AU749175B2 - Thrombopoietin - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): GENENTECH, INC.

Invention Title:

THROMBOPOIETIN

The following statement is a full description of this invention, including the best method of performing it known to me/us: 05/04 2002 19:05 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA @]007 la r i

THROMBOPOIETIN

FIELD OF THE INVENTION This invention relates to the isolation, purification and recombinant or chemical synthesis of proteins that Influence survival, proliferation, differeritiation or maturation of hematopoietic cells, especially platelet progenitor cells. This invention specifically relates to the cloning and expression of nucleic acids encoding a protein Ilgand capable of binding to and activating mpl, a member of the cytoklne receptor superfamily. This invention further relates to the use of these proteins alone or in combination with other cytokines to treat immune or hematopoletic disorders including thrombocytopenia.

BACKGROUND OF THE INVENTION All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

05/04 2002 19:05 FAX 61 3 92438333 GRIFFITH HACK 4IPAUSTRALIA a 008 Ib I. The Hematopoletic System The hematopoletic system produces the mature highly specialized blood cells known to be necessary for survival of all mammals. These mature cells include; erythrocytes, specialized to transport oxygen and carbon dioxide, T- and Blymphocytes, responsible for cell- and antibody-mediated immune responses, platelets or thrombocytes, specialized to form blood clots, and granulocytes and macrophages, specialized as scavengers and as accessory cells to combat Infection.

Granulocytes are further subdivided into; neutrophils, eosinophils, basophils-and mast cells, specialized cell types having discrete functions. Remarkably, all of these specialized mature blood cells are derived from a single common primitive cell type, referred to as the pluripotent (or totipotent) stem cell, found primarily in bone marrow (Dexter et Ann. Rev. Cell Biol, 3:423-441 [1987]).

The mature highly specialized blood cells must be produced In large numbers continuously throughout the life of a mammal. The vast majority of these specialized blood cells are destined to remain functionally active for only a few hours to weeks (Cronkite et al., Blood Cells, 2:263-284 [1976J). Thus, continuous renewal of the mature blood cells, the primitive stem cells themselves, as well as any intermediate or lineage-committed progenitor cell lines lying between the primitive and mature cells, Is necessary in order to maintain the normal steady state blood cell needs of the mammal.

At the heart of the hematopoietic system lies the pluripotent stem cell(s).

These cells are relatively few in number and undergo self-renewal by proliferation to produce daughter stem cells or are transformed, in a series of differentiation steps, WO 95/18858 PCT/US94/14553 into increasingly mature lineage-restricted progenitor cells, ultimately forming the highly specialized mature blood cell(s).

For example, certain multipotent progenitor cells, referred to as CFC-Mix, derived from stem cells undergo proliferation (self-renewal) and development to produce colonies containing all the different myeloid cells; erythrocytes, neutrophils, megakaryocytes (predecessors of platelets), macrophages, basophils, eosinophils, and mast cells. Other progenitor cells of the lymphoid lineage undergo proliferation and development into T-cells and B-cells.

Additionally, between the CFC-Mix progenitor cells and myeloid cells lie 1 0 another rank of progenitor cells of intermediate commitment to their progeny. These lineage-restricted progenitor cells are classified on the basis of the progeny they produce. Thus, the known immediate predecessors of the myeloid cells are: erythroid colony-forming units (CFU-E) for erythrocytes, granulocyte/macrophage colonyforming cells (GM-CFC) for neutrophils and macrophages, megakaryocyte colony- 1 5 forming cells (Meg-CFC) for megakaryocytes, eosinophil colony-forming cells (Eos- CFC) for eosinophils, and basophil colony-forming cells (Bas-CFC) for mast cells.

Other intermediate predecessor cells between the pluripotent stem cells and mature blood cells are known (see below) or will likely be discovered having varying degrees of lineage-restriction and self-renewal capacity.

The underlying principal of the normal hematopoietic cell system appears to be decreased capacity for self-renewal as multipotency is lost and lineage-restriction and maturity is acquired. Thus, at one end of the hematopoietic cell spectrum lies the pluripotent stem cell possessing the capacity for self-renewal and differentiation into all the various lineage-specific committed progenitor cells. This capacity is the basis of bone marrow transplant therapy where primitive stem cells repopulate the entire hematopoietic cell system. At the other end of the spectrum lie the highly lineagerestricted progenitors and their progeny which have lost the ability of self-renewal but have acquired mature functional activity.

The proliferation and development of stem cells and lineage-restricted progenitor cells is carefully controlled by a variety of hematopoietic growth factors or cytokines. The role of these growth factors in vivo is complex and incompletely understood. Some growth factors, such as interleukin-3 are capable of stimulating both multipotent stem cells as well as committed progenitor cells of several lineages, including for example, megakaryocytes. Other factors such as granulocyte/macrophage colony-stimulating factor (GM-CSF) was initially thought to be restricted in its action to GM-CFC's. Later, however, it was discovered GM-CSF also influenced the proliferation and development of interalia megakaryocytes. Thus, IL-3 and GM-CSF were found to have overlapping biological activities, although with -2- WO 95/18858 PCT/US94/14553 differing potency. More recently, both interleukin-6 (IL-6) and interleukin-11 (IL-11), while having no apparent influence on meg-colony formation alone, act synergistically with IL-3 to stimulate maturation of megakaryocytes (Yonemura et al., Exp. Hematol., 20:1011-1016 [1992]).

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

It also appears that hematopoietic growth factors can exhibit their effect at different stages of cell development from the totipotent stem cell through various 1 0 committed lineage-restricted progenitors to the mature blood cell. For example, erythropoietin (epo) appears to promote proliferation only of mature erythroid progenitor cells. IL-3 appears to exert its effect earlier influencing primitive stem cells and intermediate lineage-restricted progenitor cells. Other growth factors such as stem cell factor (SCF) may influence even more primitive cell development.

1 5 It will be appreciated from the foregoing that novel hematopoietic growth factors that affect survival, proliferation, differentiation or maturation of any of the blood cells or predecessors thereof would be useful, especially to assist in the reestablishment of a diminished hematopoietic system caused by disease or after radiation- or chemo-therapy.

II. Megakaryocytopolesis Platefet Production Regulation of megakaryocytopoiesis and platelet production has been reviewed by: Mazur, Exp. Hematol., 15:248 [1987] and Hoffman, Blood, 74:1196-1212 [1989]. Briefly, bone marrow pluripotent stem cells differentiate into megakaryocytic, erythrocytic, and myelocytic cell lines. It is believed there is a hierarchy of committed megakaryocytic progenitor cells between stem cells and megakaryocytes. At least three classes of megakaryocytic progenitor cells have been identified, namely; burst forming unit megakaryocytes (BFU-MK), colony-forming unit megakaryocytes (CFU-MK), and light density megakaryocyte progenitor cells (LD-CFU-MK). Megakaryocytic maturation itself is a continuum of development that has been separated into stages based on standard morphologic criteria. The earliest recognizable member of the megakaryocyte (MK or meg) family are the megakaryoblasts. These cells are initially 20 to 30 ±pm in diameter having basophilic cytoplasm and a slightly irregular nucleus with loose, somewhat reticular chromatin and several nucleoli. Later, megakaryoblasts may contain up to 32 nuclei (ployploid), but the cytoplasm remains sparse and immature. As maturation proceeds, the nucleus becomes more lobulate and pyknotic, the cytoplasm increases in quantity and becomes more acidophilic and granular. The most mature cells of this family may give the WO 95/18858 PCT/US94/14553 appearance of releasing platelets at their periphery. Normally, less than 10% of megakaryocytes are in the blast stage and more than 50% are mature. Arbitrary morphologic classifications commonly applied to the megakaryocyte series are megakaryoblast for the earliest form; promegakaryocyte or basophilic megakaryocyte for the intermediate form; and mature (acidophilic, granular, or platelet-producing) megakaryocyte for the late forms. The mature megakaryocyte extends filaments of cytoplasm into sinusoidal spaces where they detach and fragment into individual platelets (Williams et al., Hematology, 1972).

Megakaryocytopoiesis is believed to involve several regulatory factors 1 0 (Williams et al., Br. J. Haematol., 52:173 [1982] and Williams et al., J. Cell Physiol., 110:101 [1982]). The early level of megakaryocytopoiesis is postulated as being mitotic, concerned with cell proliferation and colony initiation from CFU-MK but is not affected by platelet count (Burstein et al., J. Cell Physiol., 109:333 [1981] and Kimura et al:, Exp. Hematol., 13:1048 [1985]). The later stage of 1 5 maturation is non-mitotic, involved with nuclear polyploidization and cytoplasmic maturation and is probably regulated in a feedback mechanism by peripheral platelet number (Odell et al., Blood, 48:765 [1976] and Ebbe et al., Blood, 32:787 [1968]).

The existence of a distinct and specific megakaryocyte colony-stimulating factor (MK-CSF) has been disputed (Mazur, Exp. Hematol., 15:340-350 [1987]).

However most authors believe that a process so vital to survival as platelet production would be regulated by cytokine(s) exclusively responsible for this process. The hypothesis that megakaryocyte/platelet specific cytokine(s) exist has provided the basis for more than 30 years of search but to date no such cytokine has been S2 5 purified, sequenced and established by assay as a unique MK-CSF (TPO).

*Although it has been reported that MK-CSF's have been partly purified from experimentally produced thrombocytopenia (Hill et al., Exp. Hematol., 14:752 [1986]) and human embryonic kidney conditioned medium [CM] (McDonald et al., J.

Lab. Clin. Med., 85:59 [1975]) and in man from a plastic anemia and idiopathic thrombocytopenic purpura urinary extracts (Kawakita et al., Blood, 6:556 [1983]) and plasma (Hoffman et al., J. Clin. Invest., 75:1174 [1985]), their physiological function is as yet unknown In most cases.

The conditioned medium of pokeweed mitogen-activated spleen cells (PWM- SpCM) and the murine myelomonocyte cell line WEHI-3 (WEHI-3CM) have been used as megakaryocyte potentiators. PWM-SpCM contains factors enhancing CFU-MK growth (Metcalf et al., Pro. Natl. Acad. Sci., USA, 72:1744-1748 [1975]; Quesenberry et al., Blood, 65:214 [1985]; and Iscove, in Hematopoietic Cell Differentiation, ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. 10, Golde WO 95/18858 PCTIUS94/14553 et al., eds. (New York, Academy Press] pp 37-52 [1978]), one of which is interleukin-3 a multilineage colony stimulating factor (multi-CSF [Burstein, Blood Cells, 11:469 [1986]). The other factors in this medium have not yet been identified and isolated. WEHI-3 is a murine myelomonocytic cell line secreting relatively large amounts of IL-3 and smaller amounts of GM-CSF. IL-3 has been found to potentiate the growth of a wide range of hematopoietic cells (Ihle et al., J.

Immunol., 13:282 [1983]). IL-3 has also been found to synergize with many of the known hematopoietic hormones or growth factors (Bartelmez et al., J. Cell Physiol., 122:362-369 [1985] and Warren et al., Cell, 46:667-674 [1988]), including both erythropoietin (EPO) and interleukin-1 in the induction of very early multipotential precursors and the formation of very large mixed hematopoietic colonies.

Other sources of megakaryocyte potentiators have been found in the conditioned Smedia of murine lung, bone, macrophage cell lines, peritoneal exudate cells and human i 1 5 embryonic kidney cells. Despite certain conflicting data (Mazur, Exp. Hematol., 15:340- 350 [1987]), there is some evidence (Geissler at al., Br. J. Haematol., 60:233-238 [1985]) that activated T lymphocytes rather than monocytes play an enhancing role in megakaryocytopoiesis. These findings suggest that activated Tlymphocyte secretions such as interleukins may be regulatory factors in MK 20 development (Geissler et al., Exp. Hematol., 15:845-853 [1987]). A number of studies on megakaryocytopoiesis with purified erythropoietin EPO (Vainchenker et al., Blood, 54:940 [1979]; McLeod et al., Nature, 261:492-4 [1976]; and Williams et al., Exp. Hematol., 12:734 [1984]) indicate that this hormone has an enhancing effect on MK colony formation. This has also been demonstrated in both serum-free 2 5 and serum-containing cultures and in the absence of accessory cells (Williams et al., 7 Exp. Hematol., 12:734 [1984]). EPO was postulated to be involved more in the single and two-cell stage aspects of megakaryocytopoiesis as opposed to the effect of PWM- SpCM which was involved in the four-cell stage of megakaryocyte development. The interaction of all these factors on both early and late phases of megakaryocyte 3 0 development remains to be elucidated.

Data produced from several laboratories suggests that the only multi-lineage factors that individually have MK-colony stimulating activity are GM-CSF and IL-3 and, to a lesser extent, the B-cell stimulating factor IL-6 (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 to increase megakaryocyte size and ploidy (Yonemura et al., British Journal of Hematology, 84:16-23 [1993]; Burstein et al., J. Cell. Physiol., 153:305-312 [1992]; Metcalf et al., Blood, 76:50-56 [1990]; Metcalf et al., Blood, 77:2150-2153 [1991]; WO 95/18858 PCTIUS94/14553 Bruno et al., Exp. Hematol., 19:378-381 [1991]; and Yonemura et al., Exp.

Hematol., 20:1011-1016 [1992]).

Other documents of interest include: Eppstein et al., U.S. Patent No.

4,962,091; Chong, U.S. Patent No. 4,879,111; Fernandes et al., U.S. Patent No.

4,604,377; Wissler et al., U.S. Patent No. 4,512,971; Gottlieb, U.S. Patent No.

4,468,379; Bennett et al., U.S. Patent No. 5,215,895; Kogan et al., U.S. Patent No.

5,250,732; Kimura et al., Eur. J. Immunol., 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].

Ill. Thrombocytopenia Platelets are critical elements of the blood clotting mechanism. Depletion of the circulating level of platelets, called thrombocytopenia, occurs in various clinical conditions and disorders. Thrombocytopenia is commonly defined as a platelet count S: 20 below 150 X 109 per liter. The major causes of thrombocytopenia can be broadly divided into three categories on the basis of platelet life span, namely; impaired production of platelets by the bone marrow, platelet sequestration in the/spleen (splenomegaly), or increased destruction of platelets in the peripheral circulation autoimmune thrombocytopenia or chemo- and radiation-therapy).

Additionally, in patients receiving large volumes of rapidly administered platelet-poor blood products, thrombocytopenia may develop due to dilution.

The clinical bleeding manifestations of thrombocytopenia depend on the severity of thrombocytopenia, its cause, and possible associated coagulation defects. In general, patients with platelet counts between 20 and 100 X 10 9 per liter are at risk of excessive post traumatic bleeding, while those with platelet counts below 20 X 109 per liter may bleed spontaneously. These latter patients are candidates for platelet transfusion with attendant immune and viral risk. For any given degree of thrombocytopenia, bleeding tends to be more severe when the cause is decreased production rather than increased destruction of platelets. In the latter situation, accelerated platelet turnover results in the circulation of younger, larger and hemostatically more effective platelets. Thrombocytopenia may result from a variety of disorders briefly described below. A more detailed description may be found in Schafner, A. "Thrombocytopenia and Disorders of Platelet Function," Internal -6- WO 95/18858 PCT/US94/14553 Medicine, 3rd Ed., John J. Hutton et al., Eds.. Little Brown and Co., Boston/Toronto/ London [1990].

Thrombocytopenia due to impaired platelet production Causes of congenital thrombocytopenia include constitutional aplastic anemia (Fanconi syndrome) and congenital amegakaryocytic thrombocytopenia, which may be associated with skeletal malformations. Acquired disorders of platelet production are caused by either hypoplasia of megakaryocytes or ineffective thrombopoiesis.

Megakaryocytic hypoplasia can result from a variety of conditions, including marrow aplasia (including idiopathic forms or myelosuppression by chemotherapeutic agents 1 0 or radiation therapy), myelfibrosis, leukemia, and invasion of the bone marrow by metastatic tumor or granulomas. In some situations, toxins, infectious agents, or drugs may interfere with thrombopoiesis relatively selectively; examples include transient thrombocytopenias caused by alcohol and certain viral infections and mild thrombocytopenia associated with the administration of thiazide diuretics. Finally, 1 5 ineffective thrombopoiesis secondary to megaloblastic processes (folate or B1 2 deficiency) can also cause thrombocytopenia, usually with coexisting anemia and leukopenia.

Current treatment of thrombocytopenias due to decreased platelet production depends on Identification and reversal of the underlying cause of the bone marrow failure. Platelet transfusions are usually reserved for patients with serious bleeding complications, or for coverage during surgical procedures, since isoimmunization may lead to refractoriness to further platelet transfusions. Mucosal bleeding resulting from severe thrombocytopenia may be ameliorated by the oral or intravenous administration of the antifibrinolytic agents. Thrombotic complications may develop, 25 however, if antifibrinolytic agents are used in patients with disseminated intravascular coagulation

(DIC).

Thrombocytopenia due to splenic sequestration Splenomegaly due to any cause may be associated with mild to moderate thrombocytopenia. This is a largely passive process (hypersplenism) of splenic 3 0 platelet sequestration, in contrast to the active destruction of platelets by the spleen in cases of immunomediated thrombocytopenia discussed below. Although the most common cause of hypersplenism is congestive splenomegaly from portal hypertension due to alcoholic cirrhosis, other forms of congestive, infiltrative, or lymphoproliferative splenomegaly are also associated with thrombocytopenia. Platelet counts generally do not fall below 50 X 10 9 per liter as a result of hypersplenism alone.

-7- WO 95/18858 PCT/US94/14553 Thrombocytopenia due to nonimmune-mediated platelet destruction Thrombocytopenia can result from the accelerated destruction of platelets by various nonimmunologic processes. Disorders of this type include disseminated intravascular coagulation, prosthetic intravascular devices, extra corporeal circulation of the blood, and thrombotic microangiopathies such as thrombotic thrombocytic purpura. In all of these situations, circulating platelets that are exposed to either artificial surfaces or abnormal vascular intima either are consumed at these sites or are damaged and then prematurely cleared by the reticuloendothelial system.

Disease states or disorders in which disseminated intravascular coagulation (DIC) may 1 0 arise are set forth in greater detail in Braunwald at al. (eds), Harrison's Principles of Internal Medicine, 11th Ed., p.1478, McGraw Hill [1987]. Intravascular prosthetic devices, including cardiac valves and intra-aortic balloons can cause a mild to moderate destructive thrombocytopenia and transient thrombocytopenia in patients undergoing cardiopulmonary bypass or hemodialysis may result from consumption or 1 5 damage of platelets in the extra corporeal circuit.

Drug-induced immune thrombocytopenia More than 100 drugs have been implicated in immunologically mediated thrombocytopenia. However, only quinidine, quinine, gold, sulfonamides, cephalothin, and heparin have been well characterized. Drug-induced thrombocytopenia is S" 20 frequently very severe and typically occurs precipitously within days while patients are taking the sensitizing medication.

Immune (autoimmune) thrombocytopenic purpura (ITP) ITP in adults is a chronic disease characterized by autoimmune platelet destruction. The autoantibody is usually IgG although other immunoglobulins have also 2 5 been reported. Although the autoantibody of ITP has been found to be associated with platelet membrane GPIIbllla, the platelet antigen specificity has not been identified in o. most cases. Extravascular destruction of sensitized platelets occurs in the reticuloendothelial system of the spleen and liver. Although over one-half of all cases of ITP are idiopathic, many patients have underlying rheumatic or autoimmune 30 diseases systemic lupus erythematosus) or lymphoproliferative disorders S;"i chronic lymphocytic leukemia).

HIV-lnduced ITP ITP is an increasingly common complication of HIV infection (Morris et al., Ann. Intern. Med., 96:714-717 [1982]), and can occur at any stage of the disease progression, both in patients diagnosed with the Acquired Immune Deficiency Syndrome (AIDS), those with AIDS-related complex, and those with HIV infection but without AIDS symptoms. HIV infection is a transmissible disease ultimately characterized by a profound deficiency of cellular immune function as well as the -8- WO 95/18858 PCT/US94/14553 occurrence of opportunistic infection and malignancy. The primary immunologic abnormality resulting from infection by HIV is the progressive depletion and functional impairment of T lymphocytes expressing the CD4 cell surface glycoprotein (Lane et al., Ann. Rev. Immunol., 3:477 [1985]). The loss of CD4 helper/inducer T cell function probably underlies the profound defects in cellular and humoral immunity leading to the opportunistic infections and malignancies characteristic of AIDS Lane supra).

Although the mechanism of HIV-associated ITP is unknown, it is believed to be different from the mechanism of ITP 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. Current Therapy for Thrombocytopenia The therapeutic approach to the treatment of patients with thrombocytopenia is 1 5 dictated by the severity and urgency of the clinical situation. The treatment is similar for HIV-associated and non-HIV-related thrombocytopenia, and although a number of different therapeutic approaches have been used, the therapy remains controversial.

Platelet counts in patients diagnosed with thrombocytopenia have been successfully increased by glucocorticoid prednisolone) therapy, however in most *i 20 patients, the response is incomplete, or relapse occurs when the glucocorticoid dose is reduced or its administration is discontinued. Based upon studies with patients having HIV-associated ITP, some investigators have suggested that glucocorticoid therapy may result in predisposition to AIDS. Glucocorticoids are usually administered if platelet count falls below 20 X 10 9 /liter or when spontaneous bleeding occurs.

2 5 For patients refractory to glucocorticoids, the compound: 4 2 -chlorphenyl)-9-methyl-2-[3-(4-morpholinyl)-3-propanon-1-yl]6H thieno[3,2,f][1,2,4]triazolo[4,3,a,][1,4]diazepin (WEB 2086) has been successfully used to treat a severe case of non HIV-associated ITP. A patient having platelet counts of 37 ,000-58,000/ l was treated with WEB 2086 and after 1-2 weeks treatment platelet counts increased to 140,000-190,000/pl.

(EP

361,077 and Lohman et al., Lancet, 1147 [1988]).

Although the optimal treatment for acquired amegakaryocytic thrombocytopenia purpura (AATP) is uncertain, antithymocyte globulin (ATG), a horse antiserum to human thymus tissue, has been shown to produce prolonged complete remission (Trimble et al., Am. J. Hematol., 37:126-127 [1991]). A recent report however, indicates that the hematopoietic effects of ATG are attributable to thimerosal, where presumably the protein acts as a mercury carrier (Panella et al., Cancer Research, 50:4429-4435 [1990]).

WO 95/18858 PCTfUS94/14553 Good results have been reported with splenectomy. Splenectomy removes the major site of platelet destruction and a major source of autoantibody production in many patients. This procedure results in prolonged treatment-free remissions in a large number of patients. However, since surgical procedures are generally to be avoided in immune compromised patients, splenectomy is recommended only in severe cases of thrombocytopenia severe HIV-associated ITP), in patients who fail to respond to 2 to 3 weeks of glucocorticoid treatment, or do not achieve sustained response after discontinuation of glucocorticoid administration. Based upon current scientific knowledge, it is unclear whether splenectomy predisposes patients to AIDS.

1 0 In addition to prednisolone therapy and splenectomy, certain cytotoxic agents, vincristine, and azidothimidine (AZT, zidovudine) also show promise in treating HIV-induced ITP; however, the results are preliminary.

It will be appreciated from the foregoing that one way to treat thrombocytopenia would be to obtain an agent capable of accelerating the 1 5 differentiation and maturation of megakaryocytes or precursors thereof into the platelet-producing form. Considerable efforts have been expended on identifying such an agent, commonly referred to as "thrombopoietin" (TPO). Other names for TPO commonly found in the literature include; thrombocytopoiesis stimulating factor (TSF), megakaryocyte colony-stimulating factor (MK-CSF), megakaryocyte- 20 stimulating factor and megakaryocyte potentiator. TPO activity was observed as early 0as 1959 (Rak et al., Med. Exp., 1:125) and attempts to characterize and purify this agent have continued to the present day. While reports of partial purification of(TPOactive polypeptides exist (see, for example, Tayrien et al., J. Biol. Chem., 262:3262 [1987] and Hoffman et al., J. Clin. Invest. 75:1174 [1985]), others have- postulated 25 that TPO is not a discrete entity in its own right but rather is simply the polyfunctional manifestation of a known hormone (IL- 3, Sparrow et al., Prog. Clin.

Biol. Res., 215:123 [1986]). Regardless of its form or origin, a molecule possessing thrombopoietic activity would be of significant therapeutic value. Although no protein has been unambiguously identified as TPO, considerable interest surrounds the recent discovery that mpl, a putative cytokine receptor, may transduce a thrombopoietic i signal.

V. Mpl is a Megakaryocytopoietic Cytokine Receptor It is believed that the proliferation and maturation of hematopoietic cells is tightly regulated by factors that positively or negatively modulate pluripotential stem cell proliferation and multilineage differentiation. These effects are mediated through the high-affinity binding of extracellular protein factors to specific cell surface receptors. These cell surface receptors share considerable homology and are generally WO 95/18858 PCT/US94/14553 classified as members of the cytokine receptor superfamily. Members of the superfamily include receptors for: IL-2 (p and y chains) (Hatakeyama et al., Science, 244:551-556 [1989); Takeshita et al., Science, 257:379-382 [1991]), IL-3 (Itoh et al., Science, 247:324-328 [1990]; Gorman et al., Proc. Natl. Acad. Sci. USA, 87:5459-5463 [1990]; Kitamura et al., Cell, 66:1165-1174 [1991a]; Kitamura et al., Proc. Natl. Acad. Sci. USA, 88:5082-5086 [1991b]), IL-4 (Mosley et al., Cell, 59:335-348 [1989], IL-5 (Takaki et al., EMBO 9:4367-4374 [1990]; Tavernier et al., Cell, 66:1175-1184 [1991]), IL-6 (Yamasaki et al., Science, 241:825-828 [1988]; Hibi et al., Cell, 63:1149-1157 [1990]), IL-7 (Goodwin 1 0 et al., Cell, 60:941-951 [1990]), IL-9 (Renault et al., Proc. Natl. Acad. Sci. USA, 89:5690-5694 [1992]), granulocyte-macrophage colony-stimulating factor (GM- CSF) (Gearing et al., EMBO 8:3667-3676 [1991]; Hayashida et al, Proc. Natl.

Acad. Sci. USA, 244:9655-9659 [1990]), granulocyte colony-stimulating factor (G-CSF) (Fukunaga et al., Cell, 61:341-350 [1990a]; Fukunaga et al., Proc. Natl.

1 5 Acad. Sci. USA, 87:8702-8706 [1990b]; Larsen et al., J. Exp. Med., 172:1559- 1570 [1990]), EPO (D'Andrea et al., Cell, 57:277-285 [1989]; Jones et al., Blood, 76:31-35 [1990]), Leukemia inhibitory factor (LIF) (Gearing et al., EMBO J., 10:2839-2848 [1991]), oncostatin M (OSM) (Rose et al., Proc. Natl. Acad. Sci.

USA, 88:8641-8645 [1991]) and also receptors for prolactin (Boutin et/al., Proc.

Natl. Acad. Sci. USA, 88:7744-7748 [1988]; Edery et al., Proc. Natl. Acad. Sci. USA, 86:2112-2116 [1989]), growth hormone (GH) (Leung et al., Nature, 330:537- 543 [1987]) and ciliary neurotrophic factor (CNTF) (Davis et al., Science, 253:59-63 [1991].

Members of the cytokine receptor superfamily may be grouped into three 25 functional categories (for review see Nicola et al., Cell, 67:1-4 [1991]). The first class comprises single chain receptors, such as erythropoietin receptor (EPO-R) or granulocyte colony stimulating factor receptor (G-CSF-R), which bind ligand with high affinity via the extracellular domain and also generate an intracellular signal. A second class of receptors, so called a-subunits, includes interleukin-6 receptor 30 (IL6-R), granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R), interleukin-3 receptor (IL3-Ra) and other members of the cytokine receptor superfamily. These a-subunits bind ligand with low affinity but cannot transduce an intracellular signal. A high affinity receptor capable of signaling is generated by a heterodimer between an a-subunit and a member of a third class of cytokine receptors, termed P-subunits, Pc, the common P-subunit for the three asubunits IL3-Ra and GM-CSF-R.

Evidence that mpt is a member of the cytokine receptor superfamily comes from sequence homology (Gearing, EMBO 8:3667-3676 [1988]; Bazan, Proc.

1 1 WO 95/18858 PCT/US94/14553 Natl. Acad. Sci. USA, 87:6834-6938 [1990]; Davis et al., Science, 253:59-63 [1991) and Vigon et al., Proc. Natl. Acad. Sci. USA, 89:5640-5644 [1992]) and its ability to transduce proliferative signals.

Deduced protein sequence from molecular cloning of murine c-mpl reveals this protein is homologous to other cytokine receptors. The extracellular domain contains 465 amino acid residues and is composed of two subdomains each with four highly conserved cysteines and a particular motif in the N-terminal subdomain and in the Cterminal subdomain. The ligand-binding extracellular domains are predicted to have similar double 3-barrel fold structural geometries. This duplicated extracellular 1 0 domain is highly homologous to the signal transducing chain common to IL-3, IL-5 and GM-CSF receptors as well as the low-affinity binding domain of LIF (Vigon et al., Oncogene, 8:2607-2615 [1993]). Thus mpl may belong to the low affinity ligand binding class of cytokine receptors.

A comparison of murine mpl and mature human mpl P, reveals these two 1 5 proteins show 81% sequence identity. More specifically, the N-terminus and Cterminus extracellular subdomains share 75% and 80% sequence identity respectively. The most conserved mpl region is the cytoplasmic domain showing 91% amino acid identity, with a sequence of 37 residues near the transmembrane domain Sbeing identical in both species. Accordingly, mpl is reported to be one of the most 20 conserved members of the cytokine receptor superfamily (Vigon supra).

Evidence that mpl is a functional receptor capable of transducing a proliferative signal comes from construction of chimeric receptors containing an extracellular domain from a cytokine receptor having high affinity for a known cytokine with the mpl cytoplasmic domain. Since no known ligand for mpl has been 25 reported, it was necessary to construct the chimeric high affinity ligand binding extracellular domain from a class one cytokine receptor such as IL-4R or G-CSFR.

Vigon et al., supra fused the extracellular domain of G-CSFR with both the transmembrane and cytoplasmic domain of c-mpl. An IL-3 dependent cell line, BAF/B03 (Ba/F3) was transfected with the G-CSFR/mpl chimera along with a full S* 30 length G-CSFR control. Cells transfected with the chimera grew equally well in the presence of cytokine IL-3 or G-CSF. Similarly, cells transfected with G-CSFR also grew well in either IL-3 or G-CSF. All cells died in the absence of growth factors. A similar experiment was conducted by Skoda et al., EMBO 12(7):2645-2653 [1993] in which both the extracellular and transmembrane domains of human IL-4 receptor (hlL-4-R) were fused to the murine mpl cytoplasmic domain, and transfected into a murine IL-3 dependent Ba/F3 cell line. Ba/F3 cells transfected with wild type hlL-4-R proliferated normally in the presence of either of the species specific IL-4 or IL-3. Ba/F3 cells transfected with hlL-4R/mp/ proliferated -12- WO 95/18858 PCT/US94/14553 normally in the presence of hlL-4 (in the presence or absence of IL-3) demonstrating that in Ba/F3 cells the mpl cytoplasmic domain contains all the elements necessary to transduce a proliferative signal.

These chimeric experiments demonstrate the proliferation signaling capability of the mpl cytoplasmic domain but are silent regarding whether the mpl extracellular domain can bind a ligand. These results are consistent with at least two possibilities, namely, mpl is a single chain (class one) receptor like EPO-R or G-CSFR or it is a signal transducing 1-subunit (class three) requiring an a-subunit like IL-3 (Skoda et al. supra).

VI. Mpl Ligand is a Thrombopoietin

(TPO)

As described above, it has been suggested that serum contains a unique factor, sometimes referred to as thrombopoietin (TPO), that acts synergistically with various other cytokines to promote growth and maturation of megakaryocytes. No such natural 1 5 factor has ever been isolated from serum or any other source even though considerable effort has been expended by numerous groups. Even though it is not known whether mpl is capable of directly binding a megakaryocyte stimulating factor, recent experiments demonstrate that mpl is involved in proliferative signal transduction from a factor or factors found in the serum of patients with aplastic bone marrow 20 (Methia et al., Blood, 82(5):1395-1401 [1993]).

Evidence that a unique serum colony-forming factor distinct from IL-la, IL- P--7 3, IL-4, IL-6, IL-11, SCF, EPO, G-CSF, and GM-CSF transduces a proliferative signal through mpl comes from examination of the distribution of c-mpl expression in primitive and committed hematopoietic cell lines and from mpl antisense studies in one 2 5 of these cell lines.

Using reverse transcriptase (RT)-PCR in immuno-purified human hematopoietic cells, Methia et al., supra demonstrated that strong mpl mRNA messages were only found in CD34+ purified cells, megakaryocytes and platelets. CD34+ cells purified from bone marrow (BM) represents about 1% of all BM cells and are 30 enriched in primitive and committed progenitors of all lineages erythroid, granulomacrophage, and megakaryocytic).

Mpl antisense oligodeoxynucleotides were shown to suppress megakaryocytic colony formation from the pluripotent CD34 cells cultured in serum from patients with aplastic marrow (a rich source of megakaryocyte colony-stimulating activity 3 5 [MK-CSA]). These same antisense oligodeoxynucleotides had no effect on erythroid or granulomacrophage colony formation.

Whether mpl directly bound a ligand and whether the serum factor shown to cause megakaryocytopoiesis acted through mpl was still unknown. It had been -13- WO 95/18858 PCT/US94/ 14 5 53 suggested, however, that if mpl did directly bind a ligand, its amino acid sequence was likely to be highly conserved and have species cross-reactivity owing to the considerable sequence identity between human and murine mp extracellular domains (Vigon et al., supra [1993]).

In view of the foregoing, it will be appreciated there is a current and continuing need in the art to isolate and identify molecules capable of stimulating proliferalion, dierentiation d e ad detiy m o c u es c a p a b le of stimulating roliferation differentiation and maturation of hematopoietic cells, especially megakaryocytes or their predecessors for therapeutic use in the treatment of thromboctopenia. It is believed such a molecule is a mpl ligand and thus there exists a further need to isolate such ligand(s) to evaluate their role(s) in cell growth and differentiation.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", means "including but not limited to" and is not intended to exclude other additives, components, integers or steps.

0 .~r 4S* p..

S

-14- 2 7 APR 1998 27 APR 1998 02/05 2002 12:39 FAX 61 3 92438333 GRIFFITH HACK 1005 15 SUMMARY OF THE INVENTION The invention provides an isolated mpl ligand polypeptide as obtainable by a process which comprises: screening a human genome library with an oligonucleotide based on the genomic sequence depicted in Fig 9 hereof to isolate genomic DNA which includes the mpl ligand exon coding sequence depicted in Fig 9 hereof together with the remaining exons of the gene, (ii) inserting the DNA into an expression vector, (iii) transfecting a mammalian cell with the vector and expressing the gene, and (iv) recovering mpl ligand polypeptide from the cell culture medium.

The invention further provides an isolated mammalian megakaryocytopoietic proliferation and maturation promoting protein, denominated the "mpl ligand" (ML) or "thrombopoietin" (TPO), capable of stimulating proliferation, maturation and/or differentiation of megakaryocytes into the mature platelet-producing form.

This substantially homogeneous protein may be purified from a natural source by a method comprising; contacting a source plasma containing the mpl ligand molecules to be purified with an immobilized receptor polypeptide, specifically mpt or a mpl fusion polypeptide immobilized on a support, under conditions whereby the mpl ligand molecules to be purified are selectively adsorbed onto the immobilized receptor polypeptide, washing the immobilized receptor polypeptide and its support to remove non-adsorbed material, and eluting the mpt ligand molecules from the immobilized receptor polypeptide to which they are adsorbed with an elution buffer.

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

Optionally, the preferred megakaryocytopoietic proliferation and maturation promoting protein is an isolated substantially homogeneous mpl ligand polypeptide made by synthetic or recombinant means.

05/04 2002 19:06 FAX 61 3 92438333 GRIFFITH HACK 4 IJPAUSTRALIA Idjolo 15a The "mpl ligand" polypeptide or TPO' of this invention preferably his'at least overall sequence identity with the amino acid sequence of the highly purified substantially homogeneous porcine mpl ligand polypeptide and at least 80% sequence Identity with the "EPO-domain" of the porcine mpl ligand polypeptide. Optionally, the mpl ligand of this invention is mature human mpl ligand (hML), having the mature amino acid sequence provided' in Fig. 1 (SEQ ID NO; or a variant or posttranscrlptionally modified form thereof or a protein having about 80% sequence identity with mature human mpl ligand. Optionally the mpl ligand variant is a fragment, especially an amino-terminus or "EPO-domain" fragment, of the mature human mp/ ligand (hML). Preferably the amino terminus fragment retains substantially all of the human ML sequence between the first and fourth cysteine residues but may contain substantial additions, deletions or substitutions outside that region. According to this embodiment, the fragment polypeptide may be represented by the formula:" X-hML(7-151)-Y Where hML(7-151) represents the human TPO (hML) amino acid sequence from Cys 7 throuah Cys 15 inclusive; X represents, the amino group of Cys 7 or one or more of the amino-terminus amino acid residue(s) of the mature hML or amino acid residue extensions thereto such as Met, Tyr or leader sequences containing, for example, proteobytic cleavage sites Factor Xa or thrombin); and Y represents the carboxy terminal group of Cys 1 5 1 or one or more carboxy-terminus amino acid residue(s) of the mature hML or extensions thereto.

Optionally the mpl ligand polypeptide or fragment thereof may be fused to a heterologous polypeptide (chimera). A preferred heterologous polypeptide is a cytokine, colony stimulating factor or interleukin or fragment thereof, especially kitligand IL-1, IL-3, IL-6, IL-11, EPO, GM-CSF or LIF. An optional preferred heterologous polypeptide is an -immunoglobin chain, especially human IgG1, lgG2, lgG3, lgG4, IgA, IgE, IgD, IgM or fragment thereof, especially comprising the constant domain of an IgG heavy chain.

Another aspect of this invention provides a composition comprising an isolated mpl agonist that is biologically active and is preferably capable of stimulating the 1 5 incorporation of labeled nucleotides 3 H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected with human mpl. Optionally the mpl agonist is biologically active mpl ligand and is preferably capable of stimulating the incorporation of 3 5 S into circulating platelets in a mouse platelet rebound assay.

Suitable agonists include hML 1 5 3 hML(R153A, R154A), hML2, hML3, hML4, mML, mML2, mML3, pML, and pML2 or fragments thereof.

In another embodiment, this invention provides an isolated antibody capable of binding to the mpl ligand. The isolated antibody capable of binding to the-mnpl ligand may optionally be fused to a second polypeptide and the antibody or fusion thereof may be used to isolate and purify mpl ligand from a source as described above for 2 5 immobilized mpl. In a further aspect of this embodiment, the invention provides a method for detecting the mpl ligand in vitro or in vivo comprising contacting the antibody with a sample, especially a serum sample, suspected of containing the ligand and detecting if binding has occurred.

In still further embodiments, the invention provides an isolated nucleic acid 3 0 molecule, encoding the mpl ligand or fragments thereof, which nucleic acid molecule may optionally be labeled with a detectable moiety, and a nucleic acid molecule having a sequence that is complementary to, or hybridizes under moderate to highly stringent conditions with, a nucleic acid molecule having a sequence encoding a mpl ligand.

Preferred nucleic acid molecules are those encoding human, porcine, and murine mpl 3 5 ligand, and include RNA and DNA, both genomic and cDNA. In a further aspect of this embodiment, the nucleic acid molecule is DNA encoding the mpl ligand and further comprises a replicable vector in which the DNA is operably linked to control sequences -1 6o .3 WO 95/18858 PCT/US94/14553 recognized by a host transformed with the vector. Optionally the DNA is cDNA having the sequence provided in Fig. 1 (SEQ ID NO: or a fragment thereof.

This aspect further includes host cells, preferably CHO cells, transformed with the vector and a method of using the DNA to effect production of mpl ligand, preferably comprising expressing the cDNA encoding the mpl ligand in a culture of the transformed host cells and recovering the mpl ligand from the host cells or the host cell culture. The mpl ligand prepared in this manner is preferably human mpl ligand.

The invention further includes a method for treating a mammal having a hematopoietic disorder, especially thrombocytopenia, comprising administering a 1 0 therapeutically effective amount of a mpl ligand to the mammal. Optionally the mpl ligand is administered in combination with a cytokine, especially a colony stimulating factor or interleukin. Preferred colony stimulating factors or interleukins include; kit-ligand LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-3, IL-6, and IL-11.

The invention further includes a process for isolating and purifying TPO (ML) from a TPO producing microorganism comprising: disrupting or lysing cells containing TPO, optionally seperating soluble material from insoluble material containing

TPO,

S(3) solublizing TPO in the insoluble material with a solublizing buffer, 20 separating solublized TPO from other soluble and insoluble material, refolding TPO in a redox buffer, and separating properly folded TPO from misfolded TPO.

The process provides for solubilizing the insoluble material containing TPO with a chaotropic agent where the chaotropic agent is selected from a salt of guanidine, 2 5 sodium thiocyanate, or urea. The process further provides that solublized TPO is seperated from other soluble and insoluble material by one or more steps selected from centrafugation, gel filtration and reverse phase chromotography. The refolding step of the process provides for a redox buffer containing both an oxidizing and reducing agent. Generally, the oxidizing agent is oxygen or a compound containing at 3 0 least one 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. Most. preferably the oxidizing agent is oxidized glutathione(GSSG) and the reducing agent is reduced glutathione(GSH). It is also prefered that the molar ratio of the oxidizing agent is equal to or greater then that of the reducing agent. The redox buffer additionally contains a detergent, preferably selected from CHAPS and CHAPSO, present at a level of at least1%. The redox buffer additionally contains NaCI preferably at a concentration range of about 0.1-0.5M, and -17- 2 7 APR 1998 WO 95/18858 PCT/US94/14553 glycerol preferably at a concentration greater than 15%. The pH of the redox buffer preferably ranges from about pH 7.5-pH 9.0. and the refolding step is conducted at 4 degrees for 12-48hr. The refolding step produces biologically active TPO in which a disulfide bond is formed between the Cys nearest the amino-terminus with the Cys nearest the carboxy-terminus of the EPO domain.

The invention further includes a process for purifying biologically active TPO from a microorganism comprising: lysing at least the extracellular membrane of the microorganism, treating the lysate containing TPO with a chaotropic agent, refolding the TPO, and separating impurities and misfolded TPO from properly folded

TPO.

1 5 BRIEF DESCRIPTION OF THE FIGURES Fig. 1 shows the deduced amino acid sequence (SEQ ID NO: 1) of human mpl ligand i. (hML) cDNA and the coding nucleotide sequence (SEQ ID NO: Nucleotides are numbered at the beginning of each line. The 5' and 3' untranslated regions are indicated in lower case letters. Amino acid residues are numbered above the sequence 20 starting at Ser 1 of the mature mpl ligand (ML) protein sequence. The boundries of presumed exon 3 are indicated by the arrows and the potential N-glycosylation sites are boxed. Cysteine residues are indicated by a dot above the sequence. The underlined sequence corresponds to the N-terminal sequence determined from mpl ligand purified from porcine plasma.

Fig. 2 shows the procedure used for the mpl ligand 3 H-thymidine incorporation assay. To determine the presence of mpl ligand from various sources, the mpl P Ba/F3 cells were starved of IL-3 for 24 hours in a humidified incubator at 370C in 5% 002 and air. Following IL-3 starvation the cells were plated out'in 96 well culture dishes with or without diluted samples and cultured for 24 hrs in a cell culture incubator.

gl of serum free RPMI media containing 1 ICi of 3 H-thymidine was added to each well for the last 6-8 hours. The cells were then harvested on 96 well filter plates and washed with water. The filters were then counted.

Fig. 3 shows the effect of pronase, DTT and heat on the ability of APP to stimulate Ba/F3-mpl cell proliferation. For pronase digestion of APP, pronase (Boehringer Mannheim) or bovine serum albumin was coupled to Affi-gellO (Biorad) and incubated individually with APP for 18hrs. at 370C. Subsequently, the resins were -18removed by centrifugation and supernatants assayed. APP was also heated to 800C for 4 min..o- made 100 M DTT followed by dialysis against PBS.

Fig. 4 shows the elution of mpl ligand activity from Phenyl-Toyopearl, Blue- Sepharose and Ultralink-mp/ columns. Fractions 4-8 from the mpl affinity column were the peak activity fractions eluted from the column.

Fig. 5 shows the SDS-PAGE of eluted Ultralink-mpl fractions. To 200 P! of each fraction 2-8, 1 ml of acetone containing 1mM HCI at -20°C was added. After 3hrs. at 1 0 -20"C samples were centrifuged and resultant pellets were washed 2x with acetone at The acetone pellets were subsequently dissolved in 30 gI of SDS-solubilization buffer, made 100 pM DTT and heated at 90C for 5 min. The samples were then resolved on a 4-20% SDS-polyacrylamide gel and proteins were visualized by silver staining.

Fig. 6 shows elution of mpl ligand activity from SDS-PAGE. Fraction 6 from the mpl-affinily column was resolved on a 4-20% SDS-polyacrylamide gel under nonreducing conditions. Following electrophoresis the gel was sliced into 12 equal regions and electroeluted as described in the examples. The electroeluted samples were 20 dialyzed into PBS and assayed at a 1/20 dilution. The Mr standards used to calibrate the gel were Novex Mark 12 standards.

Fig. 7 shows the effect of mpl ligand depleted APP on human megakaryocytopoiesis.

mpl ligand depleted APP was made by passing 1 ml over a 1 ml mpl-affinity column 25 (700 ag mpl-lgG/ml NHS-superose, Pharmacia). Human peripheral stem cell i cultures were made 10% APP or 10% mpl ligand depleted APP and cultured for 12 days. Megakaryocytopoiesis was quantitated as described in the examples.

Fig. 8 shows the effect of mpl-lgG on the stimulation of human megakaryocytopoiesis by APP. Human peripheral stem cell cultures were made 10% with APP and cultured for 12 days. At day 0, 2 and 4, mpl-lgG (0.5 pg) or ANP-R-IgG (0.5 gg) was added.

After 12 days megakaryocytopoiesis was quantitated as described in the examples. The average of duplicate samples is graphed with the actual duplicate data in parenthesis.

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

-19- WO 95/18858 PCT/US94/14553 Fig. 10 shows deduced amino acid sequence of mature human mpl ligand (hML) (SEQ ID NO: 6) and mature human erythropoietin (hEPO) (SEQ ID NO: The predicted amino acid sequence for the human mpl ligand is aligned with the human erythropoietin sequence. Identical amino acids are boxed and gaps introduced for optimal alignment are indicated by dashes. Potential N-glycosylation sites are underlined with a plain line for the hML and with a broken line for hEPO. The two cysteines important for erythropoietin activity are indicated by a large dot.

Fig. 11 shows deduced amino acid sequence of mature human mpl ligand isoforms hML 1 0 (SEQ ID NO: hML2 (SEQ ID NO: hML3 (SEQ ID NO: and hML4 (SEQ ID NO: Identical amino acids are boxed and gaps introduced for optimal alignment are indicated by dashes.

Figs. 12A, 12B and 12C show the effect of human mpl ligand on Ba/F3-mpl cell p proliferation in vitro human megakaryocytopoiesis quantitated using a radiolabeled murine IgG monoclonal antibody specific to the megakaryocyte glycoprotein GPIIbllla and murine thrombopoiesis measured in a platelet rebound assay Two hundred ninety-three cells were transfected by the CaPO4 method (Gorman, C in DNA Cloning: A New Approach 2:143-190 [1985]) with pRK5 vector alone, pRK5-hML or with pRK5-ML 1 5 3 overnight (pRK5-ML 1 5 3 was generated by introducing a stop codon after residue 153 of hML by PCR). Media was then conditioned for 36h and assayed for stimulation of cell proliferation of Ba/F3-mpl as described in Example 1 or in vitro human megakaryocytopoiesis 25 Megakaryocytopoiesis was quantitated using a 1251 radiolabeled murine IgG monoclonal antibody (HP1-1D) to the megakaryocyte specific glycoprotein GPIIbllla as described (Grant et al., Blood 69:1334-1339 [1987]). The effect of partially purified recombinant ML (rML) on in vivo platelet production was determined using the rebound thrombocytosis assay described by McDonald, T.P. Proc. Soc. Exp.

Biol. Med. 144:1006-10012 (1973). Partially purified rML was prepared from 200ml of conditioned media containing the recombinant ML. The media was passed through a 2ml Blue-Separose column equilabrated in PBS and the column was washed with PBS and eluted with PBS containing 2M each of urea and NaCI. The active fraction was dialyzed into PBS and made 1mg/ml with endotoxin free BSA. The sample 3 5 contained less than one unit of endotoxin /ml. Mice were injected with either 64,000, 32,000 or 16,000 units of rML or excipient alone. Each group consisted of six mice.

The mean and standard deviation of each group is shown, p values were determined by a 2 tailed T-test comparing medians.

Fig. 13_compares the effect of human mpl ligand isoforms and variants in the Ba/F3mpl cell proliferation assay. hML, mock, hML2, hML3, hML(R153A, R154A), and hML 15 3 were assayed at various dilutions as described in Example 1.

Figs. 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 human genomic DNA coding sequence (SEQ ID NO: 11). Nucleotides and amino acid residues are numbered at the beginning of each line.

Fig. 15 shows a SDS-PAGE of purified 293-rhML 3 32 and purified 293-rhML 1 53 Fig. 16 shows the nucleotide sequence: cDNA coding (SEQ ID NO: 12) and deduced amino acid sequence (SEQ ID NO: 13) of the open reading frame of a murine ML 1 5 isoform. This mature murine mpl ligand isoform contains 331 amino acid residues, four fewer than the putative full length mML, and is therefore designated mML2.

Nucleotides are numbered at the beginning of each line. Amino acid residues are numbered above the sequence starting with Ser 1. The potential N-glycosylation sites are underlined. Cysteine residues are indicated by a dot above the sequence.

Fig. 17 shows the cDNA sequence (SEQ ID NO: 14) and predicted protein sequence (SEQ ID NO: 15) of this murine ML isoform (mML). Nucleotides are numbered at the beginning of each line. Amino acid residues are numbered above the sequence starting with Ser 1. This mature murine mpl ligand isoform contains 335 amino acid residues 2 5 and is believed to be the full length mpl ligand, designated mML. The signal sequence is indicated with a dashed underline and the likely cleavage point is denoted with an arrow. The 5' and 3' untranslated regions are indicated with lower case letters. The two deletions found as a result of alternative splicing (mML2 and mML3) are underlined. The four cysteine residues are indicated by a dot. The seven potential Nglycosylation sites are boxed.

Fig. 18 compares the deduced amino acid sequence of the human ML isoform hML3 (SEQ ID NO: 9) and a murine ML isoform designated mML3 (SEQ ID NO: 16). The predicted amino acid sequence for the human mpl ligand is aligned with the murine mpl 3 5 ligand sequence. Identical amino acids are boxed and gaps introduced for optimal alignment are indicated by dashes. Amino acids are numbered at the beginning of each line.

-21- WO 95/18858 PCT/US94/14553 Fig. 19 compares the predicted amino acid sequences of mature ML isoforms from mouse-ML (SEQ ID NO: 17), porcine-ML (SEQ ID NO: 18) and human-ML (SEQ ID NO: Amino acid sequences are aligned with gaps, indicated by dashes, introduced for optimal alingment. Amino acids are numbered at the beginning of each line with Identical residues boxed. Potential N-glycosylation sites are indicated by a shaded box and cysteine residues are designated with a dot. The conserved di-basic amino acid motif that presents a potential protease cleavage site is underlined. The four amino acid deletion found to occur in all three species (ML2) is outlined with a bold box.

1 0 Fig. 20 shows the cDNA sequence (SEQ ID NO: 19) and predicted mature protein sequence (SEQ ID NO: 18) of a porcine ML isoform (pML). This porcine mpl ligand isoform contains 332 amino acid residues and is believed to be the full length porcine mpl ligand, designated pML. Nucleotides are numbered at the beginning of each line.

Amino acid residues are numbered above the sequence starting with Ser 1.

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

Fig. 22 compares the deduced amino acid sequence of the full length porcine ML isoform pML (SEQ ID NO: 18) and a porcine ML isoform designated pML2 (SEQ ID NO: 25 21). The predicted amino acid sequence for the pML is aligned with pML2 sequence.

Identical amino acids are boxed and gaps introduced for optimal alignment are indicated by dashes. Amino acids are numbered at the beginning of each line.

Fig. 23 shows the pertinent features of plasmid pSVI5.ID.LL.MLORF ("full length" or 3 0 TP0 3 3 2 used to transfect host CHO-DP12 cells for production of CHO-rhTPO 3 3 2 Fig. 24 shows the pertinent features of plasmid pSVI5.ID.LL.MLEPO-D ("truncated" or TPO 15 3 used to transfect host CHO-DP12 cells for production of CHO-rhTPO 1 5 3 Figs. 25A, 25B, and 25C show the effect of E. coli-rhTPO(Met-1, 153) on platelets red blood cells and white blood cells in normal mice. Two groups of 6 female C57 B6 mice were injected daily with either PBS buffer or 0.3p.g E. coli-rhTPO(Met-1, 153) (100l1 On day 0 and on days 3-7 40pl of blood was -22- WO 95/18858 PCT/US94/14553 taken from the orbital sinus. This blood was immediately diluted in 10 ml of commercial diluant and complete blood counts were obtained on a Serrono Baker Hematology Analyzer 9018. The data are presented as means Standard error of the mean.

Figs. 26A, 26B and 26C show the effect of E. coli-rhTPO(Met-1 153) on platelets red blood cells and white blood cells in sublethally irradiated mice. Two groups of 10 female C57 B6 mice were sublethally irradiated with 750 cGy of gamma radiation from a 1 3 7 Cs source and injected daily with either PBS buffer or 3.Og E.

coli-rhTPO(Met- 1 153) (1001 On day 0 and at subsequent intermediate time points 40p1 of blood was taken from the orbital sinus. This blood was immediately diluted in 10 ml of commercial diluant and complete blood counts were obtained on a Serrono Baker Hematology Analyzer 9018. The data are presented as means Standard error of the mean.

Figs. 27A, 27B and 27C show the effect of CHO-rhTPO 3 3 2 on platelets (thrombocytes), red blood cells (erythrocytes) and white blood cells (leukocytes) in normal mice. Two groups of 6 female C57 B6 mice were injected daily with either PBS buffer or 0.3g CHO-rhTPO 3 3 2 (1001l On day 0 and on 20 days 3-7 401l of blood was taken from the orbital sinus. This blood was immediately diluted in 10 ml of commercial diluant and complete blood counts were obtained on a Serrono Baker Hematology Analyzer 9018. The data are presented as rieans Standard error of the mean.

25 Fig. 28 shows dose response curves for various forms of rhTPO obtained from various cell lines. Dose response curves were constructed to rhTPO from the following cell lines: hTPO 3 3 2 from CHO (full length from Chinese hamster ovary cells); hTPOMet 153 co/i-derived truncated form with an N-terminal methionine); hTPO 3 3 2 (full length TPO from human 293 cells); Met-less 155 E-Coli (the truncated form [rhTPO 1 5 5 without the terminal methionine from E. coli).

Groups of 6 female C57B6 mice were injected daily for 7 days with rhTPO depending upon group. Each day 40.d of blood was taken from the orbital sinus for complete blood counts. The data presented above are the maximal effects seen with the various treatments and with the exception of (met 153 E-Coli) this occurred on day 7 of treatment. In the aforementioned "met 153 E-Col' group the maximal effect was seen on day 5. The data are presented as means Standard error of the mean.

-23- Fig. 29 shows dose response curves comparing the activity of full length and "cliped" forms of rhTPO produced in CHO cells with the truncated form from E. coli. Groups of 6 female C57B6 mice were injected daily with 0.3p.g rhTPO of various types. On days 2-7 40 41 of blood was taken from the orbital sinus for complete blood counts.

Treatment groups were TPO 1 5 3 the truncated form of TPO from E. coli; TPO 3 3 2 (Mix fraction) Full length TPO containing approximately 80-90% full length and 10-20% clipped forms; TP0332(30K fraction) purified clipped fraction from the original "mix" preparation; TP0332(70K fraction) purified full length TPO fraction from the original "mix" preparation. The data are presented as means Standard error of 1 0 the mean.

Fig. 30 is a cartoon showing the KIRA ELISA assay for measuring TPO. The figure shows the MPURse.gD chimera and relavant parts of the parent receptors as well as Sthe final construct (right portion of the figure) and a flow diagram (left portion of the 1 5 figure) showing relevant steps of the assay.

Fig. 31 is a flow chart for the KIRA ELISA assay showing each step in the procedure.

Figs. 32A-32L provide the nucleotide sequence (SEQ ID NO: 22) of the pSVI17.ID.LL 2 0 expression vector used for expression of Rse.gD in Example 17.

Fig. 33 is a schematic representation of the preparation of plasmid pMP1'.

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

*2 Fig. 35 is a schematic representation of the preparation of plasmid pMP151.

Fig. 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.

Fig. 39 is a table of the five best expressing TPO clones from the pMP210 plasmid 3 5 bank (SEQ ID NOS: 23, 24, 25, 26, 27 and 28).

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

-24- WO 95/18858 PCTIUS94/14553 Fig. 41 is a schematic representation of the preparation of plasmid pMP57.

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

DETAILED DESCRIPTION OF THE INVENTION I. Definitions In general, the following words or phrases have the indicated definition when used in the description, examples, and claims.

"Chaotropic agent" refers to a compound which, in aqueous solution and in suitable concentrations, can cause a change in the spatial configuration or conformation of a protein by at least partially disrupting the forces responsible for maintaining the normal secondary and tertiary structure of the protein. Such compounds include, for example, urea, guanidineHCI, and sodium thiocyanate. High concentrations, usually 4-9M, of these compounds are normally required to exert the 15 conformational effect on proteins.

"Cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone, insulin-like growth factors, human growth hormone, 20 N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and o leutinizing hormone hematopoletic growth factor, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-a 25 (TNF-a and TNF-p) mullerian-inhibiting substance, mouse gonadotropin-associated i peptide, inhibin, activin, vascular endothelial growth factor, integrin, nerve growth factors such as NGF-, platelet-growth factor, transforming growth factors (TGFs) such as TGF-a and TGF-0, insulin-like growth factor-I and -I1, erythropoietin (EPO), osteoinductive factors, interferons such as interferon-a, and colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocytemacrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (IL's) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 and other polypeptide factors including LIF, SCF, and kit-ligand. As used herein the foregoing terms are meant to include proteins from natural sources or from recombinant cell culture. Similarly, the terms are intended to include biologically active equivalents; differing in amino acid sequence by one or more amino acids or in type or extent of glycosylation.

WO 95/18858 PCT/US94/14553 "mpl ligand", "mpl ligand polypeptide", "thrombopoietin" or "TPO" are used interchangeably herein and comprise any polypeptide that possesses the property of binding to mpl, a member of the cytokine receptor superfamily, and having a biological property of the ML as defined below. An exemplary biological property is the ability to stimulate the incorporation of labeled nucleotides 3 H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected with human mpl P. Another exemplary biological property is the ability to stimulate the incorporation of 35S into circulating platelets in a mouse platelet rebound assay. This definition encompasses the polypeptide isolated from a mpl ligand source such as aplastic porcine plasma 1 0 described herein or from another source, such as another animal species, including humans or prepared by recombinant or synthetic methods and includes variant forms including functional derivatives, fragments, alleles, isoforms and analogues thereof.

A "mpl ligand fragment" or "TPO fragment" is a portion of a naturally occurring mature full length mpl ligand or TPO sequence having one or more amino 1 5 acid residues or carbohydrate units deleted. The deleted amino acid residue(s) may occur anywhere in the peptide including at either the N-terminal or C-terminal end or internally. The fragment will share at least one biological property in common with mpl ligand. Mpl ligand fragments typically will have a consecutive sequence of at least 10, 15, 20, 25, 30, or 40 amino acid residues that are identical to the sequences of 20 the mpl ligand isolated from a mammal including the ligand isolated from aplastic porcine plasma or the human or murine ligand, especially the EPO-domain thereof.

Representative examples of N-terminal fragments are hML 1 5 3 or TPO(Met- 11 53).

S"Mpl ligand variants" or "mpl ligand sequence variants" as defined herein means a biologically active mplligand as defined below having less than 100% 25 sequence identity with the mpl ligand isolated from recombinant cell culture or aplastic porcine plasma or the human ligand having the deduced sequence described in Fig. 1 (SEQ ID NO: Ordinarily, a biologically active mpl ligand variant will have an amino acid sequence having at least about 70% amino acid sequence identity with the mpl ligand isolated from aplastic porcine plasma or the mature murine or human ligand or fragments thereof (see Fig. 1 [SEQ ID NO: preferably at least about more preferably at least about 80%, still more preferably at least about even more preferably at least about 90%, and most preferably at least about A "chimeric mpl ligand" is a polypeptide comprising full length mpl ligand or one or more fragments thereof fused or bonded to a second heterologous polypeptide or one or more fragments thereof. The chimera will share at least one biological property in common with mpl ligand. The second polypeptide will typically be a cytokine, immunoglobin or fragment thereof.

-26-

I

WO 95/18858 PCT/US94/14553 "Isolated mpl ligand", "highly purified mpl ligand" and "substantially homogeneous mpl ligand" are used interchangeably and mean a mpl ligand that has been purified from a mpl ligand source or has been prepared by recombinant or synthetic methods and is sufficiently free of other peptides or proteins to obtain at least and preferably 20 amino acid residues of the N-terminal or of an internal amino acid sequence by using a spinning cup sequenator or the best commercially available amino acid sequenator marketed or as modified by published methods as of the filing date of this application, or to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Homogeneity here means 1 0 less than about 5% contamination with other source proteins.

"Biological property" when used in conjunction with either the "mpl ligand" or "Isolated mpl ligand" means having thrombopoietic activity or having an in vivo effector or antigenic function or activity that is directly or indirectly caused or performed by a mpl ligand (whether in its native or denatured conformation) or a 1 5 fragment thereof. Effector functions includempl binding and any carrier binding activity, agonism or antagonism of mpl, especially transduction of a proliferative signal including replication, DNA regulatory function, modulation of the biological activity of other cytokines, receptor (especially cytokine) activation, deactivation, up- or down regulation, cell growth or differentiation and the like. An antigenic 20 function means possession of an epitope or antigenic site that is capable of crossreacting with antibodies raised against the native mpl ligand. The principal antigenic function of a mpl ligand polypeptide is that it binds with an affinity of at least/about 106 I/mole to an antibody raised against the mpl ligand isolated from aplastic porcine plasma. Ordinarily, the polypeptide binds with an affinity of at least-about 107 25 I/mole. Most preferably, the antigenically active mpl ligand polypeptide is a polypeptide that binds to an antibody raised against the mpl ligand having one of the above described effector functions. The antibodies used to define "biologically activity" are rabbit polyclonal antibodies raised by formulating the mpl ligand isolated from recombinant cell culture or aplastic porcine plasma in Freund's complete adjuvant, subcutaneously injecting the formulation, and boosting the immune response by intraperitoneal injection of the formulation until the titer of mpl ligand antibody plateaus.

"Biologically active" when used in conjunction with either the "mpl ligand" or "Isolated mpl ligand" means a mpl ligand or polypeptide that exhibits thrombopoietic activity or shares an effector function of the mpl ligand isolated from aplastic porcine plasma or expressed in recombinant cell culture described herein. A principal known effector function of the mpl ligand or polypeptide herein is binding to mpl and stimulating the incorporation of labeled nucleotides 3 H-thymidine) into the DNA of -27- WO 95/18858 PCT/US94/14553 IL-3 dependent Ba/F3 cells transfected with human mpl P. Another known effector function of the mpl ligand or polypeptide herein is the ability to stimulate the incorporation of 3 5 S into circulating platelets in a mouse platelet rebound assay. Yet another known effector function of mpl ligand is the ability to stimulate in vitro human megakaryocytopoiesis that may be quantitated by using a radio labeled monoclonal antibody specific to the megakaryocyte glycoprotein GPIlbllla.

"Percent amino acid sequence identity" with respect to the mpl ligand sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the mpl ligand sequence isolated from aplastic 1 0 porcine plasma or the murine or human ligand having the deduced amino acid sequence described in Fig. 1 (SEQ ID NO: after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the mpl ligand 1 5 sequence shall be construed as affecting sequence identity or homology. Thus exemplary biologically active mpl ligand polypeptides considered to have identical sequences include; prepro-mpl ligand, pro-mpl ligand, and mature mpl ligand.

"Mpl ligand microsequencing" may be accomplished by any appropriate standard procedure provided the procedure is sensitive enough. In one such method, 20 highly purified polypeptide obtained from SDS gels or from a final HPLC step are sequenced directly by automated Edman (phenyl isothiocyanate) degradation using a model 470A Applied Biosystems gas phase sequencer equipped with a 120A phenylthiohydantion (PTH) amino acid analyzer. Additionally, mpl Ilgand fragments prepared by chemical CNBr, hydroxylamine, 2 -nitro-5-thiocyanobenzoate) or 25 enzymatic trypsin, clostripain, staphylococcal protease) digestion followed by fragment purification HPLC) may be similarly sequenced. PTH amino acids are analyzed using the ChromPerfect data system (Justice Innovations, Palo Alto, CA).

Sequence interpretation is performed on a VAX 11/785 Digital Equipment Co.

computer as described by Henzel et J. Chromatography, 404:41-52 [1987].

Optionally, aliquots of HPLC fractions may be electrophoresed on 5-20% SDS-PAGE, electrotransferred to a PVDF membrane (ProBlott, AIB, Foster City, CA) and stained with Coomassie Brilliant Blue (Matsurdiara, J. Biol. Chem., 262:10035-10038 [1987]. A specific protein identified by the stain is excised from the blot and Nterminal sequencing is carried out with the gas phase sequenator described above. For Internal protein sequences, HPLC fractions are dried under vacuum (SpeedVac), resuspended in appropriate buffers, and digested with cyanogen bromide, the Lysspecific enzyme Lys-C (Wako Chemicals, Richmond, VA), or Asp-N (Boehringer Mannheim, Indianapolis, IN). After digestion, the resultant peptides are sequenced as a -28- WO 95/18858 PCT/US94/14553 mixture or after HPLC resolution on a C4 column developed with a propanol gradient in 0.1% TFA prior to gas phase sequencing.

"Thrombocytopenia" is defined as a platelet count below 150 X 10 9 per liter of blood.

"Thrombopoietic activity" is defined as biological activity that consists of accelerating the proliferation, differentiation and/or maturation of megakaryocytes or megakaryocyte precursors into the platelet producing form of these cells. This activity may be measured in various assays including an in vivo mouse platelet rebound synthesis assay, induction of platelet cell surface antigen assay as measured by an anti-platelet immunoassay (anti-GPlbllla) for a human leukemia megakaryoblastic cell line (CMK), and induction of polyploidization in a megakaryoblastic cell line (DAMI).

"Thrombopoietin" (TPO) is defined as a compound having thrombopoietic *s activity or being capable of increasing serum platelet counts in a mammal. TPO is 1 5 preferably capable of increasing endogenous platelet counts by at least 10%, more preferably by 50%, and most preferably capable of elevating platelet counts in a human to greater that 150X10 9 per liter of blood.

"Isolated mpl ligand nucleic acid" is RNA or DNA containing greater than 16 and preferably 20 or more sequential nucleotide bases that encode biologically active mpl 2 0 ligand or a fragment thereof, is complementary to the RNA or DNA, or hybridizes to the RNA or DNA and remains stably bound under moderate to stringent conditions. This RNA or DNA is free from at least one contaminating source nucleic acid with which it is S* normally associated in the natural source and preferably substantially free of any other mammalian RNA or DNA. The phrase "free from at least one contaminating source 25 nucleic acid with which it is normally associated" includes the case where the nucleic acid is present in the source or natural cell but is in a different chromosomal location or is otherwise flanked by nucleic acid sequences not normally found in the source cell.

An example of isolated mpl ligand nucleic acid is RNA or DNA that encodes a biologically active mpl ligand sharing at least 75% sequence identity, more preferably at least 80%, still more preferably at least 85%, even more preferably 90%, and most preferably 95% sequence identity with the human, murine or porcine mpl ligand.

"Control sequences" when referring to expression means DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood sequences. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

-29- WO 95/18858 PCT/US94/14553 "Operably linked" when referring to nucleic acids means that the nucleic acids are placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as 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 operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.

"Exogenous" when referring to an element means a nucleic acid sequence that is foreign to the cell, or homologous to the cell but in a position within the host cell 15 nucleic acid in which the element is ordinarily not found.

"Cell," "cell line," and "cell culture" are used interchangeably herein and such designations include all progeny of a cell or cell line. Thus, for example, terms like "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where .distinct designations are intended, it will be clear from the context.

"Plasmids" are autonomously replicating circular DNA molecules possessing independent origins of replication and are designated herein by a lower case "p" preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from such available plasmids in accordance with published procedures. In addition, other equivalent plasmids are known in the art and will be apparent to the ordinary artisan.

"Restriction enzyme digestion" when referring to DNA means catalytic cleavage of internal phosphodiester bonds of DNA with an enzyme that acts only at certain locations or sites in the DNA sequence. Such enzymes are called "restriction endonucleases". Each restriction endonuclease recognizes a specific DNA sequence called a "restriction site" that exhibits two-fold symmetry. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements as established by the enzyme suppliers are used.

Restriction enzymes commonly are designated by abbreviations composed of a capital WO 95/18858 PCT/US94/14553 letter followed by other letters representing the microorganism from which each restriction enzyme originally was obtained and then a number designating the particular enzyme. In general, about 1 pg of plasmid or DNA fragment is used with about 1-2 units of enzyme in about 20 gl of buffer solution. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation of about 1 hour at 370C is ordinarily used, but may vary in accordance with the supplier's instructions. After incubation, protein or polypeptide is removed by extraction with phenol and chloroform, and the digested nucleic acid is recovered from the aqueous fraction by precipitation with ethanol. Digestion with a 1 0 restriction enzyme may be followed with bacterial alkaline phosphatase hydrolysis of the terminal 5' phosphates to prevent the two restriction-cleaved ends of a DNA fragment from "circularizing" or forming a closed loop that would impede insertion of another DNA fragment at the restriction site. Unless otherwise stated, digestion of plasmids is not followed by 5' terminal dephosphorylation. Procedures and reagents 15 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].

"Recovery" or "isolation" of a given fragment of DNA from a restriction digest means separation of the digest on polyacrylamide or agarose gel by electrophoresis, 20 identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the gel from DNA. This procedure is known generally. For example, see Lawn et al., Nucleic Acids Res., 9:6103-6114 [1981], and Goeddel et al., Nucleic Acids Res., 8:4057 [1980].

25 "Southern analysis" or "Southern blotting" is a method by which the presence i. of DNA sequences in a restriction endonuclease digest of DNA or DNA-containing composition is confirmed by hybridization to a known, labeled oligonucleotide or DNA fragment. Southern analysis typically involves electrophoretic separation of DNA digests on agarose gels, denaturation of the DNA after electrophoretic separation, and 3 0 transfer of the DNA to nitrocellulose, nylon, or another suitable membrane support for analysis with a radiolabeled, biotinylated, or enzyme-labeled probe as described 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 probe such as an oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNA fragment. The probe is labeled with a radioisotope such as 3 2 P, or by biotinylation, or with an enzyme. The RNA to be analyzed is usually electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe, -31- WO 95/18858 PCT/US94/14553 using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., supra.

"Ligation" is the process of forming phosphodiester bonds between two nucleic acid fragments. For ligation of the two fragments, the ends of the fragments must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary first to convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation. For blunting the ends, the DNA is treated in a suitable buffer for at least 15 minutes at 15°C with about 10 units of the Klenow fragment of DNA 1 0 polymerase I or T4 DNA polymerase in the presence of the four deoxyribonucleotide triphosphates. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation. The DNA fragments that are to be ligated together are put in solution in about equimolar amounts. The solution will also contain ATP, ligase buffer, and a ligase such as T4 DNA ligase at about 10 units per 0.5 gg of DNA. If the DNA is to be 1 5 ligated into a vector, the vector is first linearized by digestion with the appropriate restriction endonuclease(s). The linearized fragment is then treated with bacterial alkaline phosphatase or calf intestinal phosphatase to prevent self-ligation during the ligation step.

"Preparation" of DNA from cells means isolating the plasmid DNA from a culture of the host cells. Commonly used methods for DNA preparation are the largeand small-scale plasmid preparations described in sections 1.25-1.33 of Sambrook et al., supra. After preparation of the DNA, it can be purified by methods well known in the art such as that described in section 1.40 of Sambrook et al., supra.

"Oligonucleotides" are short-length, single- or double-stranded .2 5 polydeoxynucleotides that are chemically synthesized by known methods (such as phosphotriester, phosphite, or phosphoramidite chemistry, using solid-phase techniques such as described in EP 266,032 published 4 May 1988, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al., Nucl.

Acids Res., 14:5399-5407 [1986]). Further methods include the polymerase chain 3 0 reaction defined below and other autoprimer methods and oligonucleotide syntheses on solid supports. All of these methods are described in Engels et al., Agnew. Chem. Int.

Ed. Engl., 28:716-734 (1989). These methods are used if the entire nucleic acid sequence of the gene is known, or the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, one may infer potential nucleic acid sequences using known and preferred coding residues for each amino acid residue. The oligonucleotides are then purified on polyacrylamide gels.

-32- WO 95/18858 PCTJUS94/14553 "Polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Patent No. 4,683,195 issued 28 July 1987. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage 1 0 or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp.

Quant. Biol., 51:263 [1987]; Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample comprising the use of a known nucleic acid as a primer and a nucleic acid polymerase 15 to amplify or generate a specific piece of nucleic acid.

"Stringent conditions" are those that employ low ionic strength and high temperature for washing, for example, 0.015 M NaCI/0.0015 M sodium citrate/0.1% NaDodSO 4 (SDS) at 50°C, or employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum 20 albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCI, 75 mM sodium citrate at 420C. Another example is use of formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mMl sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ig/ml), 0.1% SDS, and 10% dextran sulfate at 420C, with 25 washes at 42"C in 0.2 x SSC and 0.1% SDS.

:i "Moderately stringent conditions" are described in Sambrook et al., supra, and include the use of a washing solution and hybridization conditions temperature, ionic strength, and %SDS) less stringent than described above. An example of moderately stringent conditions are conditions such as overnight incubation at 37°C in a solution comprising: 20% formamide, 5 X SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 5 X Denhardt's solution, 10% dextran sulfate, and 20 ll/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 X SSC at about 37-50 0 C. The skilled artisan will recognize how to adjust the temperature, ionic strength etc. as necessary to accommodate factors such as probe length and the like.

"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like -33- WO 95/18858 PCT/US94/14553 molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.

"Native antibodies and immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a 1 0 variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one and (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the 15 light and heavy chain variable domains (Clothia et al., J. Mol. Biol., 186:651-663 [1985]; Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-4596 [1985]).

The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the 20 variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called the framework The variable domains of native heavy and light chains each comprise four FR 25 regions, largely adopting a P-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the p-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD [1987]). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab') 2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

-34- WO 95/18858 PCT/US94/14553 "Fv" is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab') 2 antibody fragments originally were produced 1 5 as pairs of Fab' fragments which have hinge cysteines between them. Other, chemical couplings of antibody fragments are also known.

i. The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), IgG-1, lgG-2, IgG-3, and lgG-4; IgA- 1 and lgA-2. The heavy chain constant domains that correspond to the different classes 25 of immunoglobulins are called a, delta, epsilon, 7, and respectively. The subunit S. structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments Fab, F(ab') 2 and Fv), so long as they exhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each WO 95/18858 PCT/US94/14553 monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, U.S. Patent No.

1 0 4,816,567 [Cabilly et The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of 1 5 the chain(s) is identical with or homologous to corresponding sequences in antibodies Sderived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity Patent No. 4,816,567 (Cabilly et and Morrison et al., Proc. Natl.

Acad. Sci. USA, 81:6851-6855 [1984]).

"Humanized" forms of non-human murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain-minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.

These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region typically that of a human immunoglobulin. For further details see: Jones -36- WO 95/18858 PCT/US94/14553 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 a human" means that upon contacting the polypeptide in a pharmaceutically acceptable carrier and in a therapeutically effective amount with the appropriate tissue of a human, no state of sensitivity or resistance to the polypeptide is demonstratable upon the second administration of the polypeptide after an appropriate latent period 8 to 14 days).

II. Preferred Embodiments of the Invention Preferred polypeptides of this invention are substantially homogeneous polypeptide(s), referred to as mpl ligand(s) or thrombopoietin (TPO), that possesse Sthe property of binding to mpl, a member of the receptor cytokine superfamily, and having the biological property of stimulating the incorporation of labeled nucleotides 3 H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected with human 1 5 mpl P. More preferred mpl ligand(s) are isolated mammalian protein(s) having hematopoietic, especially megakaryocytopoietic or thrombocytopoietic activity namely, being capable of stimulating proliferation, maturation and/or differentiation of immature megakaryocytes or their predecessors into the mature platelet-producing form. Most preferred polypeptides of this invention are human mpl ligand(s) including fragments thereof having hematopoietic, megakaryocytopoietic or thrombopoietic activity. Optionally these human mpl ligand(s) lack glycosylation.

Other prefered human mpl ligands are the "EPO-domain" of hML refered to as hML 1 53 or hTPO 1 5 3 a truncated form of hML refered to as hML 2 4 5 or hTPO 2 4 5 and the mature full length polypeptide having the amino acid sequence shown in Fig. 1 (SEQ S 25 ID NO: refered to as hML, hML 3 3 2 or hTPO 3 3 2 and the biolocically active substitutional variant hML(R153A, R154A).

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

Optional preferred polypeptides of this invention are biologically active mpl ligand variant(s) that have an amino acid sequence having at least 70% amino acid sequence identity with the human mpl ligand (see Fig. 1 [SEQ ID NO: the murine mpl ligand (see Fig. 16 [SEQ ID NOS: 12 the recombinant porcine mpl ligand (see Fig. 19 [SEQ ID NO: 18]) or the porcine mpl ligand isolated from aplastic 3 5 porcine plasma, preferably at least 75%, more preferably at least 80%, still more preferably at least 85%, even more preferably at least 90%, and most preferably at least -37- WO 95/18858 PCT/US94/14553 The mpl ligand isolated from aplastic porcine plasma has the following characteristics: The partially purified ligand elutes from a gel filtration column run in either PBS, PBS containing 0.1% SDS or PBS containing 4M MgCl2 with Mr of 60,000-70,000; 2 The ligand's activity is destroyed by pronase; The ligand is stable to low pH SDS to and 2M urea; The ligand is a glycoprotein, based on its binding to a variety of lectin columns; 1 0 The highly purified ligand elutes from non-reduced SDS-PAGE with a Mr of 25,000-35,000. Smaller amounts of activity also elute with Mr of -18,000- 22,000 and 60,000; The highly purified ligand resolves on reduced SDS-PAGE as a doublet with Mr of 28,000 and 31,000; 1 5 The amino-terminal sequence of the 18,000-22,000, 28,000 and 31,000 bands is the same SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO: 29); and S( 8 The ligand binds and elutes from the following affinity 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, and Phenyl-Sepharose.

More preferred mpl ligand polypeptides are those encoded by human genomic or cDNA having an amino acid sequence described in Fig. 1 (SEQ ID NO: 1).

Other preferred naturally occurring biologically active mpl ligand polypeptides of this invention include prepro-mpl ligand, pro-mpl ligand, mature mpl ligand, mpl ligand fragments and glycosylation variants thereof.

Still other preferred polypeptides of this invention include mpl ligand sequence variants and chimeras. Ordinarily, preferred mpl ligand sequence variants and chimeras are biologically active mpl ligand variants that have an amino acid sequence having at least 70% amino acid sequence identity with the human mpl ligand or the mpl ligand isolated from aplastic porcine plasma, preferably at least 75%, more -38- WO 95/18858 PCT/US94/14553 preferably at least 80%, still more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%. An exemplary preferred mpl ligand variant is a N-terminal domain hML variant (refered to as the "EPO-domain" because of its sequence homology to erythropoletin). The prefered hML EPO-domain comprises about the first 153 amino acid residues of mature hML and is refered to as hML 1 5 3 An optionally prefered hML sequence variant comprises one in which one or more of the basic or dibasic amino acid residue(s) in the C-terminal domain is substituted with a non-basic amino acid residue(s) hydrophobic, neutral, acidic, aromatic, Gly, Pro and the like). A prefered hML C-terminal domain sequence variant 1 0 comprises one in which Arg residues 153 and 154 are replaced with Ala residues.

This variant is refered to as hML332(R153A, R154A). An alternative prefered hML variant comprises either hML 3 3 2 or hML153 in which amino residues 111-114 (QLPP or LPPQ) are deleted or replaced with a diferent tetrapeptide sequence(e.g.

AGAG or the like). The foregoing deletion mutants are refered to as A4hML332 or A4hML15 3 A preferred chimera is a fusion between mpl ligand or fragment (defined below) thereof with a heterologous polypeptide or fragment thereof. For example, hML153 may be fused to an IgG fragment to improve serum half-life or to IL-3, G- CSF or EPO to produce a molecule with inhanced thrombopoietic or chimeric hematopoietic activity.

An alternative preferred human mpl ligand chimera is a "ML-EPO domain chimera" that consists of the N-terminus 153 to 157 hML residues substituted with one or more, but not all, of the human EPO residues approximately aligned as shown in Fig. 10 (SEQ ID NO: In this embodiment, the hML chimera would be about 153- 166 residues in length in which individual or blocks of residues from the human EPO sequence are added or substituted into the hML sequence at positions corresponding to the alignment shown in Fig. 10 (SEQ ID NO: Exemplary block sequence inserts into the N-terminus portion of hML would include one or more of the N-glycosylation sites at positions (EPO) 24-27, 38-40, and 83-85; one or more of the four predicted amphipathic a-helical bundles at positions (EPO) 9-22, 59-76, 90-107, and 132-152; and other highly conserved regions including the N-terminus and Cterminus regions and residue positions (epo) 44-52 (see Wen et al., Blood, 82:1507-1516 [1993] and Boissel et al., J. Biol. Chem., 268(21):15983-15993 [1993]). It is contemplated this "ML-EPO domain chimera" will have mixed thrombopoietic-erythropoietic (TEPO) biological activity.

Other preferred polypeptides of this 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 sequences of the mpl ligand isolated from aplastic -39- WO 95/18858 PCT/US94/14553 porcine plasma or the human mpl ligand described herein (see e.g. Table 14, Example 24). A preferred mpl ligand fragment is human ML[1-X] where X is 153, 164, 191, 205, 207, 217, 229, or 245 (see Fig. 1 [SEQ ID NO: 1] for the sequence of residues Other preferred mpl ligand fragments include those produced as a result of chemical or enzymatic hydrolysis or digestion of the purified ligand.

Another preferred aspect of the invention is a method for purifying mpl ligand molecules comprises contacting a mpl ligand source containing the mpl ligand molecules with an immobilized receptor polypeptide, specifically mpl or a mpl fusion polypeptide, under conditions whereby the mpl ligand molecules to be purified are selectively adsorbed onto the immobilized receptor polypeptide, washing the immobilized support to remove non-adsorbed material, and eluting the molecules to be S. purified from the immobilized receptor polypeptide with an elution buffer. The source .i containing the mpl ligand may be plasma where the immobilized receptor is preferably S. a mpl-lgG fusion.

1 5 Alternatively, the source containing the mpl ligand is recombinant cell culture where the concentration of mpl ligand in either the culture medium or in cell lysates is generally higher than in plasma or other natural sources. In this case the above described mpl-lgG immunoaffinity method, while still useful, is usually not necessary and more traditional protein purification methods known in the art may be applied.

Briefly, the preferred purification method to provide substantially homogeneous mpl ligand comprises: removing particulate debris, either host cells or lysed fragments by, for example, centrifugation or ultrafiltration; optionally, protien may be concentrated with a commercially available protein concentration filter; followed by separating the ligand from other impurities by one or more steps selected from; 25 immunoaffinity, ion-exchange DEAE or matricies containing carboxymethyl or sulfopropyl groups), Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose, WGA-Sepharose, Con A-Sepharose, Ether Toypearl, Butyl Toypearl, Phenyl Toypearl, protein A Sepharose, SDS-PAGE, reverse phase HPLC silica gel with appended aliphatic groups) or Sephadex molecular seive or size exclusion chromatography, and ethanol or ammonium sulfate precipitation. A protease inhibitor such as methylsulfonylfluoride (PMSF) may be included in any of the foregoing steps to inhibit proteolysis.

In another preferred embodiment, this invention provides an isolated antibody capable of binding to the mpl ligand. A preferred mpl ligand isolated antibody is monoclonal (Kohler and Milstein, Nature, 256:495-497 [1975]; Campbell, Laboratory Techniques in Biochemistry and Molecular Biology, Burdon et al., Eds, Volume 13, Elsevier Science Publisrers, Amsterdam [1985]; and Huse et al., Science, 246:1275-1281 [1989]). Preferred mpl ligand isolated antibody is one that binds WO 95/18858 PCT/US94/14553 to mpl ligand with an affinity of at least about 106 I/mole. More preferably the antibody binds with an affinity of at least about 107 I/mole. Most preferably, the antibody is raised against the mpl ligand having one of the above described effector functions. The isolated antibody capable of binding to the mpl ligand may optionally be fused to a second polypeptide and the antibody or fusion thereof may be used to isolate and purify mpl ligand from a source as described above for immobilized mpl polypeptide. In a further preferred aspect of this embodiment, the invention provides a method for detecting the mpl ligand in vitro or in vivo comprising contacting the antibody with a sample, especially a serum sample, suspected of containing the ligand 1 0 and detecting if binding has occurred.

In still further preferred embodiments, the invention provides an isolated nucleic acid molecule encoding the mpl ligand or fragments thereof, which nucleic acid i molecule may be labeled or unlabeled with a detectable moiety, and a nucleic acid molecule having a sequence that is complementary to, or hybridizes under stringent or 1 5 moderately stringent conditions with, a nucleic acid molecule having a sequence encoding a mpl ligand. A preferred mpl ligand nucleic acid is RNA or DNA that encodes a biologically active mpl ligand sharing at least 75% sequence identity, more preferably at least 80%, still more preferably at least 85%, even more preferably and most preferably 95% sequence identity with the human mpl ligand. More 2 0 preferred isolated nucleic acid molecules are DNA sequences encoding biologically active mpl ligand, selected from: DNA based on the coding region of a mammalian mpl ligand gene DNA comprising the nucleotide sequence provided in Fig. 1 (SEQ ID NO: or fragments thereof); DNA capable of hybridizing to a DNA of (a) under at least moderately stringent conditions; and DNA that is degenerate to a DNA defined in or which results from degeneracy of the genetic code. It is contemplated that the novel mpl ligands described herein may be members of a family of ligands or cytokines having suitable sequence identity that their DNA may hybridize with the DNA of Fig. 1 (SEQ ID NO: 2) (or the complement or fragments thereof) under low to moderate stringency conditions. Thus a further aspect of this invention 3 0 includes DNA that hybridizes under low to moderate stringency conditions with DNA encoding the mpl ligand polypeptides.

In a further preferred embodiment of this invention, the nucleic acid molecule is cDNA encoding the mpl ligand and further comprises a replicable vector in which the cDNA is operably linked to control sequences recognized by a host transformed with the vector. This aspect further includes host cells transformed with the vector and a method of using the cDNA to effect production of mpl ligand, comprising expressing the cDNA encoding the mpl ligand in a culture of the transformed host cells and recovering the mpl ligand from the host cell culture. The mpl ligand prepared in this manner is -41 WO 95/18858 PCT/US94(14553 preferably substantially homogeneous human mpl ligand. A preferred host cell for producing mpl ligand is Chinese hamster ovary (CHO) cells.

The invention further includes a preferred method for treating a mammal having an immunological or hematopoietic disorder, especially thrombocytopenia comprising administering a therapeutically effective amount of a mpl ligand to the mammal. Optionally, the mpl ligand is administered in combination with a cytokine, especially a colony stimulating factor or interleukin. Preferred colony stimulating factors or interleukins include; kit-ligand, LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 or IL-11.

III. Methods of Making Platelet production has long been thought by some authors to be controlled by multiple lineage specific humoral factors. It has been postulated that two distinct cytokine activities, referred to as megakaryocyte colony-stimulating factor (meg- 15 CSF) and thrombopoietin, regulate megakaryocytopoiesis and thrombopoiesis (Williams et al., J. Cell Physiol., 110:101-104 [1982]; Williams et al., Blood Cells, 15:123-133 [1989]; and Gordon at al., Blood, 80:302-307 [1992]).

According to this hypothesis, meg-CSF stimulates the proliferation of progenitor megakaryocytes while thrombopoietin primarily affects maturation of more differentiated cells and ultimately platelet release. Since the 1960's the induction and appearance of both meg-CSF and thrombopoietin activities in the plasma, serum andurine of animals and humans following thrombocytopenic episodes has been well documented (Odell et al., Proc. Soc. Exp. Biol. Med., 108:428-431 [1961]; Nakeff et al., Acta Haematol., 54:340-344 [1975]; Specter, Proc. Soc. Exp. Biol., 108:146- 25 149 [1961]; Schreiner et al., J.Clin.Invest., 49:1709-1713 [1970]; Ebbe, Blood, 44:605-608 [1974]; Hoffman et al., N. Engl. J. Med., 305:533 [1981]; Straneva et al., Exp. Hematol., 17:1122-1127 [1988]; Mazur et al., Exp. Hematol., 13:1164 [1985]; Mazur et al., J.Clin. Invest., 68:733-741 [1981]; Sheiner et al., Blood, 56:183-188 [1980]; Hill et al., Exp. Hematol., 20:354-360 [1992]; and Hegyi et al., Int. J. Cell Cloning, 8:236-244 [1990]). These activities were reported to be lineage specific and distinct from known cytokines (Hill R.J. et al., Blood 80:346 (1992); Erickson-Miller C.L. et al., Brit. J. Haematol., 84:197-203 (1993); Straneva J.E. et al., Exp. Hematol. 20:4750(1992); and Tsukada J. et al., Blood 81:866-867 [1993]). Heretofore, attempts to purify meg-CSF or thrombopoietin 3 5 from thrombocytopenic plasma or urine have been unsuccessful.

Consistent with the above observations describing thrombocytopenic plasma, we have found that aplastic porcine plasma (APP) obtained from irradiated pigs stimulates human megakaryocytopoiesis in vitro. We have found that this stimulatory -42- WO 95/18858 PCT/US94/14553 activity is abrogated by the soluble extracellular domain of c-mpl, confirming APP as a potential source of the putative mpl ligand We have now successfully purified the mpl ligand from APP and amino acid sequence information was used to isolate murine, porcine and human ML cDNA. These ML's have sequence homology to erythropoietin and have both meg-CSF and thrombopoietin-like activities.

1. Purification and Identification of mpl Ligand from Plasma As set forth above, aplastic plasma from a variety of species has been reported to contain activities that stimulate hematopoiesis in vitro, however no hematopoietic 1 0 stimulatory factor has previously been reported isolated from plasma. One source of aplastic plasma is that obtained from irradiated pigs. This aplastic porcine plasma (APP) stimulates human hematopoiesis in vitro. To determine if APP contained the .i mpl ligand, its effect was assayed by measuring 3 H-thymidine incorporation into Ba/F3 cells transfected with human mpl P (Ba/F3-mp/ by the procedure shown in 1 5 Fig. 2. APP stimulated 3 H-thymidine incorporation into Ba/F3-mpl cells but not Ba/F3 control cells not transfected with human mpl Additionally, no such activity was observed in normal porcine plasma. These results indicated that APP contained a factor or factors that transduced a proliferative signal through the mpl receptor and therefore might be the natural ligand for this receptor. This was futher supported by the finding that treatment of APP with soluble mpl-lgG blocked the stimulatory effects of APP on Ba/F3-mp/ cells.

The activity in APP appeared to be a protein since pronase, DTT, or heat destroy the activity in APP (Fig. The activity was also non-dialyzable. The activity was, however, stable to low pH (pH 2.5 for 2 hrs.) and was shown to bind and 25 elute from several lectin-affinity columns, indicating that it was a glycoprotein. To further elucidate the structure and identity of this activity it was affinity purified from APP using a mpl-lgG chimera.

APP was treated according to the protocol set forth in Examples 1 and 2.

Briefly, the mpl ligand was purified using hydrophobic interaction chromatography (HIC), immobilized dye chromatography, and mpl-affinity chromatography. The recovery of activity from each step is shown in Fig. 4 and the fold purification is provided in Table 1. The overall recovery of activity through the mpl-affinity column was approximately 10%. The peak activity fraction (F6) from the mplaffinity column has an estimated specific activity of 9.8 x10 6 units/mg. The overall purification from 5 liters of APP was approximately 4 x10 6 fold (0.8 units/mg to 3.3 x 106 units/mg) with a 83 x 10 6 -fold reduction in protein (250 gms to 3 gg).

We estimated the specific activity of the ligand eluted from the mp/-affinity column to be -3x10 6 units/mg.

-43- WO 95/18858 PCT/US94/14553 TABLE 1 n 4 Pnrifinfi Ir u ii m ,I c iLan I h pI i i aiu Sample Volume Protein Units/ml Units Acitivity Yield Fold mis jm/ml Units/mq Purification APP 5000 50 40 200,000 0.8 1 Phenyl 4700 0.8 40 200,000 50 94 62 Blue-Sep. 640 0.93 400 256,000 430 128 538 mpl (pI) (Fxns 5-7 12 5x10" 4 1666 20,000 3,300,000 10 4,100,000 Protein was determined by the Bradford assay. Protein concentration of mpl-eluted fractions 5-7 are estimates based on staining intensity of a silver stained SDS-gel. One unit is defined as that causing 50% maximal stimulation of Ba/F3-mpl cell proliferation.

Analysis of eluted fractions from the mpl affinity column by SDS-PAGE (4- 1 0 20%, Novex gel) run under reducing conditions, revealed the presence of several proteins (Fig. Proteins that silver stained with the strongest intensity resolved with apparent Mr of 66,000, 55,000, 30,000, 28,000 and 18,000-22,000. To determine which of these proteins stimulated proliferation of Ba/F3-mp cell cultures, the proteins were eluted from the gel as described in Example 2.

The results of this experiment showed that most of the activity eluted from a gel slice that included proteins with Mr 28,000-32,000, with lesser activity eluting in the 18,000-22,000 region of the gel (Fig. The only proteins visible in these regions had Mr of 30,000, 28,000 and 18,000-22,000. To identify and obtain protein sequence for the proteins resolving in this region of the gel bands at 28 and 18-22 kDa), these three proteins were electroblotted to PVDF and sequenced as described in Example 3. Amino-terminus sequences obtained are provided in Table 2.

jl -44- WO 95/18858 PCTIUS94/14553 TABLE 2 Mpl Ligand Amino-Terminus Sequences kDa 1 5 10 15 20 PAP PA(C)DPRLLNKLLRDD(H/S) VLH(G) RL 28 kDa 1 5 10 15 20 (S)PAPPAXDPRLLNKLLRDD(H)VL(H)GR (SEQ IDNO:31) 18-22 kDa 1 5 XPAPPAXDPRLX(N)(K) _(SEQ ID NO: 32) Computer-assisted analysis revealed these amino acid sequences to be novel.

Because all three sequences were the same, it was believed the 30 kDa, 28 kDa and 18-22 kDa proteins were related and might be different forms of the same novel protein. Futhermore, this protein(s) was a likely candidate as the natural mpl ligand because the activity resolved on SDS-PAGE in the same region (28,000-32,000) of a 4-20% gel. In addition, the partially purified ligand migrated with a Mr of 17,000- 1 0 30,000 when subjected to gel filtration chromatography using a Superose 12 (Pharmacia) column. It is believed the different Mr forms of the ligand are a result of proteolysis or glycosylation differences or other post or pre-translational modifications.

As described earlier, antisense human mpl RNA abrogated 1 5 megakaryocytopoiesis in human bone marrow cultures enriched with CD 34+ progenitor cells without affecting the differentiation of other hematopoietic cell lineages (Methia et al., supra). This result suggested that the mpl receptor might play a role in the differentiation and proliferation of megakaryocytes in vitro. To further elucidate the role of the mpl ligand in megakaryocytopoiesis, the effects of APP and mpl ligand depleted APP on in vitro human megakaryocytopoiesis was compared. The effect of APP on human megakaryocytopoiesis was determined using a modification of the liquid suspension megakaryocytopoiesis assay described in Example 4. In this assay, human peripheral stem cells (PSC) were treated with APP before and after mpl-lgG affinity chromatography. GPIlbllla stimulation of megakaryocytopoiesis was quantitated with an 1 2 5 1-anti-llbllla antibody (Fig. Shown in Fig. 7, 10% APP caused approximately a 3-fold stimulation while APP depleted of mpl ligand had no effect. Significantly, the mpl ligand depleted APP did not induce proliferation of the Ba/F3-mpl cells.

WO 95/18858 PCT/US94/14553 In another experiment, soluble human mpl-lgG added at days 0, 2 and 4 to cultures containing 10% APP neutralized the stimulatory effects of APP on human megakaryocytopoiesis (Fig. These results indicate that the mpl ligand plays a role in regulating human megakaryocytopoiesis and therefore may be useful for the treatment of thrombocytopenia.

2. Molecular Cloning of the mpl Ligand Based on the amino-terminal amino acid sequence obtained from the 30 kDa, 28 kDa and 18-22 kDa proteins (see Table 2 above), two degenerate oligonucleotide 1 0 primer pools were designed and used to amplify porcine genomic DNA by PCR. It was reasoned that If the amino-terminal amino acid sequence was encoded by a single exon then the correct PCR product was expected to be 69 bp long. A DNA fragment of this size was found and subcloned into pGEMT. The sequences of the oligonucleotide PCR primers and the three clones obtained are shown in Example 5. The amino acid 1 5 sequence (PRLLNKLLR [SEQ ID NO: 33]) of the peptide encoded between the PCR primers was identical to that obtained by amino-terminal protein sequencing of the porcine ligand (see residues 9-17 for the 28 and 30 kDa porcine protein sequences above).

A synthetic oligonucleotide based on the sequence of the PCR fragment was used to screen a human genomic DNA library. A 45-mer oligonucleotide, designated was designed and synthesized based on the sequence of the PCR fragment. This oligonucleotide had the following sequence: 5' GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-T--ATT-TAG-GAG-TCG 3' (SEQ ID NO: 34) 2 5 This deoxyoligonucleotide was used to screen a human genomic DNA library in Xgeml2 under low stringency hybridization and wash conditions according to Example 6. Positive clones were picked, plaque purified and analyzed by restriction mapping and southern blotting. A 390 bp EcoRI-Xbal fragment that hybridized to the was subcloned into pBluescript SK-. DNA sequencing of this clone confirmed that DNA encoding the human homolog of the porcine mpl ligand had been isolated. The human DNA sequence and deduced amino acid sequence are shown in Fig. 9 (SEQ ID NOS: 3 The predicted positions of introns in the genomic sequence are also indicated by arrows, and define a putative exon ("exon Based on the human "exon 3" sequence (Example 6) oligonucleotides corresponding to the 3' and 5' ends of the exon sequence were synthesized. These 2 primers were used in PCR reactions employing as a template cDNA prepared from various human tissues. The expected size of the correct PCR product was 140 bp.

After analysis of the PCR products on a 12% polyacrylamide gel, a DNA fragment of the -46- WO 95/18858 PCT/US94/14553 expected size was detected in cDNA libraries prepared from human adult kidney, 293 fetal kidney cells and cDNA prepared from human fetal liver.

A fetal liver cDNA library (7x10 6 clones) in lambda DR2 was next screened with the same 45-mer oligonucleotide used to screen the human genomic library and the fetal liver cDNA library under low stringency hybridization conditions. Positive clones were picked, plaque purified and the insert size was determined by PCR. One clone with a 1.8 kb insert was selected for further analysis. Using the procedures described in Example 7 the nucleotide and deduced amino acid sequence of the human mpl ligand (hML) were obtained. These sequences are presented in Fig. 1 (SEQ ID 1 0 NOS: 1 2).

3. Structure of the Human mpl Ligand (hML) The human mpl ligand (hML) cDNA sequence (Fig. 1 [SEQ ID NO: 2]) comprises 1774 nucleotides followed by a poly(A) tail. It contains 215 nucleotides of 1 5 5' untranslated sequence and a 3' untranslated region of 498 nucleotides. The presumed initiation codon at nucleotide position (216-218) is within a consensus i. sequence favorable for eukaryotic translation initiation. The open reading frame is 1059 nucleotides long and encodes a 353 amino acid residue polypeptide, beginning at nucleotide position 220. The N-terminus of the predicted amino acid sequence is 2 0 highly hydrophobic and probably corresponds to a signal peptide. Computer analysis of the predicted amino acid sequence (von Heijne et Eur. J. Biochem., 133:17.-21 [1983]) indicates a potential cleavage site for signal peptidase between residues 21 and 22. Cleavage at that position would generate a mature polypeptide of 332 amino acid residues beginning with the amino-terminal sequence obtained from mpl ligand 25 purified from porcine plasma. The predicted non-glycosylated molecular weight of the ,I.i 332 amino acid residue ligand is about 38 kDa. There are 6 potential N-glycosylation sites and 4 cysteine residues.

Comparison of the mpl ligand sequence with the Genbank sequence database revealed 23% identity between the amino terminal 153 residues of mature human mpl ligand and human erythropoietin (Fig. 10 [SEQ ID NOS: 6 When conservative substitutions are taken into account, this region of hML shows 50% similarity to human erythropoietin (hEPO). Both hEPO and the hML contain four cysteines. Three of the 4 cysteines are conserved in hML, including the first and last cysteines. Sitedirected mutagenesis experiments have shown that the first and last cysteines of erythropoietin form a disulfide bond that is required for function (Wang, F.F.et al., Endocrinology 116:2286-2292 [1983]). By analogy, the first and last cysteines of hML may also form a critical disulfide bond. None of the glycosylation sites are -47- WO 95/18858 PCT[US94/14553 conserved in hML. All potential hML N-linked glycosylation sites are located in the carboxy-terminal half of the hML polypeptide.

Similar to hEPO, the hML mRNA does not contain the consensus polyadenylation sequence AAUAAA, nor the regulatory element AUUUA that is present in 3' untranslated regions of many cytokines and is thought to influence mRNA stability (Shawet al., Cell, 46:659-667 [1986]). Northern blot analysis reveals low levels of a single 1.8 kb hML RNA transcript in both fetal and adult liver. After longer exposure, a weaker band of the same size could be detected in adult kidney. By comparison, human erythropoietin is expressed in fetal liver and, in response to hypoxia, the adult kidney 1 0 and liver (Jacobs et al., Nature, 313:804-809 [1985] and Bondurant et al., Molec.

Cell. Biol., 6:2731-2733 [1986]).

The importance of the C-terminal region of the hML remains to be elucidated.

Based on the presence of the six potential sites for N-linked glycosylation and the ability of the ligand to bind lectin-affinity columns, this region of the hML is likely 1 5 glycosylated. In some gel elution experiments, we observed activity resolving with a Mr around 60,000 which may represent the full length, glycosylated molecule. The C-terminal region may therefore act to stabilize and increase the half-life of circulating hML. In the case of erythropoietin, the non-glycosylated form has full in vitro biological activity, but has a significantly reduced plasma half-life relative to 20 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-basic amino acid sequences [Arg-Arg motifs at positions 153-154 and 245-246] that could serve as potential processing sites. Cleavage at these sites may be responsible for generating the 30, 28 and 18-22 kDa forms of the ML isolated from APP.

.i Significantly, the Arg153-Arg 1 5 4 sequence occurs immediately following the erythropoietin-like domain of the ML. These observations indicate that full length ML may represent a precursor protein that undergoes limited proteolysis to generate the mature ligand.

4. Isoforms and Variants of the Human mpl Ligand Isoforms or alternatively spliced forms of human mpl ligand were detected by PCR in human adult liver. Briefly, primers were synthesized corresponding to each end as well as selected internal regions of the coding sequence of hML. These primers were used in RT-PCR to amplify human adult liver RNA as described in Example In addition to the full length form, designated hML, three other forms, designated hML2, hML3 and hML4, were observed or deduced. The mature deduced amino acid sequences of all four isoforms is presented in Fig. 11 (SEQ ID NOS: 6, 8, 9 -48- WO 95/18858 PCT/US94/14553 hML3 has a 116 nucleotide deletion a position 700 which results in both an amino acid deletion and a frameshift. The cDNA now encodes a mature polypeptide that is 265 amino acid long and diverges from the hML sequence at amino acid residue 139.

Finally, hML4 has both a 12 nucleotide deletion following nucleotide position 618 (also found in the mouse and the pig sequences [see below]) and the 116 bp deletion found in hML3. Altough no clones with only the 12 bp deletion (following nucleotide 619) have been isolated in the human (designated hML2), this form is likely to exist because such a isoform has been identified in both the mouse and pig (see below), and because it has been identified in conjunction with the116 nucleotide deletion in hML4.

Both a substitutional variant of hML in which the dibasic Arg1 5 3 -Arg 1 5 4 sequence was replaced with two alanine residues and a "EPO-domain" truncated form of hML were constructed to determine whether the full length ML was necessary for biological activity. The Argi 5 3 -Arg 1 5 4 dibasic sequence substitutional variant, r. efered to as hML(R153A, R154A), was constructed using PCR as described in 1 5 Example 10. The "EPO-domain" truncated form, hML 1 5 3 was also made using PCR by introducing a stop codon following Arg153.

Expression of Recombinant Human mpl Ligand (rhML) in Transiently Transfected Human Embryonic Kidney (293) Cells To confirm that the cloned human cDNA encoded a ligand for mpl, the ligand was expressed in mammalian 293 cells under the control of the cytomegalovirus immediate early promoter using the expression vectors pRK5-hML or pRK5-hML 1 53.

Supernatants from transiently transfected human embryonic kidney 293 cells were 25 found to stimulate 3 H-thymidine incorporation in Ba/F3-mpl cells, but not in parental Ba/F3 cells (Fig. 12A). Media from the 293 cells transfected with the pRK vector alone did not contain this activity. Addition of mpl-lgG to the media abolished the stimulation (data not shown). These results show that the cloned cDNA encodes a functional human ML (hML).

To determine if the "EPO-domain" alone could bind and activate mpl, the truncated form of hML, rhML 1 5 3 was expressed in 293 cells. Supernatants from transfected cells were found to have activity similar to that present in supernatants from cells expressing the full length hML (Fig. 12A), indicating that the C-terminal domain of ML is not required for binding and activation of c-mpl.

-49- WO 95/18858 PCT/US94/14553 6. mpl Ligand Stimulates Megakaryocytopolesis and Thrombopoiesis Both the full length rhML and the truncated rhML 1 53 forms of recombinant hML stimulated human megakaryocytopoiesis in vitro (Fig. 12B). This effect was observed in the absence of other exogenously added hematopoietic growth factors. With the exception of IL-3, the ML was the only hematopoietic growth factor tested that exhibited this activity. IL-11, IL-6, IL-1, erythropoietin, G-CSF, IL-9, LIF, kit ligand M-CSF, OSM and GM-CSF had no effect on megakaryocytopoiesis when tested separately in our assay (data not shown). This result demonstrates that the ML 1 0 has megakaryocyte-stimulating activity, and indicates a role for ML in regulating megakaryocytopoiesis.

Thrombopoietic activities present in plasma of thrombocytopenic animals have been shown to stimulate platelet production in a mouse rebound thrombocytosis assay (McDonald, Proc. Soc. Exp. Bio/. Med., 14:1006-1001 [1973] and McDonald et al., 1 5 Scand. J. Haematol., 16:326-334 [1976]). In this model mice are made acutely thrombocytopenic using specific antiplatelet serum, resulting in a predictable rebound thrombocytosis. Such immuno-thrombocythemic mice are more responsive to exogenous thrombopoietin-like activities than are normal mice (McDonald, Proc. Soc.

SExp. Biol. Med., 14:1006-1001 [1973]), just as exhypoxic mice are more sensitive i* 20 to erythropoietin than normal are mice (McDonald, et al., J. Lab. Clin. Med., 77:134- 143 [1971]). To determine whether the rML stimulates platelet production in vivo, mice in rebound thrombocytosis were injected with partially purified rhML. Platelet counts and incorporation of 3 5 S into platelets were then quantitated. Injection of mice with 64,000 or 32,000 units of rML significantly increased platelet production, as evidenced by a -20% increase in platelet counts (p=0.0005 and 0.0001, respectively) and a -40% increase in 3 5 S incorporation into platelets (p=0.003) in the treated mice versus control mice injected with excipient alone (Fig. 12C). This level of stimulation is comparable to that which we have observed with IL-6 in this model (data not shown). Treatment with 16,000 units of rML did not significantly stimulate platelet production. These results indicate that ML stimulates platelet production in a dose-dependent manner and therefore possesses thrombopoietin-like activity.

293 cells were also transfected with the other hML isoform constructs described above and the supernatants were assayed using the Ba/F3-mpl proliferation assay (see Fig. 13). hML2 and hML3 showed no detectable activity in this assay, however the activity of hML(R153A, R154A) was similar to hML and hML153 indicating that processing at the Arg153-Arg 1 5 4 di-basic site is neither required for nor detrimental to activity.

WO 95/18858 PCTIUS94/14553 7. Megakaryocytopoiesis and the mpl Ligand It has been proposed that megakaryocytopoiesis is regulated at multiple cellular levels (Williams et al., J.Cell Physiol., 110:101-104 [1982] and Williams et al., Blood Cells, 15:123-133 [1989]). This is based largely on the observation that certain hematopoietic growth factors stimulate proliferation of megakaryocyte progenitors while others appear to primarily affect maturation. The results presented here suggest that the ML acts both as a proliferative and maturation factor. That ML stimulates proliferation of megakaryocyte progenitors is supported by several lines of 1 0 evidence. First, APP stimulates both proliferation and maturation of human megakaryocytes in vitro, and this stimulation is completely inhibited by mpl-IgG (Figs. 7 and Furthermore, the inhibition of megakaryocyte colony formation by c-mpl antisense oligonucleotides (Methia et al., Blood, 82:1395-1401 [1993]) and the finding that c-mpl can transduce a proliferative signal in cells into which it is 1 5 transfected (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 stages of megakaryocyte differentiation (Methia et al., Blood, 82:1395-1401 [19931) and the ability of recombinant ML to rapidly stimulate platelet production in vivo indicate that ML also affects maturation. The 20 availability of recombinant ML makes possible a careful evaluation of its role in regulating megakaryocytopoiesis and thrombopolesis as well as its potential to influence other hematopoietic lineages.

8. Isolation of the Human mpl Ligand (TPO) Gene Human genomic DNA clones of the TPO gene were isolated by screening a human genomic library in X-Gem12 with pR45, under low stringency conditions or under high stringency conditions with a fragment corresponding to the 3' half of human cDNA coding for the mpl ligand. Two overlapping lambda clones spanning 35 kb were isolated. Two overlapping fragments (BamH1 and EcoRI) containing the entire TPO gene were subcloned and sequenced (see Figs. 14A, 14B and 14C).

The structure of the human gene is composed of 6 exons within 7 kb of genomic DNA. The boundaries of all exon/intron junctions are consistent with the consensus motif established for mammalian genes (Shapiro, M. et al., Nucl. Acids Res.

15:7155 [1987]). Exon 1 and exon 2 contain 5' untranslated sequence and the initial 3 5 four amino acids of the signal peptide. The remainder of the secretory signal and the first 26 amino acids of the mature protein are encoded within exon 3. The entire carboxyl domain and 3' untranslated as well as -50 amino acids of the erythropoietin- -51- WO 95/18858 PCT/US94/14553 like domain are encoded within exon 6. The four amino acids involved in the deletion observed within hML-2 (hTPO-2) are encoded at the 5' end of exon 6.

Analysis of human genomic DNA by Southern blot indicated the gene for TPO is present in a single copy. The chromosomal location of the gene was determined by fluorescent in situ hybridization (FISH) which mapped to chromosome 3q27-28.

9. Expression and Purification of TPO from 293 Cells Preperation and purification of ML or TPO from 293 cells is described in detail in Example 19. Briefly, cDNA corresponding to the TPO entire open reading frame 1 0 was obtained by PCR using pRK5-hmp/ 1. The PCR product was purified and cloned between the restriction sites Clal and Xbal of the plasmid pRK5tkneo (a pRK5 derived vector modified to express a neomycin resistance gene under the control of the thymidine kinase promote) to obtain the vector pRK5tkneo.ORF(a vector coding for the entire open reading frame).

S1 5 A second vector coding for the EPO homologous domain was generated the same but using different PCR primers to obtain the final construct called

D.

These two constructs were transfected into Human Embryonic Kidney cells by the CaPO 4 method and neomycin resistant clones were selected and allowed to grow to 20 confluency. Expression of ML 1 5 3 or ML 3 3 2 in the conditioned media from these clones was assessed using the Ba/F3-mpl proliferation assay.

Purification of rhML 3 3 2 was conducted as described in Examplle 19.

Briefly, 293-rhML 3 3 2 conditioned media was applied to a Blue-Sepharose (Pharmacia) column that was subsequently washed with a buffer containing 2M urea.

The column was eluted with a buffer containing 2M urea and 1M NaCI. The Blue- Sepharose elution pool was then directly applied to a WGA-Sepharose column, washed with 10 column volumes of buffer containing 2M urea and 1 M NaCI and eluted with the same buffer containing 0.5M N-acetyl-D-glucosamine. The WGA-Sepharose eluate was applied to a C4-HPLC column (Synchrom, Inc.) and eluted with a discontinuous propanol gradient. By SDS-PAGE the purified 293-rhML 332 migrates as a broad band in the 68-80 kDa region of the gel (see Fig. Purification of rhML 1 5 3 was also conducted as described in Example 19.

Briefly, 293-rhML 1 5 3 conditioned media was resolved on Blue-Sepharose as described for rhML 3 3 2 The Blue Sepharose eluate was applied directly to a mplaffinity column as described above. RhML1 5 3 eluted from the mpl-affinity column was purified to homogeneity using a C4-HPLC column run under the same conditions used for rhML332. By SDS-PAGE the purified rhML 1 5 3 resolves into 2 major and 2 minor bands with Mr of -18,000-22,000 (see Fig. -52- WO 95/18858 PCT/US94/14553 The Murine mplLigand A DNA fragment corresponding to the coding region of the human mpl ligand was obtained by PCR, gel purified and labeled in the presence of 32 P-dATP and 32 P-dCTP.

This probe was used to screen 106 clones of a mouse liver cDNA library in XGT10.

A

murine clone (Fig. 16 [SEQ ID NOS: 12 13]) containing a 1443 base pair insert was isolated and sequenced. The presumed initiation codon at nucleotide position 138- 141 was within a consensus sequence favorable for eukaryotic translation initiation (Kozak, M. J.Cell Bio., 108:229-241 [1989]). This sequence defines an open 1 0 reading frame of 1056 nucleotides, which predicts a primary translation product of 352 amino acids. Flanking this open reading frame are 137 nucleotides of 5' and 247 nucleotides of 3' untranslated sequence. There is no poly(A) tail following the 3' untranslated region indicating that the clone is probably not complete. The N-terminus of the predicted amino acid sequence is highly hydrophobic and probably represents a 15 signal peptide. Computer analysis (von Heijne, G. Eur. J. Biochem. 133:17-21 S[1983]) indicated a potential cleavage site for signal peptidase between residues 21 and 22. Cleavage at that position would generate a mature polypeptide of 331 amino acids (35 kDa) identified as mML 33 1 (or mML2 for reasons described below). The sequence contains 4 cysteines, all conserved in the human sequence, and seven 20 potential N-glycosylation sites, 5 of which are conserved in the human sequence.

Again, as with hML, all seven potential N-glycosylation sites are located in the Cterminal half of the protein.

When compared with the human ML, considerable identity for both nucleotide and deduced amino acid sequences were observed in the "EPO-domains" of these ML's.

2 5 However, when deduced amino acid sequences of human and mouse ML's were aligned, the mouse sequence appeared to have a tetrapeptide deletion between residues 111- 114 corresponding to the 12 nucleotide deletion following nucleotide position 618 seen in both the human (see above) and pig (see below) cDNA's. Accordingly, additional clones were examined to detect possible murine ML isoforms. One clone encoded a 335 amino acid deduced sequence polypeptide containing the "missing" tetrapeptide

LPLQ.

This form is believed to be the full length murine ML and is refered to as mML or mML3 3 5 The nucleotide and deduced amino acid sequence for mML are provided in Fig. 17 (SEQ ID NOS: 14 15). This cDNA clone consists of 1443 base pairs followed by a poly(A) tail. It possesses an open reading frame of 1068 bp flanked by 134 bases of 5' and 241 bases of 3' untranslated sequence. The presumed initiation codon lies at nucleotide position 138-140. The open reading frame encodes a predicted protein of 356 amino acids, the first 21 of which are highly hydrophobic and likely function as a secretion signal.

-53- WO 95/18858 PCT/US94/14553 Finally, a third murine clone was isolated, sequenced and was found to contained the 116 nucleotide deletion corresponding to hML3. This murine isoform is therefore denominated mML3. Comparison of the deduced amino acid sequences of these two isoforms is shown in Fig. 18 (SEQ ID NOS: 9 16).

The overall amino acid sequence identity between human and mouse ML (Fig.

19 [SEQ ID NOS: 6 17]) is 72% but this homology is not evenly distributed. The region defined as the "EPO-domain" (amino acids 1-153 for the human sequence and 1-149 for the mouse) is better conserved (86% homology) than the carboxyterminal region of the protein (62% homology). This may further indicate that only the "EPO-domain" is important for the biological activity of the protein.

Interestingly, of the two di-basic amino acid motifs found in hML, only the di-basic motif immediately following the "EPO-domain" (residue position 153-154) in the .human sequence is present in the murine sequence. This is consistent with the S. possibility that the full length ML may represent a precursor protein that undergoes 1 5 limited proteolysis to generate the mature ligand. Alternatively, proteolysis between Arg153-Arg1 5 4 may facilitate hML clearance An expression vector containing the entire coding sequence of mML was transiently transfected into 293 cells as described in Example 1. Conditioned media *from these cells stimulated 3 H-thymidine incorporation into Ba/F3 cells expressing 0 either murine or human mpl but had no effect on the parental (mpl-less) cell line.

This indicates that the cloned murine ML cDNA encodes a functional ligand that is able to activate both the murine and human ML receptor (mpl).

11. The Porcine mpl Ligand 2 5 Porcine ML (pML) cDNA was isolated by RACE PCR as described in Example S: 13. A PCR cDNA product of 1342 bp was found in kidney and subcloned. Several clones were sequenced and found to encode a pig mpl ligand of 332 amino acid resudues referred to as pML (or pML 3 3 2 having the nucleotide and deduced amino acid sequence shown in Fig. 20 (SEQ ID NOS: 18 19).

3 0 Again, a second form, designated pML2, encoding a protein with a 4 amino acid residue deletion (228 amino acid residues) was identified (see Fig. 21 [SEQ ID NO: Comparison of pML and pML2 amino acid sequences shows the latter form is identical except that the tetrapeptide QLPP corresponding to residues 111-114 inclusive have been deleted (see Fig. 22 [SEQ ID NOS: 18 The four amino acid deletions observed in both murine and porcine ML cDNA occur at precisely the same position within the predicted proteins.

Comparison of the predicted amino acid sequences of the mature ML from human, mouse, and pig (Fig. 19 [SEQ ID NOS: 6, 17 18]) indicates that overall -54- WO 95/18858 PCT/US94/14553 sequence identity is 72 percent between mouse and human, 68 percent between mouse and pig and 73 percent between pig and human. The homology is substantially greater in the amino-terminal half of the ML (EPO homologous domain). This domain is 80 to 84 percent identical between any two species whereas the carboxy-terminal half (carbohydrate domain) is only 57 to 67 percent identical. A di-basic amino acid motif that could represent a protease cleavage site is present at the carboxyl end of the erythropoeitin homology domain. This motif is conserved between the three species at this position (Fig. 19 [SEQ ID NOS: 6, 17 A second di-basic site present at position 245 and 246 in the human sequence is not present in the mouse or pig 1 0 sequences. The murine and the pig ML sequence contain 4 cysteines, all conserved in the human sequence. There are seven potential N-glycosylation sites within the mouse ligand and six within the porcine ML, 5 of which are conserved within the human sequence. Again, all the potential N-glycosylation sites are located in the C-terminal half of the protein.

12. Expression and Purification of TPO from Chinese Hamster Ovary (CHO) Cells The expression vectors used to transfect CHO cells are designated: pSVI5.ID.LL.MLORF (full length or TPO 3 3 2 and pSVI5.ID.LL.MLEPO-D (truncated or

TPO

1 5 3 The pertinent features of these plasmids are presented in Fig. 23 and 24.

The transfection procedures are described in Example 20. Briefly, cDNA corresponding to the entire open reading frame of TPO was obtained by PCR. The PCR product was purified and cloned between two restriction sites (Clal and Sail) of the plasmid pSVI5.ID.LL to obtain the vector pSVI5.ID.LL.MLORF. A second construct corresponding to the EPO homologous domain was generated the same way but using a different reverse primer(EPOD.Sal). The final construct for the vector coding for the EPO homologous domain of TPO is called pSVI5.1D.LL.MLEPO-D.

These two constructs were linearized with Notl and transfected into Chinese Hamster Ovary Cells (CHO-DP12 cells, EP 307,247 published 15 March 1989) by electroporation. 107 cells were electroporated in a BRL electroporation apparatus (350 Volts, 330 mF, low capacitance) in the presence of 10, 25 or 50 mg of DNA as described (Andreason, G.L. J. Tissue Cult. Meth. 15,56 [1993]). The day following transfection, cells were split in DHFR selective media (High glucose DMEM-F12 50:50 without glycine, 2mM glutamine, 2-5% dialyzed fetal calf serum). 10 to 3 5 days later individual colonies were transferred to 96 well plates and allowed to grow to confluency. Expression of ML 1 5 3 or ML 3 3 2 in the conditioned media from these clones was assessed using the Ba/F3-mpl proliferation assay (described in Example I).

WO 95/18858 PCT/US94/14553 The process for purifying and isolating TPO from harvested CHO cell culture fluid is described in Example 20. Briefly, harvested cell culture fluid (HCCF) is applied to a Blue Sepharose column (Phamacia) at a ratio of approximately 100L of HCCF per liter of resin. The column is then washed with 3 to 5 column volumes of buffer followed by 3 to 5 column volumes of a buffer containing 2.0M urea. TPO is then eluted with 3 to 5 column volumes of buffer containing both 2.0M urea and 1.OM NaCI.

The Blue Sepharose eluate pool containing TPO is then applied to a Wheat Germ Lectin Sepharose column (Pharmacia) equilibrated in the Blue Sepharose eluting 1 0 buffer at a ratio of from 8 to 16 ml of Blue Sepharose eluate per ml of resin. The column is then washed with 2 to 3 column volumes of equilibration buffer. TPO is then eluted with 2 to 5 column volumes of a buffer containing 2.0M urea and 0.5M Nacetyl-D-glucosamine.

The Wheat Germ Lectin eluate containing TPO is then acidified and C12E8 is 1 5 added to a final concentration of 0.04%. The resulting pool is applied to a C4 reversed phase column equilibrated in 0.1% TFA, 0.04% C12E8 at a load of approximately 0.2 to 0.5 mg protein per ml of resin.

The protein is eluted in a two phase linear gradient of acetonitrile containing 0.1% TFA and 0.04% C12E 8 and a pool is made on the basis of SDS-PAGE.

20 The C4 Pool is then diluted and diafilitered versus approximately 6 volumes of buffer on an Amicon YM or like ultrafiltration membrane having a 10,000 to 30,000 Dalton molecular weight cut-off. The resulting diafiltrate may be then directly a. processed or further concentrated by ultrafiltration. The diafiltrate/concentrate is usually adjusted to a final concentration of 0.01% All or a portion of the diafiltrate/concentrate equivalent to 2 to 5% of the Scalculated column volume is then applied to a Sephacryl S-300 HR column (Pharmacia) equilibrated in a buffer containing 0.01% Tween-80 and chromatographed. The TPO containing fractions which are free of aggregate and proteolytic degradation products are then pooled on the basis of SDS-PAGE. The resulting pool is filtered and stored at 2-8 0

C.

13. Methods for Transforming and Inducing TPO Synthesis in a Microorganism and Isolating, Purifying and Refolding TPO Made Therein 3 5 Construction of E. coli TPO expression vectors is described in detail in Example 21. Briefly. plasmids pMP21, pMP151, pMP41, pMP57 and pMP202 were all designed to express the first 155 amino acids of TPO downstream of a small leader which varies among the different constructs. The leaders provide primarily for -56- WO 95/18858 PCT/US94/14553 high level translation initiation and rapid purification. The plasmids pMP210-1, -T8, -21, -22, -24, -25 are designed to express the first 153 amino acids of TPO downstream of an initiation methionine and differ only in the codon usage for the first 6 amino acids of TPO, while the plasmid pMP251 is a derivative of pMP210-1 in which the carboxy-terminal end of TPO is extended by two amino acids. All of the above plasmids will produce high levels of intracellular expression of TPO in E. coli upon induction of the tryptophan promoter (Yansura, D. G. et. al. Methods in Enzymology Goeddel, D. Ed.) 185:54-60, Academic Press, San Diego [1990]).

The plasmids pMP1 and pMP172 are intermediates in the construction of the above 1 0 TPO intracellular expression plasmids.

The above TPO expression plasmids were used to transform the E. coli using the CaCI2 heat shock method (Mandel, M. et al. J. Mol. Biol., 53:159-162, [1970]) and other procedures described in Example 21. Briefly, the transformed cells were grown first at 37 0 C until the optical density (600nm) of the culture reached 1 5 approximately 2-3. The culture was then diluted and, after growth with aeration, acid was added. The culture was then allowed to continue growing with aeration for another 15 hours after which time the cells were harvested by centrifugation.

The Isolation, Purification and Refolding procedures given below for production of biologically active, refolded human TPO or fragments thereof is described in Examples 22 and 23 can be applied for the recovery of any TPO variant including N and C terminal extended forms. Other procedures suitable for refolding recombinant or synthetic TPO can be found in the following patents; Builder et al., U.S. Patent S* 4,511,502; Jones et al., U.S. Patent 4,512,922; Olson U.S. Patent 4,518,526 and Builder et U.S. Patent 4,620,948; for a general description of the recovery and refolding process for a variety of recombinant proteins expressed in an insoluble form Sin E. coli.

A Recovery of non-soluble

TPO

A microorganism such as E. coli expressing TPO encoded by any suitable plasmid is fermented under conditions in which TPO is deposited in insoluble "refractile bodies". Optionally, cells are first washed in a cell disruption buffer.

Typically, about 100g of cells are resuspended in about 10 volumes of a cell disruption buffer 10 mM Tris, 5 mM EDTA, pH 8) with, for example, a Polytron homogenizer and the cells centrifuged at 5000 x g for 30 minutes. Cells are then lysed using any conventional technique such as tonic shock, sonication, pressure cycling, 3 5 chemical or enzymatic methods. For example, the washed cell pellet above may be resuspended in another 10 volumes of a cell disruption buffer with a homogenizer and the cell suspension is passed through an LH Cell Disrupter (LH Inceltech, Inc.) or through a Microfluidizer (Microfluidics International) according to the manufactures' -57- WO 95/18858 PCT/US94/14553 instructions. The particulate matter containing TPO is then separated from the liquid phase and optionally washed with any suitable liquid. For example, a suspension of cell lysate may be centrifuged at 5,000 X g for 30 minutes, resuspended and optionally centrifuged a second time to make a washed refractile body pellet. The washed pellet may be used immediately or optionally stored frozen (at e.g. B. Solubilization and Purification of Monomeric TPO Insoluble TPO in the retractile body pellet is then solubilized with a solublizing buffer. The solublizing buffer contains a chaotropic agent and is usually buffered at a basic pH and contains a reducing agent to improve the yield of monomeric TPO.

1 0 Representative chaotropic agents include urea, guanidine-HCI, and sodium thiocyanate.

A preferred chaotropic agent is guanidine.HCI. The concentration of chaotropic agent is usually 4-9M, preferably 6-8M. The pH of the solublizing buffer is maintained by any suitable buffer in a pH range of from about 7.5-9.5, preferably 8.0-9.0 and most .S preferably 8.0. Preferably the solubilizing buffer also contains a reducing agent to 1 5 aid formation of the monomeric form of TPO. Suitable reducing agents include organic compounds containing a free thiol (RSH). Representative reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), mercaptoethanol, glutathione (GSH), cysteamine and cysteine. A preferred reducing agent is dithiothreitol

(DTT).

Optionally, the solubilizing buffer may contain a mild oxidizing agent molecular 20 oxygen) and a sulfite salt to form monomeric TPO via sulfitolysis. In this embodiment, the resulting TPO-S-sulfonate is later refolded in the presence of the redox buffer GSH/GSSG) to form the properly folded TPO.

The TPO protein is usually further purified using, for example, centrifugation, gel filtration chromatography and reversed phase column chromatography.

25 By way of illustration, the following procedure has produced suitable yields of monomeric TPO. The refractile body pellet is resuspended in about 5 volumes by weight of the solubilizing buffer (20 mM Tris, pH 8, with 6-8 M guanidine and mM DTT) and stirred for 1-3 hr., or overnight, at 4°C to effect solubilization of the TPO protein. High concentrations of urea (6-8M) are also useful but generally result in somewhat lower yields compared to guanidine. After solubilization, the solution is centrifuged at 30,000 x g for 30 min. to produce a clear supernatant containing denatured, monomeric TPO protein. The supernatant is then chromatographed on a Superdex 200 gel filtration column (Pharmacia, 2.6 x 60 cm) at a flow rate of 2 ml/min. and the protein eluted with 20 mM Na phosphate, pH 6.0, with 10 mM DTT.

Fractions containing monomeric, denatured TPO protein eluting between 160 and 200 ml are pooled. The TPO protein is further purified on a semi-preparative C4 reversed phase column (2 x 20 cm VYDAC). The sample is applied at 5 ml/min. to a column equilibrated in 0.1% TFA (trifluoroacetic acid) with 30% acetonitrile. The protein is -58- WO 95/18858 PCTIUS94/14553 eluted with a linear gradient of acetonitrile (30-60% in 60 min.). The purified reduced protein elutes at approximately 50% acetonitrile. This material is used for refolding to obtain biologically active TPO variant.

C Refolding TPO to Generate the Biologically Active Form Following solubilization and further purification of TPO, the biologically active form is obtained by refolding the denatured monomeric TPO in a redox buffer. Because of the high potency of TPO (half maximal stimulation in the Ba/F3 assay is achieved at approximately 3 pg/ml), it is possible to obtain biologically active material utilizing many different buffer, detergent and redox conditions. However, under most conditions 1 0 only a small amount of properly folded material is obtained. For commercial manufacturing processes, it is desirable to have refolding yields at least 10%, more preferably 30-50% and most preferably Many different detergents including Triton X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, sarkosyl, Tween 20 and Tween 80, Zwittergent 3-14 and others were found suitable for producing at least 15 some properly folded material.. Of these however, the most preferred detergents were Sthose of the CHAPS family (CHAPS and CHAPSO) which were found to work best in the i" refolding reaction and to limit protein aggregation and improper disulfide formation.

Levels of CHAPS greater than about 1% were most preferred. Sodium chloride was Srequired for the best yields, with the optimal levels between 0.1 M and 0.5M. The 20 presence of EDTA (1-5 mM) in the redox buffer was preferred to limit the amount of metal-catalyzed oxidation (and aggregation) which was observed with some preparations. Glycerol concentrations of greater than 15% produced the optimal refolding conditions. For maximum yields, it was essential to have a redox pair in the redox buffer consisting of both an oxidized and reduced organic thiol (RSH). Suitable 25 redox pairs include mercaptoethanol, glutathione (GSH), cysteamine, cysteine and their corresponding oxidized forms. Preferred redox pairs were glutathione(GSH):oxidized glutathione(GSSG) or cysteine:cystine. The most preferred redox pair was glutathione(GSH):oxidized glutathione(GSSG). Generally higher yields were observed when the mole ratio of oxidized member of the redox pair was equal to or in excess over the reduced member of the redox pair. pH values between 7.5 and about 9 were optimal for refolding of these TPO variants. Organic solvents (e.g.

ethanol, acetonitrile, methanol) were tolerated at concentrations of 10-15% or lower.

Higher levels of organic solvents increased the amount of improperly folded forms.

Tris and phosphate buffers were generally useful. Incubation at 4 OC also produced higher levels of properly folded TPO.

Refolding yields of 40-60% (based on the amount of reduced and denatured TPO used in the refolding reaction) are typical for preparations of TPO that have been purified through the first C4 step. Active material can be obtained when less pure -59.

WO 95/18858 PCT/US94/14553 preparations directly after the Superdex 200 column or after the initial refractile body extraction) although the yields are less due to extensive precipitation and interference of non-TPO proteins during the TPO refolding process.

Since TPO contains 4 cysteine residues, it is possible to generate three different disulfide versions of this protein: version 1: disulfides between cysteine residues 1-4 and 2-3 version 2: disulfides between cysteine residues 1-2 and 3-4 version 3: disulfides between cysteine residues 1-3 and 2-4.

During the initial exploration in determining refolding conditions, several 1 0 different peaks containing the TPO protein were separated by C4 reversed phase chromatography. Only one of these peaks had significant biological activity as determined using the Ba/F3 assay. Subsequently, the refolding conditions were optimized to yield preferentially that version. Under these conditions, the misfolded versions were less than 10-20% of the total monomeric TPO obtained from the .i 15 solubilizing step.

The disulfide pattern for the biologically active TPO has been determined to be 1-4 and 2-3 by mass spectrometry and protein sequencing, where the cysteines are numbered sequentially from the amino-terminus. This cysteine cross-linking pattern is consistent with the known disulfide bonding pattern of the related molecule 20 erythropoietin.

D. Biological Activity of Recombinant, Refolded TPO Refolded and purified TPO has activity in both in vitro and in vivo assays. For example, in the Ba/F3 assay, half-maximal stimulation of thymidine incorporation into the Ba/F3 cells for TPO (Met-1 1-153) was achieved at 3.3 pg /ml (0.3 pM).

25 In the mpl receptor-based ELISA, half-maximal activity occurred at 1.9 ng/ml (120 pM). In normal and myelosuppressed animals produced by near-lethal X-radiation, refolded TPO (Met-1 1-153) was highly potent (activity was seen at doses as low as ng/mouse) to stimulate the production of new platelets. Similar biological activity was observed for other forms of TPO refolded in accordance with the above described procedures (see Figs. 25, 26 and 28).

14. Methods for Measurement of Thrombopoletic Activity Thrombopoietic activity may be measured in various assays including the Ba/F3 mp/ ligand assay described in Example 1, an in vivo mouse platelet rebound synthesis assay, induction of platelet cell surface antigen assay as measured by an anti-platelet immunoassay (anti-GPIlbllla) for a human leukemia megakaryoblastic cell line (CMK) (see Sato et Brit. J. Heamatol., 72:184-190 [1989])(see also the liquid suspension megakaryocytopoiesis assay described in Example and WO 95/18858 PCT/US94/14553 induction of polyploidization in a megakaryoblastic cell line (DAMI) (see Ogura et al., Blood, 72(1):49-60 [1988]). Maturation of megakaryocytes from immature, largely non-DNA synthesizing cells, to morphologically identifiable megakaryocytes involves a process that includes appearance of cytoplasmic organelles, acquisition of membrane antigens (GPIIbllla), endoreplication and release of platelets as described in the background. A lineage specific promoter the mpl ligand) of megakaryocyte maturation would be expected to induce at least some of these changes in immature megakaryocytes leading to platelet release and alleviation of thrombocytopenia. Thus, assays were designed to measure the emergence of these parameters in immature 1 0 megakaryocyte cell lines, CMK and DAMI cells. The CMK assay (Example 4) measures the appearance of a specific platelet marker, GPlIblla, and platelet shedding.

The DAMI assay (Example 15) measures endoreplication since increases in ploidy are hallmarks of mature megakaryocytes. Recognizable megakaryocytes have ploidy values of 2N, 4N, 8N, 16N, 32N, etc. Finally, the in vivo mouse platelet rebound 15 assay (Example 16) is useful in demonstrating that administration of the test compound (here the mpl ligand) results in elevation of platelet numbers.

Two additional in vitro assays have been developed to measure TPO activity.

The first is a kinase receptor activation (KIRA) ELISA in which CHO cells are transfected with a mpl-Rse chimera and tyrosine phosphorylation of Rse is measured 20 by ELISA after exposure of the mpl portion of the chimera to mpl ligand (see Example 17). The second is a receptor based ELISA in which ELISA plate coated rabbit antihuman IgG captures human chimeric receptor mpl-lgG which binds the mpl ligand being assayed. A biotinylated rabbit polyclonal antibody to mpl ligand (TPO 1 55) is used to detect bound mpl ligand which is measured using streptavidin-peroxidase as 25 described in Example 18.

In Vivo Biological Response of Normal and Sublethally Irradiated Mice Treated with TPO Both normal and sublethally irradiated mice were treated with truncated and full length TPO isolated from Chinese hamster ovary (CHO) cells, E. coli, and human embryonic kidney (293) cells. Both forms of TPO produced in these three hosts stimulated platelet production in mice, however, full length TPO isolated from CHO apeared to produce the greatest in vivo response. These results indicate that proper glycosylation of the carboxy-terminal domain may be necessary for optimal in vivo activity.

E. coli-rhTPO(Met-',153) The "Met" form of the EPO domain (Met in the -1 position plus the first 153 residues of human TPO) produced in E. coli (see Example 23) was injected daily into -61 WO 95/18858 PCT/US94/14553 normal female C57 B6 mice as described in the legends to Figs. 25A, 25B and These figures show that the non-glycosylated truncated form of TPO produced in E. coli and refolded as described above is capable of stimulating about a two-fold increase in platelet production in normal mice with out effecting the red or white blood cell population.

This same molecule injected daily into sublethally irradiated (137Cs female C57 B6 mice as described in the legends to Figs. 26A, 26B and 26C stimulated platelet recovery and diminished nadir but had no effect on erythrocytes or leukocytes.

CHO-rhTPO 33 2 1 0 The full length form of TPO produced in CHO and injected daily into normal female C57 B6 mice as described in the legends to Figs. 27A, 27B and 27C produced about a five-fold increase in platelet production in normal mice with out effecting the erythrocyte or leukocyte population.

CHO-rhTPO 3 3 2 E. coli-rhTPO(Met-,15 3 293-rhTPO 3 3 2 and E.

.i 15 coli-rhTP01 5 Dose response curves were constructed for treatment of normal mice with rhTPO from various cell lines (CHO-rhTP0 3 3 2 E. co/i-rhTPO(Met-1,153); 293rhTPO 3 3 2 and E. coli-rhTP01 5 5 as described in the legend to Fig. 28. This figure shows that all tested forms of the molecule stimulate platelet production, however the 2 0 full length form produced in CHO has the greatest in vivo activity.

CHO-rhTPO 1 5 3 CHO-rhTPO"clipped,, and CHO-rhTPO 3 32 Dose response curves were also constructed for treatment of normal mice with Svarious forms of rhTPO produced in CHO (CHO-rhTPO153, CHO-rhTPO"clipped" and CHO-rhTPO 3 3 2 as described in the legend to Fig. 29. This figure shows that all 2 5 tested CHO forms of the molecule stimulate platelet production,but that the full length "70 Kda form has the greatest in vivo activity.

16. General Recombinant Preparation of mpl Ligand and Variants 3 0 Preferably mpl ligand is prepared by standard recombinant procedures which involve production of the mpl ligand polypeptide by culturing cells transfected to express mpl ligand nucleic acid (typically by transforming the cells with an expression vector) and recovering the polypeptide from the cells. However, it is optionally envisioned that the mpl ligand may be produced by homologous recombination, or with recombinant production methods utilizing control elements introduced into cells already containing DNA encoding the mpl ligand. For example, a powerful promoter/enhancer element, a suppressor, or an exogenous transcription modulatory element may be inserted in the genome of the intended host cell in -62- WO 95/18858 PCT/US94/14553 proximity and orientation sufficient to influence the transcription of DNA encoding the desired mpl ligand polypeptide. The control element does not encode the mpl ligand, rather the DNA is indigenous to the host cell genome. One next screens for cells making the receptor polypeptide of this invention, or for increased or decreased levels of expression, as desired.

Thus, the invention contemplates a method for producing mpl ligand comprising inserting into the genome of a cell containing the mpl ligand nucleic acid molecule a transcription modulatory element in sufficient proximity and orientation to the nucleic acid molecule to influence transcription thereof, with an optional further step 1 0 comprising culturing the cell containing the transcription modulatory element and the nucleic acid molecule. The invention also contemplates a host cell containing the indigenous mpl ligand nucleic acid molecule operably linked to exogenous control sequences recognized by the host cell.

A. Isolation of DNA Encoding mpl ligand Polypeptide .1 5 The DNA encoding mpl ligand polypeptide may be obtained from any cDNA library prepared from tissue believed to possess the mpl ligand mRNA and to express it at a detectable level. The mpl ligand gene may also be obtained from a genomic

DNA

library or by in vitro oligonucleotide synthesis from the complete nucleotide or amino acid sequence.

2 0 Libraries are screened with probes designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries, suitable probes include monoclonal or polyclonal antibodies that recognize and specifically bind to the mpl ligand. For cDNA libraries suitable probes include oligonucleotides of about 20-80 bases in length that encode known or suspected 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. Appropriate probes for screening genomic DNA libraries include, but are not limited to, oligonucleotides, cDNAs, or fragments thereof that encode the same or a similar gene, and/or homologous genomic DNAs or fragments thereof. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures as described in Chapters 10-12 of Sambrook et al., supra.

An alternative means to isolate the gene encoding mpl ligand is to use PCR methodology as described in section 14 of Sambrook et al., supra. This method requires the use of oligonucleotide probes that will hybridize to DNA encoding the mpl ligand. Strategies for selection of oligonucleotides are described below.

A preferred method of practicing this invention is to use carefully selected oligonucleotide sequences to screen cDNA libraries from various tissues, preferably human or porcine kidney (adult or fetal) or liver cell lines. For example, human fetal -63- WO 95/18858 PCT/US94/14553 liver cell line cDNA libraries are screened with the oligonucleotide probes.

Alternatively, human genomic libraries may be screened with the oligonucleotide probes.

The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The actual nucleotide sequence(s) is usually designed based on regions of the mpl ligand which have the least codon redundancy. The oligonucleotides may be degenerate at one or more positions.

The use of degenerate oligonucleotides is of particular importance where a library is screened from a species in which preferential codon usage is not known.

The oligonucleotide must be labeled such that it can be detected upon hybridization to DNA in the library being screened. The preferred method of labeling is to use ATP y 3 2 p) and polynucleotide kinase to radiolabel the 5' end of the oligonucleotide. However, other methods may be used to label the oligonucleotide, including, but not limited to, biotinylation or enzyme labeling.

1 5 Of particular interest is the mpl ligand nucleic acid that encodes a full-length mpl ligand polypeptide. In some preferred embodiments, the nucleic acid sequence includes the native mpl ligand signal sequence. Nucleic acid having all the protein coding sequence is obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence.

20 B. Amino Acid Sequence Variants of Native mpl ligand 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, deletions from, or Insertions or substitutions of, residues within the amino acid sequence for the porcine 25 mpl ligand. For example, carboxy terminus portions of the mature full length mpl ligand may be removed by proteolytic cleavage, either in vivo or in vitro, or by cloning and expressing a fragment or the DNA encoding full length mpl ligand to produce a biologically active variant. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired biological activity. The amino acid changes also may alter posttranslational processes of the mpl ligand, such as changing the number or position of glycosylation sites. For the design of amino acid sequence variants of the mpl ligand, the location of the mutation site and the nature of the mutation will depend on the mpl ligand characteristic(s) to be modified. The sites for mutation can be modified individually or in series, by substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.

-64- WO 95/18858 PCT/US94/14553 A useful method for identification of certain residues or regions of the mpl ligand polypeptide that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Science, 244:1081- 1085 [1989]. Here, a residue or group of target residues are identified charged residues such as arg, asp, his, lys, and glu) and replaced by any, but preferably a neutral or negatively charged, amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at or for the 1 0 sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed mpl ligand variants are screened for the optimal combination of desired 15 activity.

There are two principal variables in the construction of amino acid sequence variants: the location of the mutation site and the nature of the mutation. For example, variants of the mpt ligand polypeptide include variants from the mpl ligand sequence, Iand may represent naturally occurring alleles (which will not require manipulation of 2 0 the mpl ligand DNA) or predetermined mutant forms made by mutating the DNA, either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the mpl ligand characteristic to be modified.

Amino acid sequence deletions generally range from about 1 to 30 residues, 25 more preferably about 1 to 10 residues, and typically are contiguous. Alternatively, amino acid sequence deletions for the mpl ligand may include a portion of or the entire carboxy-terminus glycoprotein domain. Amino acid sequence deletions may also include one or more of the first 6 amino-terminus residues of the mature protein.

Optional amino acid sequence deletions comprise one or more residues in one or more of the loop regions that exist between the 'helical bundels". Contiguous deletions ordinarily are made in even numbers of residues, but single or odd numbers of deletions are within the scope hereof. Deletions may be introduced into regions of low homology among the mpl ligands that share the most sequence identity to modify the activity of the mpl ligand. Or deletions may be introduced into regions of low homology among human mpl ligand and other mammalian mpl ligand polypeptides that share the most sequence identity to the human mpl ligand. Deletions from a mammalian mpl ligand polypeptide in areas of substantial homology with other mammalian mpl ligands will be more likely to modify the biological activity of the mpl ligand more WO 95/18858 PCT/US94/14553 significantly. The number of consecutive deletions will be selected so as to preserve the tertiary structure of mpl ligands in the affected domain, beta-pleated sheet or alpha helix.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence Insertions of single or multiple amino acid residues. Intrasequence insertions insertions within the mature mpl ligand sequence) may range generally from about 1 to 10 residues, more preferably 1 to most preferably 1 to 3. An exemplary preferred fusion is that of mpl ligand or 1 0 fragment thereof and another cytokine or fragment thereof. Examples of terminal insertions include mature mpl ligand with an N-terminal methionyl residue, an artifact of the direct expression of mature mpl ligand in recombinant cell culture, and fusion of a heterologous N-terminal signal sequence to the N-terminus of the mature mpl ligand molecule to facilitate the secretion of mature mpl ligand from recombinant S1 5 hosts. Such signal sequences generally will be obtained from; and thus homologous to, the intended host cell species. Suitable sequences include STII or Ipp for E. coli, alpha factor for yeast, and viral signals such as herpes gD for mammalian cells.

Other insertional variants of the mpl ligand molecule Include the fusion to the N- or C-terminus of mpl ligand of immunogenic polypeptides not endogenous to 2 0 the host to which the fusion is administered), bacterial polypeptides such as betalactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, and Cterminal fusions with 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 6 April 1989.

25 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 inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as the active site(s) of mpl ligand and sites where the amino acids found in other analogues are substantially different in terms of sidechain bulk, charge, or hydrophobicity, but where there is also a high degree of sequence identity at the selected site among various mpl ligand species and/or within the various animal analogues of one mpl ligand member.

Other sites of interest are those in which particular residues of the mpl ligand obtained from various family members and/or animal species within one member are 3 5 identical. These sites, especially those falling within a sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 3 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more -66- WO 95/18858 PCT/US94/14553 substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened.

TABLE 3 Original Exemplary Preferred Residue Substitutions Substitutions Ala Val; Leu; lie Val Arg Lys; Gin; Asn Lys 1 0 Asn Gin; His; Lys; Arg Gin Asp Glu Glu Cys Ser Ser *Gin Asn Asn Glu Asp Asp S. 15 Gly Pro Pro His Asn; Gin; Lys; Arg Arg lie Leu; Val; Met; Ala; Phe; norleucine Leu Leu norleucine; Ile; Val; 20 Met; Ala; Phe Ile Lys Arg; Gin; Asn Arg r. Met Leu; Phe; lie Leu Phe Leu; Val; lie; Ala Leu Pro Gly Gly 25 Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Trp; Phe; Thr; Ser Phe Val lie; Leu; Met; Phe; Ala; norleucine Leu Substantial modifications in function or immunological identity of the mpl ligand are accomplished by selecting substitutions that differ significantly in their effect on maintaining the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain.

Naturally occurring residues are divided into groups based on common side-chain properties: -67- WO 95/18858 PCT/US94/14553 hydrophobic: norleucine, Met, Ala, Val, Leu, lie; neutral hydrophilic: Cys, Ser, Thr; acidic: Asp, Glu; basic: Asn, Gin, His, Lys, Arg; residues that influence chain orientation: Gly, Pro; and aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non- 1 0 conserved) sites.

In one embodiment of the invention, it is desirable to inactivate one or more protease cleavage sites that are present in the molecule. These sites are identified by inspection of the encoded amino acid sequence, in the case of trypsin, for an arginyl or lysinyl residue. When protease cleavage sites are identified, they are 1 5 rendered inactive to proteolytic cleavage by substituting the targeted residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine; by deleting the residue; or by inserting a prolyl residue immediately after the residue.

In another embodiment, any methionyl residues other than the starting 2 0 methionyl residue of the signal sequence, or any residue located within about three residues N- or C-terminal to each such methionyl residue, is substituted by another residue (preferably in accordance with Table 3) or deleted. Alternatively, about 1- 3 residues are inserted adjacent to such sites.

Any cysteine residues not involved in maintaining the proper conformation of 2 5 the mpl ligand also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. It has been found that the first and forth cysteines in the epo domain, numbered from the amino-terminus, are necessary for maintaining proper conformation but that the second and third are not.

Accordingly, the second and third cysteines in the epo domain may be substituted.

Nucleic acid molecules encoding amino acid sequence variants of mpl ligand are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared 3 5 variant or a non-variant version of mpl ligand polypeptide.

Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution, deletion, and insertion variants of mpl ligand DNA. This technique is well known in the art as described by Adelman et al., DNA, 2:183 [1983]. Briefly, mpl -68- WO 95/18858 PCT/US94/14553 ligand DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of mpl ligand. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the mpl ligand DNA.

Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the 1 0 mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al., Proc. Natl.

Acad. Sci. USA, 75:5765 [1978].

The DNA template can be generated by those vectors that are either derived 15 from bacteriophage M13 vectors (the commercially available M13mp18 and M13mp19 vectors are suitable), or those vectors that contain a single-stranded phage origin of replication as described by Viera et al., Meth. Enzymol., 153:3 [1987].

V: Thus, the DNA that is to be mutated may be inserted into one of these vectors to generate single-stranded template. Production of the single-stranded template is described in Sections 4.21-4.41 of Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY 1989).

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

SFor alteration of the native DNA sequence (to generate amino acid sequence variants, for example), the oligonucleotide is hybridized to the single-stranded template under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis. A heteroduplex molecule is thus formed such that one strand of DNA encodes 3 0 the mutated form of the mpl ligand, and the other strand (the original template) encodes the native, unaltered sequence of the mpl ligand. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli JM101.

After the cells are grown, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabeled with 32-phosphate to identify the bacterial 3 5 colonies that contain the mutated DNA. The mutated region is then removed and placed in an appropriate vector for protein production, generally an expression vector of the type typically employed for transformation of an appropriate host.

-69.

WO 95/18858 PCT/US94/14553 The method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s). The modifications are as follows: The single-stranded oligonucleotide is annealed to the single-stranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP), is combined with a modified thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from the Amersham Corporation). This mixture is added to the template-oligonucleotide complex. Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template, except for the 1 0 mutated bases is generated. In addition, this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion.

After the template strand of the double-stranded heteroduplex is nicked with an appropriate restriction enzyme, the template strand can be digested with Exolll nuclease or another appropriate nuclease past the region that contains the site(s) to be 1 5 mutagenized. The reaction is then stopped to leave a molecule that is only partially single-stranded. A complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates,

ATP,

and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell such as E. coli JM101, as described above.

DNA encoding mpl ligand mutants with more than one amino acid to be substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than 25 about ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.

In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.

The alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: wild-type DNA is used for the template, an oligonucleotide encoding the first desired amino acid 3 5 substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid WO 95/18858 PCT/US94/14553 substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on.

PCR mutagenesis is also suitable for making amino acid variants of mpl ligand polypeptide. While the following discussion refers to DNA, it is understood that the technique also finds application with RNA. The PCR technique generally refers to the following procedure (see Erlich, supra, the chapter by R. Higuchi, p. 61-70): When small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be 1 0 used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template. For introduction of a mutation into a plasmid DNA, one of the primers is designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer must be identical to a stretch of sequence of the opposite 1 5 strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 200 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced. PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone.

If the ratio of template to product material is extremely low, the vast majority of product DNA fragments incorporate the desired mutation(s). This product material is used to replace the corresponding region in the plasmid that served as PCR template 25 using standard DNA technology. Mutations at separate positions can be introduced simultaneously by either using a mutant second primer, or performing a second PCR with different mutant primers and ligating the two resulting PCR fragments simultaneously to the vector fragment in a three (or more)-part ligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 pg) is linearized by digestion with a restriction endonuclease that has a unique recognition site in the plasmid DNA outside of the region to be amplified. Of this material, 100 ng is added to a PCR mixture containing PCR buffer, which contains the four deoxynucleotide triphosphates and is included in the GeneAmp® kits (obtained from Perkin-Elmer Cetus, Norwalk, CT and Emeryville, CA), and 25 pmole of each oligonucleotide primer, to a final volume of 50 gl. The reaction mixture is overlayed with 35 Il mineral oil. The reaction mixture is denatured for five minutes at 100oC, placed briefly on ice, and then 1 il Thermus aquaticus (Taq) DNA polymerase units/pl, purchased from Perkin-Elmer Cetus) is added below the mineral oil layer.

-71 WO 95/18858 PCT/US94/14553 The reaction mixture is then inserted into a DNA Thermal Cycler (purchased from Perkin-Elmer Cetus) programmed as follows: 2 min. sec. 72 0 C, then 19 cycles of the following: 30 sec. 94°C sec. 55 0 C, and sec. 72*C.

At the end of the program, the reaction vial is removed from the thermal cycler and the aqueous phase transferred to a new vial, extracted with phenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA is recovered by standard procedures. This material is subsequently subjected to the appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based on the .i technique described by Wells et al., Gene, 34:315 [1985]. The starting material is 1 5 the plasmid (or other vector) comprising the mpl ligand DNA to be mutated. The codon(s) in the mpl ligand DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate 2 0 locations in the mpl ligand DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard 25 techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3' and 5' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated mpl ligand DNA sequence.

C. Insertion of Nucleic Acid into a Replicable Vector The nucleic acid cDNA or genomic DNA) encoding native or variant mpl ligand polypeptide is inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. Many vectors are available, and selection of the appropriate vector will depend on whether it is to be used for DNA amplification or for DNA expression, the size of the nucleic acid to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell with which it is compatible. The vector components generally include, but are not limited to, one or more of the following: a -72- WO 95/18858 PCT/US94/14553 signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

Signal Sequence Component The mpl ligand of this invention may be expressed not only directly, but also as a fusion with a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the mpl ligand DNA that is inserted into the vector. The heterologous signal sequence selected should be one that is recognized and processed cleaved by 1 0 a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native mpl ligand signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeast .i secretion the native signal sequence may be substituted by, the yeast invertase, 1 5 alpha factor, or acid phosphatase leaders, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression the native signal sequence the mpl ligand presequence that normally directs secretion of mpl ligand from its native mammalian cells in vivo) is satisfactory, although other 2 0 mammalian signal sequences may be suitable, such as signal sequences from other mpl ligand polypeptides or from the same mpl ligand from a different animal species, signal sequences from a mpl ligand, and signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex gD signal.

25 (ii) Origin of Replication Component Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 p plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).

Most expression vectors are "shuttle" vectors, they are capable of replication in at least one class of organisms but can be transfected into another -73- WO 95/18858 PCT/US94/14553 organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This is readily accomplished using Bacillus species as hosts, for example, by including in the vector a DNA sequence that is complementary to a sequence found in Bacillus genomic DNA.

Transfection of Bacillus with this vector results in homologous recombination with the genome and insertion of mpl ligand DNA. However, the recovery of genomic

DNA

encoding mpl ligand is more complex than that of an exogenously replicated vector because restriction enzyme digestion is required to excise the mpl ligand DNA.

(iii) Selection Gene Component Expression and cloning vectors should contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not 15 transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, ampicillin, neomycin, methotrexate, or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media, the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of a host cell.

Those cells that are successfully transformed with a heterologous gene express a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin (Southern et al., J. Molec. Appl.

oGenet., 1:327 [1982]) mycophenolic acid (Mulligan et al., Science, 209:1422 25 [1980]) or hygromycin Sugden et al., Mol. Cell. Biol., 5:410-413 [1985]). The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Examples of other suitable selectable markers for mammalian cells are those 3 0 that enable the identification of cells competent to take up the mpl ligand nucleic acid, such as dihydrofolate reductase (DHFR) or thymidine kinase. The mammalian cell transformants are placed under selection pressure that only the transformants are uniquely adapted to survive by virtue of having taken up the marker. Selection pressure is imposed by culturing the transformants under conditions in which the 3 5 concentration of selection agent in the medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes mpl ligand polypeptide. Amplification is the process by which genes in greater demand for the production of a protein critical for growth are reiterated in tandem within the -74- WO 95/18858 PCT/US94/14553 chromosomes of successive generations of recombinant cells. Increased quantities of mpl ligand are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasin, Proc. Natl.

Acad. Sci. USA, 77:4216 [1980]. The transformed cells are then exposed to increased levels of Mtx. This leads to the synthesis of multiple copies of the DHFR gene, and, 1 0 concomitantly, multiple copies of other DNA comprising the expression vectors, such as the DNA encoding mpl ligand. This amplification technique can be used with any otherwise suitable host, ATCC No. CCL61 CHO-K1, notwithstanding the presence of endogenous DHFR if, for example, a mutant DHFR gene that is highly resistant to Mtx is employed (EP 117,060). Alternatively, host cells [particularly wild-type hosts that 1 5 contain endogenous DHFR] transformed or co-transformed with DNA sequences encoding mpl ligand, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3' phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 [1979]; Kingsman et al., Gene, -7:141 [19791; or Tschemper et Gene, 10:157 [1980]). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 25 [1977]). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC No. 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.

(iv) Promoter Component Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the mpl ligand nucleic acid. Promoters are untranslated sequences located upstream to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence, such as the mpl ligand nucleic acid sequence, to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, the presence or absence of a nutrient or a change in WO 95/18858 PCT/US94/14553 temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to mpl ligand encoding DNA by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native mpl ligand promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the mpl ligand DNA. However, heterologous promoters are preferred, as they generally permit greater transcription and higher yields of expressed mpl ligand as compared to the native mpl ligand promoter.

Promoters suitable for use with prokaryotic hosts include the 1-lactamase and 1 0 lactose promoter systems (Chang et al., Nature, 275:615 [1978]; and Goeddel et al., Nature, 281:544 [1979]), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057 [1980] and EP 36,776) and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21- .i 25 [19831). However, other known bacterial promoters are suitable. Their 1 5 nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to DNA encoding mpl ligand (Siebenlist et al., Cell, 20:269 [1980]) using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding mpl ligand polypeptide.

Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CXCAAT region where X may be any o nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be 25 the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073 [1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg., 7:149 [1968]; and Holland, Biochemistry, 17:4900 [1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate -76-

I-

WO 95/18858 PCTIUS94/14553 dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageously used with yeast promoters.

Mpl ligand transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from 1 0 heterologous mammalian promoters, the actin promoter or an immunoglobulin promoter, from heat-shock promoters, and from the promoter normally associated with the mpl ligand sequence, provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtained as an 1 5 SV40 restriction fragment that also contains the SV40 viral origin of replication.

Fiers et al., Nature, 273:113 [1978]; Mulligan and Berg, Science, 209:1422- 1427 [1980]; Pavlakis et al., Proc. Natl. Acad. Sci. USA, 78:7398-7402 [1981].

The immediate early promoter of the human cytomegalovirus is conveniently obtained :as a Hindlll E restriction fragment. Greenaway et al., Gene, 18:355-360 [1982]. A 2 0 system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Gray et al., Nature, 295:503-508 [1982] on expressing cDNA encoding immune interferon in monkey cells; Reyes et al., Nature, 297:598-601 [1982] on expression of human B-interferon cDNA in mouse 2 5 cells under the control of a thymidine kinase promoter from herpes simplex virus; Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79:5166-5170 [1982] on expression of the human interferon 81 gene in cultured mouse and rabbit cells; and German et al., Proc. Natl. Acad. Sci. USA, 79:6777-6781 [1982] on expression of bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus long terminal repeat as a promoter.

Enhancer Element Component Transcription of a DNA encoding the mpl ligand of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector.

Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' (Laimins et al., Proc. Natl. Acad. Sci. USA, 78:993 [1981]) and 3' (Lusky et al., Mol. Cell Bio., 3:1108 [1983]) to the -77- WO 95/18858 PCTIUS94/14553 transcription unit, within an intron (Banerji et al., Cell, 33:729 [1983]), as well as within the coding sequence itself (Osborne et al., Mol. Cell Bio., 4:1293 [1984]).

Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature, 297:17-18 [1982] on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a 1 0 position 5' or 3' to the mpl ligand encoding sequence, but is preferably located at a site from the promoter.

(vi) Transcription Termination Component Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, 4 <animal, human, or nucleated cells from other multicellular organisms) will also S1 5 contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding mpl ligand.

(vii) Construction and Analysis of Vectors o* ,Construction of suitable vectors containing one or more of the above listed components employs standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required.

2 5 For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform E. col K12 strain 294 (ATCC No. 31,446) and successful transformants selected by ampicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced by the method of Messing et al., Nucleic Acids Res., 9:309 [1981] or by the method of Maxam et al., Methods in Enzymology, 65:499 [1980].

(viii) Transient Expression Vectors Particularly useful in the practice of this invention are expression vectors that provide for the transient expression in mammalian cells of DNA encoding the mpl 3 5 ligand polypeptide. In general, transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Sambrook et al., supra, pp.

-78- WO 95/18858 PCT/US94/14553 16.17 16.22. Transient expression systems, comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired biological or physiological properties. Thus, transient expression systems are particularly useful in the invention for purposes of identifying analogues and variants of mpl ligand polypeptide that have mpl ligand polypeptide biological activity.

(ix) Suitable Exemplary Vertebrate Cell Vectors Other methods, vectors, and host cells suitable for adaptation to the synthesis of mpl ligand in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 [1981]; Mantel et al., Nature, 281:40-46 [1979]; Levinson et al.; EP 117,060; and EP 117,058. A particularly useful plasmid for mammalian cell culture expression of mpl ligand is pRK5 (EP 307,247 U. S. patent no. 5,258,287) or pSVI6B (PCT Publication No. WO 91/08291).

D. Selection and Transformation of Host Cells 1 5 Suitable host cells for cloning or expressing the vectors herein are the prokaryote, yeast, or higher eukaryotic cells described above. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, E. coli, Bacilli such as B. subtilis, Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, or Serratia marcescans. One preferred E. coli cloning host 20 is E. coil 294 (ATCC No. 31,446), although other strains such as coil B, E. coli X1776 (ATCC No. 31,537), and E. coli W3110 (ATCC No. 27,325) are suitable.

These examples are illustrative rather than limiting. Preferably the host cell should secrete minimal amounts of proteolytic enzymes. Alternatively, in vitro methods of cloning, PCR or other nucleic acid polymerase reactions, are suitable.

25 In addition to 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 the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 [1981]; EP 139,383 published 2 May 1985), Kluyveromyces hosts Patent No. 4,943,529) such as, K. lactis (Louvencourt et J. Bacteriol., 737 [1983]), K. fragilis, K. bulgaricus,

K.

thermotolerans, and K. marxianus, yarrowia [EP 402,226], Pichia pastoris (EP 183,070; Sreekrishna et J. Basic Microbio., 28:265-278 [1988]), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa (Case et al., Proc. Natl. Acad.

Scl. USA, 76:5259-5263 [1979]), and filamentous fungi such as, e.g, Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.

-79- WO 95/18858 PCT/US94/14553 Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO 4:475-479 [1985]).

Suitable host cells for the expression of glycosylated mpl ligand are derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda 1 0 (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. See, Luckow et al., Bio/Technology, 6:47-55 [1988]; Miller et al., Genetic Engineering, Setlow et al., eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature, 315:592-594 [1985]. A variety of viral strains for transfection are publicly 1 5 available, the L-1 variant of Autographa californica NPV and the Bm-5 strain of .Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts. Typically, plant cells are transfected by incubation 20 with certain strains of the bacterium Agrobacterium tumefaciens, which has been previously manipulated to contain the mpl ligand DNA. During incubation of the plant cell culture with A. tumefaciens, the DNA encoding the mpl ligand is transferred to the plant cell host such that it is transfected, and will, under appropriate conditions, express the mpl ligand DNA. In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences. Depicker et al., J. Mol. Appl. Gen., 1:561 [1982].

In addition, DNA segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in recombinant DNA-containing plant tissue. EP 321,196 published 21 June 1989.

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, Academic Press, Kruse and Patterson, editors [1973]).

Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 [1980]); monkey WO 95/18858 PCT/US94/14553 kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 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 mammary 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 a human hepatoma line (Hep G2).

Host cells are transfected and preferably transformed with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or 1 0 amplifying the genes encoding the desired sequences.

Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO 4 and electroporation. Successful transfection is generally recognized when any indication of 1 5 the operation of this vector occurs within the host cell.

Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant.

Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as 20 described in section 1.82 of Sambrook et al., supra, is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw at al., Gene, 23:315 [1983] and WO 89/05859 published 29 June 1989. In addition, plants may be transfected using ultrasound treatment as described in WO 91/00358 published 10 January 1991. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 [1978] is preferred. General aspects of mammalian cell host system transformations have been described by Axel in U.S. Patent No. 4,399,216 issued 16 August 1983. Transformations into yeast are typically carried out 3 0 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]. However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or protoplast fusion may also be used.

E. Culturing the Host Cells Prokaryotic cells used to produce the mpl ligand polypeptide of this invention are cultured in suitable media as described generally in Sambrook et al., supra.

The mammalian host cells used to produce the mpl ligand of this invention may be cultured in a variety of media. Commercially available media such as Ham's -81 WO 95/18858 PCT/US94/14553 (Sigma), Minimal Essential Medium Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham and Wallace, Meth. Enz., 58:44 [1979], Barnes and Sato. Anal. Biochem., 102:255 [1980], U.S. Patent No.

4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430;

WO

87/00195; U.S. Patent Re. 30,985; or copending U.S.S.N. 07/592,107 or 07/592,141, both filed on 3 October 1990, the disclosures of all of which are incorporated herein by reference, may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth 1 0 factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin

T

drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source.

Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal.

F Detecting Gene Amplification/Expression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 [1980]), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Various labels may be employed, most commonly radioisotopes, particularly 3 2 p. However, other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a polynucleotide. The biotin then serves as the site for binding to 3 0 avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so 3 5 that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or -82- WO 95/18858 PCT/US94/14553 body fluids, to quantitate directly the expression of gene product. With immunohistochemical staining techniques, a cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product coupled, where the labels are usually visually detectable, such as enzymatic labels, fluorescent labels, luminescent labels, and the like. 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].

Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal.

1 0 Conveniently, the antibodies may be prepared against a native mpl ligand polypeptide or against a synthetic peptide based on the DNA sequences provided herein as described further below.

G. Purification of mpl ligand Polypeptide Mpl ligand preferably is recovered from the culture medium as a secreted 1 5 polypeptide, although it also may be recovered from host cell lysates when directly expressed without a secretory signal.

When mpl ligand is expressed in a recombinant cell other than one of human origin, the mpl ligand is completely free of proteins or polypeptides of human origin.

However, it is still usually necessary to purify mpl ligand from other recombinant 20 cell proteins or polypeptides to obtain preparations that are substantially homogeneous as to the mpl ligand per se. As a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions are then separated. Alternatively, a commercially available protein concentration filter Amicon or Millipore Pellicon ultrafiltration units) may be 25 used. The mpl ligand may then be purified from the soluble protein fraction and from the membrane fraction of the culture lysate, depending on whether the mpl ligand is membrane bound. Mpl ligand thereafter is purified from contaminant soluble proteins and polypeptides by salting out and exchange or chromatographic procedures employing various gel matrices. These matrices include; acrylamide, agarose, dextran, cellulose and others common to protein purification. Exemplary chromatography procedures suitable for protein purification include; immunoaffinity anti-hmpl ligand Mab), receptoraffinity mpl-IgG or protein A Sepharose), hydrophobic interaction chromatography (HIC) ether, butyl, or phenyl Toyopearl), lectin chromatography Con A-Sepharose, lentil-lectin-Sepharose), size exclusion Sephadex G-75), cation- and anion-exchange columns DEAE or carboxymethyl- and sulfopropyl-cellulose), and reverse-phase high performance liquid chromatography (RP-HPLC) (see Urdal et J. Chromatog., 296:171 [1984] where two sequential RP-HPLC steps are used to purify recombinant human -83- WO 95/18858 PCT/US94/14553 IL-2). Other purification steps optionally include; ethanol precipitation; ammonium sulfate precipitation; chromatofocusing; preparative SDS-PAGE, and the like.

Mpl ligand variants in which residues have been deleted, inserted, or substituted are recovered in the same fashion as native mpl ligand, taking account of any substantial changes in properties occasioned by the variation. For example, preparation of a mpl ligand fusion with another protein or polypeptide, a bacterial or viral antigen, facilitates purification; an immunoaffinity column containing antibody to the antigen can be used to adsorb the fusion polypeptide.

Immunoaffinity columns such as a rabbit polyclonal anti-mpl ligand column can be employed to absorb the mpl ligand variant by binding it to at least one remaining immune epitope. Alternatively, the mpl ligand may be purified by affinity chromatography using a purified mpl-lgG coupled to a (preferably) immobilized resin such as Affi-Gel 10 (Bio-Rad, Richmond, CA) or the like, by means well known in the art. A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) also may be .o 15 useful to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants. One skilled in the art will appreciate that purification methods suitable for native mpl ligand may require modification to account for changes in the character of mpl ligand or its variants upon expression in recombinant cell culture.

H. Covalent Modifications of mpl ligand Polypeptide Covalent modifications of mpl ligand polypeptides are included within the scope of this invention. Both native mpl ligand and amino acid sequence variants of the mpl ligand may be covalently modified. One type of covalent modification included within the scope of this invention is a mpl ligand fragment. Variant mpl Ilgand fragments 25 having up to about 40 amino acid residues may be conveniently prepared by chemical i synthesis or by enzymatic or chemical cleavage of the full-length or variant mpl ligand polypeptide. Other types of covalent modifications of the mpl ligand or fragments thereof are introduced into the molecule by reacting targeted amino acid residues of the mpl ligand or fragments thereof with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

-84- WO 95/18858 PCT/US94/14553 Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Parabromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing -amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; 0methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be S. 1 5 performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, with particular *interest in introducing spectral labels into tyrosyl residues by reaction with aromatic 20 diazonium compounds or tetranitromethane. Most commonly, N-acetylimidlzole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 1251 or 1311 to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable.

25 Carboxyl side groups (aspartyl or glutamyl) are selectively modified by o: reaction with carbodiimides where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morphollnyl-4-ethyl)carbodiimide or 1ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking mpl ligand to a water-insoluble support matrix or surface for use in the method for purifying antimpl ligand antibodies, and vice versa. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable WO 95/18858 PCT/US94/14553 intermediates that are capable of forming crosslinks in the presence of light.

Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated form of these residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman Co., San Francisco, pp. 79-86 [1983]), acetylation of the N-terminal amine, and amidation of any C- S1 5 terminal carboxyl group.

Another type of covalent modification of the mpl ligand polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. By altering is meant deleting one or more carbohydrate moieties found in native mpl ligand, and/or adding one or more glycosylation sites that are not 20 present in the native mpl ligand.

Glycosylation of polypeptides is typically either N-linked or O-linked. Nlinked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-Xthreonine, where X is any amino acid except proline, are the recognition sequences for 25 enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition of glycosylation sites to the mpl ligand polypeptide is conveniently accomplished by altering the amino acid sequence such that It contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the native mpl ligand sequence (for O-linked glycosylation sites). For ease, the mpl ligand amino acid sequence is preferably altered through changes at the DNA level, particularly by mutating the DNA encoding the mpl ligand polypeptide at preselected bases such that codons are generated that will translate into -86- WO 95/18858 PCT/US94/14553 the desired amino acids. The DNA mutation(s) may be made using methods described above under the heading of "Amino Acid Sequence Variants of mpl Ligand." Another means of increasing the number of carbohydrate moieties on the mpl ligand is by chemical or enzymatic coupling of glycosides to the polypeptide. These procedures are advantageous in that they do not require production of the polypeptide in a host cell that has glycosylation capabilities for N- or O-linked glycosylation.

Depending on the coupling mode used, the sugar(s) may be attached to arginine and histidine, free carboxyl groups, free sulfhydryl groups such as those of cysteine, free hydroxyl groups such as those of serine, threonine, or 1 0 hydroxyproline, aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or the amide group of glutamine. These methods are described in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev.

Biochem., pp. 259-306 [1981].

Removal of carbohydrate moieties present on the mpl ligand polypeptide may be 15 accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin, et 20 al., Arch. Biochem. Biophys., 259:52 [1987] and by Edge et al., Anal. Biochem., 118:131 [1981]. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 [1987].

Glycosylation at potential glycosylation sites may be prevented by the use of the 25 compound tunicamycin as described by Duskin et al., J. Biol. Chem., 257:3105 [1982]. Tunicamycin blocks the formation of protein-N-glycoside linkages.

Another type of covalent modification of mpl ligand comprises linking the mpl ligand polypeptide to one of a variety of nonproteinaceous polymers, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S.

Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Mpl ligand polypeptides covalently linked to the forgoing polymers are refered to herein as pegylated mpl ligand polypeptides It will be appreciated that some screening of the recovered mpl ligand variant will be needed to select the optimal variant for binding to a mpl and having the 3 5 immunological and/or biological activity defined above. One can screen for stability in recombinant cell culture or in plasma against proteolytic cleavage), high affinity to a mpl member, oxidative stability, ability to be secreted in elevated yields, and the like. For example, a change in the immunological character of the mpl ligand -87.

WO 95/18858 PCT/US94/14553 polypeptide, such as affinity for a given antibody, is measured by a competitive-type immunoassay. Other potential modifications of protein or polypeptide properties such as redox or thermal stability, hydrophobicity, or susceptibility to proteolytic degradation are assayed by methods well known in the art.

17. General Methods for Preparation of Antibodies to mpl Ligand Antibody Preparation Polyclonal antibodies Polyclonal antibodies to mpl ligand polypeptides or fragments are generally 1 0 raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the mpl ligand and an adjuvant. It may be useful to conjugate the mpl ligand or a fragment containing the target amino acid sequence to a protein that is immunogenic in the species to be immunized, keyhole limpet hemocyanin, serum albumin, bovine g* thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, 1 5 for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glytaraldehyde, succinic anhydride, SOC12, 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, immunogenic conjugates or derivatives by combining 1 mg of 1 p.g of the peptide or 20 conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for mpl ligand antibody 25 titer. Animals are boosted until the titer plateaus. Preferably, the animal boosted with the conjugate of the same mpl ligand, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are used to enhance the immune response.

3 0 (ii) Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

For example, the mpl ligand monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler Milstein, Nature, -88- WO 95/18858 PCT/US94/14553 256:495 [1975], or may be made by recombinant DNA methods Patent No.

4,816,567 [Cabilly et In the hybridoma method, a mouse or other appropriate host animal, such as hamster is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 [Academic Press, 1986]).

1 0 The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include 1 5 hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines,, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 [1984]; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987).

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

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

After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI- -89- WO 95/18858 PCT/US94/14553 1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

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 readily isolated and sequenced using conventional procedures by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine 1 0 antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA 1 5 also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, (Cabilly et al., supra; Morrison, et al., Proc. Nat. Acad. Sci., 81:6851 [1984]), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for a mpl ligand and another antigen-combining site having specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.

For example, immunotoxins may be constructed 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 typically will be labeled with a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 p, 3 5 S, or 1251, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; radioactive isotopic labels, such as, 125(, 32p, 14, or 3H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.

WO 95/18858 PCT/US94/14553 Any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods 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].

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

Competitive binding assays rely on the ability of a labeled standard (which may be a mpl ligand or an immunologically reactive portion thereof) to compete with the test sample analyte (mpl ligand) for binding with a limited amount of antibody. The amount of mpl ligand in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of 1 5 standard that becomes bound, the antibodies generally are irisolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein (mpl ligand) to be detected.

In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex. David Greene, U.S. Patent No.

4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is 2 5 labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme horseradish peroxidase).

(iii) Humanized and human antibodies Methods for humanizing non-human antibodies are well known in the art.

3 0 Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 [1986]; Riechmann et al., Nature, 332:323-327 [1988]; Verhoeyen et al., Science, 239:1534-1536 [1988]), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (Cabilly et al., supra), wherein substantially less than an intact human -91 WO 95/18858 PCT/US94/14553 variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity.

According to the so called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as S1 0 the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., S* 151:2296 [1993]; Chothia and Lesk, J. Mol. Biol., 196:901 [1987]). Another .i method uses a particular framework derived from the concensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et Proc. Natl. Acad. Sci.

USA, 89:4285 [1992]; Presta et al., J. Immnol., 151:2623 [1993]).

It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using 2 0 three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable threedimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the 2 5 residues in the functioning of the candidate immunoglobulin sequence, the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. For further details see U.S.

application Serial No. 07/934,373 filed 21 August 1992, which is a continuationin-part of application Serial No. 07/715,272 filed 14 June 1991.

Alternatively, it is now possible to produce transgenic animals mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line -92- WO 95/18858 PCT/US94/14553 immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, 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 have binding specificities for at least two different antigens. Methods 1 0 for making bispecific antibodies are known in the art.

Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature, 305:537- 539 [1983]). Because of the random assortment of immunoglobulin heavy and light 1 5 chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in PCT publication No. WO 93/08829 (published 13 May 1993), and in Traunecker et al., EMBO, 10:3655-3659 [1991].

According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and.are cotransfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance. In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this -93- WO 95/18858 PCT/US94/14553 asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in copending application Serial No.

07/931,811 filed 17 August 1992.

For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 [1986].

Heteroconjugate antibodies Heteroconjugate antibodies are also within the scope of the present invention.

1 0 Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells Patent No. 4,676,980), and for treatment of HIV infection (PCT publication Nos. WO 91/00360 and WO 92/00373; EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross- 1 5 linking agents are well known in the art, and are disclosed in U.S. Patent No.

4,676,980, along with a number of cross-linking techniques.

IV. Therapeutic Use of the Megakaryocytopoietic Protein mpl Ligand The biologically active mpl ligand having hematopoietic effector function and 20 referred to here as a megakaryocytopoietic or thrombocytopoietic protein (TPO) may be used in a sterile pharmaceutical preparation or formulation to stimulate megakaryocytopoietic or thrombopoietic activity in patients suffering from thrombocytopenia due to impaired production, sequestration, or increased destruction of platelets. Thrombocytopenia-associated bone marrow hypoplasia aplastic 2 5 anemia following chemotherapy or bone marrow transplant) may be effectively treated with the compounds of this invention as well as disorders such as disseminated intravascular coagulation (DIC), immune thrombocytopenia (including HIV-induced ITP and non HIV-induced ITP), chronic idiopathic thrombocytopenia, congenital thrombocytopenia, myelodysplasia, and thrombotic thrombocytopenia. Additionally, these megakaryocytopoietic proteins may be useful in treating myeloproliferative thrombocytotic diseases as well as thrombocytosis from inflammatory conditions and in iron deficiency.

Preferred uses of the megakaryocytopoietic or thrombocytopoietic protein (TPO) of this invention are in: myelotoxic chemotherapy for treatment of leukemia or solid tumors, myeloablative chemotherapy for autologous or allogeneic bone marrow transplant, myelodysplasia, idiopathic aplastic anemia, congenital thrombocytopenia, and immune thrombocytopenia.

-94nm WO 95/18858 PCT/US94/14553 Still other disorders usefully treated with the megakaryocytopoietic proteins of this invention include defects or damage to platelets resulting from drugs, poisoning or activation on artificial surfaces. In these cases, the instant compounds may be employed to stimulate "shedding" of new "undamaged" platelets. For a more complete list of useful applications, see the "Background" supra, especially section and references cited therein.

The megakaryocytopoietic proteins of the instant invention may be employed alone or in combination with other cytokines, hematopoietins, interleukins, growth factors, or antibodies in the treatment of the above-identified disorders and conditions.

1 0 Thus, the instant compounds may be employed in combination with other protein or peptide having thrombopoietic activity including; G-CSF, GM-CSF, LIF, M-CSF, IL-1, .i IL-3, erythropoietin (EPO), kit ligand, IL-6, and IL-11.

The megakaryocytopoietic proteins of the instant invention are prepared in a mixture with a pharmaceutically acceptable carrier. This therapeutic composition can be administered intravenously or through the nose or lung. The composition may also be administered parenterally or subcutaneously as desired. When administered systematically, the therapeutic composition should be pyrogen-free and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability.

These conditions are known to those skilled in the art. Briefly, dosage formulations of the compounds of the present invention are prepared for storage or administration by mixing the compound having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers. Such materials are non-toxic to the recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sobitol; counterions such as sodium and/or nonionic surfactants such as Tween, Pluronics or polyethyleneglycol.

About 0.5 to 500 mg of a compound or mixture of the megakaryocytopoietic protein as the free acid or base form or as a pharmaceutically acceptable salt, is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., as called for by accepted pharmaceutical practice. The amount of active ingredient in these compositions is such that a suitable dosage in the range indicated is obtained.

WO 95/18858 PCTIUS94/14553 Sterile compositions for injection can be formulated according to conventional pharmaceutical practice. For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.

Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, films, or microcapsules. Examples of 1 0 sustained-release matrices include polyesters, hydrogels poly(2-hydroxyethylmethacrylate) as described by Langer et al., J. Biomed. Mater. Res., 15:167-277 [1981] and Langer, Chem. Tech., 12:98-105 [1982] or poly(vinylalcohol)], polylactides Patent No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 [1983]), 1 5 non-degradable ethylene-vinyl acetate (Langer et al., supra), degradable lactic acidglycolic acid copolymers such as the Lupron Depot T M (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-)-3-hydroxybutyric acid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid 2 0 enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 0 C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for protein stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

Sustained-release megakaryocytopoietic protein compositions also include liposomally entrapped megakaryocytopoietic protein. Liposomes containing megakaryocytopoietic protein are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 [1985]; Hwang et al., Proc.

Natl. Acad. Sci. USA, 77:4030-4034 [1980]; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent Nos.

4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is -96- WO 95/18858 PCT/US94/14553 greater than about 30 mol. cholesterol, the selected proportion being adjusted for the optimal megakaryocytopoietic protein therapy.

The dosage will be determined by the attending physician taking into consideration various factors known to modify the action of drugs including severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. Typically, the daily regimen will range from 0.1-100 gg/kg body weight. Preferably the dosage will range from 0.1gg/kg body weight. More preferably, the initial dosage will range from 1 to tg/kg/day. Optionally, the dosage range will be the same as that of other cytokines, 1 0 especially G-CSF, GM-CSF, and EPO. Therapeutically effective dosages may be determined by either in vitro or in vivo methods.

EXAMPLES

Without further description, it is believed that one of ordinary skill in the art 1 5 can, using the preceding description and illustrative examples, make and utilize the Spresent invention to the fullest extent. The following working examples therefore specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way of the remainder of the disclosure.

EXAMPLE 1 Partial Purification of the Porcine mpl Ligand Platelet-poor plasma was collected from normal or aplastic anemic pigs. Pigs were rendered aplastic by irradiation with 900 cGy of total body irradiation using a 4mEV linear accelerator. The irradiated pigs were supported for 6-8 days with intramuscular injections of cefazolin. Subsequently, their total blood volume was removed under general anesthesia, heparinized, and centrifuged at 1800 x g for to make platelet-poor plasma. The megakaryocyte stimulating activity was found to peak 6 days after irradiation.

Aplastic porcine plasma obtained from irradiated pigs is made 4M with NaCI and stirred for 30 min. at room temperature. The resultant precipitate is removed by centrifugation at 3800 rpm in a Sorvall RC3B and the supernatant is loaded onto a Phenyl-Toyopearl column (220 ml) equilibrated in 10 mM NaPO 4 containing 4M NaCI. The column is washed with this buffer until A280 is <0.05 and eluted with dH 2 0. The eluted protein peak is diluted with dH20 to a conductivity of 15mS and loaded onto a Blue-Sepharose column equilibrated (240 ml) in PBS. Subsequently, the column is washed with 5 column volumes each of PBS and 10mM NaPO4 (pH 7.4) containing 2M urea. Proteins are eluted from the column with 10mM NaPO4 (pH 7.4) containing 2M urea and 1M NaCI. The eluted protein peak is made 0.01% octyl -97- WO 95/18858 PCT/US94/14553 glucoside(n-octyl 1 3 -D-glucopyranoside) and 1 mM each with EDTA and Pefabloc (Boehinger Mannheim) and loaded directly onto tandemly linked CD4-lgG (Capon, D.J.

et al. Nature 337:525-531 [1989]) and mpl-lgG Ultralink (Pierce) columns (see below). The CD4-IgG (2 ml) column is removed after the sample is loaded and the mpl-lgG (4 ml) column is washed with 10 column volumes each of PBS and PBS containing 2 M NaCI and eluted with 0.1M glycine-HCI pH 2.25. Fractions are collected into 1/10th volume 1M Tris-HCI (pH Analysis of eluted fractions from the mpl-affinity column by SDS-PAGE (4- Novex gel) run under reducing conditions, revealed the presence of several 1 0 proteins (Fig. Proteins that silver stain with the strongest intensity resolve with apparent Mr of 66,000, 55,000, 30,000, 28,000 and 14,000. To determine which of these proteins stimulate proliferation of Ba/F3-mpl cell cultures these proteins were eluted from the gel as described in Example 2 below.

Ultralink Affinity Columns 10-20 mg of mpl-lgG or CD4-lgG in PBS are coupled to 0.5 grams of Ultralink Sresin (Pierce) as described by the manufacturer's instructions.

Construction and Expression of mpl-lgG A chimeric molecule comprising the entire extracellular domain of human mpl (amino acids 1-491) and the Fc region of a human IgG1 molecule was expressed in 293 cells. A cDNA fragment encoding amino acids 1-491 of human mpl was obtained by PCR from a human megakaryocytic CMK cell cDNA library and sequenced. A Clal site was inserted at the 5' end and a BstEll site at the 3' end. This fragment was cloned .i upstream of the IgGI Fc coding region in a Bluescript vector between the Clal and the BstEII sites after partial digestion of the PCR product with BstEll because of two other BstEll sites present in the DNA encoding the extracellular domain of mpl. The BstEll site introduced at the 3' end of the mpl PCR product was designed to have the Fc region in frame with the mpl extracellular domain. The construct was subcloned into tkneo vector between the Clal and Xbal sites and transfected into 293 human embryonic kidney cells by the calcium phosphate method. The cells were selected in 0.4 mg/ml G418 and individual clones were isolated. Mpl-lgG expression from isolated clones was determined using a human Fc specific ELISA. The best expression clone had an expression level of 1-2 mg/ml of mpl-lgG.

Ba/F3 mpl P Expressing Cells A cDNA corresponding to the entire coding region of human mpl P was cloned into pRK5-tkneo which was subsequently linearized with Notl and transfected into the IL-3 dependent cell line Ba/F3 by electroporation (1 x 10 7 cells, 9605F, 250Volts).

Three days later selection was started in the presence of 2 mg/ml of G418. The cells were selected as pools or individual clones were obtained by limiting dilution in 96 -98- WO 95/18858 PCT/US94/14553 well plates. Selected cells were maintained in RPMI containing 15% FBS, 1mg /ml G418, 20mM Glutamine, 10mM HEPES and 100 gg/ml of Pen-Strep. Expression of mpl P in selected clones was determined by FACS analysis using a anti-mpl p rabbit polyclonal antibody.

Ba/F3 mpl ligand Assay The mpl ligand assay was conducted as shown is Fig. 2. To determine the presence of mpl ligand from various sources, the mpl P Ba/F3 cells were starved of IL-3 for 24 hours at a cell density of 5 x 10 5 cells/ml in a humidified incubator at 37 0 C in 5% CO 2 and air. Following IL-3 starvation the cells were plated out in 96 1 0 well culture dishes at a density of 50,000 cells in 200 il of media with or without diluted samples and cultured for 24 hrs in a cell culture incubator. 20 jl of serum free RPMI media containing 1 .Ci of 3 H-thymidine was added to each well for the last 6-8 hours. The cells were then harvested on 96 well GF/C filter plates and washed times with water. The filters were counted in the presence of 40 Al of scintillation 1 5 fluid (Microscint 20) in a Packard Top Count counter.

EXAMPLE 2 Highly Purified Porcine mpl Ligand Gel Elution Protocol Equal amounts of affinity purified mpl ligand (fraction 6 eluted from the mpl- IgG.column) and 2X Laemmli sample buffer were mixed at room temperature without reducing agent and loaded onto a Novex 4-20% polyacrylamide gel as quickly as possible. The sample was not heated. As a control, sample buffer without ligand was run in an adjacent lane. The gel was run at 4-6 0 C at 135 volts for approximately 2 1/4 hours. The running buffer was initially at room temperature. The gel was then removed from the gel box and the plate on one side of the gel removed.

A replica of the gel was made on nitrocellulose as follows: A piece of nitrocellulose was wet with distilled water and carefully laid on top of the exposed gel face so air bubbles were excluded. Fiducial marks were placed on the nitrocellulose and the gel plate so the replica could be accurately repositioned after staining. After approximately 2 minutes, the nitrocellulose was carefully removed, and the gel was wrapped in plastic wrap and placed in the refrigerator. The nitrocellulose was stained with Biorad's gold total protein stain by first agitating it in 3 x 10 ml 0.1% Tween 0.5 M NaCI 0.1 M Tris-HCI pH 7.5 over approximately 45 minutes followed by 3 x 10 ml purified water over 5 minutes. The gold stain was then added and allowed to develop until the bands in the standards were visible. The replica was then rinsed with water, placed over the plastic wrap on the gel and carefully aligned with the fiducial marks. The positions of the Novex standards were marked on the gel plate and lines -99- WO 95/18858 PCT/US94/14553 were drawn to indicate the cutting positions. The nitrocellulose and plastic wrap were then removed and the gel cut along the indicated lines with a sharp razor blade. The cuts were extended beyond the sample lanes so they could be used to determine the positions of the slices when the gel was stained. After the slices were removed, the remaining gel was silver stained and the positions of the standards and the cut marks were measured. The molecular weights corresponding to the cut positions were determined from the Novex standards.

The 12 gel slices were placed into the cells in two Biorad model 422 electroeluters. 12-14K molecular weight cutoff membrane caps were used in the cells. 1 0 mM ammonium bicarbonate 0.05% SDS (approximately pH 7.8) was the elution buffer. One liter of buffer was chilled approximately 1 hour in a 4-6 0 C coldroom before use. Gel slices were eluted at 10 ma/cell (40 v initially) in a 4-6 0 C coldroom.

Elution took approximately 4 hours. The cells were then carefully removed and the Sliquid above the frit removed with a pipet. The elution chamber was removed and any 1 5 liquid above the membrane cap removed with a pipet. The liquid in the membrane cap was removed with a Pipetman and saved. Fifty gI aliquots of purified water were then 00: placed in the cap, agitated and removed until all the SDS crystals dissolved. These washes were combined with the saved liquid above. Total elution sample volume was 300-500 P l per gel slice. Samples were placed in 10 mm Spectrapor 4 12-14K 20 cutoff dialysis tubing which had been soaked several hours in purified water. They were dialyzed overnight at 4-6 0 C against 600 ml of phosphate buffered saline (PBS is approximately 4 mM in potassium) per 6 samples. The buffer was replaced the next morning and dialysis continued for 2.5 hours. Samples were then removed from the dialysis bags and placed in microfuge tubes. The tubes were placed on ice for 1 hour, microfuged at 14K rpm for 3 min. and the supernatants carefully removed from the precipitated SDS. The supernatants were then placed on ice for approximately 1 hour more and microfuged again for 4 min. The supernatants were diluted in phosphate buffered saline and submitted for the activity assay. Remaining samples were frozen at -70 0

C.

EXAMPLE 3 Porcine mpl Ligand Microsequencing Fraction 6 (2.6 ml) from the mpl-IgG affinity column was concentrated on a (Amicon). In order to prevent the mpl ligand from absorbing to the Microcon, the membrane was rinsed with 1% SDS and 5 i1 of 10 SDS was added to fraction 6. Sample buffer (20 gl) of 2X was added to the fraction #6 after Microcon concentration (20 Il) and the total volume (40 gl) was loaded on a single lane of a 4gradient acrylamide gel (Novex). The gel was run following Novex protocol. The -100- WO 95/18858 PCT/US94/14553 gel was then equilibrated for 5 min. prior to electroblotting in 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer, pH 11.0, containing methanol. Electroblotting onto Immobilon-PSQ membranes (Millipore) was carried out for 45 min. at 250 mA constant current in a BioRad Trans-Blot transfer cell The PVDF membrane was stained with 0.1% Coomassie Blue R-250 in methanol, 0.1% acetic acid for 1 min. and destained for 2-3 min. with 10% acetic acid in 50% methanol. The only proteins that were visible in the Mr 18,000-35,000 region of the blot had Mr of 30,000, 28,000 and 22,000.

Bands at 30, 28 and 22 kDa were subjected to protein sequencing. Automated 1 0 protein sequencing was performed on a model 470A Applied Biosystem sequencer equipped with an on-line PTH analyzer. The sequencer was modified to inject 80-900/ *i of the sample (Rodriguez, J. Chromatogr., 350:217-225 [1985]). Acetone (-12 l/1) was added to solvent A to balance the UV absorbance. Electroblotted proteins were sequenced in the Blott cartridge. Peaks were integrated with Justice Innovation 1 5 software using Nelson Analytical 970 interfaces. Sequence interpretation was performed on a VAX 5900 (Henzel et al., J. Chromatogr., 404:41-52 [1987]). Nterminal sequences (using one letter code with uncertain residues In parenthesis) and quantity of material obtained (in brackets) is presented in Table 2'.

20 TABLE 2' Mpl Ligand Amino-Terminus Sequences 30 kDa [1.8 pmol] 1 5 10 15 20 (S)PAPPA(C)DPRLLNKLLRDD(H/S)VLH(G)RL (SEQ 28 kDa [0.5 pmol] 1 5 10 15 20 (S)PA P PAX D P R LLNKLLRD D (H)VL(H)G R (SEQ ID NO:31) 18-22 kDa [0.5 pmol] 1 5 X AP P AX D P R L X (SEQ ID NO: 32) EXAMPLE 4 Liquid Suspension Megakaryocytopoiesis Assay Human peripheral stem cells (PSC) (obtained from consenting patients) were diluted 5 fold with IMDM media (Gibco) and centrifuged for 15 min. at room temp. at 800 x g. The cell pellets were resuspended in IMDM and layered onto 60% Percoll (density 1.077 gm/ml (Pharmacia) and centrifuged at 800 x g for 30 min. The -101- WO 95118858 PCT/US94/14553 light density mononuclear cells were aspirated at the interface and washed 2x with IMDM and plated out at 1-2 x 106 cells/ml in IMDM containing 30% FBS (1 ml final volume) in 24 well tissue culture clusters (Costar). APP or mpl ligand depleted APP was added to 10% and cultures were grown for 12-14 days in a humidified incubator at 37 0 C in 5% CO 2 and air. The cultures were also grown in the presence of 10% APP with 0.5 gpg of mpl-lgG added at days 0, 2 and 4. APP was depleted of mpl ligand by passing APP through a mpl-lgG affinity column.

To quantitate megakaryocytopoiesis in these liquid suspension cultures, a modification of Solberg et al. was used and employs a radiolabeled murine IgG monoclonal antibody (HP1-1D) to GPIlbllla (provided by Dr. Nichols, Mayo Clinic).

100 jg of HP1-1D (see Grant, B. et al., Blood 69:1334-1339 [1987]). was i radiolabeled with 1mCi of Na 1 2 5 1 using Enzymobeads (Biorad, Richmond, CA) as described by the manufacturer's instructions. Radiolabeled HP1-1D was stored at -70 0 C in PBS containing 0.01% octyl-glucoside. Typical specific activities were 1-2 x 106 cpm/g precipated by 12.5% trichloroacetic acid Liquid suspension cultures were set up in triplicate for each experimental point. After 12-14 days in culture the iml cultures were transferred to eppendorf tubes and centrifuged at 800 x g for 10 min. at room temp. and the resultant cell pellets were resuspended in 100 pl of PBS containing 0.02% EDTA and 20 bovine calf serum. 10ng of 12 5 1-HP1-1D in 50 l1 of assay buffer was added to the resuspended cultures and incubated for 60 min. at room temperature (RT) with occasional shaking. Subsequently, cells were collected by centrifugation at 800 x g for 10 min. at RT and washed 2x with assay buffer. The pellets were counted for 1 min. in a gamma counter (Packard). Non-specific binding was determined by adding 1 p.g of unlabeled HP1-1D for 60 min. before the addition of labeled HP1-1D. Specific binding was determined as the total 1 2 5 1-HP1-1D bound minus that bound in the presence of excess unlabeled HP1-1D.

EXAMPLE Oligonucleotide PCR Primers Based on the amino-terminal amino acid sequence obtained from the 30 kDa, 28 kDa and 18-22 kDa proteins, degenerate oligonucleotides were designed for use as polymerase chain reaction (PCR) primers (see Table Two primer pools were synthesized, a positive sense 20 mer pool encoding amino acid residues 2-8 (mpl 1) and an anti-sense 21-mer pool complimentary to sequences encoding amino acids 18- 24 (mpl 2).

-102- WO 95/18858 PCTIUS94/14553 TABLE 4 Degenerate Oligonucleotide Primer Pools mpl 1:5 GGN GCN CCNCNGCN TGY GA 3 (2,048-fold degenerate) SEQ IDNO: NCC RTG NAR NAG RTG RTC RTC 3' (2,048-fold degenerate) -SEQ ID NO: 36) Porcine genomic DNA, isolated from porcine peripheral blood lymphocytes, was used as a template for PCR. The 50 gJ reaction contained: 0.8 jig of porcine genomic DNA in 10mM Tris-HCI (pH 50mM KGI, 3mM MgC1 2 100 jig/mI BSA, 400 jiM dNTPs, 1 jiM of each primer pool and 2.5 units of Taq polymerase. Initial template denaturation was at 9400 for 8 min. followed by 35 cycles of 45 seconds at 9400, 1 0 00 min. at 55'C and 1 min. at 72 0 C. The final cycle was allowed to extend for 10 min. at S1 0 72 0 G. PCR products were separated by electrophoresis on a 12% polyacrylamide gel ***and visualized by staining with ethidium bromide. It was reasoned that If the amino- 00 terminal amino acid sequence was encoded by a single exon then the correct POR S. product was expected to be 69 bp. A DNA fragment of this size was eluted from the gel 0 0 and subcloned into pGEMT (Promega). Sequences of three clones are shown below In 91 0 04 IS1 Table TABLE 00 69 bp Porcine Genomic DNA Fragments gemnT3 CAGCCTGTGA CCCGCGACTC CTAAATAAAC TGCCTC 'GTGA 3'GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG

ACGGAGCA.QT

TGACCACGTT CAGCAGGGC [69 bp] (SEQ ID NO: 37) ACTGGQTGCAA GTCGTGCCG (SEQ ID NO: 38) gemT7 GGCATGTGA CCOGACTC CTAAATAAAC

TGCTTCGTGA

3'GGTCGTGGAG GGGGTACACT GGGGGCTGAG GATTTATTTG

ACGAAGCAUI

CGACGAGGTC CATCACGGC [69 bp] (SEQ ID NO: 39) GCTGGQTGCAG GTAGTGCCG -(SEQ ID NO: -103- WO 95/18858 PCT/US94/14553 gemT9 P R L L N K L L R (SEQ ID NO: 32)

CCAGCACCGCCGGCATGTGACCCCCGACTCCTAAATAAACTGCTTCGTGACG

3' GGTCGTGGCGGCCGTACACTGGGGGCTGAGGATTTATTTGACGAAGCACTGC ATCATGTCTATCACGGT 3' (SEQ ID NO: 41) TAGTACAGATAGTGCCA 5' (SEQ ID NO: 42) The position of the PCR primers is indicated by the underlined bases. These results verify the N-terminal sequence obtained for amino acids 9-17 for the 30 kDa, 28 kDa and 18-22 kDa proteins and indicated that this sequence is encoded by a single exon of porcine DNA.

EXAMPLE 6 Human mpl Ligand Gene Based on the results from Example 5, a 45-mer deoxyoligonucleotide, called 1 0 pR45, was designed and synthesized to screen a genomic library. The 45-mer had the following sequence: GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-T-ATT-TAG-GAG-TCG 3' (SEQ ID NO: 28) This oligonucleotide was 32 p-labeled with 3 2 P)-ATP and T4 kinase and used S1 5 to screen a human genomic DNA library in Xgem12 under low stringency hybridization and wash conditions (see Example Positive clones were picked, plaque purified and analyzed by restriction mapping and southern blotting. Clone #4 was selected for additional analysis.

A 2.8 kb BamHI-Xbal fragment that hybridized to the 45-mer was subcloned into pBluescript SK-. Partial DNA sequencing of this clone was preformed using as primers oligonucleotides specific to the porcine mpl ligand DNA sequence. The sequence obtained confirmed that DNA encoding the human homolog of the porcine mpl ligand had been isolated. An EcoRI restriction site was detected in the sequence allowing us to isolate a 390 bp EcoRI-Xbal fragment from the 2.8 kb BamHI-Xbal and to subclone it in pBluescript SK-.

Both strands of this fragment were sequenced. The human DNA sequence and deduced amino acid sequence are shown in Fig. 9 (SEQ ID NOS: 3 The predicted positions of introns in the genomic sequence are also indicated by arrows, and define a putative exon ("exon -104- WO 95/18858 PCT7US94/14553 Examination of the predicted amino acid sequence confirms that a serine residue is the first amino acid of the mature mpl ligand, as determined from direct amino acid sequence analysis. Immediately upstream from this codon the predicted amino acid sequence is highly suggestive of a signal sequence involved in secretion of the mature mpl ligand. This signal sequence coding region is probably interrupted at nucleotide position 68 by an intron.

In the 3' direction the exon appears to terminate at nucleotide 196. This exon therefore encodes a sequence of 42 amino acids, 16 of which are likely to be part of a signal sequence and 26 of which are part of the mature human mpl ligand.

EXAMPLE 7 Full Length Human mpl Ligand cDNA Based on the human "exon 3" sequence (Example 6) two non-degenerate oligonucleotides corresponding to the 3' and 5' ends of the "exon 3" sequence were 15 synthesized (Table 6).

TABLE 6 .Human cDNA Non-degenerate PCR Oligonucleotid Primers Fwd primer: 5' GCT AGC TCT AGA AAT TGC TCC TCG TGG TCA TGC TTC T 3' (SEQ ID NO: 43) Rvs primer: 5' CAG TCT GCC GTG AAG GAC ATG G 3' (SEQ ID NO: 44) These two primers were used in PCR reactions employing as a template DNA Sfrom various human cDNA libraries or 1 ng of Quick Clone cDNA (Clonetech) from 20 various tissues using the conditions described in the Example 5. The expected size of the correct PCR product was 140 bp. After analysis of the PCR products on a 12% polyacrylamide gel, a DNA fragment of the expected size was detected in cDNA libraries prepared from adult kidney, 293 fetal kidney cells and cDNA prepared from human fetal liver (Clonetech cat. #7171-1).

A fetal liver cDNA library in X DR2 (Clonetech cat. HL1151x) was screened with the same 45 mer oligonucleotide used to screen the human genomic library. The oligonucleotide was labelled with (y 3 2 p)-ATP using T4 polynucleotide kinase. The library was screened under low stringency hybridization conditions. The filters were prehybridized for 2hr then hybridized with the probe overnight at 420C in formamide, 5xSSC, 10xDenhardt's, 0.05M sodium phosphate (pH 0.1% sodium pyrophosphate, 50 gg/ml of sonicated salmon sperm DNA for 16hr. Filters were then rinsed in 2xSSC and then washed once in 0.5xSSC, 0.1% SDS at 420C. Filters were exposed overnight to Kodak X-Ray film. Positive clones were picked, plaque purified -105- WO 95/18858 PCTUS94/14553 and the insert size was determined by PCR using oligonucleotides flanking the BamHI- Xbal cloning in X DR2 (Clonetech cat. #6475-1). 5 pl of phage stock was used as a template source. Initial denaturation was for 7 min. at 94°C followed by 30 cycles of amplification (1 min. at 94°C, 1 min. at 520C and 1.5 min. at 720C). Final extention was for 15 min. at 720C. Clone FL2b had a 1.8kb insert and was selected for further analysis.

The plasmid pDR2 (Clonetech, XDR2 pDR2 cloning and Expression System Library Protocol Handbook, p 42) contained within the XDR2 phage arms, was rescued as described per manufacturer's instructions (Clonetech, XDR2 pDR2 cloning and 1 0 Expression System Library Protocol Handbook, p 29-30). Restriction analysis of the I plasmid pDR2-FL2b with BamHI and Xbal indicated the presence of an internal BamHI restriction site in the insert approximately at position 650. Digestion of the plasmid with BamHI-Xbal cut the insert in two fragments, one of 0.65 kb and one of 1.15 kb.

DNA sequence was determined with three different classes of template derived from the *i 15 plasmid pDR2-FL2b. DNA sequencing of double-stranded plasmid DNA was carried out with the AB1373 (Applied Biosystems, Foster City, California) automated fluorescent DNA sequencer using standard protocols for dye-labeled dideoxy nucleoside S: triphosphate terminators (dye-terminators) and custom synthesized walking primers (Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 [1977]; Smith et al., Nature, 321:674-679 [1986]). Direct sequencing of polymerase chain reaction amplified fragments from the plasmid was done with the ABI373 sequencer using custom primers and dye-terminator reactions. Single stranded template was generated with the M13 Janus vector (DNASTAR, Inc., Madison, Wisconsin) (Burland et al., Nucl. Acids Res., 21:3385-3390 [1993]). BamHI-Xbal (1.15 kb) and BamHI (0.65 kb) fragments were isolated from the plasmid pDR2-FL2b, the ends filled in with T4 DNA polymerase in the presence of deoxynucleotides, and then subcloned into the Smal site of M13 Janus. Sequencing was carried out with standard protocols for dye-labeled M13 universal and reverse primers, or walking primers and dye-terminators.

Manual sequencing reactions were carried out on single strand M13 DNA using walking primers and standard dideoxy-terminator chemistry (Sanger et al., Proc. Natl. Acad.

Sci. USA, 74:5463-5467 [1977]), 3 3 P-labeled oc-dATP and Sequenase (United States Biochemical Corp., Cleveland, Ohio). DNA sequence assembly was carried out with Sequencher V2.1b12 (Gene Codes Corporation, Ann Arbor, Michigan). The nucleotide and deduced sequences of hML are provided in Fig. 1 (SEQ ID NO: 1).

-106- WO 95/18858 PCTfUS94/14553 EXAMPLE 8 Isolation of the Human mpl Ligand (TPO) Gene.

Human genomic DNA clones of the TPO gene were isolated by screening a human genomic library in X-Gem12 with pR45, a previously described oligonucleotide probe under low stringency conditions (see Example 7) or under high stringency conditions with a fragment corresponding to the 3' half of human cDNA coding for the mpl ligand (from the BamH1 site to the 3'end). Two overlapping lambda clones spanning 35 kb were isolated. Two overlapping fragments (BamH1 and EcoRI) containing the entire TPO gene were subcloned and sequenced. The structure of the 1 0 human gene is composed of 6 exons within 7 kb of genomic DNA (Fig. 14 A, B and C).

The boundaries of all exon/intron junctions are consistent with the consensus motif established for mammalian genes (Shapiro, M. et al., Nucl. Acids Res. 15:7155 i [1987]). Exon 1 and exon 2 contain 5' untranslated sequence and the initial four amino acids of the signal peptide. The remainder of the secretory signal and the first 1 5 26 amino acids of the mature protein are encoded within exon 3. The entire carboxyl domain and 3' untranslated as well as -50 amino acids of the erythropoietin-like domain are encoded within exon 6. The four amino acids involved in the deletion observed within hML-2 (hTPO-2) are encoded at the 5' end of exon 6.

EXAMPLE 9 Transient Expression of Human mpl Ligand (hML) In order to subclone the full length insert contained in pDR2-FL2b, the r: plasmid was digested with Xbal to completion, then partially digested with BamHI. A DNA fragment corresponding to the 1.8 kb insert was gel purified and subcloned in pRK5 (pRK5.hmp/ I) (see U.S. Patent No. 5,258,287 for construction of under the control of the cytomegalovirus immediate early promoter. DNA from the construct pRK5-hmpl I was prepared by the PEG method and transfected in Human embryonic kidney 293 cells maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with F-12 nutrient mixture, 20 mM Hepes (pH 7.4) and fetal bovine serum. Cells were transfected by the calcium phosphate method as described (Gorman, C. [1985] in DNA Cloning: A Practical Approach (Glover, D. M., ed) Vol. II, pp. 143-190, IRL Press, Washington, D. 36 h after transfection, the supernatant of the transfected cells was assayed for activity in the proliferation assay (see Example Supernatant of 293 cells transfected with pRK vector only gave no stimulation of the Ba/F3 or Ba/F3-mpl cells (Fig. 12A). Supernatant of cells transfected with pRK5-hmpl I had no effect on the Ba/F3 cells but dramatically stimulates the proliferation of Ba/F3-mpl cells (Fig. 12A), indicating that this cDNA encodes a functionally active human mpl ligand.

-107- WO 95/18858 PCT/US94/14553 EXAMPLE Human Mpl Ligand Isoforms hML2, hML3, and hML4 In order to identify alternatively spliced forms of hML, primers were synthesized corresponding to each end of the coding sequence of hML. These primers were employed in RT-PCR to amplify human adult liver RNA. Additionally, internal primers flanking selected regions of interest (see below) were constructed and similarly employed. Direct sequencing of the ends of the PCR product revealed a single 1 0 sequence corresponding exactly to the sequence of the cDNA isolated from the human fetal liver library (see Fig. 1 [SEQ ID NO: However, a region near the Cterminus of the EPO-domain (in the middle of the PCR product) exhibited a complex sequence pattern sugesting the existence of possible splice variants in that region. To isolate these splice variants, the primers provided in Table 7 flanking the region of 1 5 interest were used in a PCR as templates for human adult liver cDNA.

STABLE 7 SHuman ML Isoform PCR Primers phmpllcdna.3el: 5'TGTGGACTTTAGCTTGGGAGAATG3' (SEQ ID NO: pbx4.f2: 5'GGTCCAGGGACCTGGAGGTTTG3' (SEQ ID NO: 46) The PCR products were subcloned blunt into M13. Sequencing of individual subclones revealed the existence of at least 3 ML isoforms. One of them, hML (also refered to as hML 3 3 2 is the longest form and corresponds exactly to the sequence isolated from the fetal liver library. Sequences of the four human mpl ligand isoforms listed from longest (hML) to shortest (hML-4) are provided in (Fig. 11 [SEQ ID NOS: 6, 8, 9 EXAMPLE 11 Construction and Transient Expression of Human Mpl Ligand Isoforms and Substitutional Variants hML2, hML3, and hML(R153A, R154A) Isoforms hML2 and hML3 and substitutional variant hML(R153A, R154A) were reconstituted from hML using the recombinant PCR technique described by Russell Higuchi, in PCR Protocols, A guide to Methods and Applications, Acad. Press, M.A.Innis, D.H. Gelfand, J.J. Sninsky T.J. White Editors.

In all contructs, the "outside" primers used are shown in Table 8 and the "overlapping" primers are shown in Table 9.

-108- WO 95/18858 PCT/US94/14553 TABLE 8 Outside Primers Cla.FL.F2: GAT ATC GAT AGC CAG ACA CCC CGG CCA G3' (SEQ ID NO: 47)

HMPLL-R:

AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA3' (SEQ ID NO: 48) TABLE 9 Overlapping Primers hML-2: MLA4.F: 5'CTC CTT GGA MLA4.R 5'GGT CCT GCC ACC CAG GGC AGG ACC 3' CTG GGCT TCC AAG rr, 3 (SEQ ID NO: 49) CTG GT T AA C INU OU) hML-3: hMLA116+: 5'CTG CTC CGA GGA AAG GAC TTC TGG ATT 3' (SEQ ID NO: 51) hMLA 116-: 5'AAT CCA GAA GTC CTT TCC TCG GAG CAG 3' (SEQ ID NO: 52) hML(R153A. R154A): RR-KO-F: 5'CCC TCT GCG TCG CGG CGG CCC CAC CCA C 3' (SEQ ID NO: 53) RR-KO-R: 5'GTG GGT GGG GCC GCC GCG ACG CAG AGG G 3' (SEQ ID NO: 54) All PCR amplifications were performed with cloned Pfu DNA polymerase 1 0 (Stratagene) using the following conditions: Initial template denaturation was at 94°C for 7 min. followed by 30 cycles of 1 min. at 94°C, 1 min. at 55°C and 1.5 min. at 72 0 C. The final cycle was allowed to extend for 10 min. at 72°C. The final PCR product was digested with Clal-Xbal, gel purified and cloned in pRK5tkneo. 293 cells were transfected with the various constructs as described above and the supernatant was 1 5 assayed using the Ba/F3-mpl proliferation assay. hML-2 and hML-3 showed no detectable activity in this assay, however the activity of hML(R153A, R154A) was similar to hML indicating that processing at this di-basic site is not required for activity (see Fig. 13).

-109- WO 95/18858 PCT/US94/14553 EXAMPLE 12 Murine mpl Ligand cDNA mML, mML-2 and mML-3 Isolation of mML cDNA.

A DNA fragment corresponding to the entire coding region of the human mpl ligand was obtained by PCR, gel purified and labeled by random priming in the presence of 32 P-dATP and 3 2 P-dCTP. This probe was used to screen 106 clones of a mouse liver cDNA library in XGT10 (Clontech cat# ML3001a). Duplicate filters were hybridized in 35% formamide, 5xSSC, 10xDenhardt's, 0.1% SDS, 0.05M sodium phosphate (pH 0.1% sodium pyrophosphate, 100 Ag/ml of sonicated salmon sperm DNA overnight in the presence of the probe. Filters were rinsed in 2xSSC and then washed once in 0.5xSSC, 0.1% SDS at 42 0 C. Hybridizing phage were plaque-purified and the cDNA inserts were subcloned into the Eco R1 site of Bluescript SK- plasmid. Clone "LD" with a 1.5 kb insert was chosen for further analysis and both 1 5 strands were sequenced as descibed above for the human ML cDNA. The nucleotide and deduced amino acid sequences from clone LD are provided in Fig. 14 (SEQ ID NOS: 1 11). The deduced mature ML sequence from this clone was 331 amino acid residues long and identified as mML 3 3 1 (or mML-2 for reasons described below).

Considerable identity for both nucleotide and deduced amino acid sequences were observed in the EPO-like domains of these ML's. However, when deduced amino acid sequences of human and mouse ML's were aligned, the mouse sequence appeared to have a tetrapeptide deletion between human residues 111-114 corresponding to the 12 nucleotide deletion following nucleotide position 618 seen in both the human (see *i above) and pig (see below) cDNA's. Accordingly, additional clones were examined to detect possible murine ML isoforms. One clone, had a 1.4 kb insert with a 335 amino acid deduced sequence containing the "missing" tetrapeptide LPLQ. This form is believed to be the full length murine ML and is refered to as mML or mML335. The nucleotide and deduced amino acid sequence for mML are provided in Fig. 16 (SEQ ID NOS: 12 13). Finally, clone "L2" was isolated and sequenced. This clone has the 116 nucleotide deletion corresponding to hML3 and is therefore denominated mML-3.

Comparison of the deduced amino acid sequences of these two isoforms is shown in Fig.

16.

Expression of recombinant mML. Expression vectors for murine ML were prepared essentially as described in Example 8. Clones encoding mML and mML-2 were subcloned into pRK5tkneo, a mammalian expression vector that provides expression under the control of the CMV promoter and an SV40 polyadenylation signal.

The resulting expression vectors, mMLpRKtkneo and mML2pRKtkneo were transiently transfected into 293 cells using the calcium phosphate method. Following transient -110- WO 95/18858 PCTIUS94/14553 transfection, media was conditioned for five days. The cells were maintained in high glucose DMEM media supplemented with 10% fetal calf serum.

Expression of murine-mpl (mmpl) in Ba/F3 cells. Stable cell lines expressing c-mpl were obtained by transfection of mmpl pRKtkneo, essentially as described for human mpl in Example 1. Briefly, an expression vector (20 gg; linearized) containing the entire coding sequence of murine mpl (Skoda, R. et al., EMBO J. 12:2645-2653 [1993]) was transfected into Ba/F3 cells by electroporation (5 X 106 cells, 250 volts, 960 UF) followed by selection for neomycine resistance with 2 mg/ml G418. Expression of mpl was assessed by flow 1 0 cytometry analysis using rabbit anti-murine mpl-lgG antisera. Ba/F3 cells were maintained in RPMI 1640 media from WEHI -3B cells as a source of IL-3.

Supernatants from 293 cells transiently transfected with both mML and mML-2 were S-assayed in BaF3 cells transfected with both mmpl and hmpl as described in Example 1 V. EXAMPLE 13 Porcine mpl Ligand cDNA pML and pML-2 Porcine ML (pML) cDNA was isolated by RACE PCR. Briefly, an oligo dT primer and 2 specific primers were designed based on the sequence of the exon of the porcine ML gene encoding the amino terminus of the ML purified from the aplastic pig serum. cDNA prepared from various aplastic pig tissues was obtained and amplified. A PCR cDNA product of 1342 bp was found in kidney and subcloned. Several clones were *i sequenced and found to encode the mature pig mpl ligand (not including a complete secretion signal). The cDNA was found to encode a 332 amino acid mature protein (pML 3 3 2 having the sequence shown in Fig. 18 (SEQ ID NOS: 9 16).

Method: Isolation of pML gene and cDNA. Genomic clones of the porcine ML gene were isolated by screening a pig genomic library in EMBL3 (Clontech Inc.) with pR45. The library was screened essentially as described in Example 7. Several clones were isolated and the exon encoding amino acid sequence identical to that obtained from the purified ML was sequenced. Porcine ML cDNA were obtained using a modification of the RACE PCR protocol. Two specific ML primers were designed based on the sequence of the pig ML gene. Polyadenylated mRNA was isolated from the kidney of aplastic pigs essentially as previously described. cDNA was prepared by reverse transcription with the BamdT primer (BamdT: 5' GACTCGAGGATCCATCGA I I I I i I I l I il 3') (SEQ ID NO: -111- WO 95f18858 PCT/US94/14553 directed against the polyadenosine tail of the mRNA. An initial round of PCR amplification (28 cycles of 95 0 C for 60 seconds, 58 0 C for 60 seconds, and 72 0 C for ninety seconds) was conducted using the ML specific h-forward-1 primer (h-forward-1: 5' GCTAGCTCTAGAAATTGCTCCTCGTGGTCATGCTTCT 3') (SEQ ID NO: 43) and the BAMAD primer (BAMAD: 5' GACTCGAGGATCCATCG 3') (SEQ ID NO: 56) in a 100 ml reaction (50 mM KCI, 1.5 mM MgCI, 10 mM Tris pH 8.0, 0.2 mM 1 0 dNTPs,with 0.05 U/ml Amplitaq polymerase [Perkin Elmer Inc.]) The PCR product was then digested with Clal, extracted with phenol-chloroform ethanol precipitated, and ligated to 0.1 mg of Bluescript SK- vector (Stratagene inc.) that had been cut with Clal and Kpn 1. After incubation for two hours at room temperature, one fourth of the ligation mixture was added directly to a second round of PCR (22 1 5 cycles as described above) using a second ML specific forward-1 primer (forward-1: 5' GCTAGCTCTAGAAGCCCGGCTCCTCCTGCCTG 3') (SEQ ID NO: 57) and T3-21 (an oligonucleotide that binds to a sequence adjacent to the multiple cloning region within the Bluescript SK- vector): CGAAATTAACCCTCACTAAAG 3') (SEQ ID NO: 58).

The resulting PCR product was digested with Xbal and Clal and subcloned into Bluescript SK-. Several clones from indepedent 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: Comparison of pML and pML-2 amino acid sequences shows the latter form is identical except that the tetrapeptide QLPP corresponding to residues 111-114 inclusive have been deleted (see Fig. 22 [SEQ ID NOS: 18 The four amino acid deletions observed in murine, human and porcine ML cDNA occur at precisely the same position within the predicted proteins.

EXAMPLE 14 CMK Assay for Thrombopoietin (TPO) Induction of Platelet Antigen GPllbllla Expression CMK cells are maintained in RMPI 1640 medium (Sigma) supplemented with fetal bovine serum and 10mM glutamine. In preparation for the assay, the cells are harvested, washed and resuspended at 5x10 5 cells/ml in serum-free GIF medium supplemented with 5mg/I bovine insulin, 10mg/I apo-transferrin, 1 X trace -112- WO 95/18858 PCT/US94/14553 elements. In a 96-well flat-bottom plate, the TPO standard or experimental samples are added to each well at appropriate dilutions in 100 il volumes. 100 .pl of the CMK cell suspension is added to each well and the plates are incubated at 37°C, in a 5% C02 incubator for 48 hours. After incubation, the plates are spun at 1000rpm at 4°C for five minutes. Supernatants are discarded and 100 gl of the FITC-conjugated GPIIbllla monoclonal 2D2 antibody is added to each well. Following incubation at 4°C for 1 hour, plates are spun again at 1000rpm for five minutes. The supernatants containing unbound antibody are discarded and 200 pl of 0.1% BSA-PBS wash is added to each well. The 0.1% BSA-PBS wash step is repeated three times. Cells are then analyzed on a FASCAN using standard one parameter analysis measuring relative fluorescence intensity.

EXAMPLE DAMI Assay for Thrombopoietin (TPO) by Measuring Endomitotic Activity of DAMI Cells on 96-well Microtiter Plates DAMI cells are maintained in IMDM 10% horse serum (Gibco) supplemented with 10mM glutamine, 100ng/ml Penicillin G, and 50 p.g/ml streptomycin. In preparation for the assay, the cells are harvested, washed, and resuspended at lxl0 6 cells/ml in IMDM 1% horse serum. In a 96-well round-bottom plate, 100 p.1 of the TPO standard or experimental samples is added to DAMI cell suspension. Cells are then incubated for 48 hours at 37°C in a 5% CO2 incubator. After incubation, plates are spun in a Sorvall 6000B centrifuge at 1000rpm for five minutes at Supernatants are discarded and 200 pI of PBS-0.1% BSA wash step is repeated. Cells are fixed by the addition of 200 l1 ice-cold 70% Ethanol-PBS and resuspended by aspiration. After incubation at 4°C for 15 minutes, the plates are spun at 2000 rpm for five minutes and 150 pl of 1mg/ml RNAse containing 0.1mg/ml propidium iodide and 0.05% Tween-20 is added to each well. Following a one hour incubation at 37°C the changes in DNA content are measured by flow cytometry. Polyploidy is measured and quantitated as follows: Normalized Polyploid Ratio (NPR) (%Cells in >G2+M/%Cells in <G2+M) with TPO (%Cells in >G2+M/%Cells in <G2+M) in control -113- WO 95/18858 PCTIUS94/14553 EXAMPLE 16 Thrombopoietin (TPO) In Vivo Assay (Mouse Platelet Rebound Assay) In Vivo Assay for 3 5 S Determination of Platelet Production C57BL6 mice (obtained from Charles River) are injected intraperitoneally (IP) with 1 ml goat anti-mouse platelet serum (6 amps) on day 1 to produce thrombocytopenia. On days 5 and 6, mice are given two IP injections of the factor or PBS as the control. On day 7, thirty pCi of Na 2 3 5

SO

4 in 0.1 ml saline are injected intravenously and the percent 35S incorporation of the injected dose into circulating 1 0 platelets is measured in blood samples obtained from treated and control mice. Platelet counts and leukocyte counts are made at the same time from blood obtained from the retro-orbital sinus.

EXAMPLE 17 15 KIRA ELISA for Thrombopoietin

(TPO)

by Measuring Phosphorylation of the mpl-Rse.gD Chimeric Receptor The human mpl receptor has been disclosed 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. Chem. 269(14):10720-10728 [1994]) with a carboxyl-terminal flag polypeptide Rse.gD) was made for use in the KIRA ELISA described herein. See Fig. 30 and 31 for a diagrammatic description of the assay.

Capture agent preparation Monoclonal anti-gD (clone 5B6) was produced against a peptide from Herpes simplex virus glycoprotein D (Paborsky et Protein Engineering 3(6):547-553 [1990]). The purified stock preparation was adjusted to 3.0mg/ml in phosphate buffered saline (PBS), pH 7.4 and 1.0ml aliquots were stored at -20' C.

Anti-phosphotyrosine antibody preparation Monoclonal anti-phosphotyrosine, clone 4G10, was purchased from UBI (Lake Placid, NY) and biotinylated using long-arm biotin-N-hydroxysuccinamide (Biotin- X-NHS, Research Organics, Cleveland, OH).

Ligand The mpl ligand was prepared by the recombinant techniques described herein.

The purified mpl ligand was stored at 4 as a stock solution.

Preparation of Rse.gD nucleic acid Synthetic double stranded oligonucleotides were used to reconstitute the coding sequence for the C-terminal 10 amino acids (880 890) of human Rse and add an -114- WO 95/18858 PCT/US94/14553 additional 21 amino acids containing an epitope for the antibody 5B6 and a stop codon.

Table 10 presents the final sequence of the synthetic portion of the fusion gene.

TABLE Synthetic Double Stranded Portion of Human Rse Fusion Gene coding strand: TGGCTG ATCCAAATCGATTCCGCGGCAAAGATCTTCCGGTCCTGTAGAAGCT-3' (SEQ ID NO: 59) noncoding (anti-sense) strand:

S'-AGCTTCTACAGGACCGGAAGATCTTTGCCGCGGAATCGATTTGGATCAGCCA

TCTTG AGGCTAGCATCTGCGCAGCTCGAGTGTGGCAGTAGCCCTTGCTGCA-3' (SEQ ID NO: The synthetic DNA was ligated with the cDNA encoding amino acids 1-880 of human Rse at the Pstl site beginning at nucleotide 2644 of the published human Rse cDNA sequence (Mark et Journal of Biological Chemistry 269(14):10720- 10728 [1994]) and Hindlll sites in the polylinker of the expression vector 1 0 pSVl7.ID.LL (See Fig. 32 A-L; SEQ ID NO: 22) to create the expression plasmid pSV.ID.Rse.gD. Briefly, the expression plasmid comprises a dicistronic primary transcript which contains sequence encoding DHFR bounded by 5' splice donor and 3' splice acceptor intron splice sites, followed by sequence that encodes the Rse.gD. The full length (non-spliced) message contains DHFR as the first open reading frame and 1 5 therefore generates DHFR protein to allow selection of stable transformants.

Preparation of mpl-Rse.gD nucleic acid The expression plasmid pSV.ID.Rse.gD produced as described above was modified to produce plasmid pSV.ID.M.tmRd6 which contained the coding sequences of the ECD of human mpl (amino acids 1-491) fused to the transmembrane domain and intracellular domain of Rse.gD (amino acids 429-911). Synthetic oligonucleotides were used to join the coding sequence of a portion of the extracellular domain of human mpl to a portion of the Rse coding sequence in a two step PCR cloning reaction as described by Mark et al., J. Biol. Chem. 267:26166-26171 (1992). Primers used for the first PCR reaction were M1 (5'-TCTCGCTACCGTTTACAG-3') (SEQ ID NO: 61) and M2 -115- WO 95/18858 PCT/US94/14553 (5'-CAGGTACCCACCAGGCGGTCTCGGT-3') (SEQ ID NO: 62) with a mpl cDNA template and R1 (5'-GGGCCATGACACTGTCAA-3') (SEQ ID NO: 63) and R2 (5'-GACCGCCACCGAGACCGCCTGGTGGGTACCTGTGGTCCTT-3') (SEQ ID NO: 64) with a Rse cDNA template. The Pvull-Smal portion of this fusion junction was used for 1 0 the construction of the full-length chimeric receptor.

Cell transformation DP12.CHO cells (EP 307,247 published 15 March 1989) were electroporated with pSV.ID.M.tmRd6 which had been linearized at a unique Notl site in the plasmid backbone. The DNA was ethanol precipitated after phenol/chloroform extraction and S1 5 was resuspended in 2 0pl 1/10 Tris EDTA. Then, 10tg of DNA was incubated with 10 7 CHO DP12 cells in 1 ml of PBS on ice for 10 min. before electroporation at 400 volts and 330gf. Cells were returned to ice for 10 min. before being plated Into nonselective medium. After 24 hours cells were fed nucleoside-free medium to select for stable DHFR+ clones.

20 Selection of transformed cells for use in the KIRA ELISA Clones expressing MPL/Rse.gD were identified by western-blotting of whole cell lysates post-fractionation by SDS-PAGE using the antibody 5B6 which detects the gD epitope tag.

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

KIRA ELISA Mpl-Rse.gD transformed DP12.CHO cells were seeded (3x10 4 per well) in the wells of a flat-bottom-96 well culture plate in 100 Il media and cultured overnight at 37 'C in 5% CO 2 The following morning the well supernatants were decanted, and the plates were lightly tamped on a paper towel. 501l of media containing either experimental samples or 200, 50, 12.5, 3.12, 0.78, 0.19, 0.048 or 0 ng/ml mpl ligand was then added to each well. The cells were stimulated at 37'C for 30 min., the well supernatants were decanted, and the plates were once again lightly tamped on a paper towel. To lyse the cells and solubilize the chimeric receptors, 100 pC of lysis buffer was added to each well. Lysis buffer consisted of 150 mM NaCI containing mM HEPES (Gibco), 0.5 Triton-X 100 (Gibco), 0.01 thimerosal, 30 KIU/ml -116- WO 95/18858 PCTIUS94/14553 aprotinin (ICN Biochemicals, Aurora, OH), imM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF; ICN Biochemicals), 50 uM 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 agitated gently on a plate shaker (Bellco Instruments, Vineland, NJ) for 60 min. at room temperature.

While the cells were being solubilized, an ELISA microtiter plate (Nunc Maxisorp, Inter Med, Denmark) coated overnight at 4'C with the 5B6 monoclonal antigD antibody (5.0 pg/ml in 50 mM carbonate buffer, pH 9.6, 100 il/well) was decanted, tamped on a paper towel and blocked with 150 p.l/well of Block Buffer [PBS 1 0 containing 0.5 BSA (Intergen Company, Purchase, NY) and 0.01 thimerosal] for min. at room temperature with gentle agitation. After 60 minutes, the anti-gD 5B6 coated plate was washed 6 times with wash buffer (PBS containing 0.05 I and 0.01 thimerosal) using an automated plate washer (ScanWasher 300, Skatron Instruments, Inc, Sterling, VA).

1 5 The lysate containing solubilized MPL/Rse.gD from the cell-culture microtiter well was transferred (85 Il/well) to anti-gD 5B6 coated and blocked ELISA well and was incubated for 2 h at room temperature with gentle agitation. The unbound mpl- Rse.gD was removed by washing with wash buffer and 100 gI of biotinylated 4G10 (anti-phosphotyrosine) diluted 1:18000 In dilution buffer (PBS containing 0.5 BSA, 0.05 Tween-20, 5 mM EDTA, and 0.01 thimerosal), i.e. 56 ng/ml was added to each well. After incubation for 2 hr at room temperature the plate was washed and 100 p.l of horseradish peroxidase (HRPO)-conjugated streptavidin (Zymed Laboratories, S. 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 agitation. The free avidin-conjugate was washed away and 100 p.l freshly prepared substrate solution (tetramethyl benzidine [TMB]; 2-component substrate kit; Kirkegaard and Perry, Gaithersburg, MD) was added to each well. The reaction was allowed to proceed for 10 minutes, after which the color development was stopped by the addition of 100 Il/well 1.0 M H 3

PO

4 The absorbance at 450 nm was read with a reference wavelength of 650 nm (ABS 4 5 0/ 6 5 0 using a vmax plate reader (Molecular Devices, Palo Alto, CA) controlled with a Macintosh Centris 650 (Apple Computers, Cupertino, CA) and DeltaSoft software (BioMetallics, Inc, Princeton, NJ).

The standard curve was generated by stimulating dpl2.trkA,B or C.gD cells with 200, 50, 12.5, 3.12, 0.78, 0.19, 0.048 or 0 ng/ml mpl ligand and presented as ng/ml TPO vs. mean ABS 4 5 0 /65 0 sd using the DeltaSoft program. Sample concentrations were obtained by interpolation of their absorbance on the standard curve and are expressed in terms of ng/ml TPO activity.

-117- WO 95/18859 WO 9518858PCTfUS94/14553 The mpl-ligand was found to be able to activate the mpi-Rse.gD chimeric receptor in a concentration-dependent and ligand-specific manner. Further, the mnpl- Rse.gD KIRA-ELISA was found to be tolerant of up to 100% human serum (shown) or 100% plasma (not shown), allowing the assay to be used to readily screen patient and plK samples.

293-produced TP 332 Std Curve 1 TPO Conc (ng/ml) Summary of TPO

ECSO

TPO Form (cells) (wt/voIL) (molarity) Hu TPO 332 (293) 2.56 n/mI 67.4 PM Mu TPO 332 (293) 3.69 ng/ml 97.1 plM Hu TPO 153 (293) -41 n/mI -1.08 nM Hu TPO 155 coi 0.44 nq/ml 11.6 M Hu TPO l53met coi) 0.829 n/mI 21.8 pM -118- WO 95/18858 PCT/US94/14553 EXAMPLE 18 Receptor Based ELISA for Thrombopoletin (TPO) ELISA plates were coated with rabbit F(ab') 2 anti-human IgG (Fc) in pH 9.6 carbonate buffer at 4 0 C overnight. Plates were blocked with 0.5 bovine serum albumin in PBS at room temperature for one hour. Fermenter harvest containing the chimeric receptor, mpl-lgG, was added to the plates and incubated for 2 hours. Twofold serial dilutions (0.39-25 ng/ml) of the standard (TPO 3 3 2 produced in 293 cells with the concentration determined by quantitative amino acid analysis) and serially diluted samples in 0.5% bovine serum albumin, 0.05% tween 20 were added to the 1 0 plates and incubated for 2 hours. Bound TPO was detected with protein A purified, biotinylated rabbit antibodies to TPO155 which was produced in E. coli (1 hour S: incubation), followed by streptavidin-peroxidase (30 min. incubation) and 3,3',5,5'-tetramethyl benzidine as the substrate. The absorbance was read at 450 nm.

Plates were washed between steps. For data analysis, the standard curve is fitted using 1 5 a four-parameter curve fitting program by Kaleidagraph. Concentrations of the S.samples were calculated from the standard curve.

EXAMPLE 19 Expression and Purification of TPO from 293 Cells 2 0 1. Preperation of 293 Cell Expression Vectors A cDNA corresponding to the TPO entire open reading frame was obtained by PCR using the following oligonucleotides as primers: TABLE 11 S293 PCR Primers Cla.FL.F: 5' ATC GAT ATC GAT CAG CCA GAC ACC CCG GCC AG 3' (SEQ ID NO: hmpll-R: 5' GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA 3' (SEQ ID NO: 48) PRK5-hmpl I (described in Example 9) was used as template for the reaction in the presence of pfu DNA polymerase (Stratagene). Initial denaturation was for 7 min. at 94°C followed by 25 cycles of amplification (1 min. at 94°C, 1 min. at and 1 min. at 720C). Final extension was for 15 min. at 720C). The PCR product was purified and cloned between the restriction sites Clal and Xbal of the plasmid pRK5tkneo, a pRK5 derived vector modified to express a neomycin resistance gene under the control of the thymidine kinase promote, to obtain the vector A second construct corresponding to the epo homologous domain was -119- WO 95/18858 PCT/US94/14553 generated the same way but using Cla.FL.F as forward primer and the following reverse primer: Arg.STOP.Xba: 5' 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 These 2 constructs were transfected into Human Embryonic Kidney cells by the CaPO 4 method as described in Example 9. 24 hours after transfection selection of 1 0 neomycin resistant clones was started in the presence of 0.4 mg/ml G418.10 to days later individual colonies were transferred to 96 well plates and allowed to grow to confluency. Expression of ML 1 53 or ML 3 3 2 in the conditioned media from these clones was assessed using the Ba/F3-mp/ proliferation assay (described in Example I) 3. Purification of rhML 3 3 2 293-rhML 3 3 2 conditioned media was applied to a Blue-Sepharose (pharmacia) column that was equilibrated in 10mM sodium phosphate pH 7.4 (buffer The column was subsequently washed with 10 column volumes each of buffer A and buffer A containing 2M urea. The column was then eluted with buffer A containing 2M urea and 1M NaCI. The Blue-Sepharose elution pool was then directly applied to a WGA-Sepharose column equilibrated in buffer A. The WGA-Sepharose column was then washed with 10 column volumes of buffer A containing 2M urea and 1 M NaCI and eluted with the same buffer containing 0.5M N-acetyl-D-glucosamine. The WGA- Sepharose eluate was applied to a C4-HPLC column (Synchrom, Inc.) equilibrated in 25 0.1% TFA. The C4-HPLC column was eluted with discontinuous propanol gradient (0- 25-35%, 35-70%). rhML 3 3 2 was found to elute in the 28-30% propanol region of the gradient. By SDS-PAGE the purified rhML 332 migrates as a broad band i. n the 68-80 kDa region of the gel(see Figure 4. Purification of rhML 1 5 3 293-rhML15 3 conditioned media was resolved on Blue-Sepharose as described for rhML33 2 The Blue Sepharose eluate was applied directly to a mpl-affinity column as described above. RhML 1 5 3 eluted from the mpl-affinity column was purified to homogeneity using a C4-HPLC column run under the same conditions as described for rhML332. By SDS-PAGE the purified rhML153 resolves into 2 major and 2 minor bands with Mr of -18,000-21,000(see Figure -120- WO 95/18858 PCT/US94/14553 EXAMPLE Expression and Purification of TPO from CHO 1. Description of CHO Expression Vectors The expression vectors used in the electroporation protocols described below have been designated: (full length or hTPO 3 3 2 and (truncated or hTPO153).

The pertinent features of these plasmids are presented in Fig. 23 and 24.

2. Preperation of CHO Expression Vectors A cDNA corresponding to the hTPO entire open reading frame was obtained by PCR using the oligonucleotide primers of Table 12.

TABLE 12 CHO Expression Vector PCR Primers C l a.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 CGG CAG TGT CTG AGA ACC 3' (SEQ ID NO: 67) 15 PRK5-hmpl I (described in Example 7 and 9) was used as template for the reaction in the presence of pfu DNA polymerase (Stratagene). Initial denaturation was for 7 min. at 94 0C followed by 25 cycles of amplification (1 min. at 94°C, 1min. at 0C and 1 min. at 72°C). Final extension was for 15 min. at 720C). The PCR product was purified and cloned between the restriction sites Clal and Sail of the plasmid pSVI5.ID.LL to obtain the vector pSVI5.ID.LL.MLORF. A second construct corresponding to the EPO homologous domain was generated the same way but using Cla.FL.F2 as forward primer and the following reverse primer: EPOD.Sal 5' AGT CGA CGT CGA CTC ACC TGA CGC AGA GGG TGG ACC 3' (SEQ ID NO: 68) The final construct is called pSVI5.ID.LL.MLEPO-D. The sequence of both constructs was verified as described in Example 7 and 9.

In essence, the coding sequences for the full length and truncated ligand were introduced into the multiple cloning site of the CHO expression vector This vector contains the SV40 early promoter/enhancer region, a modified splice unit 3 0 containing the mouse DHFR cDNA, a multiple cloning site for the introduction of the gene of interest (in this case the TPO sequences described) an SV40 polyadenylation signal and origin of replication and the beta-lactamase gene for plasmid selection and amplification in bacteria.

-121- WO 95/18858 PCT/US94/14553 3. Methodology for Establishing Stable CHO Cell Lines Expressing Recombinant Human TP0 3 3 2 and TP0 15 3 a. Description of CHO parent cell line The host CHO (Chinese Hamster Ovary) cell line used for the expression of the TPO molecules described herein is known as CHO-DP12 (see EP 307,247 published March 1989). This mammalian cell line was clonally selected from a transfection of the parent line (CHO-K1 DUX-B11(DHFR-)- obtained from Dr. Frank Lee of Stanford University with the permission of Dr.L. Chasin) with a vector expressing preproinsulin to obtain clones with reduced insulin requirements. These cells are also 1 0 DHFR minus and clones can be selected for the presence of DHFR cDNA vector sequences by growth on medium devoid of nucleoside supplements (glycine, hypoxanthine, and thymidine). This selection system for stably expressing CHO cell lines is commonly used.

b. Transfection method (electroporation) 1 5 TP0 3 32 and TP0 1 5 3 expressing cell lines were generated by transfecting DP12 cells via electroporation (see e.g. Andreason, G.L. J. Tiss. Cult. Meth., 15,56 [1993]) with linearized pSVI5.1D.LL.MLORF or pSVI5.ID.LL.MLEPO-D plasmids respectively. Three restriction enzyme reaction mixtures were set up for each i' plasmid cutting; 10g, 25pg and 504g of the vector with the enzyme NOTI by standard 2 0 molecular biology methods. This restriction site is found only once in the vector in the Slinearization region 3' and outside the TPO ligand transcription units (see Fig. 23).

The 1001 reactions were set up for overnight incubation at 37 degrees. The next day the mixes were phenol-chloroform-isoamyl alcohol (50:49:1) extracted one time and ethanol precipitated on dry ice for approximately one hour. The precipitate was then collected by a 15 minute microcentrifugation and dried. The linearized DNA was resuspended into 501p of Ham's DMEM-F12 1:1 medium supplemented with standard antibiotics and 2mM glutamine.

Suspension growing DP12 cells were collected, washed one time in the medium described for resuspending the DNA and finally resuspended in the same medium at a concentration of 107 cells per 7 50pl. Aliquots of cells (750g1) and each linearized DNA mix were incubated together at room temperature for one hour and then transferred to a BRL electroporation chamber. Each reaction mix was then electroporated in a standard BRL electroporation apparatus at 350 volts set at 330AF and low capacitance. After electroporation, the cells were allowed to sit in the apparatus for 5 minutes and then on ice for an additional 10 minute incubation period.

The electroporated cells were transferred to 60mm cell culture dishes containing 5 ml of standard, complete growth medium for CHO cells (High glucose DMEM-F12 50:50 -122- WO 95/18858 PCT/US94/14553 without glycine supplemented with 1X GHT, 2mM glutamine, and 5% fetal calf serum) and grown overnight in a 5% CO 2 cell culture incubator.

c. Selection and screening method The next day, cells were trypsinized off the plates by standard methods and transferred to 150mm tissue culture dishes containing DHFR selective medium (Ham's DMEM-F12, 1:1medium described above supplemented with either 2% or 5% dialyzed fetal calf serum but devoid of glycine, hypoxanthine and thymidine this is the standard DHFR selection medium we use). Cells from each 60mm dish were subsequently replated into 5 /150 mm dishes. Cells were then incubated for 10 to 15 days( with 1 0 one medium change) at 37 degrees/5%

CO

2 until clones began to appear and reached sizes amenable to transfer to 96 well dishes. Over a period of 4-5 days, cell lines were transferred to 96 well dishes using sterile yellow tips on a pipettman set at The cells were allowed to grow to confluency (usually 3-5 days) and then the trays were trypsinized and 2 copies of the original tray were reproduced. Two of these copies were short term stored in the freezer with cells in each well diluted into 501 of 10%FCS in DMSO. 5 day conditioned serum free medium samples were assayed from .i confluent wells in the third tray for TPO expression via the Ba/F cell based activity S.assay. The highest expressing clones based on this assay were revived from storage and scaled up to 2 confluent 150mm T-flasks for transfer to the cell culture group for 2 0 suspension adaptation, re-assay and banking.

d. Amplification Protocol Several of the highest titer cell lines from the selection described above were subsequently put through a standard methotrexate amplification regime to generate higher titer clones. CHO cell clones are expanded and plated in 10cm dishes at 4 concentrations of methotrexate 50nM, 100nM, 200nM and 400nM) at two or three cell numbers (105, 5x105, and 106 cells per dish). These cultures are then incubated at 37 degree/5%

CO

2 until clones are established and amenable to transfer to 96 well dishes for further assay. Several high titer clones from this selection were again subjected to greater concentrations of methotrexate 600nM, 800 nM, 1000nM and 1200nM) and as before resistant clones are allowed to establish and then transferred to 96 well dishes and assayed.

4. Culturing Stable CHO Cell Lines Expressing Recombinant Human TP0332 and TPO 15 3 Banked cells are thawed and the cell population is expanded by standard cell 3 5 growth methods in either serum free or serum containing medium. After expansion to sufficient cell density, cells are washed to remove spent cell culture media. Cells are then cultured by any standard method including; batch, fed-batch or continuous culture at 25-40 OC, neutral pH, with a dissolved 02 content of at least 5% until the -123- WO 95/18858 PCTfUS94/14553 constitutively secreted TPO is accumulated. Cell culture fluid is then separated from the cells by mechanical means such as centrifugation.

Purification of Recombinant Human TPO from CHO Culture Fluids Harvested cell culture fluid (HCCF) is directly applied to a Blue Sepharose 6 Fast Flow column (Phamacia) equilibrated in 0.01M Na Phosphate pH7.4, 0.15M NaCI at a ratio of approximately 100L of HCCF per liter of resin and at a linear flow rate of approximately 300 ml/hr/cm 2 The column is then washed with 3 to 5 column volumes of equilibration buffer followed by 3 to 5 column volumes of 0.01M Na Phosphate pH7.4, 2.0M urea. The TPO is then eluted with 3 to 5 column volumes of 1 0 0.01M Na Phosphate pH7.4, 2.0M urea, 1.0M NaCI.

The Blue Sepharose Pool containing TPO is then applied to a Wheat Germ Lectin Sepharose 6MB column (Pharmacia) equilibrated in 0.01M Na Phosphate pH7.4, urea, and 1.0M NaCI at a ratio of from 8 to 16 ml of Blue Sepharose Pool per ml of resin at flow rate of approximately 50 ml/hr/cm 2 The column is then washed with 2 to 3 column volumes of equilibration buffer. The TPO is then eluted with 2 to column volumes of 0.01M Na Phosphate pH7.4, 2.0M urea, 0.5M N-acetyl-Dglucosamine.

SThe Wheat Germ Lectin Pool is then adjusted to a final concentration of 0.04% C12E8 and 0.1% trifluroacetic acid (TFA). The resulting pool is applied to a C4 reverse phase column (Vydac 214TP1022) equilibrated in 0.1% TFA, 0.04% C12E8 at a load of approximately 0.2 to 0.5 mg protein per ml of resin at a flow rate.of 157 ml/hr/cm 2 The protein is eluted in a two phase linear gradient of acetonitrile containing 0.1% TFA, 0.04% C1 2

E

8 The first phase is composed of a linear gradient from 0 to 25 30% acetonitrile in 15 minutes, The second phase is composed of a linear gradient from 30 to 60% acetonitrile in 60 minutes. The TPO elutes at approximately acetonitrile. A pool is made on the basis of SDS-PAGE.

The C4 Pool is then diluted with 2 volumes of 0.01M Na Phosphate pH7:4, 0.15M NaCI and diafilitered versus approximately 6 volumes of 0.01M Na Phosphate pH7.4, 0.15M NaCI on an Amicon YM or like ultrafiltration membrane having a 10,000 to 30,000 Dalton molecular weight cut-off. The resulting diafiltrate may be then directly processed or further concentrated by ultrafiltration. The diafiltrate/concentrate is adjusted to a final concentration of 0.01% All or a portion of the diafiltrate/concentrate equivalent to 2 to 5% of the calculated column volume is then applied to a Sephacryl S-300 HR column (Pharmacia) equilibrated in 0.01M Na Phosphate pH7.4, 0.15M NaCI, 0.01% Tweenand chromatographed at a flow rate of approximately 17 ml/hr/cm 2 The TPO containing fractions which are free of aggregate and proteolytic degradation products -124- WO 95/18858 PCT/US94/14553 are pooled on the basis of SDS-PAGE. The resulting pool is filtered on a 0.224 filter, Millex-GV or like, and stored at 2-8 0

C.

EXAMPLE 21 Transformation and Induction of TPO Protein Synthesis in E. coli 1. Construction of E. coli TPO expression vectors The plasmids pMP21, pMP151, pMP41, pMP57 and pMP202 are all designed to express the first 155 amino acids of TPO downstream of a small leader which varies among the different constructs. The leaders provide primarily for high level translation initiation and rapid purification. The plasmids pMP210-1, -T8, -21, -22, -24, -25 are designed to express the first 153 amino acids of TPO downstream of an initiation methionine and differ only in the codon usage for the first 6 amino acids of TPO, while the plasmid pMP251 is a derivative of pMP210-1 in which the carboxy terminal end of TPO is extended by two amino acids. All of the above plasmids will 1 5 produce high levels of intracellular expression of TPO in E. coli upon induction of the tryptophan promoter (Yansura, D. G. et. al. Methods in Enzymology Goeddel, D. V., Ed.) 185:54-60, Academic Press, San Diego [1990]). The plasmids pMP1 and pMP172 are intermediates in the construction of the above TPO intracellular expression plasmids.

2 0 Plasmid pMP S: The plasmid pMP1 is a secretion vector for the first 155 amino acids of TPO, and was constructed by ligating together 5 fragments of DNA as shown in Fig. 33. The first of these was the vector pPho21 in which the small Mlul-BamHI fragment had been removed. pPho21 is a derivative of phGH1 (Chang, C. N. et. al., Gene 55:189- 196 [1987]) in which the human growth hormone gene has been replaced with the E.

coli phoA gene, and a Mlul restriction site has been engineered into the coding sequence for the STII signal sequence at amino acids 20-21.

J The next two fragments, a 258 base pair Hinfl-Pstl piece of DNA from hmpl I(Example 9) encoding TPO amino acids 19-103, and the following synthetic 3 0 DNA encoding amino acids 1-18

TG

ATACGGTCGGGCCGAGGAGGACGAACACTGGAGGCTCAGGAGTCATTTGACGAAGC

(SEQ ID NO: 69) (SEQ ID NO: -125- WO 95/18858 PCT/US94/14553 were preligated with T4-DNA ligase, and second cut with Pstl. The fourth was a 152 base pair Pstl-Haelll fragment from pRK5hmpll encoding amino acids 104-155 of TPO. The last was a 412 base pair Stul-BamHI fragment from pdh108 containing the lambda to transcriptional terminator as previously described (Scholtissek, S. et. al., NAR 15:3185 [1987]).

Plasmid pMP21 The plasmid pMP21 is designed to express the first 155 amino acids of TPO with the aid of a 13 amino acid leader comprising part of the STII signal sequence. It was constructed by ligating together three DNA fragments as shown in Fig. 34, 1 0 the first of these being the vector pVEG31 in which the small Xbal-Sphl fragment had been removed. The vector pVEG31 is a derivative of pHGH207-1 (de Boer, H. A. et. al.

in Promoter Structure and Function (Rodriguez, R. L. and Chamberlain, M. J. Ed), 462, Praeger, New York [1982]) in which the human growth hormone gene has been replaced by the gene for vascular endothelial growth factor this identical vector 1 5 fragment can be obtained from this latter plasmid).

The second part in the ligation was a synthetic DNA duplex with the following *i sequence: (SEQ ID NO: 71) (SEQ ID NO: 72) The last piece was a 1072 base pair Mlul-Sphl fragment from pMP1 encoding 155 amino acids of TPO.

Plasmid pMP151 The plasmid pMP151 is designed to express the first 155 amino acids of TPO downstream of a leader comprising 7 amino acids of the STII signal sequence, 8 histidines, and a factor Xa cleavage site. As shown in Fig. 35, pMP151 was constructed by ligating together three DNA fragments, the first of these being the previously described vector pVEG31 from which the small Xbal-Sphl fragment had been removed. The second was a synthetic DNA duplex with the following sequence:

GTCGTAGCC

TTAATACTFTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTTC

(SEQ ID NO: 73) (SEQ ID NO: 74) -126- WO 95/18858 PCT/US94/14553 The last was a 1064 base pair Bgll-Sphl fragment from pMP11 encoding 154 amino acids of TPO. The plasmid pMP11 is identical to pMP1 with the exception of a few codon changes in the STII signal sequence( this fragment can be obtained from pMP1).

Plasmid pMP202 The plasmid pMP202 is very similar to the expression vector pMP151 with the exception that the factor Xa cleavage site in the leader has been replaced with a thrombin cleavage site. As shown in Fig. 36, pMP202 was constructed by ligating together three DNA fragments. The first of these was the previously described pVEG31 in which the small Xbal-Sphl fragment had been removed. The second was a synthetic 1 0 DNA duplex with the following sequence:

CCACGTAGCC

TTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTT

1 5 (SEQ ID NO: (SEQ ID NO: 76) The last piece was a 1064 base pair Bgll-Sphl fragment from the previously described plasmid pMP11.

Plasmid pMP172 The plasmid pMP172 is a secretion vector for the first 153 amino acids of TPO, and is an intermediate for the construction of pMP210. As shown in Fig. 37, pMP172 was prepared by ligating together three DNA fragments, the first of which was the vector pLS32lamB in which the small EcoRI-Hindll section had been removed.

25 The second was a 946 base pair EcoRI-Hgal fragment from the previously described plasmid pMP11. The last piece was a synthetic DNA duplex with the following sequence: 5'-TCCACCCTCTGCGTCAGGT (SEQ ID NO: 77) (SEQ ID NO: 78) Plasmid pMP210 The plasmid pMP210 is designed to express the first 153 amino acids of TPO after a translational initiation methionine. This plasmid was actually made as a bank of plasmids in which the first 6 codons of TPO were randomized in the third position of each codon, and was constructed as shown in Fig. 38 by the ligation of three DNA 3 5 fragments. The first of these was the previously described vector pVEG31 in which the small Xbal-Sphl fragment had been removed. The second was a synthetic DNA dupex shown below treated first with DNA polymerasel (Klenow) followed by digestion with -127- WO 95/18858 PCTIUS94/14553 Xbal and Hinfl, and encoding the initation methionine and the randomized first 6 codons of TPO.

ACACTGGAGGCT

GTTCTCAGTAAA (SEQ ID NO: 79) (SEQ ID NO: The third was a 890 base pair Hinfl-Sphl fragment from pMP172 encoding amino 1 0 acids 19-153 of TPO.

The plasmid pMP210 bank of approximately 3700 clones was retransformed onto high tetracycline (50 1g/ml) LB plates to select out high translational initiation clones (Yansura, D. G. et. al., Methods: A Companion to Methods in Enzymology 4:151- 158 [1992]). Of the 8 colonies which came up on high tetracycline plates, five of the 1 5 best in terms of TPO expression were subject to DNA sequencing and the results are shown in Fig. 39 (SEQ ID NOS: 23, 24, 25, 26, 27 and 28).

Plasmid pMP41 The plasmid pMP41 is designed to express the first 155 amino acids of TPO fused to a leader consisting of 7 amino acids of the STII signal sequence followed by a 2 0 factor Xa cleavage site. The plasmid was constructed as shown in Fig. 40 by ligating together three pieces of DNA, the first of which was the previously described vector pVEG31 in which the small Xbal-Sphl fragment had been removed. The second was the following synthetic DNA duplex: 5'-CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (SEQ ID NO: 81) 25 TTAATACTTTTTCTTATAGCGTAAATAGCTTCCAGCAT-5' (SEQ ID NO: 82) The last piece of the ligation was the 1064 base pair Bgll-Sphl fragment from the previously described plasmid pMP11.

h Plasmid pMP57 The plasmid pMP57 expresses the first 155 amino acids of TPO downstream of a leader consisting of 9 amino acids of the STII signal sequence and the dibasic site Lys- Arg. This dibasic site provides for a means of removing the leader with the protease ArgC. This plasmid was constructed as shown in Fig. 41 by ligating together three DNA pieces. The first of these was the previously described vector pVEG31 in which the small Xbal-Sphl fragment had been removed. The second was the following synthetic DNA duplex: (SEQ ID NO: 83) (SEQ ID NO: 84) -128- WO 95/18858 PCT/US94/14553 The last part of the ligation was the 1064 base pair Bgll-Sphl fragment from the previously described plasmid pMP11.

Plasmid pMP251 The plasmid pMP251 is a derivative of pMP210-1 in which two additional amino acids of TPO are included on the carboxy terminal end. As shown in Fig.42, this plasmid was constructed by ligating together two pieces of DNA, the first of these being the previously described pMP21 in which the small Xbal-Apal fragment had been removed. The second part of the ligation was a 316 base pair Xbal-Apal fragment from pMP210-1.

1 0 2. Transformation and Induction of E. coil with TPO expression vectors The above TPO expression plasmids were used to transform the E. coli strain 44C6 (w3110 tonAA rpoHts long clpPA galE) using the CaCl2 heat shock method (Mandel, M. et al., J. Mol. Bio/., 53:159-162, [1970]). The transformed cells were grown first at 37°C in LB media containing 50 .g/ml carbenicillin until the optical 1 5 density (600nm) of the culture reached approximately 2-3. The LB culture was then diluted 20x into M9 media containing 0.49% casamino acids and 50 gg/ml .i carbenicillin. After growth with aeration at 30°C for 1 hour, indole-3-acrylic acid was added to a final concentration of 50 gg/ml. The culture was then allowed to continue growing at 300C with aeration for another 15 hours at which time the cells 2 0 were harvested by centrifugation.

EXAMPLE 22 Production of Biologically Active TPO (Met-11-153) in E. coli The procedures given below for production of biologically active, refolded TPO S 25 (met- 1 1-153) can be applied in analogy for the recovery of other TPO variants including N and C terminal extended forms (see Example 23).

A Recovery of non-soluble TPO (Met- 1 1-153) E. coli cells expressing TPO (Met-1 1-153) encoded by the plasmid pMP210- 1 are fermented as described above. Typically, about 100g of cells are resuspended in 1 L (10 volumes) of cell disruption buffer (10 mM Tris, 5 mM EDTA, pH 8) with a Polytron homogenizer and the cells centrifuged at 5000 x g for 30 minutes. The washed cell pellet is again resuspended in 1 L cell disruption buffer with the Polytron homogenizer and the cell suspension is passed through an LH Cell Disrupter (LH Inceltech, Inc.) or through a Microfluidizer (Microfluidics International) according to the manufactures' instructions. The suspension is centrifuged at 5,000g for 30 min.

and resuspended and centrifuged a second time to make a washed refractile body pellet.

The washed pellet is used immediately or stored frozen at -700C.

-129- WO 95/18858 PCT/US94/14553 B. Solubilization and purification of monomeric TPO (Met- 1 1-153) The pellet from above is resuspended in 5 volumes by weight of 20 mM Tris, pH 8, with 6-8 M guanidine and 25 mM DTT (dithiothreitol) and stirred for 1-3 hr., or overnight, at 4 0 C to effect solubilization of the TPO protein. High concentrations of urea (6-8M) are also useful but generally result in lower yields compared to guanidine. After solubilization, the solution is centrifuged at 30,000 x g for 30 min.

to produce a clear supernatant containing denatured, monomeric TPO protein. The supernatant is then chromatographed on a Superdex 200 gel filtration column (Pharmacia, 2.6 x 60 cm) at a flow rate of 2 ml/min. and the protein eluted with mM Na phosphate, pH 6.0, with 10 mM DTT Fractions containing monomeric, denatured TPO protein eluting between 160 and 200 ml are pooled. The TPO protein is further purified on a semi-preparative C4 reversed phase column (2 x 20 cm VYDAC). The sample is applied at 5 ml/min. to a column equilibrated in 0.1% TFA(trifluoroacetic acid) with 30% acetonitrile. The protein is eluted with a linear 1 5 gradient of acetonitrile (30-60% in 60 min.). The purified reduced protein elutes at approximately 50% acetonitrile. This material is used for refolding to obtain biologically active TPO variant.

C Generation of biologically active TPO (Met- 1 1-153) Approximately 20 mg of monomeric, reduced and denatured TPO protein in ml 0.1% TFA/50% acetonitrile is diluted into 360 ml of refolding buffer containing optimally the following reagents: mM Tris 0.3 M NaCI 5 mM EDTA 25 2% CHAPS detergent glycerol 5 mM oxidized glutathione :1 mM reduced glutathione pH adjusted to 8.3 After mixing, the refolding buffer is gently stirred at 4 0 C for 12-48 hr to effect maximal refolding yields of the correct disulfide-bonded form of TPO (see below). The solution is then acidified with TFA to a final concentration of 0.2%, filtered through a 0.45 or 0.22 micron filter, and 1/10 volume of acetonitrile added.

This solution is then pumped directly onto a C4 reversed phase column and the purified, refolded TPO (Met-1 1-153) eluted with the same gradient program as above. Refolded, biologically active TPO is eluted at approximately 45% acetonitrile under these conditions. Improper disulfide-bonded versions of TPO are eluted earlier.

-130- WO 95/18858 PCT/US94/14553 The final purified TPO (Met-1 1-153) is greater than 95% pure as assessed by SDS gels and analytical C4 reversed phase chromatography. For animal studies, the C4 purified material was dialyzed into physiologically compatible buffers. Isotonic buffers (10 mM Na acetate, pH 5.5, 10 mM Na succinate, pH 5.5 or 10 mM Na phosphate, pH 7.4) containing 150 mM NaCI and 0.01% Tween 80 were utilized.

Because of the high potency of TPO in the Ba/F3 assay (half maximal stimulation is achieved at approximately 3 pg/ml), it is possible to obtain biologically active material utilizing many different buffer, detergent and redox conditions.

However, under most conditions only a small amount of properly folded material 1 0 is obtained. For commercial manufacturing processes, it is desirable to have refolding yields at least 10%, more preferably 30-50% and most preferably Many different detergents (Triton X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, sarkosyl, Tween 20 and Tween 80, Zwittergent 3-14 and others) were assessed for efficiency to support high refolding yields. Of these detergents, only the CHAPS family (CHAPS and CHAPSO) were found to be generally useful in the refolding reaction to limit protein aggregation and improper disulfide formation. Levels of CHAPS greater than 1% were most useful. Sodium chloride was required for best yields, with the optimal levels between 0.1 M and 0.5M. The presence of EDTA mM) limited the amount of metal-catalyzed oxidation (and aggregation) which was observed with some preparations. Glycerol concentrations of greater than V: produced the optimal refolding conditions. For maximum yields, it was essential to have both oxidized and reduced glutathione or oxidized and reduced cysteine as the redox reagent pair. Generally higher yields were observed when the mole ratio of oxidized reagent is equal to or in excess over the reduced reagent member of the redox pair. pH 25 values between 7.5 and about 9 were optimal for refolding of these TPO variants.

Organic solvents ethanol, acetonitrile, methanol) were tolerated at concentrations Sof 10-15% or lower. Higher levels of organic solvents increased the amount of improperly folded forms. Tris and phosphate buffers were generally useful.

Incubation at 4 0 C also produced higher levels of properly folded TPO.

Refolding yields of 40-60% (based on the amount of reduced and denatured TPO used in the refolding reaction) are typical for preparations of TPO that have been purified through the first C4 step. Active material can be obtained when less pure preparations directly after the Superdex 200 column or after the initial refractile body extraction) although the yields are less due to extensive precipitation and interference of non-TPO proteins during the TPO refolding process.

Since TPO (Met-1 1-153) contains 4 cysteine residues, it is possible to generate three different disulfide versions of this protein: version 1: disulfides between cysteine residues 1-4 and 2-3 -131p WO 95/18858 PCT/US94/14553 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 exploration in determining refolding conditions, several different peaks containing the TPO protein were separated by C4 reversed phase chromatography. Only one of these peaks had significant biological activity as determined using the Ba/F3 assay. Subsequently, the refolding conditions were optimized to yield preferentially that version. Under these conditions, the misfolded versions are less than 10-20% of the total monomer TPO obtained.

The disulfide pattern for the biologically active TPO has been determined to be 1-4 and 2-3 by mass spectrometry and protein sequencing(i.e. version Aliquots of the various C4-resolved peaks (5-10 nmoles) were digested with trypsin (1:25 mole ratio of trypsin to protein). The digestion mixture was analyzed by matrixassisted laser desorption mass spectrometry before and after reduction with DTT. After reduction, masses corresponding to most of the larger tryptic peptides of TPO were 1 5 detected. In the un-reduced samples, some of these masses were missing and new S masses were observed. The mass of the new peaks corresponded basically to the sum of the individual tryptic peptides involved in the disulfide pair. Thus it was possible to unequivocally assign the disulfide pattern of the refolded, recombinant, biologically active TPO to be 1-4 and 2-3. This is consistent with the known disulfide pattern of the related molecule erythropoietin.

D. Biological activity of recombinant, refolded TPO (met 1-153) Refolded and purified TPO (Met- 1 1-153) has activity in both in vitro and in vivo assays. In the Ba/F3 assay, half-maximal stimulation of thymidine incorporation into the Ba/F3 cells was achieved at 3.3 pg /ml (0.3 pM). In the mpl receptor-based ELISA, half-maximal activity occurred at 1.9 ng/ml (120 pM). In normal and myelosuppressed animals produced by near-lethal X-radiation, TPO (Met- 1 1-153) was highly potent (activity was seen at doses as low as 30 ng/mouse) to stimulate the production of new platelets.

S

EXAMPLE 23 Production of Other Biologically Active TPO Variants in E. coll Three different TPO variants produced in E. coli, purified and refolded into biological active forms are provided below.

1 MLF 13 residues from the bacterial-derived signal sequence STII are fused to the N-terminal domain of TPO (residues 1-155). The resulting sequence is MKKNIAFLLNAYASPAPPAC CVRRA (SEQ ID NO: -132- WO 95/18858 PCTIUS94/14553 where the leader sequence is underlined and represents Cys 7 through Cys 1 5 1 This variant was constructed to provide a tyrosine for radio-iodination of TPO for receptor and biological studies.

2 H8MLF 7 residues from the STII sequence, 8 histidine residues and the Factor Xa enzymatic cleavage sequence IEGR are fused to the N-terminal domain (residues 1-155) of.TPO. The sequence is MKKNIAFHHHHHHHHIEGRSPAPPAC CVRRA (SEQ ID NO: 86) where the leader sequence is underlined and represents Cys 7 through Cys 1 5 1 This variant, when purified and refolded, can be treated with the enzyme Factor Xa 1 0 which will cleave after the arginine residue of the sequence IEGR yielding a TPO .i variant of 155 residues in length with a natural serine N-terminal amino acid.

3 T-H8MLF is prepared as described above for variant except a thrombin sensitive sequence IEPR is fused to the N-terminal domain of TPO. The resulting sequence is MKKNIAFHHHHHHHHIEPRSPAPPAC.....CVRRA (SEQ ID NO: 87) where the leader sequence is underlined and and represents Cys 7 through Cysi51. This variant, after purification and refolding can be treated with the enzyme thrombin to generate a natural N-terminal variant of TPO of 155 residues in length.

A Recovery, solubilization and purification of monomeric, biologically active TPO variants (2),and *l All of the variants were expressed in E. coli. The majority of the variants were found in refractile bodies, as observed in Example 22 for TPO (Met- 1 1-153).

Identical procedures for the recovery, solubilization and purification of monomeric TPO variants was achieved as described in Example 22. Identical refolding conditions to those used for TPO (Met 1 1-153) were used with overall yields of After refolding, the TPO variants were purified by C4 reversed phase chromatography in 0.1% TFA utilizing an acetonitrile gradient as described previously. All of the TPO variants (in their unproteolyzed forms) had biological activity as assessed by the Ba/F3 assay, with half-maximal activities of 2-5 pM.

B. Proteolytic processing of Variants and to generate authentic Nterminal TPO (1-155).

TPO variants and above were designed with an enzymaticallycleavable leader peptide before the normal N-terminal amino acid residue of TPO.

After refolding and purification of variants and as described above, each was subjected to digestion with the appropriate enzyme. For each variant, the acetonitrile from the C4 reversed phase step was removed by blowing a gentle stream of nitrogen -133- WO 95/18858 PCT/US94/14553 on the solution. Thereafter the two variants were treated with either Factor Xa or thrombin as described below.

For TPO variant 1 M Tris buffer, pH 8, was added to the acetonitrile-free solution to a final concentration of 50 mM and the pH was adjusted to 8 if necessary.

NaCI and CaCI 2 were added to 0.1 M and 2 mM, respectively. Factor Xa (New England Biolabs) was added to achieve about a 1:25 to 1:100 mole ratio of enzyme to variant.

The sample was incubated at room temperature for 1-2 hr. to achieve maximal cleavage as assessed by a change in migration on SDS gels representing the loss of the leader sequence. Thereafter, the reaction mixture was purified by C4 reversed phase .1 0 chromatography using the same gradient and conditions as described above for the purification of properly folded variants. Uncleaved variant B was separated from cleaved variant by these conditions. The N-terminal amino acids were shown to be SPAPP, indicating that removal of the N-terminal leader sequence was successful.

Factor Xa also generated variable amounts of an internal cleavage within the TPO S1 5 domain; cleavage was observed after the arginine residue at position number 118 generating an additional N-terminal sequence of TTAHKDP.(SEQ ID NO: 88). On nonreducing SDS gels, a single band at approximately 17000 daltons was observed for the Factor Xa cleaved variant; on reducing gels two bands were seen of molecular weight of approximately 12000 and 5000 daltons, consistent with cleavage at arginine 118.

2 0 This observation also confirmed that the two parts of the molecule were held together by a disulfide bond between the 1st and 4th cysteine residues, as deduced from the tryptic digestion experiments described above. In the Ba/F3 biological assay, the purified TPO (1-155) variant, after removal of the N-terminal leader sequence and with the internal cleavage, had a half-maximal activity of 0.2 to 0.3 picomolar. The intact variant with the leader sequence had a half-maximal activity of 2-4 picomolar.

For variant the digestion buffer consisted of 50 mM Tris, pH 8, 2% CHAPS, 0.3 M NaCI, 5 mM EDTA and human or bovine thrombin (Calbiochem) at a 1:25 to 1:50 by weight of enzyme to TPO variant protein. Digestion was conducted at room temperature for 2-6 hours. The progress of the digestion was assessed by SDS gels as described above for the Factor Xa cleavage reaction. Generally, more than cleavage of the leader sequence was achieved in this time. The resultant TPO was purified on C4 reversed phase columns as described above and was shown to have the desired N-terminal by amino acid sequencing. Only very minor amounts of an internal cleavage at the same arginine-threonine bond as observed above with Factor Xa was obtained. The resultant TPO protein had high biological activity with halfmaximal responses in the Ba/F3 assay at 0.2-0.4 picomolar protein. In the mpl receptor based ELISA, this protein had a half-maximal response at 2-4 ng/ml purified protein (120-240 picomolar) while the intact variant containing the leader sequence -134- WO 95/18858 PCT/US94/14553 was less potent in both assays by 5-10 fold. For animal studies, the HPLC-purified cleaved protein was dialyzed into physiological acceptable buffers, with 150 mM NaCI, 0.01% Tween 80 and 10 mM sodium succinate, pH 5.5, or 10 mM sodium acetate, pH or 10 mM sodium phosphate, pH 7.4. By HPLC and SDS gels, the purified protein was stable for several weeks when stored at 4 0 C. In normal and myelosuppressed mice, this purified TPO with the authentic N-terminal sequence was highly active, stimulating the production of platelets at doses as low as 30 ng/mouse.

EXAMPLE 24 Synthetic mpl Ligand Although Human mpl ligand (hML) is usually made using recombinant methods, Sit can also be synthesized via enzymatic ligation of synthetic peptide fragments using methods described below. Synthetic production of hML allows the incorporation of unnatural amino acids or synthetic functionalities .such as polyethylene glycol.

Previously, a mutant of the serine protease subtilisin BPN, subtiligase (S221C/P225A) was engineered to efficiently ligate peptide esters in aqueous solution (Abrahmsen et al., Biochem., 30:4151-4159 [1991]). It has now been shown that synthetic peptides can be enzymatically ligated in a sequential manor to produce enzymatically active long peptides and proteins such as ribonuclease A (Jackson et al., 2 0 Science, [1994]). This technology, described in more detail below, has enabled us to chemically synthesize long proteins that previously could be made only with o: recombinant DNA technology.

A general strategy for hML 1 5 3 synthesis using subtiligase is shown (Scheme Beginning with a fully deprotected peptide corresponding to the C-terminal fragment of the protein, an N-terminal protected, C-terminal activated ester peptide is added along with subtiligase. When the reaction is complete, the product is isolated by reverse phase HPLC and the protecting group is removed from the N-terminus. The next peptide fragment is ligated, deprotected and the process is repeated using successive peptides until full length protein is obtained. The process is similar to solid phase methodology in that an N-terminal protected C-terminal activated petide is ligated to the N-terminus of the preceding peptide and protein is synthesized in a C->N direction. However because each coupling results in addition of up to 50 residues and the products are isolated after each ligation, much longer highly pure proteins can be synthesized in reasonable yields.

-135- WO 95/18858 PCTIUS94/1 4553 Scheme 1. Strategy for Synthesis of hML Using Subtiligase R-NH-Peptide 2 -CO-R'

H

2 N-Peptidel-C0 2 S1) Subtiligase R-NH-Peptide 2 -CO-NH-Peptide 1 C0 2 S2) Zn/CH 3 00 2

H

H

2 N-Peptide 2 -CO-NH-Peptide 1 -002 3) repeat 1 2 *H2N-Pe pti de 3 -CO-N HPeptide 2 .CO.N H.Pept del -002 0 0 R R= kQ0.N H

NH

2 0 (OH 2 4

NH

2 Based on our knowledge of the sequence specificity of the subtiligase as well as the amino acid sequence of the biologically active "epa-domain" of hML, we divided hML1 5 3 into seven fragments 18-25 residues in length. Test ligation tetrapeptides were synthesized to determine suitable ligation junctions for the 18-25mer's. Table 13 shows the results of these test ligations.

TABLE 13 hML Test Ligations. Donor and nucleophile peptides were dissolved at 10 mM in 100 mM tricine (pH 7.8) at 2200. Ligase was added to a final concentration of 10 RM from a 1.6 mg/mL stock (-70 j±M) and the ligation allowed to proceed overnight.

1 5 Yields are based on ligation vs. hydrolysis of the donor peptides.

-136- WO 95/18858 PCTIUS94/14:553 Sit Donor (gIc..K-NH 2 Nucleophile-NH 2 I%Hydrol sis %Ligation_ 1 (23/24) HVLH SRLS 92 08 (SEQ ID NO: 89) (SEQ ID NO: (22/23) SHVL HSRL 48 52 (SEQ ID NO: 91) (SEQ ID NO: 92) 2 (46/47) AVOF SLGE 22 78 ID NO: 93) (SEQ ID NO: 94) 3 (69/70) AVTL LLEG 53 47 (SEQ ID NO: 95) (SEQ ID NO:- 4 (89/90) LSSL LGQL 95 -(SEQ ID NO: 97) (SEQ ID NO: (88/89) C(acm)LSS -LL-GQ 0 0 0 0 (SEQ ID NO: 99) (SEQ ID NO: 100) (90/91) SSLL GQLS 45 ID NO: 101) (SEQ ID NO: 102) (88/89) CLSS LLGQ 90 ID NO: 103) (SEQ ID NO: 100) 5(107/108) LQSL LGTO 99 01 (SEQ IDN N:104) (SEQ ID NO: 105) (106/107) ALQS LLGT 70 (SEQ ID NO: 106) (SEQ ID NO: 107) 6(128/129) NAIF LSFQ 60 (SEQ ID NO: 108) 1 (SEQ ID NO: 109) Based on these experiments, the ligation peptides indicated in Table 14 should be efficiently ligated by the subtiligase. A suitable protecting group for the Nterminus of each donor ester peptide Was needed to prevent seif-ligation. We chose an isonicotinyl (iNOC) protecting group (Veber et J. Org. Chem., 42:3286-3289 f 1977]) because it is water soluble, it can be incorporated at the last stop of solid phase peptide synthesis and it is stable to anhydrous HF used to deprotect and cleave peptides from the solid phase resin. In addition, it can be removed from the peptide 1 0 after each ligation under mild reducing conditions (Zn/CH 3 C0 2 H) to afford a free Nterminus for subsequent ligations. A glycolate-lysyl-amide (glc-K-NH2) ester was used for C-terminal activation based on previous experiments which showed this to be efficiently acylated by subtiligase (Abrahmsen et Biochem., 30:4151-4159 [1991]). The iNOC-protected, glc-K-amide activated peptides can be synthesized -137- WO 95/18858 PCT/US94/14553 using standard solid phase methods as outlined (Scheme The peptides are then seqentially ligated until the full protein is produced and the final product refolded in vitro. Based on homology with EPO, disulfide pairs are believed to be formed between cysteine residues 7 and 151 and between 28 and 85. Oxidation of the disulfides may be accomplished by simply stirring the reduced material under an oxygen atmosphere for several hours. The refolded material can then be purified by HPLC and fractions containing active protein pooled and lyophilized. As an alternative, disulfides can be differentially protected to control sequential oxidation between specific disulfide pairs.

Protection of cysteines 7 and 151 with acetamidomethyl (acm) groups would ensure 10 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 acm protected and oxidized in case sequential oxidation is required for correct folding. Optoinally, Cysteins 28 and Smay be substituted with another natural or unnatural residue other than Cys to insure proper oxidation of cysteins 7 and 151.

TABLE 14.

Peptide Fragments Used For Total Synthesis of h-ML Using Subtiligase Fragment Seauence 1 (SEQ ID NO: 110) iNOC-HN-SPAPPACDLRVLSKLLRDSHVL-glc-K-NH 2 (1-22) 2 (SEQ ID NO: 111) iNOC-HN-HSRLSQCPEVHPLPTPVLLPAVDF-glc-K-NH 2 (23-46) 3 (SEQ ID NO: 112) iNOC-HN-SLGEWKTQMEETKAQDILGAVTL-gic-K-NH2 (47-69) 4 (SEQ ID NO: 113) iNOC-HN-LLEGVMAARGQLGPTCLSSLL-glc-K-NH 2 (70-90) -138- WO 95/18858 PCT/US94/14553 (SEQ ID NO: 114) iNOC-HN-GQLSGQVRLLLGALQS-glc-K-NH 2 (90-106) 6 (SEQ ID NO: 115) iNOC-HN-LLGTQLPPQGRTTAHKDPNAIF-glc-K.NH 2 (107-128) 7 (SEQ ID NO: 116) H2N-LSFQHLLRGKVRFLMLVGGSTLCVR-CO 2 (129-153) SPeptide ligations are carried out at 25 0 C in 100mM tricine, pH 8 (freshly prepared and degassed by vacuum filtration through a 5 pM filter). Typically the C- 1 5 terminal fragment is dissolved in buffer (2-5 mM peptide) and a 10x stock solution of subtiligase (1 mg/ml in 100mM tricine, pH 8) is added to bring the final enzyme concentration to 5gM. A 3-5 molar excess of the glc-K-NH 2 activated donor peptide is then added as a solid, dissolved, and the mixture allowed to stand at 25 0

C.

The ligations are monitored by analytical reverse phase C18 HPLC (CH 3

CN/H

2 0 gradient with 0.1% TFA). The ligation products are purified by preparative HPLC and lyophilized. Isonicotinyl (iNOC) deprotection was performed by stirring HCI activated o zinc dust with the protected peptide in acetic acid. The zinc dust is removed by filtration and the acetic acid evaporated under vacuum. The resulting peptide can be used directly in the next ligation and the process is repeated. Synthetic hML 1 5 3 can be ligated by procedures analogous to those described above to synthetic or recombinant hML 1 5 4 3 3 2 to produce synthetic or semisynthetic full length hML.

Synthetic hML has many advantages over recombinant. Unnatural side chains can be introduced in order to improve potency or specificity. Polymer functionalities such as polyethylene glycol can be incorporated to improve duration of action. For example, polyethylene glycol can be attached to lysine residues of the individual fragments (Table 14) before or after one or more ligation steps have been performed. Protease sensitive peptide bonds can be removed or altered to improve stability in vivo. In addition, heavy atom derivatives can be synthesized to aid in structure determination.

-139- WO 95/18858 PCTrUS94/14553 Scheme 2. Solid Phase Synthesis of Peptide Fragments for Segment Ligation.

U

HN

MBHA

RESI

R

1 0

BOC-NHA

R 0 a b c d (automated synthesis) 2-(PePideNH 0HRN e, f (cleavage) 0 0 0 N OO NH(Peptide) N O NH 2 O R O (CH 2 )4 NH2 Isonicotinyl (iNOC) glycolate-lysyl-amide (glc-K-NH 2 a) Lysyl-paramethylbenzhydrylamine (MBHA) resin 1 (0.63 meq./gm., Advanced ChemTech) is stirred with bromoacetic acid (5 eq.) and diisopropyl carbodiimide eq.) for 1 h. at 25 0 C in dimethylacetamide (DMA) to afford the bromoacetyl derivative 2. b) The resin is washed extensively with DMA and individual Boc-protected amino acids (3 eq., Bachem) are esterified by stirring with sodium bicarbonate (6 eq.) in dimethylformamide (DMF) for 24 h. at 501C to afford the corresponding glycolatephenylalanyl-amide-resin 3. The amino acetylated resin 3 is washed with DMF (3x) and dichloromethane

(CH

2 C1 2 (3x) and can be stored at room temperature for several months. The resin 3 can then be loaded into an automated peptide synthesizer (Applied Biosystems 430A) and the peptides elongated using standard solid phase procedures c) The N-a-Boc group is removed with a solution of 45% trifluoroacetic acid in

CH

2

CI

2 d) Subsequent Boc-protected amino acids (5 eq.) are preactivated using benzotriazol-1 -yl-oxy-tris-(dimethylamino) phosphonium hexafluorophosphate (BOP, 4 eq.) and N-methylmorpholine (NMM, 10 eq.) in DMA and coupled for 1-2 h.

e) The final N-a-Boc group is removed (TFA/CH 2

CI

2 to afford 4 and the isonicotinyl (iNOC) protecting group is introduced as described previously via stirring with of -140- 4-isonicotinyl-2-4-dinitrophenyl carbonate 3 eq.) and NMM (6 e) in DMA at C for_24 h. f) Cleavage and deprotecion of the peptide via treatnien with anhydrous HF anisole/ 5, ethylmethyl suLfid) at 0 C for 1 hr. affords the iN0Cprotected, glycolate-lys-amide activated peptide 5 which is purified by reverse phase C18 HPLC (CH 3

CN/H

2 0 gradient, 0.1% TFA). The identity of all substrates is confirmed by mass spectrometry.

SUPPLEMEN-AL

ENABLEMENT

The invention as claimed is enabled in accordance with the above specification 1 0 and readily available references and starting materials. Never t t heless A p plicants hve Sdeposited with the American ype Culture Collection, Rockville, Md., USA (ATCC) the cell line listed below: Escherichia coli, DH1OB.pBSK-hmp/l 1.8, ATCC accession no. CRL 69575 deposited February 24, 1994.

5 Plasmid, pSVI5.ID.LL.MLORF ATCC accession no. CRL 75958, deposited December 2, 1994; and CHO DP-12 cells, ML 1/50 MC8 (labeled #1594), ATCC accession no. CRL 11770, deposited December 6, 1994.

These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture for 30 years from date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicants and ATCC which assures unrestricted availability upon 2 5 issuance of the pertinent U.S. patent. Availability of the deposited strain is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

While the invention has necessarily been described in conjunction with preferred enbodiments and specific working examples, one of ordinary skill, after reading the foregoing specification, will be able to. effect various changes, substitutions of equivalents, and alterations to the subject matter set forth herein, without departing from the spirit and scope thereof. Hence, the invention can be practiced in ways other than those specifically described herein. It is therefore 3 5 intended that the protection granted by letters patent hereon be limited only by the appended claims and equivalents thereof.

All references cited herein are hereby expressly incorporated by reference.

1 4 1 142 SEQUENCE

LISTING

GENERAL INFORMATION: APPLICANT: Eaton, Dan L.

de Sauvage, Frederic J.

(ii) TITLE OF INVENTION:

THROMBOPOIETIN

(iii) NUMBER OF SEQUENCES: 144 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Genentech, Inc.

STREET: 460 Point San Bruno Blvd CITY: South San Francisco STATE: California COUNTRY: USA ZIP: 94080 COMPUTER READABLE

FORM:

MEDIUM TYPE: 3.5 inch, 1.44 Mb floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: WinPatin (Genentech) (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: 08/374540 FILING DATE: 18-Jan-1995

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: PCT/US94/14553 FILING DATE: 28-DEC-1994 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: 08/249376 FILING DATE: 25-MAY-1994 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: 08/223263 FILING DATE: 04-APR-1994 (vii) PRIOR APPLICATION

DATA:

:1 APPLICATION NUMBER: 08/196689 FILING DATE: 15-FEB-1994 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/348658 FILING DATE: 02-DEC-1994 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/185607 FILING DATE: 21-JAN-1994 (vii) PRIOR APPLICATION DATA: 143 APPLICATION NUMBER: 08/348657 FILING DATE: 02-DEC-1994 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: 08/176553 FILING DATE: 03-JAN-1994 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Winter, Daryl B.

REGISTRATION NUMBER: 32,637 REFERENCE/DOCKET NUMBER: P0871P5 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 415/225-1249 TELEFAX: 415/952-9881 TELEX: 910/371-7168 INFORMATION FOR SEQ ID NO:1: SEQUENCE

CHARACTERISTICS:

LENGTH: 353 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu -21 -20 -15 Ala Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys -5 1 o..

Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu 10 15 Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr S 25 30 Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys 45 Met Glu Glu Thr Lys Ala Gin Asp Ile Leu Gly Ala Val 60 Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly 70 75 Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gin Val S. 85 90 Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 i05 110 Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile 115 120 125 Leu Thr Asp Leu His Ser.

Pro Val Thr Gin Thr Leu Pro Thr Arg Leu Pro Pro Phe Leu 144 Arg Gly Lys Ser Phe Gin His Leu Leu 130 135 Val. Arg Phe Leu Met Leu 140 Val 145 Ala 160 Gay Val Gly Pro Ser Ser Thr Arg Leu 150 165 9.

S 9 I. 4999 Pro Asn 175 Ala Arg 190 Arg Ala 205 Asp Gin 220 Gly Thr 235 Ala Pro 250 Pro Asn 265 Thr Gly 280 Arg Thr Lys Ile Arg Asp Leu aln *Thr Thr Ile Pro Gly Ile Gin Tyr Ser Gly Pro Gly Leu Ser Pro Gly 180 Ser 195 Gly 210 Tyr 225 Phe 240 Ser 255 Gly 270 Cys Ser Leu Gly Leu Leu Pro Gly Tyr Val Leu Leu Leu Leu Asn Gly Thr Ser *Arg Val Giu Leu Asri Arg Pro Ser Pro Arc Let.

Thr Lys Gin Ile Ser Asp Ser ;Ala 155 Thr 170 Asn 185 Trp 200 Thr His 230 Arg 245 Thr 260 Pro 275 Pro Pro Leu Asn Phe Thr Gin Gin Ser Arg Giu Leu Arg Thr Gly Ser Thr His Th2 Gilt Ala Gly Ser Leu Leu Leu Pro -Thr ILeu Ser Phe Leu Asn Gly Pro Pro Thr I'hr Leu Phe Pro Leu Pro Pro Thr Leu Pro 285 290 Pro Val Val. Gin Leu His Pro Leu Leu Pro Asp Pro Ser Ala *295 300 305 :'.Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr Ser Tyr Thr 310 315 320 Ser Gin Asn Leu Ser Gin Giu Giy 325 330 332 INFORMATION FOR SEQ ID NO:2: SEQUENCE

CHARACTERISTICS:

LENGTH: 1795 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: :TCTTCCTACC CATCTGCTCC CCAGAGGGCT GCCTGCTGTG CACTTGGGTC P ro lis 145 CTGGAGCCCT TCTCCACCCG GATAGATTCC TCACCCTTGG CCCGCCTTTG 100 CCCCACCCTA CTCTGCCCAG AAGTGCAAGA GCCTAAGCCG CCTCCATGGC 150 CCCAGGAAGG ATTCAGGGGA GAGGCCCCAA ACAGGGAGCC ACGCCAGCCA 200 GACACCCCGG

CCAGA

ATG GAG CTG ACT GAA TTG Met Giu Leu Thr Glu Leu -22. -20 CTC CTC 239 Leu Leu GTG GTC ATG CTT CTC CTA ACT GCA AGG CTA ACG CTG TCC 278 Val Val. Met .Leu Leu Leu Thr Ala Arg Leu Thr Leu Ser AGC CCG GCT CCT CCT GCT Ser Pro Ala Pro Pro Ala 1 5 TGT GAC CTC Cys Asp Leu CGA GTC CTC AGT Arg Val. Leu Ser 317 AAA CTG CTT Lys Leu Leu is AGC CAG TGC Ser Gin Cys CGT GAC TCC Arg Asp Ser CAT GTC His Val.

20 CTT CAC AGC Leu His Ser AGA CTG 356 Arg' Leu CCA GAG Pro Glu GTT CAC CCT Val His Pro

TTG

Leu CCT ACA CCT GTC 395 Pro Thr Pro Val- GGA GAA TGG AAA 434 Gly Giu Trp, Lys CTG CTG CCT GCT GTG Leu Leu Pro Ala Val

GAC

Asp 45 TTT AGC TTG Phe Ser Leu *coo 04 9 I000 ACC CAG ATG GAG Thr Gin Met Glu 55 GCA GTG ACC.CTT Ala Val. Thr Leu GAG ACC AAG Glu Thr Lys

GCA

Ala 60 CAG GAC ATT Gin Asp Ie CTG GGA 473 Leu Gly

CTG

Leu CTG GAG GGA GTG ATG GCA GCA CGG 512 Leu Glu Gly Val. Met Ala Ala Arg GGA CAA Gly Gin CTG GGA Leu Gly CCC ACT TGC Pro Thr Cys 85 CTC TCA TCC CTC CTG GGG 551 Leu Ser Ser Leu Leu Gly 4*

'S

*4* 4s CAG CTT TCT GGA Gin Leu Ser Gly 95 CAG GTC CGT CTC CTC CTT GGG GCC CTG Gin Val Arg Leu Leu Leu Gly Ala Leu 100 590 *CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG 629 Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg 11,1 ACC ACA GCT CAC AAG GAT CCC AAT CCC ATC TTC CTG AGC 668 Thr Thr Ala His Lys Asp Pro Asn 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 Arg Gly Lys Val. Arg Phe Leu Met 135 140 CTT GTA GGA GGG TCC ACC CTC TOC GTC AGG CGG GCC CCA 746 Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro 145 150 155 CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC 785 Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu 160 165 ACA CTG AAC Thr Leu Asn 170 GAG ACA AAC Glu Thr Asn 185 GAG CTC CCA AAC Glu Leu Pro Asn 175 TTC ACT GCC TCA Phe Thr Ala Ser AGG ACT TCT Arg Thr Ser GGA TTG TTG 824 Gly Leu Leu 180

GCC

Ala 190 AGA ACT ACT GGC Arg Thr Thr Gly

TCT

Ser 195 863 902 GGG CTT Gly Leu CCT GGT Pro Gly 210 CTG AAG TGG CAG Leu Lys Trp Gin 200 CTG CTG AAC CAA Leu Leu Asn Gin CAG GGA TTC Gin Gly Phe

AGA

Arg 205 GCC AAG ATT Ala Lys Ile

ACC

Thr 215 TCC AGG TCC CTG Ser Arg Ser Leu GAC CAA 941 Asp Gin 220 .9 9 0990 0*Se *.S4 .)h4*

I

~4 9 9 1

S

9

ATC

Ile

GGA

Gly 235 CCC GGA TAC CTG Pro Gly Tyr Leu 225 ACT CGT GGA CTC Thr Arg Gly Leu AAC AGG ATA Asn Arg Ile

CAC

His 230 GAA CTC TTG AAT Glu Leu Leu Asn 980

TTT

Phe 240 CCT GGA CCC TCA Pro Gly Pro Ser CGC AGG ACC 1019 Arg Arg Thr 245 CTA GGA GCC CCG Leu Gly Ala Pro 250 GGC TCC CTG CCA Gly Ser Leu Pro GAC ATT TCC Asp Ile Ser TCA GGA Ser Gly 255 ACA TCA GAC ACA 1058 Thr Ser Asp Thr 260 CCC AAC Pro Asn 265 CTC CAG CCT Leu Gin Pro

GGA

Gly 270 TAT TCT CCT 1097 Tyr Ser Pro .4

S

9@* S S 5' 0 TCC CCA ACC Ser Pro Thr 275 CAT CCT CCT His Pro Pro

ACT

Thr 280 GGA CAG TAT ACG Gly Gin Tyr Thr CTC TTC 1136 Leu Phe 285 of* CCT 4Pro CTT CCA CCC Leu Pro Pro 290 ACC TTG CCC Thr Leu Pro ACC CCT GTG Thr Pro Val 295 GTC CAG CTC Val Gin Leu 1175 CAC CCC CTG CTT *His Pro Leu Leu 300 CCT GAC Pro Asp 305 CCT TCT GCT CCA Pro Ser Ala Pro ACG CCC ACC 1214 Thr Pro Thr 310 147 CCT ACC AGC CCT CTT CTA AAC ACA TCC TAC ACC CAC TCC 1253 Pro Thr Ser Pro Leu Leu Asri Thr Ser Tyr Thr His Ser 315 320 325: C CAG AAT CTG TCT CAG GAA GGG T AAGGT TCTCAGACAC 1290 Gin Asn Leu Ser Gin Glu GJly 330 332 TGCCGACATC AGCATTGTCT CATGTACAGC TCCCTTCCCT GCAGGGCGCC 1340 CCTGGGAGAC AACTGGACAA GATTTCCTAC TTTCTCCTGA AACCCAAAGC 1390 CCTGGTAAA GGGATACACA GGACTGAAAA GGGA.ATCATT TTTCACTGTA 1440 CATTATAA.AC 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 AAAAAA 1790 AAAAA 1795 INFORMATION FOR SEQ ID NO:3: (j)SEQUENCE

CHARACTERISTICS:

ma' LENGTH: 42 amino acids TYPE: Amino Acid 0 TOPOLOGY: Linear e.I (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala Arg Leu Thr Leu -16 -15 -10 Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys .l5 *Le u Leu Arg Asp Ser His Val Leu His Ser Arg Leu 1520 25 26 INFORMATION FOR SEQ ID NO:4: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 390 base pairs TYPE: Nucleic Acid a. STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GAATTCCTGG AATACCAGCT GACAATGATT TCCTCCTCAT CTTTCAACCT CACCTCTCCT CATCTAAGAA TTG CTC CTC GTG GTC ATG CTT 91 Leu Leu Leu Val. Val. Met Leu -16 -15 CTC CTA ACT GCA AGG CTA ACG CTG TCC AGC CCG GCT CCT 130 Leu LeU Thr Ala Arg Leu Thr Leu Ser Ser Pro Ala Pro 1 CCT GCT TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT 169 Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg 10 GAC TCC CAT GTC CTT CAC AGC AGA CTS GTGA GAACTCCCAA 21.0 Asp Ser His Val Leu His Ser Arg Leu 25 26 CATTATCCCC TTTATCCGCG TAACTGGTAA GACACCCATA CTCCCAGGAA 260 GACACCATCA CTTCCTCTAA CTCCTTGACC CAATGACTAT TCTTCCCATA 310 TTGTCCCCAC CTACTGATCA CACTCTCTGA CAAGAATTAT TCTTCACAAT 360 ACAGCCCGCA TTTAAAAGCT CTCGTCTAGA 390 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 390 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID 4* 4 S. 4* 4** 4 *4 4 4 4 *444* *TCTAGACGAG AGCTTTTAAA

TGCGGGCTGT

TCAGAGAGTG TGATCAGTAG

GTGGGGACAA

GGTCAAGGAG TTAGAGGAAG

TGATGGTGTC

seTTACCAGTTA CGCGGATAAA

GGGGATAATG

GCTGTGAAGG ACATGGGAGT

CACGAAGCAG

::.CACAAGCAGG AGGAGCCGGG

CTGGACAGCG

AGCATGACCA CGAGGAGCAA

TTCTTAGATG

.ATGAGGAGGA AATCATTGTC

AGCTGGTATT

ATTGTGAAGA

ATAATTCTTG

TATGGGAAGA

ATAGTCATTG

TTCCTGGGAG

TATGGGTGTC

TTGGGAGTTC

TCACCAGTCT

TTTACTGAGG

ACTCGGAGGT

TTAGCCTTGC

AGTTAGGAGA

AGGAGAGGTG

AGGTTGAAAG

CCAGGAATTC 390 so 100 150 200 250 300 350 149 INFORMA~TION FOR SEQ ID NO:6: SEQUENCE

CHARACTERISTICS:

LENGTk-: 332 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:' Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu 1 5 10 Leu Arg Asp Ser His Val Leu His Ser Ary Leu Ser Gln Cys Pro 25 Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp 40 Phe Ser Leu Gly Giu Trp Lys Thr Gin Met Glu Giu Thr Lys Ala 55 Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met 70 Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser teu Leu 85 Giy Gin Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gin 100 105 Ser Leu Leu Giy Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala 110 115 120O *:His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu ***125 130 135 *Arg Gly Lys Val Arg Phe Leu Met Leu Vai Gly Gly Ser Thr Leu 140 145 150 ::Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr 155 160 165 Ser Leu Vai Leu Thr Leu Asn Giu Leu Pro Asn Arg Thr Ser Gly 170 175 180 Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser 185 190 195 Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile Pro Gly ~*200 205 210 Leu Leu Asn Gin Thr Ser Arg Ser Leu Asp Gln Ile Pro Giy Tyr 215 220 225 :Leu Asn Arg Ile His Giu Leu Leu Asn Gly Thr Arg Gly Leu Phe 230 235 240 99* Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Se 245 250 Gly Thr Ser Asp Thr Gay Ser Leu*Pro Pro Asn Leu Gin Pro Gi 260 265 27 Tyr Ser Pro Se r Pro Thr His Pro Pro Thr Gay Gin Tyr Thr Le 275 280 28 Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gin Leu Hi 290 295 30 Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Se: 305 32.0 31~ Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gi: 320 325 33( Glu Gly 332 INFORMATION FOR SEQ ID NO:7: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 166 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr 1 5 10 Leu Leu Glu Ala Lys Giu Ala Glu Asn Ile Thr Thr Gly Cys Ala 25 *Glu His Cys Ser Leu Asn Giu Asn Ilie Thr Val Pro Asp Thr Lys *935 Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gay Gin Gin Ala 55 Val Glu Val Trp Gin Gly L eu Ala Leu Leu Ser Giu Ala Val Leu S.:Arg Gay Gin Ala Leu Leu Val Asn Ser Ser Gin Pro Trp, Glu Pro 809 85 *Leu Gin Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu 100 105 Thr Thr Leu Leu Arg Ala Leu Gay Ala Gin Lys Glu Ala Ile Ser 110 115 120 y '0

U

0 r 151 Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala 125 130 135 Asp Thr Phe Arg Lys Leu Phe Arg Val. Tyr Ser Asn Phe Leu Arg 140 145 150 Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 155 160 165 Arg 166 INFORMATION FOR SEQ ID NO:8: SEQUENCE

CHARACTERISTICS:

LENGTH: 328 amino acids TYPE: Amino Acid TOPOLOGY: Linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val. Leu Ser Lys Leu 1 5 10 Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro 25 Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp 3 5 4 04 Phe Ser Leu Gly Glu Trp Lys Thr Gin Met GJlu Glu Thr Lys Ala 55 6 Gin Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met 70 7 *.Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu 85 99s: Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin 100 105 Ser Leu Leu Gly Thr Gin Gly Arg Thr Thr Ala His Lys Asp Pro 110 115 120 *Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val 125 130 135 Arg Phe Leu Met Leu Val. Gly Gly Ser Thr Leu Cys Val Arg Arg 140 145 150 Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Va. Leu 155 160 165 *.:Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr 152 170 Ser Asn Phe Thr Ala Ala Arg Thr 175 Thr Gly Ser 190 Gly Leu Leu 180 Ly s 195 185 Trp Gin Thr Ser His Giu Arg Arg Thr Gly Pro Thr Pro Thr Asp Pro Gin Arg Leu Thr Ser His Leu Ser Gly Ser Leu Leu Leu Pro Pro Ala Phe 200 Leu 215 Asn 230 Gly 245 Pro 260 Pro 275 Thr 290 Pro 305 Arg Ala Asp Gin Gly Thr Ala Pro Pro Asn Thr Gly Pro Val Thr Pro Lys Ile Arg Asp Leu Gin Vai Thr Ile Pro Gly Ile Gin Tyr Gin Pro Pro 205 Gly 220 Leu 235 Ser 250 Pro 265 Thr 280 Leu 295 Thr 310 Gly Tyr Phe Ser Gly Leu His Ser Leu Leu Pro Gly Tyr Phe Pro Pro Leu Asn Gly Thr Ser Pro Leu Leu Asn Gin 210 Arg Ile 225 Pro Ser 240 Ser Asp 255 Pro Ser 270 Leu Pro 285 Leu Pro 300 Leu Asn 315 @6 6 6 6666 6666 6666 66.** 6 *6 6 6

S

Thr Ser Tyr Thr His Ser Gin Asn L 320 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 265 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ IJ Ser Pro Aia Pro Pro Ala Cys Asp L~ 1 5 Leu Arg Asp Ser His Val Leu His S~ 20 Giu Val His Pro Leu Pro Thr Pro Vz Phe Ser Leu Gly Glu Trp Lys Thr G1 Gin Asp Ile Leu Gly Ala Vai Thr Le 65 eu Ser Gin Glu Gly 325 328 66 6 6.@9 @6 *6 6 66 6 6 66. 6 DNO: 9: u Arg Val 10 r Arg Leu 25 1i Leu Leu 40 .n Met Giu 55 ~u Leu Leu 70 Leu Ser Pro Glu Glu Ser Gin Ala Thr Gly Lys Cys Val Lys Val Leu Pro Asp Ala Met 6 6* 66 6 153 Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu 85 Gly Gin Leu Ser Gly Gin Val. Arg Leu Leu Leu Gly Ala Leu Gin 100 105 Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala 110 1115 120 His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His LeU Leu 125 130 135 Arg Gly Lys Asp Phe Trp Ile Vai Giy Asp Lys Leu His Cys Leu 140 145 150 Ser Gin Asn Tyr Trp Leu Trp Ala Ser Giu Val Ala Ala Gly Ile 155 160 165 Gin Ser Gin Asp Ser Trp, Ser Ala Giu Pro Asn Leu Gin Val Pro 170 175 180 Giy Pro Asn Pro Arg Ile Pro Giu Gin Asp Thr Arg Thr Leu Glu 185 190 195 Trp Asn Ser Trp Thr Leu Ser Trp Thr Leu Thr Gin Asp Pro Arg 200 205 210 Ser Pro Gly His Phe Leu Arg Asn Ile Arg His Arg Leu Pro Ala *215 220 225 Thr Gin Pro Pro Ala Trp, Ile Phe Ser Phe Pro Asn Pro Ser Ser 230 235 240, **.Tyr Trp Thr Val Tyr Ala Leu Pro Ser Ser Thr His Leu Ala His 245 250 255 Pro Cys Gly Pro Ala Pro Pro Pro Ala Ser .:260 265 INFORMATION FOR SEQ ID NO:i0: SEQUENCE

CHARACTERISTICS:

LENGTH: 261 amino acids TYPE: Amino Acid TOPOLOGY: Linear

V,

(xi) SEQUENCE DESCRIPTION: SEQ ID Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu 1 5 10 Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro 25 Giu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp 154 Phe Ser Leu Gly Giu Trp Lys Thr Gin Asp Ile Leu Gly Ala Val Thr Ala Ala Arg Gly Gln Leu Gly Pro Gly Gin Leu Ser Gly Gin Val Arg Ser Leu Leu Gly Thr Gin Gly Arg 110 Asn Ala Ile Phe Leu Ser Phe Gin 125 Phe Trp Ile Val Gly Asp Lys Leu 140 Trp Leu Trp Ala Ser Glu Val Ala 155 Ser Trp Ser Ala Giu Pro Asn Leu 170 Arg Ile Pro Glu Gin Asp Thr Arg 185 Thr Leu Ser Trp Thr Leu Thr Gin 200 Phe Leu Arg Asn Ile Arg His Arg 1 215 Ala Trp Ile Phe Ser Phe Pro Asn E 230 Tyr.Ala Leu Pro Ser Ser Thr His L 245 Ala Pro Pro Pro Ala Ser 260 261 INFORMATION FOR SEQ ID NO:11: SEQUJENCE

CHARACTERISTICS:

LENGTH: 7849 ba 'se pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear 40 *Gin Met Giu Giu Thr Lys 55 Leu Leu Leu Giu Gly Val 70 Thr Cys Leu Ser Ser Leu 85 Leu Leu Leu Gly Ala Leu 100 Thr Thr Ala His Lys Asp 115 His Leu Leu Arg Gly Lys 130 His Cys Leu Ser Gin Asn 145 Ala Gly Ile Gin Ser Gin 160 Gin Val Pro Gly Pro Asn 175 rhr Leu Glu Trp Asn Ser 190 %sp Pro Arg Ser Pro Gly 1 2052 jeu Pro Ala Thr Gin Pro P 220 2 'ro Ser Ser Tyr Trp Thr V 235 2 ~eu Ala His Pro Cys Gly P 250 2 Ala Met Leu Gin 105 Pro 120 Asp 135 Tyr 150 Asp 165 Pro 180 rrp is 'ro 'al ro 4.

44*4 4444 4**4*4 4 4 .4 9 444 4 44.44.

4 .4 4.44 9* .4 4 4.

4. 0 9*4 44~* 4 4*

RA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

CCCAGCCTCC

GTCTCTCCCT

TCCTTCCTAA

TTTCTCTTGT

CCCACACACA

TCTTGGGAGA

GGATCTAGGC

CACACTTCTC

ACAGGAGAG

AGGTCAGGC

TGGGAAGAA

AGCAGGGAG

GGGACACATi ATGGAAGA

C

TGTGTGTGT)

CTCAGGCTT2

GACAGTCCAC

ACAGGAGAT'I

GGGAATGACP

AAGCAAG

CAT

GAAATTTGGA

TCTGTGCTTC

AAAAAACTTC

GGAAAAGATG

GAGGAGTGGA

CTTCCGGGGG

GAAGTGGCCC

CACCGCCACA

TGCACTTGGG

A GCCTGAGGAA C AGGAAGCCCC A GCAA.AGGAGC G TATTTGCGGG 3 GGCCTGGTTA 3 GAGACAGAAC

~GCCATCCAAG

SACCCAGTGCA

TCAACCCGTC

TCTCTCATGT

AGATAGGACT

CCTGTTGGAT

TAGCCAGGGA

TTCCCCAGCAI

TGCTCCTGTC

C

GATCCCCCTA

I

CCCTGGTCCA

G

CCTTCACCAG

T

AGGCAGGCGT

A

TGTCTTCCTA

C

TCCTGGAGCC

C

TCCCTGGTCA

CCCACTATCC

CATCTCGTCT

AGCAGACATG

GTTCTGGGG

TGAGGACAGA

TAGAGCCAGG

GGAGGTCCAG

TTCCTCTTGT

AAGCAAAGGA

CCACTGGACC

-GTGTGCGCA

2AAACCCTTT

GGCAATATC

'CCTAGGGGA

'TCAGCAGCA

;TGAAAACCCC

LCACAAATGT

C

'CCCTCCAGG 2 CCAAATCTT

C

GCAGGGGCT

C

GTCTGGTGG

C

TGACCTGCT

G

:CATCTGCT

C

rTCTCCACC Ci TGCCTGCCTC CCTGTCTCCT

TCCCAGCTAT

GGCTGGACGG

CCCATCCTTG

ACAGGGGGAT

GACTGTGGGG

GCCAAAGGAA

CAGCTGTCTT

CACATGTGGA

GGGCCCTGGG

CCAGCAGACG

CATACATGTG

CCCCATAACA

CGTGTTCCCA

rTACAGAAAG

GTATGATGT

:ACCAATCTT2 ~CTGCCAGAT

I

CCCAGGTTG C TCCGTGGTG

T

CAGGGAAGA

G

TCCCTTCTC

T

CTGTGGAGG G CCCAGAGGG

C

GGTGAGTGG C CCCTACACCC 100 GAAAATTCCA 150 GGGAGGAGGA 200 GATGGGATCA 250 AGACTGGGAC 300 AAGGGGGGCC 350 TCCTAAGACA 400 ACGGTAGGAG 450 CACAGAGGTC 500 AGCACCTAAG 550 CCCCGCACCT 600 CCAACCCATA 650 CTTCGAAAGG 700 kAAAGCAGGA 750 :CAGGGAAAA 800 kAACAAGACC 850 'CCTCCTGGA 900 CCATGTCCA 950 'GTGTGGGTG 1000 ~AAGGCGTCA 1050 'GATTGGGCA 1100 GCTGTGCCC 1150 TGCCTGCTG 1200 CAGCAGGGT 1250 4.

4 4 .4 4 44***4 4 .4 4 4400 GTGGGGTTAT GTGAGGGTAG AAAGGACAGC AAAGAGAAAT GGGCTCCCAG 1300 eel:% 4

CTGGGGGA

GAAGAGTG

GGGGTGGG

AG CA CAG C

AATAAGAG

GACACAGC(

GTTCTGAA]

GAGAGGGT~z TCCCAATA7

GAGGAAGCC

AGGGGGCAG

CCTAAGACA

TCCTGGGCAi

CTTGGCCC.

TCCAGTGGC?

TCTCCCATAC

TTGTGCAGT7

CAACATATTG

TTTCCACTCT

CCATCCCAGA

GCTAATTAAG

GGGTAGGTGG

AAGGAGACAC

TTTTTTTTGA

GTGCGATCTC

TCCTGTCTCA

CCCTGCTAGT

GAGGCTGGTG

.GG GGCAGGCA.

TA GCCTTCC.

GT GCTGGTTT( C TGGCTGGC( kG GGAGCTGC; :C CAATCCcCC Tc TGGTGCTGG LA TGGGAGGAG 'T GAATCAGGT 'A TGTGGGGGG G GAGGCAGGTi A GGTAAGCCC( "a CTGGTCTCA( k. GATAATGGAC P TTATGTGTGC

CGCCTTTTAP

CCCCTCCCCA

CGCCCGTTTG

CTTCTCCAGG

TTTTCTCTGA

ATATGTGTGT

CACCAAATCC

CATATGCTTT

GACGGAGTTT

GGCTCACCAC

GCCTCCCAAG

TTTTTTGTAT

GCGAACTCCT

k.A CTGGAACCTA %G AATGGGAGGA T GAGGGACTGA 'C TAAGGAAAGG LC TTAGGGCTTA ,T TCTTAGCTGG G GAAGCTGGGC G GCCCACTCAT G CAAGCCTCTT T CCTGTGAAGG 3 TGAGCTATGA TAA.GGTGGGC2 GAAGGAAGTC

C

;TATTTCAGGA

C

GGGTAGATAG

G

LTCCTGACCTCT

GCCCCACTCC

C

CCAGTTCCTC

A

CTGAAGCCAC

A

CCTAACAACC

X

ATACATATCA

T(

GTGTCCGATT C2 CTTGCTTTCT

TI

CACTCTTATT

GC

AAACCTCCGC

CT

TAGCTTGGAT

TA

TTCGTAGAGC

CG

GACCTCAGGT GA

CAGGCACTGA

GCAGGGCAGA

TCACTTACTT

GGACATGAGC

GCAAACACAG

TCATTCCTCG

CAGGCAAGCC

GTTGACAGAC

TGCACAACTT

kLACCGGAAGG

.,ACAGATATG

k~TCACCCAGC

I

~CAGAACTGT

TTGGAGTCC

~GAAAGAATAG

'AGTGGTCCC

A

CACCGCAGA

A

CCCAGGCCC

T

~TACTTTCC

T'

~GGTTGCTC

AC

3TCCTGCTG

C~

CTGAGGAG

TC

'CTTTCTTT Cl ~CCAGGCTG

G.A

'CCCAGGTA CA CAGGCATG

AG

GGGTTTCA

CC

TCCACCCG

CC

CCTTTGTCGA 1350 GCAGGGGTAG 1400 GGTGGAATAC 1450 CCAGGGAGAA 1500 TAGTAAGATG 1550 TTAGCTTAAG 1600 AGGGCGCAAG 1650 CTACAGGAAA 1700 GTGAAAGGAG 1750 .7GTTCTGCCA 1800 L'TAGTGGGCG 1850 ~GGTGCCCGT 1900 .'AGCCCATCT 1950 LAAGAAAAGC 2000 'AGGTTAATT 2050 GTTACAGCT 2100 GTTACCCCT 2150 GCATCCCAT 2200 TCTCTATCC 2250 GAATTTAAG 2300 LCTCAGCAG 2350 CTGACAAA 2400 TTCTTTTT 2450 ~GTGCAATG 2500 AGCGATTC 2550 CCACCACA 2600 ATGTTAGT 2650 TTGGACTC 2700

C.

C S *5*

C..

C

*CC..

S.

C S

C

See..

S

C.

e.g Ce .e S SC S

S

S.C.

we S.

'I

i

CCAAAGTGCT

GCTTTCATCA

GCTGGTCAG

C

GTCTCTTCCT

GGGATTACAG

CAAGAAAATG

ATCTCAAGCC

AGAAACTTGG

GATAGATTCC

TCACCCTTGG

0* S

S

Sea.

S

.5*05 *0 5 9.O 0

S.

C

044 *550 a a a

S.

S

*05* S S

AAGTGCAAG

GAGGCCCCA

GCTGACTGG

CAGAAGGGG,

GAA.CCCATTI

TCAGGTCTG(

TTCCTCCTC)

GTGGTCATGC

TCCTGCTTGI

TCCTTCACAC

GTAACTGGTA

ACTCCTTGAC

ACACTCTCTG

TCTCGTCTAG

CATAGCTCTC

CAGAGCCAGT

TGCTGTGGAC

CATCCCTAAC

CCCATGGATT

GCTTGGCCAC

ATAAGATGAT

ACCATGAAAA

CCYGTTCAGT

kGCCTAAGCCG k. ACAGGGAGCC r GAGAACACAC k GAGAGAAAGG

TCCCAAAAAT

GTCCTGAATG

TCTTTCAACC

TTCTCCTAAC

GACCTCCGAG

CAGACTGGTG

AGACACCCAT.

CCAATGACTA

ACAAGAATTA

AGATAGTACT

TCTATTTCAGC

GCCCAGAGGT

IJ

TTTAGCTTGG

C

CTTGGCTTCC

C

CTCCAACATT

C

157

GCATGAGCCA

TGAGAGAATT

CT CCC CAG CA

TTAAATGTTC

CCCGCCTTTG

CCTCCATGGC

ACGCCAGCCA

CTGAGGGGCT

AGACACGCTG

AAGGGGTCTG

GGAATTCCTG

TCACCTCTCC

TGCAAGGCTA

rCCTCAGTAA2 kGAACTCCCA

I

kCTCCCAGGA

;Z

CTCTTCCCAT

P~

'TCTTCACAA

.ATGGAGGACT

'TCCCTTCTC C 'CACCCTTTG

C

*AGAATGGAAA

TAAGTCCTG

T

TTGAGCTTT T

CTGCACCCGG

CAGGGCTTTG

TCTGTTCACC

ACTCTTCTTG

CCCCACCCTA

CCCAGGAAGG

GACACCCCGG

AGGGCCATAT

CAGGGGGCAG

AGGGGTGGAT

GAATACCAGC

rCATCTAAGA, k.CGCTGTCCA k~CTGCTTCGT

~CATTATCCC

LGACACCATC2

LTTGTCCCCAC

'ACAGCCCGC

'AGCCTGCTT 'CCCCACCAA T CTACACCTG T1 ACCCAGATG G CTTCAGTTT C TAAAAATAT C CTATGATGA T A.ATAGATTT G.

3CCATGCCT T kGCAAGACT C CACACCATAT 2750 GCAGTTCCAG 2800 CTGCCAGGCA 2850 CTACTTTCAG 2900 CTCTGCCCAG 2950 ATTCAGGGGA 3000 CCAGAATGGA 3050 GGAPACATGA 3100 GAAGCTGGGG 3150 TCCCTGGGTT 3200 TGACAATGAT 3250 ATTGCTCCTC 3300 GCCCGGCT6C 3350 GACTCCCATG 3400 :TTTATCCGC 3450 %CTTCCTCTA 3500 'CTACTGATC 3550' LTTTAAAAGC 3600 LTTAGGCTAC 3650 CTTTTTCAA 3700 'CCTGCTGCC 3750 TAAGAAAGC 3800 CCACTGCTT 3850 TCACCTTCA 3900 AGCCTGTGG 3950 AAGCTGAAC 4000 TGACCTATT 4050 kTATGTCAT 4100 CCTAACCC.A

TCTACATTCA

GGCTTGCAGG

TCCAATATGT

GCTGGAGAGA

CTTCTTAAAT

AATCGCTCAT

TGGCATGAAG

RK

I)'

(3 158 CCACAGATGA CACAAAGCTG GGAAGTACCA CTAAAATAAC AAAAGACTGA 4150 ATCAAGATTC

AAATCACTG

AGAGATATA

ACTTCTACA

GCACTTTGGG

AGGCCGAGG

AGCAGCCTGG

CCAACATGGI

TAGCCGGGCA

TGGTAGTGC

AAGCAGGAGA

ATCCCTTGAJ

ATCATGCCA6

TGCACTCCAC

AAAAGAAAAA

AAAATTCTA(

CAGCTTTCAG

GCCACAATGC

AGCACTTCCT

ACGAAAAGG;

CCATGTAACA

TTACTGAAGC

GTTTGATGTT

TAGCATCCCC

CCGAGTGGAC

TACACTTAAA

GAAGACATAT

GCTAATTTAT

CTAGAAAGCA

GCAGCCTGAA

:'.CAATAGTTTA

AAAAACTAAA

:TTCCTTCAGG

ACTGAGTCAG

:~:TAGTCCTTTC

TTTTCATCCT

*'*CATTTAGTTT

ATTTATTATT

GTGC-AGTGGC

CCGGATTCAA

GCGATTCTCC

AGGTGCCCAC

CACCATGCCC

GGTTTCACCA

TGTTGGCCAG

CCACCTGCCT

CAGCCTCCCA

qCACCCAGCCT

TCATTCAGTT

CAGAAAGAGT

AAATTTGCAG

A AAGACTAGG T GTGGGCCGG C AGGCAGATC C GAAACCCCG k. TGCCTGTAA' k CCCAGGAGG' 3 CCTGGGTGA(

ATGTGTAAA'.

'CCTGCTTCCI

TCTGAGAGA;

TGCTATTCTI

ATTGTGGAA-Z

TATACTGGCC

TAAGAGGGAC

CAGAAAGAGA

ATCTATCCTC

GGAAGAAGGG

TATGATCATT

ATTATTTGAG

ATGATCTCAA

TGCCTCAGTC

AGCTAATTTT

GCTGATCTTG

AAGTGCTGGG

TAAAAATCAA

T CAAAAACAA G GGCTCACGC A CCTGAGGGC T CTCTACTAAl T CCCAGCTAC r GGAGGTT=T

:AAGAGCAAAIU

r TAATGAGTA) k. TCATTTAAG(

STTAAATTGCC

AAAGCTAGT;

LTGCTCGTACA

TGAACACCGG

CATATTAAAC

CTAGAAGCAT

AAGAACCCTA

CAGTTCCTAT

ATGGTAGAGT

ACGGAGTCTC

CTCACTGCAA

TCCCAAGTAG

TGTATTTTTG

AACTCCTGAC

ATTACAGGCG

ATGATCCTAA

.G GTGAAACAAC 4200 C TGTAATCCCA 4250 A GGAGTTTGAG 4300 G AATACAAAAT 4350 r TGGAAGGCTG 4400 k. GTGAGCTGAG 4450 k CTCCGTCTCA 4500 k AGTCCTATTC 4550 CTCTGGCCCT 4600 :CCCAAACTTA 4650 LATTCTTGTCT 4700 GAACTCTATT 4750 ACATCCCCCT 4800 TAACATGTGT 4850 GTTTTATGGG 4900 GCGTCCCTTC 4950 GGGTCCCTTC 5000 CTCATACCTA 5050 ACTCTATCCC 5100 CCTCAGCCTC 5150 CTGGGATTAC 5200 GTAGAGATGG 5250 CTCAGGTGAT 5300 TGAGCCACTG. 5350 GGTTTTGCAG 5400 CACTAGAACC AAGAGGTAAA AGCTGTAACA 5450 :GGGCAGATTT CAGCAACGTA AGAAAAAAGG AGCTCTTCTC ACTGAAACCA 55900

AGTGTAAGA,

CTGATGACCJ

ACCTTGGTC(

TCC TAAGG C]

CTCTCTTCC;

GAGCAGTGAC

GGACCCACTT

TCTCCTCCTT

AGTCAAGGGA

GACCTGAGGG

ATCTACTAAG

TGTCTTTCCT

GCCTCAGGAC

GCTCACAAGG

AGGAAAGGTG

2CAGGCTGGAC k. GCTGTCGGGA

TGTCCAGTTC

CCCCACCCGC

TCTCTTTCTC

CCTTCTGCTG

GCCTCTCATC

GGGGCCCTGC

TCTGTAGA

AAGAAGGGCT

AGTGCTCCCT

ACTTAGACAA

CATCCTCTGC

ATCCCAATGC

CGTTTCCTGA IJ

TAGAGGACAC

GACTGTGAAG

TCAGCCTGTA

TTTTAGTGTG

AGGAGGAGAC

GAGGGAGTGA

CCTCCTGGGG

AGAGCCTCCT

CTGTTCTTTT

CTTCCAGGGA

GCCAGCCACA

3 GGAGGCCTG2

:CTCAGCTTC

:ATCTTCCTG :GCTTGTAGG A~

GGGAGTTTTT

GAATTCCTGC

TGATTCACTC

CCCTTTGAGG

CAAGGCACAG

TGGCAGCACG

CAGCTTTCTG

TGGAACCCAG

CTGACTCAGT

GCTCAAGGGC

%TGCCTGGGT

kGATCTGGCC

:TCCACAGGG

LGTTTCCAAC

I

LGGGTCCACCC

CAGAACCTC

I

GATTGTTGG

A

CTTCTGAAG

T

CCAAACCTC

C

ACGAACTCT

T

ACCCTAGGA

G

GCCACCCAA C4 TGGACAGT

A~

;TcCAGCTC

C)

rACCAGCCC

TC

0GGAAGGGT A Z AGCTCCCT

TC

TACTTTCT

CC

GAAGCAGAGG

CCTGGGTGGG

TGCTGGCTAC

CAGTGCGCTT

GACATTCTGG

GGGACAACTG

GACAGGTCCG

GTAAGTCCCC

CCCACTAGAA

AGAAGAGCTG

ACTGGCATCC

CTGGTGTTTG

5550 5600 5650 5700 5750 5800 5850 5900 5950 6000 6050 6100 GGCGGGCCCC ACCCACCACA

GCTGTCCCCA

S h 'gee

S

0 i0 Se 0 0 Begee b 0 em

C

400 *0 S. 6O 0 eas

ACACTGAACC

CACTGCCTC;

GATTCAGAGC

GACCAAATCC

TCGTGGACTC

TTTCCTCAGG

GGATATTCTC

CCCTCTTCCA

TTCCTGACCC

ACATCCTACA

GACACTGCCG

GCGCCCCTGG

;AGCTCCCAA.j

LGCCAGAACTP

CAAGATTCCI

CCGGATACCT

TTTCCTGGAC

AACATCAGAC

CTTCCCCAAC

CCCACCTTGC

TTCTGCTCCA

CCCACTCCCA

ACATCAGCAT

GAGACAACTG

LCAGGACTTCT

LCTGGCTCTGG

GGTCTGCTGA

GAACAGGATA

CCTCACGCAG

ACAGGCTCCC

CCATCCTCCT

CCACCCCTGT

ACGCCCACCC

GAATCTGTCT

TGTCTCATGT

GACAAGATTT

:AGGACCACA 6150 ~CCTGCTCCG 6200 TCTGCGTCA 6250 'CTAGTCCTC 6300 ~GACAAACTT 6350 GGCAGCAGG 6400 AGGTCCCTG 6450 GAATGGAAC 6500 CCCCGGACA 6550 CTCCAGCCT 6600 rACGCTCTT 6650 CCCCCTGC 6700 TTCTAAAC 6750 LGGTTCTCA 6800 CCTGCAGG 6850 TGAAACCC 6900

S.

Cd a AAAGCCCTGG TAAAAGGGAT ACACAGGACT GAAAAGGGAA TCATTTTTCA 6950

CTGTACATTA

TCATCAGAGC

CACTGATTCT

GCTGGCCTGG

AGAGAAAGGG

CATCCCCTTT

AGATCTTTAC

CTATACACTA

AAAAGCTCCC

AT CCC CCT CA

AGAGCCTCAC

ACCTGGATGT

GAGGCTTTGC

GAAGTGCAGG

GAACAAGTAC

AAATGCATCT

GATCTCTAAA2

TAAACCTTCA

AGCTAGCTCT

CTACATGCTC

CAGTTGAACA

TAATTTCCTT

ACTATCATTC

TCTTGAGAAA

GACAAAACTG

TAAAAAGCAA

CCCTGCCACC

ACCCCAGGTA

GACAGCTGAG

CAGGCATGGA

AAGTGGCATG

AAGTCAAGTA

kAAAAGCAGC kAGGAGTCAGC

GAAGCTATTT

TTGGTCTATT

TTTTTCTGTG

GAGGGAGAGA

TGCTTCAAAT

TCAGTGGGAC

TGAATAAGCT

AGCCTGTATA

GGGAAAGATG

CCAAACAAAA

AGGCTGTGTA

CAAACAGCTA

CGCCTGCCTC

kGTCAGGCTC4

:AAGTTGAAG

ECTGTGTGAC

CTTATGGGG

TTTTA.AGCTA

TTCTGCAGAA

ATAACTCTGC

CTAACCTTGA

TCAAGGCCTT

TCTGATCCCA

TTCTCTCAGA

AGGAATAAAT

TTCTTCGAGG

AAGCTAACAG

GACAGTTCAG

GAGCTTTGGC

CCTCCTGTGG

CTTGAGCTCA

GCTCATTTCC

:ACCATAAAC

TTTGCAAAT

TCAGCAATAC 7000 ATTTGCAACT 7050 .AAAGGCCTGG 7100 GTCAGAAAAC 7150 CCAACGCCCC 7200 TATTCTTAAC 7250 AATGCTGTCC 7300 GGG.AGCGCCG 7350 GTGGCAATAG 7400 GAAGCCTTGG 7450 TAAAGACAGG 7500 AGCTCAGCAG 7550 AGGTCAGGAG 7600 CACAGCAGGA 7650 CAGTTCCCGC 7700 TCTGCTAGGG 7750 kAGTGCTGCC 7800 9O

S

S.

S..

oe TTGGTGCTCA GGAAAAGGTT TGTGTTGCAC AAAACACAAA TTCCACTGC 7849 INFORMATION FOR SEQ ID NO:12: (i)SEQUENCE

CHARACTERISTICS:

LENGTH: 1443 base pairs TYPE: Nucleic Acid STRAkNDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2.2: *:GAGTCCTTGG CCCACCTCTC TCCCACCCGA CTCTGCCGAA AGAAGCACAG *AAGCTCAAGC CGCCTCCATG GCCCCAGGAA AGATTCAGGG GAGAGGCCCC 100 ATACAGGGAG CCACTTCAGT TAGACACCCT GGCCAGA ATG GAG 143 Met Glu

S.A

-21 CTG ACT GAT TTG CTC CTG GCG GCC Leu Thr Asp Leu Leu Leu Ala Ala ATG CTT Met Leu CTT GCA GTG 182 Leu Ala Val CCT GCC TGT 221 pro Ala Cys GCA AGA Ala Arg CTA ACT CTG TCC Leu Thr Leu Ser

AGC

Ser 1 CCC GTA GCT Pro Val Ala GAC CCC AGA CTC Asp Pro Arg Leu CTC CTT CAC AGC Leu Leu His Ser CTA AAT AAA Leu Asn Lys

CTG

Leu 15 CTG CGT GAC TCC Leu Arg Asp Ser CAC 260 His

CGA

Arg CTG AGT CAG TGT Leu Ser Gln Cys

CCC

Pro GAC GTC GAC 299 Asp Val Asp GTG GAC TTT 338 Val Asp Phe CCT TTG Pro Leu TCT ATC CCT GTT Ser Ile Pro Val

CTG

Leu 40 CTG CCT GCT Leu Pro Ala AGC CTG GGA Ser Leu Gly

GAA

Glu s0 TGG AAA ACC CAG Trp Lys Thr Gln

ACG

Thr GAA CAG AGC AAG 377 Glu Gin Ser Lys CTT CTA CTG GAG 416 Leu Leu Leu Glu

GCA

Ala CAG GAC ATT CTA Gin Asp Ile Leu

GGG

Gly 65 GCA GTG TCC Ala Val Ser GGA GTG ATG GCA Gly Val Met Ala CTC TCA TCC CTC Leu Ser Ser Leu

GCA

Ala CGA GGA CAG TTG Arg Gly Gin Leu 80 GGA CAG CTT TCT Gly Gin Leu Ser GAA CCC TCC Giu Pro Ser TGC 455 Cys 9 9 9**S 9** See 9 9 9

CTG

Leu 90

GGG

Gly CAG GTT CGC 494 Gin Val Arg CTC CTC Leu Leu 100 TTG GGG GCC CTG Leu Gly Ala Leu

CAG

Gln 105 GGC CTC CTA GGA Gly Leu Leu Gly ACC CAG Thr Gin 110 533 GGC AGO ACC Giy Arg Thr

ACA

Thr 115s GCT CAC AAG GAC Ala His Lys Asp

CCC

Pro 120 AAT GCC CTC TTC 572 Asn Ala Leu Phe

*C.

*9S9

C

*5 9 9* a 9*S

TTG

Leu 125 AGC TTG CAA CAA Ser Leu Gin Gin

CTG

Leu 130 CTT CGG GGA AAG Leu Arg Gly Lys GTG CGC TTC Val Arg Phe 135 611 CTG CTT CTG Leu Leu Leu 140 GTA GAA GGT CCC Val Glu Gly Pro

ACC

Thr 145 CTC TGT GTC AGA Leu Cys Val Arg CGG 650 Arg 150 ACC CTG CCA ACC ACA GCT GTC CCA AGC AGT ACT TCT CAA 689 foe.

f Thr Leu Pro Thr Thr Ala Val Pro Ser Ser Thr Ser Gin 155 160 CTC CTC ACA CTA AAC AAG TTC CCA AAC AGG ACT TCT GGA 728 Leu Leu Thr Leu Asn Lys Phe Pro Asn 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 As Phe Ser Val Thr Ala Arg Thr Ala 180 185 GGC CCT GGA Gly Pro Gly AAG ATT ACT Lys Ile Thr 205 CTT CTG AGC Leu Leu Ser 195 AGO CTT CAG GGA TTC AGA GTC Arg Leu Gin Gly Phe Arg Val 200 806 CCT GGT Pro Gly CAG CTA AAT CAA ACC TCC Gin Leu Asn Gln Thr Ser 210 AGG TCC 845 Arg Ser 215 CCA GTC CAA ATC TCT Pro Vai Gin Ile Ser 220 GGA TAC CTG Gly Tyr Leu AAC AGG ACA CAC GGA Asn Arg Thr His Gly 225 884 CCT GTG Pro Val 230 AAT GGA ACT CAT As Gly Thr His

GGG

Gly 235 CTC TTT GCT GGA Leu Phe Ala Gly ACC TCA 23 Thr Ser 240 CTT CAG ACC Leu Gin Thr

CTG

Leu 245 GAA GCC TCA GAC ATC TCG CCC GGA GCT Glu Ala Ser Asp Ile Ser Pro Gly Ala 250 962 TTC AAC Phe Asn 255 AAA GGC TCC Lys Gly Ser

CTG

Leu 260 GCA TTC AAC CTC Ala Phe Asn Leu CAG GGT GGA 1001 Gin Gly Gly 265

C

*c CTT CCT CCT Leu Pro Pro 270 TCT CCA AGC CTT Ser Pro Ser Leu

GCT

Ala 275 CCT GAT GGA CAC Pro Asp Gly His

ACA

Thr 280 1040 CCC TTC CCT CCT TCA CCT GCC TTG Pro Phe Pro Pro Ser Pro Ala Leu 285 TCT CCA CCC CAG CTC CAC CCC CTG Ser Pro Pro Gin Leu His Pro Leu 295 300 CCC ACC ACC CAT Pro Thr Thr His 290 TTT CCT GAC Phe Pro Asp

CCT

Pro 305 GGA 1079 Gly TCC 1118 Ser ACA 1157 Thr .t a a. ACC ACC ATG CCT AAC TCT ACC GCC CCT CAT CCA GTC Thr Thr Met Pro Asn Ser Thr Ala Pro His Pro Val 310 315 ATG TAC CCT CAT CCC AGG AAT TTG TCT CAG GAA ACA TAGCGC 1199 Met Tyr Pro His Pro Arg Asn Leu Ser Gin Glu Thr 320 325 330 331 a a lJ~ 163 G GGCACTGGCC CAGTGAGCGT CTGCAGCTTC TCTCGGGGAC 1240 AAGCTTCCCC AGGAAGGCTG AGAGGCAGCT GCATCTGCTC CAGATGTTCT 1290 GCTTTCACCT AAAAGGCCCT GGGGAAGGGA TACACAGCAC TGGAGATTGT 1.340 AAAATTTTAG GAGCTATTTT TTTTTAACCT ATCAGCAATA TTCATCAGAG 13.90 CAGCTAGCGA TCTTTGGTCT ATTTTCGGTA TAAATTTGAA AATCACTAAT .1440 TCT 1443 INFORMATION FOR SEQ ID NO:l3: SEQUENCE

CHARACTERISTICS:

LENGTH: 352 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Glu Leu Thr Asp Leu Leu Leu Ala Ala Met Leu Leu Ala Val -21 -20 -15 Ala Arg Leu Thr Leu Ser Ser Pro Val Ala Pro Ala Cys Asp Pro 1 Arg Leu Leu Asn Lys Leu Leu Arg Asp.Ser His Leu Leu His Ser 15 Arg Leu Ser Gin Cys Pro Asp Val Asp Pro Leu Ser Ile Pro Val 30 Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin 40 45 Thr Glu.Gin Ser Lys Ala Gin Asp Ile Leu Gly Ala Val Ser Leu 55 60 6 Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Glu Pro Ser 70 75 Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu 90 Leu Leu Gly Ala Leu Gin Gly Leu Leu Gly Thr Gin Gly Arg Thr 100 105 110 Thr Ala His Lys Asp Pro Asn Ala Leu Phe Leu Ser Leu Gln Gin 115 120 125 Leu Leu Arg Gly Lys Val Arg Phe Leu Leu Leu Val Glu Gly Pro 130 135 140 Thr Leu Cys Val Arg Arg Thr Leu Pro Thr Thr Ala Val Pro Ser a at a a a 9* ae.

a. a a a a.

C

a a 4* 164 145 150 155 Ser Thr Ser Gin Leu Leu Thr Leu Asn Lys Phe Pro Asn Arg 160 165 170 Ser Gly Leu Leu Glu Thr Asn Phe Ser Val. Thr Ala Arg Thr o~ 175 180 185 Gly Pro Gly Leu Leu Ser Arg Leu Gin Gly Phe Arg Val Lys 1 190 195 200 Thr Pro Gly Gin Leu Asn Gin Thr Ser Arg Ser Pro Val Gin I 205 210 215 Ser Gly Tyr Leu Asn Arg Thr His Gly Pro Val Asri Gly Thr H 220 225 230 Gly Leu Phe Ala Gly Thr Ser Leu Gin Thr Leu Glu Ala Ser A 235 240 245 Ile Ser Pro Gly Ala Phe Asn.Lys Gly Ser Leu Ala Phe Asn L 20255 260 Gin Gly Gly Leu Pro Pro Ser Pro Ser Leu Ala pro Asp Gly H-I 265 270 275 *Thr Pro Phe Pro Pro Ser Pro Ala Leu Pro Thr Thr His Gly S~ 280 285 290 *Pro Pro Gin Leu His Pro Leu Phe Pro Asp Pro Ser Thr Thr ME *295 300 305 Pro Asn Ser Thr Ala Pro His Pro Val Thr Met Tyr Pro His P1 **310 315 320 Arg Asn Leu Ser Gin Glu Thr 325 330 331 INFORMATION FOR SEQ ID NO:14: Ci) SEQUENCE

CHARACTERISTICS:

CA) LENGTH: 1536 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: *GAGTCCTTGG CCCACCTCTC TCCCACCCGA CTCTGCCGAA AGAAGCACAG 'AAGCTCAAGC CGCCTCCATG GCCCCAGGAA AGATTCAGGG GAGAGGCCCC 100 ATACAGGGAG CCACTTCAGT TAGACACCCT GGCCAGA ATG GAG 143 Met Glu ?hr la le le is sp eu is :0 9 4* 1~y* I! -21 CTG ACT GAT TTG Leu Thr Asp Leu

CTC

Leu CTG GCG GCC ATG Leu Ala Ala Met CTT CTT GCA GTG 182 Leu Leu Ala Val GCA AGA Ala Arg CTA ACT CTG TCC AGC CCC GTA GCT CCT GCC TGT 221 Leu Thr Leu Ser Ser Pro Val Ala Pro Ala Cys GAC CCC AGA CTC Asp Pro Arg Leu CTC CTT CAC AGC Leu Leu His Ser CTA AAT AAA Leu Asn Lys

CTG

Leu is CTG CGT GAC TCC Leu Arg Asp Ser CAC 260 His

CGA

Arg CTG AGT CAG TGT Leu Ser Gin Cys

CCC

Pro GAC GTC GAC Asp Val ASP 299 CCT TTG Pro Leu TCT ATC CCT GTT Ser Ile Pro Val.

CTG

Leu 40 CTG CCT GCT GTG Leu Pro Ala Val GAC TTT 338 Asp Phe AGC CTG GGA Ser Leu Gly

GAA

Glu TGG AAA ACC CAG Trp Lys Thr Gin

ACG

Thr GAA CAG AGC AAG 377 Glu Gin Ser Lys CTT CTA CTG GAG 416 Leu Leu Leu Giu

GCA

Ala CAG GAC ATT CTA Gin Asp Ile Leu

GGG

Gly 65 GCA GTG, TCC Ala Val Ser GGA GTG, ATG Gly Val Met GCA GCA CGA GGA Ala Ala Arg Gly

CAG

Gin 80 TTG GAA CCC TCC Leu Giu Pro Ser TGC 455 Cys 0 eb..

.0e* 0 4 U. 4 9.

0 9*t 04 CTC TCA TCC CTC Leu Ser Ser Leu

CTG

Leu 90 GGA CAG CTT TCT Gly Gin Leu Ser

GGG

Gly CAG GTT CGC Gin Vai Arg 494

CTC

Leu

CTC

Leu 100 TTG GGG GCC CTG Leu Gly Ala Leu

CAG

Gin 105 GGC CTC CTA GGA Gly Leu Leu Gly ACC CAG 533 Thr Gin 110 CTT CCT CTA Leu Pro Leu

CAG

Gin 115 GGC AGG ACC ACA Giy Arg Thr Thr

GCT

Al a 120 CAC AAG GAC CCC 572 His Lys Asp Pro

AAT

Asn 125 GCC CTC TTC TTG Ala Leu Phe Leu

AGC

Ser 130 TTG CAA CAA CTG Leu Gin Gin Leu

CTT

Leu 135 CGG GGA 611 Arg Gly AAG GTG CGC Lys Vai Arg TTC CTG CTT CTG Phe Leu Leu Leu

GTA

Val1 145 GAA GGT CCC ACC Giu Gly Pro Thr CTC 650 Leu TGT GTC AGA CGG ACC CTG CCA ACC ACA GCT GTC CCA AGC 689 zN* 166 Cys Val Arg Arg Thr Leu Pro Thr Thr Ala Val. Pro Ser 155 160 AGT ACT TCT CAA CTC CTC ACA CTA AAC AAG TTC CCA AAC 728 Ser Thr Ser Gin Leu Leu Thr Leu Asn Lys Phe Pro Asn 165 170 175 AGG ACT TCT GGA TTG TTG GAG ACG AAC TTC AGT GTC ACA 767 Arg Thr Ser Gly Leu Leu Giu Thr Asn *Phe Ser Val Thr 180 185 GCC AGA ACT GCT GGC CCT GGA CTT CTG AGC AGG CTT CAG 806 Ala Arg Thr Ala Gly Pro Giy Leu Leu Ser Arg Leu Gin 190 195 200 GGA TTC AGA GTC AAG ATT ACT CCT GGT CAG CTA AAT CAA Gly Phe Arg Val Lys Ile Thr Pro Gly Gin Leu Asn Gin 205 210 215 845 ACC TCC AGG TCC CCA GTC Thr Ser Arg Ser Pro Val 220 CAA ATC TCT GGA TAC CTG AAC 884 Gin Ile Ser Gly Tyr Leu Asn 225 AGG ACA CAC GGA CCT GTG AAT GGA ACT CAT GGG CTC TTT 923 Arg Thr His Gly Pro Val Asn Gly Thr His Gly Leu Phe 230 235 240 a.

a a a I. p. 4 'a a a a a a~a a a GCT GGA ACC TCA CTT CAG ACC CTG GAA GCC TCA GAC ATC 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 Ser Pro Gly Ala Phe Asn Lys Gly Ser Leu Ala Phe Asn 255 260 265 962 1002J CTC CAG GGT GGA CTT Leu Gin Gly Gly Leu 270 GAT GGA CAC ACA CCC Asp Gly His Thr Pro 285 CCT CCT TCT CCA AGC Pro Pro Ser Pro Ser 275 TTC CCT CCT TCA CCT Phe Pro Pro Ser Pro 290 CTT GCT CCT 1040 Leu Ala Pro 280 GCC TTG CCC 1079 Ala Leu Pro a.

*aa a.

a. a a.

4 ACC ACC CAT GGA TCT CCA Thr Thr His Gly Ser Pro 295 CCT GAC CCT TCC ACC ACC Pro Asp Pro Ser Thr Thr 310 CCC CAG CTC CAC CCC CTG TTT Pro Gin Leu His Pro Leu Phe 300 305 11l8 ATG CCT AAC TCT ACC GCC CCT 1157 Met Pro Asn Ser Thr Ala Pro CAT CCA GTC ACA ATG TAC CCT CAT CCC AGG AAT TTG TCT 1196 His Pro Val Thr Met Tyr Pro His Pro Arg Asn Leu Ser 320 325 330 a 167 CAG GAA ACA TAGCG CGGGCACTGG CCCAGTGAGC GTCTGCAGCT 1240 Gin Glu Thr 335 TCTCTCGGGG ACAAGCTTCC CCAGGAAGGC TGAGAGGCAG CTGCATCTGC 1290 TCCAGATGTT CTGCTTTCAC CTAAAAGGCC CT-GGGGAAGG GATACACAGC 1340 ACTGGAGATT GTAAAATTTT AGGAGCTATT TTTTTTTAAC CTATCAGCAA 1390 TATTCATCAG AGCAGCTAGC GATCTTTGGT CTATTTTCGG TATAAATTTG 1440 AAAATCACTA AAAAAAAAAA AAAAAAAAA~ AAAA AAAA AAA.AAA 1490 AAAAAAA AA AAAAA AAAAA A AA AA A A AA AAAAAA 1-536 INFORMATION FOR SEQ ID Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 356 amino acids too TYPE: Amino Acid TOPOLOGY: Linear to: (xi) SEQUENCE DESCRIPTION: SEQ ID Met Glu Leu Thr Asp Leu Leu Leu Ala Ala Met Leu Leu Ala Val. .o-21 -20 -15 Ala Ar g Leu Thr Leu Ser Ser Pro Val Ala Pro Ala Cys Asp Pro *A-rg Leu Leu Asn Lys Leu Leu Arg Asp Ser His Leu Leu His Ser .15 Arg Leu Ser Gin Cys Pro Asp Val. Asp Pro Leu Ser Ile Pro Val 25 30 3 too. 3 Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin 40 45 Thr Giu Gin Ser Lys Ala Gin Asp Ile Leu Gly Ala Val. Ser Leu 55 60 Leu Leu Giu Gly Val Met Ala Ala Arg Gly Gin Leu Glu Pro Ser 75 Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu 85 90 S:Leu Leu Gly Ala Leu Gin Gly Leu Leu Gly Thr Gin Leu Pro Leu 100 105 110 *:*Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Leu Phe Leu 115 120 125 168 Arg Gly Lys Ser Leu Gin Gin Leu Leu 130 135 Vai Ar9' Phe Leu LeU Leu 140 Val Glu 145 Ala Val 160 Pro Asn 175 Ala Arg 190 Arg Val 205 Pro Val 220 Asn Gly 235 Glu Ala 250 Glj Pro Arg Thr Lys Gin Thr Ser Pro Thr Ser Ser Thr Ser Ala Gly Ile Thr Ile Ser His Gly Asp Ile Leu 150 Thr 165 Gly 180 Pro 195 Pro 210 Gly 225 Leu 240 Ser 255 Cys Ser Leu Gly Gly Tyr Phe Pro Val Gln Leu Leu Gin Leu Al a Gly Arg Leu Glu Leu Leu Asn Gly ~Arg Leu Thr Ser Asn Arg Thr *Thr Leu Pro Thr 155 Thr Leu Asn Lys 170 Asn Phe Ser Val 185 Arg Leu Gin Gly 200 Gin Thr Ser Arg 215 Thr His Gly Pro 230 Ser Leu Gin Thr 245 Asn Lys Gly Ser 260 Thr Phe Thr Phe Ser Val Leu Leu Ala Phe Ala Phe Asn Leu Gin Gly Gly Leu Pro Pro Ser Pro Ser Leu Ala 265 270 275 4* Pro Asp Gly His Thr Pro Phe Pro Pro Ser Pro Ala Leu Pro Thr 280 285 290 **Thr His Gly Ser Pro Pro Gin Leu His Pro Leu Phe Pro Asp Pro 295 300 305 geSer Thr Thr Met Pro Asn Ser Thr Ala Pro His Pro Val Thr Met 315 320 'Tyr Pro His Pro Arg Asn Leu Ser Gin Giu Thr 325 330 335 INFORMATION FOR SEQ ID NO:16: Ci) SEQUENCE

CHARACTERISTICS:

LENGTH: 241 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: *Ser Pro Val Ala Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu 1 5 10 Leu Arg Asp Ser His Leu Leu His Ser Arg Leu Ser Gin Cys Pro 169 25 Asp Val. Asp Pro Leu Ser Ile Pro Val Leu Leu Pro Ala Vai Asp 40 Phe Ser Leu Gly G].u Trp, Lys Thr Gin Thr Glu Gin Ser Lys Ala 55 Gin Asp Ile Leu Gly Ala Val Ser Leu Leu Leu Glu Giy Vai Met 70 Ala Ala Arg Gly Gin Leu Giu Pro Ser Cys Leu Ser Ser Leu Leu 85 Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin 100 105 Gly Leu Leu Gly Thr Gin Leu Pro Leu Gin Gly Arg Thr Thr Ala 110 115 120 *His Lys Asp Pro Asn Ala Leu Phe Leu Ser Leu Gin Gin Leu Leu 125 130 135 *Arg Gly Lys Asp Phe Trp Ile Val. Giy Asp Glu Leu Gin Cys His 0.0140 145 150 Ser Gin Asn Cys Trp Pro Trp, Thr Ser Giu Gin Ala Ser Gly Ile 155 160 165 *9Gin Ser Gin Asp Tyr Ser Trp Ser Ala Lys Ser Asn Leu Gin Val 170 175 180 Pro Ser Pro Asn Leu Trp Ilie Pro Giu Gin Asp Thr Arg Thr Cys 185 190 195 Glu Trp Asn Ser Trp, Ala Leu Cys Trp Asn Leu Thr Ser Asp Pro :200 205 210 9**.*Gly Ser Leu Arg His Leu Ala Arg Ser Phe Gin Gin Arg Leu Pro 215 220 225 :Gly Ile Gin Pro Pro Gl Tp Thr Ser Ser Phe Ser Ly Pro Cy 230 235 240 Ser 241 INFORMATION FOR SEQ ID NO:17: SEQUENCE

CHARACTERISTICS:

LENGTH: 335 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 170 Ser Pro Val Ala Pro Ala Cys 1 Asp Pro Arg Leu 10 Leu Asn Lys Leu Leu Asp Phe Arg Va1 Ser Asp Asp Leu Ser His Leu Leu Pro Gly

L

G

Gin Asp Ile Ala Ala Arg Gly Gin Leu Gly Leu Leu His Lys Asp Arg Gly Lys Cys Val Arg Ser Gin Leu Leu Leu Glu Gly Leu Leu Gly Gin Leu Tyr Leu Asn 2 Phe Ala Gly J Pro Gly Ala I Leu Gly Ser Gly Pro Vai Arg Leu Thr Ser ksn %rg 7hr he E

G

G

G

T)

Ar 1 14 T1 Th 17 As 18 Al 20 Gl 21 rh 3e 44 leu Ser Ile *iu Trp Lys ly Ala Val in Leu Glu iy Gln Val hr Gln Leu 3n Ala Leu rg Phe Leu ir Leu Pro 55 ir Leu Asn '0 nf Phe Ser 5 g Leu Gin 0 n Thr Ser 5 r His Gly F 0 r Leu Gin T 5 His Ser Arg Le, 25 Pro Val Leu Lei 40 Thr Gln'Thr Gli 55 Ser Leu Leu Lei 70 Pro Ser Cys Let 85 Arg Leu Leu LeL 100 Pro Leu Gin Gly 115 Phe Leu Ser Leu 130 Leu Leu Val Glu 145 Thr Thr Ala Val 160 Lys The Pro Asn 175 al Thr Ala Arg 190 3ly Phe Arg Val 205 Irg Ser Pro Val 220 'ro Val Asn Gly 235 Ihr Leu Glu Ala 250 er Leu Ala Phe 265 u Ser Gin Cys Pro a Pro Ala Val Asp 1 Gin Ser Lys Ala i Glu Gly Val Met i Ser Ser Leu Leu L Gly Ala Leu Gin 105 Arg Thr Thr Ala 120 Gin Gin Leu Leu 135 Gly Pro Thr Leu 150 Pro Ser Ser Thr 165 Arg Thr Ser Gly 180 Thr Ala Gly Pro 195 Lys Ile Thr Pro 210 Gin Ile Ser Gly 225 Thr His Gly Leu 240 Ser Asp Ile Ser 255 Asn Leu Gin Gly 270

C

*r S Oe* 4L 6

U

595 9e*S 5 5.9 0 ~sn Lys Gly S 260 Gly Leu Pro

A,

Pro Ser Pro Ser Leu Ala Pro Asp Gly His Thr Pro 171 275 280 285 Phe Pro Pro Ser Pro Ala Leu Pro Thr Thr His Gly Ser Pro pro 290 295 300 Gin Leu His Pro Leu Phe Pro Asp Pro Ser Thr Thr Met Pro Asn 305 310 315 Ser Thr Ala Pro His Pro Val Thr Met Tyr Pro His Pro Arg Asn 320 325 330 Leu Ser Gin Glu Thr 335 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 332 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE

DESCRIPTION:

Ser Pro Ala Pro Pro Ala Cys 1 5 SEQ ID NO:18: Asp Pro Arg Leu His Gly Arg Leu 25 Leu Ser Leu Asp Phe e* *Thr Val Asp ~~His 0 0: Arg Ile Thr Asp Al a Gin Leu L~ys Asp Asn Leu Val Arg Leu Leu Ser His Val.

Pro Gly Leu Gly Ser Gly Leu Ser Glu Trp Gly Ala Gin Val Gly Gin Met Gin 110 Ser Ala 125 Arg Phe 140 Leu Thr Lys Thr Gly Val Leu Ile Leu Asn Lys5 Gin Cys Pro Thr Thr Pro Arg Pro Phe Leu Val Leu Leu Pro Ala Val 40 Gin Thr Glu Gin Thr Lys 55 Leu Leu Leu Glu Ala Val 70 Pro Cys Leu Ser Ser Leu 85 Leu Leu Leu Gly Ala Leu 100 Pro Gin Gly Arg Thr Thr 115 Leu Asn Phe Gin Gin Leu 130 1,eu Val Val Gly Pro Ser Leu is Pro Asp Ala Met Leu Gin 105 Ala 120 Leu 135 Leu 150 Asp Pro Arg Gly Lys Val 0. 0 145~ Cys Ala Lys Arg Ala 155 Pro Pro Ala Ile Ala 160 Val Pro Ser Ser Thr 165 Ser Pro Phe Leu Leu Giu His Thr Leu 170 Thr Asn Ser 185 Asn Lys Leu Ser Ile Ser Pro Asn Arg Thr Ser 175 Ala Arg Thr Thr Gly 190 Gly 180 Ser 195 Gl Leu Gin Pro Ala Glu Phe Pro PhE Leu Asn Gly Thr Ser Ser Leu Leu Lys Asn Gin Gly Thr Pro Gin Ser Gly Pro Ser Pro Ser Leu Pro Arg 200 Thr 215 His 230 Pro 245 Met 260 Pro 275 Pro 290 Leu Ser Gly Gly Giy Ala Thr Gin Arg Pro Ala Ser His Ser Ala Ser Leu Leu Arg Pro Pro Phe Leu Ser Gly Pro Ser Ser Arg 205 Asp 220 Gly 235 Ala 250 Thr 265 Pro 280 Pro 295 Ala Gin Ile Pro Tyr Gly Thr Lys Ile His Asp Leu Arg Val Ile Pro Gly Ile Gin Tyr Gin Pro Gly Leu Pro Pro Thr Leu Gly 210 His 225 Phe 240 Pro 255 Gly 270 Leu 285 Gin 300 Ser A.sp Pro Ser Ala Ile Thr Pro Asn Ser Thr 305 310 Pro Leu Leu Phe Ala Ala His Pro His Phe Gin Asn Leu Ser Gin 320 325 330, ::>Giu Glu INFORMATION FOR SEQ ID NO:i9: SEQUENCE

CHARACTERISTICS:

a LENGTH: 1026 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: AGC CCG GCT CCT CCT GCC TGT GAC CCC CGA CTC CTA 36 e~e Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu O*AAT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC GGC AGA Asn Lys Leu Leu Arg Asp Ser His Val Leu His Gly Arg 20 Ii) 1, 173 CTG AGC

CAG

Leu Ser Gin TGC CCA GAC

ATT

Cys Pro AspIle AAC CCT

TTG

Asn Pro Leu TCC ACA

CCT

Ser Thr Pro 114 153 GTC

CTG

Val Leu CTG CCT GCT

GTG

Leu Pro Ala Val.

GAC

Asp TTC ACC TTG

GGA

Phe Thr Leu Giy GAA TGG Giu Trp AAA ACC

CAG

Lys Thr Gin

ACG

Thr GAG CAG ACA

AAG

Giu Gin Thr Lys

GCA

Ala

GGA

Gly GCC ACA ACC

CTT

Ala Thr Thr Leu CAG GAT GTC CTG 1-92 Gin Asp Val Leu GTG ATG ACA GCA 231 Val Met Thr Ala

CTG

Leu CTG GAG

GCA

Leu Glu Ala CGG 3GA Arg 3 1y

CAA

Gin GTG GGA CCC

CCT

Val Gly Pro Pro

TGC

Cys CTC TCA TCC

CTG

Leu Ser Ser Leu CTG 270 Leu GTG CAG CTT

TCT

Val Gln Leu Ser

GGA

Gly CAG GTT CGC CTC CTC CTC GGG GCC 309 Gin Val Arg Leu Leu Leu Gly Ala CTG

CAG

Leu Gln 105 GAC CTC CTT

GGA

Asp Leu Leu Gly

ATG

Met CAG CTT CCT CCA Gin Leu Pro Pro CAG GGA 348 Gin Gly AGG ACC

ACA

Arg Thr Thr

GCT

Ala 120 CAC AAG GAT

CCC

His Lys Asp Pro

AGT

Ser GCC ATC TTC CTG 387 Ala Ile Phe Leu 0 4 Ga..

u.S.

04,45.

0 0

S

AAC

Asn 130 TTC CAA CAA

CTG

Phe Gin Gin Leu

CTC

Leu 135 CGA GCA AAG

GTG

Arg Gly Lys Val CGT TTC

CTG

Arg Phe Leu 426 CTC

CTT

Leu Leu

CTA

Val 145 CTG GGG CCC TCC Val Giy Pro Ser

CTC

Leu TGT CCC AAG AGG Cys Ala Lys Arg GCC 465 Ala *,*CCA CCC GCC ATA Pro Pro Ala Ile

GCT

Ala 160 GTC CCG ACC

AGC

Val Pro Ser Ser

ACC

Thr CAC ACA His Thr 170 CTG AAC AAG

CTC

Leu Asn Lys Leu

I

64 ~e

CCA

Pro AAC AGG

ACC

Asn Arg Thr TCT CCA TTC 504 Ser Pro Phe TCT GGA TTG 543 Ser Cly Leu 180 ACT ACT GGC 582 Thr Thr Gly TTG GAG ACA Leu Ciu Thr

AAC

Asn 1.85 TCC AGT ATC

TCA

Ser Ser Ile Ser

CC

Ala

AGA

Arg

TCT

Ser P0 195

OCA

Gly TTT CTC AAG Phe Leu Lys

AGG

Arg 200 CTG CAG CCA

TTC

Leu Gin Ala Phe

AGA

Arg 205 GCC AAG 621 Ala Lys ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG TCC CTA GAC Ile Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser Leu Asp 210 215 220 660 CAA ATC CCT GGA CAC CAG AAT GGG ACA CAC GGA CCC TTG 699 Gin Ile Pro Gly His Gin Asn Gly Thr His Gly Pro Leu 225 230 AGT GGA ATT CAT GGA CTC TTT CCT GGA CCC CAA Ser Gly Ile His Gly Leu Phe Pro Gly Pro Gin 235 240 CCC GGG 738 Pro Gly 245 GCC CTC GGA GCT CCA GAC ATT CCT CCA GCA ACT TCA GGC 777 Ala Leu Gly Ala Pro Asp Ile Pro Pro Ala Thr Ser Gly 250 255 ATG GGC TCC CGG CCA ACC TAC CTC CAG CCT GGA GAG TCT 816 Met Gly Ser Arg Pro Thr Tyr Leu Gin Pro Gly Glu Ser 260 265 270 CCT TCC CCA GCT CAC CCT TCT CCT GGA CGA TAC ACT Pro Ser Pro Ala His Pro Ser Pro Gly Arg Tyr Thr 275 280 CTC 855 Leu 285 TTC TCT CCT TCA CCC ACC TCG CCC TCC CCC ACA Phe Ser Pro Ser Pro Thr Ser Pro Ser Pro Thr 290 295 GTC CAG 894 Val Gin CTC CAG CCT CTG CTT CCT GAC CCC TCT GCG ATC ACA CCC 933 Leu Gln Pro Leu Leu Pro Asp Pro Ser Ala Ile Thr Pro 300 305 310 AAC TCT ACC AGT CCT CTT CTA TTT GCA GCT CAC CCT CAT 972 Asn Ser Thr Ser Pro Leu Leu Phe Ala Ala His Pro His 315 320 0O0 TTC CAG AAC CTG TCT CA Phe Gin Asn Leu Ser G1I 325 33 CCTGCCAACT TCAGCA 1026 f G GAA GAG TAAG GTGCTCAGAC 1010 n Glu Glu 0 332 *.6 *0 fe S. *0 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 1014 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID AGC CCG GCT CCT CCT GCC TGT GAC CCC CGA CTC CTA 36 Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu i AAT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC GGC AGA Asn Lys Leu Leu Arg Asp Ser His Val Leu His Gly Arg 20 CTG AGC CAG TGC CCA GAC ATT AAC CCT TTG TCC ACA CCT 114 Leu Ser Gin Cys Pro Asp Ile Asn Pro Leu Ser Thr Pro GTC CTG CTG CCT GCT Val Leu Leu Pro Ala AAA ACC CAG ACG GAG Lys Thr Gin Thr Glu GGA GCC ACA ACC CTT Gly Aia Thr Thr Leu GTG GAC TTC Val Asp Phe 45 CAG ACA AAG Gin Thr Lys ACC TTG GGA GAA TGG Thr Leu Gly Giu Trp 153 GCA CAG GAT GTC CTG 192 Ala Gin Asp Val Leu

CTG

Leu 70 CTG GAG GCA GTG ATG ACA GCA 231 Leu Giu Ala Val Met Thr Ala CGG GGA CAA GTG GGA CCC Arg Giy Gin Val Gly Pro GTG CAG CTT TCT GGA CAG Val Gin Leu Ser Gly Gin CCT TGC CTC TCA TCC CTG Pro Cys Leu 85 GTT CGC CTC Val Arg Leu Ser Ser Leu CTG 270 Leu- CTC CTC GGG GCC 309 Leu Leu Gly Ala 100 4* 9 9 q CTG CAG GAC CTC Leu Gin Asp Leu 105 CTT GGA ATG Leu Gly Met 110 CAG GGA AGG ACC ACA GCT 348 Gin Gly Arg Thr Thr Ala 115 CAC AAG GAT CCC AGT His Lys Asp Pro Ser .120 CTG CTC CGA GGA AAG Leu LeU Arg Gly Lys 130 GCC ATC TTC CTG AAC Ala Ile Phe Leu Asn 125 TTC CAA CAA Phe Gin Gin 387 GTG CGT Val1 Arg 135 TTC CTG CTC CTT GTA GTG 426 Phe Leu Leu Leu Val Val 140 9** 9 9 9 9* GGG CCC TCC CTC Gly Pro Ser Leu 145 GCT GTC CCG AGC Ala Vai Pro Ser TGT GCC AAG Cys Ala Lys AGG GCC CCA Arg Ala Pro 150 CCC GCC ATA 465 Pro Ala Ile AGC ACC TCT CCA TTC CAC ACA CTG AAC 504 Ser Thr Ser Pro Phe His Thr Leu Asn 160 165 AAG CTC CCA AAC AGG ACC TCT GGA TTG TTG GAG Lys Leu Pro Asn Arg Thr Ser Gly Leu Leu Giu 170 175 ACA AAC 543 Thr Asn TCC AGT ATC TCA GCC AGA ACT ACT GGC TCT GGA TTT CTC 582 Mmmm Ser Ser Ile Ser Ala Arg Thr Thr Gly Ser Gly Phe Leu 185 190 AAG AGG CTG CAG GCA TTC AGA GCC AAG ATT CCT GGT CTG 621 Lys Arg Leu Gin Ala Phe Arg Ala Lys Ile Pro Gly Leu 195 200 205 CTG AAC CAA ACC TCC AGG TCC CTA GAC CAA ATC CCT GGA 660 Leu Asn Gin Thr Ser Arg Ser Leu Asp Gin Ile Pro Gly 210 215 220 CAC CAG AAT GGG ACA CAC GGA CCC TTG AGT GGA ATT CAT 699 His Gin Asn Gly Thr His Gly Pro Leu Ser Gly Ile His 225 230 GGA CTC TTT CCT GGA CCC CAA CCC GGG GCC CTC GGA GCT 738 Gly Leu Phe Pro Gly Pro Gin Pro Gly Ala Leu Gly Ala 235 240 245 CCA GAC ATT CCT CCA GCA ACT TCA GGC ATG GGC TCC CGG 777 Pro Asp Ile Pro Pro Ala Thr Ser Gly Met Gly Ser Arg 250 255 CCA ACC TAC CTC CAG CCT GGA GAG TCT CCT TCC CCA GCT 816 Pro Thr Tyr Leu Gin Pro Gly Glu Ser Pro Ser Pro Ala 260 265 270 CAC CCT TCT CCT GGA CGA TAC ACT CTC TTC TCT CCT TCA 855 His Pro Ser Pro Gly Arg Tyr Thr Leu Phe Ser Pro Ser 275 280 285 CCC ACC TCG CCC TCC CCC ACA GTC CAG CTC CAG CCT CTG 894 Pro Thr Ser Pro Ser Pro Thr Val Gin Leu Gin Pro Leu 290 295 CTT CCT GAC CCC TCT GCG ATC ACA CCC AAC TCT ACC AGT 933 S* Leu Pro Asp Pro Ser Ala Ile Thr Pro Asn Ser Thr Ser 300 305 310 CCT CTT CTA TTT GCA GCT CAC CCT CAT TTC CAG AAC CTG 972 Pro Leu Leu Phe Ala Ala His Pro His Phe Gin Asn Leu 315 320 TCT CAG GAA GAG TAAGGT GCTCAGACCC TGCCAACTTC 1010 Ser Gin Glu Glu 325 328 AGCA 1014 INFORMATION FOR SEQ ID NO:21: SEQUENCE

CHARACTERISTICS:

LENGTH: 328 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu Leu Arg Asp Se: Asp Phe Gin Thr Val Asp Ser Arg Ala Thr Asn.

Arg I Thr S His G Pro G Ii Th As' Al~ G1i Let Ala Phe Pro 1,eu 3er ~eu er ~ly ly e Asn r Leu p Val a Arg SLeu ±Leu Ile Leu IPro Asn Ser Gin2 Arg Pro I Ala I Pr Gil Let Gl Ser Gly Phe Leu Ala Lys Ile kla 3er ~eu ~eu r His Va SLeu Se2 SGlu Trr Gly Ala Gin Val Gly Gin Met Gin 110 Leu Asn 125 Leu Val 140 Ile Ala 155 Leu Pro 170 Ser Ala 185 Phe Arg 200 Leu Asp .215 Ser Gly 230 Gly Ala 245 LLeu His Gly Arg Leu 25 -Thr Pro Val Leu Leu 40 Lys Thr Gin Thr Giu 55 Thr Thr Leu Leu Leu 70 Gly Pro Pro Cys Leu 85 Val Arg Leu Leu Leu 100 Gly Arg Thr Thr Ala 115 Phe Gin Gin Leu Leu 130 Val Gly Pro Ser Leu 145 Val Pro Ser Ser Thr 160 Asn Arg Thr Ser Gly 175 Arg Thr Thr Gly Ser 190 Ala Lys Ile Pro Gly I 205 Gin Ile Pro Gly His C 220 Ile His Gly Leu Phe P 235 Pro Asp Ile Pro Pro 250 Ser Gin Pro Ala Gin Thr GJlu Ala Ser Ser Gly Ala His Lys Arg Gly Cys Ala Ser Pro eu Leu .71y Phe ~eu Leu ;in Asn ~ro Giy la Thr c Cy~ Val Lys Val Leu Leu Jsp Lys Lys Phe Giu Leu ksn ?ro er Pro Asp Ala *Met ILeu *Gin 105 Pro 120 Val 135 Arg 150 His 165 Thr 180 Lys 195 Gin 210 Thr 225 Gin 240 Gly 255 4* 0 0 So...

o 0 0 0

S

005 00* 9.

0* 5 0 *50

S

S. Met Gly Ser Arg Pro Thr Tyr Leu Gin 260 Pro 265 Gly Glu Ser Pro Ser 270 178 Pro Ala His Pro Ser Pro Gly Arg Tyr Thr Leu Phe Ser Pro Ser 275 280 285 Pro Thr Ser Pro Ser Pro Thr Val Gin Leu Gin Pro Leu Leu Pro 290 295 300 Asp Pro Ser Ala Ile Thr Pro Asn Ser Thr Ser Pro Leu Leu Phe 305 310 315 Ala Ala His Pro His Phe Gin Asn Leu Ser Gin Giu Glu 320 325 328 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 5141 base pairs TYPE: Nucleic Acid STRANDEDNESS: Double TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TTCGAGCTCG CCCGACATTG ATTATTGACT AGAGTCGATC GACAGCTGTG GAATGTGTGT

CAGTTAGGGT

GAAGTATGCA

AAGCATGCAT

TCCCCAGGCT

CCCCAGCAGG

GTCAGCAACC

ATAGTCCCGC

CGCCCAGTTC CGCCCATTCT TATGCAGAGG

CCGAGGCCGC

AGGAGGCTTT

TTTGGAGGCC

CCGGGAACGG

TGCATTGGAA

GTACCGCCTA

TAGAGCGATA

TCGACCATTG AACTGCATCG AGAACGGAGA

CCTACCCTGG

CAAAGAATGA

CCACAACCTC

TATGGGTAGG

AAAACCTGGT

AGGACAGAAT

TAATATAGTT

GGAGCTCATT

TTCTTGCCAA

GTGGAAAGTC

CTCAATTAGT

CAGAAGTATG

CCCTAACTCC

CCGCCCCATG

CTCGGCCTCT

TAGGCTTTTG

CGCGGATTCC

AGAGGATTTT

TCGCCGTGTC

CCTCCGCTCA

TTCAGTGGAA

CCCAGGCTCC CCAGCAGGCA CAGCAACCAG GTGTGGAAAG CAAAGCATGC ATCTCAATTA GCCCATCCCG CCCCTAACTC GCTGACTAAT TTTTTTTATT GAGCTATTCC AGAAGTAGTG CAAAAAGCTA GCTTATCCGG CCGTGCCAAG AGTGACGTAA ATCCCCGCTG CCATCATGGT CCAAAATATG GGGATTGGCA GGAACGAGTT CAAGTACTTC GGTAAACAGA ATCTGGTGAT 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 TCTCCATTCC TGAGAAGAAT CTCAGTAGAG AACTCAAAGA AAGTTTGGAT GATGCCTTAA

CGACCTTTAA

ACCACCACGA

GACTTATTGA

:I'

ACAACCGGAA TTGGCAAGTA AAGTAGACAT GGTTTGGATA GTCGGAGGCA 850 f 0S Aw'e e

GTTCTGTTTA

GTGACAAGGA

TGATTTGGGG

AGGTCCAGGA

AAAGACTAAC

ATGCATTTTT

CTTCGTTAGA

ATACGATTTA

CAGGTGTCCA

GTCGACTCTA

AACTTGTTTA

AAATTTCACA

CCAAACTCATC

CGGCGCAGCA

C

GTACCTTCTGP

TGTGGAAAGT

C

TCTCAATTAG

T

GCAGAAGTAT

G

CCCCTAACTC

C

TCCGCCCCAT Gi CCTCGGCCTC

T(

CTAGGCTTTT

G(

CGCCATTTAA

A'

ACGTCGTGAC

TC

CACATCCCCC

C']

CGCCCTTCCC

AP~

GTATTTTCTC

CT

CCAGGAAGCC

TCATGCAGGA

AAATATAAAC

GGAAAAAGGC

AGGAAGATGC

ATAAGAC CAT

ACGCGGCTAC

GGTGACACTA

CTCCCAGGTC

GAGGATCCCC

rTGCAGCTTA

ATAAAGCAT

:AATGTATCT

:CATGGCCTG

LGGCGGAAAG

CCCAGGCTC

CAGCAACCA(

CAAAGCATGC

GCCCATCCC

C

GCTGACTAA U

GAGCTATTCC

CAAAAAGCT G rcCTGCAGGT ;GGAAAACC C rTCGCCAGC T LCAGTTGCG T 'TACGCATC

T(

ATGAATCAAC

ATTTGAAAGT

CTCTCCCAGA

ATCAAGTATA

TTTCAAGTTC

GGGACTTTTG

AATTAATACA

TAGATAACAT

CAACTGCACC

GGGGAATTCA

TAATGGTTAC

TTTTTTCACT.

TATCATGTCT

.kAATAACCTC k.ACCAGCTGTC

:CCAGCAGGC

.,GTGTGOAAA

C

ATCTCAATT

JCCCCTAACT

C

'TTTTTTTAT

T

AGAAGTAGTG

*TTACCTCGA

G

AACAGCTTG

G

TGGCGTTAC

C

GGCGTAATA

G

~GCCTGAAT

G

GTGCGGTAT T'

CAGGCCACC

GACACGTT2

ATACCCAGC

AGTTTGAACG

TCTGCTCCC

CTGGCTTTA

TAAC CT TAT

CCACTTTGC

TCGGTTCTA.

ATCGATGGC(

AAATAAAGC2

GCATTCTAG',

GGATCGATCC

TGAAAGAGG;

7GAATGTGTC k~GAAGTATGC

TCCCCAGGC

LGTCAGCAAC

~CGCCCAGTT

'TATGCAGAG

,AGGAGGCTT

CGGCCGCTT

CACTGGCCG

CAACTTAAT

CGAAGAGGC

GCGAATGGC

rCACACCGC TAGACTCTTT g00 TCCCAGAAAT 950 GTCCTCTCTG 1000 CTACGAGAAG 1050 TCCTAAAGCT 1100 ATCCCCTTGG 1150 TATCATACAC 1200 TTTCTCTCCA 1250 GCTTCTGCAG 1300 GCCATGGCCC 1350 ATAGCATCAC 1400 TGTGGTTTGT 1450 GGAATTAATT 1500 ACTTGGTTAG 1550 ECAGTTAGGG 1600 FLAAGCATGCA 1650' r'CCCCAGCAG 1700 =AAGTCCCG 1750 CGCCCATTC 1800 3CCGAGGCCG 1850 TTTGGAGGC 1900 LATTAAGGCG 1950 CGTTTTACA 2000 GCCTTGCAG 2050 CGCACCGAT 2100 0 4 400 0.00 *0 0 O qo 9 0 000.

0 0 GCCTGATGCG

ATACGTCAAA

2150 2200 180

GCAACCATAG

TGGTTACGCn

CCTTTCGCTT

TCAAGCTCTA

GGCACCTCGA

CCATCGCCCT

CTTTAATAGT

CGGGCTATTC

TTAAAAAATG

ATTAACGTTT

CCGCATAGTT

CGCCCCGACA

CTCCCGGCAT

GTGTCAGAGG

GAAGACGAAA

ATAATAATGG

CGGAACCCCT

STCATGAGACA

J

SAGTATGAGTA IJ

:ATTTTGCCTTC

ATGCTGAAGA UI AACAGCGGTA A GATGAGCACT

T

.:.ACGCCGGGCA

A

TTGGTTGAGT

A

AGTAAGAGAA

T

CCAACTTACT Ti TACG CG C CCT

CAGCGTGACC

TCTTCCCTTC

PATCGGGGGC

CCC CAAAAAA

GATAGACGGT

GGACTCTTGT

TTTTGATTTA

AGCTGATTTA

ACAATTTTAT

AAGCCAACTC

CCCGCCAACA

:CGCTTACAG

rTTTCACCGT "7GGCCTCGTG

L'TTCTTAGAC

=TTGTTTAT

LTAACCCTGA

I

'TCAACATTT C 'CTGTTTTTG

C

CAGTTGGGT G

GATCCTTGAG

TTAAAGTTC T GAGCAACTC G CTCACCAGT C rATGCAGTG C :'TGACAACG A

GTAGCGGCGC

GCTACACTTG

CTTTCTCGCC

TCCCTTTAGG

CTTGATTTGG

TTTTCGCCCT

TCCAAACTGG

TAAGGGATTT

ACAAAAATTT

GGTGCACTCT

CGCTATCGCT

CCCGCTGACG

ACAAGCTGTG

CATCACCGAA

%TACGCCTAT

3TCAGGTGGC2 [TTTCTAAAT I rAAATGCTTC :CGTGTCGCC C

:TCACCCAGA

rCACGAGTGG

G

,AGTTTTCGC

C

GCTATGTGG C GTCGCCGCA T ACAGAAAAG C TGCCATAAC C TCGGAGGAC C

ATTAAGCGCG

CCAGCGCCCT

ACGTTCGCCG

GTTCCGATTT

GTGATGjGTTC

TTGACGTTGG

AACAACACTC

TGCCGATTTC

AACGCGAATT

CAGTACAATC

ACGTGACTGG

CGCCCTGACG

~CCGTCTCCG

kCGCGCGAGG rTTTATAGGT k.CTTTTCGGG

.CATTCAAAT

~aTAATATTG2

:TTATTCCCT

LACGCTGGTG ~TTACATCGA .z CCGAAGAAC C 'GCGGTATTA T ACACTATTC TI

ATCTTACGG

kTGAGTGATA

GAAGGAGCTA

GCGGGTGTGG 2250 AGCGCCCGCT 2300 GCTTTCCCCG 2350 AGTGCTTTAC 2400 ACGTAGTGGG 2450 AGTCCACGTT 2500 AACCCTATCT 2550 GGCCTATTGG 2600 TTAACAAAAT 2650 TGCTCTGATG 2700 GTCATGGCTG 2750 GGCTTGTCTG 2800 GGAGCTGCAT 2850 CAGTATTCTT 2900 TAATGTCATG 2950 GAAATGTGCG 3000 kTGTATCCGC 305-0 kAAAAGGAAG 3100 ETTTTGCGGC 3150 LkAGTAAAAG 3200 LCTGGATCTC 3250 ;TTTTCCAAT 3300 'CCCGTGATG 3350 'CAGAATGAC 3400 ,TGGCATGAC 3450 ACACTGCGG 3500 ACCGCTTTT 3550 TTGCACA.ACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGCAACCGGA 3600 181 GCTGAATGAA GCCATACCAA ACGACGAGC

CAATGGCAA

GCTTCCCGG,

ACCACTTCT(

CTGGAGCCG(

GATGG'tAAG(

AACTATGGAJ

TTAAGCATTC

GATTTAAAAC

TGATAATCTC

CGTCAGACCC

CTGCGCGTAA

GGTTTGTTTG

GCTTCAGCAG

TTAGGCCACC

GCTAATCCTG

CCGGGTTGGA

TGAACGGGGG

CGAACTGAGA

AAGGGAGAAA

GAGCGCACGA

TGTCGGGTTT

CAGGGGGGCG

TTCCTGGCCT

CCCTGATTCT

CTCGCCGCAG

GAAGAGCGCC

C AACGTTGCG,

CAACAATTAA'

3CGCTCGGCC( 3 TGAGCGTGG(

CCTCCCGTAJ

r' GAACGAAATI

GTAACTGTC;

TTCATTTTT;

ATGACCAAAP

CGTAGAAAAC

TCTGCTGCII

CCGGATCAAG

AGCGCAGATA

ACTTCAAGAA

TTACCAGTGG

CTCAAGACGA

GTTCGTGCAC

TACCTACAGC

GGCGGACAGG

GGGAGCTTCC

CGCCACCTCT

GAGCCTATGG

TTTGCTGGCC

GTGGATAACC

CCGAACG-ACC

CAATACGCAA

.AAACTATTA.

r AGACTGGAT

TTCCGGCTG(

3 TCTCGCGGTj C CGTAGTTAT( kGACAGATCG(

GACCAAGTT'

LATTTAAAAGC

LTCCCTTAACC

ATCAAAGGAI

GCAAACAAAP

AGCTACCAAC

CCAAATACTG

CTCTGTAGCA

CTGCTGCCAG

TAGTTACCGG

ACAGCCCAGC

GTGAGCATTG

TATCCGGTAA

AGGGGGAAAC

GACTTGAGCG

AAAAACGCCA

TTTTGCTCAC

GTATTACCGC

GAGCGCAGCG

ACCGCCTCTC

G TGACACCACI k CTGGCGAAC'

GAGGCGGAT

3 CTGGTTTAT' :L TCATTGCAG(

TACACGACGC

STGAGATAGG9 r ACTCATATAI

;ATCTAGGTG.;

;TGAGTTTTCC

.1 CTTCTTGAGP.

LAAACCACCGC

TCTTTTTCCG

TCCTTCTAGT

*CCGCCTACAT

TGGCGATAAG

ATAAGGCGCA

TTGGAGCGAA

AGAAAGCGCC

GCGGCAGGGT

GCCTGGTATC

TCGATTTTTG

GCAACGCGGC

ATGTTCTTTC

CTTTGAGTGA

AGTCAGTGAG

CCCGCGCGTT

3 ATGCCAGCAG 3650 r ACTTACTCTA 3700 AAGTTGCAGG 3750 r GCTGATAAAT 3800 :ACTGGGGCCA 38,50 GGAGTCAGGC 3900 ?GCCTCACTGA 3950 'ACTTTAGATT 4000 L AGATCCTTTT 4050 TTCCACTGAG 4100 TCCTTTTTTT 4150 TACCAGCGGT 4200 AAGGTAACTG 4250 GTAGCCGTAG 4300 ACCTCGCTCT 4350 TCGTGTCTTA 4400 GCGGTCGGGC 4450' CGACCTACAC 4500 ACGCTTCCCG 4550 CGGAACAGGA 4600 TTTATAGTCC 4650 TGATGCTCGT 4700 CTTTTTACGG 4750 CTGCGTTATC 4800 GCTGATACCG 4850 CGAGGAAGCG 4900 GGCCGATTCA 4950 de 9 .9~9 9tt*99 9 9* 94* 9 9

A..

C' 9 99 9 9.

9 9 999 9999 9 9

N.,,TTAATCCAGC

IA

TGGCACGACA GGTTTCCCGA CTGGAAAGCG GGCAGTGAGC 5000 182 GCAACGCAAT TAATGTGAGT TACCTCACTC ATTAGGCACC CCAGGCTTTA 5050 CACTTTATGC TTCCGGCTCG TATGTTGTGT GGAATTGTGA GCGGATAACA 5100 ATTTCACACA GGAAACAGCT ATGACCATGA TTACGAATTA A 5141 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ATGTCNCCNG CNCCNCCNGC N 21 INFORMATION FOR SEQ ID NO:24: SEQUENCE

CHARACTERISTICS:

.i LENGTH: 21 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: ATGTCTCCAG CGCCGCCAGC G 21 S INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID ATGTCGCCTG CTCCACCTGC T 21 INFORMATION FOR SEQ ID NO:26: SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: a 183 ATGTCGCCAG CGCCACCAGC C 21 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: ATGTCCCCAG CCCCACCCGC A 21 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: ATGTCGCCAG CGCCGCCAGC G 21 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids S TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu 1 5 10 Leu Arg Asp Asp His Val Leu His Gly Arg INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu 1 5 10 S Leu Arg Asp Asp Xaa Val Leu His Gly Arg Leu 25 26 INFORMATION FOR SEQ ID NO:31: SEQUENCE

CHARACTERISTICS:

LENGTH: 25 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Ser Pro Ala Pro Pro Ala Xaa Asp Pro Arg Leu Leu Asn Lys Leu 1 5 10 Leu Arg Asp Asp His Val Leu His Gly Arg INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Xaa Pro Ala Pro Pro Ala Xaa Asp Pro Arg Leu Xaa Asn Lys 1 5 10 14 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: S' Pro Arg Leu Leu Asn Lys Leu Leu Arg 1 5 9 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GCCGTGAAGG ACGTGGTCGT CACGAAGCAG TTTATTTAGG AGTCG 185 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID CCNGCNCCNC CNGCNTGYGA INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: NCCRTGNARN ACRTGRTCRT C 21 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA TGACCACGTT CAGCACGGC 69 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: GCCGTGCTGA ACGTGGTCAT CACGAGGCAG TTTATTTAGG AGTCGGGGGT CACAGGCTGG CGGCGCTGG 69 A4.

186 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA CGACCACGTC CATCACGGC 69 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID GCCGTGATGG ACGTGGTCGT CACGAAGCAG TTTATTTAGG AGTCGGGGGT CACATGCCGG AGGTGCTGG 69 S INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: CCAGCACCGC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA CGATCATGTC TATCACGGT 69 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 187 ACCGTGATAG ACATGATCGT CACGAAGCAG TTTATTTAGG AGTCGGGGGT CACATGCCGG CGGTGCTGG 69 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: GCTAGCTCTA GAAATTGCTC CTCGTGGTCA TGCTTCT 37 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: Nucleic Acid STRANDEDNESS:. Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CAGTCTGCCG TGAAGGACAT. GG 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID TGTGGACTTT AGCTTGGGAG AATG 24 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GGTCCAGGGA CCTGGAGGTT TG 22 188 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: ATCGATATCG ATAGCCAGAC ACCCCGGCCA G 31 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GCTAGCTCTA GACAGGGAAG GGAGCTGTAC ATGAGA 36 INFORMATION FOR SEQ ID NO:49: SEQUENCE

CHARACTERISTICS:

LENGTH: 24 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 44 CTCCTTGGAA CCCAGGGCAG GACC 24 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID GGTCCTGCCC TGGGTTCCAA GGAG 24 INFORMATION FOR SEQ ID NO:51:

**U

SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: CTGCTCCGAG GAAAGGACTT CTGGATT 27 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: AATCCAGAAG TCCTTTCCTC GGAGCAG 27 S* INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: CCCTCTGCGT CGCGGCGGCC CCACCCAC 28

S

S INFORMATION FOR SEQ ID NO:54: i: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: GTGGGTGGGG CCGCCGCGAC GCAGAGGG 28 INFORMATION FOR SEQ ID

SEQUENCE-CHARACTERISTICS:

0 LENGTH: 35 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID GACTCGAGGA TCCATCGATT TTTTTTTTTT TTTTT INFORMATION FOR SEQ ID NO:56: SEQUENCE

CHARACTERISTICS:

LENGTH: 17 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: GACTCGAGGA TCCATCG 17 INFORMATION FOR SEQ ID NO:57: SEQUENCE

CHARACTERISTICS:

LENGTH: 32 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: GCTAGCTCTA GAAGCCCGGC TCCTCCTGCC TG 32 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE-DESCRIPTION: SEQ ID NO:58: CGAAATTAAC CCTCACTAAA G 21 INFORMATION FOR SEQ ID NO:59: SEQUENCE

CHARACTERISTICS:

LENGTH: 103 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:

T

S 7n .j

V-

J

191 TGCAGCAAGG GCTACTGCCA CACTCGAGCT GCGCAGATGC TAGCCTCAAG ATGGCTGATC CAAATCGATT CCGCGGCAAA GATCTTCCGG TCCTGTAGAA 100 GCT 103 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 103 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID AGCTTCTACA GGACCGGAAG ATCTTTGCCG CGGAATCGAT TTGGATCAGC *.i CATCTTGAGG CTAGCATCTG CGCAGCTCGA GTGTGGCAGT AGCCCTTGCT 100 GCA 103 INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: TCTCGCTACC GTTTACAG 18 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: CAGGTACCCA CCAGGCGGTC TCGGT INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: Nucleic Acid 192 STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: GGGCCATGAC ACTGTCAA 18 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: GACCGCCACC GAGACCGCCT GGTGGGTACC TGTGGTCCTT S(2) INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 32 base pairs TYPE: Nucleic Acid S(C) STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID ATCGATATCG ATCAGCCAGA CACCCCGGCC AG 32 INFORMATION FOR SEQ ID N0:66: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: TCTAGATCTA GATCACCTGA CGCAGAGGGT GGACC INFORMATION FOR SEQ ID NO:67: S SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear 193 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: AGTCGACGTC GACGTCGGCA GTGTCTGAGA ACC 33 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: AGTCGACGTC GACTCACCTG ACGCAGAGGG TGGACC 36 INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: CGCGTATGCC AGCCCGGCTC CTCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TG 62 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs S(B) TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID AGTCACGAAG CAGTTTACTG AGGACTCGGA GGTCACAAGC AGGAGGAGCC

V,

GGGCTGGCAT A 61 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single D) TOPOLOGY: Linear

A/

194 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTAA 37 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: CGCGTTAAGA AGAAATGCGA TATTCTTTTT CATAATT 37 INFORMATION FOR SEQ ID NO:73: SEQUENCE

CHARACTERISTICS:.

LENGTH: 69 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: CTAGAATTAT GAAAAAGAAT ATCGCATTTC ATCACCATCA CCATCACCAT CACATCGAAG GTCGTAGCC 69 INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: TACGACCTTC GATGTGATGG TGATGGTGAT GGTGATGAAA TGCGATATTC TTTTTCATAA TT 62 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear i /4 vA"\ 195 (xi) SEQUENCE DESCRIPTION: SEQ ID CTAGAATTAT GAAAAAGAAT ATCGCATTTC ATCACCATCA CCATCACCAT CACATCGAAC CACGTAGCC 69 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: TACGTGGTTC GATGTGATGG TGATGGTGAT GGTGATGAAA TGCGATATTC TTTTTCATAA TT 62 INFORMATION FOR SEQ ID NO:77: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: TCCACCCTCT GCGTCAGGT 19 INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: AGCTACCTGA CGCAGAGG 18 INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear 196 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: GCAGCAGTTC TAGAATTATG TCNCCNGCNC CNCCNGCNTG TGACCTCCGA GTTCTCAGTA AA 62 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 49 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID GAAGGACATG GGAGTCACGA AGCAGTTTAC TGAGAACTCG GAGGTCACA 49 INFORMATION FOR SEQ ID NO:81: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear

:S

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: CTAGAATTAT GAAAAAGAAT ATCGCATTTA TCGAAGGTCG TAGCC INFORMATION FOR SEQ ID NO:82: k SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: TACGACCTTC GATAAATGCG ATATTCTTTT TCATAATT 38 INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear SEQUENCE DESCRIPTION: SEQ ID NO:83: i

N,.

CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTAAACG TAGCC INFORMATION FOR SEQ ID NO:84: Wi SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: Nucleic Acid STRANDEDNESS: Single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: TACGTTTAAG AAGAAATGCG ATATTCTTTT TCATAATT 38 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 168 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Lys Asn Ile Ala Phe Leu Leu Asn Ala Tyr Ala Ser Pro 3* 0 *040 p 4 p .4 0 .00 p

S

*eeap~ Ala Asp His Leu Ile Arg Leu Leu Pro Ser Pro Gly Leu Giy Ser Gly Pro Ala Cys His Val Leu 35 Leu, Pro Thr 50 Glu Trp, Lys 65 Gly Ala Val Gin Leu Gly Gly Gin Val 110 Thr Gin Leu 125- Asp His Pro Thr Thr Pro Arg Pro Leu Arg Val Ser Arg Leu Val Leu, Leu Gin-Met Giu Leu Leu Leu, Thr Cys Leu Leu Leu, Leu Pro Gin Gly Leu 25 Ser Pro 55 Giu 70 Giu.

85 Ser 100 Gly 115 Arg 130 Ser Gin Aia Thr Gly Ser Ala Thr Lys Leu Cys Pro Val Asp Lys Ala Val Met Leu Leu Leu Gin Thr Ala Leu Arg Glu Val.

Phe Ser Gin Asp Ala Ala Gly Gin 105 Ser Leu.

120 His Lys 135 0@ p p 4P* em..

0g -S p @0

C

top p Asp Pro Asn Ala Ile Phe Leu Ser 140 Phe Gin 145 His Leu Leu Arg Gly 150

O..

'I

198 Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val 155 160 165 Arg Arg Ala 168 INFORMATION FOR SEQ ID NO:86: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPT Met Lys Lys Asn Ile Glu Gly Leu Ser Lys Ser Gin Cys Pro Ala Val Glu Thr Lys Glu Gly Val Ser Ser Leu Gly Ala Leu Arg.Thr Thr Gin His Leu Gly Ser Thr Arg Leu Pro Asp Ala Met Leu Gin Ala Leu Ile Ala Ser Pro Leu Arg Glu Val Phe Ser Gin Asp Ala Ala 95 Gly Gin 110 Ser Leu 125 His Lys 140 Arg Gly 155 ION: SEQ ID NO:86: Phe His His His His 10 Ala Pro Pro Ala Cys 25 Asp Ser His Val Leu 40 His Pro Leu Pro Thr 55 Leu Gly Glu Trp Lys 70 Ile Leu Gly Ala Val 85 Arg Gly Gin Leu Gly 100 Leu Ser Gly Gin Val 115 Leu Gly Thr Gin Leu 130 Asp Pro Asn Ala Ile 145 Lys Val Arg Phe Leu 160 Arg Arg Ala 174 His Asp His Pro Thr Thr Pro Arg Pro Phe Met His His Leu Arg Ser Arg Val Leu Gin Met Leu Leu Thr Cys Leu Leu Pro Gin Leu Ser Leu Val His Val Leu Leu Glu Leu Leu 105 Leu 120 Gly 135 Phe 150 Gly 165

C

C 0

OOCC

*0Se a Ste

S

@9CSC..

B.

Sr *Se

CCC.

C

Leu Cys Val 170 INFORMATION FOR SEQ ID NO:87: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE

DESCRIPTION:

SEQ ID NO:87: Met 1 Lys Lys Asn Ile Ala Phe His His His His His His His His Ile Giu Prc Leu Ser Pro Glu Giu Ser Gly Arg Gin Ser Gin Al a Thr Gly Ser Ala Thr Hius Lys Cys Val Lys Val Leu Leu Thr ELeu Arg Ser Pro Leu Leu Arg Pro Glu Val Asp Phe Ser Ala Gin Asp Met Ala Ala Leu Gly Gin 110 Gin Ser Leu 125 Ala His Lys 140 Leu Arg Gly 1~55 Ala Asp His Leu Ile Arg Leu Leu Asp Lays Pro Ser Pro Gly Leu Gly Ser Gly Pro Val.

Pro His Leu Gi u Gly Gin Gly Thr Asn 10 Ala 25 Val 40 Pro 55 Trp 70 Al a 85 Leu 100 Gin 115 Gin 130 Ala 145 Cys Leu Thr Lys Val Gly Val Leu Ile Asp His Pro Thr Thr Pro Arg Pro Phe Leu Ser Val Gin Leu Thr Leu Pro Leu Arg Arg Leu Met Leu Cys Leu Gin Ser Val1 Leu Leu Glu Leu Leu 105 Leu 120 Gly 135 Phe 150 Gly 165 so*@ a Arg Phe Leu Met Leu Val Gly Ser Thr Leu Cys Val Arg Arg Ala 174 INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: Amino Acid TOPOLOGY: Linear SEQUENCE DESCRIPTION: SEQ ID NO:88: Thr Thr Ala His Lys'Asp Pro 1 5 7 INFORM4ATION FOR SEQ ID NO:89: 17 200 SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: His Val Leu His 1 4 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Arg Leu Ser 1 4 0* INFORMATION FOR SEQ ID NO:91: SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: Ser His Val Leu o 1 4 9 INFORMATION FOR SEQ ID NO:92: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: His Ser Arg Leu 1 4 INFORMATION FOR SEQ ID NO:93: SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:

N"

Ala Val Asp Phe 201 1 4 INFORMATION FOR SEQ ID NO:94: SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: Ser Leu Gly Glu 1 4 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Val Thr Leu 1 4 INFORMATION FOR SEQ ID NO:96: SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: S 1 4 INFORMATION FOR SEQ ID NO:97: SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear S (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: Leu Ser Ser Leu 1 4 INFORMATION FOR SEQ ID NO:98: SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids 4 202 TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: Leu Gly Gin Leu 1 4 INFORMATION FOR SEQ ID NO:99: SEQUENCE

CHARACTERISTICS:

LENGTH: 5 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: Cys Xaa Leu Ser Ser 1 INFORMATION FOR SEQ ID NO:100: SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: Leu Leu Gly Gin 1 4 INFORMATION FOR SEQ ID NO:101: SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: Ser Ser Leu Leu 1 4 INFORMATION FOR SEQ ID NO:102:

V"

SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: Gly Gin Leu Ser 1 4 7 -Emwwn 203 INFORMATION FOR SEQ ID NO:103: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: Cys Leu Ser Ser 1 4 INFORMATION FOR SEQ ID NO:104: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: Leu Gin Ser Leu 1 4 INFORMATION FOR SEQ ID NO:105: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: Leu Gly Thr Gin 1 4 INFORMATION FOR SEQ ID NO:106: SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: Ala Leu Gin Ser S 1 4 INFORMATION FOR SEQ ID NO:107: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear 4 204 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: Leu Leu Gly Thr 1 4 INFORMATION FOR SEQ ID NO:108: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: Asn Ala Ile Phe 1 4 INFORMATION FOR SEQ ID NO:109: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: Leu Ser Phe Gin 1 4 INFORMATION FOR SEQ ID NO:110: SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu 1 5 10 Leu Arg Asp Ser His Val Leu 22 INFORMATION FOR SEQ ID NO:111: 4 SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr 1 5 10 C. C 205 Pro Val Leu Leu Pro Ala Val Asp Phe 24 INFORMATION FOR SEQ ID NO:112: SEQUENCE

CHARACTERISTICS:

LENGTH: 23.amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin 1 5 10 Asp Ile Leu Gly Ala Val Thr Leu 23 INFORMATION FOR SEQ ID NO:113: SEQUENCE CHARACTERISTICS: LENGTH: 21 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr 1 5 10 Cys Leu Ser Ser Leu Leu 20 21 INFORMATION FOR SEQ ID NO:114: SEQUENCE CHARACTERISTICS: A) LENGTH: 16 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin 1 5 10 Ser 16 INFORMATION FOR SEQ ID NO:115: SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: Amino Acid TOPOLOGY: Linear 206 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115: Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His 1 5 10 Lys Asp Pro Asn Ala Ile Phe 22 INFORMATION FOR SEQ ID NO:116: SEQUENCE

CHARACTERISTICS:

LENGTH: 25 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116: Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met .1 5 10 Leu Val Gly Gly Ser Thr Leu Cys Val Arg INFORMATION FOR SEQ ID NO:117: SEQUENCE

CHARACTERISTICS:

LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117: Met Pro Pro Ala 1 4 INFORMATION FOR SEQ ID NO:118: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids S(B) TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: Met Ala Pro Pro Ala 1 INFORMATION FOR SEQ ID NO:119: SEQUENCE CHARACTERISTICS: LENGTH: 6.amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119: i o 207 Met Pro Ala Pro Pro Ala 1 5 6 INFORMATION FOR SEQ ID NO:120: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120: Met Ser Pro Ala Pro Pro Ala 1 5 7 INFORMATION FOR SEQ ID NO:121: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121: Ala Pro Pro Ala S1 4 INFORMATION FOR SEQ ID NO:122: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122: Pro Ala Pro Pro Ala 1 «4 INFORMATION FOR SEQ ID NO:123: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123: Ser Pro Ala Pro Pro Ala 1 5 6 INFORMATION FOR SEQ ID NO:124: SEQUENCE CHARACTERISTICS: A) LENGTH: 4 amino acids -t 208 TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124: Val Arg Arg Ala 1 4 INFORMATION FOR SEQ ID NO:125: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125: Val Arg Arg Ala Pro 1 INFORMATION FOR SEQ ID NO:126: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126: Val Arg Arg Ala Pro Pro 1 5 6 INFORMATION FOR SEQ ID NO:127: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids S TYPE: Amino Acid TOPOLOGY: Linear S (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127: S Val Arg Arg Ala Pro Pro Thr 1 5 7 INFORMATION FOR SEQ ID NO:128: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128: Val Arg Arg Ala Pro Pro Thr Thr 1 5 8 S. .^o r 209 INFORMATION FOR SEQ ID NO:129: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129: Val Arg Arg Ala Pro Pro Thr Thr Ala 1 5 9 INFORMATION FOR SEQ ID NO:130: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130: Val Arg Arg Ala Pro Pro Thr Thr Ala Val S1 5 S' INFORMATION FOR SEQ ID NO:131: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: Amino Acid TOPOLOGY: Linear S:(xi) SEQUENCE DESCRIPTION: SEQ ID NO:131: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro 5 10 11 INFORMATION FOR SEQ ID NO:132: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: Amino Acid S TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser 1 5 10 12 INFORMATION FOR SEQ ID NO:133: SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids TYPE: Amino Acid TOPOLOGY: Linear i ita

(I.

210 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg 1 5 10 13 INFORMATION FOR SEQ ID NO:134: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr 1 5 10 14 INFORMATION FOR SEQ ID NO:135: SEQUENCE

CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135: S' Val.Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser 1 5 10 INFORMATION FOR SEQ ID NO:136: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser 1* 5 10 Leu S 16 INFORMATION FOR SEQ ID NO:137: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser 1 5 10

S

9. 211 Leu Val 17 INFORMATION FOR SEQ ID NO:138: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser 1 5 10 Leu Val Leu 18 INFORMATION FOR SEQ ID NO:139: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: Amino Acid TOPOLOGY: Linear To (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser 1 5 10 Leu Val Leu Thr 19 INFORMATION FOR SEQ ID NO:140: 4** SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser 1 5 10 Leu Val Leu Thr Leu INFORMATION FOR SEQ ID NO:141: SEQUENCE CHARACTERISTICS: LENGTH: 21 amino acids TYPE: Amino Acid TOPOLOGY: Linear 1^I <0i 212 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser 1 5 10 Leu Val Leu Thr Leu Asn 21 INFORMATION FOR SEQ ID NO:142: SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser 1 5 10 Leu Val Leu Thr Leu Asn Glu 20 22 INFORMATION FOR SEQ ID NO:143: SEQUENCE CHARACTERISTICS: LENGTH: 23 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser S1 5 10 Leu Val Leu Thr Leu Asn Glu Leu 23 INFORMATION FOR SEQ ID NO:144: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: Amino Acid TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144: Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser 1 5 10 Leu Val Leu Thr Leu-Asn Glu Leu Pro 24 The entire disclosure in the complete specification of our Australian Patent Application No. 15146/95 is by this crossreference incorporated into the present specification.

Claims (13)

1- An isolated mpl ligand polypeptide as obtainable by a process which comprises: screening a human genomic library with an oligonucleotide based on the genomic sequence depicted in Fig 9 hereof to isolate genomic DNA which includes the mpl ligand exon coding sequence depicted in Fig 9 hereof together with the remaining exons of the gene, (ii) inserting the DNA into an expression vector, (iii) transfecting a mammalian cell with the vector and expressing the gene, and (iv) recovering mpl ligand polypeptide from the cell culture medium.

2. An isolated mpl ligand polypeptide as obtainable by a process which comprises: isolating from a genomic library genomic DNA which hybridises under low stringency with the DNA sequence GCCCTGAAGGACGTGGTCGTCACGAAGCAGTTTATTTAGGAGTCG and which contains the exons encoding an mpl ligand polypeptide, (ii) inserting the DNA into an expression vector, 25 (iii) transfecting a mammalian cell with the vector and expressing the gene, and (iv) recovering mpl ligand polypeptide from the cell culture medium.

3. An isolated mpl ligand polypeptide as obtainable by a process which comprises: identifying a suitable cell source of RNA for the human mpl ligand and preparing one or more cDNA libraries from said RNA, (ii) hybridisation-screening the library or libraries with an oligonucleotide based on the coding sequence shown in Fig 9 hereof to identify and isolate a clone containing \\mN.Ei\ I-$\1t \cinte \Kep\Peci4 OZ7s99.doc 02/05M02 02/05 2002 12:40 FAX 61 3 92438333 GRIFFITH HACK @007 214 the coding sequence of mpl ligand, (iii) inserting the encoding DNA into an expression vector suitable for expression in mammalian cells, (iv) transfecting a mammalian cell with the vector and expressing the cDNA, and recovering mpl ligand polypeptide from the cell culture medium-

4. An isolated mpl ligand polypeptide as obtainable by a process which comprises: preparing a cDNA library or libraries from mRNA extracted from human kidney cells, (ii) hybridisation-screening the library or libraries with an oligonucleotide based on the coding sequence shown 15 in Fig 9 hereof to identify an isolated clone containing the coding sequence of mpl ligand, (iii) inserting the encoding DNA into an expression vector suitable for expression in mammalian cells, (iv) transfecting a mammalian cell with the vector and 20 expressing the cDNA, and recovering mpl ligand polypeptide from the cell culture medium. a a oo g*• 25 wherein: An isolated substantially homogeneous mpl ligand the ligand stimulates the incorporation of labelled nucleotides 3 H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected with human mpl P; the ligand stimulates 35 S incorporation into circulating platelets in a platelet rebound assay; the ligand is stable to pH 2.5, SDS at and 2M urea; the ligand is a glycoprotein; the amino terminal sequence of the polypeptide is SPAPPACDPRLLNKLLRDDHVLHGR or SPAPPACDLRVLSKLLRDSHVLHSRL. \\~\*rerilaa\h~~*9\sincr*\r.m\s~cci\1000 (111081')1 02/05 2002 12:40 FAX 61 3 92438333 GRIFFITH HACK 1008 215

6. An isolated human mpl ligand polypeptide comprising the amino acid sequence SPAPPACDLRVLSKLLRDSHVLHSRL and is biologically active.

7. An isolated mpl ligand as obtainable by purification from aplastic plasma by hydrophobic interaction chromatography, immobilised dye chromatography and mpl-affinity chromatography, which has an apparent molecular weight, as revealed by SDS-PAGE run under reducing conditions, in the region of 18-22kD, 28kD or 30kD, and has an amino acid sequence which has at least about 70% sequence identity with the sequence set out in SEQ ID NO:1.

8. An isolated mpl ligand polypeptide encoded by a nucleic acid having a sequence that hybridises under moderately stringent conditions to nucleic acid having a sequence from nucleotide 119 to 196 of Pig 9 (SEQ ID NO:4 or SEQ ID

9- An isolated mpl ligand of any one of claims 1 to S* 8 that is biologically active.

10. An isolated mpl ligand polypeptide which shares at least 70% sequence identity with a polypeptide of any one of claims 1 to 9.

11. An isolated mpl ligand polypeptide which is an allele or variant of a polypeptide of any one of claims 1 to 10, or a fragment of said mpl ligand, allele or variant, which shares at least one biological property in common with mpl ligand.

12. An mpl ligand of claim 11, wherein one or more potential protease cleavage sites are modified so as to make it resistant to cleavage. \\ats.1b~iTsl.\hsui\eint*n.\antmp~vpic\Q9.doc 02/051i)2 02/05 2002 12:40 FAX 61 3 92438333 GRIFFITH HACK 0~009 216

13. An mpl ligand of claim 12, wherein said potential cleavage site is at the amino acid position 153-154 and/or

245-246 of the mpl ligand as depicted in Fig 14- 14. An mpl ligand of claim 13, wherein the cleavage site at position 153-154 is inactivated by substitution. An mpl ligand of claim 14, wherein the Arg-Arg residues at 153-154 is substituted by Ala residues. 16. A variant mpl ligand of claim 11, which comprises a fusion to a heterologous polypeptide. 9 15 17. An mpl ligand of any one of claims 11 to 16, which comprises a fusion to one or more amino acid residues at the amino- and/or carboxy terminus. 18. An mpl ligand of claims 17, which has an N- terminal methionine. 19. An mpl ligand polypeptide of any one of claims 1 to 6 and 8 to 18 wherein the mpl ligand is derived from a human species. 20. An mpl ligand polypeptide of any one of claims 2, and 7 to 18 wherein the mpl ligand is derived from a non-human species. 21. An mpl ligand polypeptide according to any one of claims 1 to 20 which is unglycosylated. 22. An mpl ligand of any one of claims 1 to 21 linked to a non-proteinaceous polymer. 23. An mpl ligand of claim 22, wherein said polymer is polyethylene glycol, polypropylene glycol or a \\mcThtiLz\hoc \airae\Keop\~ec±c420 02/O5/Q2 02/05 2002 12:41 FAX 61 3 92438333 GRIFFITH HACK 0010 217 polyoxyalkylene. 24. An isolated nucleic acid molecule encoding a polypeptide of any one of claims 1 to 21. An isolated nucleic acid molecule selected from: a cDNA clone when obtained from step (ii) of claims 3 or step (ii) of claim 4; a DNA sequence capable of hybridizing under stringent conditions to a clone of and A genetic variant of any of the DNA sequences of and which encodes a polypeptide possessing a biological property of a naturally occurring mpl ligand polypeptide. 26. An isolated nucleic acid having a sequence that hybridises under moderately stringent conditions to nucleic acid having a sequence from nucleotide 119-196 of Fig 9 (ID NO:4 or ID NO:5) and encodes an mpl ligand. 27. An isolated nucleic acid of claim 26 that hybridises to nucleotide 119 to nucleotide 196 of Figure 9 (SEQ ID NO:4) or SEQ ID NO:5) and encodes an mpl ligand or fragment thereof which shares at least one biological 25 property with mpl ligand. 28. An isolated genomic DNA or cDNA molecule corresponding to at least part of the mpl ligand gene, and which hybridises under moderate to highly stringent conditions with a naturally occurring nucleic acid molecule having a sequence encoding an mpl ligand, wherein the mpl ligand is a ligand of any one of claims 1 to 21. 29. A nucleic acid of any one of claims 24 to 28 operably linked to a promoter. An expression vector comprising a nucleic acid of %%aelofiie- \hcmes\ciraf\Kcfp\,pcci\4027.99.de 02/05/12 02/05 2002 12:41 FAX 61 3 92438333 GRIFFITH HACK @1011 218 any one of claims 24 to 29 operably linked to control sequences recognised by a host cell. 31. A host cell transformed with a nucleic acid of any one of claims 24 to 30 so as to be capable of expressing said nucleic acid to produce an mpl ligand polypeptide. 32. A process which comprises the expression of a recombinant nucleic acid of any one of claims 24 to 30 in a suitable host cell to produce an mpl ligand polypeptide. 33 A process of claim 32 wherein the mpl ligand polypeptide is recovered from the host cell or host cell 15 culture medium. 34. A process according to claim 32 or claim 33 in which the host cell is a CHO cell line. 35. A process which comprises the expression in a cell of an indigenous mpl ligand gene under the control of a promoter/enhancer element, a suppressor, or an exogenous transcription modulatory element which has been inserted i: into the genome of the cell in proximity and orientation 25 sufficient to influence the transcription of DNA encoding the mpl ligand polypeptide, wherein the mpl ligand is as defined in any one of claims 1 to 23 or is obtainable by the process of any one of claims 32 to 34. 36. A composition comprising the mpl ligand of any one of claims 1 to 23 or as obtainable by the process of any one of claims 32 to 34, and a pharmaceutically acceptable carrier 37. A process which comprises the use of an mpl ligand polypeptide of any one claims 1 to 23 or obtained by the process of any one claims 32 to 34 in the m-lb-ilte \omes \cinrzeep\preci\40207.!9. doc 03105132 02/05 2002 12:41 FAX 61 3 92438333 GRIFFITH HACK I012 219 preparation of a medicament. 38. A composition according to claim 36 or when obtained by the process of claim 37 which further comprises a therapeutically effective amount of an agent selected from cytokines, colony stimulating factors and interleukins. 39. A composition according to claim 38 wherein the agent is selected from LIF, G-CSF, GM-CSF, M-CSF, Epo, IL- 1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 and IL-11. 40. A method of amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase reaction with 15 nucleic acid corresponding to at least part of the genomic or cDNA of claim 28. 41. A method of determining the presence of mpl ligand polypeptide, comprising hybridising nucleic acid of claim 28 to a test sample nucleic acid and determining the presence of mpl ligand polypeptide nucleic acid. 42. A process which comprises the use of an mpl ligand polypeptide of any one of claims 1 to 23 or 25 obtained by the process of any one of claim 32 to 34 in the preparation of mpl ligand specific antibody. 43. A process of claim 42, wherein a monoclonal antibody is prepared. 44. An antibody as obtainable by the process of claim 42 or claim 43. An isolated antibody that is capable of binding an mpl ligand polypeptide of any one of claims 1 to 23. 46. An antibody according to claim 45 which is a \\melhfl e2 \honfrS\cIntac\Kepcc\ apa 402 .99.ao 03/05/32 02/05 2002 12:41 FAX 61 3 92438333 GRIFFITH HACK @1013 220 monoclonal antibody. 47. A hybridoma cell line producing an antibody of any one of claims 44 to claim 46. 48. An mpl ligand, according to any one of claims 1 to 8, substantially as herein described with reference to any of the examples or figures. 49. An isolated nucleic acid molecule according to claim 25 or claim 26, substantially as herein described with reference to any of the examples or figures. 50. Genomic DNA or cDNA according to claim 15 substantially as herein described with reference to the examples or figures- 51. A method of determining the presence of mpl ligand, polypeptide according to any one of claims 40 to 42, substantially as herein described with reference to any of the examples or figures. *4 52. An expression vector comprising nucleic acid of claim 49, substantially as herein described with reference 25 to any of the examples or figures. 4* 53. A host cell according to claim 31, substantially as herein described with reference to any of the examples or figures. 54. A process according to claim 32 or claim substantially as herein described with reference to any of the examples or figures. 55. A pharmaceutical according to claim 36, substantially as herein described with reference to any of the examples or figures. \\melhfile=homeS\cintao\Kcep\spcci\40207.99.doc 02/05/02 02/05 2002 12:42 FAX 61 3 92438333 GRIFFITH HACK @014 221 56. An mpl ligand-specific antibody according to claim 45, substantially as herein described with reference to any of the examples or figures. 57. An isolated polypeptide comprising the amino acid sequence SPAPPACDPRLLNKLLRDDHVLHGR or SPAPPACDLRVLSKLLRDSHVLHSRL. 58. An isolated DNA encoding a polypeptide according to claim 57. 59. A polypeptide according to claim 57, substantially as herein described with reference to any of 15 the examples or figures. Dated this 2nd day of May 2002 GENENTECH, INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia \\melbJfilec\hcceS\cincae\Keep\-eci\40207.99.doc 02/05/02