Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/524—Thrombopoietin, i.e. C-MPL ligand
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to the isolation, purification and recombinant or chemical synthesis of proteins that influence survival, proliferation, differentiation or maturation of hematopoietic cells, especially platelet progenitor cells.
• This invention specifically relates to the cloning and expression of nucleic acids encoding a protein iigand capable of binding to and activating mpl, a member of the cytokine receptor superfamily.
• This invention further relates to the use of these proteins alone or in combination with other cytokines to treat immune or hematopoietic disorders including thrombocytopenia.
• the Hematopoietic system produces the mature highly specialized blood cells known to be necessary for survival of all mammals. These mature cells include; erythrocytes, specialized to transport oxygen and carbon dioxide, T- and B- lymphocytes, responsible for cell- and antibody-mediated immune responses, platelets or thrombocytes, specialized to form blood clots, and 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.
• neutrophils eosinophils
• basophils eosinophils
• mast cells specialized cell types having discrete functions.
• 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 al., 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 [1976]).
• 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.
• pluripotent stem cell(s) 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, into increasingly mature lineage-restricted progenitor cells, ultimately forming the highly specialized mature blood cell(s).
• CFC-Mix 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.
• CFU-E erythroid colony-forming units
• GM-CFC granulocyte/macrophage colony- forming cells
• Meg-CFC megakaryocyte colony- forming cells
• Eos-E eosinophil colony-forming cells
• CFC basophil colony-forming cells
• Bas-CFC basophil colony-forming 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.
• 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.
• primitive stem cells repopulate the entire hematopoietic cell system.
• progenitors At the other end of the spectrum lie the highly lineage- restricted progenitors and their progeny which have lost the ability of self-renewal but have acquired mature functional activity.
• IL-3 interleukin-3
• GM-CSF granulocyte/macrophage colony-stimulating factor
• IL-3 and GM-CSF were found to have overlapping biological activities, although with differing potency.
• interleukin-6 IL-6
• interleukin-1 1 IL-11
• 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.
• hematopoietic growth factors can exhibit their effect at different stages of cell development from the totipotent stem cell through various committed lineage-restricted progenitors to the mature blood cell.
• erythropoietin epo
• 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.
• 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 re- establishment of a diminished hematopoietic system caused by disease or after radiation- or chemo-therapy.
• megakaryocytic progenitor cells 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 ⁇ m in diameter having basophilic cytoplasm and a slightly irregular nucleus with loose, somewhat reticular chromatin and several nucleoli.
• 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 appearance of releasing platelets at their periphery. Normally, less than 10% of megakaryocytes are in the blast stage and more than 50% are mature.
• megakaryoblast for the earliest form
• promegakaryocyte or basophilic megakaryocyte for the intermediate form
• 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 (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 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 [ 1 968]) .
• MK-CSF megakaryocyte colony-stimulating factor
• 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.
• PWM- SpCM pokeweed mitogen-activated spleen cells
• WEHI-3CM murine myelomonocyte cell line WEHI-3
• 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, N.N., in Hematopoietic Cell Differentiation, ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. 10, Golde et al., eds.
• interleukin-3 IL-3
• multilineage colony stimulating factor multi-CSF [Burstein, Blood Cells, 11 :469 [1986]
• 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 (IL-1 ), in the induction of very early multipotential precursors and the formation of very large mixed hematopoietic colonies.
• EPO erythropoietin
• IL-1 interleukin-1
• megakaryocyte potentiators have been found in the conditioned media of murine lung, bone, macrophage cell lines, peritoneal exudate cells and human embryonic kidney cells. Despite certain conflicting data (Mazur, Exp. Hematol.,
• 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 development remains to be elucidated.
• 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 below 150 X 10 9 per liter. The major causes of thrombocytopenia can be broadly divided into three categories on the basis of platelet life span, namely; (1) impaired production of platelets by the bone marrow, (2) platelet sequestration in the spleen (splenomegaly), or (3) increased destruction of platelets in the peripheral circulation (e.g., 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.
• thrombocytopenia 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 10 9 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.
• 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 or radiation therapy), myelfibrosis, leukemia, and invasion of the bone marrow by metastatic tumor or granulomas.
• 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.
• ineffective thrombopoiesis secondary to megaloblastic processes can also cause thrombocytopenia, usually with coexisting anemia and leukopenia.
• 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 antifibrinoiytic agents. Thrombotic complications may develop, however, if antifibrinoiytic agents are used in patients with disseminated intravascular coagulation (DIC).
• DIC disseminated intravascular coagulation
• Splenomegaly due to any cause may be associated with mild to moderate thrombocytopenia. This is a largely passive process (hypersplenism) of splenic platelet sequestration, in contrast to the active destruction of platelets by the spleen in cases of immunomediated thrombocytopenia discussed below.
• hypersplenism 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.
• 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.
• 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 arise are set forth in greater detail in Braunwald et al.
• 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 damage of platelets in the extra corporeal circuit.
• ITP in adults is a chronic disease characterized by autoimmune platelet destruction.
• the autoantibody is usually IgG although other immunoglobulins have also been reported.
• the autoantibody of ITP has been found to be associated with platelet membrane GPIIblMa- the platelet antigen specificity has not been identified in most cases.
• Extravascular destruction of sensitized platelets occurs in the reticuloendothelial system of the spleen and liver.
• rheumatic or autoimmune diseases e.g., systemic lupus erythematosus
• lymphoproliferative disorders e.g., chronic lymphocytic leukemia.
• 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 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.
• 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 (H. Lane supra).
• the therapeutic approach to the treatment of patients with thrombocytopenia is dictated by the severity and urgency of the clinical situation.
• the treatment is similar for HIV-associated and non-HIV-related thrombocytopenia, and although a number of different therapeutic approaches have been used, the therapy remains controversial.
• glucocorticoid e.g., prednisolone
• the response is incomplete, or relapse occurs when the glucocorticoid dose is reduced or its administration is discontinued.
• glucocorticoids are usually administered if platelet count falls below 20 X 10 /liter or when spontaneous bleeding occurs.
• the compound For patients refractory to glucocorticoids, the compound:
• AATP amegakaryocytic thrombocytopenia purpura
• 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.
• surgical procedures are generally to be avoided in immune compromised patients, splenectomy is recommended only in severe cases of thrombocytopenia (e.g. 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.
• cytotoxic agents e.g., vincristine, and azidothimidine (AZT, zidovudine) also show promise in treating HIV-induced ITP; however, the results are preliminary.
• AZT azidothimidine
• TPO thrombopoietin
• TPO- active polypeptides While reports of partial purification of TPO- active 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 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 signal.
• V. Mpl is a Megakaryocytopoietic Cytokine Receptor
• IL-2 ⁇ and ⁇ chains
• IL-3 Itoh et al., Science, 247:324-328 [1990]
• IL-4 Mosley et al., Cell, 59:335-348 [1989]
• IL-5 Teakaki et al., EMBO J., 9 :4367-4374 [1990]
• IL-6 Yamamoto et al., Science, 241 :825-828 [1988]
• IL-7 Goodwin et al., Cell, 60:941-951 [1990]
• IL-9 Renault et al., Proc.
• GM- CSF granulocyte-macrophage colony-stimulating factor
• G-CSF granulocyte colony-stimulating factor
• 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]
• GH growth hormone
• CNTF ciliary neurotrophic factor
• the cytokine receptor superfamily may be grouped into three 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 intraceliular signal.
• a second class of receptors, so called ⁇ -subunits includes interleukin-6 receptor (IL6-R), granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R), interleukin-3 receptor (IL3-R ⁇ ) and other members of the cytokine receptor superfamily.
• IL6-R interleukin-6 receptor
• GM-CSF-R granulocyte-macrophage colony stimulating factor receptor
• IL3-R ⁇ interleukin-3 receptor
• ⁇ -subunits bind ligand with low affinity but cannot transduce an intraceliular signal.
• a high affinity receptor capable of signaling is generated by a heterodimer between an o -subunit and a member of a third class of cytokine receptors, termed ⁇ -subunits, e.g., ⁇ c , the common ⁇ -subunit for the three ⁇ - subunits IL3-R ⁇ and GM-CSF-R.
• mpl is a member of the cytokine receptor superfamily comes from sequence homology (Gearing, EMBO J., 8:3667-3676 [1988]; Bazan, Proc. Natl. Acad. Sci. USA, 87:6834-6938 [1990]; Davis et al., Science, 253:59-63 [1991] and Vigon et al., Proc. Natl. Acad. Sci. USA, 89:5640-5644 [1992]) and its ability to transduce proliferative signals.
• 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 C- terminal subdomain.
• the ligand-binding extracellular domains are predicted to have similar double ⁇ -barrel fold structural geometries.
• mpl may belong to the low affinity ligand binding class of cytokine receptors.
• mpl is reported to be one of the most conserved members of the cytokine receptor superfamily (Vigon supra).
• 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 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/mp/ chimera along with a full length G-CSFR control.
• Cells transfected with the chimera grew equally well in the presence of cytokine IL-3 or G-CSF.
• cells transfected with G-CSFR also grew well in either IL-3 or G-CSF. All cells died in the absence of growth factors.
• mpl is a single chain (class one) receptor like EPO-R or G-CSFR or it is a signal transducing ⁇ -subunit (class three) requiring an ⁇ -subunit like IL-3 (Skoda et al. supra).
• Mpl Ligand is a Thrombopoietin (TPO)
• TPO thrombopoietin
• CD34+ cells purified from bone marrow (BM) represents about 1% of all BM cells and are enriched in primitive and committed progenitors of all lineages (e.g., 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 [MK-CSA]). These same antisense oligodeoxynucleotides had no effect on erythroid or granulomacrophage colony formation.
• nucleic acid molecules encoding such protein ligands and to use these nucleic acid molecules to produce mpl binding ligands in recombinant cell culture for diagnostic and therapeutic use. It is yet another object to provide derivatives and modified forms of the protein ligands including amino acid sequence variants, variant giycoprotein forms and covalent derivatives thereof.
• the objects of the invention are achieved by providing 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.
• ML low ligand
• TPO thrombopoietin
• This substantially homogeneous protein may be purified from a natural source by a method comprising; (1) contacting a source plasma containing the mpl ligand molecules to be purified with an immobilized receptor polypeptide, specifically mpl 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, (2) washing the immobilized receptor polypeptide and its support to remove non-adsorbed material, and (3) eluting the mpl ligand molecules from the immobilized receptor polypeptide to which they are adsorbed with an elution buffer.
• the natural source is mammalian plasma or urine containing the mpl ligand.
• the mammal is aplastic and the immobilized receptor is a mpl- ⁇ gG fusion.
• the prefered megakaryocytopoietic proliferation and maturation promoting protein is an isolated substantially homogeneous mpl ligand polypeptide made by synthetic or recombinant means.
• the "mpl ligand" polypeptide or "TPO” of this invention preferably has at least 70% overall sequence identity with the amino acid sequence of the highly purified substantially homogeneous porcine mpl ligand polypeptide and at least 80% sequence identity with the "EPO-domain" of the porcine mpl ligand polypeptide.
• 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: 1), or a variant or posttranscriptionally modified form thereof or a protein having about 80% sequence identity with mature human mpl ligand.
• the mpl ligand variant is a fragment, especially an amino-terminus or "EPO-domain" fragment, of the mature human mpl ligand (hML).
• the amino terminus fragment retains substantially all of the human ML sequence between the first and forth cysteine residues but may contain substantial additions, deletions or substitutions outside that region.
• the fragment polypeptide may be represented by the formula:
• hML(7-151) represents the human TPO (hML) amino acid sequence from Cys 7 through Cys 151 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, proteolytic cleavage sites (e.g. Factor Xa or thrombin)
• Y represents the carboxy terminal group of Cys 1 51 or one or more carboxy-terminus amino acid residue(s) of the mature hML or extensions thereto.
• 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 kit- iigand (KL), IL-1 , IL-3, IL-6, IL-11 , EPO, GM-CSF or LIF.
• An optional preferred heterologous polypeptide is an immunoglobin chain, especially human lgG1 , lgG2, lgG3, lgG4, IgA, IgE, IgD, IgM or fragment thereof, especially comprising the constant domain of an IgG heavy chain.
• compositions comprising an isolated mpl agonist that is biologically active and is preferably capable of stimulating the incorporation of labeled nucleotides (e.g., ⁇ H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected with human mpl.
• labeled nucleotides e.g., ⁇ H-thymidine
• the mpl agonist is biologically active mpl ligand and is preferably capable of stimulating the incorporation of 355 j n t 0 circulating platelets in a mouse platelet rebound assay.
• Suitable mpl agonist include hMLi 53, hML(R153A, R154A), hML2, hML3, hML4, mML, mML2, mML3, pML, and pML2 or fragments thereof.
• this invention provides an isolated antibody capable of binding to the mpl ligand.
• 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.
• 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.
• the invention provides an isolated nucleic acid 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 ligand, and include RNA and DNA, both genomic and cDNA.
• 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 recognized by a host transformed with the vector.
• the DNA is cDNA having the sequence provided in Fig. 1 5'-3' (SEQ ID NO: 2), 3'-5' 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 therapeutically effective amount of a mpl ligand to the mammal.
• a 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 (KL), 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:
• 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, 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.
• the oxidizing agent is oxygen or a compound containing at least one disulfide bond and the reducing agent is a compound containing at least one free sulfhydryl.
• the oxidizing agent is selected from oxidized glutathione(GSSG) and cystine and the reducing agent is selected from reduced glutathione(GSH) and cysteine.
• 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 Ieast1%.
• the redox buffer additionally contains NaCl preferably at a concentration range of about 0.1-0.5M, and 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:
• Fig. 1 shows the deduced amino acid sequence (SEQ ID NO: 1) of human mpl ligand (hML) cDNA and the coding nucleotide sequence (SEQ ID NO: 2). 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 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 ⁇ H-thymidine incorporation assay.
• the mpl P Ba/F3 cells were starved of IL-3 for 24 hours in a humidified incubator at 37°C in 5% CO2 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.
• Fig. 3 shows the effect of pronase, DTT and heat on the ability of APP to stimulate Ba/F3-mpl cell proliferation.
• pronase Boehringer Mannheim
• bovine serum albumin was coupled to Affi-gel10 (Biorad) and incubated individually with APP for 18hrs. at 37°C. Subsequently, the resins were removed by centrifugation and supernatants assayed.
• APP was also heated to 80°C for 4 min. or 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-mp/ fractions.
• acetone containing 1mM HCI at -20°C was added. After 3hrs. at -20°C samples were centrifuged and resultant pellets were washed 2x with acetone at
• Fig. 6 shows elution of mpl ligand activity from SDS-PAGE.
• Fraction 6 from the mp/-affinity column was resolved on a 4-20% SDS-polyacrylamide gel under non ⁇ reducing conditions. Following electrophoresis the gel was sliced into 12 equal regions and electroeluted as described in the examples. The electroeluted samples were 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 / ⁇ .p/-affinity column (700 ⁇ g p/-lgG/ml NHS-superose, Pharmacia).
• Human peripheral stem cell 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 mp/-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, mp/-lgG (0.5 ⁇ g) or ANP-R-lgG (0.5 ⁇ g) 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.
• 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: 3), the coding sequence (SEQ ID NO: 4), and its compliment (SEQ ID NO: 5) are shown.
• 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: 7).
• 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-glycosyiation 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
• Fig. 11 shows deduced amino acid sequence of mature human mpl ligand isoforms hML (SEQ ID NO: 6), hML2 (SEQ ID NO: 8), hML3 (SEQ ID NO: 9), and hML4 (SEQ ID NO:
• Figs. 12A, 12B and 12C show the effect of human mpl ligand on Ba/F3-mp/ cell proliferation (A), in vitro human megakaryocytopoiesis quantitated using a radiolabeled murine IgG monoclonal antibody specific to the megakaryocyte glycoprotein GPIIbMla (B), and murine thrombopoiesis measured in a platelet rebound assay (C).
• rML partially purified recombinant ML
• C in vivo platelet production
• 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 NaCl.
• the active fraction was dialyzed into PBS and made 1 mg/ml with endotoxin free BSA.
• the sample contained less than one unit of endotoxin /ml. Mice were injected with either 64,000,
• Fig. 13 compares the effect of human mpl ligand isoforms and variants in the Ba/F3- mpl cell proliferation assay.
• hML, mock, hML2, hML3, hML(R153A, R154A), and hML-) 53 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-rhML332 and purified 293-rhML-
• 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 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 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 deleations found as a result of alternative splicing (mML2 and mML3) are underlined.
• the four cysteine residues are indicated by a dot. The seven potential N- glycosylation 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 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.
• 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: 6).
• 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.
• 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.
• Fig. 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 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: 21). The predicted amino acid sequence for the pML is aligned with pML2 sequence.
• Fig. 23 shows the pertinent features of plasmid pSVI5.ID.LL.MLORF ("full length” or TPO332) used to transfect host CHO-DP12 cells for production of CHO-rhTP ⁇ 332-
• Fig. 24 shows the pertinent features of plasmid pSVI5.ID.LL.MLEPO-D ("truncated" or TPO153) used to transfect host CHO-DP12 cells for production of CHO-rhTPOi53.
• Figs. 25A, 25B, and 25C show the effect of E. co//-rhTPO(Met " 1 , 153) on platelets (A), red blood cells (B) and (C) white blood cells in normal mice.
• Two groups of 6 female C57 B6 mice were injected daily with either PBS buffer or 0.3 ⁇ g E. co/ -rhTPO(Met "1 , 153) (100 ⁇ l sc).
• Figs. 26A, 26B and 26C show the effect of E. co//-rhTPO( et " 1 , 153) on platelets (A), red blood cells (B) and (C) 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 37 Cs source and injected daily with either PBS buffer or 3.0 ⁇ g E. co/// ' -rhTPO(Met "1 , 153) (100 ⁇ l sc).
• PBS buffer 3.0 ⁇ g E. co/// ' -rhTPO(Met "1 , 153) (100 ⁇ l sc).
• 40 ⁇ l of blood was taken from the orbital sinus. This biood 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
• Figs. 27A, 27B and 27C show the effect of CHO-rhTP ⁇ 332 on (A) platelets (thrombocytes), (B ) red blood cells (erythrocytes) and (C ) white blood cells (leukocytes) in normal mice.
• Two groups of 6 female C57 B6 mice were injected daily with either PBS buffer or 0.3 ⁇ g CHO-rhTP ⁇ 332 (100 ⁇ l sc). On day 0 and on days 3-7 40 ⁇ l 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.
• 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: hTP0332 from CHO (full length from Chinese hamster ovary cells); hTPOMet " 1 153 (E- co//-derived truncated form with an N-terminal methionine); hTP ⁇ 332 (full length TPO from human 293 cells); Met-less 155 E-Coli (the truncated form [rhTPO-i ss] without the terminal methionine from E. coli) .
• TPO-j 53 the truncated form of TPO from E. coli
• TPO332 (Mix fraction) Full length TPO containing approximately 80-90% full length and 10-20% clipped forms
• TPO332(30K fraction) purified clipped fraction from the original "mix” preparation
• TPO332(70K fraction) purified full length TPO fraction from the original "mix” preparation.
• the data are presented as means ⁇ Standard error of 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 the final construct (right portion of the figure) and a flow diagram (left portion of the figure) showing relavant 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 expression vector used for expression of Rse.gD in Example 17.
• Fig. 33 is a schematic representation of the preperation of plasmid pMP1.
• Fig. 34 is a schematic representation of the preperation of plasmid pMP21.
• Fig. 35 is a schematic representation of the preperation of plasmid pMP151.
• Fig. 36 is a schematic representation of the preperation of plasmid pMP202.
• Fig. 37 is a schematic representation of the preperation of plasmid pMP172.
• Fig. 38 is a schematic representation of the preperation of plasmid pMP210.
• Fig. 39 is a table of the five best expressing TPO clones from the pMP210 plasmid bank (SEQ ID NOS: 23, 24, 25, 26, 27 and 28).
• Fig. 40 is a schematic representation of the preperation of plasmid pMP41.
• Fig. 41 is a schematic representation of the preperation of plasmid pMP57.
• Fig. 42 is a schematic representation of the preperation of plasmid pMP251.
• 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, guanidine-HCI, and sodium thiocyanate. High concentrations, usually 4-9M, of these compounds are normally required to exert the conformational effect on proteins.
• Cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
• cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone, insulin-like growth factors, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and leutinizing hormone (LH), hematopoietic growth factor, hepatic growth factor, fibrobiast growth factor, prolactin, placental lactogen, tumor necrosis factor- ⁇ (TNF- ⁇ and TNF- ⁇ ) mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, nerve growth factors such as N
• mpl 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; e.g., differing in amino acid sequence by one or more amino acids or in type or extent of glycosylation.
• mpl ligand “mpl ligand polypeptide”, “ML”, “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 (e.g., ⁇ 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 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 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 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.
• N-terminal fragments are hML-
• Mpl ligand variants or "mpl ligand sequence variants” as defined herein means a biologically active mpl ligand as defined below having less than 100% 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: 1).
• 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: 1]), 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.
• 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 (1 ) to obtain at least 15 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 (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Homogeneity here means 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 fragment thereof.
• Effector functions includemp/ 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 function means possession of an epitope or antigenic site that is capable of cross- reacting 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 10 6 l/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 10 7 i/mole.
• 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 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 35 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 GPIIbllla-
• 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 porcine plasma or the murine or human ligand having the deduced amino acid sequence described in Fig. 1 (SEQ ID NO: 1), 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 sequence shall be construed as affecting sequence identity or homology.
• exemplary biologically active mpl ligand polypeptides considered to have identical sequences include; prepro-mp/ ligand, pro- p/ ligand, and mature mpl ligand.
• Mpl ligand microsequencing may be accomplished by any appropriate standard procedure provided the procedure is sensitive enough.
• 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.
• PTH phenylthiohydantion
• mpl ligand fragments prepared by chemical (e.g., CNBr, hydroxylamine, 2-nitro-5-thiocyanobenzoate) or enzymatic (e.g., trypsin, clostripain, staphylococcal protease) digestion followed by fragment purification (e.g., 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 al., J. Chromatography, 404:41 -52 [1987].
• 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 N- terminal sequencing is carried out with the gas phase sequenator described above.
• HPLC fractions are dried under vacuum (SpeedVac), resuspended in appropriate buffers, and digested with cyanogen bromide, the Lys- specific enzyme Lys-C (Wako Chemicals, Richmond, VA), or Asp-N (Boehringer Mannheim, Indianapolis, IN). After digestion, the resultant peptides are sequenced as a mixture or after HPLC resolution on a C4 column developed with a propanol gradient in 0.1% TFA prior to gas phase sequencing.
• Lys-C Lys-specific enzyme
• Asp-N Boehringer Mannheim, Indianapolis, IN
• 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-GPII Ml a ) for a human leukemia megakaryoblastic cell line (CMK), and induction of polyploidization in a megakaryoblastic cell line (DAMI).
• TPO Thrombopoietin
• TPO is defined as a compound having thrombopoietic activity or being capable of increasing serum platelet counts in a mammal. TPO is 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 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 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 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.
• isolated mpl ligand nucleic acid is RNA or DNA that encodes a biologically active tTp/ 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.
• Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
• "Operably linked” when referring to nucleic acids means that the nucleic acids are placed in a functional relationship with another nucleic acid sequence.
• 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.
• "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 nucleic acid in which the element is ordinarily not found.
• Cell Cell
• cell line cell line
• cell culture are used interchangeably herein and such designations include all progeny of a cell or cell line.
• 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.
• 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.
• restriction enzymes commonly are designated by abbreviations composed of a capital letter followed by other letters representing the microorganism from which each restriction enzyme originally was obtained and then a number designating the particular enzyme.
• plasmid or DNA fragment is used with about 1-2 units of enzyme in about 20 ⁇ l of buffer solution.
• buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer.
• Incubation of about 1 hour at 37 C C 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 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.
• digestion of plasmids is not followed by 5' terminal dephosphorylation.
• Procedures and reagents for dephosphorylation are conventional as described in sections 1.56-1.61 of Sambrook et al., Molecular Cloning: A Laboratory Manual [New York: Cold Spring Harbor Laboratory Press, 1989].
• 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, 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., 9:6103-61 14 [1981], and Goeddel et al., Nucleic Acids Res., 8:4057 [1980].
• Southern analysis or “Southern blotting” is a method by which the presence 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 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.
• 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 32 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, 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.
• 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.
• the DNA is treated in a suitable buffer for at least 15 minutes at 15°C with about 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of the four deoxyribonucleotide triphosphates.
• the DNA is then purified by phenol-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 ⁇ g of DNA.
• 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 large- and 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 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 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.
• oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified.
• the 5' 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 or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant.
• 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 to amplify or generate a specific piece of nucleic acid.
• “Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCI/0.0015 M sodium citrate/0.1% NaDodS ⁇ 4 (SDS) at 50°C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
• a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
• Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC and 0.1 % SDS.
• Modely stringent conditions are described in Sambrook et al., supra, and include the use of a washing solution and hybridization conditions (e.g., 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 NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 X Denhardt's solution, 10% dextran sulfate, and 20 ⁇ l/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 X SSC at about 37-50°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 are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like 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 (L) chains and two identical heavy (H) 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 variable domain (V
• Each light chain has a variable domain at one and (V 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 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]).
• 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 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 (FR).
• CDRs complementarity determining regions
• FR framework
• the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -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.
• 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 V
• the six CDRs confer antigen binding specificity to the antibody.
• 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 as pairs of Fab' fragments which have hinge cysteines between them. Other, chemical couplings of antibody fragments are also known.
• immunoglobulins 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), e.g., lgG-1 , lgG-2, lgG-3, and lgG-4; IgA- 1 and lgA-2.
• immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., l
• the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , delta, epsilon, ⁇ , and ⁇ , respectively.
• the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
• 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 (e.g., Fab,
• the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., 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 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.
• 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, e.g., U.S. Patent No. 4,816,567 [Cabilly et al.]).
• 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 the chain(s) is identical with or homologous to corresponding sequences in antibodies derived 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 (U.S. Patent No. 4,816,567 (Cabilly et al.); and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851 -6855 [1984]).
• 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 the chain(s) is
• Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
• 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.
• CDR complementary determining region
• donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
• humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• 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 ail 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 (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• 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 (e.g., 8 to 14 days).
• Preferred polypeptides of this invention are substantially homogeneous polypeptide(s), referred to as mpl ligand(s) or thrombopoietin (TPO), that possesse the 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 mpl P.
• mpl ligand(s) or thrombopoietin (TPO) that possesse the 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 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.
• these human mpl ligand(s) lack glycosylation.
• hML human mpl ligands
• EPO-domain of hML refered to as .1ML153 or hTPO-
• a truncated form of hML refered to as hML245 or hTP0245
• the mature full length polypeptide having the amino acid sequence shown in Fig. 1 (SEQ ID NO: 1), refered to as hML, hML332 or hTP0332 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: 1]), the murine mpl ligand (see Fig. 16 [SEQ ID NOS: 12 & 13]), the recombinant porcine mpl ligand (see Fig.
• the porcine mpl ligand isolated from aplastic porcine plasma has the following characteristics:
• the ligand's activity is destroyed by pronase;
• the ligand is stable to low pH (2.5), SDS to 0.1%, and 2M urea;
• the ligand is a glycoprotein, based on its binding to a variety of lectin columns;
• 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;
• mpl ligand polypeptides are those encoded by human genomic or cDNA having an amino acid sequence described in Fig. 1 (SEQ ID NO: 1).
• mpl l igand polypeptides of this invention include prepro-mp/ ligand, pro-mp/ ligand, mature mpl ligand, mpl iigand fragments and glycosylation variants thereof.
• preferred polypeptides of this invention include mpl ligand sequence variants and chimeras.
• preferred mpl ligand sequence variants and chimeras are biologically active mpl iigand 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 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 erythropoietin).
• the prefered hML EPO-domain comprises about the first 153 amino acid residues of mature hML and is refered to as hMLi53.
• 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) (e.g., hydrophobic, neutral, acidic, aromatic, Gly, Pro and the like).
• a prefered hML C-terminal domain sequence variant comprises one in which Arg residues 153 and 154 are replaced with Ala residues.
• hML332 This variant is refered to as hML332(R153A, R154A).
• An alternative prefered hML variant comprises either hML332 or hML-
• the foregoing deletion mutants are refered to as ⁇ 4hML332 or ⁇ 4hML-
• a preferred chimera is a fusion between mpl ligand or fragment (defined below) thereof with a heterologous polypeptide or fragment thereof.
• hMLi 53 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: 7).
• 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: 6).
• 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 ⁇ -helical bundles at positions (EPO) 9-22, 59-76, 90-107, and 132-152; and other highly conserved regions including the N-terminus and C- terminus regions and residue positions (epo) 44-52 (see e.g., Wen et al., Blood, 82:1507-1516 [1993] and Boissel et al., J. Biol. Chem., 268(21 ): 15983-15993 [1993]). It is contemplated this "ML-EPO domain chimera" will have mixed thrombopoietic-erythropoietic (TEPO) biological activity.
• TEPO thrombopoietic-erythropoietic
• 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 iigand isolated from aplastic 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 1-X).
• Other preferred mpl ligand fragments include those produced as a result of chemical or enzymatic hydrolysis or digestion of the purified ligand.
• the source containing the mpl iigand may be plasma where the immobilized receptor is preferably a mp/-lgG fusion.
• 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.
• concentration of mpl ligand in either the culture medium or in cell lysates is generally higher than in plasma or other natural sources.
• the above described mpl gG immunoaffinity method while still useful, is usually not necessary and more traditional protein purification methods known in the art may be applied.
• 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; immunoaffinity, ion-exchange (e.g., 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 (e.g., silica gel with appended aliphatic groups) or Sephadex molecular seive or size ex
• this invention provides an isolated antibody capable of binding to the mpl ligand.
• a preferred mpl iigand 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 Publishers, Amsterdam [1985]; and Huse et al., Science, 246:1275-1281 [1989]).
• Preferred mpl ligand isolated antibody is one that binds to mpl ligand with an affinity of at least about 10 6 l/mole. More preferably the antibody binds with an affinity of at least about 10 7 l/mole.
• 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.
• 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.
• the invention provides an isolated nucleic acid molecule encoding the mpl ligand or fragments thereof, which nucleic acid 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 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 90%, and most preferably 95% sequence identity with the human mpl ligand.
• More preferred isolated nucleic acid molecules are DNA sequences encoding biologically active mpl ligand, selected from: (a) DNA based on the coding region of a mammalian mpl ligand gene (e.g., DNA comprising the nucleotide sequence provided in Fig.
• 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.
• a further aspect of this invention includes DNA that hybridizes under low to moderate stringency conditions with DNA encoding the mpl ligand polypeptides.
• the nucleic acid molecule is cDNA encoding the mpl ligand and further comprises a repiicable 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 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.
• the mpl ligand is administered in combination with a cytokine, especially a colony stimulating factor or interleukin.
• a colony stimulating factor or interleukin 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.
• 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- CSF) and thrombopoietin, regulate megakaryocytopoiesis and thrombopoiesis
• meg- CSF megakaryocyte colony-stimulating factor
• thrombopoietin thrombopoietin
• meg-CSF stimulates the proliferation of progenitor megakaryocytes while thrombopoietin primarily affects maturation of more differentiated cells and ultimately platelet release.
• aplastic porcine plasma obtained from irradiated pigs stimulates human megakaryocytopoiesis in vitro. We have found that this stimulatory activity is abrogated by the soluble extracellular domain of c-mpl, confirming APP as a potential source of the putative mpl ligand (ML).
• ML mpl ligand
• aplastic plasma from a variety of species has been reported to contain activities that stimulate hematopoiesis in vitro, however no hematopoietic 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.
• APP contained the mpl ligand To determine if APP contained the 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 Fig. 2.
• APP stimulated 3 H-thymidine incorporation into Ba/F3-mpl cells but not
• the activity in APP appeared to be a protein since pronase, DTT, or heat destroy the activity in APP (Fig. 3). 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 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- ⁇ gG 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 mp/-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 mpl- affinity column has an estimated specific activity of 9.8 x10 6 units/mg.
• HIC hydrophobic interaction chromatography
• immobilized dye chromatography immobilized dye chromatography
• mp/-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 mpl- affinity column has an estimated specific activity of 9.8 x10 6 units/mg.
• Blue-Sep. 640 0.93 400 256 , 000 430 1 28 538 mpl ( ⁇ l ) (Fxns 5-7) 1 2 5x 1 0- 4 1 666 20 , 000 3 , 300 ,000 1 0 4, 1 00 ,000
• Protein was determined by the Bradford assay. Protein concentration of mp/-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-mp/ cell proliferation.
• the amino acid 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 pR45, was designed and synthesized based on the sequence of the PCR fragment. This oligonucleotide had the following sequence:
• SEQ ID NO: 34 This deoxyoligonucleotide was used to screen a human genomic DNA library in ⁇ gem12 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 45-mer 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 & 4). The predicted positions of introns in the genomic sequence are also indicated by arrows, and define a putative exon ("exon 3").
• 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.
• a fetal iiver 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 Iiver 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 NOS: 1 & 2).
• 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 5' untranslated sequence and a 3' untranslated region of 498 nucleotides.
• the presumed initiation codon at nucleotide position (216-218) is within a consensus 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 highly hydrophobic and probably corresponds to a signal peptide.
• 332 amino acid residue ligand is about 38 kDa. There are 6 potential N-glycosylation sites and 4 cysteine residues.
• 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 Iiver. After longer exposure, a weaker band of the same size could be detected in adult kidney.
• human erythropoietin is expressed in fetal Iiver and, in response to hypoxia, the adult kidney and iiver (Jacobs et al., Nature, 313:804-809 [1985] and Bondurant et al., Molec. Cell. Biol., 6:2731 -2733 [1986]).
• M r 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.
• the non-glycosylated form has full in vitro biological activity, but has a significantly reduced plasma half-life relative to glycosylated erythropoietin (Takeuchi et al., J. Biol. Chem., 265: 12127 '-12130
• 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.
• Argi 53-Argi 54 sequence occurs immediately following the erythropoietin-like domain of the ML.
• Isoforms and Variants of the Human mpl Ligand Isoforms or alternatively spliced forms of human mpl ligand were detected by PCR in human adult iiver. 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 Iiver RNA as described in Example 10.
• hML 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 & 10).
• 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.
• 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.
• 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-hMLi 53.
• Supematants from transiently transfected human embryonic kidney 293 cells were 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 mp/-lgG to the media abolished the stimulation (data not shown).
• mice 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. Biol. Med., 14:1006-1001 [1973] and McDonald et al., Scand. J. Haematol., 16:326-334 [1976]).
• 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. Exp. Biol.
• mice just as exhypoxic mice are more sensitive to erythropoietin than normal are mice (McDonald, et al., J. Lab. Clin. Med., 77:134-
• hML2 and hML3 showed no detectable activity in this assay, however the activity of hML(R153A, R154A) was similar to hML and hML-1 53 indicating that processing at the Argi53-Arg-
• 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 evidence.
• APP stimulates both proliferation and maturation of human megakaryocytes in vitro, and this stimulation is completely inhibited by mpl- IgG (Figs. 7 and 8). 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 transfected (Skoda et al., EMBO, 12:2645-2653 [1993] and Vigon et al., Oncogene,
• TPO Human mpl Ligand
• 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. B., et al., Nucl. Acids Res. 15:7155 [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 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.
• FISH fluorescent in situ hybridization
• cDNA corresponding to the TPO entire open reading frame 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 pRK ⁇ tkneo (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.
• a second vector coding for the EPO homologous domain was generated the same but using different PCR primers to obtain the final construct called pRK5-tkneoEPO- D.
• rhML332 Purification of rhML332 was conducted as described in Example 19. Briefly, 293-rhML332 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 1 M NaCl. The 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 1 M NaCl. 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 NaCl and eluted with the same buffer containing 0.5M N-acetyl-D-glucosamine.
• the WGA-Sepharose eluate was applied to a C4-HPLC column (Synchrom, Inc.) and eluted with a discontinuous propanol gradient.
• SDS-PAGE the purified 293-rhML332 migrates as a broad band in the 68-80 kDa region of the gel (see Fig. 15).
• rhML-153 Purification of rhML-153 was also conducted as described in Example 19. Briefly, 293-rhML-
• 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 2p-dCTP. This probe was used to screen 10 ⁇ clones of a mouse Iiver cDNA library in ⁇ GT10.
• 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 Biol., 108:229-241 [1989]). This sequence defines an open reading frame of 1056 nucleotides, which predicts a primary translation product of
• 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. 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.
• 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 is shown in Fig. 18 (SEQ ID NOS: 9 & 16).
• Argi 53-Argi54 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 H-thymidine incorporation into Ba/F3 cells expressing either murine or human mpl but had no effect on the parental (mp/-less) cell line.
• pML pig mpl ligand of 332 amino acid resudues
• Fig. 20 SEQ ID NOS: 18 & 19
• pML2 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: 21]).
• a second di-basic site present at position 245 and 246 in the human sequence is not present in the mouse or pig sequences.
• the murine and the pig ML sequence contain 4 cysteines, all conserved in the human sequence.
• Ovary (CHO) Cells The expression vectors used to transfect CHO cells are designated: pSVI5.ID.LL.MLORF (full length or TPO332). and pSVI5.ID.LL.MLEPO-D (truncated or TPO153). The pertinent features of these plasmids are presented in Fig. 23 and 24.
• Example 20 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.ID.LL.MLEPO-D.
• Example 20 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 1.0M NaCl.
• HCCF harvested cell culture fluid
• Phamacia Blue Sepharose column
• TPO is then eluted with 3 to 5 column volumes of buffer containing both 2.0M urea and 1.0M NaCl.
• the Blue Sepharose eluate pool containing TPO is then applied to a Wheat Germ Lectin Sepharose column (Pharmacia) equilibrated in the Blue Sepharose eluting buffer at a ratio of from 8 to 16 ml of Blue Sepharose eluate per ml of resin.
• the column is then washed with 2 to 3 column volumes of equilibration buffer.
• TPO is then eluted with 2 to 5 column volumes of a buffer containing 2.0M urea and 0.5M N- acetyl-D-glucosamine.
• the Wheat Germ Lectin eluate containing TPO is then acidified and Ci 2 E 8 - s 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% C12E8 and a pool is made on the basis of SDS-PAGE.
• 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 processed or further concentrated by ultrafiltration.
• the diafiltrate/concentrate is usually adjusted to a final concentration of 0.01% Tween-80.
• 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 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 intraceliular 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 intraceliular expression plasmids.
• TPO expression plasmids were used to transform the E. coli using the CaCl2 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°C until the optical density (600nm) of the culture reached approximately 2-3. The culture was then diluted and, after growth with aeration, acid was added. The culture was then allowed to continue growing with aeration for another 15 hours after which time the cells were harvested by centrifugation.
• CaCl2 heat shock method Mondel, M. et al. J. Mol. Biol., 53:159-162, [1970]
• a microorganism such as E. coli expressing TPO encoded by any suitable plasmid is fermented under conditions in which TPO is deposited in insoluble "retractile bodies".
• cells are first washed in a cell disruption buffer.
• a cell disruption buffer e.g. 10 mM Tris, 5 mM EDTA, pH 8
• a Polytron homogenizer for example, a Polytron homogenizer
• 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, chemical or enzymatic methods.
• 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' instructions.
• the particulate matter containing TPO is then separated from the liquid phase and optionally washed with any suitable liquid.
• 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 retractile body pellet.
• the washed pellet may be used immediately or optionally stored frozen (at e.g. -70°C).
• 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.
• 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 preferably 8.0.
• the solubilizing buffer also contains a reducing agent to 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).
• the solubilizing buffer may contain a mild oxidizing agent (e.g. molecular oxygen) and a sulfite salt to form monomeric TPO via sulfitolysis.
• a mild oxidizing agent e.g. molecular oxygen
• a sulfite salt to form monomeric TPO via sulfitolysis.
• the resulting TPO-S-sulfonate is later refolded in the presence of the redox buffer (e.g. 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.
• the following procedure has produced suitable yields of monomeric TPO.
• the retractile 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 25 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.
• the solution is centrifuged at 30,000 x g for 30 min.
• 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.
• TFA trifluoroacetic acid
• the protein is 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.
• TPO Refolding TPO to Generate the Biologically Active Form
• 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 only a small amount of properly folded material ( ⁇ 10%) is obtained. For commercial manufacturing processes, it is desirable to have refolding yields at least 10%, more preferably 30-50% and most preferably >50%.
• Triton X-100 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 some properly folded material.
• the most preferred detergents were those of the CHAPS family (CHAPS and CHAPSO) which were found to work best in the refolding reaction and to limit protein aggregation and improper disulfide formation.
• Levels of CHAPS greater than about 1% were most preferred.
• Sodium chloride was required for the best yields, with the optimal levels between 0.1 M and 0.5M.
• EDTA metal-catalyzed oxidation (and aggregation) which was observed with some preparations.
• Glycerol concentrations of greater than 15% produced the optimal refolding conditions.
• RSH reduced organic thiol
• Suitable 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
• Tris and phosphate buffers were generally useful. Incubation at 4 °C also produced higher levels of properly folded TPO.
• Refolding yields of 40-60% are typical for preparations of TPO that have been purified through the first C4 step.
• Active material can be obtained when less pure preparations (e.g. directly after the Superdex 200 column or after the initial retractile body extraction) although the yields are less due to extensive precipitation and interference of non-TPO proteins during the TPO refolding process.
• TPO contains 4 cysteine residues
• version 1 disulfides between cysteine residues 1-4 and 2-3
• version 2 disulfides between cysteine residues 1-2
• 3-4 version 3 disulfides between cysteine residues 1-3 and 2-4.
• 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 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 erythropoietin.
• Refolded and purified TPO has activity in both in vitro and in vivo assays.
• 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).
• the mpl receptor-based ELISA half-maximal activity occurred at 1.9 ng/ml (120 pM).
• refolded 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. Similar biological activity was observed for other forms of TPO refolded in accordance with the above described procedures (see Figs. 25, 26 and 28).
• Thrombopoietic activity may be measured in various assays including the Ba/F3 mpl iigand 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-GPIIbllla) or a human leukemia megakaryoblastic cell line (CMK) (see Sato et al., Brit. J.
• 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 (i.e., the mpl ligand) of megakaryocyte maturation would be expected to induce at least some of these changes in immature megakaryocytes leading to platelet release and alleviation of thrombocytopenia.
• assays were designed to measure the emergence of these parameters in immature megakaryocyte cell lines, i.e., CMK and DAMI cells.
• the CMK assay (Example 4) measures the appearance of a specific platelet marker, GPIIbllla. 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.
• the in vivo mouse platelet rebound assay (Example 16) is useful in demonstrating that administration of the test compound (here the mpl ligand) results in elevation of platelet numbers.
• kinase receptor activation (KIRA) ELISA in which CHO cells are transfected with a mpl-Rse chimera and tyrosine phosphorylation of Rse is measured by ELISA after exposure of the mpl portion of the chimera to mpl ligand (see Example
• the second is a receptor based ELISA in which ELISA plate coated rabbit anti- human IgG captures human chimeric receptor mpMgG which binds the mpl ligand being assayed.
• a biotinylated rabbit polyclonal antibody to mpl iigand (TPO155) is used to detect bound mpl ligand which is measured using streptavidin-peroxidase as described in Example 18.
• mice 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.
• CHO Chinese hamster ovary
• E. coli E. coli
• 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 normal female C57 B6 mice as described in the legends to Figs. 25A, 25B and 25C.
• 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.
• Dose response curves were constructed for treatment of normal mice with rhTPO from various cell lines (CHO-rhTP ⁇ 332; E. co// ' -rhTPO(Met "1 ,153); 293- rhTP0332; and E. co//-rhTPO-
• This figure shows that all tested forms of the molecule stimulate platelet production, however the full length form produced in CHO has the greatest in vivo activity.
• Dose response curves were also constructed for treatment of normal mice with various forms of rhTPO produced in CHO (CHO-rhTPO-
• 70 Kda form has the greatest in vivo activity.
• 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.
• 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 iigand.
• 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 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.
• 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 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.
• 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. 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.
• 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
• 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 Iiver cell lines.
• tissues preferably human or porcine kidney (adult or fetal) or Iiver cell lines.
• human fetal liver cell line cDNA libraries are screened with the oligonucleotide probes.
• 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 (e.g., 32 ?) and polynucleotide kinase to radiolabel the 5' end of the oligonucleotide.
• ATP e.g., 32
• polynucleotide kinase to radiolabel the 5' end of the oligonucleotide.
• other methods may be used to label the oligonucleotide, including, but not limited to, biotinylation or enzyme labeling.
• the mpl ligand nucleic acid that encodes a full-length mpl ligand polypeptide.
• 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.
• 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 mpl ligand.
• 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 post- translational processes of the mpl ligand, such as changing the number or position of glycosylation sites.
• 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, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
• 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].
• a residue or group of target residues are identified (e.g., 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 sites of substitution.
• 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 activity.
• variants of the mpl ligand polypeptide include variants from the mpl ligand sequence, and may represent naturally occurring alleles (which will not require manipulation of 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.
• 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, more preferably about 1 to 10 residues, and typically are contiguous.
• 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.
• 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 iigand more significantly.
• the number of consecutive deletions will be selected so as to preserve the tertiary structure of mpl ligands in the affected domain, e.g., 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 i.e., insertions within the mature mpl ligand sequence
• An exemplary preferred fusion is that of mpl ligand or fragment thereof and another cytokine or fragment thereof.
• 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 hosts.
• 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.
• insertional variants of the mpl ligand molecule include the fusion to the N- or C-terminus of mpl ligand of immunogenic polypeptides (i.e., not endogenous to the host to which the fusion is administered), e.g., bacterial polypeptides such as beta- lactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, and C- terminal fusions with proteins having a long half-life such as immunoglobulin constant regions (or other immunoglobulin regions), albumin, or ferritin, as described in WO 89/02922 published 6 April 1989.
• a third group of variants are amino acid substitution variants.
• These variants have at least one amino acid residue in the mpl ligand molecule removed and a different residue 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 side- chain 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.
• 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 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 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.
• Substantial modifications in function or immunological identity of the mpl ligand are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
• Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, He;
• 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- conserved) sites.
• 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, e.g., for an arginyl or iysinyl residue.
• protease cleavage sites are identified, they are 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.
• any methionyl residues other than the starting 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.
• another residue preferably in accordance with Table 3
• about 1- 3 residues are inserted adjacent to such sites.
• Any cysteine residues not involved in maintaining the proper conformation of 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 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 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.
• 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 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 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].
• 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
• single-stranded DNA template may be generated by denaturing double-stranded plasmid (or other) DNA using standard techniques.
• 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 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.
• the cells are grown, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabeled with 32-phosphate to identify the bacterial 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.
• 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.
• dCTP-(aS) deoxyriboadenosine
• dGTP deoxyriboguanosine
• dTTP deoxyribothymidine
• 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 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 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.
• 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 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.
• this template already contains one or more mutations.
• the oligonucleotide encoding the additional desired amino acid 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 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.
• 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 strand of the plasmid, but this sequence can be located anywhere along the plasmid
• 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.
• template plasmid DNA (1 ⁇ g) 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.
• 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 ⁇ l.
• the reaction mixture is overlayed with 35 ⁇ l mineral oil.
• Thermus aquaticus (Taq) DNA polymerase (5 units/ ⁇ l, purchased from Perkin-Elmer Cetus) is added below the mineral oil layer.
• the reaction mixture is then inserted into a DNA Thermal Cycler (purchased from Perkin-Elmer Cetus) programmed as follows:
• 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.
• the starting material is 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.
• 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 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.
• nucleic Acid e.g., cDNA or genomic DNA
• 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 (1) whether it is to be used for DNA amplification or for DNA expression, (2) the size of the nucleic acid to be inserted into the vector, and (3) 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 signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
• 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.
• 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 (i.e., cleaved by a signal peptidase) by the host cell.
• 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.
• a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders.
• yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase, alpha factor, or acid phosphatase leaders, the C. albicans glucoamylase leader (EP
• the native signal sequence i.e., the mpl ligand presequence that normally directs secretion of mpl ligand from its native mammalian cells in vivo
• the native signal sequence i.e., the mpl ligand presequence that normally directs secretion of mpl ligand from its native mammalian cells in vivo
• 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.
• Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
• 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.
• origins of replication or autonomously replicating 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 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
• 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, i.e., they are capable of replication in at least one class of organisms but can be transfected into another organism for expression.
• 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
• Selection genes 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 transformed with the vector containing the selection gene will not survive in the culture medium.
• Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., 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. Genet., 1 :327 [1982]) mycophenolic acid (Mulligan et al., Science, 209: 1 422 [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.
• Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the mpl ligand nucleic acid, such as dihydrofolate reductase (DHFR) or thymidine kinase.
• DHFR dihydrofolate reductase
• 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 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 chromosomes of successive generations of recombinant cells. Increased quantities of mpl ligand are synthesized from the amplified DNA.
• 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.
• Mtx methotrexate
• 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, 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, e.g., 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).
• host cells 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, e.g., kanamycin, neomycin, or G418.
• a suitable selection gene for use in yeast is the trp.
• the trp1 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: 1 2 [1977]).
• ATCC No. 44076 or PEP4-1 Jones, Genetics, 85: 1 2 [1977]
• the presence of the ttpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
• Leu2-deficient yeast strains ATCC No.
• 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 (5') 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, e.g., the presence or absence of a nutrient or a change in 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 iigand as compared to the native mpl ligand promoter.
• Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems (Chang et al., Nature, 275:615 [1978]; and Goeddel et al., Nature, 281 :544 [1979]), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057 [1980] and EP 36,776) and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21 - 25 [1983]).
• trp tryptophan
• hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21 - 25 [1983]).
• other known bacterial promoters are suitable.
• 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 nucleotide.
• X may be any nucleotide.
• At the 3' end of most eukaryotic genes is an AATAAA sequence that may be 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.
• 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.
• enolase such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho
• 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 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 ceils 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 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., 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.
• viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papillom
• the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
• 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.
• 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 position 5' or 3' to the mpl ligand encoding sequence, but is preferably located at a site 5' from the promoter.
• Transcription Termination Component Expression vectors used in eukaryotic host cells will also 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.
• Construction and Analysis of Vectors 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.
• Plasmids 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. For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform E. coli 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,
• 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 iigand polypeptide.
• 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.
• 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.
• 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.
• 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 ]; Mantei et al., Na ture, 281 :40-46 [1 979]; 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.
• 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.
• E. coli cloning host is E. coli 294 (ATCC No. 31 ,446), although other strains such as E. coli B, E. coli
• X1776 ATCC No. 31 ,537), and E. coli W3110 (ATCC No. 27,325) are suitable. These examples are illustrative rather than limiting.
• the host cell should secrete minimal amounts of proteolytic enzymes.
• in vitro methods of cloning e.g., PCR or other nucleic acid polymerase reactions, are suitable.
• 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.
• K. lactis (Louvencourt et al., J. Bacteriol., 737 [1983]), K. fragilis, K. bulgaricus, K. thermotolerans, and K. marxianus, yarrowia [EP 402,226], Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa (Case et al., Proc. Natl. Acad.
• 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. 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 J., 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 (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
• Bombyx mori NPV 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.
• plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens, which has been previously manipulated to contain the mpl ligand DNA.
• Agrobacterium 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.
• regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences.
• 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.
• 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 cellsADHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 [1980]); mouse sertoli cells (TM4, Mather, Biol.
• monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat Iiver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human Iiver 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 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, CaP ⁇ 4 and electroporation. Successful transfection is generally recognized when any indication of 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 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 et al., Gene, 23:315 [1983] and WO 89/05859 published 29 June 1989.
• plants may be transfected using ultrasound treatment as described in WO 91/00358 published 10 January 1991.
• 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 F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are suitable for culturing the host cells.
• any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as GentamycinTM 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.
• 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 32 P.
• other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a poiynucleotide.
• the biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like.
• 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 that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
• Gene expression may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
• 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. 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.
• Mpl ligand preferably is recovered from the culture medium as a secreted polypeptide, although it also may be recovered from host cell lysates when directly expressed without a secretory signal.
• the mpl ligand 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 iigand from other recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous as to the mpl ligand per se.
• the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions are then separated.
• a commercially available protein concentration filter e.g., Amicon or Millipore Pellicon ultrafiltration units
• 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 (e.g., anti-hmp/ ligand Mab), receptoraffinity (e.g., mpMgG or protein A Sepharose), hydrophobic interaction chromatography (HIC) (e.g., ether, butyl, or phenyl Toyopearl), lectin chromatography (e.g., Con A-Sepharose, lentil-lectin-Sepharose), size exclusion (e.g., Sephadex G-75), cation- and anion-exchange columns (e.g., DEAE or carboxymethyl- and sulfopropyl-cellulose), and reverse-phase high performance liquid chromatography (RP-HPLC) (see e.g., Urdal et al., J.
• immunoaffinity e.g., anti-hmp/ ligand Mab
• receptoraffinity e.g., mpMgG or protein A Sepharose
• HIC
• Mpl ligand variants in which residues have been deleted, inserted, or substituted are recovered in the same fashion as native mpl iigand, taking account of any substantial changes in properties occasioned by the variation.
• preparation of a mpl ligand fusion with another protein or polypeptide e.g., 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-mp/ ligand column can be employed to absorb the mpl ligand variant by binding it to at least one remaining immune epitope.
• the mpl ligand may be purified by affinity chromatography using a purified mp/-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 useful to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants.
• 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.
• mpl ligand polypeptides 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 ligand fragments having up to about 40 amino acid residues may be conveniently prepared by chemical synthesis or by enzymatic or chemical cleavage of the full-length or variant mpl ligand polypeptide.
• Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
• Oysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ x - b ro m o - ⁇ - ( 5 - imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2- pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2- chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1 ,3-diazole.
• 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.
• Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
• 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.
• Suitable reagents for derivatizing -amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O- methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.
• imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O- methylisourea; 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 performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
• tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
• aromatic diazonium compounds or tetranitromethane Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
• Tyrosyl residues are iodinated using 1 25
• R and R' are different alkyl groups, such as 1 -cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1 - ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.
• 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 anti- mpl 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 intermediates that are capable of forming crosslinks in the presence of light.
• 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 ⁇ -amino groups of lysine, arginine, and histidine side chains (T.E. 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- 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.
• 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 present in the native mpl ligand.
• N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
• the tripeptide sequences asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
• 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).
• 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 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.
• the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.
• Removal of carbohydrate moieties present on the mpl ligand polypeptide may be 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 al., Arch. Biochem. Biophys., 259:52 [1987] and by Edge et al., Anal. Biochem.,
• 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 compound tunicamycin as described by Duskin et al., J. Biol. Chem., 257:3105
• 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, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192 or 4,179,337.
• Mpl ligand polypeptides covalently linked to the forgoing polymers are refered to herein as pegylated mpl ligand polypeptides
• mpl ligand variant some screening of the recovered mpl ligand variant will be needed to select the optimal variant for binding to a mpl and having the immunological and/or biological activity defined above.
• a change in the immunological character of the mpl ligand 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.
• Animals are immunized against the mpl ligand polypeptide or fragment, immunogenic conjugates or derivatives by combining 1 mg of 1 ⁇ g of the peptide or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
• 1 mg of 1 ⁇ g of the peptide or conjugate for rabbits or mice, respectively
• 3 volumes of Freund's complete adjuvant injecting the solution intradermally at multiple sites.
• 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 titer. Animals are boosted until the titer plateaus.
• 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.
• Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
• the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
• the mpl ligand monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler & Milstein, Nature, 256:495 [1975], or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567 [Cabilly et al.]).
• lymphocytes 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. Altematively, 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]).
• a suitable fusing agent such as polyethylene glycol
• 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.
• a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• the culture medium for the hybridomas typically will include 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.
• 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, 1 987) .
• Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against mpl ligand.
• 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).
• RIA radioimmunoassay
• ELISA enzyme-linked immunoabsorbent assay
• the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson & Pollard, Anal. Biochem., 107:220 [1980].
• 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- 1640 medium.
• 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 (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
• the hybridoma cells of the invention serve as a preferred source of such DNA.
• 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 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.
• 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.
• immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond.
• suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
• 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.
• the detectable moiety may be a radioisotope, such as 3 H, 1 4 C, 3 P, 35 S, or 25 l, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; radioactive isotopic labels, such as, e.g., 125 l, 32 P, 14 C, or 3 H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
• any method known in the art 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.
• the antibodies generally are insolubilized 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.
• 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.
• 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 labeled with a detectable moiety (indirect sandwich assay).
• sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme (e.g., horseradish peroxidase).
• 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: 1 534- 1 536 [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 variable domain has been substituted by the corresponding sequence from a non-human species.
• 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.
• 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 the human framework (FR) for the humanized antibody (Sims et al., J. Immunol.,
• Another 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 al., Proc. Natl. Acad. Sci. USA, 89:4285 [1992]; Presta et al., J. Immnol., 151 :2623 [1993]).
• humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using 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 three- dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
• 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.
• the CDR residues are directly and most substantially involved in influencing antigen binding.
• transgenic animals e.g., mice
• transgenic animals e.g., mice
• antibody heavy chain joining region
• 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 ]).
• Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. Methods for making bispecific antibodies are known in the art.
• bispecific antibodies are 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 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 ].
• antibody variable domains with the desired binding specificities 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
• 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.
• 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 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.
• Heteroconjugate antibodies are also within the scope of the present invention.
• Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. 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- linking agents are well known in the art, and are disclosed in U.S. Patent No.
• the biologically active mpl ligand having hematopoietic effector function and 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.
• TPO megakaryocytopoietic or thrombocytopoietic protein
• Thrombocytopenia-associated bone marrow hypoplasia 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.
• DIC disseminated intravascular coagulation
• immune thrombocytopenia including HIV-induced ITP and non HIV-induced ITP
• chronic idiopathic thrombocytopenia congenital thrombocytopenia
• myelodysplasia myelodysplasia
• thrombotic thrombocytopenia e.g., myelodysplasia
• these megakaryocytopoietic proteins may be useful in treating myeloproliferative thrombocytotic diseases as well as thrombocytos
• 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.
• 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.
• 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.
• 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 , 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.
• 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.
• 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.
• buffers such as phosphate, citrate, acetate and other organic acid salts
• antioxidants such as
• 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.
• sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
• sustained-release matrices include polyesters, hydrogels [e.g., poly(2-hydroxyethyl- methacrylate) as described by Langer et al., J. Biomed. Mater. Res., 15:167-277 [1981] and Langer, Chem. Tech., 1 2 :98-105 [1982] or poly(vinylalcohol)], polylactides (U.S. Patent No.
• 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.
• the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal megakaryocytopoietic protein therapy.
• 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.
• the daily regimen will range from 0.1-100 ⁇ g/kg body weight.
• the dosage will range from 0.1- 50 ⁇ g/kg body weight.
• the initial dosage will range from 1 to 5 ⁇ g/kg/day.
• the dosage range will be the same as that of other cytokines, especially G-CSF, GM-CSF, and EPO.
• Therapeutically effective dosages may be determined by either in vitro or in vivo methods.
• 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 30min. 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 NaCl 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 NaP ⁇ 4 containing 4M NaCl. The column is washed with this buffer until A28O is ⁇ 0.05 and eluted with dH2 ⁇ .
• the eluted protein peak is diluted with dH2 ⁇ 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 NaP04 (pH 7.4) containing 2M urea. Proteins are eluted from the column with 10mM NaP ⁇ 4 (pH 7.4) containing 2M urea and 1 M NaCl.
• the eluted protein peak is made 0.01% octyl glucoside(n-octyl ⁇ -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-lgG (2 ml) column is removed after the sample is loaded and the mp/-lgG (4 ml) column is washed with 10 column volumes each of PBS and PBS containing 2 M NaCl and eluted with 0.1 M glycine-HCI pH 2.25. Fractions are collected into 1/10th volume 1 M Tris-HCl (pH 8.0).
• mpl-lgG A chimeric molecule comprising the entire extracellular domain of human mpl (amino acids 1-491) and the Fc region of a human lgG1 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 BstEII site at the 3' end.
• This fragment was cloned 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 BstEII because of two other BstEII sites present in the DNA encoding the extracellular domain of mpl.
• the BstEII 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 pRK5- 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- ⁇ gG 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 mp/-lgG.
• IL-3 dependent cell line Ba/F3 by electroporation (1 x 10 7 cells, 9605F, 250Volts).
• the mpl ligand assay was conducted as shown is Fig. 2.
• 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°C in 5% CO2 and air.
• the cells were plated out in 96 well culture dishes at a density of 50,000 cells in 200 ⁇ l of media with or without diluted samples and cultured for 24 hrs in a cell culture incubator. 20 ⁇ l 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 5 times with water.
• the filters were counted in the presence of 40 ⁇ l of scintillation fluid (Microscint 20) in a Packard Top Count counter.
• 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 20 + 0.5 M NaCl + 0.1 M Tris-HCl 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 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 electro- eluters. 12-14K molecular weight cutoff membrane caps were used in the cells. 50 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°C coldroom before use. Gel slices were eluted at 10 ma/cell (40 v initially) in a 4-6°C coldroom. Elution took approximately 4 hours. The cells were then carefully removed and the liquid above the frit removed with a pipet. The elution chamber was removed and any liquid above the membrane cap removed with a pipet. The liquid in the membrane cap was removed with a Pipetman and saved.
• the tubes were placed on ice for 1 hour, microfuged at 14K rpm for 3 min. and the supematants carefully removed from the precipitated SDS. The supematants were then placed on ice for approximately 1 hour more and microfuged again for 4 min. The supematants were diluted in phosphate buffered saline and submitted for the activity assay. Remaining samples were frozen at -70°C.
• EXAMPLE 3 Porcine mpl Ligand Microsequencing Fraction 6 (2.6 ml) from the mp/-lgG affinity column was concentrated on a Microcon-10 (Amicon). In order to prevent the mpl ligand from absorbing to the Microcon, the membrane was rinsed with 1% SDS and 5 ⁇ l of 10 % SDS was added to fraction 6. Sample buffer (20 ⁇ l) of 2X was added to the fraction #6 after Microcon concentration (20 ⁇ l) and the total volume (40 ⁇ l) was loaded on a single lane of a 4- 20 % gradient acrylamide gel (Novex). The gel was run following Novex protocol. The gel was then equilibrated for 5 min.
• 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.
• PSC peripheral stem cells
• the light density mononuclear cells were aspirated at the interface and washed 2x with IMDM and plated out at 1-2 x 10 6 cells/ml in IMDM containing 30% FBS (1 ml final volume) in 24 well tissue culture clusters (Costar).
• APP or mpl iigand depleted APP was added to 10% and cultures were grown for 12-14 days in a humidified incubator at 37°C in 5% CO2 and air.
• the cultures were also grown in the presence of 10% APP with 0.5 ⁇ g of mp/-lgG added at days 0, 2 and 4.
• APP was depleted of mpl ligand by passing APP through a mpl- ⁇ gG affinity column.
• Radiolabeled HP1-1 D was stored at -70°C in PBS containing 0.01% octyl-glucoside. Typical specific activities were 1-2 x 10 6 cpm/ ⁇ g (>95% 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 1ml cultures were transferred to 1.5ml eppendorf tubes and centrifuged at 800 x g for 10 min. at room temp, and the resultant cell pellets were resuspended in 100 ⁇ l of PBS containing 0.02% EDTA and 20% bovine calf serum. 10ng of 1 25
• RT room temperature
• oligonucleotides were designed for use as polymerase chain reaction (PCR) primers (see Table 4). Two primer pools were synthesized, a positive sense 20 mer pool encoding amino acid residues 2-8 (mpl 1) and an anti-sense 21-mer pool complimentary to sequences encoding amino acids 18- 24 (mpl 2).
• PCR polymerase chain reaction
• Porcine genomic DNA isolated from porcine peripheral blood lymphocytes, was used as a template for PCR.
• the 50 ⁇ l reaction contained: 0.8 ⁇ g of porcine genomic DNA in 10mM Tris-HCl (pH 8.3), 50mM KCl, 3mM MgCl2, 100 ⁇ g/ml BSA, 400 ⁇ M dNTPs, 1 ⁇ M of each primer pool and 2.5 units of Taq polymerase.
• Initial template denaturation was at 94°C for 8 min. followed by 35 cycles of 45 seconds at 94°C, 1 min. at 55°C and 1 min. at 72°C. The final cycle was allowed to extend for 10 min. at 72°C.
• 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- terminal amino acid sequence was encoded by a single exon then the correct PCR product was expected to be 69 bp. A DNA fragment of this size was eluted from the gel and subcloned into pGEMT (Promega). Sequences of three clones are shown below in Table 5.
• pR45 45-mer deoxyoligonucleotide
• This oligonucleotide was 32 P-labeled with (T ⁇ PJ-ATP and T4 kinase and used to screen a human genomic DNA library in ⁇ gem12 under low stringency hybridization and wash conditions (see Example 7). 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-.
• 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 synthesized (Table 6).
• HL1151x was screened with the same 45 mer oligonucleotide used to screen the human genomic library.
• the oligonucleotide was labelled with ( ⁇ PJ-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 42°C in 20% formamide, 5xSSC, lOxDenhardt's, 0.05M sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 50 ⁇ g/ml of sonicated salmon sperm DNA for 16hr.
• the plasmid pDR2 (Clonetech, ⁇ DR2 & pDR2 cloning and Expression System Library Protocol Handbook, p 42) contained within the ⁇ DR2 phage arms, was rescued as described per manufacturer's instructions (Clonetech, ⁇ DR2 & pDR2 cloning and Expression System Library Protocol Handbook, p 29-30). Restriction analysis of the 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 plasmid pDR2-FL2b. DNA sequencing of double-stranded plasmid DNA was carried out with the ABI373 (Applied Biosystems, Foster City, California) automated fluorescent DNA sequencer using standard protocols for dye-labeled dideoxy nucleoside 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]).
• ABI373 Applied Biosystems, Foster City, California
• 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.
• Human genomic DNA clones of the TPO gene were isolated by screening a human genomic library in ⁇ -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 BamHI site to the 3'end). Two overlapping lambda clones spanning 35 kb were isolated. Two overlapping fragments (BamHI and EcoRI) containing the entire TPO gene were subcloned and sequenced. The structure of the human gene is composed of 6 exons within 7 kb of genomic DNA (Fig. 14 A, B and C).
• 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 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.
• the 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 pRK5) under the control of the cytomegalovirus immediate early promoter.
• DNA from the construct pRK5-hmp/ 1 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 10% 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. C). 36 h after transfection, the supernatant of the transfected cells was assayed for activity in the proliferation assay (see Example I).
• primers were employed in RT-PCR to amplify human adult Iiver 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 sequence corresponding exactly to the sequence of the cDNA isolated from the human fetal Iiver library (see Fig. 1 [SEQ ID NO: 1]). However, a region near the C- terminus of the EPO-domain (in the middle of the PCR product) 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 interest were used in a PCR as templates for human adult live ⁇ cDNA.
• hML also refered to as hML332
• hML332 is the longest form and corresponds exactly to the sequence isolated from the fetal Iiver 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 & 10]).
• 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 & TJ. White Editors.
• 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) .
• EXAMPLE 12 Murine mpl Ligand cDNA mML, mML-2 and mML-3
• the deduced mature ML sequence from this clone was 331 amino acid residues long and identified as mML33i (or mML-2 for reasons described below).
• mML33i 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.
• 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 above) and pig (see below) cDNA's. Accordingly, additional clones were examined to detect possible murine ML isoforms.
• 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 pRK ⁇ tkneo, 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 transfection, media was conditioned for five days. The cells were maintained in high glucose DMEM media supplemented with 10% fetal calf serum.
• murine-mpl 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 ⁇ g ; linearized) containing the entire coding sequence of murine mpl (Skoda, R. C, et al., EMBO J. 1 2 :264 ⁇ -26 ⁇ 3 [1993]) was transfected into Ba/F3 cells by electroporation ( ⁇ X 10 6 cells, 2 ⁇ 0 volts, 960 ⁇ F) followed by selection for neomycine resistance with 2 mg/ml G418.
• mpl was assessed by flow 0 cytometry analysis using rabbit anti-murine mp/-lgG antisera.
• Ba/F3 cells were maintained in RPMI 1640 media from WEHI -3B cells as a source of IL-3.
• Supematants from 293 cells transiently transfected with both mML and mML-2 were assayed in BaF3 cells transfected with both mmpl and hmpl as described in Example 1 .
• Porcine mpl Ligand cDNA pML and pML-2 Porcine ML (pML) cDNA was isolated by RACE PCR. Briefly, an oligo dT 0 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 sequenced and found to encode the mature pig mpl ligand (not including a complete 5 secretion signal). The cDNA was found to encode a 332 amino acid mature protein
• Genomic clones of the porcine ML gene were isolated by screening a pig genomic library in EMBL3 (Clontech Inc.) with pR45. The 0 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
• T3-21 an oligonucleotide that binds to a sequence adjacent to the multiple cloning region within the Bluescript SK- vector: ( ⁇ ' CGAAATTAACCCTCACTAAAG 3')
• pML-2 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:
• GPIIbllla Expression CMK cells are maintained in RMPI 1640 medium (Sigma) supplemented with
• the cells are harvested, washed and resuspended at 5x10 5 cells/ml in serum-free GIF medium supplemented with 5mg/l bovine insulin, 10mg/l apo-transferrin, 1 X trace elements.
• the TPO standard or experimental samples are added to each well at appropriate dilutions in 100 ⁇ l volumes.
• 100 ⁇ l of the CMK cell suspension is added to each well and the plates are incubated at 37°C, in a 5% CO2 incubator for 48 hours. After incubation, the plates are spun at lOOOrpm at 4°C for five minutes.
• Supematants are discarded and 100 ⁇ l 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 lOOOrpm for five minutes. The supematants containing unbound antibody are discarded and 200 ⁇ l 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 15 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 60 ⁇ g/ml streptomycin.
• the cells are harvested, washed, and resuspended at 1 x10 6 ce!ls/ml in IMDM + 1% horse serum.
• 100 ⁇ l of the TPO standard or experimental samples is added to DAMI cell suspension. Cells are then incubated for 48 hours at 37 C C in a ⁇ % CO2 incubator. After incubation, plates are spun in a Sorvall 6000B centrifuge at lOOOrpm for five minutes at 4°C.
• NPR Normalized Polyploid Ratio
• IP IP with 1 ml goat anti-mouse platelet serum (6 amps) on day 1 to produce thrombocytopenia.
• mice are given two IP injections of the factor or PBS as the control.
• thirty ⁇ Ci of Na2 35 S ⁇ 4 in 0.1 ml saline are injected intravenously and the percent 35 S incorporation of the injected dose into circulating 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.
• the human mpl receptor has been disclosed by Vigon et al., PNAS, USA 89:6640-6644 (1992).
• a chimeric receptor comprising the extracellular domain (ECD) of the mpl receptor and the transmembrane (TM) and intraceliular domain 0 (ICD) of Rse (Mark et al., J. of Biol. Chem. 269(14):10720-10728 [1994]) with a carboxyl-terminal flag polypeptide (i.e. Rse.gD) was made for use in the KIRA ELISA described herein. See Fig. 30 and 31 for a diagrammatic description of the assay.
• Monoclonal anti-gD (clone ⁇ B6) was produced against a peptide from Herpes ⁇ simplex virus glycoprotein D (Paborsky et al., Protein Engineering 3(6):647-553 [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.
• PBS phosphate buffered saline
• Monoclonal anti-phosphotyrosine, clone 4G10 was purchased from UBI (Lake 0 Placid, NY) and biotinylated using long-arm biotin-N-hydroxysuccinamide (Biotin- X-NHS, Research Organics, Cleveland, OH).
• the mpl ligand was prepared by the recombinant techniques described herein. The purified mpl ligand was stored at 4 °C. as a stock solution. ⁇ (d) 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 additional 21 amino acids containing an epitope for the antibody ⁇ B6 and a stop codon.
• Table 10 presents the final sequence of the synthetic portion of the fusion gene.
• 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 al., Journal of Biological Chemistry 269(14): 1 0720- 10728 [1994]) and Hindll I sites in the polylinker of the expression vector 0 pSVI7.ID.LL (See Fig. 32 A-L; SEQ ID NO: 22) to create the expression plasmid pSV. ID. Rse.gD.
• the expression plasmid comprises a dicistronic primary transcript which contains sequence encoding DHFR bounded by ⁇ ' 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 ⁇ therefore generates DHFR protein to allow selection of stable transformants.
• 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 0 intraceliular 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 ⁇ ( ⁇ '-TCTCGCTACCGTTTACAG-3')
• DP12.CHO cells (EP 307,247 published 1 ⁇ March 1989) were electroporated with pSV.ID.M.tmRd ⁇ which had been linearized at a unique Notl site in the plasmid backbone.
• the DNA was ethanol precipitated after phenol/chloroform extraction and ⁇ was resuspended in 20 ⁇ l 1/10 Tris EDTA. Then, 10 ⁇ g of DNA was incubated with 10 7
• Clones expressing MPL/Rse.gD were identified by western-blotting of whole cell lysates post-fractionation by SDS-PAGE using the antibody 6B6 which detects the gD epitope tag.
• the media was supplemented with 10% diafiltered FBS (HyClone, Logan, Utah), 2 ⁇ mM HEPES and 2mM L-glutamine.
• Mp/-Rse.gD transformed DP12.CHO cells were seeded (3x10 4 per well) in the 0 wells of a flat-bottom-96 well culture plate in 100 ⁇ l media and cultured overnight at 37 °C in ⁇ % C02- The following morning the well supematants were decanted, and the plates were lightly tamped on a paper towel. ⁇ O ⁇ l of media containing either experimental samples or 200, 60, 12. ⁇ , 3.12, 0.78, 0.19, 0.048 or 0 ng/ml mpl iigand was then added to each well.
• the cells were stimulated at 37°C for 30 min., the ⁇ well supematants were decanted, and the plates were once again lightly tamped on a paper towel. To lyse the cells and solubilize the chimeric receptors, 100 ⁇ l of lysis buffer was added to each well.
• Lysis buffer consisted of 160 mM NaCl containing 60 mM HEPES (Gibco), O. ⁇ % Triton-X 100 (Gibco), 0.01 % thimerosal, 30 KlU/ml aprotinin (ICN Biochemicals, Aurora, OH), 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF; ICN Biochemicals), 60 ⁇ M leupeptin (ICN Biochemicals), and 2 mM sodium orthovanadate (Na3V ⁇ 4; Sigma Chemical Co, St.
• the plate was then agitated gently on a plate shaker (Bellco ⁇ Instruments, Vineland, NJ) for 60 min. at room temperature.
• an ELISA microtiter plate (Nunc Maxisorp, Inter Med, Denmark) coated overnight at 4°C with the ⁇ B6 monoclonal anti- gD antibody ( ⁇ .O ⁇ g/ml in 60 mM carbonate buffer, pH 9.6, 100 ⁇ l/well) was decanted, tamped on a paper towel and blocked with 1 ⁇ O ⁇ l/well of Block Buffer [PBS 0 containing O. ⁇ % BSA (Intergen Company, Purchase, NY) and 0.01 % thimerosal] for 60 min. at room temperature with gentle agitation.
• Block Buffer [PBS 0 containing O. ⁇ % BSA (Intergen Company, Purchase, NY) and 0.01 % thimerosal] for 60 min. at room temperature with gentle agitation.
• the anti-gD 5B6 coated plate was washed 6 times with wash buffer (PBS containing 0.05 % Tween-20 and 0.01 % thimerosal) using an automated plate washer (ScanWasher 300, Skatron Instruments, Inc, Sterling, VA). 5 The lysate containing solubilized MPURse.gD from the cell-culture microtiter well was transferred (8 ⁇ ⁇ l/well) to anti-gD 6B6 coated and blocked ELISA well and was incubated for 2 h at room temperature with gentle agitation.
• wash buffer PBS containing 0.05 % Tween-20 and 0.01 % thimerosal
• ScanWasher 300 Skatron Instruments, Inc, Sterling, VA
• the unbound mpl- Rse.gD was removed by washing with wash buffer and 100 ⁇ l of biotinylated 4G10 (anti-phosphotyrosine) diluted 1 :18000 in dilution buffer (PBS containing O. ⁇ % 0 BSA, O.O ⁇ % Tween-20, ⁇ mM EDTA, and 0.01 % thimerosal), i.e. 66 ng/ml was added to each well. After incubation for 2 hr at room temperature the plate was washed and 100 ⁇ l of horseradish peroxidase (HRPO)-conjugated streptavidin (Zymed Laboratories, S.
• HRPO horseradish peroxidase
• the mpMigand was found to be able to activate the mp/-Rse.gD chimeric receptor in a concentration-dependent and ligand-specific manner. Further, the mpl- 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 pK samples.
• TPO Form (cells) (wt/vol) (molarity)
• Hu TPO 156 E. coli 0.44 ng/ml 11.6 pM
• ELISA plates were coated with rabbit F(ab')2 anti-human IgG (Fc) in pH 9.6 carbonate buffer at 4°C overnight. Plates were blocked with O. ⁇ % bovine serum albumin in PBS at room temperature for one hour. Fermenter harvest containing the chimeric receptor, mpl- ⁇ gG, was added to the plates and incubated for 2 hours. Twofold serial dilutions (0.39-26 ng/ml) of the standard (TPO332 produced in 293 cells with the concentration determined by quantitative amino acid analysis) and serially diluted samples in 0.6% bovine serum albumin, 0.06% tween 20 were added to the 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 incubation), followed by streptavidin-peroxidase (30 min. incubation) and 3,3',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 a four-parameter curve fitting program by Kaleidagraph. Concentrations of the samples were calculated from the standard curve.
• a cDNA corresponding to the TPO entire open reading frame was obtained by PCR using the following oligonucleotides as primers:
• PRK ⁇ -hmp/ 1 (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 2 ⁇ cycles of amplification (1 min. at 94°C, 1 min. at 55°C and 1 min. at 72°C). Final extension was for 16 min. at 72°C).
• the PCR product was purified and cloned between the restriction sites Clal and Xbal of the plasmid pRK ⁇ tkneo, a pRK ⁇ derived vector modified to express a neomycin resistance gene under the control of the thymidine kinase promote, to obtain the vector pRK ⁇ tkneo.ORF.
• a second construct corresponding to the epo homologous domain was generated the same way but using Cla.FL.F as forward primer and the following reverse primer:
• Arg.STOP.Xba 5'TCTAGATCTAGATCACCTGACGCAGAGGGTGGACC3'
• 293-rhM L332 conditioned media was applied to a Blue-Sepharose (pharmacia) column that was equilibrated in 10mM sodium phosphate pH 7.4 (buffer A). The column was subsequently washed with 10 column volumes each of buffer A and buffer A containing 2M urea. The column was then eluted with buffer A containing 2M urea and 1 M NaCl. The Blue-Sepharose elution pool was then directly applied to a Blue-Sepharose (pharmacia) column that was equilibrated in 10mM sodium phosphate pH 7.4 (buffer A). The column was subsequently washed with 10 column volumes each of buffer A and buffer A containing 2M urea. The column was then eluted with buffer A containing 2M urea and 1 M NaCl. The Blue-Sepharose elution pool was then directly applied to a Blue-Sepharose (pharmacia) column that was equilibrated in 10mM sodium phosphate pH
• 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 NaCl and eluted with the same buffer containing O. ⁇ M N-acetyl-D-glucosamine.
• the WGA- Sepharose eluate was applied to a C4-HPLC column (Synchrom, Inc.) equilibrated in 0.1% TFA.
• the C4-HPLC column was eluted with discontinuous propanol gradient (0-
• rhML332 was found to elute in the 28-30% propanol region of the gradient. By SDS-PAGE the purified rhML332 migrates as a broad band in the 68-80 kDa region of the gel(see Figure 15). 4. Purification of rhMLts3 293-rhML-
• 53 eluted from the mp/-affinity column was purified to homogeneity using a C4-HPLC column run under the same conditions as described for rhML332.
• SDS-PAGE the purified rhML-j 53 resolves into 2 major and 2 minor bands with Mr of _18,000-21 ,000(see Figure 15) .
• EXAMPLE 20 Expression and Purification of TPO from CHO
• pSVI ⁇ .lD.LL.MLORF full length or hTP0332
• pSVI ⁇ .lD.LLMLEPO-D truncated or hTPO-153.
• Fig. 23 and 24 The pertinent features of these plasmids are presented in Fig. 23 and 24.
• ⁇ PRK ⁇ -h/np/ 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°C followed by 26 cycles of amplification (1 min. at 94°C, 1 min. at ⁇ °C and 1 min. at 72°C). Final extension was for 15 min. at 72°C).
• the PCR product was purified and cloned between the restriction sites Clal and Sail of the 0 plasmid pSVI5.ID.LL to obtain the vector pSVI ⁇ .lD.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:
• the coding sequences for the full length and truncated ligand were introduced into the multiple cloning site of the CHO expression vector pSVI5.ID.LL.
• This vector contains the SV40 early promoter/enhancer region, a modified splice unit 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. 3.
• 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 16 March 1989).
• This mammalian cell line was clonally selected from a transfection of the parent line (CHO-K1 DUX-B1 1 (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.
• 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.
• nucleoside supplements glycine, hypoxanthine, and thymidine.
• DP12 cells via electroporation (see e.g. Andreason, G.L. J. Tiss. Cult. Meth., 16,66 [1993]) with linearized pSVI ⁇ .lD.LL.MLORF or pSVI ⁇ .lD.LL.MLEPO-D plasmids respectively.
• Three (3) restriction enzyme reaction mixtures were set up for each plasmid cutting; 10 ⁇ g, 2 ⁇ g and ⁇ O ⁇ g of the vector with the enzyme NOTI by standard 0 molecular biology methods. This restriction site is found only once in the vector in the linearization region 3' and outside the TPO ligand transcription units (see Fig. 23). The 100 ⁇ l reactions were set up for overnight incubation at 37 degrees.
• 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 360 volts set at 330 ⁇ F and low capacitance. After electroporation, the cells were allowed to sit in the 5 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 without glycine supplemented with 1X GHT, 2mM glutamine, and ⁇ % fetal calf serum) and grown overnight in a ⁇ % CO2 cell culture incubator. c. Selection and screening method
• DHFR selective medium Ham's DMEM-F12, 1 :1 medium described above supplemented with either 2% or 6% 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 ⁇ /1 ⁇ 0 mm dishes. Cells were then incubated for 10 to 16 days( with 0 one medium change) at 37 degrees/6% CO2 until clones began to appear and reached sizes amenable to transfer to 96 well dishes.
• CHO cell clones are expanded and plated in 10cm dishes at 4 6 concentrations of methotrexate (i.e.. ⁇ OnM, 100nM, 200nM and 400nM) at two or three cell numbers (10 ⁇ , 6x106, and 106 cells per dish). These cultures are then incubated at 37 degree/ ⁇ % CO2 until clones are established and amenable to transfer to 96 well dishes for further assay.
• methotrexate i.e. 600nM, 800 nM, 0 1000nM and 1200nM
• Banked cells are thawed and the cell population is expanded by standard cell 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 26-40 °C, neutral pH, with a dissolved O2 content of at least 6% until the constitutively secreted TPO is accumulated. Cell culture fluid is then separated from the cells by mechanical means such as centrifugation.
• HCCF Harvested cell culture fluid
• HCCF Harvested cell culture fluid
• a Blue Sepharose 6 Fast Flow column Pulacia
• the column is then washed with 3 to 5 column volumes of equilibration buffer followed by 3 to 5 column volumes of 0.01 M Na Phosphate pH7.4, 2.0M urea.
• the TPO is then eluted with 3 to 5 column volumes of 0.01 M Na Phosphate pH7.4, 2.0M urea, 1.0M NaCl.
• the Blue Sepharose Pool containing TPO is then applied to a Wheat Germ Lectin Sepharose 6MB column (Pharmacia) equilibrated in 0.01 M Na Phosphate pH7.4, 2.0M urea, and 1.0M NaCl at a ratio of from 8 to 16 ml of Blue Sepharose Pool per ml of resin at flow rate of approximately 60 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 5 column volumes of 0.01 M Na Phosphate pH7.4, 2.0M urea, 0.6M N-acetyl-D- glucosamine.
• the protein is eluted in a two phase linear gradient of acetonitrile containing 0.1% TFA, 0.04% C12E8.
• the first phase is composed of a linear gradient from 0 to 30% acetonitrile in 15 minutes
• the second phase is composed of a linear gradient from 30 to 60% acetonitrile in 60 minutes.
• the TPO elutes at approximately 50% acetonitrile.
• a pool is made on the basis of SDS-PAGE.
• the C4 Pool is then diluted with 2 volumes of 0.01 M Na Phosphate pH7.4, 0.16M NaCl and diafilitered versus approximately 6 volumes of 0.01 M Na Phosphate pH7.4, 0.16M NaCl 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% Tween-80.
• the plasmids pMP21 , pMP161 , pMP41 , pMP67 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, -2 ⁇ are designed to express the first 163 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 pMP261 is a derivative of pMP210-1 in which the carboxy terminal end of TPO is extended by two amino acids.
• Plasmid pMP1 Plasmid pMP1
• 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.
• SEQ ID NO: 69 (SEQ ID NO: 70) were preligated with T4-DNA ligase, and second cut with Pstl.
• the fourth was a 152 base pair Pstl-Haelll fragment from pRK ⁇ hmpll 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]).
• the plasmid pMP21 is designed to express the first 156 amino acids of TPO with the aid of a 13 amino acid leader comprising part of the STII signal sequence. It was constructed by ligating together three (3) DNA fragments as shown in Fig. 34, the first of these being the vector pV ⁇ G31 in which the small Xbal-SphI fragment had been removed.
• the vector pVEG31 is a derivative of pHGH207-1 (de Boer, H. A. et. al. , in Promoter Structure and Function (Rodriguez, R. L. and Chamberlain, M. J. , Ed), 462, Praeger, New York [1982]) in which the human growth hormone gene has been replaced by the gene for vascular endothelial growth factor (this identical vector fragment can be obtained from this latter plasmid).
• the second part in the ligation was a synthetic DNA duplex with the following sequence:
• the plasmid pMP151 is designed to express the first 165 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-SphI fragment had been removed. The second was a synthetic DNA duplex with the following sequence:
• Plasmid pMP202 The plasmid pMP202 is very similar to the expression vector pMP161 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-SphI fragment had been removed. The second was a synthetic DNA duplex with the following sequence:
• 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-Hindlll section had been removed. The second was a 946 base pair EcoRI-Hgal fragment from the previously described plasmid pMP1 1 . The last piece was a synthetic DNA duplex with the following sequence:
• the plasmid pMP210 is designed to express the first 163 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 fragments.
• the first of these was the previously described vector pVEG31 in which the small Xbal-SphI fragment had been removed.
• the second was a synthetic DNA dupex shown below treated first with DNA polymerasel (Klenow) followed by digestion with Xbal and Hinfl, and encoding the initation methionine and the randomized first 6 codons of TPO.
• the third was a 890 base pair Hinfl-Sphl fragment from pMP172 encoding amino acids 19-163 of TPO.
• the plasmid pMP210 bank of approximately 3700 clones was retransformed onto high tetracycline (50 ⁇ g/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]).
• Yansura, D. G. et. al. Methods: A Companion to Methods in Enzymology 4:151- 158 [1992]
• Fig. 39 SEQ ID NOS: 23, 24, 2 ⁇ , 26, 27 and 28.
• the plasmid pMP41 is designed to express the first 156 amino acids of TPO fused to a leader consisting of 7 amino acids of the STII signal sequence followed by a 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-SphI fragment had been removed.
• the second was the following synthetic DNA duplex: ⁇ '-CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (SEQ ID NO: 81) TTAATACTTTTTCTTATAGCGTAAATAGCTTCCAGCAT- ⁇ ' (SEQ ID NO: 82)
• the last piece of the ligation was the 1064 base pair Bgll-Sphl fragment from the previously described plasmid pMP11.
• the plasmid pMP67 expresses the first 166 amino acids of TPO downstream of a leader consisting of 9 amino acids of the STII signal sequence and the dibasic site Lys-
• 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-SphI fragment had been removed. The second was the following synthetic DNA duplex: ⁇ '-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAAACGTAGCC (SEQ ID NO: 83)
• the plasmid pMP261 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 . 2. Transformation and Induction of E. coli with TPO expression vectors
• TPO expression plasmids were used to transform the E. coli strain 44C6 (w31 10 tonA ⁇ rpoHts IO ⁇ clpP ⁇ galE) using the CaCl2 heat shock method
• the transformed cells were grown first at 37°C in LB media containing 50 ⁇ g/ml carbenicillin until the optical density (600nm) of the culture reached approximately 2-3.
• the LB culture was then diluted 20x into M9 media containing 0.49% casamino acids (w/v) and 50 ⁇ g/ml carbenicillin.
• indole-3-acrylic acid was added to a final concentration of 50 ⁇ g/ml.
• the culture was then allowed to continue growing at 30°C with aeration for another 15 hours at which time the cells were harvested by centrifugation.
• 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
• 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°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.
• 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 0 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 6 ml/min. to a column equilibrated in 0.1% TFA(trifluoroacetic acid) with 30% acetonitrile.
• the protein is eluted with a linear ⁇ gradient of acetonitrile (30-60% in 60 min.).
• the purified reduced protein elutes at approximately 50% acetonitrile. This material is used for refolding to obtain biologically active TPO variant.
• the refolding buffer is gently stirred at 4°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 0.46 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 5 purified, refolded TPO (Met" 1 1-153) eluted with the same gradient program as above.
• TPO Refolded, biologically active TPO is eluted at approximately 45% acetonitrile under these conditions. Improper disulfide-bonded versions of TPO are eluted earlier.
• the final purified TPO (Met "1 1-153) is greater than 95% pure as assessed by SDS gels and analytical C4 reversed phase chromatography. For animal studies, the C4 purified material was dialyzed into physiologically compatible buffers. Isotonic buffers (10 mM Na acetate, pH 5.6, 10 mM Na succinate, pH ⁇ . ⁇ or 10 mM Na ⁇ phosphate, pH 7.4) containing 160 mM NaCl and 0.01% Tween 80 were utilized.
• CHAPS 5 CHAPS 5 family
• CHAPS 5 CHAPS 5 family
• EDTA metal-catalyzed oxidation (and aggregation)
• Glycerol concentrations produced the optimal refolding conditions.
• Organic solvents e.g. ethanol, acetonitrile, methanol
• Tris and phosphate buffers were generally useful.
• Incubation at 4°C also produced higher levels of properly folded TPO.
• 0 Refolding yields of 40-60% are typical for preparations of TPO that have been purified through the first C4 step.
• Active material can be obtained when less pure preparations (e.g. directly after the Superdex 200 column or after the initial retractile body extraction) although the yields are less due to extensive precipitation ⁇ and interference of non-TPO proteins during the TPO refolding process.
• TPO (Met" 1 1-163) contains 4 cysteine residues
• version 1 disulfides between cysteine residues 1-4 and 2-3
• version 2 disulfides between cysteine residues 1-2
• 3-4 version 3 disulfides between cysteine residues 1-3 and 2-4.
• 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(/.e. version 1). Aliquots of the various C4-resolved peaks ( ⁇ -10 nmoles) were digested with trypsin (1 :26 mole ratio of trypsin to protein). The digestion mixture was analyzed by matrix- assisted laser desorption mass spectrometry before and after reduction with DTT. After reduction, masses corresponding to most of the larger tryptic peptides of TPO were detected. In the un-reduced samples, some of these masses were missing and new masses were observed. The mass of the new peaks corresponded basically to the sum of the individual tryptic peptides involved in the disulfide pair.
• TPO Refolded and purified TPO (Met" 1 1-163) has activity in both in vitro and in vivo assays.
• 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).
• the mpl receptor-based ELISA half-maximal activity occurred at 1.9 ng/ml (120 pM).
• 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.
• TPO variants produced in E. coli, purified and refolded into biological active forms are provided below.
• MKKNIAFLLNAYASPAPPAC CVRRA (SEQ ID NO: 85) where the leader sequence is underlined and C C represents Cys 7 through Cys 151 .
• This variant was constructed to provide a tyrosine for radio-iodination of TPO for receptor and biological studies.
• MKKNIAFHHHHHHHHIEGRSPAPPAC CVRRA (SEQ ID NO: 86) where the leader sequence is underlined and C C represents Cys 7 through Cys 151 .
• This variant when purified and refolded, can be treated with the enzyme Factor Xa 0 which will cleave after the arginine residue of the sequence IEGR yielding a TPO variant of 1 ⁇ residues in length with a natural serine N-terminal amino acid.
• T-H8MLF - is prepared as described above for variant (2), 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 C C represents Cys 7 through
• 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. 0 B. Proteolytic processing of Variants (2) and (3) to generate authentic N- terminal TPO (1-155).
• TPO variants (2) and (3) above were designed with an enzymatically- cleavable leader peptide before the normal N-terminal amino acid residue of TPO.
• variants (2) and (3) were ⁇ subjected to digestion with the appropriate enzyme.
• the acetonitrile from the C4 reversed phase step was removed by blowing a gentle stream of nitrogen on the solution. Thereafter the two variants were treated with either Factor Xa or thrombin as described below.
• TPO variant (2) 1 M Tris buffer, pH 8, was added to the acetonitrile-free solution to a final concentration of 60 mM and the pH was adjusted to 8 if necessary. NaCl and CaCl2 were added to 0.1 M and 2 mM, respectively.
• Factor Xa New England Biolabs was added to achieve about a 1 :26 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 chromatography using the same gradient and conditions as described above for the purification of properly folded variants.
• the purified TPO (1-156) 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.
• the digestion buffer consisted of 50 mM Tris, pH 8, 2% CHAPS, 0.3 M NaCl, 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 90% 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.
• TPO protein had high biological activity with half- maximal responses in the Ba/F3 assay at 0.2-0.4 picomolar protein.
• 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 was less potent in both assays by 5-10 fold.
• the HPLC-purified cleaved protein was dialyzed into physiological acceptable buffers, with 160 mM NaCl, 0.01% Tween 80 and 10 mM sodium succinate, pH ⁇ . ⁇ , or 10 mM sodium acetate, pH 6.5, or 10 mM sodium phosphate, pH 7.4.
• the purified protein was stable for several weeks when stored at 4°C.
• 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.
• hML Human mpl ligand
• a general strategy for hML-153 synthesis using subtiligase is shown (Scheme 1 ). 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.
• hMLi53 Based on our knowledge of the sequence specificity of the subtiligase as well as the amino acid sequence of the biologically active "epo-domain" of hML, we divided hMLi53 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.
• the ligation peptides indicated in Table 14 should be efficiently ligated by the subtiligase.
• a suitable protecting group for the N- terminus of each donor ester peptide was needed to prevent self-ligation.
• iNOC isonicotinyl
• the peptides are then seqentially ligated until the full protein is produced and the final product refolded in vitro.
• disulfide pairs are believed to be formed between cysteine residues 7 and 161 and between 28 and 86. 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 iyophilized.
• 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 oxidation of 28 and 85.
• residues 28 and 85 could be acm protected and oxidized in case sequential oxidation is required for correct folding.
• Cysteins 28 and 85 may be substituted with another natural or unnatural residue other than Cys to insure proper oxidation of cysteins 7 and 151.
• H2N-LSF Q HLLRGKVRFLMLVGGSTLCVR-CO2 (129-153)
• Peptide ligations are carried out at 25°C in 100mM tricine, pH 8 (freshly prepared and degassed by vacuum filtration through a ⁇ ⁇ M filter).
• the C- terminal fragment is dissolved in buffer (2-6 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 - 5 ⁇ M.
• a 3-6 molar excess of the glc-K-NH2 activated donor peptide is then added as a solid, dissolved, and the mixture allowed to stand at 25°C.
• the ligations are monitored by analytical reverse phase C18 HPLC (CH3CN/H2O gradient with 0.1% TFA).
• the ligation products are purified by preparative HPLC and
• Synthetic hML-.53 can be ligated by procedures analogous to those described above to synthetic or recombinant 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. Scheme 2. Solid Phase Synthesis of Peptide Fragments for Segment
• INOC Isonicotinyl glycolate-lysyl-amide
• the amino acetylated resin 3 is washed with DMF (3x) and dichloromethane (CH2CI2) (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 6 (5). c) The N- ⁇ -Boc group is removed with a solution of 46% trifluoroacetic acid in
• Boc-protected amino acids ( ⁇ 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.
• BOP benzotriazol-1 -yl-oxy-tris-(dimethylamino) phosphonium hexafluorophosphate
• NMM N-methylmorpholine

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  • 1994-12-28 WO PCT/US1994/014553 patent/WO1995018858A1/en active IP Right Grant
  • 1994-12-28 JP JP7518499A patent/JPH09508262A/ja active Pending
  • 1994-12-28 CA CA2178482A patent/CA2178482C/en not_active Expired - Lifetime
  • 1994-12-28 CN CNB941947513A patent/CN1134539C/zh not_active Expired - Lifetime
  • 1994-12-28 HU HU9601805A patent/HUT75657A/hu not_active Application Discontinuation
  • 1994-12-29 IL IL11218494A patent/IL112184A/en not_active IP Right Cessation
  • 1994-12-30 PT PT101627A patent/PT101627B/pt not_active IP Right Cessation
  • 1994-12-30 ES ES09402681A patent/ES2114786B1/es not_active Expired - Fee Related
• 1995
  • 1995-01-02 IT IT95TO000002A patent/IT1283770B1/it active IP Right Grant
  • 1995-01-02 FR FR9500007A patent/FR2714670B1/fr not_active Expired - Lifetime
  • 1995-01-03 NL NL9500010A patent/NL9500010A/nl active Search and Examination
  • 1995-01-03 IE IE950002A patent/IE72517B1/en not_active IP Right Cessation
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• 1996
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  • 1996-07-02 FI FI962723A patent/FI121573B/fi not_active IP Right Cessation
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  • 1996-07-24 LV LVP-96-315A patent/LV11632B/xx unknown
• 1997
  • 1997-03-27 JP JP9074906A patent/JPH10113186A/ja active Pending

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