer. , up to a final volume of 50 gl. The surface of the reaction mixture was covered with 35 g of mineral oil. Then the reaction mixture is denatured for 5 minutes at 100 ° C, placed on ice for a while, and then 1 g of Thermus aquaticus (Taq) DNA polymerase

- 86 (5 units / μΐ, purchased from Perkin-Elmer Cetus) is added below the mineral oil layer. The reaction mixture is inserted into a DNA Thermal Cycler (purchased from Perkin-Elmer Cetus), which is programmed as follows:

min55 ° C with 72 ° C, then 19 cycles with the following program:

s94 ° C with 55 ° Ca with 72 ° C.

At the end of the program, the reaction vial is removed from the DNA Thermal Cycler and the aqueous phase is transferred to a new vial, extracted with phenol / chloroform (50:50 v / v), precipitated with ethanol, and the DNA is separated by standard procedures. This material is then subjected to suitable processing for introduction into the vector.

Another method for preparing variants, cassette mutagenesis, is based on the procedure described by Wells et al., Gene, 34: 315 (1985). The starting material is a plasmid (or other vector) containing the mpl ligand DNA to be mutated. The code (s) of the mpl Uganda DNA to be mutated is identified. The position of the specific restriction endonuclease must be located on each side of the identified mutation position (s). If such restriction sites do not exist, then they must be generated using the mutagenic method described above by means of an ohgonucleotide to be introduced at the appropriate positions of the mpl Uganda DNA. When these restriction positions are introduced into the plasmid, the plasmid is cut at these positions and linearized. A double stranded oligonucleotide encoding the DNA sequence between the restriction positions but containing the desired mutation (s) is synthesized using standard procedures. Two strands are synthesized separately and then hybridized together using standard procedures. Such a double-stranded oligonucleotide is referred to as a cassette. This cassette is designed to have 3 'and 5' ends that are compatible with the ends of the Unearized plasmid so that it can be directly ligated to the plasmid. This plasmid then contains the mutated mpl ligand DNA sequence.

C. Insertion of a nucleic acid into a replicable vector

A nucleic acid (e.g., cDNA or genomic DNA) encoding a natural or variant mpl ligand polypeptide is inserted into a replicable vector for further cloning (DNA amplification) or for expression. Many vectors are available and the choice of the appropriate one will depend on (1) whether it is useful for DNA enlargement

or for the expression of DNA, (2) from the size of the inserted nucleic acid into the vector, and (3) from the host cell to be transformed with the vector. Each vector contains different components, depending on its function (DNA doubling or DNA expression) and the host cell with which it is compatible. The r generally comprises one or more of the following components: a signal sequence, a replication source, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence, and others.

(i) Signal sequence component

The Mpl ligand of the invention may be expressed not only directly, but also by association with a heterologous polypeptide, preferably a signal sequence, or other polypeptide having a specific cleavage position in the N-terminal of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector or may be part of the mpl ligand DNA that is inserted into the vector. The selected heterologous signal sequence may be recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequence of the native mpl ligand, the signal sequence is substituted with a selected prokaryotic signal sequence, for example, from the group of alkaline phosphatase, penicillinase, Ipp or heat stable enterotoxic Π conductors. In the case of yeast secretion, the natural signal sequence may be substituted, e.g. by yeast invertase, alpha factor or by conducting acid phosphatases, by C. albicans conducting glucoamylase (EP 362,179 published April 4, 1990) or by the signal disclosed in WO 90/13646, published November 15, 1990. In mammalian cell expression, a signal sequence (e.g. mpl ligand sequence that normally directs secretion of mpl ligand from its native mammalian cells in vivo), although other mammalian signal sequences may be appropriate, such as signal sequences from other mpl ligand polypeptides or from the same mpl ligand from different animal species , signal sequences from mpl Ugand, and signal sequences from secreted polypeptides of the same or related species, as well as viral secretion wires, for example, the herpes simplex gD signal.

(ii) Source of replication component

Both the expression and cloning vector contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells.

In general, for cloning vectors, this sequence allows the vector to replicate independently of the host chromosomal DNA and includes a source of replication or autonomously replicating sequences. Such sequences are well we have for a number of bacteria, yeast and viruses. The source of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the 2 µ plasmid source is suitable for yeast and various viral sources (SV40, polyoma, adenovirus, VSV or BPV) are used for cloning vectors in mammalian cells. Generally, the source of the replication component is not needed for mammalian expression vectors (the SV40 source can typically be used only because it contains an initial promoter).

Most expression vectors are shuttle vectors, that is, they are capable of replicating in at least one group of organisms and can be transferred to other organisms for expression. For example, the vector is cloned in E. coli and then the same vector is transferred to or into yeast. of mammalian cells for expression, although it is not capable of replication independently of the host cell chromosomes.

DNA can also be amplified by insertion into the host genome. This is achieved by using Bacillus species as hosts, for example by inserting into the vector a DNA sequence that is complementary to a sequence found in the genomic DNA of the respective Bacillus species. Transfection of Bacillus together with this vector induces homologous recombination with the genome and insertion of the mpl ligand DNA. However, the separation of the genomic DNA encoding the mpl ligand is more complex than its separation from the exogenously replicated vector, since the restriction enzyme digestion is required to remove the mpl ligand DNA.

(iii) Selection of gene component

Expression and cloning vectors may contain a selection gene, also referred to as a selectable marker. This gene encodes a protein that is necessary for the survival or growth of transformed host cells growing in a selective culture. Such host cells which have not been transformed with the vector containing the selection gene in culture will not survive. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline; (b) supplement auxotrophic deficiencies; or (c) supply critical nutrients unavailable from a complex medium, e.g. a gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of a host cell. These cells, which are successfully transformed with a heterologous gene, express the protein, induce drug resistance and thus survive the selection regimen. Examples of such dominant selection include: using neomycin as a drug (Southem et al., J. Molec. Appl. Genet., 1: 327 [1982]), using mycophenolic acid (Mulligan et al., Science, 209: 1422 [1980]) or hygromycin (Sugden et al., Mol. Cell. Biol. 5: 410-413 [1985]). In the above three examples, eukaryotic-regulated bacterial genes are used to confer resistance to suitable drugs such as G418 or neomycm (geneticin), xgpl (mycophenolic acid) or hygromycin.

Examples of other suitable selection-capable markers of mammalian cells are those capable of identifying cells competent to pick up the mpl nucleic acid ligand, such as dihydrofolic acid salt reductase (DHFR) or thymidine kinase. Mammalian cell transformants are placed at a selected pressure such that only the transformants are specifically adapted to survival due to the pick up effect by a marker. The selected pressure is introduced by culturing the transformants under conditions where the concentration of the selection agent in the medium gradually changes and thereby results in amplification of both the selection gene and the DNA that encodes the mpl ligand polypeptide. Amplification is the process by which genes, in most of the requirements for the preparation of a protein critical for growth, are repeatedly iterated (gene repeat) in tandem with the chromosomes of successful generations of recombinant cells. Larger amounts of mpl Uganda are then synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture containing methotrexate (Mtx) and a competing DHFR antagonist. A suitable host cell when wild-type DHFR is used is the Chinese Hamster Seed (CHO) cell line, which has a deficient DHFR activity, prepared and propagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216 (1980). The transformed cells are then exposed to an increased concentration of Mtx. This results in the synthesis of multiple copies of the DHFR gene and consequently multiple copies of additional DNA containing an expression vector, such as the DNA encoding mpl Ugand. Amplification techniques may be used with any other suitable host, e.g. ATCC No.

CCL61 CHO-K1, regardless of the presence of endogenous DHFR, if, for example, a mutant of the DHFR gene that is highly resistant to Mtx is used (EP 117,060). Host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with a DNA sequence encoding an mpl ligand, wild-type DE1FR protein, and other selectable signaling moieties such as aminoglycoside 3 phosphotransferase (APH) may also be selected by cell growth in a medium that contains a selection agent for a selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418 (see US Patent 4,965,199).

A suitable selection gene for use in yeast is the trp1 gene that is present in the YRp7 yeast plasmid (Stinchcomb et al., Nature, 282: 39 [1979]; Kingsman et al., Gene, 7: 141 [1979]; or Tschemper and et al., Gene, 10: 157 [1980]). The trp1 gene acts as a selectable marker for a mutant strain of yeast that has lost its ability to grow in tryptophan, e.g. 44076 or PEP4-1 (Jones, Genetics, 85:12 [1977]). The presence of damage suffers in the genome of the yeast host cell then ensures effective development of detection of transformation by growth in the absence of tryptophan. Similarly, Ĺeu2-deficient yeast strains (ATCC No. 20,622 or 38,626) become complementary with known plasmids carrying the Leu2 gene.

(iv) Promoter component

Expression and cloning vectors usually contain a promoter that the host organism can recognize and is usefully attached to the mpl nucleic acid ligand. Promoters are untranslated sequences upstream (5) of the design gene start code (generally, at a distance of about 100 to 1000 bp) that regulate the transcription and translation of a special nucleic acid sequence such as the mpl ligand nucleic acid sequence to which are usable. Such promoters are usually divided into two groups, inductive and basic. Inductive promoters are those that initiate an increase in the level of transcription from DNA and regulate this activity in response to certain changes in culture conditions, such as the presence or absence of nutrients or a change in temperature. A large number of promoters recognized by a variety of potential host cells are currently known. These promoters are usefully associated with the mpl ligand encoding DNA by removing the promoter from the DNA source by

restriction enzyme digestion and insertion of the isolated promoter sequence into the vector. Both the natural mpl ligand promoter sequence and a number of heterologous promoters can be used to directly amplify and / or express the mpl ligand DNA. However, heterologous promoters are preferred because they generally allow for greater transcription and give higher yields of the expressed mpl ligand compared to the native mpl ligand promoter.

Promoters suitable for use with prokaryotic hosts include β-lactamase and lactose promoter systems (Chang et al., Nature 275: 615 [1978]; and Goeddel et al., Nature 281: 544 [1979]). alkaline phosphatase and the tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 [1980] and EP 36,776) and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80 : 21-25. Other bacterial promoters are also suitable. Their nucleotide sequences have been published, allowing the skilled worker to operatively link them to DNA encoding the mpl ligand (Siebenlist et al., Cell, 20: 269 [1980]) using linkers or adapters to provide any desired restriction positions. Promoters for use in bacterial systems will also contain a Shine-Dalgam (S.D.) sequence operably linked to DNA encoding the mpl ligand polypeptide.

Promoter sequences are known for eukaryotes. In fact, all eukaryotic genes have an AT-rich region located approximately 25 to the base upstream of the transcription initiation site. Another sequence, found 70 to 80 bases upstream from the start of transcription of most genes, is the CXCAAT region where X can be any nucleotide. At the 3 end of most eukaryotic genes is an AATAAA sequence that may be a signal for the addition of the poly A end to the 3 end of the coding sequence. All of these sequences are conveniently inserted into eukaryotic expression vectors.

Examples of suitable activation sequences for use in a yeast host include the 3-phosphbglycerate kinase promoters (Hitzeman et al., J. Biol. Chem., 255: 2073 [1980]) or other glycolysis enzymes (Hees et al., J. Adv. Enzyme Reg., 7: 149 [1968] and Holland, Biochemistry, 17: 4900 [1978]) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphatoisomerase, 3-phosphoglycerate mutase , pyruvantinase, triosophosphate isomerase, phosphoglucose isomerase and glucocmase.

Other yeast promoters that are inducible promoters and have the added benefit of growth-regulated transcription are the promoter regions of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradation enzymes associated with the metabolism of nitrogen, phosphoryl thionatedehydrogenase, metallothionine dehydrogenase, gyeryceral thionate, responsible for the use of maltose and galactose. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al., EP 73,657A. Yeast enhancers are also preferably used with yeast promoters

Transcription of mpl ligand from vectors in mammalian host cells is regulated, for example, by promoters derived from viral genomes such as polyoma virus, chicken pox virus (UK 2 211 504, published July 5, 1989), adenovirus (such as Adenovirus 2), virus bovine papilloma, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis-B virus, and preferably Simian Virus 40 (SV40), from a heterologous mammalian promoter, for example, an actin promoter or an immunoglobulin promoter, from a heatshock promoter, and from a promoter ligand to the promoter normally ensure that such promoters are compatible with the host cell system.

Early and later SV40 virus promoters are conveniently obtained as SV40 restriction fragments that also contain the SV40 viral replication source (Fiers et spoL, Nature, 273: 113 [1978]; Mulligan and Berg, Science, 209: 1422-1427 [1980]; Pavlakis et al., Proc. Natl. Acad. Sci. USA, 78: 7398-7402 [1981]). The closest early promoter of the human cytomegalovirus is conveniently obtained as a restriction E restriction fragment (Greenaway et al., Gene, 18: 355-360 [1982]). A system for expressing DNA in hosts (mammals) using bovine papilloma virus as a vector is described in U.S. Pat. Reference may also be made to Gray et al., Nature 295: 503-508 [1982] regarding the expression of cDNA encoding immune interferon in monkey cells; to Reyes et al., Nature 297: 598-601 [1982] regarding the expression of human β-interferon cDNA in mouse cells by regulation of the herpes simplex virus thymidine kinase promoter; to Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79: 5166-5170 [1982] regarding the expression of the human interferon β gene in cultured mouse and rabbit cells; and Gorman et al., Proc. Natl. Acad. Sci. USA, 79: 6777-6781 [1982] regarding expression of bacterial CAT sequences into monkey kidney CV-1, hen embryo fibroblasts, Chinese hamster semen cells, HeLa cells, and NIH mice

3 to 3 cells, reusing the long terminal of Rous sarcoma virus as a promoter.

(v) Amplifier component

Transcription of the DNA encoding the mpl ligand of the invention for higher eukaryotes can often be enhanced by insertion of the enhancer sequence into the vector. Amplifiers are cis-acting elements of DNA, usually about 10 to 300 bp, that act on the promoter and enhance its transcription. Amplifiers are independent of relative orientation and position and may have as found 5 (Laimins et al., Proc. Natl. Acad. Sci. USA, 78: 993 [1981]) and 3 '(Lusky et al., Mol. Cell. Bio., 3: 1108 [1983]) against the transcriptional unit, within the intron (Banerji et al., Cell, 33: 729 [1983]), as well as within the coding sequence itself (Osbome et al., Mol. Cell Bio., 4: 1293 [1984]). Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, an enhancer from a eukaryotic cell virus is used. Examples include the SV40 upstream replication source (bp 100-270), the cytomegalovirus initial promoter enhancer, the upstream replication polyoma enhancer, and the adenovirus enhancer. See also Yaniv, Nature, 297: 17-18 [1982] for enhancers for activation of eukaryotic promoters. The amplifiers can be attached to the vector at position 5 or 3 to the mpl ligand coding sequence, but are preferably located at position 5 from the promoter.

(vi) Transcription termination component

Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans or nucleated cells from other multicellular organisms) will also contain sequences that are required for transcription termination and mRNA stabilization. Such sequences are commonly available from 5 as well as 3 untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments, transcribed as polyadenylated fragments in the untranslated portions of the mpl ligand encoding mRNA.

(vii) Construction and analysis of vectors

The construction of suitable vectors containing one or more of the above components utilizes standard attachment techniques. Isolated plasmids or DNA fragments are cleaved, cut, and reamed in the form required to generate the desired plasmids.

To confirm that the correct sequences were constructed in the plasmids, analysis was performed using the blended mixtures for transformation of E. coli K12 strain 294 (ATCC No. 31,446) and successful transformants selected for ampicillin or tetracycline resistance, as appropriate. Plasmids were prepared from transformants, analyzed by restriction endonuclease digestion, and / or sequenced by Messing et al., Nucleic Acids Res., 9: 309 [1981] or by Maxam et al., Methods in Enzymology, 65: 499 [1980], (viii) Transient expression vectors

Especially useful in the practice of this invention are expression vectors that provide for transient expression in mammalian cells with DNA that encodes the mpl ligand polypeptide. Generally, transient expression involves the use of an expression vector that is capable of replicating efficiently in a host cell such that the host cell accumulates multiple copies of the expression vector and, on the other hand, synthesizes high concentrations of the desired polypeptide encoded by the expression vector. Sambrook et al. p. 16.17 - 16.22 Transient expression systems comprising a suitable expression vector and a host cell allow for appropriate positive identification of the polypeptides encoded by the cloned DNA, as well as rapid screening of such polypeptides for the desired biological or physiological property. Thus, transient expression systems are particularly useful within the meaning of this invention for the purpose of identifying analogs and variants of the mpl ligand polypeptide having the biological activity of the mpl ligand polypeptide.

(ix) Suitable examples of vertebrate cell vectors in

Other methods, vectors and host cells suitable for adaptation to mpl ligand synthesis in recombinant vertebrate cell culture are described by Gething et al., Nature, 293: 620-625 [1981]; Mantei et al., Nature, 281: 40-46 [1979]; Levinson et al; EP 117,060; and EP 117,058. A particularly useful plasmid for the expression of a cell culture of mpl Uganda mammals is pRK5 (EP 307,247; U.S. Pat. 5,258,287) or pSV10B (PCT WO 91/08291).

D. Selection and transformation of host cells

Suitable host cells for cloning or expressing the vectors of the invention are prokaryotes, yeast or higher eukaryotic cells, which are already described above. Suitable prokaryotes include eubacteria such as Gram-negative or Gram-positive organisms, for example, E. coli, Bacilli such as B. subtilis, Pseudomonas

sp. such as P. aeruginosa, Salmonella typhimurium or Serratia marcescans. One preferred E. coli cloning host cell is E. coli 294 (ATCC No. 31,446), although other strains such as E. coli B, E. coli-6 (ATCC No. 31.537) and

E. coli W3110 (ATCC No. 27.325) are suitable. These examples are illustrative only and do not limit the scope of use of the respective host cells. A preferred host cell is to secrete minimal amounts of proteolytic enzymes. Similarly, in vitro methods of cloning, i.e., PCR or other nucleic acid polymerase reactions, are suitable.

Along with prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for mpl ligand encoding vectors. Saccharomyces cerevisiae or common baker's yeast is most commonly used among lower eukaryotic host microorganisms. However, a number of other commonly available genera, species and strains, such as Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published May 2, 1985), hosts of the genus Kluyveromyces (US U.S. Patent 4,943,529) such as K. lactis (Louvencourt 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 [1988]), Candida, Trichoderma reesia ( EP 244,234), Neurospora crassa (Cas et al., Proc. Natl. Acad. Sci. USA, 76: 5259-5263 [1979]) and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991) and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 [1983]; Tilbum et al., Gene, 26: 205- 221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 147-1474 [1984]) and A. niger (Kelly and Hynes, 4: 475-479 [1985]).

Suitable host cells for the expression of glycosylated mpl ligand were derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is processable, whether from vertebrate or invertebrate culture. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains and variants and corresponding allowed insect host cells have been identified from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx

Sea. See, for example, Luckow et al., Bio / Technology, 6: 47-55 [1988]; Miller et al., Genetic Engineering, Setlow et al., Eds., Vol. 8 (Plenum Publishing, 1986), p. 277-279; and Maeda et al., Nature, 315: 592-594 [1985], A number of viral strains for transfection are publically available, for example, the L1 variant from Autographa californica NPV and the Bm-5 strain from Bombyx mori NPV and such viruses can be used as viruses. according to the invention, in particular for the transfection of Spodoptera frugiperda cells.

Cell cultures of cotton, wheat, potato, soybean, petunia, tomato and tobacco plants can be used as hosts. Typically, the procedure involves transfection of plant cells by incubation with certain strains of Agrobacterium tumefaciens which have been first engineered to contain mpl Uganda DNA. During incubation of the plant cell culture with A. tumefaciens, the DNA encoding mpl Ugand is transferred to the host cell of the plant such that infection occurs and the host cell will express the mpl Uganda DNA under appropriate conditions. In addition, regulatory and signal sequences compatible with plant cells such as the nopal synthase promoter and polyadenylation signal sequences are available, Depicker et al., J. Mol. Appl. Gen., 1: 561 [1982], DNA segments isolated from the upstream region of the T-DNA 780 gene are also capable of activating and increasing the transcriptional level of plant-expressing genes in recombinant plant tissues containing DNA. EP 321,196 published June 21, 1989.

However, more attention has been paid to vertebrate cells and the reproduction of vertebrate cells in culture (tissue culture) has become a routine matter in recent years (Tissue Culture, Academic Press, Kruse and Patterson, editors [1973]). Examples of useful mammalian host cell lines include the monkey log CV1 cell line transformed with SV4O (COS-7, ATCC CRL 1651); human embryonic lineage line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol., 36:59 [1977]); hamster chick cells (BHK, ATCC CCL 10); Chinese hamster testicular cells / -HFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 [1980]); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver

cells (Hep G2, HB 8065); mouse breast tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 [1982]); MRC 5 cells; FS4 cells; and the human hepatoma cell line (Hep G2).

Host cells are infected and preferably transformed with the expression or cloning vectors of the invention described above and cultured in conventional nutrient media, modified as necessary for induction by promoters, selection by transformers, or for amplification by genes encoding the desired sequences.

Transfection is a process by which an expression vector enters a host cell, and in no way is any coding sequence expressed. Those skilled in the art will recognize a variety of methods of infection, such as CaPO ₄ and electroporesis. Successful transfection is generally known by referring to any activity of this vector within the host cell.

Transformation means the introduction of DNA into the organism such that the DNA is replicated, either as an extrachromosomal element or by means of a chromosomal integrant. Depending on the host cell used, transformation is performed using standard techniques appropriate to the cells. For prokaryotes or other cells containing substantial cell wall impediments, it is generally recommended to use calcium treatment using calcium chloride as described in section 1.82 of Sambrook et al., Supra. Agrobacterium tumefaciens infection is used to transform some plant cells as described by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859, published June 29, 1989. In addition, plants can be transfected by ultrasound, such as This is described in WO 91/00358, published January 10, 1991. If cell walls do not create such barriers as, for example, mammalian cells, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52: 456-457 (1978). General aspects of transformations in mammalian cell host systems are described by Axel in U.S. Pat. No. 4,399,216, issued August 16, 1983. Yeast transformations are typically performed according to the method of Van Solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. USA 76: 3829 (1979). Other methods for introducing DNA into cells, such as nuclear injection, electroporesis, or protoplast fusion, may also be used.

E. Culturing Host Cells

The prokaryotic cells used to prepare the mpl ligand polypeptide of the invention are cultured in an appropriate medium as generally described by Sambrook et al., Supra.

Mammalian host cells used to prepare mpl ligands of the 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 medium described in Ham and WaUace, Meth. Enz., 58:44 [1979], Bames 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. Patent Re. 30,985 or commonly discussed by U.S.S.N. 07 / 592,107 or 07 / 592,141, both filed on October 3, 1990, which are incorporated herein by reference, can be used as a culture medium for host cells. 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 and magnesium phosphates), buffers (such as is HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Uek Gentamycin ™), trace elements (defined as inorganic compounds, which are usually present at final concentrations in the micromole range) and glucose or an equivalent energy source. Any other necessary additions may also be added at appropriate concentrations, as is known to those skilled in the art. Culture conditions such as temperature, pH and the like. are as previously used in the selected host cell for expression and are known to those skilled in the art.

In relation to the foregoing, the host cells include both cells in vitro culture and cells that are in the host animal.

F. Detection of gene amplification / expression

Gene amplification and / or expression can be measured directly in the sample, for example, by conventional methods such as Southem blotting, northem blotting, to quantify mRNA transcription (Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 [1980]) , dot blotting or in situ hybridization using a suitably labeled sample based on the sequences disclosed herein. Various substances may be used for labeling,

most preferred are radioisotopes, especially ³² P. However, other methods, such as hintin-modified nucleotides for introduction into a polynucleotide, may also be used. Biotin then serves as a position for binding to avidin or antibodies, which can be labeled with a wide variety of labeling agents, such as radionucleotides, enzymes, fluorescent agents and the like. Similarly, antibodies that can recognize specific duplexes or DNA-protein duplexes can be used. Antibodies, in turn, can be labeled and assayed to determine if the duplex is bound to the surface, so that by determining the binding of the duplex to the surface, the presence of bound antibody to the duplex is determined.

Gene expression can be measured by immunological methods such as immunohistochemical staining of tissue sections and cell or body fluid culture assays to directly quantify expression of the gene product. An immunohistochemical staining technique is used to prepare a cell sample by a typical procedure of dehydration and fixation and then by reaction with labeled specific antibodies for the linked gene product; visually detectable labels, such as enzymes, fluorescent substances, himiniscent substances and the like, are usually used. A particularly sensitive staining technique suitable for use in the present invention is described by Hsu et al., Am. J. Clin. Path., 75: 734-738 (1980).

Monoclonal or polyclonal antibodies, which can be prepared in any mammal, are used for immunohistochemical staining and / or fluid sample assay. Suitably, the antibodies are prepared against a native mpl ligand polypeptide or a synthetic peptide based on DNA sequences within the meaning of the invention as described below.

G. Purification of mpl Uganda Polypeptide

The Mpl ligand is preferably recovered from the culture medium as a secreted polypeptide, although it can also be obtained from host cell lysates after direct expression without the use of a secretory signal.

When the mpl ligand is expressed in a recombinant cell other than a cell of human origin, then the mpl ligand is completely devoid of proteases or polypeptides of human origin. However, it is usually necessary to purify the mpl ligand from other recombinant cell proteins or polypeptides in order to obtain preparations substantially homogeneous to the mpl ligand per se. In a first step, the culture medium or lysate is centrifuged to remove individual cell debris. The membrane and the appropriate protein fractions are then separated. Alternatively, a commercially available filter may be used

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(e.g., Amicon or Milipore Pellicon ultrafiltration unit) to thicken (concentrate) the protein. Further, the mpl ligand is purified from soluble protein fractions and culture lysate membrane fractions, depending on whether the mpl ligand is membrane bound. Then, the mpl ligand is purified by salting out of contaminating soluble proteins and polypeptides and by exchange or chromatographic techniques using various gel matrices. These matrices include: acrylamide, agarose, dextran, cellulose, and other commonly used protein purifying agents. Examples of chromatographic procedures suitable for protein purification include: immunoaffinity (i.e., anti-hwp / Mab ligand), receptor affinity (i.e., np / -IgG or protein A Sepharose), hydrophobic interaction chromatography (i.e. ether, butyl or phenyl Toyopearl), lectin chromatography (i.e. Con A-Sepharose, lentil-lectin-Sepharose), size exclusion method (i.e. Sephadex G-75), ion exchangers (cation exchanger and anion exchange resin) ( i.e. DEAE or carboxymethyl- and sulfopropylcellulose) and reverse phase high performance liquid chromatography (RP-HPLC) (see, e.g., Urdal et al., J. Chromatog., 296: 171 (1984), where two sequential RPv steps were used).

HPLC to purify recombinant human IL-2). Additional purification steps optionally include: ethanol precipitation, ammonium sulfate precipitation, chromatofocusing, preparative SDS-PAGE, and the like.

Variants of mpl ligand in which residues removed, inserted or substituted are obtained as well as the natural mpl ligand, taking into account any substantial changes in properties due to variation. For example, preparing the fusion of an mpl ligand with another protein or polypeptide, such as a bacterial or viral antigen, will facilitate purification; an immunoaffinity column containing an antibody to the antigen is used to adsorb the bound polypeptide. Immunoaffinity columns, such as a rabbit polyclonal anti-mpl Uganda column, can be used to absorb a variant of mpl Uganda by binding it to at least one remaining immune epitope. Alternatively, mpl Ugand can be purified by affinity chromatography using purified mplAgfj in pair with (preferably) immobilized resin, such as Affi-Gel 10 (Βίο-Rad, Richmond, CA) or the like, in a manner well known to those skilled in the art. . A protease inhibitor such as phenylmethylsulfonyl fluoride (PMSF) can also be used to inhibit proteolytic degradation during purification, and antibiotics can also be used to prevent the growth of foreign accidental impurities.

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One of skill in the art will recognize that purification methods suitable for the native mpl ligand require modification due to changes in the nature of the mpl ligand or variants thereof for expression in recombinant cell culture.

H. Covalent Modifications of mpl Uganda Polypeptide

Covalent modifications of the mpl ligand polypeptide are within the scope of this invention. Both natural mpl Ugand and amino acid sequence variants of mpl Ugand can be covalently modified. One type of covalent modification that is encompassed within the scope of this invention is, for example, the mpl ligand fragment. Fragments of mpl ligand variants having up to 40 amino acid residues may conveniently be prepared by chemical synthesis or by enzymatic or chemical cleavage of the full length or mpl ligand variant polypeptide. Other types of covalent modifications of mpl ligand or fragments thereof are introduced into the molecule by reacting the target residues of mpl ligand or fragments thereof with an organic derivatizing agent capable of reacting with selected side chains or N- or C-terminal residues.

Cysteinyl residues most commonly react with α-haloacetates (and the corresponding amines), such as chloroacetic acid or chloroacetamide, to form carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues can also be derivatized by reaction with bromotrifluoroacetone, α-bromo-β- (5-imidazolyl) propionic acid, chloroacetylphosphate, N-alkyl maleaimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyldisulfide, p-chlorosuromurur, p-chloromethylsuromurec 4-nitrophenol or chloro-7-nitrobenzo-2-oxo-1,3-diazole.

The histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 since this agent is relatively specific for the side chain of these residues. Para-bromophenylacyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodylane at pH 6.0.

Lysyl and amino-terminal residues react with carboxylic acid anhydrides such as butanedioic acid, but also with others Derivatization with these agents affects the change in charge of lysyl residues. Other suitable reagents for derivatizing amino-containing moieties include imidoesters such as methyl picolinimidane, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, pentane-2,4-dione, and a transaminase catalyzed reaction with glyoxylate.

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Arginyl residues are modified by reaction with one or more conventional reagents, for example phenylglyoxal, butane-2,3-dione, cyclohexane-1,2-dione, and ninhydrin. Derivatization of argjnyl residues requires that the reaction be carried out in an alkaline medium because of the high pK value _{and the} guanidine functionality. Furthermore, these reagents can react with the lysine group as well as the arginine epsilonamino group.

Specific modification of the tyrosyl residues may be performed with respect to the introduction of spectral labeling into the tyrosyl residues by reaction with aromatic diazonium compounds or with tetranithromethane. Most commonly, Nacetylimidazole and tetranitromethane can be used to give O-acetyl tyrosyl compounds and 3-nitro derivatives. Tyrosyl residues are iodinated using ¹²⁵ L or ¹³¹ L and labeled proteins are prepared for use in a radioimmunoassay, the chloramine T method described above is also suitable.

The side carboxyl groups (aspartyl or gutamyl) are selectively modified by reaction with carbodiimides (RN = C = N-R '), where R and R' are different alkyl groups than 1-cyclohexyl-3- (2-moifolin-4-ethyl) ) carbodiimide or 1-ethyl-3- (4-azo-4,4-dimethylpentyl) carbodiimide. Further, aspartyl and glutamyl residues were converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional reagents is suitable for cross-linking the mpl ligand with a water-insoluble carrier or surface matrix for further use in the purification of anti-znp / ligand antibodies and vice versa. Commonly used crosslinking agents include: 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxybutanedioic acid imide esters, for example 4-azidosalicilic acid esters, homobifunctional imidoesters, including dibutanedioic acid esters such as 3,3'- dithiobis (succinimidyl propionate); and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents, such as methyl 3 - [(p-azidophenyl) dithio] propioimidane, provide intermediates with photoactivating capability that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated hydrocarbons and reactive substrates described in U.S. Patents 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537 and 4,330,440 can also be used to immobilize protein.

Glutaminyl and asparaginyl residues are often deamidated to the corresponding ghitamoyl and aspartoyl residues. These residues are formed by deamidation at

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neutral or alkaline conditions. This deamidated form of the residues is also within the scope of the invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl and threonyl residues, methylation of α-amino groups of tyrosine, arginine and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & 79-86 [1983]), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

in

Other types of covalent modifications of the mpl ligand polypeptide encompassed within the scope of this invention include adaptation of the natural model of polypeptide glycosylation. By adaptation is meant removal of one or more carbohydrate moieties that are present in the natural mpl ligand and / or addition of one or more glycosylation positions that are not present in the natural mpl ligand.

Glycosylation of polypeptides typically goes either to the N or O position. When it goes to the N position, this refers to the attachment of the hydrocarbon moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-Xtreonine, where X is any amino acid except proline, are distinctive sequences in the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of each of these tripepidide sequences in the polypeptide creates a potential glycosylation position. O-position glycosylation refers to the attachment of one of the sugars - N-acetylgalactosamm, galactose or xylose - to a hydroxyamino acid, most commonly to serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the mpl ligand polypeptide is suitably accompanied by the adaptation of the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for glycosylation to the N-positions). Adaptation can also be accomplished by the addition or substitution of one or more serine or threonine residues to the natural mpl ligand sequences (in glycosylation to O-positions). For simplicity, the amino acid sequence of the mpl ligand is preferably adapted by changes at the DNA level, in particular by mutating the DNA encoding the mpl ligand polypeptide in predetermined bases such that the codes are generated for translation into the desired amino acids. The DNA mutation (s) can be performed using the methods described above under the heading "amino acid sequence variants of mpl Uganda".

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Another method of increasing the number of carbohydrate moieties in an mpl ligand is by chemical or enzymatic attachment of glycosides to the polypeptide. These methods have the advantage that they do not require the preparation of a polypeptide in a host cell that has glycosylation capabilities for glycosylation into N- or O-positions. Depending on the coupling method used, the sugar (s) can be attached to (a) argiinine and histidine, (b) free carboxyl groups, (c) free sulfohydryl groups, for example in cysteine, (d) free hydroxyl groups, for example in serine , threonine or hydroxyproline, (e) aromatic residues, for example in phenylalanine, tyrosine or tryptophan, or (f) glutamine amide groups. These methods are described in WO 87/05330 published September 11, 1987 and in Aplin and Wriston, CRC Crit. Rev. Biochem., P. 259-306 (1981).

Removal of the carbohydrate moieties present in the mpl ligand polypeptide can be accomplished chemically or enzymatically. Chemical deglycosylation requires treatment of the polypeptide with trifluoromethanesulfonic acid or a similar equivalent compound. This cleaves most or all of the sugars, except the linking sugar (N-acetylghicosamine or N-acetylgalactosamine), which leaves the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., Ar. Biochem. Biophys., 259: 52 (1987) and Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of the carbohydrate moiety in a polypeptide can be achieved by various endo- and exo-glycosidases, as described by Thotakura et al., Meth. Enzymol., 138: 350 (1987).

Glycosylation at potential glycosylation positions can be prevented by the use of tunicamycin as described by Duskin et al., J. Biol. Chem., 257: 3105 (1982). Tunicamycin blocks protein-N-glycoside binding.

in

Another type of covalent modification of mpl ligand comprises binding of the mpl ligand polypeptide to one of a plurality of non-proteinaceous polymers, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, as described in U.S. Pat. U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. mpl Uganda polypeptides covalently linked to the above polymers are referred to herein as "pegylated" mpl Uganda polypeptides.

It will be appreciated that screening of the obtained variants of mpl Uganda will be necessary to select the optimal variant for binding to mpl and having the immunological and / or biological activity as defined above. Screening can also be performed in terms of

105 stability in recombinant cell culture or plasma (i.e., against proteolytic cleavage), in view of high affinity for the mpl member, oxidative stability, ability to be secreted in increased yields and the like. For example, a change in the immunological character of an mpl ligand polypeptide, such as affinity for a given antibody, is measured by a suitable type of immunoassay. Other potential modifications of the properties of a protein or polypeptide such as redox or thermal stability, hydrophobicity, or sensitivity to proteolytic degradation are measured by methods well known in the art.

17. General methods for preparing antibodies to mpl Uganda

Preparation of Antibodies (i) Polyclone Antibodies

Polyclone antibodies to mpl ligand polypeptides or fragments generally arise in animals following multiple subcutaneous (sc) or intraperitoneal (ip) injections of mpl Ugand and adjuvant. It may be useful to conjugate mpl Ugand or a fragment containing a target amino acid sequence to a protein that is immunogenic in those species that are immunized, i.e., keyhole limpet hemocyanine, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, using a bifunctional or derivatizing agents such as maleimide-benzoylsulfosuccinimide ester (conjugated via cystem residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, butanedioic anhydride, SOCl ₂ , or R ¹ N = C = NR, where R and R ¹ are different alkyl groups.

Animals are immunized against the mpl ligand polypeptide or fragment with immunogenic conjugates or derivatives by combining 1 mg of 1 µg peptide or conjugate (for rabbits or mice) with 3 volumes of complete Freund's adjuvant and injecting the solution intradermally into multiple sites. One month later, animals are injected subcutaneously at multiple sites using only 1/5 to 1/10 of the original amount of peptide or conjugate in complete Freund's adjuvant. 7-14 days later, animals are bled and the serum is assayed for mpl Uganda antibody titer. Animals are injected until the titer reaches a stabilized level. Preferably, animals are injected with a conjugate of the same mpl Ugand but conjugated to a different protein and / or

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with a different crosslinking agent. Conjugates can also be prepared in recombinant cell culture as a mixture of proteins. Aggregating agents, such as alum, are also used to enhance the immunological response.

(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations, which may be present in minimal amounts. Thus, the term "monoclonal" indicates the nature of the antibody, which is not a mixture of individual antibodies.

For example, the monoclonal antibodies of mpl Uganda of the invention may be prepared using the hybridoma method first described by Kohler & Milstein, Nature, 256: 495 (1975), or may be prepared by recombinant DNA method (US 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 a reaction of lymphocytes that produce or are capable of producing antibodies that then specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then mixed with myeloma cells using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 [Academic Press, 1986]).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, which preferably contains one or more components inhibiting the growth or survival of unattached parental myeloma cells. For example, if the parent myeloma cells do not have hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the hybridoma culture medium will typically contain hypoxanthine, aminopterin and thymidine (HAT medium) that prevent the growth of deficient HGPRT cells.

Preferred myeloma cells are those that combine efficiently, help stabilize high levels of antibody expression with selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among them, further preferred myeloma cell lines are mouse myeloma lines such as those that are derived from MOPC-21 and MPC-11 mouse tumors available from

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Salk Cell Distribution Center, San Diego, Califomia USA and SP-2 cells, available from the American Type Culture Collection of Rockville, Mairyland USA. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described in the preparation of human monoclonal antibodies (Kozbor, J. ImmunoL, 133: 3001 [1984]; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, In c., New York, 1987).

The culture medium in which the hybridoma cells are growing is tested for the production of monoclonal antibodies against mpl Uganda. 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 a radioimmunoassay (RIA) or an immunoabsorbent binding enzyme assay (ELISA).

The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis of Munson & PoUard, Anal. Biochem., 107: 220 [1980].

After it has been determined that the hybridoma cells produce antibodies of the desired specificity, affinity and / or activity, the clones can be further subcloned by limiting dilution procedures and ensured to grow by standard procedures (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. In addition, hybridoma cells can grow in vivo as ascites tumors in animals.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes 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. Once isolated, the DNA can be placed in expression vectors which are then transfected into host cells, such as: monkey COS cells, Chinese hamster testicle (CHO) cells, or myeloma cells that otherwise do not produce immunoglobulin protein to synthesize monoclonal antibodies in recombinant host cells cells. The DNA can also be modified, e.g. i

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substitution of the human heavy and light chain constant domain coding sequences at homologous mouse sequence sites (Cabilly et al., supra; Morrison et al., Proc. Nat. Acad. Sci., 81: 6851 [1984]) or by covalent attachment of all or a portion polypeptide non-immunoglobulin polypeptide coding sequences are typically replaced by constant domains of an antibody of the invention or are replaced by variable domains of an antibody-binding position of an antigen of the invention to produce a chimeric bivalent antibody comprising a single antigen-binding position that is specific to an mpl ligand and another position joining an antigen specific to a different antigen.

Chimeric or hybrid antibodies can also be prepared in vitro using methods known in protein synthesis chemistry, including those involving cross-linking agents. For example, immunotoxins can be prepared using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents 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 moiety may be any moiety capable of eliciting, either directly or indirectly, a detectable signal. The detectable moiety may be, for example, a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I, a fluorescent or chemihiminiscent compound such as fiorescein isothiocyanate, rhodamine or hiciferin; radioactive isotopic markers such as ¹²⁵ L, ³² P, ¹⁴ C or ³ H or an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art may be used to separately conjugate the antibody to the detectable moiety, including those described by 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).

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

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Competitive binding assays are based on the ability of a labeled standard (which may be an mpl ligand or an immunologically reactive portion thereof) to compete with a test sample (mpl ligand) to bind with a limited amount of antibody. The amount of mpl ligand in the test sample is inversely proportional to the amount of standard that binds to the antibodies. To facilitate the determination of the amount of standard to be bound, the antibodies are generally rendered insoluble before or after the assay so that the standard and test sample that bind to the antibodies can be conveniently separated from the standard and test component remaining unbound.

Indirect sandwich assays involve the use of two antibodies, each capable of binding to different immunogenic portions, or an epitope of the protein (mpl ligand) that is detected. In the sandwich assay, the test sample binds with the first antibody, which is immobilized on a solid support, and then the second antibody binds to it, forming an insoluble three-part complex (David & Greene, U.S. Patent 4,376,110). The second antibody itself may be labeled with a detectable moiety (direct sandwich assay) or may be determined using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay in which the detectable portion is an enzyme (i.e., horseradish peroxidase).

(iii) Humanized and human antibodies

Methods for humanizing non-human antibodies are well known in the art. Generally, the humanized antibody has one or more amino acid residues introduced into the antibody from a non-human source. These non-human amino acid residues often refer to "imported" residues, which are typically selected from an "imported" variable domain. Humanization can essentially be performed according to the method of Winter et al. (Jones et al., Nature, 321: 522-525 [1986]; Riechmann et al., Nature, 332: 323-327 [1988]; Verhoeyen et al., Science, 239: 1534-1536 [1988]), replacing the rodent CDRs and CDR sequences with the corresponding human antibody sequences. Such "humanized" antibodies are chimeric antibodies (Cabilly et al., Supra) in which substantially less than the intact human variable domain has been replaced by corresponding sequences from non-human species. In practice, this means that humanized antibodies are typically human antibodies in which they are

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replaced some CDR residues and possibly some FR residues with residues from analogous positions in rodent antibodies.

The selection of human variable domains, both light and heavy, used to prepare humanized antibodies is very important in reducing antigenicity. According to the so-called. In the best-fit method, the rodent antibody variable domain sequence is screened against an entire library of known human variable domain sequences. The human sequence closest to that rodent sequence is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 [1993]; Chothia and Lesk, J. Mol. Biol., 196: 901 [1987]). Another method utilizes a special structure derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for some different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 [1992]; Presta et al., J. Immunol., 151: 2623 [1993]) .

It is further important that the antibodies are humanized in such a way as to retain high affinity for the antigen and other necessary biological properties. In order to achieve this effect, the humanized antibodies are prepared by a process of parental sequence analysis and various conceptual humanized products in accordance with a preferred method using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are commercially available and are known to those skilled in the art. Available computer programs illustrate and visually depict the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these images allows to analyze the likely role of residues in the functionality of the candidate immunoglobulin sequence, i.e., to analyze residues that affect the ability of the candidate immunoglobulin to bind its antigen. Thus, FR residues can be selected and combined from a consensus and important sequence to achieve the desired antibody characteristic, such as increased affinity for the target (s) antigen (s). In general, CDR residues directly and substantially affect antigen binding. Further details are given in U.S. Pat. application 07/934 373 filed August 21, 1992, which is a continuation of application 07/715 272 filed June 14, 1991.

Alternatively, it is now possible to prepare transgenic animals (e.g., mice) that are capable of producing a full spectrum of human antibodies after immunization without producing an endogenous immunoglobulin. For example, it is described to be homzygous

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the removal of the antibody heavy chain joining the gene region (Jh) into chimeric and germline mutant mice is complete; inhibiting endogenous antibody production. Transfer of a group of human germline immunoglohulin genes to such germline mutant mice will produce human antibodies induced by the presence of antigen. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-255 [1993]; Jakobovits et al., Nature, 362: 255-258 [1993]; Bruggermann et al., Year in Immuno., 7:33 [1993], Human antibodies can also be produced in phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227, 381 [1991]; Marks et al. , J. Mol. Biol., 222, 581 [1991]).

(iv) Bispecific antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that bind the specificities of at least two different antigens. Methods for making bispecific antibodies are known to those skilled in the art.

Traditional recombinant preparation of bispecific antibodies is based on the coexpression of two immunoglobulin pairs (heavy chain-light chain), where the two heavy chains have different specificities (Millstein and Cuello, Nature, 305: 537-539 [1983]). Because of the random distribution of immunoglobulin heavy and light chains, these hybridomas (quadromas) yield a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually accomplished by several affinity chromatography steps, is rather cumbersome and yields are low. Similar procedures are disclosed in PCT Application WO 93/08829 (published May 13, 1993) and are also referred to by Traunecker et al., EMBO, 10: 3655-3659 [1991].

According to a different and more preferred approach, the variable domains of an antibody with the desired binding specificities (antibody-antigen-binding positions) are linked to immunoglobulin constant domain sequences. Coupling is preferably performed with an immunoglobulin heavy chain constant domain comprising at least a portion of the attachment site of the CH2 and CH3 regions. Suitably, the first heavy chain constant region (CH1) comprises a position that is required for light chain binding present in at least one linkage product. DNA molecules encoding the immunoglobulin heavy chain junction products are introduced into separate expression vectors and co-transfected into a suitable host organism. This provides greater flexibility in adjusting the relative proportions of the three

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polypeptide fragments when incorporated in the examples, when unequal ratios of the three polypeptide chains were used to produce optimal yields. It is also possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when the expression of at least two polypeptide chains in the same ratios gives high yields or when the ratios are not of specific significance. In a preferred method, the bispecific antibodies consist of a hybrid immunoglobulin heavy chain with a first bound specificity on one strand and a hybrid immunoglobulin heavy and light chain pair (providing binding of a second specificity) in the other strand. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from unexpected combinations of immunoglobulin chains, since the presence of the immunoglobulin light chain is only half the bispecific molecule, facilitating the separation pathway. This procedure is set out in application 07/931 811, filed August 17, 1992, pending.

in

Further details of generating bispecific antibodies are given, for example, in Suresh et al., Methods in Enzymology, 121: 210 [1986].

(v) Heteroconjugate antibodies

Heteroconjugate antibodies are also within the scope of this invention. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies have been proposed against the target immune system of cells, against unwanted cells (U.S. Patent 4,676,980) and for the treatment of HIV infection (PCT WO 91/00360 and WO 92/00373; EP 03089). Heteroconjugate antibodies can be made using any conventional cross-linking method. Suitable crosslinking agents are well known to those skilled in the art and are disclosed in U.S. Patent 4,676,980 along with a number of crosslinking procedures.

IV. Therapeutic use of megakaryocytopoietic protein mpl ligand

A biologically active mpl ligand having a hematopoietic executive function, and in the sense of the present invention a megakaryocytopoietic or thrombocytopoietic protein (TPO), is used in sterile pharmaceutical preparation or formulations to stimulate megakaryocytopoietic or thrombocytopoietic activity in patients suffering from thrombocytopoiesis or thrombocytopenia formation, increased breakdown of platelets. - Thrombocytopenia associated with bone marrow hypoplasia (ie

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aplastic anemia with subsequent chemotherapy or bone marrow transplantation) can be effectively treated with the compounds of the invention, as well as other diseases such as disseminated intravascular coagulation (DIC), immune thrombocytopenia (including HIV-induced ITP and non-HIV-induced, idiopathic, thrombocytopenia, congenital thrombocytopenia, myelodysplasia, and thrombotic thrombocytopenia. In addition, these megakaryocytopoietic proteins may be useful in the treatment of myeloproliferative thrombocyte diseases, as well as thrombocytoses resulting from non-inflammatory conditions and iron deficiency.

Preferred uses of the megakaryocytopoietic and thrombocytopoietic protein (TPO) of the present invention are in: myelotoxic chemotherapy in the treatment of leukemias or solid tumors, myeloahlative chemotherapy in autologous and allogenetic bone marrow transplantations, myelodisplasia, idiopathic anthropoiesia, thiopathic aplastitemia, and thiopathic aplastitemia.

Other diseases that can be effectively treated with the megakaryocytopoietic proteins of the invention include platelet defects and platelet damages caused by drugs, poisoning or activation on artificial surfaces. In these cases, instant compounds of the invention that stimulate the "release" of new "undamaged" platelets can be used. A more complete list of applicable applications is provided in the "Prior Art" section (see above), in particular in sections (a) - (f) and references cited therein.

The megakaryocytopoietic proteins for soluble use of the invention may be used alone or in combination with other cytokines, hematopoietins, interleukins, growth factors or antibodies in the treatment of the above-identified diseases and conditions. Thus, instant compounds may be used in combination with other proteins or peptides having thrombopoietic activity, including: G-CSF, GM-CSF, LIF, M-CSF, IL-1, IL-3, erythropoietin (EPO), kit ligand , IL-6 and IL-11.

The instant instant megakaryocytopoietic proteins of the invention are prepared in admixture with a pharmaceutically acceptable carrier. The therapeutic composition may be administered intravenously or via a nose or lung. The composition may also be administered parenterally or subcutaneously, if necessary. When administered systemically, the therapeutic composition must be pyrogen-free and have appropriate pH, isotonicity and stability in parenterally acceptable solutions. These conditions are known to those skilled in the art. Briefly, batches of dosage forms

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The compounds of this invention are prepared for storage or administration by mixing a compound having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers. Such materials are nontoxic at the dosages and concentrations employed, and also include buffer solutions such as phosphate, citrate , acetate and salts of other organic acids; antioxidants such as ascorbic acid; low molecular weight peptides (less than about ten residues) such as polyarginine, proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides and other hydrocarbons, including cellulose and its derivatives, glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; antionic agents such as sodium and / or nonionic surfactants such as Tween, Pluronics or polyethylene glycol.

About 0.5 to 500 mg of the compound or mixture of the megakaryocytopoietic protein in free acid or base form or a pharmaceutically acceptable salt is mixed with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, colorant, etc. as required by accepted pharmaceutical practice. The amount of active agent in these compositions is such that appropriate dosages within the indicated range are obtained.

The sterile composition for injection may be formulated in accordance with conventional pharmaceutical practice. For example, dissolution or suspension of the active compound in a vehicle such as water or a natural vegetable oil such as sesame, peanut or cotton seed oil or in a synthetic fatty vehicle such as ethyl oleate and the like is required. Also buffers, preservatives, antioxidants, etc. may be used in accordance with accepted pharmaceutical practice.

Suitable examples of sustained and uniform release formulations include semipermeable matrices of solid hydrophobic polymers containing polypeptides, which matrices are in the form of shaped articles, for example, thin layers or microcapsules. Examples of long-term and uniformly acting matrices include polyesters, hydrogels [for example, poly (2-hydroxyethyl methacrylate) as described by 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. Patent 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman &amp;

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et al., Biopolymers, 22: 547-556 [1983]), non-degradable ethylene vinyl acetate (Langer et al., supra), degradable lactic acid-glycolic acid copolymers such as Lupron Depot ™ (injectable microspheres composed of lactic acid copolymer). and glycolic acid and leucoprolidate acetate) and poly-D - (-) - 3-hydroxybutyric acid (EP 133 988).

While polymers such as ethylene-vinyl acetate and butyric-glycolic acid copolymers allow release of molecules for more than 100 days, some hydrogels release proteins for a shorter time. When encapsulated proteins remain in the body for a long time, they may deform or aggregate due to exposure to moisture at 37 ° C, resulting in a loss of biological activity and likely changes in immunogenicity. Various procedures may be used to stabilize the protein, depending on the expected mechanism of loss of its biological activity. For example, if an aggregation mechanism by intra-molecular S-S linkages is expected due to disulfide exchange, in this case stabilization is achieved by modifying the sulfohydryl residues, lyophilizing from acidic solutions, controlling moisture content, using suitable additives, and developing a composition with a specific polymer matrix.

The long-term and uniformly active megakaiyocytopoietic protein compositions also include a liposome-linked megakaryocytopoietic protein. Liposomes containing the megakaryocytopoiesis protein are prepared by the methods described in: DE 3 218 121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 36883692 [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 83-118008; U.S. Patent 4,485,045 and 4,544,545; and EP 102 324. Liposomes are of a small (about 200-800 Angstroms) single-lamellar type in which the lipid content is greater than about 30 moL% cholesterol in terms of the selected dose, which is adjusted to optimal megakaryocytopoiesis protein therapy.

The dose will be determined by the attending physician taking into account various known factors that may affect the performance of the medicines, including severity and type of the disease, weight of the patient, sex, diet, time and mode of administration, other medicines it is taking and other relevant clinical factors. Typically, the daily regimen comprises a dose in the range of 0.1-100 gg / kg patient weight. A preferred dose will be in the range of 0.1-50 gg / kg patient weight. It is preferred that the initial dose be in the range of 1 to 5 gg / kg / day. The dose range will be the same as for other cytokines, especially G-CSF,

- 116 GM-CSF and EPO. Therapeutically effective doses can be determined by either an in vitro or in vivo method.

Examples

Without further elaboration, it is understood that one skilled in the art, using the preceding description and illustrative examples, will make and utilize the present invention to the fullest extent. The following working examples therefore emphasize preferred embodiments of the present invention and are in no way designed to limit the other data in any way.

Example 1

Partial purification of porcine mpl ligand

Platelet-depleted plasma was collected from normal pigs or from pigs with aplastic anemia. Aplastic anemia was induced in pigs by irradiation of 900 cGy of total body radiation using a 4 mEV linear accelerator. Irradiated pigs were injected intramuscularly for 6-8 days with cefazoline. Subsequently, under general anesthesia, whole blood was collected, blood was heparinized and centrifuged at 1800g for 30 minutes. The megakaryocyte stimulating effect was found to peak (peak) 6 days after irradiation

Aplastic porcine plasma, obtained from irradiated pigs, was treated with 4M NaCl (sodium choride) and stirred for 30 minutes at room temperature. The resulting precipitate was removed by centrifugation at 3800 rpm in Sorvall RC3B and the supernatant was loaded onto a phenyl-toyopearl column (220 mL) equilibrated with 10 mM NaPO ₄ containing 4M NaCl. The column was washed with buffer up to A ₂₈₀ <0.05 and eluted with dH ₂ O. The washed protein zone was diluted with dH ₂ O to a conductivity value of 15 mS and loaded onto a Blue-Sepharose column (240 mL), equilibrated. in PBS. Subsequently, the column was washed with a 5-fold (based on column volume) volume of both PBS and NaPO ₄ (pH 7.4) containing 2 M urea. Proteases were eluted from the column with 10 mM NaPO ₄ (pH 7.4) containing 2 M urea and 1 M NaCL The protein eluate was treated with 0.01% octylglucoside (n-octyl-3-D-glucopyranoside), 1mM EDTA and 1mM Pefabloc (Boehringer Mannheim) and introduced directly onto tandem-bound CD4-IgG (Capon,

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D.J. et al., Nature 337: 525-531 [1989]) and w / R-IgG Ultralink (Pierce) columns (see below). The CD4-IgG (2 ml) column was removed after the sample was loaded and the mp / -IgG (4 ml) column was washed with 10-fold both PBS and PBS containing 2 M NaCl and eluted with 0.1M glycine-HCl pH 2.25. Fractions were collected in 1/10 volume of 1M tris-HCl (pH 8.0).

Analysis of the fractions eluted from the mp / -affinity column by SDS-PAGE (4-20%, Novex gel), measured under reducing conditions, showed the presence of several proteins (Fig. 5). The most potent silver dye proteins were digested with apparent Mr 66000, 55000, 30000, 28000, and 14000. To determine which of these proteins stimulate proliferation of Ba / F3-mpZ cell cultures, these proteins were eluted from the gel as indicated. in Example 2 below.

Ultra - affinity columns

10-20 mg of wpZ-IgG or CD4-IgG in PBS is combined with 0.5 g of Ultralink resin (Pierce) as described in the manufacturer's instructions.

Construction and expression of mpl-IgG

A chimeric molecule comprising the entire extracellular domain of human mpl (amino acids 1-491) and the Fc region of the human IgG1 molecule was expressed into 293 cells. The cDNA fragment encoding amino acids 1-491 of human mpl was obtained by PCR from the human megakaryocyte CMK cell library cDNA and sequenced. The ClaI position was inserted at the 5 end and the BstEII position at the 3 end. This fragment was cloned upstream of the IgG1 Fc coding region in the Bluescript vector between the ClaI and BstEII sites after partial digestion of the PCR product with BstEII for two additional BstEII positions present in the DNA encoding the extracellular domain of mpl. The BstEII position introduced at the 3 end of the mpl PCR product was labeled to have the Fc region in frame with the mpl extracellular domain. The construct was subcloned into the pRK5-tkneo vector between the ClaI and XbaI sites and infected into 293 human embryonic kidney cells by 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 best expressed clone had an expression level of 1-2 mg / ml mpl-YgG.

- 118 Ba / F3 mpl P expression cDNA cells corresponding to the entire coding region of human mpl P were cloned into pRK5-tkneo, which was then linearized with Not1 and transfected into EL-3 dependent Ba / F3 cell line Ba / F3 by electroporation (1x10 ⁷ cells) , 9605 F, 250 volts). After three days, selection was started in the presence of 2 mg / ml G418. Cells were selected as a whole or individual clones were obtained by limiting (limiting) dilution in 96-well microtiter plates. The selected cells were maintained in RPMI containing 15% FBS, 1 mg / ml G418, 20 mM glutamine, 10 mM HEPES and 100 µg / ml Pen-Strep. Expression of mpl P in selected clones was determined by FACS analysis using anti-zwpZ P rabbit polyclonal antibody.

TestBa / F3 mpl ligand

The mpl ligand assay was performed as shown in Figure 2. To determine the presence of mpl ligand from various sources, mpl P Ba / F3 were exposed to IL-3 deficiency for 24 hours at a cell density of 5 x 10 ⁵ cells / ml in a humidified incubator at 37 ° C, 5% CO ₂ and air. After IL-3 deficiency, cells were seeded in 96 culture microtubes to a density of 50,000 cells in 200 µL of diluted or undiluted sample medium and cultured for 24 hours in a cell culture incubator. 20 μΐ serum, without RPMI medium containing 1 pCi of ³ H-thymidine, was added to each microtube for the last 6-8 hours. Cells were then harvested into 96-well GF / C filter microplates and washed 5 times with water. The filtrates were evaluated in the presence of 40 μΐ scintillation solution (Mikroscint 20) in a Packard Top Count.

Example 2

Highly purified porcine mpl ligand

Protocol on gel elution

Equal amounts of affinity purified mpl ligand (fraction 6 eluted from the nPZ-IgG column) and a 2X sample of Laemmli buffer were mixed at room temperature without reducing agent and loaded onto a 4-30% polyacrylamide Novex gel as quickly as possible. The sample was not heated. As a control, a sample of buffer without Ugandan was applied. The gel was run at 4-6 ° C at 135 V for about 2 1/4 hours. The displacement buffer was initially at room temperature. Gel sa

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then removed from the electrophoretic chamber (gel box) and the plate was removed from one side of the gel.

A copy of the gel was made on nitrocellulose as follows: a piece of nitrocellulose was moistened with distilled water and gently placed on top of the exposed gel side so as not to form air bubbles. Base markers were placed on nitrocellulose and gel plate so that the copy can be accurately positioned after staining. After approximately 2 minutes, the nitrocehilose was carefully separated and the gel was wrapped in a plastic container and stored in a refrigerator. Nitrocellulose was stained with Biorad's gold total protein dye by first shaking it in 3x10 ml 0.1% Tween 20 + 0.5 M NaCl + 0.1 M tris-HCl pH 7.5 for approximately 45 minutes, followed by 5 minutes 3x10 ml purified water . The gold dye was then added and allowed to act until the bands were visible in the standards. The copy was then rinsed with water, placed in a plastic gel container, and carefully assigned to the base markers. The positions of Novex standards were marked on the gel plate and lines were drawn to indicate the locations of the sections. The nitrocellulose and the plastic sheath were then separated, and the gel was cut into sharp lines with a sharp razor blade. The sections were stretched beyond the samples so that they could be used to determine the positions of the strips when the gel was stained After separating the strips, the remaining gel was stained in silver and the locations of the standards and the cut marks were measured. The molecular weights corresponding to the cut sites are determined from Novex standards.

The gel strips were placed in cuvettes in two Biorad 422 electric generators. Treated ultrafiltration membranes with a molecular weight of 12-14K were used in the cuvettes. The eluting timing solution was 50 mM ammonium bicarbonate + 0.05% SDS (pH about 7.8). One liter of buffer was cooled in a 4-6 ° C refrigeration chamber for about 1 hour before use. Gel strips were digested at 10mA / cuvette (original 40V) in a 4-6 ° C refrigeration chamber. The elution lasted approximately 4 hours. The cuvettes were then carefully separated and the supernatant was removed by pipette. The elution chamber was removed and all of the liquid above the membrane was removed by pipette. The liquid in the membrane was removed by pipetman and discarded. Fifty μΐ of purified water was added to the membrane, shaken and separated until all SDS crystals had dissolved. These washings were combined with the above mentioned discarded fluid. The total elution volume of the sample was 300-500 μΐ per gel band. Samples were placed in 10 mm cut Spectrapor 4 12-14K dialysis tubes, which were soaked for several hours in purified water.

They were dialyzed overnight at 4-6 ° C against 600 ml of phosphate buffered saline (PBS contains approximately 4 mM potassium) for 6 samples. The buffer was changed the following morning and dialysis was continued for 2.5 hours. Samples were separated from dialysis bags and stored in microcentrifuge tubes. The tubes were stored on ice for 1 hour, microcentrifuged for 3 minutes at 14K rpm, and the supernatants were carefully separated from the precipitated SDS. The supernatants were then stored on ice for approximately an additional 1 hour and microcentrifuged again for 4 minutes. Supernatants were diluted in phosphate buffered saline and used to determine efficacy. The remaining samples were frozen at -70 ° C.

Example 3

Microsquencing of porcine mpl ligand

Fraction 6 (2.6 ml) from the τζιρΖ-IgG affinity column was concentrated to Microcone-10 (Amicon). To prevent the absorption of mpl ligand on the Microcon membrane, rinse with 1% SDS and 5 μΐ 10% SDS was added to fraction 6. A 20X buffer sample was added to fraction # 6 after concentration on Microcone (20 μΐ) and total volume (40 μΐ) was applied to one corridor of acrylamide gel (4-20% gradlient) of Novex. The gel was processed according to the Novex protocol. The gel was then equilibrated for 5 minutes prior to electroblotting in 10 mM 3- (cyclohexylamino) -1-propanesulfonic acid (CAPS) buffer, pH 11.0, containing 10% methanol. Electroblotting was performed on Immobilon-PSQ membranes (Millipore) for 45 minutes at 250 mA constant current in a portable BioRad Trans-Blot cuvette (32). The PVDF membrane was stained with 0.1% Coomassie Bhie R-250 in 40% methanol, 0.1% acetic acid for 1 minute and stained for 2-3 minutes with 10% acetic acid in 50% methanol. Only those proteins that were visible in the Mr 18,00035,000 spot had Mr 30,000, 28,000 and 22,000.

The bands (strips) at 30, 28 and 22 kDa were due to the protein sequence. Automatic protein shifting was performed on a 470A Applied Biosystem controller equipped with an on-line PTH analyzer. The controller was modified to inject 80-90% of the sample (Rodriguez, J .: Chromatogr., 350: 217-225 [1985]). Acetone (~ 12 μΐ / ΐ) was added to the solvent and to equalize the UV absorbance. The electroblotted proteins were sequenced in a Blott cassette. The peaks were integrated with Justice Innovation

- 121 sofhvare using Nelson Analytical 970 interfaces. Sequence interpretation was performed on VAX 5900 (HenzeL, et al., J. Chromatogr., 404: 41-52 [1987]). The N-terminal sequences (using a single letter code with indeterminate residues in parentheses) and the amount of material obtained (in parentheses) are. listed in Table 2.

Table 2

The amino terminal sequences of the mpl ligand

30 kD [1.8 pmol] 1 2 3 4 5 6 7 8 9 10 11 12 (S) P A P P A (C) D P R L L K K L D (H / S) V L H (G) R L (SEQ ID NO: 29) 28 kD [0.5 pmol] 1 5 .10 15 '20 25 (S) P A P P A X D P L L N K L L D D (H) V L (H) G R (SEQ ID NO: 32) 18-22 kD [0.5 pmol] 1 5 10 X P A P P A D D L L (N) (K) (SEQ ID NO: 33)

Example d 4

Liquid suspension test of megakaryocytopoiesis

Human peripheral stem cells (PSC) (obtained from voluntary patients) were diluted 5x with IMDM medium (Gibco) and centrifuged for 15 minutes at room temperature and 800 x g. Cell sediment (pellets) was resuspended in IMDM and cells were propagated to 60% Percoll (density 1.077 gm / ml) (Pharmacia) and centrifuged at 800 xg for 30 min. The light density of the mononuclear cells was aspirated at the interface and washed 2x with IMDM and inoculated at 1-2x10 ⁶ cells / ml in IMDM containing 30% FBS (1 ml final volume) in 24 well tissue culture clusters (Costar). APP or APP emptied mpl ligand 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. The cultures were also grown in the presence of 10% APP with 0.5 µg mpl-IgG added on days 0, 2 and 4. APP was emptied from the mpl ligand by passing APP through the mpl-IgG G affinity column.

The modification of Solberg et al. and using a radiolabeled mouse IgG monoclonal antibody (HPI-1D) on GPIIblIIa

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(performed by Dr. Nichols, Mayo Clinic). 100 µg HP1-1D (see Grant, R et al. \ Blood 69: 1334-1339 [1987]) was radiolabeled with 1 mCi Na ¹²⁵ I using Enzymobeads (Biorad, Richmond, CA) as described in the manufacturer's instructions . Radiolabelled HP1-1D was stored at -70 ° C in PBS containing 0.01% octylglucoside. Typical specific activities were 1-2x10 ⁶ cpm / pg (> 95% precipitated with 12.5% trichloroacetic acid).

Liquid suspension cultures were run in triplicate for each experimental point. After 12-14 days of culture, 1 ml cultures were transferred to 1.5 ml microtubes ("eppendorfs"), which were centrifuged at 800 xg for 10 minutes at room temperature, and the resulting cell pellets (sediment) were resuspended in 100 μΐ PBS containing 0, 02% EDTA and 20% bovine calf serum. 10 ng of ¹²⁵ I-HP1-1D in a 50 μΐ buffer sample was added to the resuspended cultures and incubated for 60 minutes at room temperature (RT) with occasional shaking. Subsequently, the cells were collected by centrifugation at 800 xg for 10 minutes at RT and washed twice with buffer. The pellets were counted in a gamma counter (Packard) over 1 minute. Non-specific binding was determined by adding 1 µg of unlabeled HP1-1D for 60 minutes before adding labeled HP1-1D. Specific binding was determined as the total threshold (value) of ¹²⁵ I-HP1-1D minus the threshold (value) in the presence of excess unlabeled HP1-1D.

Example 5

Primers of PCR oligonucleotides

Based on the amino-terminal amino acid sequence obtained from the 30 kDA, 28 kDa and 18-22 kDa proteins, degenerate oligonucleotides were determined for use as polymerase chain reaction (PCR) primers (see Table 4). Two primer pools were synthesized: a positive sense 20 mer pool encoding amino acid residues 2-8 (mpl 1) and an anti-positive sense 21-mer pool encoding complementary amino acid sequencing 18-24 (mpl 2).

Table 4

Degenerate oligonucleotide primer pools

mp / 1: 5'CCN GCN CCN GCN TGC GA 3 '(2,048χ · γΙρπρπργ (SEQ ID NO: 36) mpl 2: 5 'NCC RTG RTC RTC 3 ‘(2,048 x deqener. (SEQ ID NO: 35)

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Porcine genomic DNA, isolated from porcine peripheral, blood lymphocytes, was used as a template for PCR. 50 μΐ reactions contained: 0.8 μg of porcine genomic DNA in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 100 μg / ml BSA, 400 μΜ dNTPs, 1 μΜ each from each primer pool and 2.5 Taq polymerase units. The initial template denaturation was at 94 ° C for 8 minutes, followed by 35 45-second cycles at 94 ° C, 1 min at 55 ° C and 1 min at 72 ° C. The final cycle followed a 10 minute extension at 72 ° C. PCR products were separated by 12% polyacrylamide gel electrophoresis and visualized by staining with ethidium bromide. It was found that if the amino-terminal amino acid sequence was encoded by a single exon, then 69 bp is expected for the correct PCR product. A DNA fragment of this size was eluted from the gel and subcloned into pGEMT (Promega). The sequences of the three clones are shown in Table 5 below.

Table 5 bp gemT3 genomic DNA fragments

5'CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA 3'GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG ACGGAGCAQT

TGACCACGTT CAGCACGGC [69 bp] (SEQ ID NO: 37)

ACTGGTGCAA GTCGTGCCG (SEQ ID NO: 38) gemT7

5'CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 3'GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCACT

CGACCACGTC CATCACGGC [69 bp] (SEQ ID NO: 39)

GCTGGTGCAG GTAGTGCCG _______ (SEQ ID NO: 40) ______ gemT9

P L L N K L L R (SEQ ID NO: 32)

5 'ccagcaccgccggcatgtgacccccgactcctaaataaagtggttggtgacg 3' ggtcgtggcggccgtacactgggggctgaggatttatttgaggaagcactgc

ATCATGTCTATCACGGT 3 '(SEQ ID NO: 41)

TAGTACAGATAGTGCCA 5 '(SEQ ID NO: 42)

The position of the PCR primers is indicated by the underlined base. These results, confirmed by the N-terminal sequence, were obtained for amino acids 9-17 of 30 kDa, 29 kDa, and 18-22 kDa proteins and showed that this sequence is encoded by a single exon of pork DNA.

Example 6

Human mpl ligand gene

Based on the results of Example 5, a 45-mer deoxynucleotide, called pR45, was determined and synthesized for genomic library screening. The 45-mer had the following sequence: 5 GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG 3 (SEQ ID NO: 28)

This oligonucleotide was designated ³² P with (γ ³² Ρ) -αΤΡ and T4 kinase and used to display the human genomic DNA library in ygem12 under mild hybridization and wash conditions (see Example 7). Positive clones were harvested, plaques were stained and analyzed by restriction mapping and southern blotting. Clone # 4 was selected for additional analysis.

The 2.8 kb BamHI-XbaI fragment that hybridized to the 45-mer was subcloned into pBhiescript SK-. The partial DNA sequence of this clone was preformed for use as the nucleotide primers specific for the mpl ligand sequence of the porcine DNA. The sequence obtained confirmed that DNA encoding a human porcine mpl ligand homolog was isolated. The EcoRI restriction site was found in a sequence allowing the 390 bp EcoRI-XbaI fragment to be isolated from the 2.8 kb BamHI-XbaI and subcloned in pBhiescript SK-.

Both strands of this fragment are sequenced. The human DNA and deduced amino acid sequences are shown in FIG. 9 (SEQ ID NOS: 3 and 4). Predicted intron positions in the genomic sequence are also indicated by arrows and defined by the putative exon ("exon 3").

Examination of the sequence of the predicted amino acid sequence confirms that the sermal residue is the first amino acid of mature mpl Uganda as determined from direct amino acid sequence analysis. Just upstream of this codon, the derived amino acid sequence highly resembles the signal sequence contained in the secretion

- 125 mature mpl Uganda. This signal sequence, coding region, is likely to be interrupted at nucleotide position 68 by an inron.

The exon appears to terminate at nucleotide 196 in 3 directions. This exon therefore encodes a sequence of 42 amino acids, of which 16 appear to be part of the signal sequence and 26 is part of the mature human mpl Uganda.

Example 7 / t

Full length mpl Uganda human 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 cDNA primers

Fwd primer: 5 'GCT TCT AGC AAT TGC TCC TCG TCG TCA TGC TTC T 3' (SEQ ID NO: 43) Rvs primer: 5 'CAG TCT GCCGTG (SEQ ID NO: 44)

These two primers are used in PCR reactions using DNA from different human cDNA libraries or 1 ng of Quick Clone cDNA (Clonetech) from different tissues using the conditions described in Example 5. The expected size of the correct PCR product was 140 bp . After analyzing the PCR products on a 12% polyacrylamide gel, the expected DNA fragment was detected in cDNA libraries prepared from adult, adult fetal fetal cells and cDNA prepared from human fetal liver (Clonetech cat. # 7171-1).

The fetal liver cDNA library in γ DR2 (Clonetech cat. # HL1151x) was screened with the same 45 mer ogonucleotide and utilized a human genomic library for screening. The oligonucleotide was labeled with (γ ³² Ρ) -ΑΤΡ using a T4 polynucleotide kinase. The library was sorted under mild hybridization conditions. The filtrates were prehybridized for 2 hours and then hybridized with the probe overnight at 42 ° C. in 20% formamide, 5xSSC, 10x Denhardts, 0.05M sodium phosphate (ph 6.5), 0.1% sodium pyrophosphate, 50 µg / ml ultrasonic salmon sperm DNA for 16 hours. The filters were then rinsed in 2xSSC and then washed with hexane

126 -

0.5xSSC, 0.1% SDS at 42 ° C. The filters were then exposed overnight to radioactive film (Kodak X-Ray film). Positive clones were harvested, plaque was purified and insert size was determined by PCR, using BamHI-XbaI flanking oligonucleotides encoding in λ DR2 (Clonetech cat. # 6475-1). 5 μΐ of bacteriophage culture was used as template source. Initial denaturation was performed for 7 minutes at 94 ° C, followed by 30 cycles of amplification (1 min at 94 ° C, 1 min at 52 ° C and 1.5 min at 72 ° C). The final extension was 15 min at 72 ° C. Clone # FL2b had a 1.8 kb insert and was selected for further analysis.

The plasmid pDR2 (Clonetech, yDR2 & pDR2 cloning and Expression System Library Library Handbook, p.42) contained in the yDR2 bacteriophage arms was obtained as described in the manufacturer's instructions (Clonetech, Clonetech, yDR2 & pDR2 cloning and Expression System Library). Protocol Handbook, pp. 29-32). Restriction analysis of plasmid pDR2-F2b with BamHI and XbaI showed the presence of an internal BamHI restriction site in the insert at approximately position 650. Digestion of the plasmid with BamHI-XbaI cut the insert into two fragments: one 0.65 kb and the other 1.15 kb. The DNA sequence was determined with three different classes of template derived from plasmid pDR2-FL2b. DNA sequence determination of double stranded plasmid DNA was performed with ABI373 (Applied Biosystems, Foster City, Califomia) automated fluorescent sequencer using standard dye-labeled dideoxynucleoside triphosphate terminators (stained terminators) and custom synthesized primers (Sanger et al., Proc. Acad Sci USA, 74: 5463-5467 [1977]; Smith et al., Nature, 321: 674-679 [1986]. Direct sequencing of the fragments amplified by the polymerase chain reaction from the plasmid was performed with an ABI373 sequencer using selected primer reactions and dye-labeled terminators. A single stranded template was created with the M13 Janus vector (DNASTAR, Inc., Madison, Wisconsin) (Burland et al., Nucl. Acids Res., 21: 3385-3390 [1993]). The BamHI-XbaI (1.15 kb and BamHI (0.65 kb) fragments) were isolated from plasmid pDR2-FL2b, blunted with T4 DNA polymerase in the presence of deoxynucleotides, and then subcloned into the SmaI site of M13 Janusa. for dye-labeled M13 universal and reverse primers, or moving primers and dye-labeled terminators, manually controlled sequencing reactions were performed on single-stranded M13 DNA using moving primers and standard dideoxyterminator chemistry

127 (Sanger et al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467 [1977]), ³³ P-labeled α-dATP and sequencing (United States Biochemical Corp., Cleveland, Ohio). The pooled DNA sequence was performed with Sequencher V2.1b12 (gene Codes Corporation, Ann Arbor, Michigan). The nucleotide and deduced hML sequences 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 human sorting

genomic library in γ-Gem 12 with pR45, an oligonucleotide probe already described under mild conditions (see Example 7) or under very stringent conditions with a fragment corresponding to 3 half of the human cDNA coding for mpZ-ligand (from BamHI site to 3 terminus). Two overlapping lambda clones measuring 35 kb were isolated. Two overlapping fragments (bamH1 and EcoRI) containing the full TPO gene, v

were subcloned and sequenced. The structure of the human gene consists of 6 exons in the 7 kb range of genomic DNA (Fig. 14 A, B and C). The boundaries of all exon / intron junction are consistent with the conventional motif introduced for mammalian genes (Shapiro, M.B., et al., Nucl. Acids Res. 15: 7155 [1987]). Exon 1 and exon 2 contain 5

the untranslated (untranslated) sequence and the initial amino acids of the signal peptide. The rest of the secretion signal and the first 26 amino acids of the mature protein are encoded in exon 3. The entire carboxyl domain (region) and 3 are untranslated as well as about 50 in

The amino acids of the erythropoietin-like region are encoded in exon 6. The four amino acids contained in the deletion, observed in the hML-2 (hTPO-2) range, are encoded at the 5 end of exon 6.

Example 9

Transient expression of human mpl ligand (hML)

To clone the full length of the insert contained in pDR2-FL2b, the plasmid was digested with XbaI for termination, then partially digested with BamH1. The DNA fragment corresponding to the 1.8 kb insert was gel purified and subcloned in pRK5 (pRK5-hmp / 1) (see US Pat. No. 5,258,287 for the construction of pRK5) under direct control

- 128 -

early cytomegalovirus promoter. DNA from the pRK5-hw /> / 1 construct was prepared by PEG method and transfected into 293 human germline kidney cells maintained in modified Dulbecco's Eagle with medium (DMEM) supplemented with F-12 nutritional mix, 20 mM Hepes and 10% fetal. borin serum. Cells were transfected with a calcium phosphate method as described (Gorman, C. [1985] in DNA Cloning: A Practical Approach (Glover, D.M., ed.) Vol. 143-190, IRL Press, Washington, D.C.). 36 hours after transfection, the proliferation assay activity was determined in the supernatant of the transfected cells (see Example 1). Supernatant of 293 cells transfected with only the pRK vector gave no stimulation of Ba / F3 or Ba / F3- / npZ cells (Fig. 12A). The supernatant of cells transfected with pRK5-lnnp / 1 had no effect on Ba / F3 cells but incredibly stimulated proliferation of BaZF3-wp / cells (Fig. 12A), suggesting that this cDNA encodes functionally active human mpl Ugand.

Example 10

HML2, hML3 and hML4 isoforms of human Mpl ligand

To identify alternatively spliced forms of hML, primers corresponding to each end of the hML coding sequence were synthesized. These primers were used in RT-PCR to amplify human adult liver RNA. Additionally, internal primers flanking the regions of interest (see below) were constructed and used similarly. Direct sequencing of the ends of the PCR product showed a separate sequence corresponding exactly to the cDNA sequence isolated from the human fetal liver library (see Fig. 1 [SEQ ID NO: 1]). However, a region near the C-terminus of the EPO-domain (in the middle of the PCR product) showed a complex template sequence, suggesting the existence of possible spliced variants in this region. To isolate these splice variants, the primers provided in Table 7 flanking the region of interest were used in PCR as templates for human adult liver cDNAs.

Table 7

Human ML isoform of PCR primers

phmpllcdna.3e1: '5’TGTGGACTTTAGCTTGGGAGAATG3' (SEQ ID NO: 44) pbx4.f2: 5'GGTCCAGGGACCTGGAGGTTTG3 ' (SEQ ID NO: 45)

The 129 PCR products were bluntly subcloned into M13. Sequencing of separate subclones showed the existence of at least 3 ML isoforms. One of these, hML (also referred to as I1ML332), is the longest form and exactly matches the sequence isolated from the fetal liver library. The sequences of the four isoforms of human mpl ligand, listed from longest (hML) to shortest (hML-4), are shown in FIG. 11 (SEQ ID NOS: 6,8,9 and 10).

Example 11

Construction and transient expression of human Mpl ligand isoforms and surrogate variants φ hML2, hML3 and hML4 (R153A, R154A)

The hML2 and hML3 isoforms and the hML surrogate variant (R153A, R154A) were reconstructed from hML using the recombinant PCR technique described by Russell Higuchi in: PCR Protocols, A guide to Methods and Applications, Acad. Press, M.A.Innis, D.H. Gelfand, J. J. Sninsky & T.J. White Editors.

In all designs, the "outer" primers used are listed in Table 8 and the "overlapping" primers are listed 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 (SEQ ID NO: 48)

- 130 Table 9

Overlapping primers

hML-2: MLA4.F: 5'C CTT GGA ACC CAG GGC AGG ACC 3 ' MLA4.R 5'GGT CCT GCC CTG GGT TCC AAG GAG 3 ' (SEQ ID NO: 50) (SEQ ID NO: 51) h M L - 3: h M LA 116+: 5'CTG CTC GGA AAG GAC TTC TGG ATT 3 'hMLAl16-: 5ΆΑΆ CCA GAA GTC CTT TCC TCG GAG CAG 3 3 (SEQ ID NO: 52) (SEQ ID NO: 51) hML (R154A, R154A): RR-KO-F: 5'CCC TCT GCG TCG RR-KO-R: 5'GTG GGT GGG (SEQ ID NO: 52) (SEQ ID NO: 55)

All PCR amplifications were performed with cloned Piu DNA polymerase (Stratagene) under the following conditions: initial template denaturation was at 94 ° C for 7 minutes, followed by 30 1-minute cycles at 94 ° C, 1 minute at 55 ° C and 1.5 min at 72 ° C. The final cycle was extended to 10 minutes at 72 ° C. The final PCR product was digested with ClaI-XbaI, gel purified, and cloned into pRK5tkneo. 293 cells were transfected with various constructs as described above, and supernatant activity was determined using the Ba (F3-β) proliferation assay. hML-2 and hML-3 did not show any detectable activity in this assay, but the activity of hML (R153A, R154A) was similar to hML, indicating that processing at the basic position is not required to achieve activity (see Figure 13).

Example 12 Mouse mpl ligand cMLNAs mML, mML-2 and mML-3

Isolation of mML cDNA

The DNA fragment corresponding to the entire coding region of human mpl Uganda was obtained by PCR, gel purification, and random loading in the presence of ³² P-dATP and ³² P-dCTP. This assay was used to examine 10 ⁶ clones of the mouse liver cDNA library in yGT10 (Clontech cat # ML3001a). Double filtrates were hybridized in 35% formamide, 5x SSC, 10x Denhardt, 0.1% SDS, 0.05M sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 100 ng / ml DNA from

- 131 -

ultrasonically treated salmon sperm overnight, in the presence of a probe. The filtrates were rinsed 2x SSC and then washed once with 0.5xSSC, 0.1% SDS at 42 ° C. The hybridized phages were plaque purified and the cDNA inserts were subcloned into the Eco R1 site of the Bluescript SK-plasmid. The 1.5 kb LD clone was selected for further analysis and both strands were sequenced as above for human ML cDNA. The nucleotide and deduced amino acid sequences from the LD clone are shown in FIG. 14 (SEQ ID NOS: 1 & 11). The deduced mature ML sequence from this clone god 331 amino acid residues and that God identifikovabé mML and ₃₃ (or mML-2 for reasons described above). The identity of both nucleotide and amino acid sequences was found in the EPO-like ML domains. However, when the deduced amino acid sequences of human and murine MLs were aligned, the mouse sequence was shown to have a tetrapeptide deletion between human residues 111-114, corresponding to a 12 nucleotide deletion of the following nucleotide position 618 apparent in both human (see above) and porcine (see above). below) cDNA. Therefore, other clones were investigated to determine possible murine ML isoforms. One clone, L7, had a 1.4 kb insert with a derived 335 amino acid acid containing the missing LPLQ tetrapeptide. This form is considered to be the full length form of murine ML and is referred to as mML or mML ₃₃ µ. The nucleotide and deduced amino acid sequences for mML are shown in FIG. 16 (SEQ ID NOS: 12 & 13). Finally, L2 clone was isolated and sequenced. This clone has a 116 nucleotide deletion corresponding to hML3 and is therefore termed mML-3. A comparison of the deduced amino acid sequences of the two isoforms is shown in FIG. 16th

Expression of recombinant mML

Mouse ML expression vectors were prepared essentially as described in Example 8. Clones encoding mML and mML-2 were subcloned into pRK5tkneo, a mammalian expression vector that allows expression under the control of the CMW promoter and SV40 polyadenylation signal. The resulting expression vectors, mMLpRKtkneo and mML2pRKtkneo, were transiently infected into 293 cells using the calcium phosphate method. After transient transfection, the medium was conditioned for 5 days. Cells were maintained in high DMEM glucose medium supplemented with 10% calf serum.

- 132 -

Mouse-mpl (mmpl) expression in Ba / F3 cells

Stable expression c-mpl cell lines were obtained by infecting vampl pRKtkneo, based on that described for human mpl in Example 1. Briefly, an expression vector (20 gg; linearized) containing the full murine mpl coding sequence (Skoda, RC., Et al., EMBO J. 12: 2645-2653 [1993]), was transfected into Ba / F3 cells by electroporation (5 x 10 ⁶ cells, 250 volts, 960 gF), followed by selection for neomycin resistance with 2 mg / ml G418. Mpl expression was determined by flow cytometric analysis using a rabbit anti-mouse znpZ-IgG antiserum. Ba / F3 cells were maintained in RPMI medium from WEH1 -3B cells as a source of IL-3. Supernatants from 293 cells transiently infected with both mML and mML-2 were tested in Ba / F3 cells infected with both mmpl and µmpl as described in Example 1.

Example 13

Porcine mpl Ligand cDNA pML and pML-2

Porcine ML (pML) cDNA was isolated by RAČE PCR Briefly, the oligo dT primer and 2 specific primers were labeled based on the exon sequence of the porcine ML gene encoding the amino terminus of ML, purified from aplastic porcine serum. CDNA prepared from various aplastic porcine tissues was obtained and amplified. A 1342 bp PCR cDNA product was found in the kidneys and subcloned. Some clones were sequenced and found to encode a mature porcine mpl ligand (not including the complete secretion signal). A cDNA encoding a 332 amino acid mature protein (pML ₃ 32) having the sequence shown in FIG. 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 sorted as described in Example 7. Some clones were isolated and an exon encoding an amino acid sequence identical to that obtained from purified ML was sequenced. Porcine ML cDNAs were obtained using a modification of the RAČE PCR protocol. Two specific primers

- 133 were labeled based on the porcine ML gene sequence. Polyadenylate mRNA was isolated from the kidney of aplastic pigs essentially as previously described. The cDNA was prepared by reverse transcription with a BamdT primer (BamdT: 5 GACTCGAGGATCCATCGATTTTTTTTTTTTTTTTT 3) (SEQ ID NO: 55) directed against the polyadenosine tail (end) of the mRNA. The initial PCR amplification circuit (28 cycles at 95 ° C for 60 seconds, at 58 ° C for 60 seconds, and at 72 ° C for ninety seconds) was conducted using ML specific h-forward-1 primer (h-forward-1: 5 GCTAGCTCTAGAAATTGCTTCFTCGTGGTCATGCTTCT 3) (SEQ ID NO: 43) and BAMAD primer (BAMAD: 5 'GACTCGAGGATCCATCG 3) (SEQ ID NO: 56) in 100m reaction (50mM Kel, 1.5mM MgCl, 10mM Tris pH 8.0, 0 , 2 mM dNTPs, p

0.05U / ml Amplitaq polymerase [Perkin Elmer Inc.]). The PCR product was then digested with Clal, extracted with phenol-chloroform (1: 1), precipitated with ethanol and ligated to 0.1 mg.

Bluescript SK- vector (Stratagene inc.) To be cut with Clal and Kpn 1. After a two hour incubation at room temperature, a quarter of the ligated mixture was added directly to the second PCR circuit (22 cycles as described above) using a second ML specific forward -1 primer (forward-1: 5 GCTAGCTCTAGAAGCCCGGCTCCTCCTGCCTG 3) (SEQIDNO: 57) and T3-21 (an oligonucleotide that connects to the adjacent sequence of the multicloned region in the 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-. Some clones from independent PCR reactions were sequenced.

Again, a second form, designated pML-2, encoding a protein with a 4 amino acid residue deletion (328 amino acid residues) was identified (see FIG. 21 [SEQ ID NO: 21]). Comparison of pML and pML-2 amino acid sequences shows that the latter form is identical except that the tetrapeptide QLPP, corresponding to

134 residues 111-114 inclusive were deleted (see Fig. 22 [SEQ ID NOS: 18 & 21]). The four amino acid deletions observed in the murine, human and porcine ML cDNA occur at exactly the same position in the predicted proteins.

Example 14

CMK assay of thrombopoietin (TPO) induction of platelet antigen expression

GPIhJHa

CMK cells are maintained in RMPI medium (Sigma) supplemented with 10% fetal bovine serum and 10mM glutamine. In preparation for the assay, cells are harvested, washed and resuspended at 5x10 ⁵ cells / ml serum-free GIF medium supplemented with 5 mg / L bovine insulin, 10 mg / L apo-transferrin, 1 X trace elements. The TPO standard or experimental samples are added to 96 wells of the plate, each well with the appropriate solvent (100 μΐ volumes). Add 100 μΐ of cell suspension to each well and incubate the plates at 37 ° C, in a 5% CO ₂ incubator for 48 hours. After incubation, the plates are centrifuged at 1000 rpm at 4 ° C for 5 minutes. The supernatants are separated and 100 μΐ of FITC-conjugated GPIIi, III _and monoclonal 2D2 antibody is added to each well. Following incubation at 4 ° C for 1 hour, the plates are again centrifuged at 1000 rpm for 5 minutes. Supernatants containing unbound antibody are separated by a

200 μΐ 0.1% BSA-PBS is added to each well. The washing step with 0.1% BSA-PBS was repeated three times. Cells were then analyzed in FASCAN using a single parameter of the standard by measuring the relative fluorescence intensity.

Example 15

Thrombopoietin (TPO) DAMI assay by measuring the endomitotic activity of DAMI cells in microtiter plate wells

DAMI cells were maintained in IMDM + 10% horse serum (Gibco) supplemented with 10 mM glutamine, 100 ng / ml Penicillin G and 50 µg / ml streptomycin. In preparation for the experiment, cells were harvested, washed and resuspended at 1x10 ⁶ cells / ml in IMDM + 1% horse serum. To 96 wells of the microtiter plate, 100 μΐ of the TPO standard or experimental samples was added to the DAMI cell suspension. The cells were then incubated for 48 hours at 37 ° C in a 5% CO ₂ incubator. After incubation, the plates

- 135 -

were centrifuged in a Sorvall 6000B centrifuge at 1000 ipm for 5 minutes at 4 ° C. The supernatants were separated and the 200 μΐ PBS-0.1% BSA wash step was repeated. Cells were fixed by adding 200 µL of ice-cold 70% ethanol-PBS and resuspended by aspiration. After incubation at 4 ° C for 15 minutes, the plates are centrifuged at 2000 rpm for 5 minutes and 150 µl of 1 mg / ml RNAse containing 0.1 mg / ml propidium iodide and 0.05 Tween-20 are added to each well. After one hour incubation at 37 ° C, changes in DNA content were measured by flow cytometry. Polyploidy was measured and quantified as shown:

(% cells in> G2 + M /% cells in <G2 + M) with TPO

Norm. polyploid. ratio = ------------------------------------------------ ------------ (NPR) (% cells in> G2 + M /% cells in <G2 + M) in control

Example 16

Thrombopoietin (TPO) in vivo test (Spontaneous mouse platelet test)

In vivo ³⁵ S assay to determine platelet production

C57BL6 mice (obtained from Charles River) are injected intraperitoneally (IP) with 1 ml of goat anti-mouse platelet serum (6 amps) on day 1 to induce thrombocytopenia. On days 5 and 6, mice were given two IP injections of factor or PBS as a control. On day 7, thirty pCi Na 2 ³⁵ SO 4 is injected intravenously in 0.1 ml saline and the percentage of ³⁵ S incorporation of the injected dose in circulating platelets is measured in blood samples obtained from treated and control mice. Platelet and leukocyte counting is performed at the same time from blood obtained from the retro-orbital (orbital) cavity.

Example 17

KIRA ELISA for thrombopoietin (TPO) by measuring the phosphorylation of the mpl- Rse.gD chimeric receptor

The human mpl receptor has been described by Vigon et al., PNAS, USA 89: 5640-5644 (1992). A chimeric receptor comprising the extracellular domain (ECD) of the mpl receptor and the transmembrane (TM) and intracellular domain (ICD) of Rse (Mark et al., J. of Biol.

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Chem. 269 (14): 10720-10728 [1994]) with a carboxy-terminal polypeptide (i.e. Rse.gD) was created for use in the KIRA ELISA described herein, see Figures 30 and 31 for a schematic description of the experiment.

(a) Preparation of the capture reagent

A monoclonal anti-gD (clone 5B6) was prepared against a peptide from the Herpes simplex virus glycoprotein D (Paborsky et al., Protein Engineering 3 (6): 547-553 [1990]). The purified product was treated with 3.0 mg / ml phosphate buffered saline (PBS), pH 7.4 and 1.0 ml samples were stored at -20 ° C.

(b) Preparation of an anti-phosphotyrosine antibody

Monoclonal anti-phosphotyrosine, 4G10 clone, was obtained from UBI (Lake Placid, NY) and biotinylated using long-arm biotin-N-hydroxysuccinamide (Biotin-XNHS, Research Organics, Cleveland, OH).

(c) The mpl ligand ligand was prepared by the recombinant techniques described herein. The purified mpl ligand was stored at 4 ° C as a solution of microorganisms.

(d) Preparation of Rse.gD nucleic acid

Synthetic double-stranded oligonucleotides were used to reconstitute the coding sequence for the C-terminal of 10 amino acids (880-890) of human Rse and to enlarge to another 21 amino acids containing the 5B6 antibody epitope and the stop codon. Table 10 shows the final sequence of the synthetic portion of the synthesized gene.

Table 10

A synthetic double-stranded portion of the human Rse synthetic gene

coding thread: 5 'TGCAGCAAGGGCTACTGCCACACTCGAGCTGCGCAGATGCTAGCCTCAAGA TGGCTG ATCCAAATCGATTCCGCGGCAAAGATCTTCCGGTCCTGTAGAAGCT-3 ' (SEQ ID NO: 59) non-coding (anti-sensory) fiber: Sichuan AGCTTČTAČAGGACCGGAAGATCTTTGCCGCGGAATČGÁTŤŤGGATCAGCCA TCTTG AGGCTAGCATCTGCGCAGCTCGAGTGTGGCAGTAGCCCTTGCTGCA-3 ' (SEQ ID NO: 60)

Synthetic DNA was ligated with cDNA encoding amino acids 1-880 of human

Rse at the PstI position beginning at nucleotide 2644 of the published human Rse cDNA

137 sequences (Mark et al., Joumal of Biological Chemistry 269 (14): 10720-10728 [1994]) and HindIII positions in the polylinker of the expression vector pSVI7.ID.LL (see.

Fig. 32 A-L; SEQ ID NO: 22) to generate the expression plasmid pSV.ID.Rse.gD. Briefly, an expression plasmid comprises a primary dicistron transcript that comprises a DHFR coding sequence linked to a 5 donor region and an 3 acceptor region of intron positions, followed by a sequence that encodes Rse.gD. The full length (uncut) message contains DHFR as the first open reading frame and thus generates a DHFR protein to allow for the selection of stable transformants.

(e) Preparation of mpl- Rse.gD nucleic acid

The expression plasmid pSV.ID.Rse.gD, produced as described above, was modified to prepare plasmid pSV.ID.M.tmRdo, which contained the ECD coding sequences of human mpl (amino acids 1-491) linked to the transmembrane domain and the intracellular domain. Rse.gD (amino acids 429-911). Synthetic oligouucleotides were used to link the coding sequence of a portion of the extracellular domain of human mpl to a portion of the Rse coding sequence in two steps of the PCR cloning reaction as described by Mark et al., J. Biol. Chem. 267: 26166-26171 (1992). The primers used for the first PCR reaction were Boh M1 (5-TCTCGCTACCGTTTACAC-3) (SEQ ID NO: 61) and M2 (5-CAGGTACCCACCAGGCGGTCTCGGT-3) (SEQ ID NO: 62) with mpl cDNA template and R1 (5-GGGCCCCATGAC) SEQ ID NO: 63) and R2 (5-GACCGCCACCGAGACCGCCTGGTGGGTACCTGTGGTCCTT-3) (SEQ ID NO: 64) with the Rse cDNA template. The pull-Smal portion of that fusion junction was used to construct a full-length chimeric receptor.

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(f) Cell transformation

DP12.CH0 cells (EP 307,247, published March 15, 1989) were electroporated with pSV.1D.M.tmRd6, which was linearized at a particular NotI position in the plasmid backbone. DNA was ethanol precipitated after phenol / chloroform extraction and resuspended in 20 μΐ of 1/10 tris EDTA. Then, 10 µg of DNA was incubated with 10 ⁷ CHO DP12 cells in 1 ml PBS on ice for 10 minutes, before electroporation at 400 volts and 330 µL. The cells were re-placed on ice for 10 minutes before being transferred (inoculated) to a non-selective the media. After 24 hours, cells were supplied with nucleoside-free medium for selection of stable DHFR + clones.

(g) Selection of transformed cells for use in KIRA ELISA

Expression ΛΥΡΛ / RSE.gD clones were identified by western blotting of whole post-fractionating cell lysates by SDSPAGE, using a 5B6 antibody that detects the gD lobe of the epitope.

(h) Medium

Cells were grown at F12 / DMEM 50:50 (Gibco / BRL, Life Technologies, Grand Island, NY). Medium was supplemented with 10% diafiltered FBS (HyClone, Logan, Utah), 25 mM HEPES, and 2 mM L-glutamine.

(i) KIRA ELISA

Λ / ρΖ-Rse.gD transformed DP12.CHO cells were seeded (3x10 ⁴ per well) into 96-well microtiter plate wells in 100 μΐ medium and cultured overnight at 37 ° C in 5% CO 2. The following morning, the supernatants were decanted and the plates lightly pressed onto a paper towel. 50 μΐ of medium containing either experimental samples or 200, 50, 12.5, 3.12, 0.78, 0.19, 0.048 or 0 ng / ml mpl Uganda was then added to each well. The cells were stimulated at 37 ° C for 30 minutes, the supernatants were decanted, and the plates were gently pressed again onto a paper towel. For cell lysis and chimeric receptor solubilization, 100 µl of lysed buffer was added to each well. The lysis buffer included 150 mM NaCl containing 50 mM HEPES (Gibco), 0.5% Triton-X 100 (Gibco), 0.01% timerosal, 30 KUJ / ml aprotinin (ICN Biochemicals, Aurora, OH), 1 mM 4- (2-aminoethyl) -benzenesulfonyl hydrochloride (AEBSF; ICN Biochemicals), 50 μΜ of leupeptin (ICN Biochemicals) and 2 mM sodium orthovanadate (Na ₃ VO ₄ ; Sigma Chemical Co., St. Louis, MO), pH 7.5. The plate was then gently shaken to

139 plate shaker (Bellco Instruments, Vineland, NJ) for 60 minutes at room temperature.

While the cells were solubilized, an ELISA microtiter plate (Nunc Maxisorp, Inter Med, Denmark) coated overnight at 4 ° C with 5B6 monoclonal anti-gD antibody (5.0 µg / ml in 50 mM carbonate buffer, pH 9, 6, 100 μΐ / well) was decanted, pressed onto a paper towel and blocked with 150 μΐ / well Block Buffer [PBS containing 0.5% BSA (Intergen Company, Purchase, NY) and

0.01% thiomersal] for 60 minutes at room temperature with gentle shaking. After 60 minutes, the anti-gD 5B6-coated plate was washed 6 times with buffer (PBS containing 0.05% Tween-20 and 0.01% thiomersal) using an automated plate washer (ScanWasher 300, Skatron Instruments, Inc., Sterling, VA).

The lysate containing solubilized MPL / Rse.gD from the cell isulture of the microtiter well was transferred (85 μΐ / well) to wells coated with anti-gD 5B6 and blocked by ELISA and incubated for 2 hours at room temperature with gentle shaking. Unbound wp / -Rse.gD was removed by washing with wash buffer and 100 µL biotinylated 4G10 (anti-phosphotyrosine) diluted 1: 18000 with dilution buffer (PBS containing 0.5% BSA, 0.05% Tween-20). 5 mM EDTA and 0.01% thiomersal), i. 56 ng / ml was added to each well. After incubation for 2 hours at room temperature, the plate was washed and 100 µL horseradish peroxidase (HRPO) -conjugated streptavidin (Zymed Laboratories, San Francisco, CA), diluted 1: 60000 in dilution buffer, was added to each well. The plate was incubated for 30 minutes at room temperature with gentle shaking. The avidin-free conjugate was removed by washing and 100 μΐ of freshly prepared substrate solution (tetramethylbenzidine [TMBJ; a two-component substrate substrate; Kirkegaard and Perry, Gaithersburg, MD) was added to each well. The reaction was allowed to run for 10 minutes, after which the color development was stopped by adding 100 μΐ / well of 1.0 M H ₃ PO ₄ . Absorbance at 450 nm was read at a 650 nm reference wavelength (ABS45o / 65o) using a vmax reader (Molecular Devices, Palo

Alto, CA), managed with Macintosh Centris 650 (Apple Computers, Cupertino, CA) and

DeltaSoft software (BioMetallics, Inc., Princeton, NJ).

A calibration curve was constructed 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 shown as ng / ml TPO versus ABS450 / 650 ± sd using the DeltaSoft program. concentrations

140 samples were obtained by interpolating their absorbance with a calibration curve and are expressed in ng / ml TPO activity.

An mpl ligand capable of activating the β / β-Rse.gD chimeric receptor was found depending on the concentration and specific type of ligand. Further, mp / -Rse.gD KIRA-ELISA was found, resistant to up to 100% human serum (shown) or 100% plasma (not shown), allowing the use of an experiment to screen patient and pK samples.

Overview TPOEC50

TPO form, (cells) EC50 (W. / V). EC50 (Molarity) Hu TPO 332 2.56 ng / ml 67.4 pM Mu TPO 332 3.69 ng / ml 97.1 pM Hu TPO 153 (294) -41 ng / ml -1.08 nM Hu TPO 155 (E co) 0.44 ng / ml 11.6 pM Hu TPO 153met (E coli) 0.829 ng / ml 21.8 pM

141 Example 18

ELISA-based thrombopoietin (TPO) receptor

ELISA plates were coated with rabbit F (ab) ₂ anti-human IgG (Fc) in carbonate buffer, pH 9.6 at 4 ° C, overnight. Plates were blocked with 0.5% bovine serum albumin in PBS at room temperature for 1 hour. The fermentation harvest containing the chimeric receptor, mpl-IgG, was added to the plates and incubated for 2 hours. Double dilution (0.39-25 ng / rnl) of the standard (TPO332 produced in 293 cells at a concentration determined by Irvantitative amino acid analysis) and subsequently diluted samples in 0.5% bovine serum albumin, 0.05% Tween 20 were added to the plates and incubated for 2 hours. Bound TPO was detected with Protein A, purified biotinylated rabbit antibodies TPO155, which was highly produced in E. coli (one hour incubation), followed by streptavidin-peroxidase (30-minute incubation) and 3,3, 5,5-tetramethylbenzidine as substrate. Absorbance was read at 450 nm. Plates were washed between steps. For data analysis, a calibration curve is constructed using a four-parameter curve generated by the Kaleidagraph program. The sample concentrations were calculated from the calibration curve.

Example 19

Expression and purification of TPO from 293 cells

1. Preparation of expression vectors from 293 cDNA cells corresponding to the entire open reading frame of TPO was obtained by PCR using the following oligonucleotides as primers:

Table 11

293 PCR primers

Cla.FL.F; 5 'ATC GAT ATC GAT CAG (SEQ ID NO. 65) hmpll-R: 5 'GCT AGC TCT AGA CAG (SEQ ID NO: 48)

PRK5-hwp / I (described in Example 9) was used as a template for the reaction in the presence of pfu DNA polymerase (Stratagene). The initial denaturation was during

- 142 -

minutes at 94 ° C, followed by 25 cycles of amplification (1 minute at 94 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C). The final elongation was 15 minutes at 72 ° C). The PCR product was purified and cloned between the ClaI and XbaI restriction sites of plasmid pRK5tkneo, the pRK5 derived vector was modified to express a neomycin-resistant gene, while regulating thymidine kinase, to obtain the vector pRK5tkneo.ORF. The second construct, corresponding to the epo homology domain, was generated in the same manner, but using Cla.FL.F as the forward primer and the following reverse (reverse) primer:

TCT AGA TCT AGA TCA CCT GAC GCA GAG GGT GGA CC 3 (SEQ ID NO: 66)

The final construct is called pRK5-tkneoEPO-D. The sequence of both constructs was verified as described in Example 7.

2. Transfection of human embryonic kidney cells

The two constructs were infected with human embryonic kidney cells by the CaPO 4 method as described in Example 9. 24 hours after transfection, selection of neomycin-resistant clones was initiated in the presence of 0.4 mg / ml G418. 10-15 days later, individual colonies were transferred to 96 wells and allowed to grow in parallel. Expression of ML153 or ML332 in conditioned media from these clones was determined using the B and -3-mpl proliferation assay (described in Example 1).

3. Purification of rhML132

293-rhML ₃₃ 2 conditioned medium was applied to a Blue-Sepharose (Pharmacia) column which was equilibrated in 10 mM sodium phosphate pH 7.4 (Buffer A). The column was additionally washed with 10 volumes (based on column) of Buffer A and Buffer A containing 2 M urea. The column was then eluted with buffer A containing 2 M urea and 1 M NaCl. The Blue-Sepharose elution bath was then directly applied to a WGA-Sepharose column equilibrated with Buffer A. The WGA-Sepharose column was then washed with 10 column volumes of Buffer A containing 2 M urea and 1 M NaCl and eluted with the same buffer containing 0.5 M N-acetyl-D-glucosamine. The WGA-Sepharose eluate was applied to a C4-HPLC column (Synchrom, Inc.) equilibrated in 0.1% TFA. The C4-HPLC column was eluted discontinuously

143 propanol gradient (0-25%, 25-35%, 35-70%). rhML ₃₃ 2 was selected to elute in the 28-30% propanol region of the gradient. By SDS-PAGE, purified rhML ₃₃₂ migrates as a broad band in the 68-80 kDa region of the gel (see Figure 15).

4. Purification of rhMLus

293-rhMLi ₅₃ conditioned medium was digested on Blue-Sepharose as described for rhML ₃₃₂ . The Blue-Sepharose eluate was directly applied to the wpZ-affinity column as described above. RhMLi5 ₃ // ^ eluted from the affinity column-purified to homogeneity is the use of a C4-HPLC column and run under the same conditions as described for rhML _332nd SDS-PAGE of purified rhMLi ₅₃ is decomposed into two larger and two smaller bands with Mr ~ 18,000 - 21,000 (see Figure 15).

Example 20

Expression and purification of TPO from CHO

I. Description of CHO expression vectors

Expression vectors, used in the electroporation protocols described below, are termed god:

pSVI5.ID.LL.MLORF (full length or hTPO ₃₃₂ ) and pSVI5.ĽD.LL.MLEPO-D (truncated or hTPO ₁₅₃ ).

The features relating to these plasmids are presented in FIG. 23 and 24.

2. Preparation of cDNA expression vectors corresponding to the entire TPO open reading frame was obtained by PCR using the oligonucleotide primers of Table 12.

Table 12

PCR primers of 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. Sal 5 'AGT CGA CGT CGA CGT CAG TGT CTG AGA ACC 3' (SEQ ID NO: 6.7)

144 -

PRK5-h / wpZ I (described in Examples 7 and 9) was used as a template for the reaction in the presence of DNA polymerase (Stratagene). Initial denaturation was for 7 minutes at 94 ° C, followed by 25 cycles of amplification (1 minute at 94 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C). The final elongation was 15 minutes at 72 ° C). The PCR product was purified and cloned between the ClaI and SalI restriction positions of plasmid pSVI5.ID.LL, yielding the vector pSVI5.1D.LL.MLORF. The second construct, corresponding to the epo homology domain, was generated in the same way, but using Cla.FL.F2 as the forward primer and the following reverse (reverse) primer:

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 pSVI5.1D.LL.MLEPO-D. The sequence of both constructs was verified as described in Examples 7 and 9.

Essentially, the coding sequences of the full length and truncated ligand were introduced 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 comprising a mouse DHFR cDNA, a multiple cloning site to introduce the gene of interest (TPO sequences described in this case) of the SV40 polyadenylation and origin of replication signal, and the beta-lactamase gene for plasmid selection and amplification. bacteria.

3. Methodology for the introduction of stable CHO cell lines of expression of recombinant TPO332 and TPO 153

a. Description of the parent CHO cell line

The host CHO (Chinese Hamster Ovary) cell line used to express the TPO molecules described herein is known as CHO-DP12 (see EP 307,247, published March 15, 1989). This mammalian cell line was cloned selected from an infected parental line (CHO-K1 DUX-B11 (DHFR -) - obtained from Dr. Frank Lee of Stanford University with the permission of Dr. L. Chasin) with a preproinsulin expression vector to obtain clones with reduced insulin requirements. These cells are also DHFR minus and clones can be selected for the presence of the DHFR cDNA sequence of the vector by growth on a medium depleted of nucleoside supplements (glycine, hypoxanthine and

- 145 thymidine). This selection system for stable expression of CHO cell lines is commercially used.

b. Transfection method (electroporation)

TPO332 and TPO153 expressed cell lines were generated by transfecting DP12 cells by electroporation (see, e.g., Andreason, GL, J. Tiss. Cult. Meth., 15.56 [1993]) with the linearized plasmid pSVI5.ID.LL.MLORF or pSVI5.ID .LL.MLEPO-D. Three (3) restriction enzyme reaction mixtures were prepared for each plasmid shear; 10 pg, 25pg and 50 pg of vector with NÔTI enzyme by standard molecular biology methods. This restriction site is only found once in the vector within the linearized 3 region and outside the TPO ligand transcription units (see Fig. 23). 100 µl reactions were allowed to incubate overnight at 37 ° C. The following day, the mixtures were extracted once with phenol-chloroform-isoamyl alcohol (50: 49: 1) and precipitated with ethanol on dry ice for about one hour. The precipitate was collected by microcentrifugation for 15 minutes and dried. Linearized DNA was resuspended in 50 µl of 1: 1 Hams DMEM-F12 medium supplemented with standard antibiotics and 2 mM glutamine.

The growing DP12 cell suspension was separated, washed once with the medium described for DNA resuspension, and finally resuspended in the same medium at 10 ⁷ cells per 750 µl. Equal portions of cells (750 µl) and each of the linearized DNAs were incubated together at room temperature for one hour and then transferred to a BRL electroporation chamber. Each reaction mixture was then electroporated in a standard BRL electroporation apparatus at 350 volts 330 pF and low capacitance. After electroporation, the cells were allowed to stand in the instrument for 5 minutes and then on ice for an additional 10-minute incubation period. Electroporated cells were transferred to 60mm cell culture flasks containing 5 ml of standard, complete growth medium for CHO cells (High glucose DMEM-F12 50:50 without glycine supplement with 1X GHT, 2 mM glutamine and 5% fetal calf serum) and

They were grown overnight in a 5% CO 2 cell culture incubator.

c. Method of selection and screening

On the next day, cells were trypsin-free by standard methods and transferred to 150 mm tissue culture flasks containing DHFR selective medium (Hams &apos;

- 146 -

DMEM-F12, 1: 1 medium described above, supplemented with either 2% or 5% dialyzed fetal calf serum but devoid of glycine, hypoxanthine and thymidine, is a standard DHFR selection medium used herein). Cells from each 60mm vial were then transferred to five 150mm vials. Cells were then incubated for 10 to 15 days (with one medium change) at 37 ° C / 5% CO 2 until clones were formed and sized to allow transfer to 96-well microtiter flames. Four to five days later, cell lines were transferred to 96 wells using sterile yellow tips of a 50 ml micropipetting kit. Cells were allowed to grow concurrently (usually 3-5 days) and then the plates were trypsinized and two copies of the original pad were reproduced. Two of these copies were briefly stored in a cell freezer in each well diluted with 50 μΐ 10% FCS in DMSO. Samples of 5 days treated serum-free media were tested from parallel wells in a third substrate for TPO expression by Ba / F baseline cell activity assay. The highest expressed clones based on this assay were recovered from stock and stored in 2 parallel 150mm T-flasks to transfer culture group. cells for suspension adaptation, retest and storage.

d. Amplification protocol

Some of the highest titer cell lines from the selection described above were subsequently subjected to a standard methotrexate amplification regime to generate higher titer clones. CHO cell clones were propagated and seeded in 10 cm dishes at 4 concentrations of methotrexate (i.e., 50 nM, 100 nM, 200 nM and 400 nM) and two or three cell counts (105, 5x10 5 and 106 cells per plate). These cultures were then incubated at 37 degrees / 5% CO 2 until the clones developed and were capable of transferring to 96 well microtiter plates for the next assay. Several high titer clones from this selection were re-exposed to higher concentrations of methotrexate (i.e. 600 nM, 800 nM, 1000 nM and 1200 nM) and, as before, resistant clones were allowed to grow and then transferred to 96 well microtiter wells. and tested.

4. Cultivation of stable CHO cell lines expressing recombinant

TPO332 and TPO1S3

The stored cells were thawed and the population of cells multiplied by standard cell growth methods, either in serum-free or serum-containing medium. After

147 multiplication to a sufficient cell density, the cells were washed to remove residual culture medium. The cells were then cultured by any standard method, including: loading, supplementing or continuously dosing the culture at 25-40 ° C, neutral pH, with an O2 removal of at least 5%, until a substantial pool of secreted TPO was collected. The cell culture fluid is then separated from the cells by mechanical means such as centrifugation.

Purification of recombinant human TPO from CHO culture fluids

The harvested liquid cell culture (HCCF) is directly applied to a Blue Sepharose 6 Fast Flow column (Pharmacia) equilibrated in 0.01M Na phosphate, pH 7.4, 0.15M NaCl at a ratio of about 1001 HCCF per liter resin and at a linear flow rate of about 300 ml / h / cm ² . The column is then washed with 3 to 5 times volumes of equilibration buffer, followed by a 3 to 5 times volume wash (relative to column volume) of 0.0 M Na phosphate pH 7.4, 2.0 M urea. TPO is then eluted with three to five times column volumes of 0.0M Na phosphate pH 7.4, 2.0M urea, 1.0M NaCl.

The Blue Sepharose Pool containing TPO is then applied to a Wheat Germ Lecithin Sepharose 6MB column (Pharmacia), equilibrated in 0.01M Phosphate pH 7.4, 2.0M urea and 1.0M NaCl at a ratio of 8 to 16 ml Blue Sepharose Pool per ml and a flow rate of approximately 50 ml / h / cm. The column is then washed with two to three column volumes of equilibrated buffer. The TPO is then eluted with two to five column volumes of 0.0 M Na phosphate pH 7.4, 2.0 M urea, 0.5 M N-acetylghicosamine.

The Wheat Germ Lecithin Pool is then adjusted to a final concentration of 0.04% C ₁₂ Eg and 0.1% trifluoroacetic acid (TFA). The resulting pool (amount) is applied to a C4 reverse phase column (Vydac 214TP1022), equilibrated in 0.1% TFA, 0.04% Ci ₂ Eg and at a load of approximately 0.2 to 0.5 mg protein per ml resin at a flow rate of 157 ml / h / cm ² .

The protein is eluted with a two-phase linear gradient of acetonitrile containing

0.1% TFA, 0.04% CnEg. The first phase is a linear gradient from 0 to 30% acetonitrile over 15 minutes, the second phase is a linear gradient from 30 to 60% acetonitrile over 60 minutes. TPO elutes at about 50% acetonitrile. The fund is made on SDS-PAGE basis.

148 C4 flask is then diluted with two volumes of 0.0IM Na phosphate pH 7.4, 0.15M NaCl and diafiltered against approximately 6 volumes 0.0M Na phosphate pH 7.4, 0.15M NaCl on Amicon YM or a similar ultrafiltration membrane having a cut-off molecular weight of 10,000 up to 30,000 Daltons. The resulting diafiltrate may be directly processed or further concentrated by ultrafiltration. The diafiltrate / concentrate is adjusted to a final concentration using 0.01% Tween-80.

All or part of the diafiltrate / concentrate, equivalent to 2-5% of the calculated column volume, is then applied to a Sephacryl S-300 HR column (Pharmacia) equilibrated in 0.01M Na phosphate pH 7.4, 0.15M NaCl, 0.01% Tween-80 and chromatographed at a flow rate of about 17 ml / h / cm ² . Fractions containing aggregate-free TPO and proteolytic degradation products were pooled based on SDS-PAGE. Filter the resulting flask with a 0.22μ filter, Millex-GV or the like, and store at 2-8 ° C.

Example 21

Transformation and induction of TPO protein synthesis in E. coli

Construction of E. coli TPO expression vectors

The plasmids pMP21, pMP151, pMP41, pMP57 and pMP202 are all engineered to express the first 155 amino acids of TPO downstream of the short structural genes,

that change between different structures. In particular, structural genes provide initiation for a high level of translation and rapid purification. The plasmids pMP210-1, -T8, -21, -24, -25 are designed to express the first 153 amino acids of TPO in the direction of methionine initiation and differ only in the codon used for the first 6 amino acids of TPO, while plasmid pMP251 is derived from pMP210- 1, wherein the carboxyterminal terminus of TPO is extended by two amino acids. All of the above plasmids will produce high levels of intracellular expression of TPO in E. coli by inducing a tryptophan promoter (Yansura, DG et al. Methods in Enzymology (Goeddel, DV, Ed.) 185: 54-60, Academic Press, San Diego [1990] ]). Plasmids pMP1 and pMP172 are intermediates in the construction of the above TPO intracellular expression plasmids.

149 - (a) Plasmid pMP1

Plasmid pMP1 is the secretion vector for the first 155 amino acids of TPO and was constructed by ligating 5 DNA fragments as shown in FIG. The first was the vector pPho21, in which the short MluI-BamHI fragment was removed. pPho21 is a derivative of phGH1 (Chang, CN et al., Gene 55: 189-196 [1987]) in which the human growth hormone gene has been replaced with the E. coli phoA gene and the MluI restriction site has been incorporated into the coding sequence for the STU signal sequence. amino acids 20-21.

The two following fragments, the 258 base pair Hinf1-Pst1 portion of pRK5 'mpl I (Example 9), encoding TPO amino acids 19-103 and the following synthetic DNA encoding amino acids 1-18 • 5-CGCGTATGCCAGCCCGGCTCCTCCTGCTTGTGACCTGGTTCCTCTCAGT AAACT

ATACGGTCGGGCCGAGGAGGACGAACACTGGAGGCTCAGGAGTCA TTTGACGAAGCACTGA-5 (SEQ ID NO: 69) (SEQ ID NO: 70)

god was pre-ligated with T4-DNA ligase, and the other cut with Psd. The fourth was a PstI-HaelU fragment of pRK5hmpU with 152 base pairs, encoding TPO amino acids 104-155. The last was a 412 base pair Stul-BamHI fragment from pgh108 containing the lambda transcriptional terminator as previously reported (Scholtissek, S. et. Al., NAR15: 3185 [1987]).

(b) Plasmid pMP21

Plasmid pMP21 is designed to express the first 155 amino acids of TPO using 13 amino acid structural fragments containing part of the STU signal sequence. It was constructed by ligating three (3) DNA fragments as shown in Fig. 34, the first being the pVEG31 vector, in which the short XbalSph1 fragment was removed. The vector pVEG31 is derived from pHGH207-1 (de Boer, H. A. et. Al., In Promoter Structure and Function (Rodriguez, R. L. and Chamberlain, M. J., Ed.), 462, Praeger,

150 New York [1982]), in which the human growth hormone gene has been replaced by the vascular endothelial growth factor gene (this identically vector fragment can be obtained from this last plasmid).

The second part of hgace was a duplex of synthetic DNA with the following sequence:

5-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAA

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

The last part was the MluI-SphI fragment of p721 with 1072 base pairs, encoding 155 amino acids of TPO.

(C) Plasmid pMP151

Plasmid pMP151 is designed to express the first 155 amino acids of TPO downstream of a structural fragment comprising 7 amino acids of the STU signal sequence, 8 histidines, and a Factor Xa cleavage site. As shown in Fig. 35, pMP151 was made by ligating three DNA fragments, the first of which is the previously described pVEG31 vector from which the short XbaI-SphI fragment was removed. The second was a synthetic DNA duplex with the following sequence:

-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCA TCACATCGAAGGTCGTAGCC (D TTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTA

GTGTAGCTTCCAGC AT-5 (SEQ ID NO: 73)

The last was the 1064 base pair BgU-Spg1 fragment of pMP11, encoding 154 amino acids of TPO. Plasmid pMP11 is identical to pMP1, except for a few codon changes in the STU signal sequence (this fragment can be obtained from pMP1).

(d) Plasmid pMP202

Plasmid pMP202 is very similar to the expression vector pMP151 except that the factor Xa cleavage site in the structural fragment has been replaced

151 - thrombin cleavage site. As seen in Fig. 36, pMP202 was made by ligating three DNA fragments. The first one was previously described pVEG31, in which the short XbaI-SphI fragment was removed. The second was a synthetic DNA duplex with the following sequence:

5-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCA

TCACATCGAACCACGTAGCC

TTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTA

GTGTAGCTTGGTGCAT-5 (SEQ ID NO: 75) (SEQ. NO. 76)

The last part was the Bgä-Sph1 fragment from the previously described plasmid pMP11 s

1064 base pairs (e) Plasmid pMP172

Plasmid pMP172 is the secretory vector for the first 153 amino acids of TPO and is an intermediate of the construction of pMP210. As seen in Fig. 37, pMP172 was prepared by ligating three DNA fragments, the first of which was the pLS321amB vector, in which a small EcoRI-HindIII fragment was removed. The second was an EcoRI-Hgal fragment from the previously described plasmid pMP11, with 946 base pairs. The last part was a synthetic DNA duplex with the following sequence:

-TCCACCCTCTGCGTCAGGT (SEQ ID NO: 77)

A GGAGACGCAGTCCATCGA-5 (SEQ ID NO: 78) (f) Plasmid pMP210

Plasmid pMP210 is designed to express the first 153 amino acids of TPO after translation initiation of methionine. This plasmid was actually made as a bank of plasmids in which the first 6 codons were randomized at the third position of each codon and were constructed as shown in FIG. 38, by ligating three DNA fragments. The first was the previously described pVEG31 vector, from which the short XbaI-SphI fragment had been removed. The second was a synthetic DNA duplex, shown below, first treated with DNA polymerase (Klenow) and then

- 152 digested with XbaI and Hinf1 and encoding the initiation of methionine and the randomized first 6 TPO codons.

-GCAGCAGTTCTAGAATTATGTCNCCNGCNCCNCCNGCNTGTGACCTCC

GAACACTGGAGGCTGTTCTCAGTAAA (SEQ ID NO: 79)

CAAGAGTCATTTGACGAAGCACTGAGGGTACAGGAAG-5 '(SEQIDNO: 80)

The third was the 890 base pair Hinf1-Sph1 fragment from pMP172, encoding TPO amino acids 19-153.

Plasmid flask pMP210 with approximately 3700 clones was re-transferred to LB plasma with tetracycline (50 gg / ml) for selection of high translation initiation clones (Yansura, DG et al., Methods: A Companion to Methods in Enzymology 4: 151-158 [1992]). Of the 8 colonies grown on tetracycline plates, five of the best TPO expression conditions were subjected to DNA sequence and the results are shown in Figure 39 (SEQ ID NOS: 23, 24, 25, 26, 27 and 28).

(g) Plasmid pMP41

Plasmid pMP41 is designed to express the first 155 amino acids of TPO fused to a structural fragment consisting of the 7 amino acids of the STU signal sequence, followed by a Factor Xa cleavage site. The plasmid was constructed, as shown in FIG. 40, by ligation of three portions of DNA, the first of which was the pVEG31 vector from which the short XbaI-SphI fragment was removed. The second was the following synthetic DNA duplex: 5-CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (SEQ ID NO: 81) TTAATACTTTTTCTTATAGCGTAAATAGCTTCCAGCAT-5 (SEQ ID NO: 82)

The last part of the ligation was the Bgl1-SphI fragment from the 1064 base pair pMP11 plasmid described above.

(h) Plasmid pMP57

Plasmid pMP57 expresses the first 155 amino acids of TPO downstream of a structural fragment consisting of the 9 amino acids of the STU signal sequence and dibasic.

153 place Lys-Arg. This dibasic site provides a means for removing a structural fragment with an ArgC protease. The plasmid was made, as shown in Figure 41, by ligation of three portions of DNA. The first one was the pVEG31 vector from which the short XbaI-SphI fragment was removed. The second was the following synthetic DNA duplex: 5-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAAACGTAGCC (SEQIDNO: 83) TTAATACTTTTTCTTATAGCGTAAAGAAGAATTTGCAT-5 (SEQIDNO: 84)

The last part of the ligation was the Bgl1-SphI fragment from the 1064 base pair pMP11 plasmid described above.

(i) Plasmid pMP251

Plasmid pMP251 is derived from pMP210-1 in which two additional TPO amino acids are inserted at the carboxyterminal end. As shown in Fig. 42, this plasmid was made by continuous ligation of two portions of DNA, the first of which is already described pMP21, from which the short XbaI-ApaI fragment was removed. The second part of the ligation was the XbaI-ApaI fragment of pMP210-1 with 316 base pairs.

2. Transformation and induction of E. coli with TPO expression vectors

The above TPO expression plasmids were used to transform E. coli strain 44C6 (W3110 _ίοηΑ Δ ιροΗ ^ Ιοπδ clpP _and galE) using the CaCl2 with hot shock method (Mandel, M. et. Al., J. Mol. Biol., 53: 159-162 [1970]). The transformed cells were first propagated at 37 ° C on LB medium containing 50 µg / ml carbenicillin up to the optical density (600 nm) of the culture reached about 2-3. The LB culture was then diluted 20x with M9 medium containing 0.49% casaminic acid (w / v) and 50 µg / ml carbenicillin. After growth with aeration at 30 ° C for 1 hour, indole-3-acrylic acid was added up to a final concentration of 50 µg / ml. The culture was then allowed to grow at 30 ° C with aeration for an additional 15 hours and then cells were harvested by centrifugation.

Example 154

Production of biologically active TPO (Me1-153) in E. coli

The procedures described below for the production of biologically active, reloaded TPO (m ^. 11-153) can be applied analogously to obtain other variants of TPO, including N and C terminal extension forms (see Example 23).

A. Obtaining insoluble TPO (Me 1-153)

E. coli cells expressing TPO (Me ¹ 1-153) encoded by the pMP210-1 plasmid are fermented as above. Typically, approximately 100 g of cells were resuspended in 1 L (10 volumes) of cell disruption buffer (10 mM Tris, 5 mM EDTA, pH 8) with a Polytron homogenizer, and the cells were centrifuged at 5000 x g for 30 minutes. The washed cell sediment was resuspended in 1 µl cell disruption buffer with a Polytron homogenizer and the cell suspension was transferred to an LH Cell Disrupter (LH Inceltech, Inc.) or to a Microfluidizer (Microfluidics Intemational) according to the manufacturing instructions. The suspension is centrifuged at 5000g for 30 minutes and resuspended and centrifuged a second time to obtain a washed fragile sediment. The washed sediment is used immediately or stored at -70 ° C.

B. Solubilization and purification of monomeric TPO (Me 1-153)

The above sediment is resuspended in 5 volumes of 20 mM Tris, pH 8 with 6-8 M guanidine and 25 mM DTT (dithiothreitol) and stirred for 1-3 hours or overnight at 4 ° C to solubilize the TPO protein. High concentrations of urea (6-8M) are also useful, but generally result in lower yields compared to guanidine. After solubilization, the solution is centrifuged at 30,000 x g for 30 minutes to give a clear supernatant containing denatured monomeric TPO protein. The supernatant is then chromatographed on Superdex 200 by column gel filtration (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, with 10 mM DTT. Fractions containing monomeric denatured TPO protein, eluting between 160 and 200 ml, were pooled. The TPO protein is further purified on a semi-preparative C4 reverse phase column (2 x 20 cm, VYDAC). The sample is applied at a rate of 5 ml / min per

- 155 column equilibrated in 0.1% TFA (trifluoroacetic acid) with 30% acetonitrile. The protein is eluted with a linear gradient of acetonitrile (30-60% over 60 minutes). The purified reduced protein elutes at about 50% acetonitrile. This material is used to interleave (multiply) to obtain a biologically active TPO variant.

C. Generation of biologically active TPO (Mef ¹ 1-153)

Approximately 20 mg of monomeric, reduced and denatured TPO protein in 40 ml of 0.1% TFA / 50% acetonitrile is diluted in 360 ml buffer containing optimally the following reagents:

mM Tris

0.3M NaCl mM EDTA

2% Understand With Detergent

25% glycerol mM oxidized glutathione mM reduced glutathione pH adjusted to 8

After mixing, the reloading buffer was stirred gently at 4 ° C for 12-48 hours to obtain the maximum reload (fold) yield of the correct disulfide form of TPO (see below). The solution is then acidified with TFA to a final concentration of 0.2%, filtered through a 0.45 or 0.22 micron filter and 1/10 volume of acetonitrile is added. The solution is then pumped directly onto a C4 reversed phase column and purified, the folded (expanded) TPO (Me ¹ 1-153) eluting with the same gradient program as above. The multiplied biologically active TPO elutes at approximately 45% acetonitrile according to the above conditions. Unsuitable disulfide versions of TPO elute early. The final purified TPO (Me ¹ 1-153) has a purity of greater than 95% as determined by SDS gels and C4 reverse phase analytical chromatography. For animal purposes, the C4 purified material was dialyzed in physiologically compatible buffers. Isotonic buffers (10 mM Na acetate, pH 5.5, 10 mM Na succinate, pH 5.5 or 10 mM Na phosphate, pH 7.4) containing 150 mM NaCl and 0.01% Tween 80 were used.

Given the high TPO efficacy in the Ba / F3 assay (half maximal stimulation is achieved at approximately 3 pg / ml), it is possible to obtain a biologically active material using several different buffers, detergent and redox conditions. However, under several conditions, only a small amount (<10%) of the folded material is obtained. For commercial production processes, it is desirable to achieve multiplied yields of at least 10%, more preferably 30-50%, and most preferably> 50%. A number of different detergents (Triton X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, sarcosyl, Tween 20 and Tween 80, Zwittergent 3-14 et al.) Have been found effective in achieving high multiplying yields. Of these detergents, only the CHAPS family (CHAPS and CHAPSO) were found to be generally useful in the translation reaction to limit protein aggregation and inappropriate disulfide formulations. CHAPS levels greater than 1% were more useful. For best yields sodium chloride was required, with optimum values between 0.1 M and 0.5 M. The presence of EDTA (1-5 mM) limited the amount of metal catalyzed oxidation (and aggregation) observed in some preparations. Concentrations of glycerol of greater than 15% create optimal translation conditions. For maximum yield, it was essential to have both glutathione, both oxidized and reduced, or both cysteines, both oxidized and reduced, as a redox reagent pair. In general, higher yields were observed when the molar ratio of the oxidized reagent is equal to or higher than the reduced reagent member of the redox pair. pH values between 7.5 and about 9 god optimally to multiply these TPO variants. Organic solvents (e.g., ethanol, acetonitrile, methanol) are well tolerated at concentrations of 10-15% or less. Higher levels of organic solvents increased the amount of improperly folded forms. Tris and phosphate buffers are generally applicable. Incubation at 4 ° C also produced higher levels of suitably multiplied TPO.

Multiplying yields of 40-60% (based on the amount of reduced and denatured TPO used in the translation reaction) are typical in the preparation of TPO to be purified by the first C4 step. The active material can be obtained when the preparations are less pure (e.g. directly after the Superdex 200 column or after the initial fragile body extraction), although yields are lower than the intense precipitation and interference of non-TPO proteins during the TPO reloading process.

Since TPO (Me ¹ 1-153) contains 4 cysteine residues, it is possible to generate three different disulfide versions of this protein:

- 157 version 1: disulfides between cysteine residues 1-4 and 2-3 version 2: disulfides between cysteine residues 1-2 and 3-4 version 3: disulfides between cysteine residues 1-3 and 2-4.

During the initial screening under determinative translation conditions, some different peaks (peaks) containing the TPO protein were separated by C4 reverse phase chromatography. Only one of these peaks had significant biological activity as determined using the Ba / F3 assay. Consequently, the multiplication conditions were optimized to obtain preferably that version. Under these conditions, the incorrectly translated (multiplied) versions are less than 10-20% of the total TPO monomer recovered.

The disulfide character of biologically active TPO was determined to be 1-4 and 2-3 by mass spectrometer and protein sequencing (i.e. version 1). Equal portions of the various C4-spaced peaks (5-10 nmoles) were digested with trypsin (trypsin to protein molar ratio of 1:25). The digested mixture was analyzed by laser desorption mass spectrometry using a matrix before and after reduction with DTT. After reduction, weights were detected corresponding to most of the larger tryptic TPO peptides. In the non-reduced samples some of these weights were missing and new weights were found. The weight of the new peaks essentially corresponds to the sum of the individual tryptic peptides contained in the disulfide pair. Therefore, the disulfide character of the translated, recombinant, biologically active TPO could be clearly determined as 1-4 and 2-3. This is compatible with the known disulfide character of the related erythropoietin molecule.

D. Biological activity of recombinant, translated TPO (met 1-153)

The interleaved and purified TPO (Me ¹ 1-153) is effective in both in vitro and in vivo assays. In the Ba / F3 assay, half maximal stimulation of thymidine incorporation into Ba / F3 cells was achieved at 3.3 pg / ml (0.3 pM). In the receptor-based mpl ELISA, half of the maximal efficacy occurred at 1.9 ng / ml (120 pM). In normal and myelosuppressed animals produced by near-lethal X-radiation, TPO (Met ¹ 1-153) was highly potent (activity was seen at doses below 30 ng / mouse) to stimulate the production of new blood vessels.

158 Example 23

Production of other biologically active TPO variants in E. coli

Three different TPO variants produced in E. coli, purified and translated into biologically active forms are listed below.

(1) MLF-13 residues from the bacterial derived ST11 signal sequence are linked by the N-terminal domain of TPO (residues 1-155). The resulting sequence is

MKKNIAFLLNAYASPAPPAC ...... CVRRA (SEQ ID NO: 85) wherein the structural fragment sequence is underlined and C ...... C represents Cys ⁷ to Cys ¹⁵¹ . This variant was designed to obtain tyrosine for radio-iodinating TPO for the receptor and biological studies.

(2) H8MLF-7 residues from the ST11 sequence, 8 histidine residues, and the factor Xa enzymatic cleavage of the IEGR sequence were joined to the N-terminal domain (residues 1-155) of TPO. The sequence is

MKKNIAFHHHHHHHHIEGRSPAPPAC ...... CVRRA (SEQ ID NO: 86) wherein the structural fragment sequence is underlined and C ....... C represents Cys ⁷ to Cys ^151. This variant when purified and translated (multiplied), can be treated with an enzyme factor Xa that will cleave after the arginine residue the generation of the IEGR sequence of TPO variant 155 residues in length with the natural serine N-terminal amino acid.

(3) T-H8MLF - is prepared as described above for variant (2), except that the thrombin sensitive IEPR sequence is linked to the N-terminal TPO domain. The resulting sequence is

MKKNIAFHHHHHHHHIEPRSPAPPAC ...... CVRRA (SEQ ID NO: 87) wherein the structural fragment sequence is underlined and C ... C represents Cys ⁷ to Cys ¹⁵¹ . This variant, after purification and multiplication, can be treated with a thrombin enzyme to produce a native N-terminal TPO variant with a length of 155 residues.

- 159 A. Isolation, solubilization and purification of monomeric, biologically active TPO variants (1), (2) and (3)

All of these variants were expressed in E. coli. Most of the variants were found in the form of fragile bodies, as shown in Figure 22 of TPO (Me ¹ 1-153). Identical procedures for isolation, solubilization and purification of monomeric TPO variants were performed as described in Example 22. Identical translation conditions were used as for TPO (Me ¹ 1-153) with yields exceeding 30-50%. After multiplying, the TPO variants were purified by C4 reverse phase chromatography in 0.1% TFA, using an acetonitrile gradient as previously described. All of the TPO variants (in their non-proteolyzed forms) had a biological effect, as demonstrated by the Ba / F3 assay, with half the maximum potency of 2-5 pM.

B. Proteolytic procedures of variants (2) and (3) for generating N-terminal TPO (1-155)

The TPO variants (2) and (3) above were constructed with an enzymatically cleavable peptide structural fragment in front of the normal N-terminal amino acid residue of TPO. After multiplying and purifying variants (2) and (3) as described above, each was digested with the appropriate enzyme. For each variant, acetonitrile was removed from the reverse phase step C4 by blowing a slight stream of nitrogen into the solution. Then, two variants were treated with either Factor X or thrombin as described above.

For TPO variant (2), IM Tris buffer, pH 8, was added to the acetonitrile-free solution to a final concentration of 50 mM, and the pH was adjusted, if necessary, to 8. NaCl and CaCl ₂ were added to 0.1 M resp. 2 mM. Factor Xa (New England Biolabs) was added to achieve a molar ratio of about 1:25 to 1: 100 enzyme: variant. The sample was incubated at room temperature for 1-2 hours to maximize cleavage as evidenced by a change in migration on SDS gels representing loss of leader sequence. Thereafter, the reaction mixture was purified by C4 reverse phase chromatography, using the same gradient and conditions as described above, to purify the appropriately translated variants. The uncleaved variant B was separated from the cleaved variant (2) under these conditions. N-terminal

- 160 -

amino acids appeared to be SPAPP, indicating that the removal of the N-terminal leader sequence was successful. Factor Xa also generated different amounts of internal cleavages of the TPO domain; cleavage was observed after the argmine residue at position number 118 creating an additional N-terminal sequence of TTAHKDP (SEQ ID NO: 88). One band of approximately 17000 daltons was observed on non-reducing SDS gels, for the Factor Xa cleaved variant; on reducing gels, two bands of molecular weight of approximately 12,000 and 5,000 daltons were visible, consistent with the cleavage at arginine 118. These observations also confirmed that the two parts of the molecule were held together by a disulfide bond between the first and fourth cysteine residues, as shown by tryptic experiments. of the digestion described above. In the Ba / F3 bioassay, the purified TPO (1-155) variant, after removal of the N-terminal leader sequence and with internal cleavage, had half the maximum potency of 0.2 to 0.3 picomoles. The intact variant with a leader sequence had half the maximum activity at 2-4 picomoles.

For variant (3), the digest buffer contained 50 mM Tris, pH 8, 2% CHAPS, 0.3 M NaCl, 5 mM EDTA, and human or bovine thrombin (Calbiochem) at a weight ratio of 1:25 to 1:50 based on the enzyme and TPO variant protein. Digestion was performed at room temperature for 2-6 hours. The development of digestion was assessed by SDS gels as described above for the Factor Xa cleavage reaction. In general, more than 90% of the leader sequence cleavage was achieved at this time. The resulting TPO was purified on C4 reverse phase columns as described above, and amino acid sequencing was shown to have the desired N-terminal. Only a very small amount (<5%) of internal cleavage was obtained, with the same arginine threonine bridge as found above with factor Xa. The resulting TPO protein was biologically highly active, with half maximal responses in the Ba / F3 assay at 0.2-0.4 picomoles of protein. In the ELISA-based mpl receptor, this protein had half the maximal response at 2-4 ng / ml purified protein (120-140 picomoles), while the intact variant containing the leader sequence was less efficient in both assays at 5-10 folds (multiplications) ). For animal tests, HPLC-purified cleaved protein was dialyzed in physiologically acceptable buffers, with 150 mM NaCl, 0.01% Tween 80 and 10 mM sodium succinate, pH 5.5, or 10 mM sodium acetate, pH 5.5 or 10 mM sodium phosphate, pH 4.7 The purified protein was stabilized by HPLC and SDS gels

- 161 for several weeks and then stored at 4 ° C. In normal and myelosuppressive mice, this purified TPO with an authentic N-terminal sequence was highly effective, stimulating platelet production at doses less than 30 ng / mouse.

Example 24

Synthetic mpl ligand

Although human mpl ligand (hML) is usually produced using recombinant methods, it can also be synthesized via enzymatic ligation of synthetic peptide fragments using the methods described above. Synthetic preparation of hML allows the incorporation of unnatural amino acids or synthetic functional dependencies such as polyethylene glycol. Previously, a serine protease mutant, subtilisin BPN, subtiligase (S221C / P225A) was prepared to efficiently ligate peptide esters in aqueous solution (Abrahmsen et al., Biochem. 4151-4159 [1991]). It has now been shown that synthetic peptides can be enzymatically ligated in a sequential lock to produce enzymatically active long peptides and proteins such as ribonuclease A (Jackson et. Al., Science, [1994]). This technology, which is described in more detail below, made it possible to chemically synthesize long proteins that previously could only be produced by DNA recombinant technology.

The general strategy for the synthesis of hMLi ₅₃ using subtiligase is shown (Scheme 1). Starting with the completely deprotected peptide corresponding to the C-terminal fragment of the protein, the N-terminally protected, C-terminally activated peptide ester is added together with the subtiligase. When the reaction is complete, the product is isolated by reverse phase HPLC and the protecting group is removed from the N-terminus. The following peptide fragment is ligated, deprotected, and the process is repeated using the subsequent peptides until a full length protein is obtained. The procedure is similar to solid phase methodology in which the N-terminally protected C-terminally activated peptide is ligated to the N-terminus of the passing peptide and the protein is synthesized in the C? N direction. However, since each combined result, except for the 50 residues, and the products are isolated after each ligation, much longer high purity proteins can be synthesized in quite good yields.

162 R-NH-Peptide ₂ -CO-R + H ₂ N-Peptide-CO ₂

Φ 1) subtiligase

R-NH-Peptide ₂ -CO-NH-Peptide-CO ₂

Φ 2) Zn / CH ₃ CO ₂ H

HZN-NH-PeptidrCO PeptidrCOz

Φ 3) repetition 1 + 2

H ₂ N-Peptide ₃ -CO-NH-Peptide ₂ -CO-NH-Peptide-CO ₂

Scheme 1

HML synthesis strategy using subtihgase

On the basis of knowledge of sequence specificity subtihgase and the amino acid sequence of the biologically active EPO-domain hML hMLi5 ₃ it was divided into seven fragments 18-25 residues in length. Test ligation tetrapeptides were synthesized to determine the suitability of ligation junctions for 18-25mers. Table 13 shows the results of these test ligations.

Table 13 hML test ligation. The donor and nucleophilic peptides were diluted to 10 mM in 100 mM tricine (pH 7.8) at 22 ° C. Ligase was added to a final concentration of 10 μΜ from a 1.6 mg / ml stock solution (~ 70 μΜ) and the ligation was allowed to proceed overnight. Yields are given in% ligation to hydrolysis of donor peptides.

- 163 -

location Donor (glc-K-NH1) Nucleophile-NH _n % · Hydrolysis % rígácie 1 (23/24) HVLH (SEQ ID NO: 90) SRLS (SEQ ID NO: 90) 92 08 (22/23) SHVL (SEQ ID NO: 92) HSRL (SEQ ID NO: 91) 48 52 2 (45/47) AVDF (SEQ ID NO: 92) SLGE (SEQ ID NO: 95) 22 78 3 (70/70) AVTL (SEQ ID NO: 96) llego (SEQ ID NO: 97) 53 47 4 (90/90) LSSL (SEQ ID NO: 96) LGQL (SEQ ID NO: 98) 95 05 (88/89) C (acm) LSS (SEQ ID NO: 99) LLGQ (SEQ ID NO: 100) 00 ⁰⁰ (90/91) SSLL (SEQ ID NO: 101) GQLS (SEQ ID NO: 101) 45 55 (88/89) CLSS (SEQ ID NO: 103) LLGQ (SEQ ID NO: 100) 90 1 0 5 (107/108) LQSL (SEQ ID NO: 104) LGTQ (SEQ ID NO: 105) 99. 01 (106/1 08) ALQS (SEQ ID NO: 106) LLGT (SEQ ID NO: 107) 70 30 6 (128/129) NAIF (SEQ ID NO: 108) LSFQ. (SEQ ID NO: 110) 60 40

As shown by these results, the ligation peptides listed in Table 14 were to be effectively ligated with subtiligase. A suitable protecting group for the N-terminus of each peptide ester donor was needed to prevent self-ligation. The isonicotinyl (iNOC) protecting group was selected here (Veber et al., J. Org. Chem., 42: 32863289 [1977]), since it is water-soluble, can be incipient in the last step of the solid phase peptide synthesis, is stable to anhydrous HF used to remove and cleave peptides from the solid phase resin. In addition, it can be removed from the peptide after each ligand under mild reducing conditions (Z11 / CH 3 CO 2 H) to provide a free N-terminus for subsequent ligations. Glycolate-lysyl amide (glc-K-NH ₂ ) ester was used for C-terminal activation based on previous experiments, which proved to be effectively acylated by subtiligase (Abrahmsen et al., Biochem., 30:

- 164 -

4145-4159 [1991]). INOC-protected peptides activated by glc-K-amide can be synthesized using standard solid phase methods as shown (Scheme 2). The peptides are then sequentially ligated until complete protein preparation and the end product multiplied in vitro. Based on homology with EPO, disulfide vapors are considered to be formed between cysteine residues 7 and 151 and between 28 and 85. Oxidation of disulfides can be accomplished simply by stirring the reduced material under an oxygen atmosphere for several hours. The multiplied material can then be purified by HPLC and fractions containing the active protein can be separated and lyophilized. Alternatively, the disulfides may be differential protected to control the progressive oxidation between specific disulfide pairs. Protection of cysteines 7 and 151 with acetamidomethyl groups (acm) could provide oxidation of 28 and 85. The acm groups could then be removed and residues 7 and 151 oxidized. Conversely, residues 28 and 85 could be protected with acm groups and oxidized as required in the case of sequential oxidation. Cysteines 28 and 85 may be optionally substituted with additional natural or artificial residues other than Cys to provide appropriate oxidation of cysteines 7 and 151.

Table 14

Peptide fragments used for complete synthesis of h-ML using subtiligase

fragment

sequence

1 (SEQ ID NO: 110) iNOC-HN-SPAPPACDLRVLSKLLRDSHVL-glc-K-NH ₂ (1-22) (SEQ ID NO: 111) iNOC-HN-HSRLSQCPEVHPLPTPVLLPAVDF-glc-K-NH ₂ (23-46) (SEQ ID NO. NO: 112) iNOC-HN-SLGEWKTQMEETKAQDILGAVTL-glc-KHz (47-69)

(SEQ ID NO: 113) iNOC-HN-LLEGVMAARGQLGPTCLSSLL-glc-K-NH ₂ (70-90) (SEQ ID NO: 114) iNOC-HN-GQLSGQVRLLLGALQS-glc-K-NH ₂ (90-106) ) (SEQ ID NO: 115) iNOC-HN-LLGTQLPPQGRTTAHKDPNAIF-glc-K-NH ₂ (102-128)

(SEQ ID NO: 115)

H ₂ N-LSFQHLLRGKVRFLMLVGGSTLCVR-CO ₂ (129-153)

Peptide ligation is performed at 25 ° C in a 100 mM triac, pH 8 (freshly prepared and degassed by vacuum filtration through a 5 µΜ filter). Typically, the C-terminal fragment is dissolved in buffer (2-5 mM peptide) and a 10x stock solution of subtiligase (1 mg / ml in 100 mM Tricine, pH 8) is added to achieve a final enzyme concentration of up to 5μΜ. A 3-5 molar excess of glc-K-NH ₂ activated peptide donor is then added as a solid, dissolved and allowed to stand at 25 ° C. Ligations are monitored by analytical reverse C18 phase HPLC (CH ₃ CN / H ₂ O gradient with 0.1% TFA). The ligation products were purified by preparative HPLC and lyophilized. Isonicotinyl (iNOC) deprotection was performed by mixing zinc powder-activated HCl with the protected peptide in acetic acid. The zinc dust was removed by filtration and acetic acid evaporated in vacuo. The resulting peptide can be used directly in the following leagues and the procedure is repeated. Synthetic ILML153 can be ligated by procedures analogous to those described above for synthetic or recombinant hML154. ₃₃₂ for preparing synthetic or semi-synthetic full length hML.

Synthetic hML has many advantages over recombinant. In order to improve efficiency or specificity, the unnatural side of the chains may be introduced. Polymeric functionalities, such as polyethylene glycol, may be incorporated to improve the duration of action. For example, polyethylene glycol can be attached to lysine residues of individual fragments (Table 14), before or after one or more ligation steps to be performed. Protease-sensitive peptide bridges are

166 may be removed or modified to improve in vivo stability. Further, heavy atom derivatives can be synthesized to aid in the determination of structures.

Scheme 2

Solid phase synthesis of peptide fragments for segment ligation

e, f (cleavage)

----------, -

OR O _{(CH2) 4}

I__nh ₂ [isonicotinyl (iNOC) glycolate-lysyl amide (glc-K-NH 2)]

ChemTech) was stirred with bromoacetic acid (5 eq.) And diisopropylcarbodiimide (5 eq.) For 1 h at 25 ° C in dimethylacetamide (DMA) to give the bromoacetyl derivative 2.

b) The resin is washed extensively with DMA and the individual Boc-protected amino acids (3 eq., Bachem) are esterified by mixing with sodium carbonate (6 eq.) in dimethylformamide (DMF) for 24 hours at 50 ° C to provide the corresponding glycolate phenylalanyl amide. resin 3. The amino acetylated resin 3 is washed with DMF (3x) and dichloromethane (CH 2 Cl 2) (3x) and can be stored for several months at room temperature.

room temperature. Resin 3 can also be loaded into an automated synthesizer

167 -

peptides (Applied Biosystems 430A) and peptides can be extended using standard solid phase procedures (5).

c) The Ν-α-Boc group is removed with a solution of 45% trifluoroacetic acid in CH 2 Cl 2.

d) The following Boc-protected amino acids (5 eq.) were pre-activated using benzotriazol-1-yl-oxy-tris- (dimethylamino) phosphonium hexafluorophosphate (BOP, 4 eq.) and N-methylmorpholine (NMM, 10 eq.) in 10 eq. DMA and joining for 1-2 hours.

e) The final Ν-α-Boc group is removed (TFA / CH ₂ Cl ₂ ) to give 4 and the isonicotinyl (iNOC) protecting group is introduced as described above (4) by mixing 4-isonicotinyl-2,4-dinitrophenyl carbonate (3 eq). and NMM (6 eq.) in DMA at 25 ° C for 24 hours.

f) Cleavage and deprotection of the peptide by treatment with anhydrous HF (5% anisole / 5% ethylmethylsulfide) at 0 ° C for 1 hour provides iNOC-protected, glycolate-lysamide amide activated peptide 5, which is purified by reverse phase C18 HPLC ( CH 3 CN / H 2 O gradient, 0.1% TFA). The identity of all substrates is confirmed by mass spectrometry.

ADDITIONAL IMPLEMENTATION

The invention as claimed is feasible on the basis of the above description and readily available references and starting materials. Nevertheless, the Applicant has deposited in the American Type Culture Collection, Rockville, Md., USA (ATCC) cell line below:

Escherichia coli, DH10B-pBSK-hwp / 11 1.8, ATCC accession number CRL 69575, deposited February 24, 1994.

Plasmid pSVI5.ID.LL.MLORF, ATCC Accession No. CRL 75958, deposited Dec. 2, 1994; and

CHO DP-12 cells, ML 1/50 MCB (labeled # 1594), ATCC accession number CRL 11770, deposited Dec. 6, 1994.

This deposit was carried out under the Budapest Agreement on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure and Implementing Rules (Budapest Agreement). This ensures that the life of the culture is maintained for 30 years from the date of deposit. The organisms shall be usable by the ATCC under the terms of the Budapest Agreement and the subject of the Agreement between the

168 applicants and an ATCC that provides unlimited applicability until the relevant US patent is issued. The usability of the deposited strain is not meant to be a license to use the invention in violation of the rights granted by a governmental authority in relation to its patent rights.

Since the invention is necessarily described in conjunction with preferred embodiments and specific working examples, one skilled in the art, having read the foregoing description, will be able to make various changes, substitutions of equivalents and alternatives herein.

Without departing from the spirit of the invention. Accordingly, the invention may be practiced in ways other than those specifically described herein. It is therefore expected that the protection conferred by the patent will be limited only by the appended claims and their equivalents

All references herein are hereby incorporated by reference.

- 1 SEQUENCE LIST (1) General information:

(i) Applicant: Eaton, Dan L.

Frederic J. de Sauvage

(ii) Title of the invention: Thrombopoietin (iii) Number of sequences: 144 (iv) Mailing address:

(A) Address: Genentech, Inc. (B) Street: 460 Point San Bruno Blvd (C) City: South San Francisco (D) Country: Calif (E) Country: USA (F) ZIP: 94080.

(v) Computerized form:

(A) Media Type: 3.5 inches, 1.44 Mb Floppy Disk (B) Computer: IBM PC Compatible (C) Operating System: PC-DOS / MS-DOS (D) Software: WinPatin (Genentech) (vi) Current Application Data :

(A) Application Number: 08/374540 (B) Filing Date: January 18, 1995 (C)

(vii) Priority Application Date:

(A) Application Number: PCT / US94 / 14553 (B) Filing Date: 28 December 1994 (vii) Priority Application Date:

(A) Application Number: 08/2493 76 (B) Filing Date: 25 May 1994 (vii) Priority Application Date:

(A) Application Number: 08/223263 (B) Filing Date: April 04, 1994 (vii) Priority Application Date:

(A) Application Number: 08/196689 (B) Filing Date: February 15, 1994 •

(vii) Priority Application Date:

(A) Application Number: 08/348658 (B) Filing Date: 02 December 1994 (vii) Priority Application Date:

(A) Application Number: 08/185607 (B) Filing Date: 21 January 1994

(vii) Priority Application Date:

(A) Application Number: 08/348657 (B) Filing Date: 02 December 1994 (vii) Priority Application Date:

(A) Application Number: 08/176553 (B) Filing Date: January 03, 1994 (viii) Agent Representative:

(A) Name: Winter, Daryl B.

(B) Registration number: 32,637 (C) Brand: P0871P5 (ix) Telecommunication information:

(A) Telephone: 415 / 225-1249 (B) Fax: 415 / 952-9881 (C) Telex: 910 / 371-7168 (2) Information for SEQ ID NO: 1 (i) Sequence characteristics:

(A) Length: 353 amino acids (B) Type: amino acid (C) Topology: linear (xi) Sequence description: SEQ ID NO: 1

Φ

Met -21 Glu -20 Leu Thr Glu Leu Leu -15 Leu wall wall Met Leu -10 Leu Leu Thr Ala Arg Leu Thr Leu Ser . Ser for Ala for for Ala Cys Asp Leu -5 1 5 Arg wall Leu Ser Lys Leu Leu Arg Asp Ser His wall Leu His Ser 10 15 20 Arg Leu Ser gin Cys for Glu wall His for Leu for Thr for wall 25 30 35 Leu Leu for Ala wall Asp Phe Ser Leu Gly Glu Trp Lys Thr gin 40 45 50 Met Glu Glu Thr Lys Ala gin Asp Ile Leu Gly Ala wall Thr Leu 55 60 65 Leu Leu Glu Gly wall Met Ala Ala Arg Gly gin Leu Gly for Thr 70 ⁷⁵ 80 Cys Leu Ser Ser Leu Leu Gly gin Leu Ser Gly gin wall Arg Leu 85 90 95 Leu Leu Gly Ala Leu gin Ser Leu Leu Gly Thr gin Leu for for 100 105 110 gin Gly Arg Thr Thr Ala His Lys Asp for same time Ala Ile Phe Leu 115 120 125 Ser 130 Phe gin His Leu Leu 135 Arg Gly Lys wall Arg 140 Phe Leu Met Leu

wall 145 Gly Gly Ser Thr Leu 150 Cys Val Arg Arg Ala 15 5 for for Thr Thr Ala wall for Ser Arg Thr Ser Leu wall Leu Thr Leu same time Glu Leu 160 165 170 for same time Arg Thr Ser Gly Leu Leu Glu Thr same time Phe Thr Ala Ser 175 180 185 Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp gin gin Gly Phe 190 195 200 Arg Ala Lys Ile for Gly Leu Leu same time gin Thr Ser Arg Ser Leu 205 210 215 Asp gin Ile for Gly Tyr Leu same time Arg Ile Hi.s Glu Leu Leu same time 220 225 230 Gly Thr Arg Gly Leu Phe for Gly for Ser Arg Arg Thr Leu Gly 235 240 245 Ala for Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu for 250 255 260 for same time Leu gin for Gly Tyr Ser for Ser for Thr His for for 265 270 275 Thr Gly gin Tyr Thr Leu Phe for Leu for for Thr Leu for Thr 280 285 290 for wall wall gin Leu His for Leu Leu for Asp for Ser Ala for 295 300 3 05 Thr for Thr for Thr Ser for Leu Leu same time Thr Ser Tyr Thr His 310 315 32 0 Ser gin same time Leu Ser gin Glu Gly 325 330 332

(2) Information for SEQ ID NO: 2 (i) Sequence characteristics:

(A) Length: 1795 bases (B) Type: nucleic acid (C) Fiber: single (D) Topology: linear (xi) Sequence description: SEQ ID NO: 2

CTGGAGCCCT TCTCCACCCG GATAGATTCC TCACCCTTGG CCCGCCTTTG CCCCACCCTA CTCTGCCCAG AAGTGCAAGA GCCTAAGCCG CCTCCATGGC CCCAGGAAGG ATTCAGGGGA GAGGCCCCAA ACAGGGAGCC ACGCCAGCCA GACACCCCGG CCAG ATG GAG CTG ACT GAA TTG CTC CTC 239 Met Glu Leu Thr Glu Leu Leu Leu -21 -20 -15 GTG GTC ATG CTT CTC CTA ACT GCA AGG CTA ACG CTG TCC 278 wall wall Met Leu Leu Leu Thr Ala Arg Leu Thr Leu Ser -10 -5 AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC CTC AGT 317 Ser for Ala for for Ala Cys Asp Leu Arg wall Leu Ser 1 5 10 AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG 356 Lys Leu Leu Arg Asp Ser His wall Leu His Ser Arg Leu 15 20 25 AGC CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA CCT GTC 395 Ser gin Cys for Glu wall His for Leu for Thr for wall 30 35 CTG CTG CCT GCT GTG GAC TTT AGC TTG GGA GAA TGG AAA 434 Leu Leu for Ala wall Asp Phe Ser Leu Gly Glu Trp Lys 40 45 50 ACC CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA 473 Thr gin Met Glu Glu Thr Lys Ala gin Asp Ile Leu Gly 55 60 65 GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG GCA GCA CGG 512 Ala wall Thr Leu Leu Leu Glu Gly wall Met Ala Ala Arg 70 75 GGA CAA CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG 551 Gly gin Leu Gly for Thr Cys Leu Ser Ser Leu Leu Gly 80 85 90 CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GGG GCC CTG 590 gin Leu Ser Gly gin wall Arg Leu Leu Leu Gly Ala Leu 95 100 CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG 629 gin Ser Leu Leu Gly Thr gin Leu for for gin Gly Arg 105 110 115 ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC 668 Thr Thr Ala His Lys Asp for same time Ala Ile Phe Leu Ser 120 125 130 TTC CAA CAC CTG CTC CGA GGA AAG GTG CGT TTC CTG ATG 707

Phe gin His Leu Leu 135 Arg Gly Lys wall Arg 140 Phe Leu Met CTT GTA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC CCA 746 Leu wall Gly Gly Ser Thr Leu Cys wall Arg Arg Ala for 145 150 155 CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC 785 for Thr Thr Ala wall for Ser Arg Thr Ser Leu wall Leu 160 165 ACA CTG AAC GAG CTC CCA AAC AGG ACT TCT GGA TTG TTG 824 Thr Leu same time Glu Leu for same time Arg Thr Ser Gly Leu Leu 170 175 180 GAG ACA AAC TTC ACT GCC TCA GCC AGA ACT ACT GGC TCT 863 Glu Thr same time Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser 185 190 195 GGG CTT CTG AAG TGG CAG CAG GGA TTC AGA GCC AAG ATT 902 Gly Leu Leu Lys Trp gin gin Gly Phe Arg Ala Lys Ile 200 205 CCT GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG GAC CAA 941 for Gly Leu Leu same time gin Thr Ser Arg Ser Leu Asp gin 210 215 220 ATC CCC GGA TAC CTG AAC AGG ATA CAC GAA CTC TTG AAT 980 Ile for Gly Tyr Leu same time Arg Ile His Glu Leu Leu same time 225 230 GGA ACT CGT GGA CTC TTT CCT GGA CCC TCA CGC AGG ACC 1019 Gly Thr Arg Gly Leu Phe for Gly for Ser Arg Arg Thr 235 240 245 CTA GGA GCC CCG GAC ATT TCC TCA GGA ACA TCA GAC ACA 1058 Leu Gly Ala for Asp Ile Ser Ser Gly Thr Ser Asp Thr 250 255 260 GGC TCC CTG CCA CCC AAC CTC CAG CCT GGA TAT TCT CCT 1097 Gly Ser Leu for for same time Leu gin for Gly Tyr Ser for 265 270 TCC CCA ACC CAT CCT CCT ACT GGA CAG TAT ACG CTC TTC 1136 Ser for Thr His for for Thr Gly gin Tyr Thr Leu Phe 275 280 285 CCT CTT CCA CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC 1175 for Leu for for Thr Leu for Thr for wall wall gin Leu 290 295 CAC CCC CTG CTT CCT GAC CCT TCT GCT CCA ACG CCC ACC 1214 His for Leu Leu for Asp for Ser Ala for Thr for Thr 300 305 310

CCT ACC AG C CCT CTT CTA AAC ACA TCC TAC ACC CAC TCC 1253 Pro Thr Ser Pro Leu Leu Asn Thr; Ser Tyr Thr His Ser 315 320 325 CAG AAT CTG TCT. CAG GAA GGG T AAGGT TCTCAGACAC 12 90 Gin Asn Leu Ser Gin Glu 330 332 TGCCGACATC AGCATTGTCT CATGTACAGC TCCCTTCCCT GCAGGGCGCC 1340 CCTGGGAGAC AACTGGACAA GATTTCCTAC TTTCTCCTGA AACCCAAAGC 1390 CCTGGTAAAA GGGATACACA GGACTGAAAA GGGAATCATT TTTCACTGTA 1440 CÄTTATAAAC CTTCAGAAGC TATTTTTTTA AGCTATCAGC AATACTCATC 1490 AGAGCAGCTA GCTCTTTGGT CTATTTTCTG CAGAAATTTG CAACTCACTG 1540 ATTCTCTACA TGCTCTTTTT CTGTGATAAC TCTGCAAAGG CCTGGGCTGG 1590 CCTGGCAGTT GAACAGAGGG AGAGACTAAC CTTGAGTCAG AAAACAGAGA 1640 AAGGGTAATT TCCTTTGCTT CAAATTCAAG GCCTTCCAAC GCCCCCATCC 1690 CCTTTACTAT CATTCTCAGT GGGACTCTGA TCCCATATTC TTAACAGATC 1740 TTTACTCTTG AGAAATGAAT AAGCTTTCTC TCAGAAAAAA áááááááááá .1790

ΑΑΑΑΑ 1795 (2) Information for SEQ ID NO: 3

(i) Sequence characteristics:

(A) Length: 42 amino acids (B) Type: amino acid (D) Topology: linear (xi) Sequence description: SEQ ID NO: 3

Leu -16 Leu -15 Leu wall wall Met Leu -10 Leu Leu Thr Ala Arg -5 Leu Thr Leu Ser Ser 1 for Ala for for 5 Ala Cys Asp Leu Arg 10 wall Leu Ser Lys Leu 15 Leu Arg Asp Ser His 20 wall Leu His Ser Arg 25 Leu 26

(2) Information for SEQ ID NO: 4 (i) Sequence characteristics:

(A) Length: 390 base pairs (B) Type: nucleic acid (C) Fiber: single (D) Topology: linear (xi) Sequence description: SEQ ID NO: 4

GAATTCCTGG AATACCAGCT GACAATGATT TCCTCCTCAT CTTTCAACCT 50

CACCTCTCCT CATCTAAGAA TTG Leu CTC Leu -15 CTC GTG GTC ATG 91 Leu Val wall Met Leu -10 CTC CTA ACT GCA AGG CTA ACG CTG TCC AGC CCG GCT CCT 130 Leu Leu Thr Ala Arg Leu Thr Leu Ser Ser for Ala for -5 1 CCT GCT TGT GAC CTC CGA GTC CTC AGT ΑΑΆ CTG CTT CGT 169 for Ala Cys Asp Leu Arg wall Leu Ser Lys Leu Leu Arg 5 10 15 GAC TCC CAT GTC CTT CAC AGC AGA CTG GTGA GAACTCCCAA 210 Asp Ser His wall Leu His Ser Arg Leu 20 25 26

CTCCCAGGAA

CATTATCCCC

TTTATCCGCG

TAACTGGTAA

GACACCCATA

260

GACACCATCA

CTTCCTCTAA

CTCCTTGACC

CAATGACTAT

TCTTCCCATA

310

TTGTCCCCAC

CTACTGATCA

CACTCTCTGA

CAAGAATTAT

TCTTCACAAT

360

ACAGCCCGCA

TTTAAAAGCT

CTCGTCTAGA

390 (2) Information for SEQ ID NO: 5 (i) Sequence characteristics: (A) Length:

390 base pairs

Type:

nucleic acid

Thread:

one

Topology:

linear (xi) Sequence description: SEQ ID NO: 5

TCTAGACGAG AGCTTTTAAA TGCGGGCTGT ATTGTGAAGA ATAATTCTTG 50 TCAGAGAGTG TGATCAGTAG GTGGGGACAA TATGGGAAGA ATAGTCATTG 100 GGTCAAGGAG TTAGAGGAAG TGATGGTGTC TTCCTGGGAG tatgggtGtc 150 TTACCAGTTA CGCGGATAAA GGGGATAATG TTGGGAGTTC TCACCAGTCT 200 GCTGTGAAGG ACATGGGAGT CACGAAGCAG TTTACTGAGG ACTCGGAGGT 250 CACAAGCAGG AGGAGCCGGG CTGGACAGCG TTAGCCTTGC AGTTAGGAGA 300 AGCATGACCA CGAGGAGCAA TTCTTAGATG AGGAGAGGTG AGGTTGAAAG 350

ATGAGGAGGA AATCATTGTC AGCTGGTATT CCAGGAATTC 390

- 9 (2) Information for SEQ ID NO: 6 (i) Sequence characteristics:

(A) Length: 332 amino acids

Type: amino acid

Topology: linear (xi) Sequence description: SEQ ID NO: 6

Ser 1 for Ala for for 5 Ala Asp Leu Arg Val 10 Leu Ser Lys Leu 15 Leu Arg Asp Ser His wall Leu His Ser Arg Leu Ser gin Cys for 20 25 30 Glu wall His for Leu for Thr for wall Leu Leu for Ala wall Asp 35 40 45 Phe Ser Leu Gly Glu Trp Lys Thr gin Met Glu Glu Thr Lys Ala 50 55 60 gin Asp Ile Leu Gly Ala wall Thr Leu Leu Leu Glu Gly wall Met 65 70 75 Ala Ala Arg Gly gin Leu Gly for Thr Cys Leu Ser Ser Leu Leu 80 85 90 Gly gin Leu Ser Gly gin wall Arg Leu Leu Leu Gly Ala Leu gin 95 100 105 Ser Leu Leu Gly Thr gin Leu for for gin Gly Arg Thr Thr Ala 110 115 120 His Lys Asp for same time Ala Ile Phe Leu Ser Phe gin His Leu Leu 125 130 135 Arg Gly Lys wall Arg Phe Leu Met Leu wall Gly Gly Ser Thr Leu 140 145 150 Cys wall Arg Arg Ala for for Thr Thr Ala wall for Ser Arg Thr 155 160 165 Ser Leu wall Leu Thr Leu same time Glu Leu for same time Arg Thr Ser Gly 170 175 180 Leu Leu Glu Thr same time Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser 185 190 195 Gly Leu Leu Lys Trp gin gin Gly Phe Arg Ala Lys Ile for Gly 200 205 210 Leu Leu same time gin Thr Ser Arg Ser Leu Asp gin Ile for Gly Tyr 215 220 225 Leu same time Arg Ile His Glu Leu Leu same time Gly Thr Arg Gly Leu Phe

230 235 240

for Gly for Ser Arg 245 Arg Thr Leu Gly Ala 250 for Asp Ile Ser Ser 255 Gly Thr Ser Asp Thr 260 Gly Ser Leu for for 265 same time Leu gin for Gly 270 Tyr Ser for Ser for 275 Thr His for for Thr 280 Gly gin Tyr Thr Leu 285 Phe for Leu for for 290 Thr Leu for Thr for 295 wall wall gin Leu His 300 for Leu Leu for Asp 305 for ' Ser Ala for Thr 310 for Thr for Thr Ser 315 for Leu Leu same time Thr 320 Ser Tyr Thr His Ser 325 gin same time Leu Ser gin 330

Glu Gly

332 (2) Information for SEQ ID NO: 7 (i) Sequence characteristics:

(A) Length: 166 amino acids

Type: amino acid

Topology: linear (xi) Sequence description: SEQ ID NO: 7

Ala 1 for for Arg Leu 5 Ile Cys Asp Ser Arg Val 10 Leu Glu Arg Tyr 15 Leu Leu Glu Ala Lys 20 Glu Ala Glu same time Ile 25 Thr Thr Gly Cys Ala 30 Glu His Cys Ser Leu 35 same time Glu same time Ile Thr 40 wall for Asp Thr Lys 45 wall same time Phe Tyr Ala 50 Trp Lys Arg Met Glu 55 wall Gly gin gin Ala 60 wall Glu wall Trp gin 65 Gly Leu Ala Leu Leu 70 Ser Glu Ala wall Leu 75 Arg Gly gin Ala Leu 80 Leu wall same time Ser Ser 85 gin for Trp Glu for 90 Leu gin Leu His wall 95 Asp Lys Ala wall Ser 100 Gly Leu Arg Ser Leu 105 Thr Thr Leu Leu Arg Ala Leu Gly Ala gin Lys Glu Ala Ile Ser

lio 115 120

- 11 Pro Pro Asp

Asp Thr Phe

Gly Lys Leu

Ala Ala Ser

125

Arg Lys Leu

140

Lys Leu Tyr

155

Ala Ala Pro

Phe Arg Val

Thr Gly Glu

Leu Arg Thr

130

Tyr Ser Asn

145

Ala Cys Arg

160 clay Thr-Ala

135

Phe Leu Arg

150

Thr Gly Asp

165

Arg

166

(2) Information for SEQ ID NO: 8 (i) Sequence characteristics:

(A) Length: 328 amino acids

Type: amino acid

Topology: linear (xi) Sequence description: SEQ ID NO: 8

Ser 1 for Ala for for 5 Ala Cys Asp Leu Arg 10 wall Leu Ser Lys Leu 15 Leu Arg Asp Ser His wall Leu His Ser Arg Leu Ser gin Cys for 20 25 30 Glu wall His for Leu for Thr for wall Leu Leu for Ala wall Asp 35 40 45 Phe Ser Leu Gly Glu Trp Lys Thr gin Met Glu Glu Thr Lys Ala 50 55 60 gin Asp Ile Leu Gly Ala wall Thr Leu Leu Leu Glu Gly wall Met 65 70 75 Ala Ala Arg Gly gin Leu Gly for Thr Cys Leu Ser Ser Leu Leu 80 85 90 Gly gin Leu Ser Gly gin wall Arg Leu Leu Leu Gly Ala Leu gin 95 100 105 Ser Leu Leu Gly Thr gin Gly Arg Thr Thr Ala His Lys Asp for 110 115 120 same time Ala Ile Phe Leu Ser Phe gin His Leu Leu Arg Gly Lys wall 125 130 135 Arg Phe Leu Met Leu wall Gly Gly Ser Thr Leu Cys wall Arg Arg 140 145 150 Ala for for Thr Thr Ala wall for Ser Arg Thr Ser Leu wall Leu 155 160 165 Thr Leu same time Glu Leu for same time Arg Thr Ser Gly Leu Leu Glu Thr 170 175 180

same time Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys 195 185 190 Trp gin gin Gly Phe Arg Ala Lys Ile for Gly Leu Leu same time gin 200 205 210 Thr Ser Arg Ser Leu Asp gin Ile for Gly Tyr Leu same time Arg Ile 215 220 225 His Glu Leu Leu same time Gly Thr Arg Gly Leu Phe for Gly for Ser 230 235 240 Arg Arg Thr Leu Gly Ala for Asp Ile Ser Ser Gly Thr Ser Asp 245 250 255 Thr Gly Ser Leu for for same time Leu gin for Gly Tyr Ser for Ser 260 265 270 for Thr His for for Thr Gly gin Tyr Thr Leu Phe for Leu for 275 280 285 for Thr Leu for Thr for wall wall gin Leu His for Leu Leu for 290 295 300 Asp for Ser Ala for Thr for Thr for Thr Ser for Leu Leu same time 305 310 315 Thr Ser Tyr Thr His Ser gin same time -leu Ser gin Glu Gly

320 325 328 (2) Information for SEQ ID NO: 9 (i) Sequence characteristics:

(A) Length • 265 amino acids Type: amino acid Topology: straight (xi) Sequence description: SEQ ID NO: 9 Ser for Ala for for Ala Cys Asp Leu Arg wall Leu Ser Lys Leu 1 5 10 15 Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser gin Cys for 20 25 30 Glu wall His for Leu Pro Thr for wall Leu Leu. for Ala wall Asp 35 40 45 Phe Ser Leu Gly Glu Trp Lys Thr gin Met Glu Glu Thr Lys Ala 50 55 60 gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly wall Met 65 70 75

Ala Ala Arg Gly Gin Leu Gly For Thr Cys Leu Ser Ser Leu Leu 90 80 85 Gly gin Leu Ser Gly gin wall Arg Leu Leu Leu Gly Ala Leu gin 95 100 105 Ser Leu Leu Gly Thr gin Leu for for gin Gly Arg Thr Thr Ala 110 115 120 His Lys Asp for same time Ala Ile Phe Leu Ser Phe gin His Leu Leu 125 130 135 Arg Gly Lys Asp Phe Trp Ile wall Gly Asp Lys Leu His Cys Leu 140 145 150 Ser gin same time Tyr Trp Leu Trp Ala Ser Glu wall Ala Ala Gly Ile 155 160 165 gin Ser gin Asp Ser Trp Ser Ala Glu for same time Leu gin wall for 170 175 180 Gly for same time for Arg Ile for Glu gin Asp Thr Arg Thr Leu Glu 185 190 195 Trp same time Ser Trp Thr Leu Ser Trp Thr Leu Thr gin Asp for Arg 200 205 210 Ser for Gly His Phe Leu Arg same time Ile Arg His Arg Leu for Ala 215 220 225 Thr gin for for Ala Trp Ile Phe Ser Phe for same time for Ser Ser 230 235 240 Tyr Trp Thr wall Tyr Ala Leu for Ser Ser Thr His Leu Ala His 245 250 255 for Cys Gly for Ala for for for Ala Ser

260 265 (2) Information for SEQ ID NO: 10 (i) Sequence characteristics:

(A) Length: 261 amino acids Type: amino acid Topology: straight (xi) Sequence description: SEQ JD NO: 10 Ser Pro Ala Pro Pro 1 5 Ala Cys Asp Leu Arg 10 wall Leu Ser Lys Leu 15 Leu Arg Asp Ser His 20 Val Leu His Ser Arg 25 Leu Ser gin Cys for 30 Glu Val His Pro Leu 35 Pro Thr Pro wall Leu 40 Leu for Ala wall Asp 45

Phe Ser Gly Glu 50 Trp Lys Thr Gin Met 55 Glu Glu Thr Lys Ala 60 gin Asp Ile Leu Gly Ala wall Thr Leu Leu Leu Glu Gly wall Met 65 70 75 Ala Ala Arg Gly gin Leu Gly for Thr Cys Leu Ser Ser Leu Leu 80 85 90 Gly gin Leu Ser Gly gin wall Arg Leu Leu Leu Gly Ala Leu gin 95 100 105 Ser Leu Leu Gly Thr gin Gly Arg Thr Thr Ala His Lys Asp for 110 115 120 same time Ala Ile Phe Leu Ser Phe gin His Leu Leu Arg Gly Lys Asp 125 130 135 Phe Trp Ile wall Gly Asp Lys Leu His Cys Leu Ser gin same time Tyr 140 145 150 Trp Leu Trp Ala Ser Glu wall Ala Ala Gly Ile gin Ser gin Asp 155 160 165 Ser Trp Ser Ala Glu for same time Leu gin wall for Gly for same time for 170 175 180 Arg Ile for Glu gin Asp Thr Arg Thr Leu Glu Trp same time Ser Trp 185 190 195 Thr Leu Ser Trp Thr Leu Thr gin Asp for Arg Ser for Gly His 200 205 210 Phe Leu Arg same time Ile Arg His Arg Leu for Ala Thr gin for for 215 220 225 Ala Trp Ile Phe Ser Phe for same time for Ser Ser Tyr Trp Thr wall 230 235 240 Tyr Ala Leu for Ser Ser Thr His Leu Ala His for Cys Gly for 245 250 255 Ala for for for Ala Ser

260 261 (2) Information for SEQ ED NO: 11 (i) Sequence characteristics:

(A) Length: 7849 base pairs Type : nucleic acid Topic: one

Topology:

straight

(xi) Sequence description: SEQ ID NO: 11

CCCAGCCTCC TTTCTCTTGT TCCCTGGTCA TGCCTGCCTC CCTGTCTCCT 50 GTCTCTCCCT CCCACACACA CCCACTATCC TCCCAGCTAT CCCTACACCC 100 TCCTTCCTAA TCTTGGGAGA CATCTCGTCT GGCTGGACGG GAAAATTCCA 150 GGATCTAGGC CACACTTCTC AGCAGACATG CCCATCCTTG GGGAGGAGGA 200 ACAGGAGAGA GCCTGÄGGAA GTTCTGGGGG ACAGGGGGAT GATGGGATCA 250 AGGTCAGGCC AGGAAGCCCC TGAGGACAGA GACTGTGGGG AGACTGGGAC 300 TGGGAAGAAA GCAAAGGAGC TAGAGCCAGG GCCAAAGGAA AAGGGGGGCC 350 AGCAGGGAGG TATTTGCGGG GGAGGTCCAG CAGCTGTCTT TCCTAAGACA 400 GGGACACATG GGCCTGGTTA TTCCTCTTGT CACATGTGGA ACGGTAGGAG 450 ATGGAAGACG GAGACAGAAC AAGCAAAGGA GGGCCCTGGG CACAGAGGTC 500 TGTGTGTGTA GCCATCCAAG CCACTGGACC CCAGCAGACG AGCACCTAAG 550 CTCAGGCTTA ACCCAGTGCA CGTGTGCGCA CATACATGTG CCCCGCÁCCT 600 GACAGTCCAC TCAACCCGTC CAAACCCTTT CCCCATAACA CCAACCCATA 650 ACAGGAGATT TCTCTCATGT GGGCAATATC CGTGTTCCCA CTTCGAAAGG 700 GGGAATGACA AGATAGGACT CCCTAGGGGA TTACAGAAAG AAAAGCAGGA 750 AAGCAAGCAT CCTGTTGGAT TTCAGCAGCA GGTATGATGT CCAGGGAAAA 800 GAAATTTGGA TAGCCAGGGA GTGAAAACCC CACCAATCTT AAACAAGACC 850 TCTGTGCTTC TTCCCCAGCA ACACAAATGT CCTGCCAGAT TCCTCCTGGA 900 AAAAÄACTTC TGCTCCTGTC CCCCTCCAGG TCCCAGGTTG CCCATGTCCA 950 GGAAAAGATG GATCCCCCTA TCCAAATCTT CTCCGTGGTG TGTGTGGGTG 1000 GAGGAGTGGA CCCTGGTCCA GGCAGGGGCT CCAGGGAAGA GAAGGCGTCA 1050 CTTCCGGGGG CCTTCACCAG TGTCTGGTGG CTCCCTTCTC TGATTGGGCA 1100 GAAGTGGCCC AGGCAGGCGT ATGACCTGCT GCTGTGGAGG GGCTGTGCCC 1150 CACCGCCACA TGTCTTCCTA CCCATCTGCT CCCCAGAGGG CTGCCTGCTG 1200 TGCACTTGGG TCČTGGAGCC CTTCTCCACC CGGTGAGTGG CCAGCAGGGT 1250

GTGGGGTTAT GTGAGGGTAG AAAGGACAGC AAAGAGAAAT GGGCTCCCAG 1300

CTGGGGGAGG GGCAGGCAAA CTGGAACCTA CAGGCACTGA CCTTTGTCGA 1350 GAAGAGTGTA GCCTTCCCAG AATGGGAGGA GCAGGGCAGA GCAGGGGTAG 1400 GGGGTGGGGT GCTGGTTTCT GAGGGACTGA TCACTTACTT GGTGGAATAC 1450 AGCACAGCCC TGGCTGGCCC TAAGGAAAGG GGACATGAGC CCAGGGAGAA 1500 AATAAGAGAG GGAGCTGCAC TTAGGGCTTA GCAAACACAG TAGTAAGATG 1550 GACACAGCCC CAATCCCCAT TCTTAGCTGG TCATTCCTCG TTAGCTTAAG 1600 GTTCTGAATC TGGTGCTGGG GAAGCTGGGC CAGGCAAGCC AGGGCGCAAG 1650 GAGAGGGTAA TGGGAGGAGG GCCCACTCAT GTTGACAGAC CTACAGGAAA 1700 TCCCAATATT GAATCAGGTG CAAGCCTCTT TGCACAACTT GTGAAAGGAG 1750 GAGGAAGCCA TGTGGGGGGT CCTGTGAAGG AACCGGAAGG GGTTCTGCCA 1800 AGGGGGCAGG GAGGCAGGTG TGAGCTATGA GACAGATATG TTAGTGGGCG 1850 CCTAAGACAA GGTAAGCCCC TAAGGTGGGC ATCACCCAGC AGGTGCCCGT 1900 TCCTGGGCAG CTGGTCTCAG GAAGGAAGTC CCAGAACTGT TAGCCCAŤCT 1950 CTTGGCCTCA GATAATGGAG TATTTCAGGA CTTGGAGTCC AAAGAAAAGC 2000 TCCAGTGGCT TTATGTGTGG GGGTAGATAG GGAAAGAATA GAGGTTAATT 2050 TCTCCCATAC CGCCTTTTAA TCCTGACCTC TAGTGGTCCC AGTTACAGCT 2100 TTGTGCAGTT CCCCTCCCCA GCCCCACTCC CCACCGCAGA AGTTACCCCT 2150 CAACATATTG CGCCCGTTTG CCAGTTCCTC ACCCAGGCCC TGCATCCCAT 2200 TTTCCACTCT CTTCTCCAGG CTGAAGCCAC AATACTTTCC TTCTCTATCC 2250 CCATCCCAGA TTTTCTCTGA CCTAACAACC AAGGTTGCTC AGAATTTAAG 2300 GCTAATTAAG ATATGTGTGT ATACATATCA TGTCCTGCTG CTCTCAGCAG 2350 GGGTAGGTGG CACCAAATCC GTGTCCGATT CACTGAGGAG TCCTGACAAA 2400 AAGGAGACAC CATATGCTTT CTTGCTTTCT TTCTTTCTTT CTTTCTTTTT 2450 TTTTTTTTGA GACGGAGTTT CACTCTTATT GCCCAGGCTG GAGTGCAATG 2500 GTGCGATCTC GGCTCACCAC AAACCTCCGC CTCCCAGGTA CAAGCGATTC 2550 TCCTGTCTCA GCCTCCCAAG TAGCTTGGAT TACAGGCATG AGCCACCACA 2600 CCCTGCTAGT TTTTTTGTAT TTCGTAGAGC CGGGGTTTCA CCATGTTAGT 2650

GAGGCTGGTG GCGAACTCCT GACCTCAGGT GATCCACCCG CCTTGGACTC 2700

CCAAAGTGCT GGGATTACAG GCATGAGCCA CTGCACCCGG CACACCATAT 2750 GCTTTCATCA CAAGAAAATG TGAGAGAATT CAGGGCTŤTG GCAGTTCCAG 2800 GCTGGTCAGC ATCTCAAGCC CTCCCCAGCA TCTGTTCACC CTGCCAGGCA 2850 GTCTCTTCCT AGAAACTTGG TTAAATGTTC ACTCTTCTTG CTACTTTCAG 2900 GATAGATTCC TCACCCTTGG CCCGCCTTTG CCCCACCCTA CTCTGCCCAG 2950 AAGTGCAAGA GCCTAAGCCG CCTCCATGGC CCCAGGAAGG A.TTCAGGGGA 3000 GAGGCCCCAA ACAGGGAGCC ACGCCAGCCA GACACCCCGG CCAGAATGGA 3050 GCTGACTGGT GAGAAGACAC CTGAGGGGCT AGGGCCATAT GGAAACATGA 3100 CAGAAGGGGA GAGAGAAAGG AGACACGCTG CAGGGGGCAG GAAGCTGGGG 3150 GAACCCATTC TCCCAAAAAT AAGGGGTCTG AGGGGTGGAT TCCCTGGGTT 3200 TCAGGTCTGG GTCCTGAATG GGAATTCCTG GAATACCAGC TGACAATGAT 3250 TTCCTCCTCA TCTTTCAACC TCACCTCTCC TCATCTAAGA ATTGCTCCTC 3300 GTGGTCATGC TTCTCCTAAC TGCAAGGCTA ACGCTGTCCA gcccggctCc 3350 TCCTGCTTGT GACCTCCGAG TCCTCAGTAA ACTGCTTCGT GACTCCCATG 3400 TCCTTCACAG CAGACTGGTG AGAACTCCCA ACATTATCCC CTTTATCCGC 3450 GTAACTGGTA AGACACCCAT ACTCCCAGGA AGACACCATC ACTTCCTCTA 3500 ACTCCTTGAC CCAATGACTA TTCTTCCCAT ATTGTCCCCA CCTACTGATC 3550 ACACTCTCTG ACAAGAATTA TTCTTCACAA TACAGCCCGC ATTTAAAAGC 3600 TCTCGTCTAG AGATAGTACT CATGGAGGAC TAGCCTGCTT ATTAGGCTAC 3650 CATAGCTCTC TCTATTTCAG CTCCCTTCTC CCCCCACCAA TCTTTTTCAA 3700 CAGAGCCAGT GCCCAGAGGT TCACCCTTTG CCTACACCTG TCCTGCTGCC 3750 TGCTGTGGAC TTTAGCTTGG GAGAATGGAA AACCCAGATG GTAAGAAAGC 3800 CATCCCTAAC CTTGGCTTCC CTAAGTCCTG TCTTCAGTTT CCCACTGCTT 3850 CCCATGGATT CTCCAACATT CTTGAGCTTT TTAAAAATAT CTCACCTTCA 3900 GCTTGGCCAC CCTAACCCAA TCTACATTCA CCTATGATGA TAGCCTGTGG 3950 ATAAGATGAT GGCTTGCAGG TCCAATATGT GAATAGATTT GAAGCTGAAC 4000 ACCATGAAAA GCTGGAGAGA AATCGCTCAT GGCCATGCCT TTGACCTATT 4050

CCYGTTCAGT CTTCTTAAAT TGGCATGAAG AAGCAAGACT CATATGTCAT 4100

CCACAGATGA CACAAAGCTG GGAAGŤACCA CTAAAATAAC AAAAGACTGA 4150 ATCAAGATTC AAATCACTGA AAGACTAGGT CAAAAACAAG GTGAAACAAC 4200 AGAGATATAA ACTTCTACAT GTGGGCCGGG GGCTCACGCC TGTAATCCCA 4250 GCACTTTGGG AGGCCGAGGC AGGCAGATCA CCTGAGGGCA GGAGTTTGAG 4300 AGCAGCCTGG CCAACATGGC GAAACCCCGT CTCTACTAAG AATACAAAAT 4350 TAGCCGGGCA TGGTAGTGCA TGCCTGTAAT CCCAGCTACT TGGAAGGCTG 4400 AAGCAGGAGA ATCCCTTGAA CCCAGGAGGT GGAGGTTGTA GTGAGCTGAG 4450 ATCATGCCAA TGCACTCCAG CCTGGGTGAC AAGAGCAAAA CTCCGTCTCA 4500 AAAAGAAAAA AAAATTCTAC ATGTGTAAAT TAATGAGTAA AGTCCTATTC 4550 CAGCTTTCAG GCCACAATGC CCTGCTTCCA TCATTTAAGC CTCTGGCCCT 4600 AGCACTTCCT ACGAAAAGGA TCTGAGAGAA TTAAATTGCC CCCAAACTTA 4650 CCATGTAACA TTACTGAAGC TGCTATTCTT AAAGCTAGTA ATTCTTGTCT 4700 GTTTGATGTT TAGCATCCCC ATTGTGGAAA TGCTCGTACA GAACTCTATT 4750 CCGAGTGGAC TACACTTAAA TATACTGGCC TGAACACCGG ACATCCCCCT 4800 GAAGACATAT GCTAATTTAT TAAGAGGGAC CATATTAAAC TAACATGTGT 4850 CTAGAAAGCA GCAGCCTGAA CAGAAAGAGA CTAGAAGCAT GTTTTATGGG 4900 CAATAGTTTA AAAAACTAAA ATCTATCCTC AAGAACCCTA GCGTCCCTTC 4950 TTCCTTCAGG ACTGAGTCAG GGAAGAAGGG CAGTTCCTAT GGGTCCCTTC 5000 TAGTCCTTTC TTTTCATCCT TATGATCATT ATGGTAGAGT CTCATACCTA 5050 CATTTAGTTT ΑΤΤΤΆΤΤΑΤΤ ATTATTTGAG ACGGAGTCTC ACTCTATCCC 5100 CCAGGCTGGA GTGCAGTGGC ATGATCTCAA CTCACTGCAA CCTCAGCCTC 5150 CCGGATTCAA GCGATTCTCC TGCCTCAGTC TCCCAAGTAG CTGGGATTAC 5200 AGGTGCCCAC CACCATGCCC AGCTAATTTT TGTATTTTTG GTAGAGATGG 5250 GGTTTCACCA TGTTGGCCAG GCTGATCTTG AACTCCTGAC CTCAGGTGAT 5300 CCACCTGCCT CAGCCTCCCA AAGTGCTGGG ATTACAGGCG TGAGCCACTG 5350 CACCCAGCCT TCATTCAGTT TAAAAATCAA ATGATCCTAA GGTTTTGCAG 5400 CAGAAAGAGT AAATTTGCAG CACTAGAACC AAGAGGTAAA AGCTGTAACA 5450

GGGCAGATTT CAGCAACGTA AGAAAAAAGG AGCTCTTCTC ACTGAAACCA 5500

AGTGTAAGAC CAGGCTGGAC TAGAGGACAC GGGAGTTTTT GAAGCAGAGG _5550 CTGATGACCA GCTGTCGGGA GACTGTGAAG GAATTCCTGC CCTGGGTGGG 5600 ACCTTGGTCC TGTCCAGTTC TCAGCCTGTA TGATTCACTC TGCTGGCTAC 5650 TCCTAAGGCT CCCCACCCGC TTTTAGTGTG CCCTTTGAGG CAGTGCGCTT 5700 CTCTCTTCCA TCTCTTTCTC AGGAGGAGAC CAAGGCACAG GACATTCTGG 5750 GAGCAGTGAC CCTTCTGCTG GAGGGAGTGA TGGCAGCACG GGGACAACTG 5800 GGACCCACTT GCCTCTCATC CCTCCTGGGG CAGCTTTCTG GACAGGTCCG 5850 TCTCCTCCTT GGGGCCCTGC AGAGCCTCCT TGGAACCCAG GTAAGTCCCC 5900 AGTCAAGGGA TCTGTAGAAA CTGTTCTTTT CTGACTCAGT CCCACTAGAA 5950 GACCTGAGGG AAGAAGGGCT CTTCCAGGGA GCTCAAGGGC AGAAGAGCTG 6000 ATCTACTAAG AGTGCTCCCT GCCAGCCACA ATGCCTGGGT ACTGGCATCC 6050 TGTCTTTCCT ACTTAGACAA GGGAGGCCTG AGATCTGGCC CTGGTGTTTG 6100 GCCTCAGGAC CATCCTCTGC CCTCAGCTTC CTCCACAGGG CAGGACCACA 6150 GCTCACAAGG ATCCCAATGC CATCTTCCTG AGTTTCCAAC ACCTGCTCCG 6200 AGGAAAGGTG CGTTTCCTGA TGCTTGTAGG AGGGTCCACC CTCTGCGTCA 6250 GGCGGGCCCC ACCCACCACA GCTGTCCCCA GCAGAACCTC TCTAGTCCTC 6300 ACACTGAACG AGCTCCCAAA CAGGACTTCT GGATTGTTGG AGACAAACTT 6350 CACTGCCTCA GCCAGAACTA CTGGCTCTGG GCTTCTGAAG TGGCAGCAGG 6400 GATTCAGAGC CAAGATTCCT GGTCTGCTGA ACCAAACCTC CAGGTCCCTG 6450 GACCAAATCC CCGGATACCT GAACAGGATA CACGAACTCT TGAATGGAAC 6500 TCGTGGACTC TTTCCTGGAC CCTCACGCAG GACCCTAGGA GCCCCGGACA 6550 TTTCCTCAGG AACATCAGAC ACAGGCTCCC TGCCACCCAA CCTCCAGCCT 6600 GGATATTCTC CTTCCCCAAC CCATCCTCCT ACTGGACAGT ATACGCTCTT 6650 CCCTCTTCCA CCCACCTTGC CCACCCCTGT GGTCCAGCTC CACCCCCTGC 6700 TTCCTGACCC TTCTGCTCCA ACGCCCACCC CTACCAGCCC TCTTCTAAAC 6750 ACATCCTACA CCCACTCCCA GAATCTGTCT CAGGAAGGGT AAGGTTCTCA 6800 GACACTGCCG ACATCAGCAT TGTCTCATGT ACAGCTCCCT TCCCTGCAGG 6850 GCGCCCCTGG GAGACAACTG GACAAGATTT CCTACTTTCT CCTGAAACCC 6900

AAAGCCCTGG TAAAAGGGAT ACACAGGACT GAAAAGGGAA TCATTTTTCA 6950 CTGTACATTA TAAACCTTCA GAAGCTATTT TTTTÄAGCTA TCAGCAATAC 7000 TCATCAGAGC AGCTAGCTCT TTGGTCTATT TTCTGCAGAA ATTTGCAACT 7050 CACTGATTCT CTACATGCTC TTTTTCTGTG ATAACTCTGC AAAGGCCTGG 7100 GCTGGCCTGG CAGTTGAACA GAGGGAGAGA CTAACCTTGA GTCAGAAAAC 7150 AGAGAAAGGG TAATTTCCTT TGCTTCAAAT TCAAGGCCTT CCAACGCCCC 7200 CATCCCCTTT ACTATCATTC TCAGTGGGAC TCTGATCCCA TATTCTTAAC 7250 AGATCTTTAC TCTTGAGAAA TGAATAAGCT TTCTCTCAGA AATGCTGTCC 7300 CTATACACTA GACAAAACTG AGCCTGTATA AGGAATAAAT GGGAGCGCCG 7350 AAAAGCTCCC TAAAAAGCAA GGGAAAGATG TTCTTCGAGG GTGGCAATAG 7400 ATCCCCCTCA CCCTGCCACC CCAAACAAAA AAGCTAACAG GAAGCCTTGG 7450 AGAGCCTCAC ACCCCAGGTA AGGCTGTGTA GACAGTTCAG TAAAGACAGG 7500 ACCTGGATGT GACAGCTGAG CAAACAGCTA GAGCTTTGGC AGCTCAGCÄG 7550 GAGGCTTTGC CAGGCATGGA CGCCTGCCTC CCTCCTGTGG AGGTCAGGAG 7600 GAAGTGCAGG AAGTGGCATG AGTCAGGCTC CTTGAGCTCA CACAGCAGGA 7650 GAACAAGTAC AAGTCAAGTA CAAGTTGAAG GCTCATTT.CC CAGTTCCCGC 7700 AAATGCATCT AAAAAGCAGC TCTGTGTGAC CACCATAAAC TCTGCTAGGG 7750 GATCTCTAAA AAGGAGTCAG GCTTATGGGG CTTTGCAAAT AAGTGCTGCC 7800 TTGGTGCTCA GGAAAAGGTT TGTGTTGCAC AAAACACAAA TTCCACTGC 7849

(2) Information for SEQ ID NO: 12 (i) Sequence characteristics;

(A) Length: 1443 base pairs Type: nucleic acid Thread: one Topology: straight

(xi) Sequence description: SEQ ID NO: 12

GAGTCCTTGG CCCACCTCTC TCCCACCCGA CTCTGCCGAA AGAAGCACAG 50

AAGCTCAAGC CGCCTCCATG GCCCCAGGAA AGATTCAGGG GAGAGGCCCC 100

ATACAGGGAG CCACTTCAGT TAGACACCCT GGCCAGA

-21 -20 CTG ACT GAT TTG CTC CTG GCG GCC ATG CTT CTT GCA GTG 182 Leu Thr Asp Leu Leu Leu Ala Ala Met Leu Leu Ala wall -15 -10 GCA AGA CTA ACT CTG TCC AGC CCC GTA GCT CCT GCC TGT 221 Ala Arg Leu Thr Leu Ser Ser for wall Ala for Ala Cys -5 1 5 GAC CCC AGA CTC CTA AAT AAA CTG CTG CGT GAC TCC CAC 260 Asp for Arg Leu Leu same time Lys Leu Leu Arg Asp Ser His 10 15 20 CTC CTT CAC AGC CGA CTG AGT -CAG TGT CCC GAC GTC GAC 299 Leu Leu His Ser Arg Leu Ser gin Cys for Asp wall Asp 25 30 CCT TTG TCT ATC CCT GTT CTG CTG CCT GCT GTG GAC TTT 338 for Leu Ser Ile for wall Leu Leu for Ala wall Asp Phe 35 40 45 AGC CTG GGA GAA TGG AAA ACC CAG ACG GAA CAG AGC AAG 377 Ser Leu Gly Glu Trp Lys Thr gin Thr Glu gin Ser Lys 50 55 GCA CAG GAC ATT CTA GGG GCA GTG TCC CTT CTA CTG GAG 416 Ala gin Asp Ile Leu Gly Ala wall Ser Leu Leu Leu Glu 60 65 70 GGA GTG ATG GCA GCA CGA GGA CAG TTG GAA CCC TCC TGC 455 Gly wall Met Ala Ala Arg Gly gin Leu Glu for Sei * Cys 75 80 85 CTC TCA TCC CTC CTG GGA CAG CTT TCT GGG CAG GTT CGC 494 Leu Ser Ser Leu Leu Gly gin Leu Ser Gly gin wall Arg 90 95 CTC CTC TTG GGG GCC CTG CAG GGC CTC CTA GGA ACC CAG 533 Leu Leu Leu Gly Ala Leu gin Gly Leu Leu Gly Thr gin 100 105 110 GGC AGG ACC ACA GCT CAC AAG GAC CCC AAT GCC CTC TTC 572 Gly Arg Thr Thr Ala His Lys Asp for same time Ala Leu Phe 115 120 TTG AGC TTG CAA CAA CTG CTT CGG GGA AAG GTG CGC TTC 611 Leu Ser Leu gin gin Leu Leu Arg Gly Lys wall Arg Phe 125 130 135 CTG CTT CTG GTA GAA GGT CCC ACC CTC TGT GTC AGA CGG 650 Leu Leu Leu wall Glu Gly for Thr Leu Cys wall Arg Arg 140 145 150 ACC CTG CCA ACC ACA GCT GTC CCA AGC AGT ACT TCT CAA 689

Thr Leu for Thr Thr 155 for Ser Ser 160 Thr Ser Gin CTC CTC ACA CTA AAC AAG TTC CCA AAC AGG ACT TCT GGA 728 Leu Leu Thr Leu same time Lys Phe for same time Arg Thr Ser Gly 165 170 175 TTG TTG GAG ACG AAC TTC AGT GTC ACA GCC AGA ACT GCT 767 Leu Leu Glu Thr same time Phe Ser wall Thr Ala Arg Thr Ala 180 185 GGC CCT GGA CTT CTG AGC AGG CTT CAG GGA TTC AGA GTC 806 Gly for Gly Leu Leu Ser Arg Leu gin Gly Phe Arg wall 190 195 200 AAG ATT ACT CCT GGT CAG CTA AAT CAA ACC TCC AGG TCC 845 Lys Ile Thr for Gly gin Leu same time gin Thr Ser Arg Ser 205 210 215 CCA GTC CAA ATC TCT GGA TAC CTG AAC AGG ACA CAC GGA 884 for wall gin Ile Ser Gly Tyr Leu same time Arg Thr His Gly 220 225 CCT GTG AAT GGA ACT CAT GGG CTC TTT GCT GGA ACC TCA 923 for wall same time Gly Thr His Gly Leu Phe Ala Gly Thr Ser 230 235 240 CTT CAG ACC CTG GAA GCC TCA GAC ATC TCG CCC GGA GCT 962 Leu gin Thr Leu Glu Ala Ser Asp Ile Ser for Gly Ala 245 250 TTC AAC AAA GGC TCC CTG GCA TTC AAC CTC CAG GGT GGA 1001 Phe same time Lys Gly Ser Leu Ala Phe same time Leu gin Gly Gly 255 260 265 CTT CCT CCT TCT CCA AGC CTT GCT CCT GAT GGA CAC ACA 1040 Leu for for Ser for Ser Leu Ala for Asp Gly His Thr 270 275 280 CCC TTC CCT CCT TCA CCT GCC TTG CCC ACC ACC CAT GGA 1079 for Phe for for Ser for Ala Leu for Thr Thr His Gly 285 290 TCT CCA CCC CAG CTC CAC CCC CTG TTT CCT GAC CCT TCC 1118 Ser for for gin Leu His for Leu Phe for Asp for Ser 295 300 305 ACC ACC ATG CCT AAC TCT ACC GCC CCT CAT CCA GTC ACA 1157 Thr Thr Met for same time Ser Thr Ala for His for wall Thr 310 315 ATG TAC CCT CAT CCC AGG AAT TTG TCT CAG GAA ACA TAGCGC 1199 Met Tyr for His for Arg same time Leu Ser gin Glu Thr 320 325 330 33.1

G GGCACTGGCC CAGTGAGCGT CTGCAGCTTC TCTCGGGGAC 1240

AAGCTTCCCC AGGAAGGCTG AGAGGCAGCT GCATCTGCTC CAGATGTTCT 1290

GCTTTCACCT AAAAGGCCCT GGGGAAGGGA TACACAGCAC TGGAGATTGT 1340

AAAATTTTAG GAGCTATTTT TTTTTAACCT ATCAGCAATA TTCATCAGAG 1390

CAGCTAGCGA TCTTTGGTCT ATTTTCGGTA TAAATTTGAA AATCACTAAT 144 0 TCT 1443 (2) Information for SEQ ID NO: 13 (i) Sequence characteristics:

(A) Length: 352 amino acids

Type: amino acid

Topology: linear (xi) Sequence description: SEQ ID NO: 13

Met -21 Glu -20 Leu Thr Asp Leu Leu -15 Leu Ala Ala Met Leu -10 Leu Ala wall Ala Arg Leu Thr Leu Ser Ser for wall Ala for Ala Cys Asp for -5 1 5 Arg Leu Leu same time Lys Leu Leu Arg Asp Ser His Leu Leu His Ser 10 15 20 Arg Leu Ser gin Cys for Asp wall Asp for Leu Ser Ile for wall 25 30 35 Leu Leu for Ala wall Asp Phe Ser Leu Gly Glu Trp Lys Thr gin 40 45 50 Thr Glu gin Ser Lys Ala gin Asp Ile Leu Gly Ala wall Ser Leu 55 60 65 Leu Leu Glu Gly wall Met Ala Ala Arg Gly gin Leu Glu for Ser 70 75 80 Cys Leu Ser Ser Leu Leu Gly gin Leu Ser Gly gin wall Arg Leu 85 90 95 Leu Leu Gly Ala Leu gin Gly Leu Leu Gly Thr gin Gly Arg Thr 100 105 110 Thr Ala His Lys Asp for same time Ala Leu Phe Leu Ser Leu gin gin 115 120 125 Leu Leu Arg Gly Lys wall Arg Phe Leu Leu Leu wall Glu Gly for 130 135 140 Thr Leu Cys wall Arg Arg Thr Leu for Thr Thr Ala wall for Ser 145 150 155

Ser 160 Thr Ser gin Leu Leu 165 Thr Leu Asn Lys Phe 170 for same time Arg Thr Ser Gly Leu Leu Glu Thr same time Phe Ser wall Thr Ala Arg Thr Ala 175 180 185 Gly for Gly Leu Leu Ser Arg Leu gin Gly Phe Arg wall Lys Ile 190 195 200 Thr for Gly gin Leu same time gin Thr Ser Arg Ser for wall gin Ile 205 210 215 Ser Gly Tyr Leu same time Arg Thr His Gly for wall same time Gly Thr His 220 225 230 Gly Leu Phe Ala Gly Thr Ser Leu gin Thr Leu Glu Ala Ser Asp 235 240 245 Ile Ser for Gly Ala Phe same time Lys Gly Ser Leu Ala Phe same time Leu 250 255 260 gin Gly Gly Leu for for Ser for Ser Leu Ala for Asp Gly His 265 270 275 Thr for Phe for for Ser for Ala Leu for Thr Thr His. Gly Ser 280 285 290 for for gin Leu His for Leu Phe for Asp for Ser Thr Thr Met 295 300 305 for same time Ser Thr Ala for His for wall Thr Met Tyr for His for 310 315 320 Arg same time Leu Ser gin Glu Thr 325 330 331

(2) Information for SEQ ID NO: 14 (i) Sequence characteristics:

(A) Length: 1536 base pairs

Type: nucleic acid

Thread: one

Topology: linear (xi) Sequence description: SEQ ID NO: 14

GAGTCCTTGG

CCCACCTCTC

TCCCACCCGA

CTCTGCCGAA

AGAAGCACAG

AAGCTCAAGC

CGCCTCCATG

GCCCCAGGAA

ATACAGGGAG

CCACTTCAGT

TAGACACCCT

GGCCAGA

AGATTCAGGG

GAGAGGCCCC 100

ATG GAG

Met Glu

- 25 -21 -20

CTG Leu ACT Thr GAT Asp TTG Leu CTC Leu -15 CTG GCG Leu Ala GCC Ala ATG Met CTT Leu -10 CTT Leu GCA Ala GTG wall 182 GCA AGA CTA ACT CTG TCC AGC CCC GTA GCT CCT GCC TGT 221 Ala Arg Leu Thr Leu Ser Ser for wall Ala for Ala Cys -5 1 5 GAC CCC AGA CTC CTA AAT AAA CTG CTG CGT GAC TCC CAC 260 Asp for Arg Leu Leu same time Lys Leu Leu Arg Asp Ser His 10 15 20 CTC CTT CAC AGC CGA GTG AGT CAG TGT CCC GAC GTC GAC 299 Leu Leu His Ser Arg Leu Ser gin Cys for Asp wall Asp 25 30 CCT TTG TCT ATC CCT GTT CTG CTG CCT GCT GTG GAC TTT 338 for Leu Ser Ile for wall Leu Leu for Ala wall Asp Phe 35 40 4.5 AGC CTG GGA GAA TGG AAA ACC CAG ACG GAA CAG AGC AAG 377 Ser Leu Gly Glu Trp Lys Thr gin Thr Glu gin Ser Lys 50 55 GCA CAG GAC ATT CTA GGG GCA GTG TCC CTT CTA CTG GAG 416 Ala gin Asp Ile Leu Gly Ala wall Ser Leu Leu Leu Glu 60 65 70 GGA GTG ATG GCA GCA CGA GGA CAG TTG GAA CCC TCC TGC 455 Gly wall Met Ala Ala Arg Gly gin Leu Glu for Ser Cys 75 80 85 CTC TCA TCC CTC CTG GGA CAG CTT TCT GGG CAG GTT CGC 494 Leu Ser Ser Leu Leu Gly gin Leu Ser Gly gin wall Arg 90 95 CTC CTC TTG GGG GCC CTG CAG GGC CTC CTA GGA ACC CAG 533 Leu Leu Leu Gly Ala Leu gin Gly Leu Leu Gly Thr gin 100 105 110 CTT CCT CTA CAG GGC AGG ACC ACA GCT CAC AAG GAC CCC 572 Leu for Leu gin Gly Arg Thr Thr Ala His Lys Asp for 115 120 AAT GCC CTC TTC TTG AGC TTG CAA CAA CTG CTT CGG GGA 611 same time Ala Leu Phe Leu Ser Leu gin gin Leu Leu Arg Gly 125 130 135 AAG GTG CGC TTC CTG CTT CTG GTA GAA GGT CCC ACC CTC 650 Lys wall Arg Phe Leu Leu Leu wall Glu Gly for Thr Leu 140 145 150

TGT GTC AGA CGG ACC CTG CCA

Cys wall Arg Arg Thr 155 Leu for Thr Thr Ala 160 wall for Ser - * · AGT ACT TCT CAA CTC CTC ACA CTA AAC AAG TTC CCA AAC 728 Ser Thr Ser gin Leu Leu Thr Leu same time Lys Phe for same time 165 170 175 AGG ACT TCT GGA TTG TTG GAG ACG AAC TTC AGT GTC ACA 767 Arg Thr Ser Gly Leu Leu Glu Thr same time Phe Ser wall Thr 180 185 GCC AGA ACT GCT GGC CCT GGA CTT CTG AGC AGG CTT CAG 806 Ala Arg Thr Ala Gly for Gly lime Leu Ser Arg Leu gin 190 195 200 GGA TTC AGA GTC AAG ATT ACT CCT GGT CAG CTA AA.T CAA 845 Gly Phe Arg wall Lys Ile Thr for Gly gin Leu same time gin 205 210 215 ACC TCC AGG TCC CCA GTC CAA ATC TCT GGA TAC CTG AAC 884 Thr Ser Arg Ser for wall gin Ile Ser Gly Tyr Leu same time 220 225 AGG ACA CAC GGA CCT GTG AAT GGA ACT CAT GGG CTC TTT 923 Arg Thr His Gly for wall same time Gly Thr His Gly Leu Phe · 230 235 240 GCT GGA ACC TCA CTT CAG ACC CTG GAA GCC TCA GAC ATC 962 Ala Gly Thr Ser Leu gin Thr Leu Glu Ala Ser Asp Ile 245 250 TCG CCC GGA GCT TTC AAC AAA GGC TCC CTG GCA TTC AAC 1001 Ser for Gly Ala Phe same time Lys Gly Ser Leu Ala Phe same time 255 260 265 CTC CAG GGT GGA CTT CCT CCT TCT CCA AGC CTT GCT CCT 1040 Leu gin Gly Gly Leu for for Ser for Ser Leu Ala for 270 275 280 GAT GGA CAC ACA CCC TTC CCT CCT TCA CCT GCC TTG CCC 1079 Asp Gly His Thr for Phe for for Ser for Ala Leu for 285 290 ACC ACC CAT GGA TCT CCA CCC CAG CTC CAC CCC CTG TTT 1118 Thr Thr His Gly Ser for for gin Leu His for Leu Phe 295 300 305 CCT GAC CCT TCC ACC ACC ATG CCT AAC TCT ACC GCC CCT 1157 for Asp for Ser Thr Thr Met for same time Ser Thr Ala for 310 315 CAT CCA GTC ACA ATG TAC CCT CAT CCC AGG AAT TTG TCT 1196 His for wall Thr Met Tyr for His for Arg same time Leu Ser 320 325 330

- 27 CAG GAA ACA TAGCG CGGGCACTGG CCCAGTGAGC GTCTGCAGCT 1240

Gin Glu Thr

335

TCTCTCGGGG ACAAGCTTCC CCAGGAAGGC TGAGAGGCAG CTGCATCTGC 1290

TCCAGATGTT CTGCTTTCAC CTAAAAGGCC CTGGGGAAGG GATACACAGC 1340

ACTGGAGATT GTAAAATTTT AGGAGCTATT TTTTTTTAAC CTATCAGCAA 1390

TATTCATCAG AGCAGCTAGC GATCTTTGGT CTATTTTCGG TATAAATTTG 1440

AAAATCACTA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAA 1490

AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAA AAAAAA 153 6 (2) Information for SEQ ID NO: 15 (i) Sequence characteristics:

(A) Length: 356 amino acids

Type: amino acid

Topology: linear (xi) Sequence description: SEQ ID NO: 15

Met -21 Glu -20 Leu Thr Asp Leu Leu Leu Ala -15 Ala Met Leu -10 Leu Ala wall Ala Arg Leu Thr Leu Ser Ser for wall Ala for Ala Cys Asp for -5 1 5 Arg Leu Leu same time Lys Leu Leu Arg Asp Ser His Leu Leu His Ser 10 15 20 Arg Leu Ser gin Cys for Asp wall Asp for Leu Ser Ile for wall 25 30 35 Leu Leu for Ala wall Asp Phe Ser Leu Gly Glu Trp Lys Thr gin 40 45 50 Thr Glu gin Ser Lys Ala gin Asp Ile Leu Gly Ala wall Ser Leu 55 60 65 Leu Leu Glu Gly wall Met Ala Ala Arg Gly gin Leu Glu for Ser 70 75 80 Cys Leu Ser Ser Leu Leu Gly gin Leu Ser Gly gin wall Arg Leu 85 90 95 Leu Leu Gly Ala Leu gin Gly Leu Leu Gly Thr gin Leu for Leu 100 105 110 gin Gly Arg Thr Thr Ala His you with Asp for same time Ala Leu Phe Leu

115 120 125

Ser 130 Leu gin gin Leu Leu 135 Arg Gly Lys wall Arg 140 Phe Leu Leu Leu wall Glu Gly for Thr Leu Cys wall Arg Arg Thr Leu for Thr Thr 145 150 155 Ala wall for Ser Ser Thr Ser gin Leu Leu Thr Leu same time Lys Phe 160 165 170 for same time Arg Thr Ser Gly Leu Leu Glu Thr same time Phe Ser wall Thr 175 180 185 Ala Arg Thr Ala Gly for Gly Leu Leu Ser Arg Leu gin Gly Phe 190 195 200 Arg wall Lys Ile Thr for Gly gin Leu same time gin Thr Ser Arg Ser 205 210 215 for wall gin Ile Ser Gly Tyr Leu same time Arg Thr His Gly for wall 220 225 230 same time Gly Thr His Gly Leu Phe Ala Gly Thr Ser Leu gin Thr Leu 235 240 245 Glu Ala Ser Asp Ile Ser for Gly Ala Phe same time Lys Gly Ser Leu 250 255 260 Ala Phe same time Leu gin Gly Gly Leu for for Ser for Ser Leu Ala 265 270 275 for Asp Gly His Thr for Phe for for Ser for Ala Leu for Thr 280 285 290 Thr His Gly Ser for for gin Leu His for Leu Phe for Asp for 295 300 305 Ser Thr Thr Met for same time Ser Thr Ala for His for wall Thr Met 310 315 320 Tyr for His for Arg same time Leu Ser gin Glu Thr

325 330 335 (2) information for SEQ ID NO: 16 (i) Sequence characteristics:

(A) Length: 241 amino acids

Type: amino acid

Topology: linear (xi) Sequence description: SEQ ID NO: 16

Ser 1 for wall Ala for 5 Ala Cys Asp for Arg 10 Leu Leu Asn Lys Leu 15 Leu Arg Asp Ser His Leu Leu His Ser Arg Leu Ser Gin. Cys for 20 25 30

Asp Val Asp for Leu 35 Ser Ile Pro Val Leu 40 Leu Pro Ala wall Asp 45 Phe Ser Leu Gly Glu Trp Lys Thr gin Thr Glu gin Ser Lys Ala 50 55 60 gin Asp Ile Leu Gly Ala wall Ser Leu Leu Leu Glu Gly wall Met 65 70 75 Ala Ala Arg Gly gin Leu Glu for Ser Cys Leu Ser Ser Leu Leu 80 85 90 Gly gin Leu Ser Gly gin wall Arg Leu Leu Leu Gly Ala -leu gin 95 100 105 Gly Leu Leu Gly Thr gin Leu for Leu gin Gly Arg Thr Thr Ala 110 115 120 His Lys Asp for same time Ala Leu Phe Leu Ser Leu gin gin Leu Leu 125 130 135 Arg Gly Lys Asp Phe Trp Ile wall Gly Asp Glu Leu Gin. Cys His 140 145 150 Ser gin same time Cys Trp for Trp Thr Ser Glu gin Ala Ser Gly Ile 155 160 165 gin Ser gin Asp Tyr Ser Trp Ser Ala Lys Ser same time Leu gin wall 170 175 180 for Ser for same time Leu Trp Ile for Glu gin Asp Thr Arg Thr Cys 185 190 195 Glu Trp same time Ser Trp Ala Leu Cys Trp same time Leu Thr Ser Asp for 200 205 210 Gly Ser Leu Arg His Leu Ala Arg Ser Phe gin gin Arg Leu for 215 220 225 Gly Ile gin for for Gly Trp Thr Ser Ser Phe Ser Lys for Cys 230 235 240

Ser

241 (2) Information for SEQ ID NO: 17 (i) Sequence characteristics:

(A) Length: 335 amino acids

Type: amino acid

Topology: linear (xi) Sequence description: SEQ ID NO: 17

Ser 1 For Val Ala for 5 Ala Leu Arg Asp Ser His 20 Leu Asp wall Asp for Leu 35 Ser Phe Ser Leu Gly Glu 50 Trp gin Asp Ile Leu Gly 65 Ala Ala Ala Arg Gly gin 80 Leu • Gly gin Leu Ser Gly 95 gin Gly Leu Leu Gly Thr 110 gin His Lys Asp for same time 125 Ala Arg Gly Lys wall Arq 140 Phe Cys wall Arg Arg Thr 155 Leu Ser gin Leu Leu Thr 170 Leu • Leu Leu Glu Thr same time 185 Phe Gly Leu Leu Ser Arg 200 Leu Gly gin Leu same time gin 215 Thr Tyr Leu same time Arg Thr 230 His Phe Ala Gly Thr Ser 245 Leu for Gly Ala Phe same time 260 Lys Gly Leu for for Ser 275 for

Cys Asp for Arg 10 Leu Leu same time Lys Leu  15 Leu His Ser Arg 25 Leu Ser gin Cys for 30 Ile for wall Leu 40 Leu for Ala wall Asp 45 Lys Thr gin Thr 55 Glu gin Ser Lys Ala 60 wall Ser Leu Leu 70 Leu Glu Gly wall Met 75 Glu for Ser Cys 85 Leu Ser Ser Leu Leu 90 wall Arg Leu Leu 100 Leu Gly Ala Leu gin 105 Leu for Leu gin 115 Gly Arg Thr Thr Ala 120 Leu Phe Leu Ser 130 Leu gin gin Leu Leu 135 Leu Leu Leu wall 145 Glu Gly for Thr Leu 150 for Thr Thr Ala 160 wall for Ser Ser Thr 165 same time Lys Phe for 175 same time Arg Thr Ser Gly 180 Ser wall Thr Ala 190 Arg Thr Ala Gly for 195 gin Gly Phe Arg 205 wall Lys Ile Thr for 210 Ser Arg Ser for 220 wall gin Ile Ser Gly 225 Gly for wall same time 235 Gly Thr His Gly Leu 240 gin Thr Leu Glu 250 Ala Ser Asp Ile Ser 255 Gly Ser Leu Ala 265 Phe same time Leu gin Gly 270 Ser Leu Ala for 280 Asp Gly His Thr for 285

Phe for for Ser for 290 Ala Leu for Thr Thr 295 His Gly Ser for for 300 gin Leu His for Leu 305 Phe for Asp for Ser 310 Thr Thr Met for same time 315 Ser Thr Ala for His 320 for wall Thr Met Tyr 325 for His for Arg same time 330 Leu Ser gin Glu Thr 335

(2) Information for SEQ ID NO: 18 (i) Sequence characteristics:

(A) Length: 332 amino acids

Type: amino acid

Topology: linear (xi) Sequence description: SEQ ID NO: 18

Ser 1 Pro Ala for for 5 Ala Cys Asp for Arg 10 Leu Leu Asn Lys Leu 15 Leu Arg Asp Ser His wall Leu His Gly Arg Leu Ser gin Cys for 20 25 30 Asp Ile same time for Leu Ser Thr for wall Leu Leu for Ala wall Asp 35 40 45 Phe Thr Leu Gly Glu Trp Lys Thr gin Thr Glu gin Thr Lys Ala 50 55 60 gin Asp wall Leu Gly Ala Thr Thr Leu Leu Leu Glu Ala wall Met 65 70 75 Thr Ala Arg Gly gin wall Gly for for Cys Leu Ser Ser Leu Leu 80 85 90 wall gin Leu Ser Gly gin wall Arg Leu Leu Leu Gly Ala Leu gin 95 100 105 Asp Leu Leu Gly Met gin Leu for for gin Gly Arg Thr Thr Ala 110 115 120 His Lys Asp for Ser Ala Ile Phe Leu same time Phe gin gin Leu Leu 125 130 135 Arg Gly Lys wall Arg Phe Leu Leu Leu wall wall Gly for Ser Leu 140 145 150 Cys Ala Lys Arg Ala for for Ala Ile Ala wall for Ser Ser Thr 155 160 165

Ser for Phe His Thr Leu 170 same time Lys Leu Pro Asn 175 Arg Thr Šergl 180 Leu Leu Glu Thr same time Ser Ser Ile Ser Ala Arg Thr Thr Gly Ser 185 190 195 Gly Phe Leu Lys Arg Leu gin Ala Phe Arg Ala Lys Ile for Gly 200 205 210 Leu Leu same time gin Thr Ser Arg Ser Leu Asp gin Ile for Gly His 215 220 225 gin same time Gly Thr His Gly for Leu Ser Gly Ile His Gly Leu Phe 230 235 240 for Gly for gin for Gly Ala Leu Gly Ala for Asp Ile for for 245 250 255 Ala Thr Ser Gly Met Gly Ser Arg for Thr Tyr Leu gin for Gly 260 265 270 Glu Ser for Ser for Ala His for Ser for Gly Arg Tyr Thr Leu 275 280 285 Phe Ser for Ser for Thr Ser for Ser for Thr wall gin Leu gin 290 295 300 for Leu Leu for Asp for Ser Ala Ile Thr for same time Ser Thr Ser 305 310 315 for Leu Leu Phe Ala Ala His for His Phe gin same time Leu Ser gin 320 325 330

Glu Glu

332 (2) Information for SEQ ID NO: 19 (i) Sequence characteristics:

(A) Length: 1026 base pairs Type : nucleic acid Thread: one Topology: straight

(xi) Sequence description: SEQ ID NO: 19

AGC Ser 1 CCG GCT CCT for CCT GCC TGT GAC CCC for CGA CTC Arg Leu 10 CTA 36 Leu for Ala for 5 Ala Cys Asp AAT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC GGC AGA 75 same time Lys Leu Leu Arg Asp Ser His wall Leu His Gly Arg 15 20 25

CTG AGC CAG gin TGC Cys CCA GAC ATT AAC same time CCT Pro TTG Leu 35 TCC Ser ACA CCT 114 Leu Ser for 30 Asp Ile Thr for GTC CTG CTG CCT GCT GTG GAC TTC ACC TTG GGA. GAA TGG 153 wall Leu Leu for Ala wall Asp Phe Thr Leu Gly Glu Trp 40 45 50 AAA ACC CAG ACG GAG CAG ACA AAG GCA CAG GAT GTC CTG 192 Lys Thr gin Thr Glu gin Thr Lys Ala gin Asp wall Leu 55 60 GGA GCC ACA ACC CTT CTG CTG GAG GCA GTG ATG ACA GCA 231 Gly Ala Thr Thr Leu Leu Leu Glu ALA wall Met Thr Ala 65 70 75 CGG 3GA CAA GTG GGA CCC CCT TGC CTC TCA TCC CTG CTG 270 Arg 31y gin wall Gly for for Cys Leu Ser Ser Leu Leu 80 85 90 GTC CAG CTT TCT GGA CAG GTT CGC CTC CTC CTC GGG GCC 309 wall gin Leu Ser Gly gin wall Arg Leu Leu Leu Gly Ala 95 100 CTG CAG GAC CTC CTT GGA ATG CAG CTT CCT CCA CAG GGA 348 Leu gin Asp Leu Leu Gly Met gin Leu for for gin Gly 105 110 115 AGG ACC ACA GCT CAC AAG GAT CCC AGT GCC ATC TTC CTG 387 Arg Thr Thr Ala His Lys Asp for Ser Ala Ile Phe Leu 120 125 AAC TTC CAA CAA CTG CTC CGA GGA AAG GTG CGT TTC CTG 426 same time Phe gin gin Leu Leu Arg Gly Lys wall Arg Phe Leu 130 135 140 CTC CTT GTA GTG GGG CCC TCC CTC TGT GCC AAG AGG GCC 465 Leu Leu wall wall Gly for Ser Leu Cys Ala Lys Arg Ala 145 150 155 CCA CCC GCC ATA GCT GTC CCG AGC AGC ACC TCT CCA TTC 504 for for Ala Ile Ala wall for Ser Ser Thr Ser for Phe 160 165 CAC ACA CTG AAC AAG CTC CCA AAC AGG ACC TCT GGA TTG 543 His Thr Leu same time Lys Leu for same time Arg Thr Ser Gly Leu 170 175 180 TTG GAG ACA AAC TCC AGT ATC TCA GCC AGA ACT ACT GGC 582 Leu Glu Thr same time Ser Ser Ile Ser Ala Arg Thr Thr Gly ¹⁸⁵ 190 TCT GGA TTT CTC AAG AGG CTG CAG GCA TTC AGA GCC AAG 621 Ser Gly Phe Leu Lys Arg Leu gin Ala Phe Arg Ala Lys 195 200 205

ATT ile CCT GGT CTG Leu CTG AAC Leu Asn CAA ACC TCC AGG TCC Ser CTA Leu GAC Asp 220 66Ό for Gly 210 gin Thr 215 Ser Arg CAA ATC CCT GGA CAC CAG AAT GGG ACA CAC GGA CCC TTG 699 gin Ile for Gly His gin same time Gly Thr His Gly for Leu 225 230 AGT GGA ATT CAT GGA CTC TTT CCT GGA CCC CAA CCC GGG 738 Ser Gly Ile His Gly Leu Phe for Gly for gin for Gly 235 240 245 GCC CTC GGA GCT CCA GAC ATT CCT CCA GCA ACT TCA GGC 777 Ala Leu Gly Ala for Asp Ile for for Ala Thr Ser Gly 250 255 ATG GGC TCC CGG CCA ACC TAC CTC CAG CCT GGA GAG TCT 816 Met Gly Ser Arg for Thr Tyr Leu gin for Gly Glu Ser 260 265 270 CCT TCC CCA GCT CAC CCT TCT CCT GGA CGA TAC ACT CTC 855 for Ser for Ala His for Ser for Gly Arg Tyr Thr Leu 275 280 285 TTC TCT CCT TCA CCC ACC TCG CCC TCC CCC ACA GTC CAG 894 Phe Ser for Ser for Thr Ser for Ser for Thr wall gin 290 295 CTC CAG CCT CTG CTT CCT GAC CCC TCT GCG ATC ACA CCC 933 Leu gin for Leu Leu for Asp for Ser Ala Ile Thr for 300 305 310 AAC TCT ACC AGT CCT CTT CTA TTT GCA GCT CAC CCT CAT 972 same time Ser Thr Ser for Leu Leu Phe Ala Ala His for His 315 320 TTC CAG AAC CTG TCT CAG GAA GAG TAAG GTGCTCAGAC 1010 Phe gin same time Leu Ser gin Glu Glu 325 330 332

CCTGCCAACT TCAGCA 1026 (2) Information for SEQ ID NO: 20 (i) Sequence characteristics:

(A) LENGTH: Type: Fiber: Topology:

(xi) Sequence description: SEQ ID NO: 20

1014 base pairs nucleic acid single linear

AGC CCG CCT CCT CCT CCT CCT CCT CCT CCT CCT CCT CCT 36 Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu

AAT AAA CTG CTT Leu CGT GAC Arg Asp TCC Ser CAT GTC CTT CAC His GGC Gly AGA Arg 25 75 Asn Lys Leu 15 His 20 wall Leu CTG AGC CAG TGC CCA GAC ATT AAC CCT TTG TCC ACA CCT 114 Leu Ser gin Cys for Asp Ile same time for Leu Ser Thr for 30 35 GTC CTG CTG CCT GCT GTG GAC TTC ACC TTG GGA GAA TGG 153 wall Leu Leu for Ala wall Asp Phe Thr Leu Gly Glu Trp 40 45 50 AAA ACC CAG ACG GAG CAG ACA AAG GCA CAG GAT GTC CTG 192 Lys Thr gin Thr Glu gin Thr Lys Ala gin Asp wall Leu 55 60 GGA GCC ACA ACC CTT CTG CTG GAG GCA GTG ATG ACA GCA 231 Gly Ala Thr Thr Leu Leu Leu Glu Ala wall Met Thr Ala 65 70 75 CGG GGA CAA GTG GGA CCC CCT TGC CTC TCA TCC CTG CTG 270 Arg Gly gin wall Gly for for Cys Leu Ser Ser Leu Leu 80 85 90 GTG CAG CTT TCT GGA CAG GTT CGC CTC CTC CTC GGG GCC 3 0 9 wall gin Leu Ser Gly gin wall Arg Leu Leu Leu Gly Ala 95 100 CTG CAG GAC CTC CTT GGA ATG CAG GGA AGG ACC ACA GCT 348 Leu gin Asp Leu Leu Gly Met gin Gly Arg Thr Thr Ala 105 110 115 CAC AAG GAT CCC AGT GCC ATC TTC CTG AAC TTC CAA CAA 387 His Lys Asp for Ser Ala Ile Phe Leu same time Phe gin gin 120 125 CTG CTC CGA GGA AAG GTG CGT TTC CTG CTC CTT GTA GTG 426 Leu Leu Arg Gly Lys wall Arg Phe Leu Leu Leu wall wall 130 135 140 GGG CCC TCC CTC TGT GCC AAG AGG GCC CCA CCC GCC ATA 465 Gly for Ser Leu Cys Ala Lys Arg Ala for for Ala Ile 145 150 155 GCT GTC CCG AGC AGC ACC TCT CCA TTC CAC ACA CTG AAC 504 Ala wall for Ser Ser Thr Ser for Phe His Thr Leu same time 160 165 AAG CTC CCA AAC AGG ACC TCT GGA TTG TTG GAG ACA AAC 543 Lys Leu for same time Arg Thr Ser Gly Leu Leu Glu Thr same time 170 175 180

TCC AGT ATC TCA GCC ACA ACT ACT GGC TCT GGA TTT CTC 582

Ser Ser Ile Ser Ala 185 Arg Thr Thr Gly 190 Ser Gly Phe Leu AAG AGG CTG CAG GCA TTC AGA GCC AAG ATT CCT GGT CTG 621 Lys Arg Leu gin Ala Phe Arg Ala Lys Ile for Gly Leu 195 200 205 CTG AAC CAA ACC TCC AGG TCC CTA GAC CAA ATC CCT GGA 660 Leu same time gin Thr Ser Arg Ser Leu Asp gin Ile for Gly 210 215 220 CAC CAG AAT GGG ACA CAC GGA CCC TTG AGT GGA ATT CAT 699 His gin same time Gly Thr His Gly for Leu Ser Gly Ile His 225 230 GGA CTC TTT CCT GGA CCC CAA CCC GGG GCC CTC GGA GCT 738 Gly Leu Phe for Gly for gin for Gly Ala Leu Gly Ala 235 240 245 CCA GAC ATT CCT CCA GCA ACT TCA GGC ATG GGC TCC CGG 777 for Asp Ile for for Ala Thr Ser Gly Met Gly Ser Arg 250 255 CCA ACC TAC CTC CAG CCT GGA GAG TCT CCT TCC CCA GCT 816 for Thr Tyr Leu gin for Gly Glu Ser for Ser for Ala 260 265 270 CAC CCT TCT CCT GGA CGA TAC ACT CTC TTC TCT CCT TCA 855 His for Ser for Gly Arg Tyr Thr Leu Phe Ser for Ser 275 280 285 ccc ACC TCG CCC TCC CCC ACA GTC CAG CTC CAG CCT CTG 894 for Thr Ser for Ser for Thr wall gin Leu gin for Leu 290 295 CTT CCT GAC CCC TCT GCG ATC ACA CCC AAC TCT ACC AGT 933 Leu for Asp for Ser Ala Ile Thr for same time Ser Thr Ser 300 305 310 CCT CTT CTA TTT GCA GCT CAC CCT CAT TTC CAG AAC CTG 972 for Leu Leu Phe Ala Ala His for His Phe gin same time Leu 315 320

TCT CAG GAA GAG TAAGGT GCTCAGACCC TGCCAACTTC 1010

Ser Gin Glu

325 328

AGCA 1014 (2) information for SEQ ID NO: 21 (i) Sequence characteristics:

(A) Length: 328 amino acids Type: amino acid Topology: straight

(xi) Sequence description: SEQ ID NO: 21

Ser 1 Pro Ala for for 5 Ala Cys Asp For Arg 10 Leu Leu same time Lys Leu 15 Leu Arg Asp Ser His wall Leu His Gly Arg Leu Ser gin Cys for 20 25 30 Asp Ile same time for Leu Ser Thr for wall Leu Leu for Ala wall Asp 35 40 4 5 Phe Thr Leu Gly Glu Trp Lys Thr gin Thr Glu gin Thr Lys Ala 50 55 60 gin Asp wall Leu Gly Ala Thr Thr Leu Leu Leu Glu Ala wall Met 65 70 75 Thr Ala Arg Gly gin wall Gly for for Cys Leu Ser Ser Leu Leu 80 85 90 wall gin Leu Ser Gly gin wall Arg Leu Leu Leu Gly Ala Leu gin 95 100 105 Asp Leu Leu Gly meth gin Gly Arg Thr Thr Ala His Lys Asp for 110 115 120 Ser Ala Ile Phe Leu same time Phe gin gin Leu Leu Arg Gly Lys wall 125 130 135 Arg Phe Leu Leu Leu wall wall Gly for Ser Leu cys Ala Lys Arg 140 145 150 Ala for for Ala Ile Ala wall for Ser Ser Thr Ser for Phe His 155 160 165 Thr Leu same time Lys Leu for same time Arg Thr Ser Gly Leu Leu Glu Thr 170 175 180 same time Ser Ser Ile Ser Ala Arg Thr Thr Gly Ser Gly Phe Leu Lys 185 190 195 Arg Leu gin Ala Phe Arg Ala Lys Ile for Gly Leu Leu same time gin 200 205 210 Thr Ser Arg Ser Leu Asp gin Ile for Gly His gin same time Gly Thr 215 220 225 His Gly for Leu Ser Gly Ile His Gly Leu Phe for Gly for gin 230 235 240 for Gly Ala Leu Gly Ala for Asp Ile for for Ala Thr Ser Gly 245 250 255 Met Gly Ser Arg for Thr TYR Leu gin for Gly Glu Ser for Ser 260 265 270

for Ala His for Ser 275 for Gly Arg Tyr Thr 280 Leu Phe Ser for Ser 285 for Thr Ser for Ser 290 for Thr wall gin Leu 295 gin for Leu Leu for 300 Asp for Ser Ala Ile 305 Thr for same time Ser Thr 310 Ser for Leu Leu Phe 315 Ala Ala His for His 320 Phe gin same time Leu Ser 325 gin Glu Glu 328

(2) Information for SEQ ID NO: 22

(i) Sequence characteristics:

(A) LENGTH: 5141 base pairs

Type: nucleic acid

Thread: double

Topology: linear (xi) Sequence description: SEQ ID NO: 22

TTCGAGCTCG CCCGACATTG ATTATTGACT AGAGTCGATC GACAGCTGTG 50 GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CGAGCAGGCA 100 GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG 150 TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA 200 GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG CCCCTAACTC 250 CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT 300 TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC AGAAGTAGTG 350 AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAAAGCTA GCTTATCCGG 400 CCGGGAACGG TGCATTGGAA CGCGGATTCC CCGTGCCAAG AGTGACGTAA 450 GTACCGCCTA TAGAGCGATA AGAGGATTTT ATCCCCGCTG CCATCATGGT 500 TCGACCATTG AACTGCATCG TCGCCGTGTC CCAAAATATG GGGATTGGCA 550 AGAACGGAGA CCTACCCTGG CCTCCGCTCA GGAACGAGTT CAAGTACTTC 600 CAAAGAATGA CCACAACCTC TTCAGTGGAA GGTAAACAGA ATCTGGTGAT 650 TATGGGTAGG AAAACCTGGT TCTCCATTCC TGAGAAGAAT CGACCTTTAA 700 AGGACAGAAT TAATATAGTT CTCAGTAGAG AACTCAAAGA ACCACCACGA 750

GGAGCTCATT TTCTTGCCAA AAGTTTGGAT GATGCCTTAA GACTTATTGA

ACAACCGGAA TTGGCAAGTA AAGTAGACAT GGTTTGGATA GTCGGAGGCA 850 GTTCTGTTTA CCAGGAAGCC ATGAATCAAC CAGGCCACCT TAGACTCTTT 900 GTGACAAGGA TCATGCAGGA ATTTGAAAGT GACACGTTTT TCCCAGAAAT 950 TGATTTGGGG AAATATAAAC CTCTCCCAGA ATACCCAGGC GTCCTCTCTG 1000 AGGTCCAGGA GGAAAAAGGC ATCAAGTATA AGTTTGAAGT CTACGAGAAG 1050 AAAGACTAAC AGGAAGATGC TTTCAAGTTC TCTGCTCCCC TCCTAAAGCT 1100 ATGCATTTTT ATAAGACCAT GGGACTTTTG CTGGCTTTAG ATCCCCTTGG 1150 CTTCGTTAGA ACGCGGCTAC AATTAATACA TAACCTTATG TATCATACAC 1200 ATACGATTTA GGTGACACTA TAGATAACAT CCACTTTGCC TTTCTCTCCA 1250 CAGGTGTCCA CTCCCAGGTC CAACTGCACC TCGGTTCTAA GCTTCTGCAG 1300 GTCGACTCTA GAGGATCCCC GGGGAATTCA ATCGATGGCC GCCATGGCCC 1350 AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC 1400 AAATTTCACA AATAAAGCAT _TTTTTTCACT GCATTCTAGT TGTGGTTTGT 1450 CCAAACTCAT CAATGTATCT TATCATGTCT GGATCGATCG GGAATTAATT 1500 CGGCGCAGCA CCATGGCCTG AAATAACCTC TGAAAGAGGA ACTTGGTTAG 1550 GTACCTTCTG AGGCGGAAAG AACCAGCTGT GGAATGTGTG. TCAGTTAGGG 1600 TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA 1650 TCTCAATTAG TCAGCAACCA GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG 1700 GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CA.TAGTCCCG 1750 CCCCTAACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC 1800 TCCGCCCCAT GGCTGACTAA ΤΤΤΊΤΤΤΤΆΤ TTATGCAGAG GCCGAGGCCG 1850 CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC 1900 CTAGGCTTTT GCAAAAAGCT GTTACCTCGA GCGGCCGCTT AATTAAGGCG 1950 CGCCATTTAA ATCCTGCAGG TAACAGCTTG GCACTGGCCG TCGTTTTACA 2000 ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 2050 CACATCCCCC CTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT 2100 CGCCCTTCCC AACAGTTGCG TAGCCTGAAT GGCGAATGGC GCCTGATGCG 2150 GTATTTTCTC CTTACGCATC TGTGCGGTAT TTCACACCGC ÄTACGTCAAA 2200

GCAACCATAG TACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG 2250 TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT 2300 CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG 2350 TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT AGTGCTTTAC 2400 GGCACCTCGA CCCCAAAAAA CTTGATTTGG GTGATGGTTC ACGTAGTGGG 2450 CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT 2500 CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC ÄACCCTATCT 2550 CGGGCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG. 2600 TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT TTAACAAAAT 2650 ATTAACGTTT ACAATTTTAT GGTGCACTCT CAGTACAATC TGCTCTGATG 2700 CCGCATAGTT AAGCCAACTC CGCTATCGCT ACGTGACTGG GTCATGGCTG 2750 CGCCCCGACA CCCGCCAACA CCCGCTGACG CGCCCTGACG GGCTTGTCTG 2800 CTCCCGGCAT CCGCTTACAG ACAAGCTGTG ACCGTCTCCG GGAGCTGCAT 2850 GTGTCAGAGG TTTTCACCGT CATCACCGAA ACGCGCGAGG CAGTATTCTT 2900 GAAGACGAAA GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG 2950 ATAATAATGG TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG 3000 CGGAACCCCT ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC 3050 TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG 3100 AGTATGAGTA TTCAACATTT CCGTGTCGCC CTTATTCCCT TTTTTGCGGC 3150 ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG AAAGTAAAAG 3200 ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC 3250 AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT 3300 GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGT.GATG 3350 ACGCCGGGCA AGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC 3400 TTGGTTGAGT ACTCACCAGT CACAGAAAAG CATCTTACGG ATGGCATGAC 3450 AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT AACACTGCGG 3500 CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT 3550 TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA 3600

GCTGAATGAA GCCATACCAA ACGACGÄGČG TGACACCACG ATGCCAGCAG 3650 CAATGGCAAC AACGTTGCGC AAACTATTAA CTGGCGAACT ACTTACTCTA 3700 GCTTCCCGGC AACAATTAAT AGACTGGATG GAGGCGGATA AAGTTGCAGG 3750 ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT GCTGATAAAT 3800 CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA .3850 GATGGŤAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC 3900 AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA 3950 TTAAGCATTG GTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATT 4000 GATTTAAAAC TTCATTTTTA ATTTAAAAGG ÄTCTAGGTGA AGATCCTTTT 4050 TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGAG 4100 CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT 4150 CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT 4200 GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG '4250 GCTTCAGCAG AGCGCAGATA CCAAATACTG TCCTTCTAGT GTAGCCGTAG 4300 TTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT 4350 GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG TCGTGTCTTA 4400 CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 4450 TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC 4500 CGAACTGAGA TACCTACAGC GTGAGCATTG AGAAAGCGCC ' ACGCTTCCCG 4550 AAGGGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA 4600 GAGCGCACGA GGGAGCTTCC AGGGGGAAAC GCCTGGTATC TTTATAGTCC 4650 TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGT 4700 CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG 4750 TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC 4800 CCCTGATTCT GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG 4850 CTCGCCGCAG CCGAACGACC GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG 4900 GAAGAGCGCC CAATACGCAA ACCGCCTCTC CCCGCGCGTT GGCCGATTCA 4950

TTAATCCAGC TGGCACGACA GGTTTCCCGA CTGGAAAGCG GGCAGTGAGC 5000

- 44 GCAACGCAAT TAATGTGAGT TACCTCACTC ATTAGGCACC CCAGGCTTTA

CACTTTATGC TTCCGGCTCG TATGTTGTGT GGAATTGTGA GCGGATAACA

ATTTCACACA GGAAACAGCT ATGACCATGA TTACGAATTA A 5141

5050

5100 (2) Information for SEQ ID NO: 23 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

base pairs nucleic acid single linear

(xi) Sequence description: SEQ ID NO: 23

ATGTCNCCNG CNCCNCCNGC N 21 (2) Information for SEQ ID NO: 24 (i) Sequence characteristics:

(A) Length: 21 base pairs (B) Type: nucleic acid (C) Fiber: single (D) Topology: linear (xi) Sequence description: SEQ ID NO: 24

ATGTCTCCAG CGCCGCCAGC G 21 (2) Information for SEQ ID NO: 25 (i) Sequence characteristics:

(A) Length: 21 base pairs (B) Type: nucleic acid (C) Fiber: single (D) Topology: linear (xi) Sequence description: SEQ ID NO: 25

ATGTCGCCTG CTCCACCTGC T21

- 43 (2) Information for SEQ ID NO: 26 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 26 base pairs nucleic acid single linear

ATGTCGCCAG CGCCACCAGC C 21 (2) Information for SEQ ID NO: 27 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 27 base pairs nucleic acid single linear

ATGTCCCCAG CCCCACCCGC A 21 (2) Information for SEQ ID NO: 28 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 28 base pairs nucleic acid single linear

ATGTCGCCAG CGCCGCCAGC G 21 (2) Information for SEQ ID NO: 29 (i) Sequence characteristics:

(A) Length: 21 amino acids

- 44 (B) Type: amino acid (D) Topology: linear (xi) Sequence description: SEQ ID NO: 29

Ser Pro Ala Pro Pro Cys Asp Pro Arg Leu Leu Asn Lys Leu

1015

Leu Arg Asp Asp His V al Leu His Gly Arg

2025 (2) Information for SEQ ID NO: 30 (i) Sequence characteristics:

(A) Length: 26 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 30

Ser Pro Ala Pro Pro Cys Asp Pro Arg Leu Leu Asn Lys Leu

1 5 10 15

Leu Arg. Asp Asp Xaa Val Leu His Gly Arg Leu

2025 26 (2) Information for SEQ ID NO: 31 (i) Sequence characteristics:

(A) Length: 25 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 31

Ser Pro Ala Pro Pro Xaa Asp Pro Arg Leu Leu Asn Lys Leu

1015

Leu Arg - Asp Asp Val Leu His Gly Arg

- 45 (2) Information for SEQ ID NO: 32 (i) Sequence characteristics:

(A) Length: 14 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 32

Xaa Pro Ala Pro Pro Ala Xaa Asp Pro Arg Leu Xaa Asn Lys

14 (2) Information for SEQ ID NO: 33 (i) Sequence characteristics:

(A) Length: 9 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 33 For Arg Leu Leu Asn Lys Leu Leu Arg

(9) Information for SEQ ID NO: 34 (i) Sequence characteristics:

(A) Length: 45 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 34

GCCGTGAAGG ACGTGGTCGT CACGAAGCAG TTTATTTAGG AGTCG 45 (2) Information for SEQ ID NO: 35 (i) Sequence characteristics:

(A) Length: 20 base pairs

(B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 35

CCNGCNCCNC CNGCNTGYGA 20 (2) Information for SEQ ID NO: 36 (i) Sequence characteristics:

(A) Length: 21 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 36

NCCRTGNARN ACRTGRTCRT C 21 (2) Information for SEQ ID NO: 37 (i) Sequence characteristics:

(A) Length: 69 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 37

CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA 50 TGACCACGTT CAGCACGGC 69 (2) Information for SEQ ID NO: 38 (i) Sequence characteristics:

(A) Length: 69 base pairs (B) Type: nucleic acid (C) Fiber: one

- 47 (D) Topology: linear (xi) Sequence description: SEQ ID NO: 38

GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG ACGGAGCACT 50 ACTGGTGCAA GTCGTGCCG 69 (2) Information for SEQ ID NO: 39 (i) Sequence characteristics:

(A) Length: 69 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 39

CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 50

CGACCACGTC CATCACGGC 69 (2) Information for SEQ ID NO: 40 (i) Sequence characteristics:

(A) Length: 69 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 40

GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCACT 50 GCTGGTGCAG GTAGTGCCG 69 (2) Information for SEQ ID NO: 41 (i) Sequence Characteristics:

(A) Length: (B) Type: 69 base pairs nucleic acid (C) Fiber: one

48 CCAGCACCGC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 50

CGATCATGTC TATCACGGT 69 (D) Topology: Linear (xi) Sequence Description: SEQ ID NO: 41 (2) Information for SEQ ID NO: 42 (i) Sequence Characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 42 base pairs nucleic acid single linear

GGTCGTGGCG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCACT 50

GCTAGTACAG ATAGTGCCA 69 (2) Information for SEQ ID NO: 43 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 43 base pairs nucleic acid single linear

GCTAGCTCTA GAAATTGCTC CTCGTGGTCA TGCTTCT 37 (2) Information for SEQ ID NO: 44 (i) Sequence characteristics:

(A) LENGTH:

(B) Type:

(C) Fiber:

(D) Topology:

base pairs nucleic acid single linear

- 49 (xi) Sequence description: SEQ ID NO: 44

CAGTCTGCCG TGAAGGACAT GG 22 (2) Information for SEQ ID NO: 45 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 45 base pairs nucleic acid single linear

TGTGGACTTT AGCTTGGGAG AATG 24 (2) Information for SEQ ID NO: 46 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 46 base pairs nucleic acid single linear

GGTCCAAGGGA CCTGGAGGTT TG 22 (2) Information for SEQ ID NO: 47 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 47 base pairs nucleic acid single linear

ATCGATATCG ATAGCCAGAC ACCCCGGCCA G 31

- 50 (2) Information for SEQ ID NO: 48 (i) Sequence characteristics:

(A) Length: 36 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 48

GCTAGCTCTA GACAGGGAAG GGAGCTGTAC ATGAGA 36 (2) Information for SEQ ID NO: 49 (i) Sequence characteristics:

(A) Length: 24 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 49

CTCCTTGGAA CCCAGGGCAG GACC 24 (2) Information for SEQ ID NO: 50 (i) Sequence characteristics:

(A) Length: 24 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 50

GGTCCTGCCC TGGGTTCCAA GGAG 24 (2) Information for SEQ ID NO: 51 (i) Sequence characteristics:

(A) Length: 27 base pairs

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 51 nucleic acid single linear

CTGCTCCGAG GAAAGGACTT CTGGATT 27 (2) Information for SEQ ID NO: 52 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 52 base pairs nucleic acid single linear

AATCCAGAAG TCCTTTCCTC GGAGCAG 27 (2) Information for SEQ ID NO: 53 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 53 base pairs nucleic acid single linear

CCCTCTGCGT CGCGGCGGCC CCACCCAC 28 (2) Information for SEQ ID NO: 54 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

base pairs nucleic acid single linear

- 52 (xi) Sequence description: SEQ ID NO: 54

GTGGGTGGGG CCGCCGCGAC GCAGAGGG 28 (2) Information for SEQ ID NO: 55 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 55 base pairs nucleic acid single linear

GACTCGAGGA TCCATCGATT TTTTTTTTTT TTTTT ₃₅ (2) Information for SEQ ID NO: 56 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 56 base pairs nucleic acid single linear

GACTCGAGGA TCCATCG 17 (2) Information for SEQ ID NO: 57 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 57 base pairs nucleic acid single linear

GCTAGCTCTA GAAGCCCGGC TCCTCCTGCC TG 32

- 53 (2) Information for SEQ ID NO: 58 (i) Sequence characteristics:

(A) Length: 21 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 58

CGAAATTAAC CCTCACTAAA G 21 (2) Information for SEQ ID NO: 59 (i) Sequence characteristics:

(A) Length: 103 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 59

TGCAGCAAGG GCTACTGCCA CACTCGAGCT GCGCAGATGC TAGCCTCAAG 50 · ATGGCTGATC CAAATCGATT CCGCGGCAAA GATCTTCCGG TCCTGTAGAA 100 GCT 103 (2) Information for SEQ ID NO: 60 (i) Sequence characteristics:

(A) Length: 103 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 60

AGCTTCTACA GGACCGGAAG ATCTTTGCCG CGGAATCGAT TTGGATCAGC 50

CATCTTGAGG CTAGCATCTG CGCAGCTCGA GTGTGGCAGT AGCCCTTGCT ¹¹⁰⁰

GCA 103

- 54 (2) Information for SEQ ID NO: 61 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description:

base pairs nucleic acid single linear

IDNO: 61

TCTCGCTACC GTTTACAG 18 (2) Information for SEQ ID NO: 62

(i) Sequence characteristics:

(A) Length: 25 base pairs (B) Type: nucleic acid (C) Fiber: single (D) Topology: linear (xi) Sequence description: SEQ ID NO: 62

CAGGTACCCA CCAGGCGGTC TCGGT 25 (2) Information for SEQ ID NO: 63 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 63 base pairs nucleic acid single linear

GGGCCATGAC ACTGTCAA 18 (2) Information for SEQ ID NO: 64 (i) Sequence characteristics:

(A) Length: 40 base pairs

(B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 64

GACCGCCACC GAGACCGCCT GGTGGGTACC TGTGGTCCTT 40 (2) Information for SEQ ID NO: 65 (i) Sequence characteristics:

(A) Length: 32 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 65

ATCGATATCG ATCAGCCAGA CACCCCGGCC AG 32 (2) Information for SEQ ID NO: 66 (i) Sequence characteristics:

(A) Length: 35 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 66

TCTAGATCTA GATCACCTGA CGCAGAGGGT GGACC 35 (2) Information for SEQ ID NO: 67 (i) Sequence characteristics:

(A) LENGTH: 33 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

- 56 (xi) Sequence description: SEQ ID NO: 67

AGTCGACGTC GACGTCGGCA GTGTCTGAGA ACC 33 (2) Information for SEQ ID NO: 68 (i) Sequence characteristics:

(A) Length: 36 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 68

AGTCGACGTC GACTCACCTG ACGCAGAGGG TGGACC 36 (2) Information for SEQ ID NO: 69 (i) Sequence characteristics:

(A) Length: 62 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 69

CGCGTATGCC AGCCCGGCTC CTCCTGCTTG TGACCTCCGA GTCCTCAGTA 50

AACTGCTTCG TG 62 (2) Information for SEQ ID NO: 70 (i) Sequence characteristics:

(A) Length: 61 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 70

- 58 AGTCACGAAG CAGTTTACTG AGGACTCGGA GGTCACAAGC AGGAGGAGCC 50

GGGCTGGCAT A 61 (2) Information for SEQ ID NO: 71 (i) Sequence characteristics:

(A) Length: 37 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 71

CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTAA. 37 (2) Information for SEQ ID NO: 72 (i) Sequence characteristics:

(A) Length: 37 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 72

CGCGTTAAGA AGAAATGCGA TATTCTTTTT CATAATT 37 (2) Information for SEQ ID NO: 73 (i) Sequence characteristics:

(A) Length: 69 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 73

CTAGAATTAT GAAAAAGAAT ATCGCATTTC ATCACCATCA CCATCACCAT 50

CACATCGAAG GTCGTAGCC 69

- 58 (2) Information for SEQ ID NO: 74 (i) Sequence characteristics:

(A) Length:

(B) Type:

(C) Fiber:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 74 base pairs nucleic acid single linear

TACGACCTTC CATGTGATGG TGATGGTGAT GGTGATGAAA TGCGATATTC 50

TTTTTCATAA TT 62 (2) Information for SEQ ID NO: 75 (i) Sequence characteristics:

(A) Length: 69 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 75

CTAGAATTAT GAAAAAGAAT ATCGCATTTC ATCACCATCA CCATCACCAT 50

CACATCGAAC CACGTAGCC 69 (2) Information for SEQ ID NO: 76 (i) Sequence characteristics:

(A) Length: 62 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 76

TACGTGGTTC GATGTGATGG TGATGGTGAT GGTGATGAAA TGCGATATTC 50

TTTTTCATAA TT 62

- 59 (2) Information for SEQ ID NO: 77 (i) Sequence characteristics:

(A) Length: 19 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 77

TCCACCCTCT GCGTCAGGT 19 (2) Information for SEQ ID NO: 78 (i) Sequence characteristics:

(A) Length: 18 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 78

AGCTACCTGA CGCAGAGG 18 (2) Information for SEQ ID NO: 79 (i) Sequence characteristics:

(A) Length: 62 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 79

GCAGCAGTTC TAGAATTATG TCNCCNGCNC CNCCNGCNTG TGACCTCCGA

GTTCTCAGTA AA 62 (2) Information for SEQ ID NO: 80

- 60 (i) Sequence characteristics:

(A) Length: 49 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 80

GAAGGACATG GGAGTCACGA AGCAGTTTAC TGAGAACTCG GAGGTCCCA 49 (2) Information for SEQ ID NO: 81 (i) Sequence characteristics:

(A) Length: 45 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 81

CTAGAATTAT GAAAAAGAAT ATCGCATTTA TCGAAGGTCG TAGCC 45 (2) Information for SEQ ID NO: 82 (i) Sequence characteristics:

(A) Length: 38 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 82

TACGACCTTC GATAAATGCG ATATTCTTTT TCATAATT 38 (2) Information for SEQ ID NO: 83 (i) Sequence characteristics:

(A) Length: 45 base pairs (B) Type: nucleic acid

- 61 - (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 83

CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTAAACG TAGCC 45 (2) Information for SEQ ID NO: 84 (i) Sequence characteristics:

(A) LENGTH: 38 base pairs (B) Type: nucleic acid (C) Fiber: one (D) Topology: straight

(xi) Sequence description: SEQ

TACGTTTAAG AAGAAATGCG ATATTCTTTT TCATAATT 38 (2) Information for SEQ ID NO: 85 (i) Sequence characteristics:

(A) Length: 168 amino acids (B) Type: amino acid (D) Topology: straight (xi) Sequence description: SEQ ID NO: 85 Met Lys Lys same time Ile Ala Phe Leu Leu same time Ala Tyr Ala Ser for 1 5 10 15 Ala Pro Pro Ala Cys Asp Leu Arg wall Leu Ser Lys Leu Leu Arg 20 25 30 Asp Ser His wall Leu His Ser Arg Leu Ser gin Cys for Glu wall 35 40 45 His Pro Leu for Thr Pro Val Leu Leu for Ala wall Asp Phe Ser 50 55 1 60 Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala gin Asp 65 70 75 Ile Leu Gly Ala wall Thr Leu Leu Leu Glu Gly wall Met Ala Ala 80 85 , · 90 Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly gin 95 100 105

Leu Ser Val Arg Leu 110 Leu Leu Gly Ala. Leu Gin Sei ^ Leu 115 120 Leu Gly Thr gin Leu for for gin Gly Arq Thr Thr Ala His Lys 125 130 135 Asp for same time Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly 140 145 150 Lys wall Arg Phe Leu Met Leu wall Gly Gly Ser Thr Leu Cys wall 155 160 Ί65 Arg Arg Ala

168 (2) Information for SEQ ID NO: 86

(i) Sequence characteristics:

(A) Length: 174 amino acids (B) Type:

(D) Topology:

amino acid linear (xi) Sequence description: SEQ ID NO: 86

Met Lys 1 Lys Asn Al Ala 5 Phe His His His His .10 His His His His 15 Ile Glu Gly Arg Ser for Ala for for Ala Cys' Asp Leu Arg wall 20 25 30 Leu Ser Lys Leu Leu Arg Asp Ser His wall Leu His Ser Arg Leu 35 40 45 Ser gin Cys for Glu wall His for Leu for Thr for wall Leu Leu 50 55 60 for Ala wall Asp Phe Ser Leu Gly Glu Trp Lys Thr gin Met Glu 65 70 75 Glu Thr Lys Ala gin Asp Ile Leu Gly Ala wall Thr Leu Leu Leu 80 85 90 Glu Gly wall Met Ala Ala Arg Gly gin Leu Gly for THR Cys Leu 95 100 105 Ser Ser Leu Leu Gly gin Leu Ser Gly gin wall Arg Leu Leu Leu 110 115 120 Gly Ala Leu gin Ser Leu Leu Gly Thr gin Leu for for gin Gly 125 130 1 135 Arg Thr Thr Ala His Lys Asp for same time Ala Ile Phe Leu 1 sera Phe 140 145 T 150 gin His Leu Leu Arg Gly Lys wall Arg Phe Leu Met Leu wall Gly 155 160 165 Gly Ser Thr Leu Cys wall Arg Arg Ala 170 174

(2) Information for SEQ ID NO: 87 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 87

174 amino acids linear amino acid

Met Lys Lys same time Ile Ala Phe His His His His His His His His 1 5 10 15 Ile Glu for Arg Ser for Ala for for Ala Cys Asp Leu Arg wall 20 25 30 Leu Ser Lys Leu Leu Arg Asp Ser His wall Leu His Ser Arg Leu 35 40 45 Ser gin Cys for Glu wall His for Leu for Thr for wall Leu Leu 50 55 60 for Ala wall Asp Phe Ser Leu Gly Glu Trp Lys Thr gin Met Glu 65 70 75 Glu Thr Lys Ala gin Asp Ile Leu Gly Ala wall Thr Leu Leu Leu 80 85 90 Glu Gly wall Met Ala Ala Arg Gly gin Leu Gly for Thr Cys Leu 95 100 105 Ser Ser Leu Leu Gly gin Leu Ser Gly gin wall Arg Leu Leu Leu 110 115 120 Gly Ala Leu gin Ser Leu Leu Gly Thr gin Leu for for gin Gly 125 130 135 Arg Thr Thr Ala His Lys Asp for same time Ala Ile Phe Leu Ser Phe 140 145 150 gin His Leu Leu Arg Gly Lys wall Arg Phe Leu Met Leu wall Gly 155 160 165 Gly Ser Thr Leu Cys wall Arg Arg Ala

170 174 (2) Information for SEQ ID NO: 88 (i) Sequence characteristics:

(A) Length: 7 amino acids (B) Type: amino acid (D) Topology: straight (xi) Sequence description: SEQ ID NO: 88

64 Thr Thr Ala His Lys Asp Pro

(7) Information for SEQ ID NO: 89 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 89 amino acid linear amino acid

His Val Leu His (2) Information for SEQ ID NO: 90 (i) Sequence Characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 90 amino acid linear amino acid

Ser Arg Leu Ser

4 (2) Information for SEQ ID NO: 91 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 91 amino acid linear amino acid

Ser His Val Leu

4

- 65 (2) Information for SEQ ID NO: 92 (i) Sequence characteristics:

(A) Length: (B) Type: 4 amino acids amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 92 His Ser Arg Leu (2) Information for SEQ ID NO: 93 (i) Sequence Characteristics:

(A) Length: 4 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 93

Ala Val Asp Phe (2) Information for SEQ ID NO: 94 (i) Sequence characteristics:

(A) Length: 4 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 94

Ser Leu Gly Glu (2) Information for SEQ ID NO: 95 (i) Sequence characteristics:

(A) Length: 4 amino acids

- 66 (B) Type: amino acid (D) Topology: linear (xi) Sequence description: SEQ ID NO: 95 Ala Val Thr Leu

4 (2) Information for SEQ ID NO: 96 (i) Sequence characteristics:

(A) Length: 4 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 96 Leu Leu Glu Gly

4 (2) Information for SEQ ID NO: 97 (i) Sequence characteristics:

(A) Length: 4 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 97

Leu Ser Ser Leu

4 (2) Information for SEQ ID NO: 98 (i) Sequence characteristics:

(A) Length: 4 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 98

- 68 Leu Gly Gin Leu

4 (2) Information for SEQ ID NO: 99 (i) Sequence characteristics:

(A) LENGTH:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 99 amino acids linear amino acid

Cys Xaa Leu Ser. Ser

5 (2) Information for SEQ ID NO: 100 (i) Sequence characteristics:

(A) LENGTH:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 100 amino acid amino acid linear

Gly Gin

4 (2) Information for SEQ ID NO: 101 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 101 amino acid amino acid linear

Ser Leu Leu

- 68 (2) Information for SEQ ID NO: 102 (i) Sequence characteristics:

(A) LENGTH: 4 amino acids (B) Type: amino acid (D) Topology: straight (xi) Sequence description: SEQ ID NO: 102

Gly Gin Leu Ser

4

(2) Information for SEQ ID NO: 103 (i) Sequence characteristics:

(A) LENGTH:

(B) Type:

(D) Topology:

amino acids amino acid linear (xi) Sequence description: SEQ ID NO: 103

Cys Leu Ser

4 (2) Information for SEQ ID NO: 104

(i) Sequence characteristics:

(A) LENGTH:

(B) Type:

(D) Topology:

amino acids amino acid linear (xi) Sequence description: SEQ ID NO: 104

Leu Gin Ser Leu (2) Information for SEQ ID NO: 105 (i) Sequence characteristics:

(A) Length: 4 amino acids

(B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 105

Leu Gly Thr Gin (2) information for SEQ ID NO: 106 (i) Sequence characteristics:

(A) Length: 4 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 106 Ala Leu Gin Ser (2) Information for SEQ ID NO: 107 (i) Sequence characteristics:

(A) Length: 4 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 107 Leu Leu Gly Thr (2) Information for SEQ ID NO: 108 (i) Sequence characteristics:

(A) Length: 4 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ

- 70 Asn Ala ile Phe

4 (2) Information for SEQ ID NO: 109 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 109 amino acid linear amino acid

Leu Ser Phe Gin (2) Information for SEQ ID NO: 110 (i) Sequence characteristics:

(A) Length: 22 amino acids (B) Type: amino acid (D) Topology:

linear (xi) Sequence description: SEQ ID NO: 110

Ser Pro Ala Pro Pro Cys Asp Leu Arg Val Leu Ser Lys Leu

Leu Arg

22 (2) Information for SEQ ID NO: 111 (i) Sequence characteristics:

(A) Length: 24 amino acids (B) Type: amino acid (D) Topology:

linear (xi) Sequence description: SEQ ID NO: 111

- 71 His Ser Arg Leu Ser Gin Cys For Glu Val His Pro Leu Thr

10 15

For Val Leu Leu For Ala Val Asp Phe (2) Information for SEQ ID NO: 112 (i)

(A) LENGTH: 23 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 112

Ser Leu Gly Glu (Hu Glu Thr Lys Ala Gin

Asp He Leu Gly Ala Val Thr Leu (2) Information for SEQ ID NO: 113 (i) Sequence characteristics:

(A) Length: 21 amino acids

(B) Type:

(D) Topology:

amino acid linear (xi) Sequence description: SEQ ID NO: 113

Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Le Th Glu Pro Thr 1 5 10 15

Cys Leu Ser Ser Leu Leu

21 (2) Information for SEQ ID NO: 114 (i) Sequence characteristics:

(A) Length: 16 amino acids (B) Type: amino acid

- 72 Gly Gin Leu Ser Gly Gin Val Arg

(D) Topology: linear (xi) Sequence description: SEQ ID NO: 114

Ser (2) Information for SEQ ID NO: 115 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 115 amino acids amino acid linear

Leu Leu Gly Thr Gin Leu Pro Gin Gly Arg Thr Thr Ala His

10 15

Lys Asp For Asn Ala He Phe

22 (2) Information for SEQ ID NO: 116 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 116 amino acids amino acid linear

Leu Ser Phe Gin His

5 10

Leu Val Gly Gly Ser Thr Leu Cys Val Arg

(2) Information for SEQ ID NO: 117

- 73 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 117 amino acid linear amino acid

Met Pro Pro Ala (2) Information for SEQ ID NO: 118 (i) Sequence Characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 118 amino acid linear amino acid

Met Ala Pro Pro Ala (2) Information for SEQ ID NO: 119 (i) Sequence Characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 119 amino acids linear amino acid

Met Pro Ala Pro Pro Ala (2) Information for SEQ ID NO: 120 (i) Sequence Characteristics:

(A) Length: 7 amino acids (B) Type: amino acid (D) Topology: linear (xi) Sequence description: SEQ ID NO: 120

Met Ser Pro Ala Pro Pro Ala

(7) Information for SEQ ID NO: 121 (i) Sequence characteristics:

(A) Length: 4 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 121

Ala Pro Pro Ala (2) Information for SEQ ID NO: 122 (i) Sequence Characteristics:

(A) Length:

(B) Type:

(D) Topology:

linear amino acids

(xi) Sequence description: SEQ ID NO: 122

Pro Ala Pro Pro Ala

5 (2) Information for SEQ ID NO: 123 (i) Sequence characteristics:

(A) Length: 6 amino acids (B) Type: amino acid (D) Topology: straight

(xi) Sequence description: SEQ ID NO: 123

- 75 Ser Pro Ala Pro Pro Ala (2) Information for SEQ ID NO: 124 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 124 amino acid amino acid linear

Val Arg Arg Ala

4 (2) Information for SEQ ID NO: 125 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 125 amino acids linear amino acid

Val Arg Arg Ala Pro

5 (2) Information for SEQ ID NO: 126 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 126 amino acids linear amino acid

Val Arg Arg Ala Pro Pro

5 6

- 76 (2) Information for SEQ ID NO: 127 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description:

amino acids amino acid linear IDNO: 127

Val Arg Arg Ala Pro Pro Thr

5 7

(2) Information for SEQ ID NO: 128 (i) Sequence characteristics:

(A) Length: 8 amino acids (B) Type: amino acid (D) Topology:

linear (xi) Sequence description: SEQ ID NO: 128

Val Arg Arg Ala Pro Pro Thr Thr

(8) Information for SEQ ID NO: 129

(i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

amino acids amino acid linear (xi) Sequence description: SEQ ID NO: 129

Val Arg Arg Ala Pro Thr Thr Ala

(9) Information for SEQ ID NO: 130 (i) Sequence characteristics:

(A) Length: 10 amino acids

- 77 amino acid linear (B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 130

Val Arg Arg Pro Pro Thr Thr Ala Val

5 10 (2) Information for SEQ ID NO: 131 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 131 amino acids linear amino acid

Val Arg Arg Thr Thr Thr Ala Val Pro

(2) Information for SEQ ID NO: 132 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 132 amino acid linear amino acid

Val Arg Arg Ala Pro Pro Thr Ala Val Pro Ser

5 10 12 (2) Information for SEQ ID NO: 133 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 133 amino acid linear amino acid

I

- 78 Val Arg Arg Ala Pro Pro |

5 10 13 (2) Information for SEQ ID NO: 134 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 134 amino acids linear amino acid

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

5 10 (2) Information for SEQ ID NO: 135 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 135 amino acids linear amino acid

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

5 10 (2) Information for SEQ ID NO: 136 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 136

Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg amino acids linear amino acid

Leu

Thr

Thr

Thr

Ser

Ser

- 79 (2) Information for SEQ ID NO: 137 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 137 amino acids amino acid linear

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

5 10

Thr

Ser

Leu Val (2) Information for SEQ ID NO: 138 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 138 amino acid linear amino acid

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

5 10

Thr

Ser

Leu Val Leu (2) Information for SEQ ID NO: 139 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 139 amino acids linear amino acid

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

5 10

Thr

Ser

I

- 80 Leu Val Leu Thr (2) Information for SEQ ID NO: 140 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 140 amino acids amino acid linear

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

5 10

Leu Val Leu Thr Leu (2) Information for SEQ ID NO: 141 (i) Sequence characteristics:

(A) LENGTH:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 141 amino acids amino acid linear

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

5 10

Leu Val Leu Thr Leu Asn

21 (2) Information for SEQ ID NO: 142 (i) Sequence characteristics:

(A) Length:

(B) Type:

(D) Topology:

(xi) Sequence description: SEQ ID NO: 142 amino acids linear amino acid

- 81 Val Arg Arg

10 15

Leu Val Leu Thr Leu Asn Glu

22 (2) Information for SEQ ID NO: 143 (i) Sequence characteristics:

(A) Length: 23 amino acids (B) Type: amino acid (D) Topology:

straight

(xi) Sequence description: SEQ ID NO: 143

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

Leu Val Leu Thr Leu Asn Glu Leu (2) Information for SEQ ID NO: 144 (i) Sequence characteristics:

(A) Length: 24 amino acids (B) Type: amino acid

(D) Topology:

linear (xi) Sequence description: SEQ ID NO: 144

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

Leu Val Leu Thr Leu Asn Glu Leu For ττ g

I

- 169 I I

Claims (39)

An isolated substantially homogeneous mpl ligand polypeptide. The mpl ligand polypeptide of claim 1, selected from the group consisting of a) a fragment polypeptide (b) a variable polypeptide; and c) a chimeric polypeptide. The mpl ligand polypeptide of claim 1, selected from the group consisting of a) a polypeptide isolated from a mammal (b) a polypeptide produced by recombinant means; and c) a polypeptide produced by a synthetic method. The mpl ligand polypeptide of claim 1, selected from the group consisting of (a) human polypeptide; and b) a polypeptide that is non-immunogenic in humans. 5. An isolated substantially homogeneous mpl agonist, characterized in that: a) the agonist stimulates the incorporation of labeled nucleotides ( ³ H-thymidine) into DNA of IL-3 dependent Ba / F3 cells infected with human mpl P; or b) the agonist stimulates ³⁵ S incorporation into circulating platelets in a spontaneous bouncing platelet assay. The polypeptide fragment of claim 2 represented X-hTPO (7-151) -Y, wherein hTPO (7-151) represents the human TPO (hML) amino acid sequence from Cys ⁷ to Cys ¹⁵¹ , inclusive; · X represents the amino group Cys ⁷ or the amino terminus of the amino acid I of the remainder (s) selected from the group M, ί MA - 170 I i I MPA, MPPA. (SEQ ID NO: 117) MAPPA. (SEQ ID NO: 120) MPAPPA. (SEQ ID NO: 120) MSPAPPA. (SEQ ID NO: 120) A. PA PPA, I APPA, (SEQ ID NO: 120) Pappas, (SEQ ID NO: 122) SPAPP. (SEQ ID NO: 122) Y represents the carboxy-terminal group of Cys ¹⁵¹ or the carboxy-terminus of the amino acid residue (s) selected from the group V, and VR, ' ^: VRR, 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: 128) NO: 129) VRRAPPTTAV, (SEQ ID NO: 130) VRRAPPTTAVP, (SEQ ID NO: 131) VRRAPPTTAVPS, (SEQ ID NO: 132) VRRAPPTTAVPSR. (SEQ ID NO: 133) VRRAPPTTAVPSRT, (SEQ ID NO: 134) VRRAPPTTAVPSRTS, (SEQ ID NO: 135) VRRAPPTTAVPSRTSL, (SEQ ID NO: 136) VRRAPPTTAVPSRTSLV, (SEQ ID NO: 137) VRRAPPTTAVPSRTSLVL, (SEQ ID NO: 137) NO: 138) VRRAPPTTAVPSRTSLVLTL, (SEQ ID NO: 139) VRRAPPTTAVPSRTSLVLTL, (SEQ ID NO: 140) VRRAPPTTAVPSRTSLVLTLN, (SEQ ID NO: 141) - 171 VRRAPPKAVPSRTSLVLTLNE, (SEQ ID NO: 142) VRRAPPTTAVPSRTSLVLTLNEL, (SEQ ID NO: 143) VRRAPPTTAVPSRTSLVLTLNELP, (SEQ ID NO: 144), the amino terminus of the amino acid residue extension, including one or more residues 176-332 of human ML, as shown in FIG. 1 (SEQ ID NO: 1) and its pegylated forms. The polypeptide fragment of claim 2, wherein the amino acid sequence of the polypeptide fragment comprises SP SPAPPACDLRVLSKLLRDSHVL, (SEQ ID NO: 110). HSRLSQCPEVHPLPTPVLLPAVDF, (SEQ ID NO: 111) SLGEWKTQMEETKAQDLLGAVTL, (SEQ ID NO: 112) LLEGVMAARGQLGPTCLSSLL, (SEQ ID NO: 113) GQLSGQVRLLLGALQS, (SEQ ID NO: 114) LLGTQLPPQGRTTAHKDPNAEF, (SEQ ID NO: 115) LSFQHLLRGKVRFLMLVGGSTLCVR (SEQ ID NO: 116) and combinations thereof. The polypeptide of claim 6, which is non-glycosylated. An isolated polypeptide encoded by a nucleic acid having a sequence that hybridizes under mild conditions to the nucleic acid molecules of the nucleic acid sequence shown in Figure 2 ((SEQ ID NO: 2)). I - 172 - I ι The polypeptide of claim 1, which is biologically active. [ 12. The polypeptide of claim 1, selected from the group hML, hMLi ₅ 3, hML (R153A, R154A), hML2,11ML3, hML4, mML, mML2, mML3, pML, and pML2. The polypeptide of claim 2, wherein the amino acid sequence of the polypeptide comprises amino acid residues 1 to X of FIG. 1 (SEQ ID NO: 1) wherein X is selected from the group of 153, 155, 164, 174, 191, 205, 207, 217, 229, 245, and 332. An isolated substantially homogeneous mpl ligand polypeptide having at least 80% sequence identity to the polypeptide of claim 13. The polypeptide of claim 13, wherein X is 153. A chimera comprising the mpl ligand of claim 13, fused to a heterologous polypeptide. The chimera of claim 16, wherein the heterologous polypeptide is an immunoglobulin polypeptide. The chimera of claim 16, wherein the heterologous polypeptide is an interleukin polypeptide. I A chimera comprising N-terminal residues 1 to about 153 to 157 hML substituted with one or more, but not all, human EPO residues added or substituted to the N-terminal residues of hML at sites corresponding to the underlines in FIG. 10th The antibody capable of binding to the mpl ligand polypeptide of claim 13. The antibody-producing hybridoma cell line of claim 20. - 173 -I and An isolated nucleic acid molecule encoding the mpl ligand polypeptide of claim 1.i An isolated nucleic acid molecule encoding the mpl ligand polypeptide of claim 13. [ I 24. An isolated nucleic acid molecule comprising the open reading frame of the nucleic acid sequence shown in FIG. 11 (SEQ ID NO: 2). The isolated nucleic acid molecule of claim 24, encoding an mpl ligand polypeptide selected from the group of hML, 111ML153, hML (R153A, R154A), hML2, hML3, hML4, mML, mML2, mML3, pML, and pML2. 26. An isolated nucleic acid molecule selected from the group consisting of I a) a cDNA clone comprising the nucleotide sequence of the coding region of the mpl ligand gene; b) a DNA sequence capable of hybridizing under mild conditions to clone (a); and c) a genetic variant of any DNA sequences of (a) or (b) that encodes a polypeptide having the biological property of a naturally occurring mpl ligand polypeptide. 27. An isolated DNA molecule having a sequence that can hybridize under mild conditions to the DNA sequence shown in FIG. 1 (SEQ ID NO. 2), wherein the DNA molecule encodes a biologically active mpl ligand polypeptide. 28. The nucleic acid molecule of claim 25, further comprising a promoter operably linked to the nucleic acid molecule. An expression vector comprising the nucleic acid sequence of claim 25 operably linked to control sequences recognized by the host cell 1 transformed with the vector. and I I 30. A host cell transformed with the vector of claim 29. - 174 - ι ι _t I A method of using a nucleic acid molecule encoding an mpl ligand polypeptide to streamline production of an mpl ligand polypeptide, comprising culturing a host cell according to claim 30. 32. The method of claim 31, wherein the mpl ligand polypeptide is isolated from a host cell. 33. The method of claim 31, wherein the mpl ligand polypeptide is isolated from the host cell culture medium. A method for determining the presence of an mpl ligand polypeptide, comprising hybridizing the DNA encoding the mpl ligand polypeptide to a nucleic acid sample to be tested and determining the presence of the mpl ligand polypeptide DNA. 35. A method for amplifying a test nucleic acid sample, comprising the polymerase reaction of the nucleic acid with a nucleic acid encoding an mpl ligand polypeptide. A composition comprising the mpl ligand polypeptide of claim 1 and a pharmaceutically acceptable carrier. A method of healing a mammal with or prone to thrombocytopenia, comprising administering to a mammal in need thereof a therapeutically effective amount of the composition of claim 36. The composition of claim 36, further comprising a therapeutically effective amount of an agent selected from the group consisting of a cytokine, a colony stimulating factor and interleukin. I The composition of claim 38, wherein the agent is selected from the group consisting of KL, LIF, G-CSF, GM-CSF, M-CSF, EPO, JL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9, and IL-11. s - 175 - 40. A method for biosynthesis of a human mpl ligand polypeptide, characterized by the amino acid sequence of a human mpl ligand polypeptide, as depicted in FIG. 1 (SEQ ID NO: 1), comprising: (a) growth of a host cell culture containing isolated DNA encoding a human mpl ligand polypeptide; I (b) separating the human mpl Uganda polypeptide from the culture; and c) purifying the mpl ligand polypeptide to obtain a substantially homogeneous biologically active human mpl ligand polypeptide. ¹ tcttcctacccatctgctccccagagggctgcctgctgtgcacttgggtcctggagcccttctccacccggatagaĽtcctcaccct Cggcccgcctt tg at Φ 0 U) at to E- • H at 5 0 x cn Φ E- rh Cn 0 0 it M 0 > cn £ O 3 it r4 cn 3 2 0 cn Φ E- 0 cn 0 o o u cn σ> 0 m Cu <0 M 0 U) U << ®>, and to < 0 AJ r- 0 C 10 <0 Cn M E- 0 Cn £ 0- at it E- < Φ <0 3 2 K. it Cn Φ E- ^ 3 Cn 0 0 Φ it 3 0 0 U Φ E- Cn at 0 0 M at O 5 E-  < at —T Φ E- M cn t 0 0 Φ cn AJ 0 WHAT at Φ E- ω it X < • r < at * -1 0 X at (0 E- 3 at > 0 Φ at r-t 0 0 at <0 E- F-H cn > 0 it u · 3 0 > cn 0 0 O -H it 3 0 ΓΜ X it Φ E- M and 0 0 φ U 3 ABOUT it U Φ E- ft Cn 0 E- w it 5 < cn 2 Cn 10 ^ 0 about M 10 M E- < at £ 0 3 Cn E- < Φ AJ 5 ABOUT 0 Cn Φ E- 3 tO 0 0 Φ it O 5 0 0 D> (M < -1 < U) it 1 0 0 at AJ 0 0 at Φ E- M at Σ Φ cn it WHAT AJ cn 3 at <0 Φ AJ at 0 at at f-i <0 cn it AJ cn > O u r- x: u cn < —T E- '< at 3 U <u Φ E- □ o 01 u Φ E- -1 u J o <0 E- 3 0 > 0 Φ E- 3 < 0 U Φ E- ω o 0 U • h M E- x u Φ U ^c * í what E- r-f 2 M U 0 u £ 0 Φ u E- < Λ E- C ' Cu E- P 0 M O << 0) 0. 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