SA95150635B1 - thrombopoietin (a platelet-forming factor) - Google Patents

Abstract

Abstract: The present invention relates to the detection of methods for isolating the clotting factor (thrombopoietin (TPO) and the DNA that carries the encoding gene for TPO and methods for preparing and purifying TPO either by recombinant DNA or synthetic construction. Also, it has been shown that there are different forms of TPO that affect In the process of building DAN replication and affecting the differentiation and maturation of blood cells, especially megakaryocytes and their megakaryocyte progenitor cells.Therefore, these compounds can be used to treat patients with thrombocytopenia.

Description

Y — _— thrombopoietin (Jule) thrombopoietin (platelet-producing) Full Description BACKGROUND This invention relates to the isolation and purification of recombinant DNA and construction by the method of recombinant DNA or the chemical synthesis of proteins that affect survival the proliferation, differentiation, or maturation of hematopoietic cells; In particular, platelet progenitor cells. This invention relates particularly to the cloning and gene expression of a protein ligand that is capable of binding to and activating empl, a member of the broad cytokine receptor superfamily. Proteins alone or in combination with other cytokines to treat immune disorders or disorders 0 blood cells and platelet formation Lay including platelet deficiency - thrombocytopenia 1 - Hematopoietic system of cells and platelets formation System: The hematopoietic system produces mature, highly specialized LAY cells known to be essential to the survival of all mammals. These mature cells include: erythrocytes; It is specialized for transporting oxygen and carbon dioxide; lymphocytes 1 and 5 ¢ which are responsible for cell-mediated immune responses and antibody-mediated immune responses and platelets or thrombocytes; It is intended for the formation of blood clots; granulocytes, and osteoclasts

Macrophages ¢ are specialized LAS scavengers and helper cells to fight infection. The granulocyte LAN is further subdivided into: LIA neutrophilis, eosinophils, basophils and mast cells” which are specialized cell types with separate functions. noticeably All of these specialized mature blood LDAs are derived from a normal single progenitor cell type referred to as a uly a pluripotent (or “totipotent”) stem cell primarily found in the bone marrow. (Dexter et al., Ann.

Rev.

Cell Biol,, 3: 423-441 [1 987]) marrow. Mature highly specialized blood cells must be produced in large numbers on a continuous basis over the life of a mammal. It is estimated that the vast majority of these specialized blood cells remain functional for only a few 0 hours. Or to weeks ([1976] 2: 263-284) (Cronkite et al., Blood Cells) Thus, the continuous renewal of mature blood cells and the primary stem cells themselves, as well as any intermediate or progenitor cell lines of a lineage between the primary and mature cells is lineage -committed progenitor cell 0 Necessary in order to maintain the needs of blood cells for the normal steady state of the mammalian organism. At the heart of the hematopoietic system lies the multigenital stem cell or cells. These ALE 1 cells are relatively numerous and undergo self-renewal By reproducing to produce daughter stem cells or transforming them in a series of differentiation steps into incrementally mature, lineage-restricted progenitor cells, eventually forming a highly specialized mature blood cell or cells. multi-lineage cells; referred to as CFC-Mix; derived from stem cells to proliferate (self-renew) and develop to produce colonies 0 containing all types of myeloid cells; and red cells

M «erythrocytes and neutrophils and megakaryocytes (lamellae progenitors) and basophils WA macrophages basophils and eosinophils and mast cells subject other progenitor cells of the lymphoid lineage lineage to reproduce and develop into T cells and cells — 3 -T-cells and B-cells © further; Between CFC-Mix progenitor cells and myeloid cells lies another order of progenitor cells with an intermediate commitment to their progeny. These lineage-restricted progenitor cells are classified based on the offspring they produce. so; Known mesenchymal progenitors of myeloid cells are: red LAY erythroid colony-forming units (CFU-E) and granulocytelmacrophage colonyforming cells (GM-CFC)0 for eosinophils and megakaryocyte cells; megakaryocyte colonyforming cells (Meg-CFC) eosinophil coiony-forming cells (EosCFC) for eosin-forming cells and basophil colony-torming LA Bas-CFC and other mast cells and intermediate predecessor cells 1 between MSCs and mature blood cells are known (see below) or likely to be discovered to have multiple degrees From the limitation of the strain by the lineage- restnctlon and the ability of self-renewal. The basic principle of the normal hematopoietic cell system appears to be reduced capacity for self-renewal, loss of polylineage and lineage determination upon acquisition of maturity. so; In one spectrum of hematopoietic cells are multi-lineage stem cells that have the ability to self-renew and differentiate to give birth

Ce —

All the different progenitor cells of a specific lineage. This ability is the basis for bone marrow transplant therapy, in which primary stem cells repopulate the total hematopoietic cell system. At the other end of the spectrum are the ancestors and offspring of the severely restricted strain that have lost the ability to self-renew but gain mature functionality.

© The proliferation and development of stem cells and progenitor cells of restricted lineages is carefully controlled by a variety of hematopoietic growth factors or cytokines. such as interleukin-3 (IL-3) ¢ is able to stimulate both multilineage stem cells as well as multilineage-specific progenitor cells; Ly in that for example; large cells

megakaryocytes 0 . Then at first the belief that other factors such as granulocyte/macrophage colony-stimulating factor (GM-CS F) are factors limiting their effect to 60-0709 only. after that; In any case, it was discovered that GM-CSF also affects the proliferation and development of large myeloid cells within other cells, interalia .megakaryocytes; 11-3 GM-CSF has been found to have synonymous biological activities; despite of

¢ interleukin-1 1 (IL-11) interleukin-6 (IL-6) They are of different potency. currently; Both yo act in cmeg-colony while neither of them alone has an effect on massive colony formation (Yonemura et megakaryocytes ddl) synergistically with 11-3 to stimulate cell maturation -al., Exp.

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

Thus hematopoietic growth factors may affect the growth and differentiation of one or more lineages and may synergistically with other growth factors in affecting a single progenitor cell lineage; Or it may work cooperatively with other agents. Lad appears that hematopoietic growth factors can exert their influence at different stages of cell development from a MSC, from a specific progenitor cell line to the mature blood cell. For example; Erythropoietin (epo) appears to stimulate proliferation only of mature erythroid progenitor cells. 3 appears to exert its effect early and thus on Lol primary stem and lineage-restricted intermediate progenitor cells. Other growth factors such as stem cell factor (SCF) may influence the initial development of the cell as well. 1 From the above it will be recognized that the single hematopoietic growth factors affecting the viability, proliferation, or maturation of any blood cell or its progenitors are ade and in particular to help re-establish a hematopoietic system that has previously been adversely affected by or following disease Radiation therapy or chemotherapy - chemo-therapy - formation of large myeloid cells - production of platelets Megakaryocytopoiesis - Platelet

Production Vo and platelet megakaryocytopoiesis Regulation of myeloid macrogenesis reviewed: by Mazur, Exp. Hematol, 15: 248 [1987] and Hoffman, Blood, 74: 1196-1212 [1 989] in summary; Bone marrow ADL-pluripotent stem cells divide into megakaryocytopoiesis

vy — red cells and myeloid cells. It is believed that there is a series of LA-megakaryocytopoiesis progenitors deposited between the stem cells and -megakaryocytopoiesis. At least three classes of megakaryocytopoiesis progenitor cells are assigned "i.e. blasting unit megakaryocytes (BFU-MK) and myeloid large colony-forming cells (CFU-MK) and mild progenitor megakaryocytes (LD-CFU-L) stages on the basis of standard morphological criteria. The early observable member of the MK species (meg) is the cells megakaryocytes These cells are initially Ye to 01 μm in diameter with basal cytoplasm and slightly irregular nuclei with chromatin; However, until VY 0 Thwa (Sls” (ployploid) the cytoplasm remains non-dense and immature. As maturation continues; the nucleus becomes more lobulate and pyknotic” and the cytoplasm increases in quantity [ji] becomes acidophilic and granular The more Laas cells of this species may give the appearance of platelet release at their periphery. Fewer than 0 out of 1416 are at the blast stage and more than 750 are mature The optional ve morphological classifications commonly applied to the MK WA series are: megakaryoblast for primary of which promegakaryocyte Lek or basal for intermediate then The mature or acidophilic, granular, or platelet-producing form of the latter forms of 1 11. MK cells provide filaments of cytoplasm into the granular compartments where they detach and break into individual platelets (1 972 (Williams et al., Hematology,

A —_ _ MK cell anogenesis is believed to involve multiple regulatory factors: Williams et al., Br.

J.

Haematol., 52: 173 [1982] and Williams et al., J. Cell Physiol. ( ) [1982] 101:110. The early level of MK cytogenesis is hypothesized to be meiotic interested in cell proliferation and colony formation by CFUMK but not affected by platelet count (Burstein et al, J.

Cell Physiol., 109: 333 [1981] and Kimura et al., Exp Hematol, 13: ) [1985] 1048. The final stage of maturation is non-mitotic involved with increased polyploidization:01 and maturation. Cytoplasmic maturation is likely to be regulated in a feedback mechanism by blood platelet count (Odell et al, Blood, 48: 765 [1976] and Ebbe et al, Blood. 32: 787). [1981 1 that the presence of a limiting factor to prepare the growth of colonies of cells (MK.

CSF) is questionable (Mazur, Exp, Hematol, 15: 340-350 [1987]) On the other hand, most authors believe that regulation of a process vital to survival such as platelet production is mediated by cytokine(s) which is specifically responsible for yo limitation of this process. dpa jill in the presence of specific cytokines) specific for large lymphocytes/platelet MK/platelet provided the basis for more than thirty years of research - but not yet purified 0m0:0m amino acid sequence determination Jia This cytokine has not been experimentally investigated as the single (TPO) MK-CSF inducer.

Although 16-0575 was partially purified from experimentally induced thrombocytopenia ([1986] 752:14), Hill et al., Exp.

Hematol., from conditioned medium of a human fetal kidney (McDonald et al., J.

lab.

Ctin.

Med. 85: 59 [1975]) [CM] From the urine of a person complaining of problematic plastic anemia and from the urine and blood serum of patients with aly oo platelets characterized by skin spots) [1983] 556:6 (Kawakita et al , Blood, (Hoffman et al., J.

Clean.

Invest., 75: 1174 [1985], and its physiological functions are still unknown in most cases. Conditioned medium of spleen cells activated by known carcinogen pokeweed (PWMSpCM) and a murine myelomonocyte strain (WEHI-3CM) 0 17121113 are used as MK WIA tonics PWM-SpCM contains promoting agents for 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, [New York, Academy press] pp 37-52 [1978]) o One is Jule 5¢ interleukin-3 (IL-3) multistrain colony stimulation (Muli-CSF [Burstein, Blood Cells, 11: 469 [1986]) Other factors have not yet been identified from this medium. WEHL3 is a mouse monocyte myeloid cell lineage that secretes relatively large amounts of IL-3 and smaller amounts of GM-CSF and 11-3 was found to promote the growth of a wide range of hematopoietic cells (Thle et al, J.

Immunol, 13: 282 [1983]) 11-3 co-operation was also discovered with many of the known blood-forming hormones or growth factors (Barteimez et al., J. growth factors).

- 1. including Cell Physiol., 122: 362-369 [1985] and Warren et al., Cell, 46: 667-674 [1988]) in ¢ interleukin-1 (IL-1) s erythropoietin (EPO) That is, both early and multi-estimated red cell formation and large colony formation for mixtures of precursors induction of primers. Hematopoietic cells conditioned in conditioned media MK potentiators cells I found other sources of propeller © 4ST macrophage cell lines and murine lung cell lineages of mouse lung cells -human embryonic kidney cells and human fetal kidney cells, peritoneal exudate cells, peritoneum exist (Mazur, Exp. Hematol, 15: 340-350 [1987]) despite certain mixed data.

Some evidence suggests that LIAN monocytes and not activated MK play a reinforcing role in Cell formation 0 secretions and 100d 108:016 may be the activated Jia T These findings show that lymphocyte secretions (Geissler et al, Exp. Hematol, 15: 845-853 [1987]) MK are regulatory factors In developing (Vainchemker et al., “erythropoietin EPO” that includes MK LDA a number of studies in malformation

Blood, 54: 940 [1979]; McLead et al., Nature, 261 492-4 [1976]; and Williams et al, "MK" showed that this hormone has a stimulating effect on colony formation Exp. Hematol, 12: 734 [1984]) 0 containing serum and in SERUM. This was also demonstrated using serum-free cultures. 0 LSHUM(Williams et al., Exp. Hematol., 12: 734 [1984]) Additional WA is more absent in single-cell stage and two-cell stage features than EPO four- which is assumed to be included It affects the four-cell stage of PWMSpCM compared to the effect of the MK cell stages

- 11 - The overlap of all these factors and their impact on the early and late stages of MK cells, Gail cells, remains in need of clarification. MK for cell growth, which are multi-lineage factors. 11-3 and to a lesser extent GM-CSF stimulating factor alone is a MK with newly colony stimulating activity; (Ikebuchi et Mela Proc. Natl. Acad Sci USA, 84: 9035 [1987]) IL-6 in 8-0 cells acts synergistically with 11-3 to increase LIF. Several authors have determined that 11- 11 and a leukemia inhibitory factor (Yonemura et al., British Journal of Hematology, 84: 16-23 MK) cell size and duplication

[1993]: Burstein Mill J. Cell. Physial., 153: 305-312 [1992]; Metcalf et al., Blood, 6 50-56 [1990]; Metcalf et al, Blood, 77:2150-2153 {1991]; Burno et al., Exp. Hematol. , and 19: 378-381 [1991]; and Yonemura et al., Exp. Hematol, 20: 1011-1016 [1992]). 0 : Other documents of interest are US Patent “Chong 5 11701” US Patent No. 0 (AT, Eppstein et al.. US Patent No. 5707/7 5738, 11715516 and others; Fernandes 5 879111

Bennelt 5 6 £1A 74 US Patent No. “Gottlieb 5 £01 Ya VY” and US Patent No.

VEY et al.; US Patent No. Kogan g <0Y 1 0Ad0 and others USP. No. yo and “© You

Kimura et al., Eur. J. Immunol, 20(9): 1927-1931 [1990]; Secor et al, J. of Immunol. , 144(4):1484-1489 [1990]; Warren et al, J. of Immunol., 140(1): 94-49 [1988]; Warren et al., Exp. Hematol, 17(11): 1095-1099 [1989], Bruno et al. Exp. Hematol., 17(10): 1038-1043 [1989]; Tanikawa et al., Exp. Hematol, 1 7(8): 883-888 [1989]; Koike et al, vy.

117 - - Blood, 75 (12): 2286-2291 [1990], Lotem, Blood, 75(5): 1545-1551 [1989]; Rennuck et Blood, 73(6): 1504-1512 His al., Blood, 73(7): 1826-1835 [1989]; and Cluterbuck et [1989] ¥ — Thrombocytopenia 43 yell : 0 Platelets are critical components of the blood clotting mechanism. Depletion of the level of circulating platelets; called thrombocytopenia "occurs in various cilnical conditions and clinical disorders. It is commonly defined as platelet count less than 0 x 10 per liter. It can be done extensively Division of the main causes of thrombocytopenia into three classifications based on platelet life span: (1) poor platelet production by the bone marrow; or (Y) platelet sequestration in the spleen (splenomegaly or (©) excessive destruction of platelets in the circulatory system (Je) circulation (JE) thrombocytopenia as a result of autoimmune or chemo- and radiation- therapy In addition to this; There are patients who receive large volumes of platelet-poor blood products given rapidly; yo 38 shows a lack of platelets due to dilution. Clinical bleeding and the associated indications of platelet deficiency depend on the severity of platelet deficiency, its causes, and possible coagulation defects related to it. In general; Patients with platelet counts between 0.01 x 0.01 and 71 per liter in pha have “traumatic” bleeding while patients with platelet counts less than 01 XY per liter may bleed spontaneously. ov. patients mentioned last are considered candidates for platelet transfusion with the risk of immunity

Q1 and viruses. For any known degree of thrombocytopenia; Bleeding tends to be more serious when the cause is gall production rather than excessive destruction of platelets. in the last Ala; It causes the blood platelet tumor production cycle day jus, and the platelets are young, larger in size, and more effective in preventing bleeding. Thrombocytopenia may cause any of a variety of disorders described briefly below. A more favorable explanation may be found in Schafner, A.

I., "Thrombocytopenia and Disorders of Platelet Function". Internal Medicine, 3" Ed., John J.

Hutton et al, Eds., Little Brown and Co., Boston /Toronto/ London [1990]. Thrombocytopenia due to 'impaired platelet 0100000002' include Congenital hemoglobinopathies include constitutionalal aplastic anemia (Fanconi syndrome) and congenital MK-cell thrombocytopenia, which may be associated with defects of skeletal formation. Acquired disorders of platelet production are induced by Either hypoplasia or ineffective platelet formation 1. Myeloid macrocytic hypoplasia can cause a variety of conditions, including bone marrow aplasia (Ly) and idiopathic forms. Or myelosuppression by chemotherapy agents or radiation therapy (myelosuppression by chemotherapeutic agents or radiation therapy), myelfibrosis and leukemia, and invasion of the bone marrow by a sf metastatic tumor, granulomas in some vy. cases; toxins or infection-causing agents may interfere; or drugs ‎with composition

M1 - platelets in a relatively facultative manner; Examples of symptomatic thrombocytopenia1 caused by alcohol, certain viral infections, and simple thrombocytopenia associated with administration of thiazide diuretics include ©02:0n:0. finally; The ineffective platelet-building processes thrombopoiesis secondary to the processes of enlarged blood cells gl folate ) megaloblastic processes © deficiency 12 (B) can cause thrombocytopenia »usually with the co-existence of anemia and leukopenia -leukopenla Current treatment of thrombocytopenia due to insufficient platelet production relies on identifying and reversing the underlying cause of the bone marrow failure. It is usually reserved for platelet transfusions for patients with severe bleeding or for cover-up during surgical procedures. An isoimmunizatio of 0 may result in no Response to additional platelet transfusion Mucosal bleeding from serious platelet deficiency may be relieved by oral or intravenous administration of Jal gal antifibrinolytic agents. Thrombotic complications may develop 0 Anyway if antifibrinolytic agents are used in patients with Jala 0 intravascular coagulation (DIC) 1 (b) thrombocytopenia due to J je splenic sequestration an enlarged spleen may be associated Spleen for any reason with mild to moderate platelet deficiency. This process (hypersplenism) is relatively ineffective for the isolation of platelets in the spleen; when compared to the effective destruction of platelets by the spleen in cases of immunomediated thrombocytopenia pais described below. Although 0 - _ The most common cause of hypersplenism is an enlarged spleen (splenomegaly).

1 and alcoholic portal hypertension due to congestive portal blood pressure

05 + Other forms of congestive, lymphoproliferative, or infiltrative splenomegaly are also associated.

They are all related to platelet deficiency. Platelet counts generally do not fall below 1 01 x mL per liter as a result of hypersplenia alone.

(C) thrombocytopenia due to platelet destruction due to nonimmune causes

mediated

Thrombocytopenia can be caused by the accelerated destruction of platelets by operations

different non-immunological. Disorders of this type include diffuse intravascular stimulation0, intravascular prosthetic devices, and corporeal supplementary blood circulation.

008 Jie thrombotic microangiopathies

thrombotic thrombocytic purpura in all of these cases; The platelets

The circle that is exposed to artificial surfaces or unusual vascular interiors

Abnormal vascular intima is either consumed at these sites or Vo is then destroyed and removed prematurely by the reticuloendothelial system. The conditions are satisfactory

Or disorders in which disseminated intravascular coagulation (DIC) may be present are described in detail

more in:

Braunwald et al, (eds), Harrison's Principles of Internal Medicine, 11" Ed., P. 1478.

McGraw Hill [1987].

11 - - Alternative intravascular methods, including heart valves and intra-aortic balloons, can cause mild to moderate thrombocytopenia and shunt platelet deficiency in patients undergoing cardiopulmonary bypass shunt or hemodialysis that may be consumed or damaged. Platelets due to the extra corporeal circuit. 0 (d) aii Drug-induced immune platelet deficiency thrombocytopenia: ad More than 1001 drugs that cause immune-induced platelet deficiency were included. In any case; The involvement of quinidine, quinine, gold and the sulfonamides cephalothin and hepann only has been well characterized. Thrombocytopenia 1 caused by the drug is often serious lan and typically occurs in a rush within days while patients are taking the drug that causes that allergy. (e) Immune (autoimmune) WA-forming purpura (ITP) thrombocytopenic purpura (ITP) 170 in adults is a chronic disease characterized by immune auto-destruction of platelets. The autoantibody1 is usually IgG although other immunoglobulins have been reported. Although the autoantibody binds to the platelet membrane, 11,111M0; The antigen is not set for the platelet in most cases. The idiopathic destruction of sensory platelets occurs outside the blood vessels in the reticuloendothelial -system of the spleen and liver, although half of all cases of ITP are idiopathic.; Most of the patients suffer mainly from rheumatic or autoimmune diseases.

117 - - (eg systemic lupus erythematosus or Iymphoproliferative disorders (eg chronic lymphocytic leukemia)). (f) Updated ITP HIV-Induced ITP 6 reports 11 is a common complication of HIV infection (Morris et al., Ann Intern. 1160, 96: 714-717 [1982]; HIV can occur at any stage as the disease progresses Either in patients diagnosed with Acquired Immune Deficiency Syndrome (AIDS), if they develop complications of the disease, or only asymptomatic carriers of HIV, that HIV is an infectious disease that is ultimately characterized by a deficiency of immune cellular work, accompanied by inflammation. Opportunistic and Cancer The primary immune impairment resulting from HIV infection is the progressive depletion and functional impairment of T lymphocytes producing cell surface glycoprotein 00104 (Lane et al, Ann.

Rev.

Immunal,, 3: 477 [1985]). It is possible that the loss of CD4 help/induces the action of tell WDA the significant impairment in cellular and humoral immunity 10 and is therefore due to opportunistic infections And cancer, which is the most important (HLane, AIDS) is high. Although the mechanism of overlap between HIV and ITP is not known, it is believed to be different from non-HIV-related ITP.

A _ \ — (Walsh et al, N.

Eng.

J.

Med., 311: 635-639 [1 984]; and Ratner, Am.

J.

Med., 86: 194- [1989] 198; Current treatment for thrombocytopenia *Current Therapy for Thrombocytopenia 4; geal} The treatment curve for patients with thrombocytopenia is dictated by the seriousness and urgency of the condition. Treatment is similar to that for HIV-related or non-HIV-related thrombocytopenia, and although a number of different treatment approaches are being used, treatment remains controversial. Then platelet counts are then successfully increased in patients diagnosed with thrombocytopenia by glucocorticoid therapy (eg prednisolone) and in plaza patients however the response is incomplete; or failure occurs when the dose of thrombocytopenia is reduced. glucocorticoids or stop them Based on studies with patients who had HIV-associated ITP some researchers suggest that glucocorticoid treatment may make them susceptible to AIDS Glucocorticoids are usually given if platelet counts fall blood to less than 71 x 10 / liter or when spontaneous bleeding occurs -spontaneous bleeding

1 for patients not responding to glucocorticoids; The compound 4-(2-chlorphenyl)-9-methyl-2-13- ) 4-morpholinyl)-3-propanon-1-ylil6H-thienol 3,2,f[1,2] has been successfully used. 4ltriazolol4,3,a,[1,4]diazepin (WEB 2086) for the treatment of severe ITP not associated with HIV. Treatment for 1 - 7 weeks increased the count

1 - - to 1500080 - 145066868 / µL. EP No. (Lohman et al., Lancet, s YY “VV [1988] 1147). Although the ideal treatment for amegakaryocytic thrombocytopenia purpura (AATP) is uncertain, globulin has been shown to antithymocyte globulin (ATG) ¢ It is an antiserum from human thymus tissue that induces complete J sha remission (Trimble et al., Am.

J.

Hematol., 37: 126-127 [1991]) On the other hand, a recent report shows that the effects on hematopoiesis of ATG are causative with thimerosal 0” as this protein supposedly acts as a carrier of mercury (Panella et al. ., mercury carrier .Cancer Research, 50: 4429-4435 [1990] In many patients, this procedure causes prolonged, treatment-free remission in a large number of patients. thrombocytopenia (eg severe HIV-related TTP) and in patients who failed to respond after ? to 0 “week of glucocorticoid treatment” or did not achieve a sustained response after interruption of administration 41 on the basis of knowledge Contemporary science; it is not clear whether splenectomy exposes patients to AIDS.

—Y.- splenectomy; There are certain agents toxic to prednisolone in addition to BAL therapy

AZT )azidothimidine vincristine Example Jy for cells also on cytotoxic agents On the other hand; The results are preliminary. HIV is caused by ITP, which is a promising drug in the treatment of (zidovudine). We estimate from the aforementioned that the methods for treating platelet deficiency are to obtain a factor capable of accelerating platelet-producing © 11 ol precursors. The excellence and maturity of Lal spent great efforts in identifying such a factor commonly referred to as TPOg sans Other names for thrombopoietin (TPO) include Lthrombocytopoiesls stimulating factor (TSF) He wrote on On: Jule smegakaryocyte colony-stimulating factor (MK-stimulating factor, LA megakaryocytestimulating factor) and LA megakaryocytestimulating factor (Rak et al.57) early in 404 TPO Y. 3_+——Smegakaryocyte potentialor activity was noted and attempts continued to characterize and purify this factor into this Lia gy. While 125 : 1 al, Med. Exp, (Tayrien et al, active; see, for example: pn polypeptides TPO on partial proteolysis by J. Clin. Invest. 75: 1174 [1985]). J. Biol. Chem., 262: 3262 [1987] and Hoffman et al. 1 and others hypothesize that TPO is not a self-inflicted presence but rather simply a phenomenon of the well-known multifunctional hormone (IL-3, Sparrow et al, Prog. Clean. Biol. Res, 215: 123 [1986]) regardless of its shape and construction; A molecule that has platelet-stimulating activity is of significant therapeutic value. Although a protein has not been definitively designated as TPO, it is receiving great attention

The 1 - putative cytokine recepts, which is a putative cytokine receptor, may provide Cmpl with the new discovery that signal platelet formation.

MP is a megakaryocytogenetic cytokine receptor mpl is - ©: Megakaryocytopoietic cytokinereceptor It is believed that the proliferation and maturation of hematopoietic cells is precisely regulated by mutational factors 0 . Positively or negatively the proliferation and differentiation of multilineage and multilineage stem cells modulate these effects through the high affinity binding of extracellular protein factors to specific receptors on the surface of cells. These cell surface receptors have a common semiconfiguration and are generally classified as members of the large family of cytokine receptors. The large family group (8 chains and/or) IL-2 includes the L0 receptor (Hatakeyama et al., Science, 244:551-556 [1989]; Takeshita et al., Science, 257:379- 382

[1991]), IL-3 (Itoh et al., Science, 247:324-328 [1990]; Gorman et al., Proc. Natl. Acad.

Sci. USA, 87:5459-5463 [1990]; Kitamura et al.. Cell, 66:1165-1174 [1991a]; Kitamura et al., Proc. Natal. Acad. Sci. USA, 88:5082-5086 [1991b]), IL-4 (Mosley et al.,

Cell, 59:335-348 [1989], IL-5 (Takaki et al, EMBO j, 9:4367-4374 [1990]; 0

Tavernier et al., Cell, 66:1175-1184 [1991]), IL-6 (Yamasaki et al., Science, 241:825-828 [1988]; Hibi et al., Cell, 63:1149-1157 [ 1990]), IL-7 (Goodwin et al., Cell, 60:941- 951 [1990]), IL-9 (Renault et al., Proc. Natl. Acad. Sci. USA, 89:5690-5694 [ 1992]) granulocyte-macrophage (GM-CSF) colony-stimulating macrophages ale (Gearing et al. EMBO J., 8: 3667-3676 [1991]; Hayashida et colony-stimulating factor 0

—_ Y Y —_ and cell colony stimulating factor al, Proc. Natl. Academic Science. USA, 244: 9655-9659 [1990]), granulocyte colony-stimulating factor (G-CSF) (Fukunaga et al, Cell 61:341-350 [1990a]: Fukunaga et al, Proc. Natl.

Acad. Sci. USA, 87:8702-8706 [1990b], Larsen et al, J. Exp. Med., 172:15591570

[1990]), EPO (D'Andrea et al. Cell, 57:277-285 [1989]; Jones et al, Blood. 0 76:31-35 [1990]) prolactin and also a receptor for the leukemia inhibitory factor ( LIF) is an inhibitor of leukemia Jule (Boutin et al, Proc. Natl. Acad. Sci. USA, 88:7744-7748 [1988]; Edery et al., Proc.

Natl. Acad. Sci. USA, 86:2112-2116 [1989]), cilia-stimulating factor (Leung et al., Nature, 330: 537-543 [1987]) (GH) and growth hormone 3 + ciliary neurotrophic factor (CNTF) Neuronal members of the major family group (Davis et al, Science, 253: 59-63 [1991]) may be grouped into three functional vertebrae (for information, see the cytokine of the receptor - (Nicola et al., Cell , 67: 1-4 [1991]( as ¢ single chain receptors Class I contains single chain receptor 1 that binds G-CSF-R or granulocyte colony-stimulating factor receptor erythropoietin (EPO-R) which is located outside the cell and also generates a domain with high affinity via its ligand QL-subunits inside the cell. and the interleukin-6 (IL6-R) granulocyte colony-stimulating factor receptor.

yy — — “(GM-CSF-R) interleukin-3 receptor (IL3-Rot) and other members of the cytokine receptor superfamily . These bind -0 subunits with their ligand factor with low affinity but do not transmit a signal into the cell. A high-affinity signaling receptor is composed of a heterotrimeric 0:006 dimer between a -subunit 0 and a class tertiary member of the 0-cytokine receptor; The B= cytokine de jb subunits are called eg Por and are subunit 8 common to all three IL3-Ro subunits 5 +011-051-1. Evidence comes that mpl is a member of de gana, the major family of cytokine receptors, the sequence homology of amino acids. (Gearing, EMBO J, 8: 3667-3676 [1988]; Bazan, Proc.

Natl.

Acad.

Sci.

USA, 87: 6834- Davis et al., Science, 253: 59-63 [1991] and Vigon et al, Proc.

Natl Academy. \ .,[1990] 6938 Sci.

USA, 89: 5640-5644 [1992]) and its ability to transduce proliferative signals. A protein sequence derived from a molecular clone of murine C.mpl reveals that this protein is similar to other cytokine receptors. . in which the extracellular domain contains £10 Vo amino acid and consists of two subdomains each with four highly conserved cysteines and a specific repeat motif in the N-terminal subdomain and in the ©-terminal subdomain. The extracellular ligand-binding domains are expected to have geometries resembling a cylindrical double fold-8. This repeating extracellular domain is dle-like to the common signal transducing chain of 11-3 and 11-5 receptors. and GM-CSF vo and also a quasi of the low-affinity binding domain of (Vigon et al., Opcogene, 6: 2607-2615 LIF).

M ([19985) with that; mpl may belong to the low-affinity class of mpl with its binding factor -cytokine receptors. Comparison of mouse mpl and mature human mpl reveals that these two proteins exhibit TAY ratio of sequence homology. More specifically, the extracellular m-terminal-17 and peripheral-© subdomains contribute 7720 and 780% of the sequence homology ratio, respectively. The mpl most dais region is the cytoplasmic domain distinguished by 7951 of the PLS order of the carcinomas; with a sequence of 1 amino acid near the transmembrane space and is identical in both species. Accordingly; mpl is one of the more conservative members of the major family of ex vigon cytokine receptors. Evidence that 201 is an active receptor capable of signaling reproduction comes with the creation of a chimera combination of a receptor containing the extracellular domain of a cytokine receptor that has a high affinity for a known cytokine. In the cytoplasmic domain of Less mpl that there is no known ligand for eml, it was necessary for the combination to include the OA domain - of the cell with high affinity from the class of one of the first-order cytokine receptors such as IL- 4R or Vigon + G-CSFR and others (above) have combined the extracellular compartment of G-CSFR0 with both cytoplasmic transmembrane compartments of -C-mpl. The combined receptor was implanted into a cell of IL-3 approved cell line. le and (:038/2 31/303) as cocultured with G-CSFR/mpl and full length G-CSFR transplanted for comparison to 00008. Cells modified with the mentioned combination grew equally well in the presence of IL-5 cytokine or 0 -087. Similarly, cells modified with 6-0877 grew well also if in Wa 11-3 or eile .G-CSF all cells in lev. -growth factors sail! A similar experiment was carried out By Skoda et al, EMBO J,

This [993 1[ 2645-2653: 12(7) in which both the extracellular and transmembrane compartments of the human IL-4 receptor (hIL-4R) were fused to the murine cytoplasmic domain and transplanted the combination into A murine IL-3-dependent Ba/F3 cell lineage Synthesized Ba/F3 cells harboring the normal type 1 hIL-4-R proliferated normally in the presence of any of the specified species. specific o4No or IL-3 Ba/F3 cells transfected with 1M111-48/0 proliferated normally in the presence of 11-4 (in the presence or absence of IL-3) indicating that in Ba/F3 WMA, the cytoplasmic domain of mpl contains all the elements necessary to transmit the proliferative signal These synthetic combination experiments demonstrate the proliferative signal formation ability of the proliferative cytoplasmic domain but are silent depending on the potential of the extracellular compartment yy these results are consistent with at least two possibilities ie the mpl is a single chain receptor such as EPO-R or 0-0578 Or be a subunit: E - 8 transponders (class three) require a Skoda subunit (11-3 Jie » - subunit et al. eg). + - the ligand of mpl is thrombopoietin (TPO) as described by JE it has been suggested that serum contains a unique factor; 1 sometimes referred to as thrombopoietin" that acts synergistically With various other cytokines to improve the growth and maturation of MK cells.No such natural factor has ever been isolated from blood serum or any other source despite great effort by many groups.Although it is not known whether mpl is able to bind directly with mpl Stimulation of cell 106; however, recent experiments showed that mpl is related to the transmission of a proliferative signal in the presence of a factor or factors present in the serum of patients with aplastic bone marrow ([1993] 1395-1401: 5( 82 Blood, Millah (Methia et

- 0*1 Evidence comes from mpl that there is a unique colony-forming factor in serum different from 11-16, 11-4, and 1-6] that can transmit a proliferative signal through GM-CSF 5 G-CSF 3 EPO 3 and 5807 IL-11 5 or primitive for 6.001 expression in primary hematopoietic cell lines Examination of the distribution of expression in one of these cell lines. Reproducible autisense Specialized mpl and use studies

0 Using reverse transcriptase (RT)-PCR in purified human Lelia hematopoietic cells, Methia et al. above showed that only strong mpl mRNA signals were found in purified 0034+ cells and 0034 cells. MK and platelets. 0034+ cells purified from bone marrow (BM) represented about 71 out of 314 cells that were abundant in primary or adherent progenitor cells of all lineages (eg, erythroid cells, AT cells, macrogranulosa cells) (MK

0 and then showing that antisense oligodeoxynucleotides in mpl are able to suppress the colony formation of MK LA from LDA +0034 multiple ADL cultured in sera from patients with aplastic marrow (a source rich in active large myeloma colony stimulator [016-05/8]). These reproducible oligo-deoxynucleotides have no effect on red cell colony formation or large granulosa osteoclasts.

Lyi directly with ligand and whether the serum factor showed the ability to prepare the formation of mpl cells whether yo binds directly to mpl and it has been suggested that if it binds large mpl it is still unknown that this was done through

with a ligature; The sequence of its amino acids is conservative in all species and has immunological cross-links with it because of this similarity, as in the extracellular space of humans and suppliers (Maurine (Vigon) and others. Previous [YAY]

Objects - Vv In looking at the above, it can be estimated that there is an urgent, current and ongoing need in the art to isolate and identify molecules capable of stimulating the proliferation, differentiation, and maturation of blood-forming cells; Especially Cell 116 or for therapeutic use in the treatment of thrombocytopenia. It is believed that such a molecule is theraputic of its ancestors, and thus there is an urgent need to isolate such a ligand or ligands (compounds ligand ligand 0

associative) to assess their cycle(s) in the growth and differentiation of cells.

Lag for that; One of the objectives of this invention is to obtain a pharmaceutically pure molecule capable of catalyzing

Proliferation, differentiation and/or maturation of myeloid macrocytes into mature platelet-producing form.

Another goal is to provide the molecule in a form for therapeutic use in the treatment of a blood formation disorder; 1, especially platelet deficiency.

An additional objective of the present invention is the isolation, purification and specific identification of protein ligands

able to bind to a major cytokine family receptor in the organism known as

mpl and transmit a propagative signal.

Still another goal is to provide nucleic acid molecules that carry genes like these 1-CPs and to use these nucleic acid molecules to produce ligands

It binds to mpl in cultured cells carrying recombinant DNA and is used diagnostically or therapeutically.

Another goal is to provide derivatives and modified forms of protein crosslinking compounds, including

Ge variants, amino acid sequences, glgloprotein forms and derivatives

covalent is an equivalent one.

An additional goal is to provide fusion polypeptide forms in which the mpl ligand is incorporated

heterologous protein and equivalent derivatives thereof.

Still an additional goal is to provide different 6m formats integrating ligands with additions

And substitutions of amino acids from the sequence, EPO, to produce a protein capable of regulating the proliferation and growth of 0 progenitors of both platelets and red blood cells.

An additional goal is still to prepare immunogens to create antibodies

antibodies against the mpl ligand or fused forms thereof; And also obtain able antibodies

to bind such ligands.

These and other aims of the invention will be apparent to those with ordinary experience in the art when the specification is considered as a whole.

General description of the invention

as set forth in the accompanying claims; The present invention provides; Among the things Al 5 isolate

A protein to proliferate and promote the maturation and formation of mammalian MK cells.

Designation of a protein seeking proliferation and maturation of MK cells “ligand (ML)” mpl or “thrombopoietin® (TPO) 0; capable of inducing proliferation and/or maturation of Ss Marking and arrival of MK cells

to the productive form of mature platelets.

This protein homogenate may be completely purified from a natural source by a method containing: (1).

Contacting a plasma source containing the ligand molecules mpl to be purified ih ug

vq - - polypeptide immobilized receptor polypeptide » specifically mpl or a polypeptide combined with mpl and fixed on a support under conditions where 5 JS selective adsorbed adsorption The ligand molecules of the mpl to be purified on the receptor immobilized polypeptide; and(?) washing the polypeptide of the immobilized receptor and its support to remove the unadsorbed material; (¥) elution of eluting © ligand molecules of mpl of the receptor stabilized polypeptide that have been adsorbed to it with an elution buffer preferably the natural source is plasma from mammalian organisms or urine containing mpl ligand Optionally the mammalian organism is genotyped aplastic and the ligand receptor is a combined combination of mpl-IgG Optionally; protein of choice for induction and promotion of MK maturation is polypeptide 0 ligand mpl is a completely homogenous synthetically manufactured or recombinant mpl La polypeptide or 100 ' of the present invention is preferably a total sequence of at least 71 7 amino acid sequences of the polypeptide Ligand 01 of porcine, fully homogenous, high purity, sequence identity of at least 7/80 with “EPO-domain” Vo 00m©0170m mpl ligand for porcine. Optionally, the mpl ligand of this invention is Mature human mpl ligand o(Hml) having the amino acid sequence of the mature ligand provided in form number (1) (sequence identity number (SEOID 100: 1 1), a variant or a modified form after Posttranscriptionally modified thereof or a protein having sequence identity of about 7860 with a mature human mpl ligand. Optionally the different form of the mpl ligand is a fragment ¢ 0 and in particular the amino-terminus of the two proteins or the "EPO-domain" fragment of the ligand

For mature human mpl (AML) the amino-terminal fragment preferentially occupies all of the human ML sequence Lila between cysteine I and IV but may contain 00:6008 additions, deletions or substitutions outside this area. according to this model; The polypeptide fragment is fragmented with the formula: X-hML(7-151)-Y ; where hML(7-151) represents the amino acid sequence of human (Hm)TPO from Cys' via Accor on the face of inclusion; 5 X represents the amino acid Cys’ or one of the amino acids of the terminal group of mature AML or its amino acid extensions such as Mel or Tyr or containing lead sequences; For example; contain proteolytic sites (on dw wt 0 factor”) thrombin 4 Xa and 7 representing the carboxy terminal of Cys! or any one or more terminal amino acids present in the KML Mature or extensions thereof. Optionally mpl day J) polypeptide 0 part may be combined with a (synthetic) heterocyclic polypeptide. The preferred heteropolypeptide is a cytokine « colony stimulating factor factor, interleukin, or part thereof; and as dials yo-activating ligand (KL) Kit, IL-1, (IL-3, IL-6, or (IL- 11 (EPO, “GM-CSF, or LIF) and the preferred optional heterologous Lad polypeptide is immunoglobin chain ¢ and in particular 1561, 1802, 1503, or 1504 IgA J or Human IgE, IgD, or IgM, or part thereof, especially the content on the constant domain of a heavy chain -1gG heavy chain

“py is bioactive and the mpl agonist is able to provide the mpl stimulus. The polypeptide ligand =H numbered (eg, preferably labeled nucleotiaes) is able to stimulate the internalization of 3 cells dependent on 11 3- Incorporating thymidine DNA into human FF circulating platelets and/or preferentially able to stimulate mpl mpl incorporation includes a platelet rebound assay in mouse blood in a platelet rebound experiment 0 mML2 5 mML s hML4 5 hML3 5 AML2 3 1101/8153, RIS44) and the appropriate combinations of pML2, PML 5 mML3, and 1 antibody this provides. The invention is an isolated antibody al in an embodiment The isolated antibody capable of binding to mpl may optionally be combined with a second ligand and the antibody may be used or combined to isolate and purify a polypeptide ligand m" with 0 From its source as described by LM “Proven. In an additional feature of this model” UFO mpl containing alin vivo or in organism 10 vitro in vitro mpl The invention is a method for detecting a ligand on contact with an antibody with a sample; In particular, serum sample 800001; expected to contain binding and detect 13) orphaned binding events; carrying a DNA gene The invention provides a nucleic acid clad molecule in additional forms 0 or parts thereof; where the nucleic acid molecule may optionally be encoded by mpl day encoding complementary and a nucleic acid molecule with a sequence that is complementary ¢ detectable for sensing the LE split to; or hybridize under moderate to severe stress conditions with; A nucleic acid molecule with a sequence that carries the preferred nucleic acid molecules are those that carry the mpl ligand gene, the ligand gene of both DNA RNA and mouse 06 and include porcine, human and porcine vy.

—- yy-

The cDNA genomic is an additional feature of this model; The DNA molecule is

DNA carries the mpl ligand gene and additionally contains a replication vector

control sequences to DNA control sequences in which the a3 replicable can be connected

A vector-modified host can be used by a vector-modified host. Optionally — DNA © is a Cdna with the sequence provided as a Y number (SEQ ID NO: 2) ¥ - 5#1 -

° or part thereof.

This attribute also includes cells of the 'host cells' family, preferably 'CHO' cells

A modified vector and a method of using DNA to induce the production of an eml ligand

Including expression of Jala cDNA ligand mpl gene in modified host cell culture and recovery of 0 m ligand from host cells or host cell culture. The ligand mpl prepared by this

The method is preferably a human mpl bond.

The invention provides a method for treating a mammal with a hematopoietic disorder pall.

in particular thrombocytopenia; It includes giving a therapeutically effective amount

from the mpl ligand to the mammalian organism. Optionally ligand 01 is administered in combination with the cytokine Vo; In particular colony stimulating factor or interleukin include stimulating factors

M- 3 GM-CSF 5 G-CSF 5 LIF and (KL) Kit- favored on colonized interleukins ligand or

JL-11 9 IL-6 3 IL-3 5 IL-1s EPO 3 CSF

The invention may include a J jal process and purification of (ML) TPO from a microorganism that produces TPO

contain:

(1) disrupting or WIA lysing containing TPO and (¥) optional separation of a soluble substance from a non-ALE soluble substance containing TPO; and (¥) dissolution of TPO insoluble by solvent in buffer solution; o 5 (?) separating dissolved TPO from other dissolved and insoluble substances (6+ and sole) (0) folding TPO refolding in a redox buffer; and (7). Separation of properly folded TPO from misfolded TPO. The process dissolves the insoluble material containing TPO by reducing chaotropic agent 0 wherein the reducing agent is selected from guanidine is sodium or thiocyanate or 8©. The process furthermore requires that soluble TPO be separated from other dissolved and insoluble substances by one or more selected steps of centrifugation, gel filtration, and reverse phase chromatography. sale) refolding process reductant buffer solution 0 oxidizing agent containing oxidizing agent and reducing agent in general; An oxidation dale is an oxygen or a compound containing at least one disulfide bond. preferably The oxidation agent is selected from oxidized glutathione (GSSG) and cysleine and the reducing agent is selected from oxidized glutathione (GSSG) and Aden. The most preferred is the oxidizing agent glutathione oxidoreductase (GSSG).

Reductase glutathione reductant (GSH) It is also desirable that the molecular ratio of the oxidizing agent be equal to or greater than that of the reducing agent. The oxidizing/reducing buffer solution additionally contains a detergent “preferably selected from SHAPSD y CHAPS” contained in a level of at least 1. The oxidizing / reducing buffer solution additionally contains NaCl preferably in a concentration range of about (= 16 M) and glycerol preferably at a concentration greater than INO. The pH of the oxidizing / reducing buffer solution ranges from about a pH of V,0 pH to a pH of 9; And the re-folding step is carried out in; degrees Celsius for EA -1 hour. The sale step produces a biologically active TPO folding in which a disulfide bond is formed between the Cys closest to the amino-terminus with the Cys 0 closest to the carboxy terminus. -terminus of the 'EPO' domain. The invention may further include a process for purifying biologically active TPO from microGIS comprising: (i) at least a lysate of the extracellular membrane of the microorganism; and (Y) treatment of the TPO-containing lysate degradation by chaotropic \o » and (V) refolding the “TPO” and (?) separation of impurities TPO Unfolded impurities 10 TPO folded properly.

A brief explanation of the graphics: Figure No. (1): shows the inferred amino acid sequence (1) (SEQ ID NO: for the mpl ligand in human cDNA (hML) and the nucleotide sequence carrying the gene 200160006 coding. (SEQ) ID NO: 2) sequence Nucleotides are numbered at the beginning of each line. Untranslated regions 5° and “ are indicated in lowercase letters. Equivalents of amino acids are numbered upstream of the sequence starting with serine 1-1-5:” of a protein sequence Mature (ML) ligand mpl. Putative exon ¥ boundaries are indicated by arrows and possible 1-7 group insertion sites:0087180000 are boxed. Cysteine sites are indicated by a dot above the sequence. Sequence 01 corresponds to the subtitled Line is the N-terminal amino-terminal sequence determined from the mpl ligand purified from pig plasma 0. Figure No. (1) shows steps for an experiment to introduce 317 - thymidine in mpl day to determine the presence of mpl day from different sources; mpl P Ba/F3 WAY was deprived of IL-3 for y¢ h in a humidified incubator at 71°C in CO;\o 70 and air. After 11-3 deprivation, cells were placed on 16-well culture plates with or without diluted samples and cultured for YE 1 h in a culture incubator. 01 μl serum-free RPMI media containing 1 microcurie 0M of 311-thymidine was added to each eye for the last 4-6 hours. The cells were then harvested on 17-well filter plates and 7 washed with water. The filters were then counted (radiation count) 0

1M Figure No. (9): The effect of pronase enzyme, 1011 and heat on the ability of APP to stimulate proliferation of Ba/F3-mpl cells to digest “pronase APP” was paired with Boehringer Mannheim pronase or albumin bovine serum albumin to Affi.gel10 (Biorad) and incubated them separately with APP for YA 1 h at 0 °LTV as a result (lla resins were removed by centrifugation and tested of gall supernatants. The APP was also heated to an Avian for 10 minutes or made in 001 μm DTT followed by dialyzed against PBS fig. (;): Shows the radioactivity of mpl ligand elution from Blue-Sepharose™ ys Phaenyl columns.

Toyopeat™ 01 August 1710. A — 4 Fractions from the mpl affinity column had the highest radioactivity. Fig. 1: SDS-PAGE showing the Ultralink mpl fractions eluted: To 100 µL of each fraction ¥ = oA acetone (Ja 1) was added containing 1 yo micromolar [110 at -10 °C. After Y hours, at -10 °C, the samples were centrifuged (53S yall) and the resulting pellets were washed twice with acetone at -10 °C. The resulting beads of acetone in 1 μl of SDS-solubilization buffer were dissolved in buffer, supplemented to 100 μm DTT and heated in Lay 7 0 C for 0 minutes. Separation of samples on SDS-polyacrylamide gel

Name - 70 The proteins were visualized by a silver staining Figure (+): shows the mpl ligand removed from SDS-PAGE Part 6 of the mpl-affinity column mpl has been dissolved On SDS-polyacrylamide gel induced non-reducing conditions after electrophoresis the DAL was dissected into 11 equal regions which were electro-rinsed as shown in the examples. The eluted samples were electrodialyzed in PBS and examined at a dilution of 0/1. The standards in partial weights Mr used for calibration of the gel were Novex Mark 12 0 standards Figure No. (7): The effect of mpl depleted the ligand in APP on megakaryocytopoiesis was made by passing (Jo 1) over an affinity column mpl-Vaffinity column + V + µg mplIgG / ml NHS- (Superpose™, Pharmacia). Human peripheral stem cell cultures were made in APP 01 or APP 0 without \o ligand mpl and cultured for 11 days The formation of MK cells was quantitatively evaluated as shown in the examples Figure 4: The effect of mpl-IgG on the stimulation of the formation of human MK cells by APP LDA stem cultures were made terminal human terminal in APP 701 and cultured for VY days.On days 0, 7 and 4; (+0,0 µg) mpl-IgG (1,0 7 µg) LANP-RIGG was added after VY One day, the formation of MK cells was quantitatively evaluated

pain as shown in the examples. The mean of the homologous samples is plotted while the actual multiples data are in the brackets. Figure No. (9): Both bands for 90 show a genomic DNA that carries the ligand gene for mpl. The amino acid sequence of exon 7 is indicated (sequence identity).

°Number *: 3 “(SEQ ID NO:; SEQ ID NO::10170)

4) and its complement (sequence identity) 0:8 5 (SEQID NO: Fig. No.: (01) showing the inferred amino acid sequence of a mature human mpl ligand (AML) (sequence identity no.: 7). The predicted amino acid sequence of the human mpl ligand is aligned with the sequence of human erythropoietin formation.

1 Matching amino acids are boxed and spacers are left for perfect alignment indicated by dashed lines. Possible N-glycosylation group insertion sites are underlined intact for Hml and dashed line for -hEPO. Two cysteines important for erythropoietin-forming activity are indicated by a large dot.

mpl from the ligand isoforms showing the amino acid sequence elicited for isoforms 11: (Fig. 1 and human mature 13012 (sequence identity) 1, 11:0 (sequence identity number:) (sequence identity number: : 4) and 11014 (hML3 sequence identity 5 (A number: leaving ayy). Identical amino acids are enclosed within squares. number: 01. Inserted spacers for perfect alignment are shown by dashed lines.

Figures 11(Y), (71b), and (01c): showing the effect of the human mpl ligand on the proliferation of Ba/F3-mpl (a) cells; the formation of human MK cells in vitro In vitro quantitatively assessed using a murine IgG IgG monoclonal antibody.

GPILII, mouse radiolabeled specific for monoclonal antibody glycoprotein 0 for MK cells (b); Mouse platelet mune generates thrombopoiesis as measured by assay of platelet aggregation (c). The information was transferred into two hundred and ninety-three cells by the method of 0.0, 0.0 (German. PrkS by means of a vector in DNA Cloning: A New Appraach 2: 143-190 [1985]) only or Prks- Hml or by Prks-MLys; overnight (Prks-MLys; 01 was generated by insertion of the stop codon after acid 153 of Hml by PCR). h and examined for stimulation of proliferation of Ba/F3-mpl cells as described in Example 1 (a) or in a dg of in vitro human MK cells (b). Using a monoclonal mouse IgG antibody cloned and tattooed with Lele) by PT (Grant et al. as described in MK of 611,111,611,111 cell specific (HP1-1D) Vo cells, the effect of a product was determined. Synthetic ligand al, Blood 69: 1334-1339 [1987]) and partially purified platelet production in (ML) genetically engineered organism (c) using a platelet-rebound proliferation experiment McDonald, T.P. Proc. Soc. Exp. Biol. Annotated by thrombocytosis (do You) Partially purified from IML prepared Med. 144: 1006-10012 (1973) a Genetically engineered plant. The medium was passed through ML on gine conditioned medium

4.0 - (Ja Y) Blue - Separos'e column was saturated with PBS and the column was washed with 58 and rinsed with PBS containing ¥ M of each of NaCl urea. Active fraction 00006 was dialyzed in PBS concentrated to 1 mM fala ml in BSA free from endotoxin. The sample contained a J of 1 unit of endotoxia per mL. Phthran was injected with either or ©0001 or 1101 units of IML or excipient only into each group of six mice. The mean, standard deviation, and standard deriation are shown. The value of da ul gy P was determined. Analysis of test 1 to compare tailed 1 - test - 2 ends. 0 Figure (VF) comparing the effect of isoforms and different isoforms of the human mpl ligand in a Ba/F3-mpl cell proliferation experiment. ; and 1141,153 at different dilutions as shown in Example No. (1). Figures (1) 5 (28 (5) 1£ 1c): Showing the inferred amino acid sequence (Sequence ID No.: 1) Ve The human mpl ligand (AML) or human TPO (0170) and gene sequence in human DNA (sequence ID no: 11). Nucleotides and amino acid components are numbered at the beginning of each line. Figure (0:) shows the SDS-PAGE for th ML332 293 purified 5 thMLiss 293 purified. Figure (V7): Showing the Sequence of Nucleotides: A cDNA Encoding (Sequence ID: (VY) and the Sequencing Amino Acid Sequence (Sequence ID: (VY)) of the Open Reading Frame

sy - - ML —! open reading frame murine ML isoform This isoform of the mature mouse mpl ligand contains “71 amino acids; less than the assumed full length of .017 and dW is designated 140012. Nucleotides are numbered at the beginning of each line. The amino acid components are numbered above the sequence beginning with serial acid 1 1 56. Possible N-glycosylation group insertion sites are underlined. Cysteine acids are indicated by placing a dot at the top of the sequence. Figure (VY): showing the cDNA sequence (sequence id: ?01) and the predicted protein sequence (J ula id: yo) for this figure. ML — mouse isoform 01 ( 241. Nucleotides are numbered at the beginning of each line. The amino acid components are numbered up the sequence beginning with serial number 1 561. This isoform of the mature mouse [mp]ligand contains 75 amino acid components and is believed to be a ligand. mpl full length” labeled ML The signal sequence signal 6 is indicated by a dashed underline and the potential fission point Vo is indicated by an arrow Untranslated regions © and “ are indicated by lower case letters The two deleations present as a result of the alternate cut and paste (mML2 and mML3) are underlined. day Ylcysteine acids are indicated by a dot. The seven possible N-glycosylation group insertion sites are boxed.

gy - - Fig. 18: Comparing the inferred amino acid sequences of the human ML isoform 3 (sequence ID: 9) and the murine ML isoform called MML3 (sequence ID: 11). The predicted amino acid sequence of the complex for the human ML ligand is aligned with the sequence of the mouse mpl ligand. Identical amino acids are placed in squares and dashes are spaced for perfect alignment. Amino acids are numbered at the beginning of each line. Figure No. (19): Comparison of predicted amino acid sequences of isoforms of mature ML from mouse ML (sequence identity No.: ML y (VV) of porcine (sequence identity No: ML (VA - Lanshuman (Seq ID no: 6). Ve amino acid sequences are aligned with commas indicated by dashes; inserted for perfect alignment. Amino acids are numbered at the beginning of each line with matching amino acids placed In boxes Possible sites for N-group insertion 70 are indicated by a shaded box and the residue — cysteine is designated by a dot De sens dibasic amino acids are underlined with \o and to the enzyme site of action are underlined A potential protease for the protein cleavage. The four deletions of amino acids that have been shown to occur in all three species (ML2) are identified by a black square. Figure No. (01) shows the cDNA sequence (Sequence ID No.: ( V4 and inferred mature protein sequence (Sequence ID No.: VA) of the porcine ML isoform (0141). This 2 mpl Allay isoform of porcine has 777 amino acid residues and is believed to be

Cozy The whole pig PML is numbered; labeled mpl This is the length of the ligament at the beginning of each line. Amino acid residues at the top of the sequence are numbered beginning with nucleotides

Ser1 (1) isoform number: (71) showing the cDNA sequence (sequence ID: Yo) and the expected mature protein sequence (Y) of the ML isoform. Porcine (pML2) This isoform of the porcine mpl-ligand contains YYA minus four amino acids from the full length of the porcine mpl-ligand designated 2. The nucleotides are numbered at the start of each line. An amino acid residue upstream of the sequence is numbered beginning with 1, Ser no. (1). (VA and pig-labeled ML isoform 0112 (sequence ID: 71). The predicted amino acid sequence of PML is aligned with the sequence of pML2 Matching amino acids are placed in a box and spacers inserted for perfect alignment by dashes . vo amino acids are numbered at the beginning of each line Fig. (“7): showing features relevant to the complete J ska’)plasmid pSVIS.ID.LLMLORF” or (TPOs3) , used to transduce the gene into 0110-0012 host cells to produce CHO- .th TPO33, fig. (; 7): showing features closely related to the truncated “” plasmid pSV15.ID.LL.MLEPO-D CHO- to produce CHO-DP/2 or 10,5) used to transduce the gene into Y. .th and 10 host cells

M - Figures (Yo) (iro) and (© 7"c): showing the effect of E.coli-rh TPO (Met, 153) on platelets (a) and red blood cells (b) and (c) white blood cells in normal OA. Two groups of six female 05786 mice were injected daily with either PBS buffer solution or 17 μg (046,153,046 (Ecoli-th TPO) °C subcutaneously). Day 0 and on days =v (0 µl) blood was taken from the orbital sinus This blood was directly diluted in (10 mL) of commercial sale dilution and a complete blood count was obtained on a Serrono. Baker Hematology Analyzer 9018 Data presented as mean + Uns standardized mean -Standard error 0 Figures (IY), (@¥), (1) and (77c): showing effect of E.coli 153 -rh-TPO (Met), on platelets (1), red blood cells (2), and (c) white blood cells in sublethally irradiated mice treated with irradiation and submorbidity two groups of 01 05736 female mice by cGy Vor of gamma radiation from a Cs source and injected daily with Lf buffer solution PBS or ©Vo 153 μg) E.coli-th TPO (Met, . On day 0 and successive intermediate time points (£4 μl) of blood was taken from the sinus. This blood was diluted directly in (0 mL) of commercial sala dilution and a complete blood count was obtained on a 9018 Baker Hematology Analyzer 560000. Data are presented as mean + standardized Tha of mean.

— fo on (a) platelet CHO-th TPO; And (Ab) and (17c): showing the effect of (fry) shapes, and (B) red blood cells (erythrocytes) ¢ (thrombocytes (clotting cells) and (C) white blood cells ( leukocytes) in normal mice, or PBS was injected daily with either C57 buffer solution 6 female mice 1 two groups of

0 ) CHO-th TPO, alas See oY ° microliter subcutaneously). Per day

On days 0 - 7 (£0 μl) blood was taken from the sinus. This blood was diluted directly in (10 mL) of a commercial diluent and a complete blood count was obtained on a 9018 Serrono Baker Hematology Analyzer. 24 Data presented as mean + standardized Wad of mean.

Figure 0 (YA): showing dose response curves of different forms of 00770 obtained from different cell strains. Dose-response curves for thTPO were generated from the following cell strains: 0770,0 from CHO (full length Chinese hamster ovary cells) and 110014,153 (truncated form attached to E.coli). with a terminal = N-methionine of which “(N-terminal methionine).

hTPO;, Vo (full length TPO from human YAY cells); met E.coli is incomplete

methionine without [th TPOjs5] (cut form Met-less 155 methionine 05786 female mice daily for 1 day) groups of terminal (E.coli) were injected from according to the group. Every day taken)£ 4 µl) blood of th TPO days by 1 eye pocket for a complete blood count. The data previously presented are the maximum effects of this event in (Met 153 E.coli) seen with the various treatments except for Y. indicated by “146: 153 E.coli from treatment. in cohort 1 day

- See Maximum Impact on Day 5.0 Present data as averages + standard error of the mean. Figure 4 shows dose-response curves comparing the activity of full-length and “clipped” forms of th TPO produced in CHO LA in a truncated form of E.coli ° injected into groups of 1 female mice 05786 per day by (0,(μg) th TPO of various types. In N= XY asl) (0 μl) blood was taken from the sinuses for a complete blood count. Treatment groups were TPO placebo, TPO truncated fragment ¢ d::7100 Ecoli (mixed fragment). Full Length TPO Content Approximately 460-860 full length and 10-100 100 cut; :100 (71 part 30165800001 ) = purified trimmed portion of the original "mix" preparation TPOss, 5 » original "mix" preparation (01 part (k) purified full length of the TPO portion of the original "mix" preparation. Data are presented as means + standard error of the mean Figure (Yh) is a cartoon showing the KIRA ELISA experiment measuring TPO \o showing the mixed MPL/Rse.gD chimera And suitable parts for the basic receptors, as well as the final assembly (all) (the right part of the figure) and a process log (the left part of the figure) indicating steps related to the experiment. Figure No. (71): A process sequence map for the KIRA ELISA experiment showing each step in the procedure.

Figures (IVY) - (7*l): availability of the nucleotide sequence (sequence ID No.: (YY) of the LL expression vector .17.112 .17.112 051771 used for expression 10 in Example No. (09) Fig. No. (v7): It is a schematic representation of the preparation of the plasmid Het 1 pMP" Figure No. (V4) It is a schematic representation of the preparation of the pPMP,; plasmid Figure No. ( ) vo is a schematic representation of the preparation of .pMPs; plasmid fig. no. ): is a schematic representation of the preparation of .pMP”, plasmid fig. no. (PV) is a schematic representation of the preparation of the plasmid : 0110 0 Figure No. (YA) is a schematic representation of the preparation of the PMP; plasmid Figure No. (39): It is a table of the five best TPO clones expressing TPO from the plasmid bank, 10 (Sequence identities of numbers: Yo YE YY, Ola, and 81). Figure No. ($): a schematic representation of the preparation of .pMP,; Figure No. (1): a schematic representation of this Lh For the preparation of the -pMPs; plasmid 0 Figure ($Y): a schematic representation of the preparation of the -pMP5;5; plasmid

Detailed description 1 - definitions: in general; The following words or expressions shall have the meanings indicated when used in the description, examples, and claims .© The term “chaotropic agent” refers to a compound that can; in aqueous solution and at appropriate concentrations; Inducing a change in the spatial configuration or conformation of a protein by disrupting at least partially the forces responsible for maintaining the normal 8:secondary and tertiary structure of the protein. Such compounds include; for example ′′ urea; And guanidine HCI ¢ and sodium thiocyanate. 0 naturally high concentrations are needed; usually ; - 4 molar; of these compounds to exert a chiral effect on the structuring of proteins. “Cytokine” is a generic expression of proteins released by one type of cell that act on another cell as intermediaries between cells. Among the A Bd these cytokines are lymphokines, monokines, and classic polypeptide hormones. The A Bd include cytokines growth hormone insulin-like growth factors human growth hormone human growth hormone —=N N-methionyl human growth saa 9 bovine growth hormone ys wa 5bovine growth hormone parathyroid hormone thyroxine extract insulin early insulin proinsulin early relaxin hormones Jie glycoprotein ovarian follicle stimulating hormone ( FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), and growth factor

Hematopoietic growth factor, hepatic growth factor, fibroblast growth factor, 2018000, placental lactogen, tumor necrosis factor (TNF-Bs TNF-0) and substance Ja afi mullerian-inhibiting substance and mouse gonadotropin-associated peptide 0: and inhibin and activin and vascular endothelial growth factor and integnn and nerve growth factors TNF-B 5s TNF-a and insulin-like growth factor 1 and 7 11- insulin-like growth factor 1 and erythropoietin (EPO) and factors interferon-a Ju interferons s osteoinductive factors, 8, 7 and colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF)0 and granulocyte macrophages. macrophage-CSF WA 5 (GM-CSF) granulocyte IL- 5 IL-1 Jie interleukins (IL's) 5 granulocyte-CSF (G-CSF) IL-2 5 la and IL-3 and IL-6 § IL-5 s IL-4 IL-7 IL-8 IL-9 J deg IL-12 5 IL-11 other polypeptide incl. LIF, 5017 and ligand group. As used in this specification, the previous expressions mean to include proteins from natural sources or from ve cell culture, the product of genetic engineering, recombinant cell culture. likewise; Expressions are meant to include biologically active equivalents ; For example; different in sequence

An amino acid with one or more amino acids or in gol i.e. the extent of the glycosylation insertion. "mpl" ligand and "polypeptide" of thrombopoietin's "ML" s "mpl" ligand or "TPO" is used interchangeably in this specification and includes any polypeptide that has the property of binding to mpl A member of the v. superfamily of the coytokine receptor and has a biosynthetic property of ML as defined below. An ideal biological property would be the ability to catalyze the incorporation of radioactive nucleotides (eg, thymidine 311) into the 11-3-dependent Ba/F3 LAI DNA transposon.

Co. = by mplP human. Another ideal biomarker is the ability to induce fusion of 33S peripheral platelets in a rebound assay of murine platelets. This definition includes a polypeptide isolated from the Jie mpl ligand source of fixed aplastic pig plasma described herein or from another source; such as sal animal species including human m or prepared by the product of recombinant genetic engineering or synthetic methods and includes various forms including clin da functional derivatives, fragments and strains of alleles and isoforms and analogues thereof. 'mpl ligand fragment' or 'mpl ligand fragment' or 'TPO fragment' means part of a normal mpl ligand with mature full length or TPO sequence deficient in one or more amino acids or 0 carbohydrate units Acid deficiency may occur Amino or acid anywhere in the peptide including Lyf at the N-terminal or C-terminal end or internally The segment meets at least one biological property with a ligand mpl will have two ligand segments mpl is typically a sequence of at least 01, 1, 70, 78, 0, or 46 amino acid components that are identical to the ligand sequences of mpl isolated from a 1y organism Including a ligand isolated from aplastic porcine plasma or a ligand of a human or mouse, and in particular its 800 domain space. The expression “mpl ligand variants” or “mpl ligand sequence variants” as defined herein means a biologically active mpl ligand as © - defined hereinafter having less than Sequence ID of 001 ligand mpl isolated from genetically engineered cell culture, aplastic porcine plasma, or human ligand having the inferred sequence described in Figure (1) (Seq ID No.: .1) normal; Different types of ligand will have an active mpl

1e - bioavailable amino acid sequence of at least 770 amino acid sequence identity with ligand mpl isolated from porcine aplastic plasma or whole mouse or human ligand or parts thereof (refer to fig. [sequence id number: 1]); preferably about at least 775; more favorable still about at least 0 and more favorable still about at least 788 and more favorable Lad about at least 140 0; and most preferred about ve at least. A “chimeric mpl ligand” is a polypeptide containing a mpl ligand of the entire length or one or more parts thereof fused or bonded with a second heterologous polypeptide or one part or more than him. The chimera will contribute at least one biological feature - in association with the ligand mpl the second polypeptide will typically be a cytokine or immunoglobin 0 or part thereof. The expressions "highly purified mpl ligand" and "l= mpl ligand mpl ddan y's" homogeneous mpl ligand substantially used are used interchangeably and mean mpl ligand has been purified from mpl ligand source or prepared by genetic engineering or synthetic methods and is sufficiently free of peptides or other vo proteins (1) to obtain at least Ve and preferably 01 A terminal amino acid or endogenous amino acid sequence using a spinning cup sequenator best commercially available sequence read device sold on the market or modified by published methods as of the date of filing of this Adal gall or (?) Homogeneity was determined by SDS-08 under non-reducing or reducing conditions using Coomassie blue 5 « 0 preferably stain silver: 51176. Homogeneity here means less than about #5 contamination with proteins from another source.

The term 'biological property' when used in Ale means either with 'Adan' or 'isolated ligand' to have thrombopoietic activity or to have on an in vivo 10 effector antigenic function or activity that is directly or indirectly induced or performed by a denatured or native conformation (whether in its natural mpl 0 structure or binding Any carrier that binds an Effector or part thereof. The functions of an effector include conformation and in particular transmission of a proliferative signal including empl, stimulation or inhibition of the carrier, assistance of cytokines and modulation of the biological activity of DNA and the regulatory function of replication, replication, cytokine activation (particularly other receptor activation; receptor activation, growth or differentiation of cells, etc.) up- or down regulation and deactivation. or antigenic site epitope generating function against having an antigenic sticky top similar. means antibodies with crossreacting antibodies that are capable of cross-reactivity antigenic site polypeptide — generation of natural antigenic base antigen . The function of the mpl antibody ligand established against at least 1/001 mol antibody ligand is that it binds with an affinity of about mpl ligand isolated from porcine aplastic plasma. normally mpl binds against las ve gvfd'm polypeptide ligand at least mol. The most preferred is 1/01 *1 affinity polypeptide against ligand (AU) that binds to an antibody. An active polypeptide is antigenically mpl having one of the effector functions described before. mpl rabbit polyclonal is a polyclonal antibody 'biologleally activity' isolated from cell culture produced from rabbit mpl created by ligand formulation of antibodies 0

Freund's aplastic porcine in the complete adjuvant Le 320 or recombinant genetically engineered skin-boosting response to the formula; Caan augmentation and injection (Freund for complete adjuvant

ov — — immunogenicity by injecting the composition into the intraperitoneal cavity until the plateaus level the antibody titer to the ligand mpl The expression 'Jad' means 'biologically active' when used in conjunction with either 'ligand' "mpl or mpl Alay; isolate"; mpl ligand or polypeptide possessing 0 thrombopoietic platelet-generating activity or contributing to the effector function of the mpl ligand isolated from aplastic or expressed porcine plasma A known basic effector function of the mpl ligand or polypeptide in this specification binds to mpl and catalyzes the incorporation of radioactive nucleotides (*H - thymidine) into DNA to 11-3-based mpl P transfected human Ba/F3 cells. Another known effector function of mpl ligand 0 or 0017060808 in this specification is the ability to induce incorporation of 58 into circulating platelets in Mouse platelet aggregation experiment Another known effect of the mpl ligand is the ability to induce the generation of human MK WA in vitro Its quantity may be assessed using a radiolabeled monoclonal antibody specifically for dual cell glycoprotein megakaryocyte glycoprotein GPIIpIII, 1 _ “Amino acid percentage” relative to mpl ligand sequence is defined in this specification as the percentage of protein components of amino acids of a chosen sequence that are identical to residues in the isolated mpl ligand sequence from non-transcriptional porcine plasma or mouse or human ligand having the inferred amino acid sequence annotated in Figure (1) (sequence ID no: of) after aligning the sequences and inserting the spacers; if necessary; to achieve sequence identity with the maximum percentage; and .oy does not consider any conservative substitutions for part of the sequence identity. No deletions, N-terminal or C= insertions, or internal extensions of 4 mpl ligand sequences will be interpreted as affecting sequence quality or

Mug - similarity. The polypeptides thus include an optimal biologically active mpl ligand that is said to have identical sequences: a mpl-pre-ligand, a pro-mpl ligand, and a mature mpl ligand. ligand Knowledge of the “microsequencing of the mpl ligand” may be achieved by any appropriate 0 measurement procedure provided that the procedure is sufficiently sensitive. In one of these ways Jie; A purified Je polypeptide chain obtained by SDS gels or from a direct A fli HPLC step is performed by Edman automated Edman phenyl isothiocyanate degradation using a Model 4708 chain method for biosystems used as a phase. Applied Biosystems gas phase sequencer equipped with als wg 120A phenylthiohydantion (PTH) amino acid analyzer | eg hydroxylamine. 2-nitro-5-thiocyanobenzoate “CNBr” or enzyme (eg; staphylococcal protease “¢ clostripain” trypsin followed by fragmentation (eg HPLC). Amino 1 using Justic innovations, (Palo Alto, CA) Chrom Perfect 0 sequence illustration performed on a VAX™ Digital Equipment Co. 11/785 computer as described by Henzel et al. ., J.

Chromatography, [1987] 404 : 41-52 optional; Samples separated by HPLC may be electrophoresis analyzed on © + SDS-PAGE 7Y and then electrophoretically transferred to a membrane (Pro Blott, AIB, PVDF Foster City, CA) stained with Coomassie™ Brilliant Blue (Matsurdiara, J.

Chem, 262: 10035-10038 [1987]) © 3 Excision of a specific dye-specific protein from the macula and a Ne-terminal chain sequencing study is performed by the gas-phase sequencing method described above. For internal protein sequences; HPLC sections are dried under a vacuum (Speed Vac™) and re-suspended in appropriate pH-regulated solutions; and digested

- 00 —

Wako Chemicals, Richmond,) Lys-c or a specialized enzyme » cyanogen bromide by (va or (Boehringer Mannhein, Indianapolis, IN) Asp-N after digestion; resulting peptides are sequenced to a mixture or after separation b HPLC on a C4 saturated propanol gradient column at TAF 70.11 before the series in the gas phase “Thrombocytopenia 0” is defined as a platelet count less than 11 101 per liter Thrombopoietic activity is defined as biological consisting of the acceleration of proliferation, differentiation and/or maturation of MK cells or precursors of MK cells into a platelet-producing form. This activity may be measured in various experiments including the in vivo mouse platelet aggregation synthesis experiment and the platelet surface LDA antigen 0 induction experiment as measured by an anti-platelet immunoassay blood anti-platelet immunoassay (Anti GPILIIL,) of a human leukemia megakaryoolastic cell line (CMK) and induced multiple mating in the CMK strain. megakaryoblastic cell line (DAMI) “thrombopoietin” (TPO) is defined as a compound that has plateletogenic activity or is 1 capable of increasing serum platelet counts in AS my breasts. 170 is preferably able to increase the endogenous platelet count by at least 701 and more preferably by 0 5 and most preferably is able to raise the human platelet count to more than 101 x 01 per liter of the blood. 'Tsolated mpl ligand nucleic acid' is RNA or 0 0 l containing more than 11 and preferably 01 or more sequential nucleotide bases bases encoding a mpl ligand or a biologically active part of it that is complementary to RNA or “DNA” or “hybridizes” RNA hybndizes or DNA and remains linked

ov — - consistently under moderate to severe stnngent conditions. This RNA or DNA shall be free from the nucleic acid of at least one contaminating source to which it is normally bound in the natural source and preferably completely free of any RNA or 0718 of a mammalian organism. The expression “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 normal cell but is in a different chromosomal location or is otherwise flanked by DNA sequences not normally found in the source cells. An example of an isolated mpl ligand DNA is RNA or DNA that encodes a biologically active mpl ligand 0 contributing at least a Ve sequence identity; and more preferably yA at least; It is still more preferable at least; Also more preferably 90 and more preferably 796 sequence identifications with the human, murine, or porcine association mpl. The expression “Control sequences” when referring to expression means the DNA sequences necessary for the expression of a functional genetic segment in a particular organism. 1 Control sequences suitable in prokaryotes ¢ include for example 'Jha a promoter' and optionally a factor sequence; the ribosome binding site and possibly other poorly understood sequences. It is known that WIA is eukaryotic using promoters, polyadenylation signals and -enhancers The expression means 'operably linked' when referring to nucleic acids in which nucleic acids are © placed In a functional relationship with another nucleic acid sequence, for example J, the DNA of a previous sequence or a secretory guide (secretory guide) is malleably spliced into the DNA of a polypeptide if it is constructed expressed as a protein contributing to Polypeptide secretion: done

Because - in practice, a 0802© enhancer or promoter is connected to a coding sequence if it affects the replication of the sequence; or functionally attach binding site 110080016 to a genetic sequence if it is positioned in such a way as to facilitate the translation ely of the proteins. in general; The term "Joa ga" operably linked means that the DNA sequences being read are continuous in meaning; In the case of secretory evidence; Continuous meaning and in the process of reading 0 continuous. Connection enhancers are implemented on the other hand; Phase inductors do not have to be at appropriate cut-off locations. if no such sites exist; Ligation is used by ligation according to synthetic oligonucleotide linkers or connectors adapters for conventional practice.

0 means the expression "exogenous" when referring to an element means; A sequence of nucleic acid that is homologous or similar to the cell but at a location within the host cell's DNA where the element is not normally present. The terms "cell", "cell line Aa" and "WA culture" are used interchangeably throughout this patent and such designations include all progeny cells or ADL cells. Benlac;

vo (JB) Jue expressions for “transformants” and “transformed cells” include the first cells and cultures derived from them regardless of the number of transformations. It is also understood that all offspring may not It is precisely identical in DNA content due to intentional or unintentional modifications. Mutant progeny with the same function or biological activity that was searched for in the originally mutated cells are included. Where you mean separate labels; this will be clear

Gee the context. “Plasmids are autonomously replication 4&1 wwe replicating circular DNA molecules possessing Independent ongins for replication and are named herein

mah - by lowercase letters "m" preceded and/or followed by uppercase letters and/or numbers. 05 the beginning in this patent is either commercially available; or is available to all on an unrestricted basis; Or these plasmids can be generated from available Jie according to published procedures. in addition to; Plasmids know other equivalents in the art that would be obvious to the average professional in the art.

0 means “pad restriction enzyme digestion” when referring to (DNA) catalytic severing of internal phosphodiester bonds of DNA by an enzyme that works only at certain sites or sites in DNA sequence Such enzymes are called "restriction endonucleases". Each restriction endonuclease enzyme recognizes a specific DNA sequence called ad sa' restricted. JIG appears two-fold sy letter be

The various limiting enzymes used herein are commercially available and their reaction conditions and cofactors are used; Other claims are as established by abbreviations consisting of a capital letter followed by other letters representing the microorganism from which each restriction enzyme was originally obtained and then the number assigned to the particular enzyme. in general; About 1 microgram is used

0 or a DNA fragment with about 1 - ? an enzyme unit in about 1 microliter of solution

1 regulator. Quantities of buffer solutions and substrate for specific bound enzymes are specified by the manufacturer. In the salad, incubation for about one hour at a temperature of VY is used, but this may change according to the supplier's instructions. after incubation; Protein or polypeptide is removed by phenol extraction and chloroform Digested DNA is recovered from the epithelium by ethanol precipitation Digestion may be followed by restriction enzyme hydrolysis phosphatase

0 bacterial alkaline phosphatase l phosphates © end to prevent both ends of the binding

The splitters of a DNA segment of the "circularizing configuration" or form a closed loop that will prevent the insertion of another DNA segment into the restriction site. if not stated otherwise; Digestion of plasmids is not followed by terminal dephosphorylation de gana © . be

oq — - Conventional stripping reaction procedures and materials as described in Sections Oma =) mo - 1 Sambrook et al., Molecular Cloning: A Laboratory Manual [New York: Cold Spring Harbor Laboratory Press, 1989] means "Recovery" or "isolate" a known DNA fragment from a restriction digest; Separation of the digest on polyacrylamide or agarose gel by Gaal 5 electrophoresis separated the fragment of interest by comparing its migration against that of distinct DNA fragments of a known molecular weight; remove the part of the gel containing the desired part; Separation of the gel from the DNA is a generally known procedure. For example, see Lawn et al., Nucleic Acids Res., 9: 6103-6114 [1981], and Goeddel et al., Jala? Nucleic Acids Res., 8: 4057 [1980] Southern analysis 'or 'Southern blotting 0' is a method by which the presence of DNA sequences in DNA digestion of a restriction endonuclease enzyme or DNA _containing a structure whose existence has been proven by hybridization with a labeled radioactive oligonucleotide or an radioactive DNA fragment. Southern analysis typically involves separation of DNA digests by electrophoresis on agarose gels and de-Naturalization of DNA after separation by electrophoresis; Transfer the DNA to nitrocellulose 00, nylon, or another support membrane suitable for analysis by radiolabelling, insertion of a biotinylated bioset, or an enzyme-labeled probe as described in Sections 4 — ©Y — 4 - YV from Sambrook et al. Jala' Southern analysis or 'Southern blotting' is a method used to identify RNA sequences that hybridize to a known probe such as an oligonucleotide, Y.DNA fragment, or cDNA or part thereof; or RNA fragment probe digitized by radioisotope FP Jie or by insertion of a biotinylation kit; or by enzyme. The RNA to be analyzed is usually separated by electrophoresis on agarose gel or polyacrylamide gel and transferred to nitrocellulose, nylon ¢ or a suitable membrane; hybridization with the probe; using

Well-known standard techniques in the Art of Jue are those described in the OF = V = FA = 1 sections of Sambrook et al. preceding. Ligation is the process of forming phosphodiester bonds between two nucleic acid molecules. For linking the two parts; The terminals of the two parts must be compatible with each other. aed 0e compatible . in some cases; The ends will be compatible immediately after the endonuclease aad enzyme. on the other hand; It may be necessary to first convert the dislocated ends commonly produced after endonuclease digestion into dull ends to make them compatible for binding. to dull the limbs; The DNA is processed in a suitable buffer solution for at least 10 minutes at 11°C with about 10 units of T4 DNA § Klenow fragment of DNA polymerase 1 & j= polymerase 0 in the presence of the four deoxyribonucleotides. tnphosphates The segments of DNA to be linked together are placed in a solution in approximately equimolar amounts. The solution will also contain ATP, a buffer ligase, and a ligase-binding enzyme such as DNA ligase 14 in about 10 units per 1.5 μg DNA. If the DNA is bound in a vector; The vector is first rendered rectified by digestion by the bound endonuclease(s)ve. The rectum is then treated with bacterial alkaline phosphatase or phosphatase in the gut of 1 calf to prevent self-ligation during the ligation step. ‘DNA Preparation’ from LAY means isolation of plasmid DNA from a plasmid host cell culture. Commonly used methods for preparing DNA are the large and small scale © plasmid preparations described in the FY - 1 - Yo - 1 sections of Sambrook et al. preceding. After preparing the 0178; It can be purified by methods well known in the art such as those described in 1 aud-40 of Sambrook et al. prior.

+1 - - "Oligonucleotides" are short-length, single-stranded, double-stranded polydeoxynucleotides that are chemically constructed by known methods (such as phosphotriester, phosphite, or phosphoramidite ¢ using Alla solid soiid-phase technologies as described in EP No. 15077 published in May VAAN or via © deoxynucleoside H-phosphonate media as described by Frochler et al., Nucl.) Acids Res., 14: 5399-5407 [1986] Additional methods include the polymerase chain reaction defined hereinafter, other autopnemer methods, and the synthesis of oligonucleotide syntheses on solid carriers. Each is described These methods are used if the entire DNA sequence of the 8600 gene is known, or a complementary 0 DNA sequence to the coding strand of the coding strand is available. Someone guesses possible DNA sequences using known and preferred coding guesses for each amino acid wish. The oligonucieotides are then purified on polyacrylamide gels. The term “polymerase chain reaction” or “PCR” refers to the procedure or method by which small amounts of nucleic acid such as RNA and/or DNA as described in US Patent No. 4,687,190 issued in YA July TAY In general, the sequence information from the terminal ends of the region of interest must be available in order to be able to Thus, the design of the oligonucleotide primers that match sequences or are similar to the reverse strands of the template sequence to opposite strands of the template to be amplified 0 to be amplified. The nucleotides may match

- a" _-

At the terminal © of two primers with the terminal ends of the material to be supplied. PCR can be used to supply specific specific RNA or DNA sequences from a total of genomic DNA or cDNA cloned from a group of cellular RNA. Or from bacteriophage viruses, or from plasmid sequences, etc., as in the following general reference, Mullis et al., Cold Spring Harbor Symp.

Quant.

Biol., 51:263 [1987]: Erlich. ed.. PCR ° Technology, (Stockton Press, NY. 1989).

As used throughout this text, PCR is one, but not the only, example of a method

“Jel DNA Polymerase for Provision of a Test Sample of Nucleic Acid” is a method that involves the use of a known nucleic acid as a primer; with acid polymerase

0 Nucleic to supply or build a specific piece of DNA.

And “Stringent conditions” means those conditions in which (1) - low ionic concentrations and high temperatures are used in the washing process” i.e., for example, a solution of sodium (0.1 M ©) sodium citrate / ( (M ++) chloride (©) / 7 from (SDS), 11200050 under

temperature of 00 m; or (Y) — in which a naturally occurring substance, formamide, is used during hybridization;

Bovine serum 0.1/aibumin of 0.1 with formamide (volume/volume) of 0 Se ve sodium micromolar buffering solution of 0+ [polyvinylpyrrolidone] 7 | Ficoll of .7 mM VO 5 ¢ sodium chloride mM of YOu of 1.0 °C; with (pH)phosphate

of 017016 sodium at a temperature of 247°C. Another example is the use of formamide 50 (sodium chloride) SSC X #a “sodium citrate” (M 1r; Al 5 +0 ml

X and sodium pyrophosphate from 7+.) 5 ¢(1,A = pH)sodium phosphate ..mol from 0

Denhard's solution and DNA from sonicated salmon sperm DNA treated with ultrasound (04 µg/mL); (5) + 7 dextran sulfate 7 01, SDS induces a temperature of 7 X 550 (8.080) at 1.1 out of 505. “Moderately stringent conditions” is described in the Sambrook reference. et al above; this includes the use of a wash solution and hybridization conditions (such as temperature; ionic concentration; dy sal for SDS) which are less severe than the sla described above. An example of moderately severe cases is the procedure An overnight incubation process at a temperature of 71°C in a solution containing + formamide 7Y «© VO) SSC X ml M of sodium chloride », 11 ml M of «(trisodium citrate, 00 . 1 mM of (pH) sodium phosphate = 1.6); that is X dextran solution} 7 + 5 “Denhard sulfate ¢ and 10 µL/mL of DNA from normal and normalized salmon sperm; follow This is washing the filters in SSC 71 under a temperature of approximately between TV = 200 and a person experienced in this field understands how to adjust the temperature; ionic concentration etc. as necessary to accommodate Jie dal so along probe, etc. ve and for 'antibodies (Abs) 00000:66m' and immunoglobulins (Igs)" are glycoproteins with similar structural properties. Although Balad bodies show specificity from association with a specific antigen; However, antibodies include both antibodies and other antibody-like molecules. But it is limited to the specific specificity towards the antigen. Polypeptides of the latter type are produced at low levels

— > -_ via the lymph system, and at high levels by myeloma cells. sale What “Native antibodies and immunoglobulins” are tetrameric heterotetrameric glycoproteins© glycoproteins with a molecular weight of about 1501000010 Daltons » Jag in two of identical identical light (L) chains and two identical heavy (H) ALAN chains. Each of the light chains is linked with a heavy chain by a covalent bond of disulfide, while the number of disulfide bonds varies between the heavy chains of different types of homologous immune bodies. Each heavy chain and light chain also includes bridges of 0 disulfide that are equidistant from the chain. Each heavy chain at its san) terminals contains a variable domain (VH) followed by a number of constant domains. Each light chain has a variable space at one end (VL) and a constant space at the other end. align the light chain constant domain with the first A LEN constant domain and the light chain variable domain align with the heavy chain variable domain. It is believed that there are specific amino acid building blocks that form an interface linking the disparate domains of the light chain and the heavy chain. (Clothia et al., J.

Mol.

Biol.. 186:651-663 [1985]: Novotny and Haber, Proc.

Natl Acad Sci.

USA. 82:4592-4596 [1985]).The term "variable" refers to the fact that some parts of the variant © domains differ widely in amino acid sequences between antibodies; And used

Ho —_ in the binding of a particular antibody and in determining its specialization with respect to a particular antigen. However, here the disparity is not uniformly distributed among the different domains of the antibodies; Rather, it is concentrated in “compartments called complementanty regions (CDRs) or hypervariable regions in each of the varying domains of the light chain and the heavy chain. The more conservative parts of contrast domains are called the framework (FR). The divergent domains of the local heavy chain and the local light chain include; Regions of the framework (FR) and these largely take the form of a B-sheet beta-sheet connected by “complementary defining regions (CDR) to form a ring that communicates with or is part of the structure of the Uy-8 sheet. In each chain, CDR regions from another chain combine to form the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, National Vo Institute of Health, Bethesda, MD). [1987]) The constant domains are not directly involved in the process of binding between the antibody and the antigen, but here they show different functions affecting Jia, the participation of the antibody in antibody-dependent cellular toxicity. 1 Digestion of the antibodies by Papain yeast leads to the production of similar antigen-binding parts called “785” parts, each located within one site for the cantigen to bind with a remaining part of (Fc) whose name reflects the ability of this part It crystallizes quickly.Treatment with Pepsin enzyme results in the production of a fragment of F(ab) with two antigen-binding sites while retaining its ability to cross-link with -antigen.

1- FY represents the minimum antibody fragment that contains a locus for antigen binding and recognition. This region includes a dimer with a variable domain of the ALE chain and another of a light chain in a contiguous position through a ligand. non-covalent. In this configuration, the three complementarity determination regions (CDR) interact in each of the varying domains to distinguish a binding locus

antigen 0 on the surface of the compound .17-17 dimers. Overall, the six complementarity determination regions (CDR) impart specific specificity to antigen-antibody binding. However, even with a single variant (gf) half of Fy comprising only three antigen-specific complementarity (CDR) regions, this single domain has the ability to discriminate and bind antigen, but to a lesser degree than Familiarity compared to the entire bonding locus.

0 The Fab fragment also contains a light chain constant domain and a first CHI heavy chain constant domain. The Fab fragments differ from the Fab fragments in terms of the addition of a few residues from the terminal ends of the CHI domain in the heavy chain, Loy, including one or more cysteine acids from the antibody adhesion region. The Fab-SH represents the Fab fragment in which the cysteine residues in the fixed domains de sane carry a free thiol group, and originally the body parts were produced

1 The F(ab)2 antagonist is in the form of pairs of Fab' parts with adjacent cysteine acids between them. There are other known cases of chemical couplings between parts of the antibody. The "light chains" of antibodies (immune bodies) from any vertebrate species can be grouped into one of two distinct types, Kappa and Kappa.

Lambda© is based on the sequence of amino acids in their fixed domains.

Vv — — Depending on the sequence of amino acids in the constant domain in the heavy chains, Lelia bodies can be classified here into different categories. There are 0 basic classes of antibodies” [IgA] IgM IgG IgE < IgD Many of which can be divided into subclasses (symmetric types) such as 180-1; 2; IgG-2 < IgA-1 4 IgG-4 dgG-3 The constant domains of the corresponding -© heavy chains of different classes of immunoglobulins are called poy » epsilon ¢ delta »a, respectively. The subunit structures and three-dimensional stereomorphisms in the different classes of immunoglobulins are well known. The term “antibody” is used in a broad sense to specifically cover monoclonal antibodies (including agonist0 and non-antigenist) and antibody combinations of specificity (pail). The polyepitopic label for “antigen” as well as antibody fragments such as F(ab”), 5 Fab 7 as long as these fragments have a desired biological activity. The term refers to “monoclonal antibody” as stated in This text refers to an antibody that is obtained from a group of homogeneous antibodies " 1 Any individual antibodies that contain a similar group except for possible mutations that may be present in small quantities. Monoclone antibodies are considered highly specific and directed towards Moreover, unlike conventional Jl polyclonal antibody preparations they typically include different antibodies directed at different epitopes; here each antibody is directed at a single epitope on the antigen. © In addition to specificity, monoclonal antibodies have the advantage of:

The possibility of creating these bodies in the medium of a hybrid tumor culture without contamination with other immune bodies. The prefix eal “monoclonal” refers to a property of an antibody that is obtained from a broadly homogeneous pool of antibodies without the antibody having to be produced in any particular way. For example, the antibodies to the hybridoma method can be prepared using the al method used according to the invention.

which was first described in ref.:

Kohler & Milstein, Nature, 256:495 (1975).

Specifically, monoclonal antibodies include chimeric antibodies (immunoglobulins) in which part of the metabolite chain and/or the light chain is homologous or homologous to the corresponding sequences in the antibodies derived from species specific or belonging to a particular class or subclass of antibodies; the remainder of the chain (or chains) is identical or homologous to the corresponding sequences in the antibodies

_ derived from other types or belonging to another class or subclass of antibody; and also on parts of such antibodies as long as they exhibit desirable biological activity.

The monoclonal antibodies herein specifically include "" antibodies () in (U.S.

Patent No. 4,816,567 (Cabilly et al.); and Morrison et al.. Proc.

Natl.

014 Non-human antibodies, Jia, those found in mice that were humanized, are immunoglobulins prepared by chimeric mixing; or chains of immune bodies; or parts of such chains (such as FV, Fab, Fab, Fab), or other LJ sequences of binding antibodies — 60p8008; It contains a minimum of 0 sequences derived from non-human immunoglobulins. For the most part, the antibodies represented in human form are human antibodies (recipient Jie¢(antibody) in which a specific complementarity amino acid locus (CDR) on the recipient antibody is replaced by one from Non-human species on the given antibody “donor antibody” which are species including mice, rats, rats, and rabbits that have a desirable degree of 0 specificity, affinity, and capacity. In some cases, frame residues are replaced. The FV of the human immune body with other non-human counterpart residues.In addition, the human antibody represented may include residues not found in any of the recipient antibody sequences; or in the imported CDR or in frame sequences.These modifications aim to further purify and optimize the performance of the antibody.vo In general, a homozygous antibody primarily in human form comprises at least one domain and typically two distinct domains in which there are limiting regions complementarity (CDR) is analogous to that of a non-human immunobody; The FR regions are a sequence of the human immune system Lede pane 5n0005©08. Also ideally, an antibody represented in a human image also contains at least one portion of an immunoglobulin constant region (FX)©, typical of that portion in a human immunobody. For more details, the following references can be found:

Ya -— — Jones et al., Nature, 321:522-525 [1986]. Reichmann et al., Nature, 332:323-329 [1988]; and Presta, Curr.

Op.

Struck.

Biol, 2:593-596 [1992]). Suitable tissues in humans do not show here a state of sensitivity or resistance to the polypeptide upon the second administration of the polypeptide after an appropriate latent period ranging between EN YEA Second: Preferred models from this Preferred Embodiments of the Invention The preferred polypeptide in this invention is essentially a homodimeric polypeptide 0 known as ligand group or groups 001; or thrombopoietin (TPO), which has the property of binding to the mother of a member of the major family of cytokine receptors; Which also has the biological property of stimulating the fusion of distinct nucleotides (*H-Thymidine) labeled on DNA in Ba [F3-dependent 11-3 cells to which the human mpl P information was transferred. As for the most detailed mpl ligand group or groups, it is those that have been isolated from a mammalian protein (or proteins) with hematopoietic activity, specifically megakaryocytopoietic activity or thrombocutopoietic activity through which these proteins can stimulate GROWTH, MATURATION AND/OR DIFFERENCE OF MEGAKARYO CELLS OR PREGNATORS OF THESE CELLS AND THEIR TRANSFORMATION INTO A MATURE PRODUCTIVE PLAETTE FORM. The most preferred polypeptide in the present invention is the human mpl ligand group(s) or parts of those groups which are characterized by megakaryocytopietic or thrombocutopoietic activity and optionally these groups

— except — human mpl lacks glycosylation . There are other associative groups favored by human mpl; It is an EPO domain in ¢hML and represents the polymerized form of IML known as 107-248 or 11000-248 with a mature full-length polypeptide containing the amino acid sequence shown in Figure 1-( Sequence No. 1- (referred to as BML, 241-332, or 700-332) and the biologically active substitutional variant (R153A, R154A) for the first time, and there is an optional polypeptide preferred in this invention. They are variants of the biologically or immunologically active mpl-ligand groups that are selected from 2t 5 ¢hML3 “mML 5 < hML4 5 ls and ligands 3 . PML2 5 “PML 0 There is also polypeptide )4,483 is preferred in this invention” and is a variant of biologically active mpl-ligand groups containing an amino acid homologue having at least Ve of an ideal amino acid identity with the human mpl-ligand group as in Figure Y = (sequence No. -1); the combination of the mouse mpl ligand as in Figure 11- (sequence No. (VF Y = de sane) the ligand mpl resulting from genetic engineering in Pigs as in Fig. 19-10 (sequence No. -81); or the related MPL group in pigs that was isolated from aplastic aplastic plasma in “jy Jal” with a preferred ratio of ©7/ at least; best at least 80; and most preferably AG at least; The preferred one is more than a percentage of at least 78; Specifically, the best is a score of at least 4905. The following are the characteristics of the ligand group mpl that was isolated from aplastic aplastic plasma in pigs:

(1)_The partially purified ligand can be sequentially separated from a gel-filtration column in either a PBS containing a solution of MgCl, (4M) with a molecular weight of 805+7. (Y) The activity of the ligand is broken down by the enzyme 0 pronase (YF) 0 The ligand is stable at a low pH of 3.5 degrees; and a ratio of SDS up to 50.1 with urea (2M) (4) The ligand is a glycoprotein dependent in its binding on different lectin columns. The highly purified aay i(0) is sequentially separated from Non-reducing SDS-PAGE with a molecular ratio of 00 To, 00 - Yo, and the Ve | sequential separation can also be performed with lower amounts of activity at partial ratios of less than 100 g (1) The purified ligand resolves with a high degree of reduction in SDS-PAGE in the form of a doublet, with molecular ratios of FV eg YA ee (V) that the terminal amino Amd whose molecular ratios range from 180000 - 0 to 10 - 1.3 KDE PE in compliance with SPAPPACDPRLLNKLLRDDHVLHGR (sequence No. - 19). (A) The associative groups are connected and sequentially separated from the following gravitational columns:

vy -— _ Blue-Sepharose. CM Blue-Sepharose MONO, MONO-S. Lentil lectin-Sepharose. WGA-Sepharose. ° Con A-Sepharose, Ether 650m Toyopearl, Butyl 650m Toyopearl. The most preferred mpl vid'm polypeptide is that found in human 10s genomic DNA (cDNA) characterized by the amino acid sequences described in Figure 1 (Seq No. - .) 1 There are other polypeptides in this invention that are preferred to the naturally occurring and biologically active mpl-ligand; these include a prepro-mpl ligand and a prepro-mpl ligand. prepro-mpl ligand; mature prepro-mpl ligand ligand; parts of the mpl ligand and variants of those glycosylation-binding groups There is in this invention another preferred polypeptide of the anpl ligand and these include Variants are genetic mixtures of the mpl ligand sequence. Typically, these variants and © mixtures are biologically active variations of the mpl ligand that contain the sequence

Amino acids of which at least 70/ of these are the ligand mpl which is isolated from aplastic aplastic plasma of pigs; It is better for this percentage to be at least 70; It is more preferable that the ratio be at least 7850; Specifically, it is preferred that the percentage be at least 785; It is more preferred that the ratio be 796, and in particular it is preferred that the aforementioned ratio be 0©25. Among the preferred models for the mpl ligand, we mention here the variant ,0341 of the N-terminal space, which is referred to as (EPO) space, due to the sequence homology with erythropoietin. The EPO space of the variable [11] includes about VOY. of clang the first amino acid structure in mature .11; here known as hML-153 A preferred variant of the hML sequence is one or more basic or dibasic amino acid moieties in the N-terminal domain © 0 replaced by non-basic amino acid building blocks such as hydrophobic building blocks, neutral building blocks, acidic building blocks, aromatic building blocks, glycine, and others. m” and the like. The variant of the preferred C-terminal sequence variant of hML includes a space in which the amino acid residues (Vo argonone (Vo 8) are replaced by the amino acid residues alanine) and this variable is referred to here as hML332 (R154A) 0,61538. A preferred replacement variant for 0,131 comprises either 111.153 or a 3m where the amino Asli cla a gl (QLPP) 114 — 111 or (LPPQ) is deleted or replaced by a sequence a different tetrapeptide AGAG ie or similar. Deletion mutants are indicated

Preceding B A4 hML153 or .A4 hML153 and with regard to the favorite of gall prepared by genetic mixing, it is a product of the fusion of a mpl ligand or 0 part of it, as will be defined later, with a heterologous polypeptide or with a part of it . For example 141,153 can be combined with IgG antibody fraction to improve serum half-life.

Vo - - half-life; or to induce either IL-3, 6-057, or EPO to produce a molecule characterized by increased activity of either hematopoietic allogenesis or thrombopoietic formation. A preferred alternative chimera containing the human ligand mpl is scrambled. ; It is a scramble in the ¢ML-EPO domain and consists of the terminal 17 of Ad AML© residues 117-117 replaced by one or more, but not entirely, conjugated human EPO Aly subunits. dligne approximately as shown in Figure - 01 (sequence No. 0). In this model, the length of the BML genotype material ranges between 1-115"” of residues; and in it is added or Substitution of individual residues or groups of residues in human sequence 0 by sequence 11, at positions corresponding to the alignment condition shown in Figure 0 (01) (sequence 7). Example inserts are ideal for block sequence inserts. The groups in Ja are the N-terminal end of (ML) and these include one or more of the N-glycosylation processing sites at EPO positions from 4 7- YY and from 0-748 cde From AO =AY the inserts may also include one or more helical - » amphipathic double solubility at EPO positions from 4 - YY, from =09 VU and from eV oY -4500 and from 1 1 OY 137 (1 OY) with other regions of dle conservatism including N'A hall end regions, "©" terminal regions, and loci of OY-44 EPO residues expected Here the transgenic material containing the scrambled ML-EPO domain has mixed biological activity related to thrombopoietic-erythropoietic (TEPO) formation. Another polypeptide is preferred in this invention; It includes parts of group 0 ligand mpl with a sequential Ll containing 01 or 11 or SY ©? or 0 or 6; of units

Amino acid structures identical to those of the mpl ligand sequences isolated from porcine non-developmental plasma6 or from the human mpl ligand ela described in this text (see for example VE Table and Example Fig. (YE

SATE aor gl EX Cua ML[1-X] is preferred and is mpl and there is a ligature of 14A or 01# or 117 or 11 or 79 or YEO (see Figure 1 Sequence #1; for residue sequences 1-7). Other preferred fractions of the mpl ligand include those resulting from ol enzymatic degradation, chemical degradation, or digestion of the purified ligand. Another preferred aspect of the invention is a method of purifying ligand 0 l© molecules; This method includes contacting an mpl ligand source containing mpl ligand molecules with 6 of a receptor other than “immobilized fie,” specifically mpl sa polypeptide or polypeptide of eml bonding, under adsorbed conditions. adsoption mpl-ligand molecules to be purified and bound to the unimmobilized receptor polypeptide; The fixed support is then washed to remove the unadsorbed material; Then the particle separating process is carried out. Materials 1 are purified from the receptor-stabilized polypeptide; By using a buffer buffer solution for sequential separation operations. The mpl-containing source may be plasma; In it, it is preferable that the stabilized receptor be a product of mpl fusion with the immune body IgG. Alternatively, the old source containing the mpl ligand is a median of alia cells resulting from recombinant genetic engineering. ; The concentration of the A ligand in any of the culture media will be extracted, or in the materials resulting from cell lysates, more than in the plasma or in the cell lysates.

- No other natural sources. In this case, the aforementioned method of co-immunoprecipitation between mpl and 186, although useful, is not necessary. Here, conventional protein purification methods known in this field can be used. In brief, the preferred virulence method to obtain a highly homogenous ligand mpl involves: removal of waste fractions represented in 0 host cells or in debris lysed cell fragments; This is done - for example - by centrifugation or ultrafiltration -ultrotilteration. JS can optionally concentrate the protein using one of the commercially available tslters for this purpose, followed by separation of the ligand from other impurities by one or more of the steps chosen from Immunoaffinity ¢ or jonic exchange (using DEAE or matrices 0 base containing methyl carboxymethy groups or “(sulfopropyl) or blue sepharoce or “CM - blue sepharore” or “MONO-Q” or “MONO-S” or lectin sepharose 0 ¢ lentil ¢ or “Con-A sepharose 0 “WGA sepharose or ether ctoyopearl or cbuty toyopead or “phenyl toyopearl” <A — protein asepherose or revese phase HPLC chromatography on a silica gel bed with 1 aliphatic groups attached; or a molecular sieve of the Sephadex model” or electromagnetically sorting the particles by size; or sedimentation using ethanol and ammonium sulfate and in any of the previous steps a protease inhibitor (PMSF) can be used ) methylsulfonyl fluoride Jie in order to prevent protein degradation, proteolysis, and in another preferred model in this invention, we present here an isolated antibody that has the ability to bind 0 with an eml ligand, and it is preferable that this antibody be monoclonal.

YA - - (kohler and Milstein, Nature, 256:495-497 [1975] : Campbell Laboratory Techniquse in Biochemistry and Molecutar Biology, Burdon et.

At., Eds., Volume 13,. Elsevier Science Publishers, Amsterdam [1985]; and Huse., Science. 246 : 1275 - 1281 [1989). 0 It is preferable that this degree reach at least 0.1 L / mol; it is more preferable that the antibody is grown against a mpl ligand characterized by one of the aforementioned effective functions. Optionally, the isolated antibody that is able to bind can be combined with mpl ligand with a second polypeptide, and that the antibody or fusion product of this antibody be used to bind the mpl linkage group 0 from the previously described source for the immobile mpl polypeptide. This model The invention provides a method for detecting the ligand mpl group in the body (in ad) Gl vivo or in vitro din vitro This method includes contacting the antibody with die, especially a sample of blood serum suspected of containing ligand, with detection of whether binding has occurred.In other preferred embodiments, the invention presents an isolated nucleic acid molecule carrying the encoding gene ve ligand mpl parts of that group. The DNA molecule may be labeled or unlabeled with a detectable moiety; And this molecule has a sequence that complements or hybridizes in severe or moderate conditions with a nucleic acid molecule that contains a sequence that carries the mpl ligand gene. One of the preferred nucleic acids that contain mpl-associative groups is RNA or acid DNA encoding a ligand of ©1c of biologically active individuals in association with at least yo proportion of sequences with the mpl ligand

ve - — humanity; It is better for this percentage to be at least 780; most preferably at least 785; Specifically, it is preferred that this percentage be at least 790; It is more preferable that the percentage be at least 740. There are molecules more preferable to an isolated nucleic acid” which are DNA sequences that carry the biologically active mpl ligand selected from: (1) °DNA based on a mammalian mpl ligand gene; i.e. DNA containing the nucleotide sequence shown in Figure No. (1) (sequence No. (Y) or parts thereof. (Y) DNA capable of hybridizing with acid nuclear DNA in 0 (at least under conditions of moderate intensity.moderately stnngent 1 (©)_ degenerate DNA that gives the known DNA in 0 or (b) as a result of degenerate Genetic code 0 genetic code It is conceivable that the new mpl Sle gana described here are members of the class of cytokines sf ligands that contain an appropriate identity of sequences that make their DNA recombinant with DNA DNA in the form of 1 (conformance number Y) or with 1 complements or dia fragments under low to medium intensity conditions. Thus, another aspect of this invention includes DNA that hybridizes Under low to medium intensity conditions with DNA encoding a polypeptide ligand .mpl and in another preferred embodiment in this invention the DNA molecule is a cDNA carrying the ligand gene mpl which includes La on a repeatable genetic vector

Ar — — Practical JSS transformed CDNA splicing with control sequences recognized by a variable host with the genetic vector. This aspect also includes host cells altered by the genetic vector; and on a way to use cDNA to activate mpl ligand production; It is a method based on the expression of 8 genotype of nucleic acid (DNA) carrying the mpl ligand gene in the medium of 0 culture of the altered host cells; With the extraction of ligand 1 from the culture medium of the host Lo. It is preferable that the mpl-ligand prepared in this way be a human homologous ligand to a large extent and that the host cells used to produce the mpl-ligand are preferably cells from Ovary ovary extracted from Chinese hamster (CHO)Chinese hamster. The invention also includes a preferred method for the treatment of a mammal suffering from immune or hematopoietic disorders; Specifically, a lack of platelet formation. The method involves giving the mammal a therapeutically effective amount of mpl ligand optionally in combination with one of the cytokines; specifically interleukins colony-stimulating factor; Preferred colony-stimulating agents include: ligand - Kit™, Tink, G-CSF, GM-CSF, (M-CSF), EPO, 1-protein, or IL- 2 or IL-3 or 5TB or IL-6 0 7TB or “IL-8” or dL-9 or 11-11 Preparation Methods of Making: ie some The authors have long held that platelet production is controlled by multiple humoral factors in the blood with specific specification to the multiple lineage and it has been envisaged that there are two distinct activities — a cytokine referred to as megakaryocyte stimulating factor. For colony formation «(meg-CSF) megakaryocyte colony-stimulating factor and factor

- AY - megakaryocytopoiesis. These two factors regulate  thrombopoietin, forming thrombopoiesis. (Williams et al., J. Cell Physiol. 110:101-109 11982]; Williams et al., Blood Cells, 15:123 -133 [1989] and Gordon et al., Blood 80:302-307 [1992]). While “progenitor megakaryocytes by stimulating meg-CSF cells, according to this theory, ° is mainly based on the growth of more distinct cells; Then thrombopoietin acts as a constituent factor that affects the release and liberation of platelets. Since the 1960s, inductions in plasma and serum urine of thrombopoietin and meg-CSF-forming factor and the emergence of activities in both humans and animals after incidental thrombin formation have been well documented; This is in the following references: (Odell et al., Proc. Soc. Exp. Biol Med.. 108.428-431 [1961]; Nakeff et al., Acta Vo

Haematol, 54:340-344 [1975]; Specter, ProSoc. Exp. Biol., 108:146-149 119611;

Schreiner et al, J.Clin.invest., 49:1709-1713 [1970]; Ebbe, Blood, 44:605-608 [1974l;

Hoffman et al., N. Engl. J. Med., 305:533 [1981]; Straneva et al., Exp. HematoL, Ye. 17:1122-1127 [19881, Mazur et al., Exp. Hematol., 13:1164 [1985]; Mazur et aL, J.Clin.

Invest, 68:733-741 119811, Sheiner et al., Blood, 56:183-188 [1980]; Hill et boredom Exp.

Hematol, 20:354-360 [1992]; and Hegyi et al, Int. J. Cell Cloning, 8:236-244 [1990]). It has been reported that these activities are characterized by specificity towards the strain and differ from the known cytokines.

AY — — (Hill RJ. et al., Blood 80 346 (1992); Erickson-Miller CL. et al, Brit.

J.

Haematol, Straneva JE. et al., Exp.

Hematol. 20:4750 (1992); and Tsukada J. et al. (1993) 84:197-203, Blood 81:866-867 [1993]). thrombopoietin 0 formation from plasma or from urine in which thrombopoietin LS formation is low. From pigs exposed to irradiation 0 stimulate the formation of human megakaryo cells in vitro. La found that this catalytic activity can be abrogated; by the soluble and extracellular domain in (Compl 0), which confirms that the APP is the source (Sas ligand (ML) mpl) and we have now succeeded in purifying the ligand mpl from (APP Amino acid sequence information was used to isolate ML cDNA from mice, porcine, and humans. The ML's contain a sequence that is homologous to thrombopoietin and has activities similar to those of meg-CSF and meg-CSF. .thrombopoietin 1 - Purification and characterization of mpl-ligand ligand from plasma:

lS from different types of aplastic As we mentioned above, references indicate that agronal plasma is 0 in the laboratory 100 0; However, hematopoiesis characterized by such aplastic hematopoietic activities was not reported on the isolation of hematopoietic factor from plasma. And from non-immunoactive plasma sources, this plasma stimulates the formation of (APP) obtained from irradiated pigs present in the A ligand (APP). ,

ve tested its effect by measuring the degree of incorporation of SH-thymidine with Ba/F3 WIA grafted or

AY --- — transfected human P mpl (Ba/F3- mpl) was transferred to it and this test was carried out according to the procedures shown in Fig. No. Je APP catalyzes *H-Thymidine fusion in Ba/F3-mpl cells but not in control or control Ba/F3 cells that were not fed human mpl-P.In addition, no such activity was seen in normal pig plasma.These results indicate that APP contains

Eh on a factor or factors that transduced the proliferative signal and proliferative

through the eml receptor and therefore it is a natural ligand with respect to this receptor. This has been endorsed

With the results reported that the treatment of the APP with a soluble mixture containing mpl and the immunoglobulin

6 inhibited the inducible effect of APP on Ba/F3-mpl cells

The activity of the APP appears to be protein-based; This is because this activity is stopped by the effect of the pronase enzyme.

non-dialyzable In addition to the fact that this activity is non-dialyzable oF, 277, or heat (Fig. No. 0

0, however, the activity was stable over a pH of 0.0Y degrees for two hours; And he showed a sequential connection and separation of many columns that comprise lectin-affinity; Which indicates that this activity is caused by a glycoprotein. To clarify this structure and to learn more details on this activity, 455 of the APP were done using mixtures 001-180 prepared with genetic mixtures 0

10 The APP was processed according to the protocol described in the examples (Yo) in short 45 mpl ligands were processed using hydrophobic interaction chromatography (HIC); and immobilized dye “chromatography” and affinity chromatography towards -mpl-affinity. It shows the shape of a number; recovery activity from each step; Table 1 shows the number of fold purification times

vy. The percentage of total activity recovered during familiarity with the mpl column was about 700 and

-_ Am -_ The fraction with apical activity (F6) produced by the affinity column towards mpl has a specific activity estimated at 9.4” X 01 units/mg. As for the total purification of © liters of APP was about 1 01 ¢ (0 A units/mg to YY units/mg), with a decrease of AY Nox times YOu (grams to ? µg) protein. We estimated the specific activity hay ll© that was sequentially separated from a column composed of activity mpl at a value of -01” XY units/mg. Table No. (1) Purification of mpl ligand group Protein units/volume times Activity Product Sample mg/ml Glad 1 de 1 Purification (ml) Specific Yield Purificatio Units Units/ | Protein Sample mls (7) n mg mg/ml Ce freee ee am Cw ele ree Le ey ee Lo [ome | oe [oe] group FY xo 11 1 mpl | 16h | Yoon Lor Yee Maghrib ligand (µl) (Wl — Examples L Protein determined by Bradford test and protein concentration estimated in mpl-separated fractions Sequentially © - 1 based on the pigmentation intensity of the SDS gel dyed with silver.

FM - 0 . A single unit is defined as the unit that causes 0 75 of the maximum stimulation of the ISS Khadia Ba/F3-mpl The presence of multiple proteins has been reported (Fig. 0) by sequentially separating fraction analysis of an affinity column mpl using a Novex® - 7100 SDS-PAGE gel (Novex®) under reductive conditions 1. The proteins that stained with silver were highly and clearly alc resolved at molecular ratios of Mr. 0 tetroot nick YA, vee, YY, 000 VA eg. To determine which of these proteins stimulate proliferation of Ba/F3-mpl cell culture medium, the proteins were sequentially separated from the gel as described in an example. No. (1). The results of this experiment showed that most of the sequentially separated activity was for a gel strip containing 0 proteins with a molecular ratio of Mr ranging between 78.0060 - FY, nee, with activity of J being sequentially separating it in a region with a molecular ratio of 18,0050 = 77,000 in the gel (Fig. Protein sequences and obtaining proteins in this region of the gel (molecular bands at YA and 81-?? 0 kilo Daltons (kDa). He carried out the electrical representation of the lines of those three proteins in PVDF and then analyzed the sequence of acids (Aad) in them as described in Example No. oF, and Table No. ? The obtained amino-terminus sequences.

— M - Table No. (Y) of the ginal end sequences of the mpl group Ligand Amino-Terminus 1m11 Sequences (sequence number: kDa 30 (ve 25 20 15 10 5 1 (S)PAPPA(CDPRLLNKLLRDD (H/S)VLH (G)R ) String number: kDa 28 1( 25 20 15 10 5 1 SYPAPPAXDPRLLNKLLRDDH)VLHGR ) String number: kDa 18-22 (YY 10 5 1 SEQ ID NO: 32 mm mm mm mm mm mm mm”

AY — - Computer aided analysis reported the novelty of these amino acid sequences. And due to the oscillation of all three sequences, it was believed here that proteins with a molecular weight of 30 KDa; kDa 28 kDa 18-22 are similar proteins or different isoforms of the same novel protein and moreover this protein(s) could have been mpl Hk dk vim 0 natural due to the bioactivity of the proteins separated on SDS -PAGE in the same region (vv pre =YA van ) for the gel? - 7460. In addition, the partially purified ligand is migrated here with a molecular ratio of 0.000 = 17.006, after being subjected to a gel filtration chromatography process using a (Pharmacia) Superose-12 column. It is believed that the different forms of the molecular ratios are due to the differences that occur. In proteolysis or treatment with glycosylation 0 | or as a result of post or pre- translational modifications. As previously described, human noncoding mpl-RNA antisense suppressed the formation of megakaryocytes in human bone marrow culture media saturated with CD34 progenitor cells without affecting other hematopoietic cell lineages. (See previous research by Supra 0. Al-Methia et al.) This result suggests that the mpl receptor may play a role in the differentiation and proliferation of megakaryocytes in vitro, and to further confirm the role of day or m Here, the effect of APP APP devoid of ligand ligands on the in vitro formation of human megakaryocytes was compared here. The effect of APP on the formation of human megakaryocytes was determined. megakaryocytes in humans by modifying the formation test of those cells using a liquid suspension, megakaryocytopoiesis 110010, which is the test described in Example No. 4. In this test, human peripherald; 3—s superficial stem cells were treated.

AM _ - CAPP—_stem cells (PSC) before and after chromatographic treatment to determine the degree of affinity with the mixture of mpl and the antibody TG, and the stimulation of the formation of megakaryocytes was quantified by (GPILIII, And that using the “L-anti-IL III, antibody (Fig. 7). Figure 0 shows that 70 07M led to ¥ times the stimulation, while the APP without the ligand of mpl It had no effect.Essentially, the APP devoid of the eml ligand induces proliferation of -Ba/F3-mpl A in another experiment the addition of soluble human mpl-IgG On day 0, EY to culture media containing APP 0 neutralized the catalytic effects of APP on the formation of human LDA megakaryocytes, as shown in Figure 8. These 0 results indicate that The mpl ligand plays a role in regulating the formation of human LDA megakaryocytes, and therefore can be used in the treatment of platelet cell deficiency. 0 thrombocytopenia — Molecular Cloning of the mpl Ligand mpl: Based on The sequence of amino acids at the terminal end “amine and” amino-terminal obtained vo from proteins kDa s 28 kDa s < 30 kDa 18-22 (Table No. ¥ (Def) two Ale were designed gana decomposing oligonucleotide primers degenerate oligonucleotide 0 primer pools and used to multiply the DNA Jill in pigs by PCR. By a single exon the length of the correct PCR product is then expected to be yy. 14 bp base nuts Such a size of DNA fragment has been found and subcloned as

— subset to .pGEMT and example 0 shows the sequences of oligonucleotide primers used for PCR with ¥ clones of it obtained. The amino acid sequence (PRLLNKLR) sequence number (YY) of the peptide encoded among the PCR 4534 materials was identical to that obtained by making a sequence of a protein with an amino-terminal terminal. for a ligand in pigs (see residues 17-4 for the porcine protein sequences 28 kDa and 1002 30 above).

The simple synthetic plant oligonucleotide, which consists mainly of the sequence of the part used by PCR, was used to screen a human genomic DNA library in humans. A 45-mer oligonucleotide expressed as Pra5 was designed and built on

This oligonucleotide takes the form of the following sequence number: GCC-GTG-MG-GAC-GTG-GTC-GTC-ACG-MG-CAG-TTT-ATT-TAG- GAG-TCG 3 '5 (SEQ ID NO: 34) This deoxyollgonucleotide was used to screen a human genomic DNA in 12 Agem under hybridization and wash conditions. The intensity of the Vo stringency is low according to Example No. + Positive copies were captured, purified and analyzed by restriction mapping and by blotting 500118:0 Subcloned from Fragment cut by EcoRI-Xbal length (+ ¥4) bp, which was hybridized with 45-mer to transform into pBluescript SK. The DNA sequences of this copy were updated as the DNA encoding the ligand mpl homologous human homolog with ligand mpl © _ ligand in pigs has been isolated 0 and Figure No. 4 shows the DNA sequence in humans and a sequence

Reductase amino acids (sequence number oF) The specific positions of the A introns of the genetic sequence are also indicated by arrows; they also characterize the conceptual 0 (AY - exon) and based on the human “exon — ©” sequence (example) “A oligonucleotides corresponding to the 7′-terminal ends of the cexon sequence were synthesized and these two primers were used in 0 PCR reactions used for Cdna as a database una template 3 from various human tissues; The expected size of the Yé+ sa PCR product was 0 bp, and after studying the PCR product on JAX polyacrylamide gel, a DNA fragment of the expected size was detected in the cDNA groups prepared from Kidney, an adult human, and also TAT. Fetal renal cell CDNA fetal prepared from human fetal liver. The same 45-mer oligonucleotide that was used in scanning the human population A ysl genomic library and the fetal liver cDNA collection under low intensity hybridization conditions. Aum gal transcripts were also captured and purified. 7 by PCR with one copy with an A0 insert selected for further analysis. Using the procedures described in 1_Example 7, the 06 and amino acid sequence of the human mpl ligand dati al (.1) were obtained; These sequences are shown in Fig. 1 (Sequences No. © 1). ¥ Human mpl ligand motif (17): The human mpl ligand (hML) cDNA includes (sequence no. - ; Figure 1 - (Y on; 179 nucleotide followed by a poly(A) tail. A Jul contains 107 of 0 06600088 which have © untranslated sequences and 7 untranslated regions she has

41 - 4 and 0 Ma'a'iyyah. The initiation codon sal at position Y11 (nucleotide = 1) is among the consensus sequences useful for initiating translation in eukaryotic 0 and has an exposed reading frame length of 0159 nucleotides; it encodes YOY ha polypeptide is an amino acid unit starting with the nucleotides at the YY position. The Lalo terminal No of the amino acid sequence is expected to be one of the hydrophobic ones and with a degree of dle, it may be a signal peptide regulator. Computer analyzes of the specific amino acid sequence suggest a possible locus of action for the signaling peptidase among the YY 71 residues (see Von Heijne et al, Eur.

J.

Biochem. 133: 17-21 (1983)) Fission at this site generates a mature polypeptide containing 77 amino acid residues starting with ddl. - 0 terminal “ginal” obtained from mpl ligand selected from porcine plasma. dug The unglycosylated molecular weight of the remaining ligand containing 1 ?* amino acid is about FA kDa; There are 1 possible positions for the N-glycosylation group insertion; with ; Possible residues for the amino acid .cysteine and by comparing the mpl sequence with the Genbank sequence database, it was revealed that there is 1 match of 777 between the VOT residues containing the “amino” terminal of the ligand mpl between human erythropoietin and human erythropoietin (Fig. Human (02500). Each hML 5 hEPO contains four cysteines “ of which are kept invariant in hML including the first amino acid cysteines© and the last amino acid cysteines. Mutagenesis 1-directed loci experiments showed that the first and last cysteine forms a bond

ay -_6 required to perform this role 2286-2292:116 (Wange, EF. et al, Endocrinology ((1983) Similarly, the first and last cysteines in .1245 may form an important Lad bond The disulfide does not retain any of the hML-processing sites (siglycosylation) and all possible glycosylation-linked -17-linked sites in Hmin are located midway through the oo-terminal 'carboxy-terminal' of Hm !polypeptide Similar to hEPO, mRNA 1101 does not contain the complex sequence bs group + which is the sequence (AAUAAA) nor does it contain the regulatory element AUUUA present in the untranslated ¥ regions of many “cytokines i is believed to be stable (Shaw et al, Cell, 46: 659-667 (1986)) mRNA and analysis of Northern blot dad; 0 indicates the presence of low levels of kb hML RNA transcript 1-8 found in the liver of both the fetus and the adult human. After exposure to exposure for a long time, a weaker band of Silas size can be detected in the kidneys of the adult human. In comparison, the process of formation of erythropoietin in the human appears in the liver of the fetus As a response to hypoxia in the circulatory system, and also in the adult human kidneys and liver. (Jacobs et al., Nature, 313: 804-809 [1985] and Bondurant et al, Molec.

Cell.

Biol.

Vo )[1986] 6:2731-2733 The importance of the "©" terminal region in BML remains to be explained. Based on the presence of 1 potential N-linked glycosylation group insertion sites and the ability of the ligand to bind to lectin-affinity columns, JY. AML and in some sequential 0 washing experiments

ay — - Jal We note that the activity dissociates at a molecular ratio of about Tose, which is a value that represents the full length of the glycosylation-containing molecule. Therefore the '©' terminal region can act here to stabilize and increase the half-life of BML circulating in the blood stream. In the case of formation of li-erythropoietin in the non-0 0-containing form, it has full biological activity in the laboratory, but has a significantly shortened plasma half-life compared to glycosylation-treated erythropoietin (Takeuchi et al. J.

Blol.

Chem., 265:12127-12130 [1990], Narhi et al, J.

Biol.

Chem, and Spivack et al., Blood. 7:90-g9 [19891]. [1991] 266:23022-23026 The “©” terminal domain of AML contains two di-basic 0 amino acid sequences (Arg- moieties). Arg at positions Not 10 and positions 40 7- (YEN), which are the positions that act as potential processing sites, and the cut in those positions may be responsible for the generation of ML forms with a molecular weight of YY -18 YA Fy kilodaltons, which were isolated from LAPP and essentially the 54 Argl153-Argl sequences lie immediately after the erythropoietin-like domain of ML and these observations indicate that the full length — Vo.111 may represent proteolysis. It undergoes a specific degree of proteolysis to generate a mature ligand.; - Analogue and multiple forms of the human mpl ligand: Analogues or, alternatively, spliced mutated forms of the human mpl ligand were detected by 00 in liver In short, the primers ely primers for each of the terminal ends as well as the inner regions selected to encode the ML sequence were used in 87-00 to provide the amplify RNA in adult liver as sla ‏

a8 - _ Described in Example .01 In addition to the full-length AML figure © there are other 40 shapes seen or researched: hML2, ¢hML3, and 14l. Figure 11 shows the inferred mature amino acid sequences for all analogues (sequences No. 3 AA (Ve) hML3 contains position 701 of a 176 nucleotide deletion and this leads to 0 deletion of an amino acid And a frameshift transformation, cDNA now encodes a mature polypeptide of 365 amino acids in length and diverges from the hML sequence at the amino acid Ava and finally 1114 contains each of the 11 nucleotide deletion loci after the 18 nucleotide (also found in sequences in mice and pigs - see section below); with a base deletion locus bp 111 found in 11.3 although no copies with only the bp 11 deletion locus ( nucleotide) were isolated 0 follows 119) in humans (03072), but it is possible that this analog form exists after it has been distinguished and identified in both mice and pigs (see part (plaid) and also given that this analog form has been distinguished and identified by conjugation With a 111-nucleotide deletion region in -hML4 a different substitutional structure of .13401 was made in which the dibasic sequence Argl53-Argls4|0 is replaced by two calanine residues and the EPO domain truncated from 1, No, to determine whether the full length of the ML is necessary to perform the biological activity. A different substitutional structure of the 54 Arg]53-Argl bibase sequence and the reference hML (R153A-R154A leu) was made using PCR as described in Example No. 01. An EPO space was also made. extracted from ¢hMLIS3 using PCR and by inserting a stop codon after the acid Argl53.

0° - expression of the genetically engineered human mpl ligand (rhML) genotype

Recombinant cells from a human embryonic kidney (YAY) in which information is transmissibly deposited:

To ensure that the cloned human cDNA carried the de sans day genome (encoded recombination) for

control, the genotype of that group was shown in 203 mammary cells under mpl control

© promoter of early cytomegalovirus using any of the format vectors

genotypes pRKS-hML or .pRKS-hML-153. It was found that supernatants from cells

YAY The transiently infected human embryonic kidney works here on

Stimulation of °H-thymidine fusion in Ba/F3-mpl WA but not in parental Ba/F3 cells (Fig.

Fig. 11a) 0 did not show this biological activity in media from YAY cells that had deposited led 0 information via the pRK vector alone; And the addition of 1180 AD to those circles led to

Gene(207-0247 Functional Human.

To determine whether the EPO domain alone can bind with and activate or not, it is shown here

The genotype of the truncated form of hML (form 153 orhML) was found in a YAY cell. It was found that the supernatants from the cells in which the information was deposited had a Silas activity similar to that produced by

Cells showing the full length of AML (Fig. 11a); indicating that the terminal space is "0"

For ML it is not required to link or activate compl

1- The mpl ligand stimulates megakaryocytopoiesis and the formation of thrombopoiesis :

Both forms of full-length or truncated 153-thML cleavage thML form human meghakaryocytes 0 stimulated by genetically engineered AML in vitro.

- 0410 (Fig. 11B). This effect was observed in the absence of other exogenously applied hematopoietic growth factors. With the exception of 11-3, ML was the only hematopoietic growth factor tested that was shown to exhibit this activity. (IL-6g IL-11 Lal-11 and IL-9 5 “G-CSF 3” erythropoietin LIF 5 and ligand GM-CSF 5 “OSM.

M-CSF 5 (KL) Kit did not affect hematopoiesis © when tested separately in our assay (data not shown). This result proves that ML had a stimulating activity for megakaryocytes, and it also indicates a role for ML in regulating the formation of megakaryocytopoiesis. Platelets stimulate platelet production in mouse rebound tests. (McDonald, Proc.

Soc.

Exp.

Biol Meo 14: 1006-1001 [1973] and McDonald et al, Scand J.

HaematoL, 16:326-334 11976]). This resulted in platelet reconstitution with a identifiable Vo pattern. These immune-thrombocythemic rats enhanced with immunostimulation are more responsive to exogenous thrombopoletin-like activity compared to normal rats. (McDonald.

Proc.

Soc.

Exp.

Biol.

Med., 14:1006-1001 [1973]) It is like a rat that suffers from a lack of external oxygen; They are more responsive to thrombopoletin compared to normal mice.

Vy — (McDonald. et al., J.

lab.

Clean.

Med, 77:134143 [1971]). And that is in Tamil al-Muttaqi in part; He then performed a quantitative count of the platelet count and of the 3*8 platelet fusion. Injecting mice with IML with M units of 14.0660 00 FY, significantly increased platelet production; This is evidenced by an increase of about 700// in platelet counts (coincidence odds of 7 - nee and 01, respectively); and by an increase of 7468 PS platelet fusions (coincidence probability 0 - 0.007). ) in treated rats compared to control level group rats that were injected only with the excipient (Fig. VY). This level of 0 stimulation approximates the level seen in 11-6 in this model (data not shown). This Treatment with 06,000 units of IML did not result in an estimated stimulation of platelet production.These results indicate that ML stimulates platelet production in a dose-dependent pattern and therefore has activity similar to that of - thrombopoletin The other form of AML described above was deposited in 797; 1 supernatants were tested using the Ba/F3-mpl reproducibility assay (see Fig. VV) and none of the 2 hML showed and 1141.3 no activity observed in this assay.” However, the activity of R154A),114108153, was similar to that in ¢hML153 5 hML indicating that processing at the dibasic Arg153-Argl54 locus is not required for activity and is It is not harmful to him.

4A — - 1 - Tikrin of Megakaryocytes and Ligand Megakaryocytopoiesis and the mpl ligand It has been suggested that the formation of megakaryocytes is regulated at multiple cellular levels. (Williams et al., J.Cell Physiol. 110:101-104 [1982] and Williams et al., Blood Cells, 5.) [1989] 133- 15:123 This suggestion is based on the observations that some Hematopoietic growth factors stimulate the proliferation of LWIA megakaryocytes progenitors cprogenitors while others mainly influence maturation. The results we present here suggest that ML acts as an All proliferative factor as well as a maturation factor. The .101 stimulation of progenitor Dla megakaryocytes is a process that is supported by several lines of evidence. The first is that APP stimulates the proliferation and maturation of human megakaryocytes in vitro and this stimulation is completely inhibited by USE. ) mplIgG No. (AY) In addition, the inhibition of colony formation of Megakaryocytes WIA by antisense oligonucleotides containing the Compl ligand as stated in: 1392-1401: 82 Blood His (Methia et al. 10 (1993). Based on the results that compl ols can transduce the proliferative signal in transfected cells (Skoda et al., EMBO. 12). 2645-2653 [1993] and Vigon et al, Oncogene 8:2607-2615.

a9 _ - This also indicates the role of .147 in stimulating the reproduction process. Likewise, the clear expression of the genotype of Compl during differentiation stages of cells (Methia et al, Blood, Megakaryocytes) (1993) 1395-1401:82; in addition to the ability of ML to rapidly stimulate the production of platelets in the body of the organism pall to point out that ML also influences maturation.Given the availability of genetically engineered ML, there can be an accurate evaluation of its role in regulating the processes of megakaryocyte formation and thrombopoiesis, as well as its potential role in influencing Other blood-forming strains A - Isolation of the Human mpl Ligand (TPO) Gene: 0 copies of the human mpl ligand genomic DNA were isolated clones carrying the TPO gene by scanning de sans human genomic library at A-Gem12 by 1645AD under low or high stress conditions; with a sragment corresponding to half 7 in CDNA the human mpl-ligand gene carrier. Two overlapping lambda copies of the kb YO domain were isolated and sub-clones were made from the two BamHI overlapping fragments (EcoRls0) comprising the entire TPO gene; Its copies and knowledge of its subcloned and 0 sequence (see Figure - Zz) Ee 4 1 The structure of the human gene consists of exons 1 within kbY of genomic DNA. The boundaries of all exon/intron junctions are in line with what is accepted for mammalian genes. (Shapiro.

M.

B.. et al., Nucl.

Acids Res. 15:7155 [1987)).

0.1. - exon 5) exon sinus ¥ on © untranslated sequences with the ; A signal peptide amino acid. The remainder of the secretory signal and the first 26 amino acids of the mature protein are encoded; Within the exon ©. The (Jal) carboxyl space, as well as the untranslated + amino acids and the 00 amino acids constituting the SS-like space erythropoietin© are encoded within the exon, and the four amino acids involved in the observed deletion are encoded within (HTPO- 2) 1141-2 at the © terminal in exon 1. Human genetic DNA analysis by Southern blot indicated that the TPO gene is present in a single copy and the chromosomal locus has been identified. The chromosomal location 0 of the gene by fluorescent in situ hybridization (FiSH) was mapped to chromosome 3027-28. 4- Expression and Purification of TPO YAY from 293 Cells: In Example No. 19, a detailed description of the process of preparing and purifying ML or TPO from YAY cells was provided. 10 Briefly, the cDNA equivalent of the fully exposed reading frame of TPO was obtained by PCR using 00011- and 00. The PCR product was purified and cloned between the xbal 5 Clal restriction loci into the pRKStkneo plasmid (a PRKS-derived genetic vector modified for BY expression of the neomycin a glial neomycin resistaut gene). Lill under thymidine kinase promote to obtain the pRKStkneo ORF #0 (Genetic vector encoding the full unfolded reading frame).

0110 - - a second transgenic vector encoding the cognate EPO domain has been generated; in the same way; However, different PCR primers were used to obtain a final structure called .pRKS5-tkneoEPO-D. Infection of these two structures was induced in human embryonic kidney cells by the p0p0 method; The neomycin-resistant clones were selected and allowed to grow. Gene expression was also evaluated in phl101153 or 101332 in media prepared from these etl using the SL-Ba/F3-mpl assay. thML332 was purified according to the method described in Example V4. Briefly, prepared media containing 393-rhML332 were used on a Blue-Sepharose column (Pharmacia); which was later washed with a buffer solution containing urea (234). Elution 0 was washed with a buffer containing (2M) urea with sodium chloride (114). After that, the downstream of the Blue-Sepharose cascade was placed directly on the WGA-Sepharose column and washed with a buffer solution of a volume equivalent to 01 times the volume of the column and containing urea (2M) and sodium chloride (134). ); Then eluting was carried out with the same buffer containing N-acetyl-D-glucosamine (0.524). The material resulting from the separating with WGA-Sepharose 0 was placed on a C4-HPLC column (from Synchrom, Inc) and the sequential separation was carried out with a gradient of propanol in a non-continuous manner. Through SDS -PAGE 299-72 was migrated in a broad band format in the region of 68-80kDa for the gel (see Fig. No. Ve). thML332153 was also purified according to the method described in Example No. . 09 In short, Av. Here, the prepared media containing 293-thML153 were sorted into Blue-Sepharose as mentioned.

1011 _ - described for 21101332 and has the output of the cascade split by Blue-Sepharose placed directly on a column of mpl as described above; To the extent of homogeneity, 20141153 BIN was sequentially removed from the affinity column towards [emp] using the C4-HPLC column under the same ag hall used for 2011.332 and through SDS-PAGE the purified 3 separates into two 0 of major and two non-major bands +0100 in a molecular ratio of 6 - 17,001 (Fig. No. (Yo mpl day, -0) in 'The Murine' mice mplLigand A piece of DNA equivalent to the human mpl ligand coding region was obtained by PCR and was gel purified and labeled in the presence of 2p-dATP and 00-007. 0 This probe was used to scan 01 copies of the cDNA library set in the liver of a mouse in AGT10. The mouse copy was isolated (form oy number of sequences number (VW OY) containing The 0 led sha insert was a base pair; it was then spliced into a sequence; the putative initiator codon was at position 11 = PA nucleotide within a consensus sequence suitable for translation initiation in 0 eukaryotic cells (Kozak, M.

J.Cell Biol., 108:229-241 [1989] Vo This sequence marks the open reading frame for 011 nucleotides; This determines the first translation product of a Yo amino acid. On the side of this exposed read frame are 01 nucleotides at position 0 4 nucleotides Y£V at position of the untranslated sequence. Is there no poly (A) terminal after the region? +0. untranslated; This indicates that the version is not complete 0006. It is considered the end

— 01 “-terminal 7” of the identified amino acid sequence is a region of high hydrophobic affinity with a cell) and potentially represents a signal peptide. Computer analysis indicates the locus of

NYY) among the residues of the signal peptidase to cut the signal peptides (Sas (von Heijne, G. Eur. J. Biochem. 133:17-21 [1983]) a 71-amino acid exudate consisting of a polypeptide and performing Cuts at this location generate © for reasons described later). mML2 (or mML331 identified as ¢(35kDa)

V are all preserved in a human sequence; with cysteines and the sequence contains; Acids from © positions conserved in the human sequence. N-glycosylation Possible sites for insertion of de sane group All possible positions at which AML can be inserted again, and as is the case for N-glycosylation 0 Here, in the middle of the "©" terminal of the protein. Compared to human ML, there was clear identification of both the nucleotide sequences and the inferred amino acids; This identity has been observed in the LalAlIEPO-domains of ML. However, when inferred amino acid sequences are aligned in human with 11, in mice, the mouse AME al is a region of tetrapeptide deletion between residues 111 1, 4101 and these correspond to a region of a VY nucleotide deletion after 018 aia gall of the nucleotide seen in both human (see section above) and porcine (see section below) cDNA. Accordingly, additional transcripts were screened revealing the possible presence of isoforms of .10 in mice. One copy carried a polypeptide sequence FO gene from which amino acids containing the missing LPLQ tetrapeptide were derived. This figure is thought to represent the full length of the ML in mice; herein denoted as mML or -mML332 and shows the shape of the AE VV number of the nucleotide

11 The V0 VE version consists (sequences number mML and amino acids inferred for and contains Poly (A) base pairs followed by a terminal end of this 047 from cDNA © base at the end 174 ext. and on its sides 180160 there are 001168 unfolded reading frames untranslated; 41 7 bases at the “7” end untranslated. The putative initiation codon is located at the nucleotide; the unfolded reading frame completes the specific protein gene of 150 1- Position 0 is an amino acid, the first twenty-one of which represent acids that have a high level of hydrophobic Fo secretion signal and work potentially as a signal for hydrophobic secretion Finally, a third mouse copy was isolated and formed in a sequence; isoform region Therefore, this murine form isoform hML3 equivalent to 111 for deletion of the nucleotide Comparison of the inferred amino acid (VA) sequences of control 003413 and illustrates Figure No. 0

The total VA of these two isoforms (sequences No. mouse (Fig. human AML) is ML). The total identity of the amino acid sequence identity between is estimated at about 777; however, this homogeneity is not uniformly distributed. 09 VER-1 and human sequence number; Vor-1 (amino acid EPO region characterizing the 7A space (score of better conserved murine Cua) is better than Adal 0 and may be in homology compared to the carboxy-terminal region of the protein (777) Homogeneity is only the important domain in the biological activity of the protein. EPO is also an indication that the domain of the two amino acid bases present in motifs It is interesting that among the two sequences —VoY (the resting position EPO immediately after the ad space) there is only one dibasic sequence 7 in the human sequence; it is also found in the murine sequence. This is consistent with the possibility that ) 100; ©

#01 first protein undergoes limited proteolysis to generate ligand ML representing the full length may assist in clearance of Arg-153-Arg154 then proteolytic between mature Jay. -hML clearance coding The genetic formula containing the complete sequence, Jil, has been accidentally deposited in (1) cell, as described in example number YAY, to mML encoded for the © sequence.

Ba/F3 with H-Thymidine* cells conditioned media from those cells to induce fusion in murine or human mpl; But it did not affect the lineage of the parental cell mpl that is genetically expressed in the cloned mouse that carries the gene of the functional ligand that ML cDNA. This indicates that (less (mpl) mouse and human ML can activate both receptors: The Porcine mpl Ligan in pigs mpl ligand -11 vy., as described in the example of RACE PCR in pigs by AML (pML) cDNA J je done in kidney and re-cloned 147 (5) For 008 cDNA, the product (1”) number was obtained in pigs, mpl. Sequences were also made for several copies, and it was found that they carry a subcloned © ligand gene (or PML, which contains 377 Of the constituent amino acid units and referred to as B, which contains the nucleotide of the inferred amino acid sequence shown in the form (PMLsy, 0 OA 19). Sequences No. (01) encodes a protein with a region deleted from; PML2 units, and again a second form was identified, which is (sequence number (YV) an amino acid unit as a whole); this appears in the form of a number (YYA) amino acids omitted elit that the last form is identical, but PML2 5 PML The comparison between sequences (71 (YY) is shown as shown in the form of the number VE-111 corresponding to the QLPP units of the tetrapeptide ©

011 = — (sequence number (YY VA) The four amino acid deletion regions seen in both murine and human MLCDNA are located at the exact same position within the identified proteins. Comparison of the amino acid sequences identified for mature ML indicates In humans, mice and pigs (Fig. No. 14 Sequences No. 08 OY 1) indicates that the similarity ratio is 0 7727 between mouse and human, 7768 between SU and pig, and 7779 between pig and human. There is a much greater degree of homogeneity in Mid of the “amino-terminal” end of ML (homologous EPO domain). The proportion of matches for this domain ranges from 88-7484 between any two species; while for the mid of the “carboxy” (carbohydrate domain) The match ratio ranges from O——0V only 717. The sequence of dibasic amino acids represents a site for the cleavage protease 0¢ and these are located at the “carboxyl” terminal of the erythropoeitin conformation domain. That sequence is between ¥ species at this locus, as shown in Figure (14) (Sequences No. 7 (VA OY) and for the second dibasic locus located at positions 745 and 16 in the human sequences, it is not present in the rat or pig sequences. The mouse and swine ML sequence contains; of acids, cysteines; All of which are preserved in the human sequence. 1 There are 1 possible places to insert the combination “817005718000-11 into a ligature in the phalanges; and 1 within ML in pigs; of which © are preserved in the human cascade. Again, all possible positions for the N-glycosylation group insertion are here in the middle of the "©" terminal of the protein. -0 Expression of TPO and its spasticity from Hamster ovary cells: (CHO)

11 - — — The genetic vectors used to insert the CHO genetic information are known as pSV15.ID LL.

MLORF (full length or Js— ll) pSV15.ID.MLEPO-D 5 ¢(TPOs3, truncated or (TPOuss), JSS shows number (YY) and figure number (Y£) Distinctive features of these ©-plasmids The procedures involved in the transfer of genetic information are described in example number L(Y). Briefly, the cDNA corresponding to the full frame TPO of the open read was obtained. reading frame, by means of “PCR”. The product was also evaluated and cloned between two clal (Sall restriction sites) in the pSVISID.LL plasmid to obtain the retroviral vector pSV15.ID.LL.MLORF. Another structure corresponding to the EPO homologous space is; This is prepared 0 - in the same way using a reverse prime sale (for 2001.58). The final structure of the vector encoding the EPO homologous domain in TPO is called the .pSV15.ID.LL MLEPO-D structure. These two structures were formed into a strain by 0008 and the information was inserted into cells from a Chinese hamster ovary (CHO-DP12). ) (see European Patent No. 07747 published on March 11, 1989), by the electroporation method. This operation was carried out (1) on “01 cells in the BRL (Yo) device, 71 volts, milliafarad 007; low capacitance, in the presence of 01, Yo, or 00 mg From 0178 as described in Andreason, G.L.

J.

Tissue cult.

Meth. 15, 56 (1993). A day after infection, cells were divided in selective DHFR media (high glucose DMEM-F12 & 0 at tO +0 without Y 5 ¢ glycine mM from Y 5 ¢ glutamine = 70

#01 = — serum from fetal calves after dialysis); After -01 days individual colonies were transferred to 976-well dishes and allowed to grow to one point confluency. The genotype of Mlys3;or blood was evaluated in media conditioned from these clones; By using the Ba/F3-mpl proliferation assay described in Example No. (1) 0. Example No. (01) describes a process for purifying and isolating TPO from the culture medium of harvested CHO cells, briefly The fluid Harvested Cell Culture Medium (HCCF) is placed on a (Phamacia) Blue Sepharose column at a ratio of approximately 100 ml of 11007 per liter of resin, after which the column is washed with an amount of buffer solution equal to =F times its volume, followed by buffer solution washing with a volume of solution equal to =F 0 times the volume of the column and this buffer contains urea (2.014).The sequential elution of TPO is carried out With an amount of buffer equal to © = © times the volume of the column The buffer here contains urea (2.014) and NaCI (1.014).The downstream of the wash out of Blue Sepharose containing TPO is placed at 1 Sepharose column contains Wheat Germ (Pharmacia) Lectin and this is neutralized in a buffer solution for washing sepharose containing Blue Sepharose at a ratio of A = 01 ml of Blue Sepharose per 1 ml of resin, and then the column is washed with an amount of buffer solution 1 - “times the volume of the column to equalize it.” The backwash is carried out with TPO with an amount of buffer solution equal to ¥ - 0 times as much as the column size; 0 and this solution contains M) Nacetyl-D-glucosamine 5 (2.0 M)urea 0.5).

011 - — Then the acidified 30 sequential separation of wheat germ lectin containing TPO is acidified and Cis is added to it to reach a final concentration of 4 est and the resulting pool is placed on the reverse phase column reversed phase©, stabilizer in TFA 7001 with 0.654 microscopes at 1000 loadings ranging from approximately 1.5 - 1 pale,” protein per mL of resin© and sequentially separated in two phases in acetonitrile A linear gradient containing 70.1 TFA and 7 05 “CoE.” A pool is made on the basis of SDS-PAGE, and then the Cs pool is sequentially separated and filtered twice diafiltered for about > Volumes of buffer solution on an ultrafiltration membrane of type AmiconYM® or similar with a molecular weight of 0.0001 = 0.0060" Dalton. The resulting material may then be treated immediately binary filtration; or concentrated by ultrafiltration. The binary filtrate/concentrate is usually adjusted to a final concentration of 0.01 ,7 Tween-80" and all or part of the binary filtration/concentrate equivalent to ¥ = 70 of the calculated column volume on a (Pharmacia) Sephacryl®S-300HR column weighed in buffer containing 780.01 (Tween-80) and then chromatographic purification was performed. 1 TPO containing the aggregates and proteolytic degradation products is then combined; This is in a pool based on “SDS-PAGE” with this hglfLu filtered and stored under ? A=

101 - 1 - Methods of transforming 28 and stimulating the synthesis of TPO Inducing a microorganism, isolating, purifying and repeating the Refolding 0 formed in this organism: Example No. (YV) detailed description of vector construction In particular, all “pMP57 3 11041 cpMP151 5” (pMP21 5 plasmids pMP2025 0) were designed to genetically express the first 0096 amino acid to the right. down 0 for a small leader that varies between different structures. This command 35 provides a high level of translation initiation and rapid repetition. “pMP210-1 plasmids 3-8 21- 2+ 24 © 25- are designed to to show the genetic formula for Vor first amino acid in TPO to the right methionine — downstream initiator” and these differ only in that codon 0 is used for the first six amino acids in “TPO” while the pMP251 plasmid is a A derivative of pMP210-1 in which the TPO __ carboxy terminal is extended by two amino acids.All of the above plasmids produce high levels of genetic expression of Jala TPO in E.Coli cells. This is when a tryptophan promoter (Yansura, D.

G.et.

AL Methods in Enzymology (Goeddel, D.

V., Ed.) 185:54-60, Academic Press, San Diego [1990]). Vo and for the pMP172 “pMP1 plasmids,” they are intermediate materials for the work of the above-mentioned plasmids, which are related to showing the genetic formula of TPO inside cells. The above plasmids for the expression of the genotype of TPO were used to translate the E.Coli transform by the heat shock method and other methods mentioned by lena © in Example No. (YY)

111 - - (Mandel.

M. et al.

J.

Mol 3101., 53:159-162, [1970]) In short, the transformed cells were first grown at a temperature of “C” until the optical density (Teo) nanometers (nm) of the culture medium reached about 0 7-17 After that, the culture medium was diluted, and after growth with aeration, acid was added, then the © culture medium was allowed to continue growing with aeration for another 11 hours, after which the cells were harvested by centrifugation. YY 77: Separation, purification, and repeated folding procedures described P601410: for the production of biologically active human TPO or parts thereof; herein used to recover any TPO variants including expanded forms with a terminal '7' or ' There are 10 other sled yals suitable for performing recombinant TPO folding or synthetic TPO.” These procedures are described in the following patents that describe in general Iterative retrieval and folding of different genetically engineered proteins expressed i in an insoluble form in E.Coli Builder et at., U.S Patent 4.511,502; Jones et al., U.S.

Patent 4,512,922; Olson U.S. Patent 4.518,526 and Builder ef al.. U.S.

Patent 4,620,948; Vo a- Recovery of non-soluble TPO: Fermented E-Coli Jie microorganism genetically expressing TPO encoded with any suitable plasmid”; This is in conditions where TPO is placed in insoluble and refractile bodies. Optionally, the LAY Jue is first dissolved in a disruption buffer

111 - - to break cells; Typically 100 gram of cells are resuspended in a solution with a volume equal to about 10 volumes of buffer (i.e. 01 mmol Tris; 05 a M = EDTA and a pH of A ); This was done by means of a “Polytron homogenizer” with a centrifugation of the cells at a speed of 10001 for a period of Yr min. The lysed cells are then dissolved using any of the methods (Jie Lali) tonic shock; or sonication; or pressure cycling; Or by chemical or enzymatic treatment methods. Jey eg sale) Uy ural WAN granules mentioned above can be suspended in 01 other volumes of buffer solution padi wal for analysis of cell disruption by homogenizer; The LAN suspension is then passed through an LH Cell Disrupter (0) (LH Inceltech) or through a Microfluidizer (Micofluidics) (International) as directed. The TPO-containing particulate matter is then separated from the liquid phase and optionally washed with an appropriate liquid E.g. the material obtained from the cell solution can be centrifuged at 500 860 x p for ye min Then it is resuspensed and optionally centrifuged again to make a pellet of 1 washed refractory body 76586016 This pellet can be used directly or optionally stored in a frozen state below -Ve m Sia B - Dissolution and purification of Monomeric TPO: The undissolved TPO is then dissolved in a refractile body disc, using a buffer solution that contains a cchaotropic disintegrating substance and is set at a number 0 alkaline pH; contains reductant to increase monomeric TPO product.

1101” - - Standard disintegrators that include guanidine-HCI urea ¢ and sodium thiocyanate ¢ and preferably guanidine HCI. The disintegration material usually ranges between; - 9 Molar, preferably between Alg.

Yea 6 - > maintain the pH of the buffer solution at a level between 4.5 vo and better between A -1; The most preferred is 4 mah degrees. It is also preferable that the buffer solution contain a reducing substance that helps in the formation of the monomeric form of TPO, and the appropriate reducing materials are those that include compounds containing free thiol (RSH) » Examples of which are Lull, dithiothreitol (DTT) ) and dithioerythritol (DTE) + and mercaptoethanol and glutathione (GSH) and cysteamine + and cysteine ¢ and dithiothreitol (DTT) is preferred. The solute solution a—aiall 0 may optionally contain a slightly oxidizing substance (eg molecular oxygen ) with salt 5021016 to form monomeric TPO by decomposition of 90106. In this embodiment it is recently Refolding of the resulting sulfitolysis 700 in the presence of the Ue redox buffer buffer GSH/GSSG to form suitably folded TPO A further purification is usually performed on the TPO protein using centrifugation - ex = 1 and chromatography gel filtration; Dissolve 10 mM Tris buffer solution A= pH with 1 - 4 M 0 of guanidine; You mM of DTT and stirred for 1 - “Hours or all the time

- 1104 -

night under a temperature of ; High concentrations of urea (1-A M) can also be used to activate the dissolution of TPO protein, but in general they lead to a somewhat lower production compared to that of Alla guanidine. After dissolution, the solution is quickly centrifuged 02000 "p Vo Saal min to produce a superuatent clear supernatant containing monomeric denatuned TPO protein. The supernatant was then chromatographically treated using a 10 cm Pharmacia 5006:0200 X 7.1 gel filter column at a flow rate of 7 mL/min; With the sequential washing of the protein elution by Yo mmol of Na phosphate at a pH of > degrees using 01 mmol of DTT and a pool of fragments containing the dissociated TPO protein is carried out. Monounic which 0 sequentially separates in an amount ranging from 101 mL to Yor. 4 Protein 170 is filtered into a C4 reversed phase column, semi-preparative 01 XY cm (VYDAC) and the sample Janay © ml/min is placed on a column calibrated in 7001 (trifluoroacetic acid) TFA With 101 acetonitrile. The protein is sequentially separated at a linear graded level of acetonitrile (0 10-7 in 0 minutes) while the selected reduced protein is sequentially separated at a ratio of acetonitrile yo of Approximately 750. This material is used in the refolding sale process to obtain

A different form of biologically active TPO.

C- TPO refolding to generate the biologically active form:

After dissolution and further purification of TPO the biologically active form is obtained by refolding of the monomeric denatured TPO; This is in a buffered oxidation-reduction solution. Due to the high strength of TPO (half-maximal stimulation in Ba/F3 selection occurs at about ¥ pg/mL

— #10 -

(pg/ml) it is not possible here to obtain a biologically active substance using several buffers, detergents, or different redox conditions. However, in most cases a small amount of The material that has been subjected to proper folding (less than + (1)) and for commercial manufacturing operations, it is preferable that the product of the re-folding be at least 701; and the most detailed that this ratio ranges between +7*- 050, and it is especially preferred that this ratio exceeds 0 75. There are many different detergents that have been found to be

suitable for producing at least some of the suitably folded material; These detergents include: Triton X-100 (Trade Mark) dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, sarkosyl (Trade Mark) Tween 20 and Tween 80, Ziwittergent 3014. 0 Among these are of gall is particularly preferred (CHAPSO 5 CHAPS) CHAPS class which has been found to perform best in the Bale folding reaction and to limit protein agglomeration or unwanted disulfide formation. It is particularly preferred to use levels of CHAPS greater than ad.) sodium chloride was required J peas on lef yield; This is ideally in the range of 0.1 - 1. Preferably the presence of 1 (EDTA - © mM) in the redox buffer solution; 1, in order to reduce oxidation and agglomeration catalyzed by metal-catalyzed metals, which have been seen in some formulations. The use of Glycerol in concentrations greater than 219 resulted in optimal refolding conditions. In order to obtain the maximum yield, it was necessary to have redox pairs present in a buffer solution containing both the oxidized amyloid thiol and the reduced organic thiol (8517). Suitable redox pairs include: glutathione (GSH) 5 « mercaptoethanol 00 ¢, cysteamine », cysteine and forms of these substances

1101 - - Corresponding oxidant. As for the preferred oxidation-reduction pairs, they are glutathione (GSH) « oxidized glutathione (GSSG) or Ll .cysteine - cystine The most preferred oxidation-reduction pairs are oxidized glutathione (GSSG) « glutathione (GSH) In general, it was obtained on a larger quantity of products when the molar ratio of the oxidized member of the 0-reduction pair was equal to or greater than the reducing member of that pair. The optimum pH for the ale] folding of the TPO variants ranged between 4-7.5 degrees. The organic solvents ethanol Jia and methanol acetonitrile were tolerated at concentrations of 10-01 or less; while increasing levels of organic solvents resulted in inappropriately folded shapes. Buffer solutions 5 and phosphate are generally considered useful; Incubation under 0 temperature of ; m yielded higher levels of properly folded TPO. The refolding process yielded 400 - 710 (based on the amount of reduced and degraded TPO used in the refolding reaction); this is typical for formulations TPO that was then regurgitated during the first Aa je © , An active substance can be obtained when there are preparations of lower purity (i.e. used immediately after sequencing on a Superdex 0 200 column or after the first extraction of the body refractory); although the product is lower at lower concentration levels in terms of precipitation and interference with non-TPO proteins during the sale process of TPO. Since TPO contains cysteine building blocks, it Here different 'forms of disulfides' can be generated for this protein: 0 - pics number 1: disulfide between residues 1 cysteine - ; and 7 - 2

11017 - Image No. (7): disulfide between residues YX - 1 cysteine - 4 Image No. (7): ediflusid between residues 1 cysteine - and 71 - 4. During the initial detection to determine Refolding cases, many different peaks containing TPO protein were separated by reversed phase Cs chromatography. There was only one crest with intrinsic biological activity according to (sald) Ba / F3 test, and therefore the clea refolding cases are ideal to produce this shape in the best way; In these conditions and cases, the distorted folded images represented less than 71/7-01 of the total monounits TPO obtained in the dissolution step. The disulfide profile of biologically active TPO has been determined; And that when a value of 0 ranges from -1 ; And the? - ¥ by means of mass spectrometry and protein sequencing » and here the cysteine was numbered sequentially starting from the “amino” terminal. The cross-linking pattern of cysteine was consistent with the known disulfide-linking pattern of the related wrythropietin moiety. D - Biological activity of genetically engineered recombinant TPO 53 subjected 10 to a refolding process: Recombined and purified TPO has been shown to be active in tests conducted on the body of the organism and in vitro, for example For example, in the Ba-F3 assay, the 1-maximal stimulation of thymidine incorporation with Ba/F3 cells for (Met-1 1-153) TPO was obtained with a value of 7.7 pg/ mL (¥ picomolar) 0 and in the receptor-based ELISA

- AA - mpl half-maximal activity occurred at a value of 0.9 ng/mL (0 picomolar VY). In normal animals and those in which the spinal cord was suppressed by near-lethal x-radiation, refolded TPO (1-153) Met-1 had activity High (activity observed at doses as low as 00 ng/mouse) to stimulate production of new platelets.0 Similar biological activity has been observed to other forms of TPO after refolding Gis The procedures described Above (see Figures No. 076 (YA YT) 6- Methods for measuring thrombopoietic activity Platelet production can be measured in various tests including the choice of ligand 3 described in Example No. (1); and the formation test. Platelets in the prothrombin; and 0 induction of antigen on the surface of platelet cells as measured by the complementary antiplatelet inmunoassay alias (alias) for a megakaryoblastic cell line of human leukemia (CMK) (see 184-190-1989: 72 Sato et al, Brit.

J.

Heamatol, as well as a test for the formation of CMK cells in a liquid suspension; which is the test mentioned in Example No. ;); and testing the induction of polyploidization in the LA of (DAM) megakaryocytes {Dw (see Ogura et al, Blood 72 (1): 49-60-1988 0). The maturation of immature, non-DNA-generating WA megakaryocytes to morphologically distinguishable cells is a process that includes the appearance of an antigen on the organocytoplasmic membrane (GPILIIL) and the release of platelets as described in the background of this invention. A specific promoter of ADL (such as the mpl ligand of maturation of megakaryocytes) would be expected to search for at least some of these variants in immature cells, thereby leading to deregulation.

- 111 -

Platelets and alleviation of Alla thrombocytopenia. Thus, tests were designed to measure the emergence of these variables in immature strains of megakaryocytes, such as CMK-DAMI cells. The CMK test (Example No.) measures the appearance of the qualitative marker of platelet M1 and the disappearance of platelets. The DAMI test (Example 10) measures endoreplication, because an increase in the degree of chromosomal redundancy indicates the presence of mature megakaryocytes. The identified megakaryocytes had replication values chromosomal amount of 32N 167 SN AN 2N etc. Finally, the platelet test in the body of mice (Example No. (V1) was useful in proving that the intake of the test compound (ligand)

It leads to an increase in platelet counts.

0 conducted two additional tests in the laboratory to measure TPO activity. The first test was the kinase receptor activation (KIRA) ELISA, in which the mpl-Rse hybrid gene information chimera was injected into CHO cells; The phosphorylatlon Rse was measured by tyrosine phosphorylation using ELISA after exposure of the mpl part of chimera ligand mpl hybrids (see Example No. (VV) and in the second test based on 21158 which

1 In it, the ELISA strips contain a layer of rabbit antibodies against the human mpl-IgG, which in turn binds to the human transgenic receptor mpl-lgG, which in turn binds to the mpl ligand to be measured. The polyclonal antibody was used in rabbits after being treated with biotinylated and prepared against the ligand (TPO 155) mpl, in order to detect the ligand of mpl; Measurement was done here using streptavidin-peroxidase as stated

AA Describe it in Example 0

AY. -— — Yo The biological response in the body of the organism J is normal and another one was exposed to X irradiated radiation at a sublethal dose sublethally and was treated with “TPO”

Normal and radiotreated were treated with full-length cleaved TPO after it was isolated from Chinese rat ovary cells (CHO), E. Coli cells, and human renal cells in the embryonic stage M0 (293). Both forms of TPO produced in these three factors stimulated platelet production in mice; however, the full-length 0 isolated from CHO appeared to produce the largest in vivo response. These results indicate that entering a set of 0 in the end field carboxy'ds shall ' may be necessary

The occurrence of the most active activity in the organism's body.

E.coli-thTPO(Met 153) = a 0 plus 1 - from position Met) “Met” of type EPO in the space “Met” the figure was injected daily by in (YY (example human E-Coli number); resulting from the first TPO in Yor residues and these are illustrated zl by (IYO) described in the footnote to the figures ela as 057 86 Ale female Ovran which resulted in TPO in non-glycosylated forms The untreated 410 cleaved form refolded as described above resulted in 2-fold increase in stimulation in E.Coli than in E.Coli. Vo

in platelet production in normal mice without affecting erythrocyte count or

white 0, and when this same molecule was injected daily into female oxen, it was exposed to 72" radiation in doses similar to

mortality (137CS) 6 as ela described in fig. 776 footnote ?a em c; led to

2-fold increase in platelet production with effect on red blood cell count 0 and white cell count.

CHO-rh TPOs3, =

The full length of TPO produced in CHO that was injected daily into 057186 female mice that

Described in the footnote to the figures ze Jyv has here led to a two-fold increase in the production of

platelets in normal mice without affecting the numbers of red blood cells and white cells. © C-E. coh-thTPO(Met™, 53); 203-thTP0332; and E. coli-thTPOyss dd0 0110-11

Dose-response curves were drawn for treating normal mice with thTPO cell lines

different as described in the footnote to Figure No. (YA).

E. coli-thTPO(Met; 153); 293thTPOs3,; and E. col,-thTPO ss)

This figure shows that all of the treated molecules stimulated platelet 0 production; However, the full-length form produced in CHO had the greatest in vivo activity.

CHO-rhTPO0;s3. CHO-rhTPO cllpped and CHo-rhTPO33; - Dr

Dose-response curves were also created for treatment of normal rats with different forms of thTPO

Resulting in both “CHO(CHO-thTPO153) and clipped “CHO- 5” CHO-thTPO

2 As described in a footnote to Figure No. (79). This figure shows that all 1 CHO forms of the tested molecule stimulated platelet production; But it was for the format

The full length CHO produced in the organism has the greatest activity in the body.

1"1 - -— - The general method for preparing mpl ligand products and its various forms by recombinant genetic engineering. By culturing the cells in which the genetic information was deposited, in order to genetically express the nucleic acid of the mpl ligand (typically by WIAD transforming with a vector of the genetic formula ¢ (vector) with the recovery of a polypeptide from However, it is optionally conceived that the mpl ligand could be produced by homologous genetic engineering; or by genetic engineering methods using controls inserted into cells containing the DNA encoding the mpl ligand eg For example, any of the “promoter/enhancer” or “suppressor” element, or an external element regulating transcription, can be inserted into the genetic structure of the target host cells with a degree of proportionality and orientation. It is sufficient to affect the transcription of the DNA encoding the polypeptide of the desired mpl ligand. The control does not encode the mpl bundle ; Rather, 0348 is Jd indigenous DNA, the genetic structure of the host cell. This is followed by a search for ve the cells that make up the 6 in the future according to this invention; or for increased or decreased levels of genotype expression as required. Thus the present invention implies a method for producing the associative ligand mpl; The method includes the insertion of 48 A transcription regulators into an Adal genome epithelial structure containing a DNA molecule for the mpl-associative ligand; And this is with a degree of proximity (proximity) and orientation (orientation 00) sufficient for the DNA molecule to influence the transcription process” with an optional step

NYY - an additive that includes a cell culture containing a transcription-regulating element and a nucleic acid molecule. The invention also implies a host cell containing a mpl-ligand DNA molecule that physically communicates with the external and recognized control sequences that are unique to the host cell.

: mpl Polypeptide ligand for Encoding DNA isolate - i group of Af from mpl Aan Polypeptide encoding DNA © can be obtained and expressed genetically mpl Ligand mRNA prepared from cells believed to contain cDNA from the mpl-ligand gene group at a detectable level. It is also possible to obtain the simple from the nucleotide or by building in the laboratory the genomic library + genetic DNA

1056 complete or amino acid sequences.

0 The groups are viewed by a specific probe that is designed to identify the gene of interest or the protein encoded by that gene. As for the ol expression groups in cDNA, the appropriate probe for it includes monoclonal or multiclonal antibodies that can distinguish and bind in a specific way with the Ul mpl ligand. For cDNA groups, the appropriate probe here includes oligonucleotides Its length ranges from A to 71 bases

10 and encodes known or suspected portions of acid 0118 of the mpl ligand; This is of the same type or of different types of creatures; And/or the probe here may be complementary cDNA, chomologous homologues, or fragments of such DNA that encode a similar gene. As for the appropriate probe for groups of genetic DNAs, it includes - without limitation - oligonucleotides or cDNAs or parts thereof encoding a similar gene or

Screening is hereditary or parts thereof. Similar DNAs review process may take place; and/or on x.

— 1714 -

genome or cDNA by a selected probe; using standard procedures

It is described in chapters 11-01 in the reference to Sambrook et al. cited above.

An alternative method is to isolate the gene encoding the mpl-associative ligand; It is the use of PCR according to the method

described in the Sambrook © al. reference above. This method requires the use of a simple oligonucleotides© probe that hybridizes the DNA encoding the .mpl ligand and there are ways to test these

oligonucleotides” will be described later.

A preferred method of exercising this invention is the use of carefully selected sequences of

oligonucleotides; In order to review the collections of CDNA from different tissues; Preferably

that tissue from a kidney in a human or in a pig (whether that organism is an adult or in its embryonic stage 0); Or that tissue is a lineage of cells in the liver. For example is a review

Embryonic human liver cells containing cDNA clusters using a probe

of simple oligonucleotides. Alternatively, human genetic groups can be filtered by

A probe of simple oligonucleotides.

As for the sequences of oligonucleotides chosen as probes, they must be of length AK and far from 10 ambiguities so that the false positives are reduced and reach a minimum of 0.

Sequences — actual nucleotide based on ligand 1 regions (which have less than

Codon redundancy as possible. The degradation of oligonucleotides may occur

simple at one or more loci and the use of these soluble oligonucleotides is considered an issue

Of particular interest when not knowing which set of species is being viewed for which 0 does not differentiate the use of a preferred codon .

110- The oligonucleotide must be distinguished so that it can be detected when hybridization with DNA in the group being presented. A preferred recognition method is the use of ATP as (Y3P) and polynucleoticie kinase to mark the ©-terminus of the radioactive oligonucleotide. However, there are other methods that can be used to characterize the oligonucleotide; These include, but are not limited to, discrimination by biotinylation group insertion or enzyme-labeling. Of particular interest is the mpl-ligand DNA that marks the full-length ligand polypeptide. In some preferred embodiments, the DNA sequence includes a local signal sequence of the ligand mpl, and the DNA containing the AS encoded sequences is obtained by browsing selected CDNA or genetic cle gana using - an inferred amino acid sequence deduced + b- types of amino acid sequences in the mpl ligand Native: These types are prepared by inserting appropriate modifications to the nucleotide in the DNA ligand empl or by building in vitro a polypeptide ligand mpl desirable. For example here A) carboxy-terminal parts can be formed for the full length of the mpl ligand by proteolysis; Whether in vivo, in vitro, or by transcribing or expressing a fragment or DNA that encodes the full length of the mpl ligand to produce a biologically active species. Any combination of deletion, insertion, and substitution can be made; To reach the final genetic structure »provided that this structure has a desired biological activity. The amino acid can also change the processes of the 005078051810081 ligand mpl protein beta, such as Me vy. A change in the number or position of the places where the glycosylation group enters. For design types

111- The a-amino acid sequence in the mpl ligand, the location and nature of the aay mutation depends here on the characteristics of the mpl ligand to be modified. It is possible to modify the locus of occurrence of the mutation individually or Liles and that is Sa by: = substituting a conservative amino acid ¢ and then with more radical choices depending on the 0 results obtained. - deletion of target residue 0 target residue 7 - insertion of residues of the same class or of a different class adjacent to the locus; or by a mixture of options (1). mpl, which are the preferred loci for mutation to occur, and it is called “mutation induced by alanine scanning mutagenesis.” It was described in the reference Cunningham and Wells; Science J] (1989) 1081-1085: 244- In this case, one or a group of the target residues (charged residues Jie and casp co dner “glu”) are labeled and then replaced with any of the amino acids that are preferably neutral or negative (and more preferably alanine or polyalanine acids) in order to influence the interaction between amino acids and the surrounding aqueous environment outside the cell. These domains that show functional sensitivity to substitutions are then refined by introducing additional variables or other variables when Substitution sites Thus, with predetermination of the insertion site of the amino acid sequence, the nature of the mutation in itself does not need to be determined.

1" - - previously. For example, to optimize mutation performance at a known locus, an alanine or random mutation at a codon or target region is presented here; then mpl-ligand types are separated to arrive at There are two basic types in the genetic structure of the amino acid sequence: the locus 0 of the mutation and the nature of the mutation. “They may represent naturally occurring variants in which there is no need to adapt the DNA to the mpl ligand,” or they may represent predetermined forms of mutation resulting from mutation in the DNA to reach a variant that is not ligand in nature. The nature of the mutation selected depends here on the characteristics of the mpl set to be modified.

- 1 The number of building blocks that are deleted from the sequence of amino acids ranges between about 0, and typically this number is close to 1 - 01, and it is preferable that this number ranges between 0 mm mpl acids The amino acid of the Lime ligand. Alternatively, residues that are deleted from the “carboxy” terminal of the glycoprotein may include part of the entire domain of those residues also containing one or more of the units. The first six constructs containing

The yo-terminal "amino" terminus of a mature protein. There are optional building blocks in the sequence of amino acids that include one or more building blocks in one or more of the loops located between the helical bundles. Convergent deletion usually occurs with equal numbers of building blocks, but the deletion is in odd numbers or in numbers Incorrect also falls within the scope of this operation. A deletion may occur in regions with little homogeneity between the Mt ligand

© - share most of the sequence identity to modify the activity of that group. Operations may also be performed

#17 -— - Deletions in regions of little homogeneity among the human mpl ligand or in other mammals that share most of the human mpl ligand identity. Deletions of mpl ligand polypeptides in mammals in regions characterized by a high degree of homogeneity with mpl groups in other Aldi are considered factors that could lead to greater modification and alteration of mpl 0 ligand biological activity. The number of successive deletions is chosen so that the 3-layer structure of the mpl ligand is preserved in the affected domain E in the beta-pleated sheet domain or the Lill domain -alpha helix and includes insertions Amino acid sequence on amino-terminal and/or carboxyl fusions “Led sha ranges from one residue to polypeptides containing 0_hundred or more residues; Lady inserts din contains one amino acid or several amino acid residues.In general, the number of residues in the insertions of the interstitial sequence (inserts within a mature sequence consisting of a ligand (mpl) ranges between about 1-01 residues. It is preferable that this number ranges between 1 - 0, and the most preferable is that this number ranges between -1”. or with part of it. Examples of terminal inclusions include those comprising a mpl ligand with a 77-terminal methionyl unit with a factor for direct expression of the genotype in the mpl ligand matured in a genetically engineered cell culture medium; And with a merger that occurs between a heterogeneous signal sequence that contains the terminal end "77" and the terminal end 7" in a fragment of a mature mpl ligand, in order to facilitate the secretion of the 1m" ligand from families in which the genetic engineering was carried out. In general, such results are obtained. These signal sequences are of host cell types

1" - - target; suitable sequences include 7 or Ipp for bacteria: 8-001; factor alpha in yeast yeast; and viral signals such as herpes virus gD in mammalian cells. Other penetration variants of this molecule are ligand mpl; this includes fusion with the terminal “C” ligand mpl in the immune-forming polypeptides (which are not secreted internally in the body of the host, but are given to it by Leia dala bacterial polypeptides such as beta-lactamase or enzymes encoded by E.Coli trplocas or yeast protein); and fusion of the terminal "©" lal with proteins with a long half-life such as the constant regions of immunoglobulins (or any other regions of immunoglobulins). ); and albumin or 0 as described in International Patent Application No. 777 published on April 1, AAA 0 The third group of variants are amino acid substitution variants; these contain at least one of Amino acid constituent units of the mpl ligand that have been removed and replaced with another one of the residues.The loci of greatest interest for substitution mutations include those known to be mpl ligand active sites in which the amino acids are found in other homologues and are substantially different in terms of 1 the size of the sidechain bulk (or the charge) or the degree of hydrophobicity; However, with the dle degree of sequence discrimination at a chosen locus between different types of mpl ligand or/or within different animal homologues in one member of the -mpl ligand group, there are other important loci, which are those in which certain building blocks are similar From ligand mpl 0 obtained from different class members and/or animal species within a single member.

AY. — — Table No. (¥) Substitutions Perfect Substitutions Preferred Substitutions Ala (A) Val; Leu; lie Val

Arg(R) Lys; Gln; Asn Lys

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

Asp(D) Glu Glu

Cys(C)Ser Ser

Gln (Q) Asn Asn

Glu (E) Asp Asp

Gly (G) Pro His (H) Asn, Gln; Lys; Arg Arg lle (I) Leu, Val; Met, Ala; Phe; Norleucine Leu

Leu (L) norleucine, ile, val, Met: Ala. Phe Lle Lys (K) Arg, Gln. Asn Arg

met (M) Leu; Phe; lie Leu

Phe (F) Leu: Val; lle; Ala Leu

Pro (P) Gly Gly

Ser (S) Thr Thr

Thr (T) Ser Ser

Trp (W) Tyr Tyr

Tyr (Y) Trp; Phe; Thr; Ser Phe

Val (V) lle; Leu; met; Phe; Ala; Norleucine Leu

111. There are substantial alterations in the function or immunological identity of the mpl ligand; These are achieved by selecting substitutions that differ fundamentally in their effect on the preservation of: i - the polypeptide structure in the substitution region; Whether it is - for example - a sheet or helical conformation structure. Mb - the charge of the molecule or the degree of its hydrophobicity at the target position. C- The size of the side chain and its properties that determine the division of naturally occurring acute residues into groups. )( Water repellents ‎norleucine, Met.

Ala.

Val, Leu.lle; : hydrophobic ¥( neutral hydrophilic materials Cys.Ser.

Thr; : neutral hydrophilic (v 01 Asp, Glu; : acidic ¢) bases: Asn, Gln, His, Lys, Arg; m) residues affecting chain orientation ly, Pro; : chain orientation (Trp, Tyr, Phe : aromatic) Non-conservative substitutions will require the replacement of a member of one vo of these classes by another. Such residues may also be inserted within substitution sites retention; or more preferably within (non-preservation) sites.

111 - In one of the examples of the invention; It is desirable to deactivate or deactivate one or more bond-breaking sites of 'protease' that are present in the molecule. These sites are identified and identified by examination of the amino acid sequence (iid) in the case of “trypsin” (enzyme in pancreatic juice); eg for an arginyl residue or ©78071a. When the sites or sites of bond-breaking by the protease enzyme are identified and identified; It is rendered inert to bond-breaking for proteolysis by replacing the target residue with another Aly, preferably a base residue such as glutamine or a water-repellent residue such as serine; by canceling the unit Ald) or by inserting a prolyl residue immediately after the residue. 0 In another embodiment, any "methionyl" residue other than the "infinitely"-beginning unit of the single chain or any residue contained within about three residues at the terminal -17 or carbon -© ends of each residue of Such residues of "6001" are either replaced by another residue (preferably lay for what is given in table number "") or eliminated. in an alternative way; From about (1) to about (©)1 residues are entered to be near or adjacent to these sites. Any cysteine residues that are not included in the conservation form may also be replaced. The appropriate structure of the mpl' bond, and in general with the serine', in order to improve the stability against oxidation of the molecule and prevent aberrant crosslinking. 800), which have been numbered starting with the 0 - ends of the amino groups, are necessary to maintain proper structural shape, but it has been found that

11# - The second and third are not necessary. accordingly; The second and third cysteine compounds in the IPO (UM) domain may be substituted. DNA molecules encoding types of ligand amino acid sequences are prepared by a variety of different methods known in this field of art. Such methods include, but are not limited to, separation from a natural source (in the case of naturally occurring amino acid sequence types) or preparation by mutation occurring via the “oligonucleotide” (or “positive site”). site-directed and PCR mutagenesis « and a mutagenesis kit of an early prepared variant or a non-variant transformation of a polypeptide” “ligand set .mpl 0 and the method of mutagenesis via Oligonucleotide” is the preferred method for preparing variants Substitution + deletion and insertion of (DNA) ligand “mpl” This technical technique has been known in this art as described in [183: 2 Adelman et al, DNA, (1983). In particular, it changes the DNA of a ligand (mpl) by hybridizing the “Oligonucleotide” encoding the required and desired mutation to become a template or a Cus (DNA) template, the mentioned ve template is The single-stranded image of a "plasmid or bacteriophage containing ie (DNA) unchanged or native to the ligand (mpl) and after hybridization is done; the "polymerase" enzyme is used. "for DNA to make a complete strand of complete strands with a complete wall of the model that will integrate the *" initiating oligonucleotide; Which will encode the second alteration that occurs in the DNA of the ligand (mpl)

174 - As a dale, the “” oligonucleotides of at least (YO) nucleotides in length are used. An ideal 'oligonucleotide' will have anywhere from (VY) to (10) nucleotides that are fully complementary to the motif on either side of the nucleotide(s)' encoding the mutation. The This ensures that the “oligonucleotide” will hybridize appropriately to the single-stranded DNA© molecule. The “oligonucleotides” are prepared pin oY using techniques known in such art as described in Crea et al, Proc.

Natl Academy.

Sci] (1978) 5765:75 bp 5ta. The DNA model can be generated and produced by vectors that Ld) are derived from bacteriophage (M13) vectors [vectors form ( [M13mp19] (1 commercially available (M13mp18) are suitable vectors]; or that they are derived from those vectors that originate from a single-stranded bacteriophage with a single 0h-replication ongin site as described in [Viera et al., Meth.

Enzymol, 153:3 (1987)] Therefore; The DNA to be modified and mutated may be inserted into one of these vectors to produce a single-stranded template. The production of the single-stranded template has been described in sections No. Sambrook "Alia (£,£1) 0 et al" Sambrook et al, Molecular Cloning; A Laboratory]. For a double-stranded (or other) plasmid, using standard technical techniques.

\Yo - — to alter or modify the homology of native DNA (to produce variants of amino acid sequences, for example); The "oligonucleotide" is then hybridized to its single-stranded form under the appropriate hybridization conditions. ususally; Then, the DNA polymerase enzyme, which is a “Klenow” fragment of the “1 DNA polymerase” 0 enzyme, is added to manufacture a complementary and integrated strand of the model using the “oligonucleotide” as a raw material Thus, a heteroduplex molecule is formed so that one strand of the DNA strand encodes the modified form of the ligand (mpl) that has had a mutation, and the other strand (the model (lal) encodes the unmodified natural sequence ligand (mpl) then; 40 this heterologous duplex is transformed into a suitable host cell; it is usual to be a prokaryote 0 such as (E.coli 1 01) and after it grows (LOAN) it is plated placed them on top of "06" plates and then separated them using an oligonucleotide "primer" radioactively stained with 71-(32-phosphate) to identify bacterial colonies containing the modified DNA that had been mutated. The modifying region in which the mutation occurred is removed and placed in a suitable vector for protein production.” In general, a transgenic 1 vector of the type transforming the appropriate host is typically used. The method just described may be modified so as to construct a homodimer molecule in which both strands of the plasmid contain the mutation(s). A single-stranded model as described.A mixture of three “0” deoxyribonucleotides, namely deoxyriboguanosine 3, deoxyriboadenosine (Datp) SL deoxyribothymidine (dTTP) 5 ¢ (dGTP) with thio-deoxyribocytosine, was made. called dCTP-).

17 - 5) (which can be obtained from the “Amersham Corporation” and then this mixture was added to the complex compound of the form - 0 oligonucleotide and when DNA polymerase was added to this mixture; A strand of DNA identical to the model, except for the modified bases (which have been mutated). In addition, the new strand of DNA will contain Yay dCTP-(0S)© from the “dCTP” It works to protect it from partial digestion by the action and effect of the enzyme restriction endonuclease After the sample strand of the double heterogeneous double strand was cut with a digesting enzyme, it became possible to digest the sample strand with an enzyme "ExollI" nuclease or an enzyme " Another suitable nuclease precedes the region containing the position(s) to be modified 0 and makes a mutation in it. The reaction is then stopped to leave the molecule that is partially single-stranded noninvasively. Formation of a complete homologous double stranded DNA molecule using DNA polymerase in the presence of all ATP “deoxynbonucleotlde” I triphosphates and dione-binding enzyme fim-1.

DNA 3) Ji can transfer this duplex fragment to a suitable host cell eg Article 0 (8601114101). As previously described Lash. DNA encoding sly mutations may be produced from A single amino acid is to be substituted in a ligand (mpl) in one of several ways.If all the amino acids are placed close together in the "polypeptide" chain they may be modified and mutated simultaneously with an "oligonucleotide" One that encodes all the desired and desirable amino acid substitutions 0. In any case, if the amino acids are at some distance from each other

1117 - the other (separated by more than about ten amino acids); However, it is difficult to produce a single 'oligonucleotide' encoding all the desired changes. Alternatively, one of the two alternative methods may be used. In the first method, a separate 'oligonucleotide' is generated for each amino acid that is desired. oo is then simultaneously recycled into the single-stranded DNA template, and the second strand of DNA synthesized from the template will encode all of the desired amino acid substitutions. The alternative method is two or more mutagenesis cycles to produce the desired desired mutation.The first cycle as described for single mutants is: 0 uses the wild-type DNA of the model; sale) the duplex structure of the "oligonucleotide" encoding the desired first amino acid substituent(s) and converted into this form; then, the heterologous duplex DNA molecule is generated and produced. The second cycle, A gl, performs the mutation using the The DNA that has been modified by mutation and produced in the first cycle of generating or forming the mutation in the form of the template. Thus; This model is configured and ready daily Yo to contain one or more mutations. then; The duplex structure of the oligonucleotide encoding the desired additional amino acid substituent(s) is reconverted into this rd gall and the resulting strand of DNA then encodes mutations through the first and second mutagenesis cycles. This resulting DNA can be used as a model in the third cycle of mutagenesis; and so on.

17 - — Also, the formation of the mutation by PCR is suitable, La, to make changes in the amino acids of the “polypeptide” ligand (mpl), although the following explanation refers to the DNA; However, it is understood that the technique (technical method) makes the application also compatible with — (RNA) and in general the PCR technique refers to the following procedure 0 [see (61-70) M.: [Erlich. supra, the chapter by R.

Higuchi, when small amounts of template DNA are used as starting material for a PCR reaction, primers that are slightly different in sequence from the corresponding region in the template DNA can be used to generate and produce relatively large amounts of po si DNA, which differs from the pattern sequence only at the positions where the primers differ from the pattern.To introduce a mutation into the plasmid DNA, the properties of one of the 0 primers primers are determined to overlap and overlap with the mutation site and to contain the mutation The other initiator strand must be the same length as the corresponding strand strand of the plasmid, but this strand can be placed at a sl position along the length of the plasmid DNA. The second primer after nucleotides 701 starting from the first terminator so that the entire elongated or amplified region of the DNA at the end is bound by a primer vo that can be easily sequenced. or PCR elongation using a primer pair such as that one primer just described yields dic numbers of DNA fragments that differ at the mutation locus labeled by the primer; It is possible at other locations; Being a copy model is somewhat error-prone. if the ratio of template to material of the product is excessively low; The vast majority© of the product of the DNA fragments incorporate the desired desired mutation(s). This product material is used to substitute the compatible region in the plasmid that served as a PCR template

vq - - using standard DNA technology. Mutations can be introduced simultaneously at the discrete loci either by the use of a second mutation initiator; Or by making a second 7018 with different mutation primers and performing “enzymatic” ligating of the two parts of the PCR product simultaneously with a ga gall vector 3 fragment ligating to three parts (or more). To generate and create a mutation by means of PCR, the plasmid DNA template “1”) micro (ng alsa) is placed with a single strip of digestion with the partial restriction endonuclease that has a unique distinct site in the plasmid DNA and outside the desired region Of this substance, “(001) nanograms ng is added to the PCR mixture containing a buffer solution for the PCR, which contains the four compounds of deoxynucleotide triphosphates”¢, and 0 has been included In kits branded (© (GeneAmp) (obtained from Perkin- (Y©) Elmer Cetus, Norwalk, CT and Emeryville, CA) picomol of each primer oligonucleotide until the final volume reaches (+0) μl ul 0 the reaction mixture is covered with (Vo) μl of mineral oil The reaction mixture is denatured for (0) minutes At a temperature of (100 "C), then it is placed 16 completely on ice; Then (1) μL of DNA polymerase enzyme of the Thermus aquaticus (Taq) type (0 units/μL) is added; It can be purchased from (Perkin- “Elmer Cetus) where the addition takes place at the bottom of the layer of mineral oil. The reaction mixture layer is then fed into a DNA Thermal Cycler (can be purchased from Wa «Perkin-Elmer» and programmed as follows:

a 003) a daa min in | 0

1501 — — “0 sec at a temperature of a YY then 9 cycles is as follows: ae sec at a temperature ¢ m ¢ Ya sec at ap temperature 00 a ¢ bv. at a temperature of a VY ¢ h at the end of the reaction; the reaction flask is removed from the thermocycler; then the aqueous phase is transferred to a new flask; the “phenol/chloroform” (by volume) is extracted ('or ion') and the precipitated ethanol is deposited; then the DNA is recovered by carrying out standard procedures. This material is then subjected to the appropriate treatments to perform insertion into a vector. There is another way to prepare the species. variants; and ikl) developed on the basis of the cassette mutagenesis method described by Wells et al, Gene,] (1985) 315:34]. The starting material is a plasmid (or other vector) It contains the DNA ligand of yall (mpl) to make a mutation in. The codon (codons) present in the (mpl) Alan) (DNA) to be mutated has been specified. There must be a unique Ve part endonuclease-digesting enzyme site on all ILs of the identified mutation site(s). If no such sites of marginal digestion exist; They may be produced and generated using the method of generation and generation of mutation that occurs through oligonucleotide-mediated mutagenesis to insert them into appropriate sites in the DNA of a ligand (mpl) and after JAY partial digestion sites are done in ; Plasmid aa is attached to these sites to make it linear. ais manufacturing "oligonucleotide" with a single strand

161 - The coding duplex of the Led DNA sequences between the partial digestion sites but containing the required mutation(s) is desired, using standard procedures. The two strands are made separately and then crossed (mixed) together using standard techniques. This double-stranded "oligonucleotide" is referred to as the cassette. and 0 is designed to be 7-pin; 0 are the terminals that correspond to plasmids af

which has been made in writing; So that it can be directly linked with a plasmid This plasmid now contains the DNA sequence of the mutated ligand (mpl). c. Insertion of Nucleic Acid into a Replicable Vector:

A “polypeptide” (a genetic) signaling DNA or cDNA DNA (eg 0 for replication for further cloning Jil the modified or natural ligand is inserted into a vector from the genotype. The There are many expressions (i) to express whether it is to be used (i) of vectors available; the selection of the appropriate vector will depend on: to express the genotype; (7) the size of the DNA (DNA) to replicate or amplify the

1 _ to be inserted into the conveyor; (7) The host cell to be transformed 0 with the vector. Each vector contains several units according to its function (replicating the 'DNA' or to express the genetic form of 'DNA' and the host cell with which it is compatible. In general, the components of the vector include, but are not limited to: One or more of each of the following: a signal sequence » origin of replication ; and one or more hh genes

- 7?1 ¢marker genes, an enhancer ¢ promoter, and the Flay transcription termination sequence. (1) Signal Sequence Component: the genotype of a ligand (mpl) may be expressed not only directly but also in the form of a fusion with a peptide of different structure or conformation; preferably in the form of a signal sequence or Another polypeptide having a bond cleavage site of particular quality at the N-terminal end of a mature 58m protein or protein. In general, the signal homologue may be a jill component or may be a fragment From the DNA ligand (mpl) inserted into the vector, the signal sequence of different structure or shape must be selected to be one of those 0 sequences that are tagged, recognized, and processed by the host cell (sf). Breaking its bond by (signal peptidase) and for Ally prokaryotic cells that do not discriminate or process a ligand signal sequence (mpl), the signal sequence is replaced by a signal sequence 06 + selected as JE from the set of alkaline phosphatase « or penicillinase « or spp directives (enterotoxin "! 1) heat-stable enterotoxin IT leaders 10. For the yeast secretion cyeast 26 the normal signal sequence is substituted.” For example; yeast invertase enzyme; or alpha factor WlSe, acid phosphatase leaders; The C.albicans glucoamylase vector has been described in [European Patent No. 79 1 (EP) which was then published in Ariel (1940 AD) or the reference sequence that was described in International Patent Application No. (WO 1 3646) 0 Published on November 11 ( 0 um). In the mammalian genotype LIAN; the

12” - normal signal sequence [i.e. ligand sequences (mpl) that normally direct JA ligand (mpl) from normal mammary cells in test tubes] are acceptable; This is despite the fact that there are mammalian signal sequences that may be suitable; Such as signal sequences from another Aas' polypeptide' (mpl) or from the same ligand (mpl) from different animal species; signal sequences 0 from one ligand (mpl) and signal sequences from ligand (mpl) a “polypeptide” secreted to the same or related species; as well as viral secretory leaders; For example; Signal “gD” of Herpes simplex 0 herpes simplex (ii) Origin of Replication Component + Both genotype expression and cloning vectors contain a DNA sequence that enables the vector to replicate in a cell One or more selected host cells. In general; For in cloning vectors, this sequence is one of the sequences that enable the carrier to independently and independently duplicate the chromosomal DNA with the inclusion of the origins of the starting place for the duplication or duplication of sequences in an independent, functionally self-ail manner. autonomously replicating such sequences are well known for different bacteria; 1, yeast, yeast and viruses. The start site of replication taken from the plasmid (Pbr322) is suitable for most Gram-negative bacteria, and the origin of the plasmid (M2) is suitable for yeast; Polyoma' (SV40 Jia) or adenoviruses, VSV or BPV (BPV) are useful cloning vectors in mammalian cells. In general, no starting site is required. Multiplication for expression vectors

1544 — - for the mammalian genotype (typically only the "5740" root would be used since it contains the promoter). Most genotype expression vectors are 'shuttle' vectors, ie they are capable of replicating in at least one class of microorganism but can be transfected to another microorganism for the purpose of genotype expression. For example; A vector is cloned in 085.001 and then transferred to yeast or mammalian cells ll of the genotype even if it is not able to replicate independently from the chromosome of the host cell. It is also possible to increase the amplification of DNA copies by inserting into the group 0 genetic factors of the host. This can already be accomplished by using Bacillus species as hosts; For example; By including it in the DNA sequence of Bacillus with this vector, it will lead to the recombination of the genetic set of genetic factors and the introduction of the Aa} DNA (mpl) and in any case; The recovery of the DNA of the set of genetic factors encoding a ligand (mpl) is much more complex than an exogenously replicated vector or ve formation, since a borderline digestion enzyme is required to excise or cut the DNA of a ligand. (mpl) 0 Selection Gene Component : the genotype and clone vectors must contain the + side of the selection gene; It is also termed as a selectable marker. This gene encodes a transformed host protein, LIA gail, which is grown in selected culture media. Host cells transformed with the vector containing the gene cannot select

- 150 -

to continue living in the culture medium. Typical selection genes encode proteins

As it gives 0 resistance to antibiotics or toxins.

ampicillin “Jie cs AY!” or neomycin ¢ or methotrexate » or tetracycline or

(b) complement auxotrophic deficiencies or (c)

© By supplying critical nutrients not available in complex media ¢ at

example; The gene encoding the enzyme D-alanine racemase of bacilli

,. Bacilli "for bacilli".

An example of a selection scheme uses a drug 8::688 to stop the growth of a host cell. get up

Cells that are successfully transformed by inclusion with a gene of different structure or shape by expressing genotype 0 of the donor protein are resistant to a drug and thus survive in the chosen environment. Jie AB

Southern et al, J.

Molec.

Appl] neomycin This predominant or dominant choice using a drug

Mulligan et al., Science, 209: 1422] or mycophenolic acid [Genet., 1: 327 (1982)

(1980) [< or [Sugden et al., Mol.

Cell.

Biol, 5: 410-413 (1985)] Examples are based on hygromycin

The previous three were put forward using bacterial genes under the control of eukaryotic vectors to transmit resistance 1a to the appropriate drug ((G418) or to “neomycin (geneticin)” i.e. “xgpt (mycophenolic acid) or

hygromycin ¢, respectively.

Examples of other appropriate selectable or selectable function of gall are specific to Lydian cells;

It is those substances that are able to recognize qualified cells that are able to internalize

Jie ligand DNA (mpl) thymidine sf dihydrofolate reductase (DHFR) kinase 0 Mammary variant is placed under selective stress so that it is uniquely primed to survive

1067 -‏ - Only variables are alive because they have the property of absorbing a function. Selective pressure is imposed by the steadiness of the variables in a static medium under conditions in which the concentration of the function factor in the medium changes sequentially; Thus, this leads to an increase in the duplication of both the function gene and the DNA that encodes the polypeptide ligand (m). Increasing replication is the process by which the demand for genes that are involved in the production of proteins important for growth are more and more present in tandem within the chromosomes of successive generations of genetically engineered cells. Increased amounts of ligand (mpl) have been made from the DNA that has been duplicated. For example, torrent; Cells transformed with function 0 (DHFR) are first identified and differentiated by culture in culture medium for all variants in culture medium containing methotrexate (Mix) ¢ and a competitive antagonist of 0 When wild-type DHFR is used ¢ the suitable host cell is a Chinese hamster ovary (CHO) ¢ deficient cell line In terms of (DHFR) activity, it is prepared and propagated as 0 described by [(1980) 4216:77 [Chasin.

Proc.

Natl.

Acad.

Sci., USA, The transformed cells are then exposed to increased levels of (Mix) This leads to the manufacture of multiple copies of the (DHFR) gene and concomitantly; This also leads to the manufacture of multiple copies of other DNA nucleic acids that include the vectors expressing the genetic formula. Say the DNA encoding a ligand (mpl) can be performed using the 0 technique (the method (Agi) For this multiplication with any suitable host of another species, for example, the article ((ATCC No.

CCL 61 0160-1) without resistance to endogenous DHFR (DHFR).

149 - for example; Using the mutant (DHFR) gene that has been mutated and is highly resistant to (Mix) (EP No. 117, 060 ("EP") in an alternative manner; the host cells [and in particular the host cells of normal type and containing [endogenous] DHFR transformed or co-transformed with o(mpl) ligand encoding (DNA)0 sequences and normal type (DHFR) protein; other Alla selectable as aminoglycoside 3' phosphotransferase (APH) 43 These cells can be selected by growing the cell in a medium containing a selection agent for an aminoglycosidic antibiotic (eg neomycin sl kanamycin) or 8M [see US Patent No. 4, 965, 199]. [US Patent No. 0] Among the selection genes suitable for use in yeast is gene (01) found in the yeast plasmid Kingsman et al., Gene, 7, 141] ol ¢ [Stinchcomb et al., Nature, 282: 39 (1979))(Yrp7) (1979)]; The gene (trpl) provides sale select function for a mutant strain of yeast that lacks the ability to grow in "¢" tryptophan eg 44076 ATCC No. or 4-1 -[Jones, Genetics, 85: 12 (1977)] PEP The presence of Ne at the (1rpl) cesion in the Jal gall genetic group of the host yeast cell will then provide effective and effective Af in detecting and determining transformation by sell in the absence of tryptophan' and along the same lines; Yeast strains lacking the ATCC No. 20, 622) "Leu2" or ATCC (No. 38, 626) by 4d y yealplasmids carrying the "2 Leu" gene.

1;8 -

‘Promoter Component’ (iv) than usual; The genotype-expressing vectors and cloning vectors contain a promoter that is recognized by a host microorganism and is operationally bound with the promoters being untranslated sequences present (mpl) ligand DNA ( Generally it is between the upstream of the structural gene start codon later (0) the © start codon and translation control the transcription and the confluence controls the transcription (bp "0001" Mbp 0 1) that binds (mpl ) DNA sequence of Jia ligand specific nucleic acid sequence; 0108 event A These catalysts fall into two categories; operationally Jie. Typically, they fall

inducible and a constitutive structural category, and the inducing or influencing materials are 0 catalysts that search for the beginning of the tate to copy copies of (DNA) and under its control and in response to some changes in the conditions and conditions of culture culture conditions; For example » exist or

The absence of food or a change in temperature. at this time; number is recognized

a large number of stimuli and their recognition by a diverse number of known potential host cells

good. These catalysts are functionally linked to a ligand (mpl) in DNA by removing the material

0 The catalyst is extracted from the DNA source by the limiting restriction enzyme and the separated promoter sequence is introduced into the carrier. Each of the catalyst sequences may use a ligand

(mpl) and many stimuli of different composition or shape in order to direct the replication frequency and/or the expression of the DNA genetic formula of the Jes (mpl) i.e. (Js)

Catalysts of different composition or shape are preferred; Since they generally allow copying

0 is greater and with a higher yield of ligand (mpl) expressing the genetic formula compared to the stimulus

for the natural (mpl) ligand.

049 - — Suitable promoters for use with the prokaryotic family include 8 — lactamase and improved lactose systems [Nature] lactose promoter systems [Chang et (1978) 615:275]; [(1979) 544 :281 {{Goeddel et al, Nature, and alkaline phosphatase enhanced system of {[Goeddel, Nucleic Acids Res. 8: 4057 (1980)] tryptophan (trp)0 and EP No. 36776 88); and hybrid catalysts such as tac i Jes [de Boer et al., Proc.

Natl.

Acad.

Sci.

USA, 80: (21-25) (1983)] HAL; There are some other known bacterial stimulants that are suitable. Its nucleotide sequences have been published, thus enabling skilled workers in this field to perform an operational process by linking it to the DNA encoding its ligand (mpl) [Siebenlist et al ., Cell, 20: 269 (1980)] 0 by using linkers or adapters 359 to supply any desired segmental digestion sites. The catalysts intended for use in bacterial systems will also contain the Shine - Dalgamo sequence (S.D.) which is functionally linked to the (DNA) encoding the polypeptide' (mpl) ligand. ci jo is the catalytic sequence of 98. Realistically, all 0 eukaryotes gardens have a region rich in 7m bases located between bases ranging from about (Yo) to (1”) approximately before upstream The site at which transcription begins. Other Ale's found among bases ranging from (V1) to (A+) a point before the onset of transcription for many genes is region 607" where it may be X is any "polypeptide". At the Cy-terminus of most eukarvotic sequences is the sequence AATAAA, which may be the signal for adding the M-methyl polypeptide "Ye" to the M-terminal end of the encoded sequence. All these sequences The sequences are appropriately inserted into the vectors used in the eukaryotic syntax.

Examples of suitable promoter sequences for use with yeast hosts include the improvers of [Hitzeman et al, J.

Bio. Chem. 255: 2073 (1980)] 3-phosphoglycerate kinase or other glycolytic enzymes](1968) 149:7 [Hess et al, J.

Adv.

Enzyme Reg, [Holland, Biochemistry, 17: 4900 (1978)] as enzymes glyceraldehyde-3- ¢ enolase phosphofructokinase ¢ pyruvate decarboxylase “hexokinase ¢” phosphate dehydrogenase © pyruvate kinase ¢ 3-phosphoglycerate mutase ¢ glucose-6-phosphate isomerase ¢ ¢ glucokinase « phosphoglucose isomerase > triosephosphate isomerase . Other yeast catalysts that are inducible catalysts with the additional advantage or benefit of transcription controlled by growth conditions are ; These are the catalytic enhancers of 2 alcohol dehydrogenase, © isocytochrome and acid phosphatase and related degradation enzymes.; With nitrogen metabolites, metallothionein, and glyceraldehyde-3-phosphate dehydrogenase, as well as the enzymes responsible for the metabolism of 6 and galactose. In addition to the foregoing, vectors and catalysts suitable for use in the expression of the yeast genotype were also described. This is in European Patent No. ve (EP 736578) by Hitzeman et al. It is also beneficial to use yeast enhancers with yeast promoters. The ligand transcription (mpl) of vectors present in mammalian host cells is controlled; For example, the JE pathway is controlled by stimuli that are obtained from a group of genetic factors for viruses such as polyoma virus and fowlpox virus [UK Patent 3 No. 2, 211, 504] (UK Posted Jul 0 (a) AA) and virus.

1110 - - ¢("adenovirus 2" Ji) adenovirus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, and retrovirus And the hepatitis B virus - "hepatitis-B" and the most preferred of them is the "+" Simian virus (40 of Simian Virus) from different mammary stimulating materials oo the composition or formation, such as the substance of ' “actin promoter or immunoglobulin stimulator”; Heat-shock promoters; It is a catalyst that is usually accompanied by a ligand sequence (mpl) provided that such a catalyst is compatible with host cell systems. appropriately The early and late 0 virus promoters (SVA40) are obtained in the form of a segment of a segmental digesting enzyme (SVA0) which also contains the viral replication site of Mulligan and [¢] Fiers et al, Nature, 273: 3 (1978)] « (SVA40) Acad., Sci, USA] « [Berg.

Science, 209: (1422-1427) (1980). Fragment image of a subcompartmental digestive enzyme (Hindill "E")0 as described in Gene [Hindill E restriction fragment Greenway et (1982) 355-360:18:18]. A system for expression has been unveiled. on the genetic formula of (DNA) in Audi families using the “JS” bovine papilloma virus, in US Patent No. (U.S.

Pat.

No. 4, 419, 446) A modification of this system is also described in US Patent No. (U.S.Pat.

No. 4, 601, 978. See also [Gray et al, Nature, 1982] 503-508:295 regarding expression of the genetic form (cDNA) of deli interferon in cells of monkey; See also [(1982) 598-601:297 [Reyes et al, Nature, regarding the expression of the formula

117 - Genetic expression of p-interferon (cDNA) in mouse cells under the control of the promoter "limidine kinase" taken from Canaani and Herpes simplex virus] » herpes simplex virus [Berg, Proc.

Natl, Acad.

Sci.

USA, 79: 5166-5170 (1982) in relation to the human B, -interferon gene in mouse and rabbit cells cultured in culture media; See also Gorman et Led [Proc Natl.

Acad.

Sci., USA, 79: 6777-6781 (1982) 0 concerning expression of the bacterial CAT sequence genotype in LDA monkey kidney 017-1; and in WIA chicken embryo fibroblasts; In WIA, Chinese hamster ovary cells; And in Hela! cells and in lll 'NTH-3T3' cells, using the "sarcoma virus" part known as (LTR) longterminal repcat as a catalyst. (v) 0 Enhancer Element Component: Copying of the DNA encoding a ligand (mpl) of this invention is often augmented by eukaryotes by cascade insertion of a promoter into a vector. An aspect of DNA (usually ranging from about (01) bp (001) Sop) that acts on the promoter by increasing its replication. It is usual. and is located at position 0 ([Laimins et al., Proc.

Natl.

Acad.

Sci.

USA, 78: 993 (1981)] and at position v a Cell Bio., 3: 1108 (1 983)], but [Lusky et al, to where the transcription unit ¢ is within the intron [ Banerji et al., Cell, 33 729 (1983)] and Osborne] ail [et al., Mol.

Cell Bio., 4: 1293 (1984) Many reinforcer sequences have been identified from Bla0 mammalian genes [including from albumin ¢ elastase ¢ globin 'lipoprotein-a'

Voy — _ le [insulin ¢ a-fetoprotein anyway; Typically; A specialist will use a booster from an eukaryotic cell virus. Examples of boosters include the SVA0 booster on the late side of the site of replication initiation ¢ (bp 770 — 001) origin and the promoter of the early-stage cytomegalovirus©; and the fibrous le polyoma booster The late aspect of the site of initiation of replication; promoters of adenovirus See also [Yaniv, Nature, Lad 17-18: 297] (1982) relates to reinforcing elements for the activation of eukaryotic promoters. May connect or couple the promoter into the vector at position 5 or 3' with the sequence-encoded ligand (mpl) but preferably located at position 5 of the promoter.(vi) | 0 terminal component of Transcription Termmaflon Component will also contain vectors for expression of the genotype used in cells — (Je) eukaryotic yeast (yeast); tungi pies; or plant, animal, or nucleated cells of other organisms. They are multicellular because they contain sequences necessary for transcription and stabilizing (mRNA) and it is common and common for these sequences ia to be 01 _ available in the untranslated regions '5 and by coincidence of the untranslated regions '3 of each of the (DNAs) or eukaryotic or viral cDNAs. These regions contain 56 fragments transcribed as polyadenylated fragments in the untranslated part of the mRNA encoding the ligand (mpl).

ot — _ (vii) Construction ana Analysis of Vectors J8 sill: Builds and configures vectors containing one or more of the aforementioned components using standard articulation techniques or methods; Or separate plasmids “Isolated plasmids or parts of DNA 5 that are tailored” and adapted (385) need or ima le and religated 0 in the desired form in order to produce the required plasmids For the purposes of conducting the analysis to confirm valid sequences in the plasmids generated and constructed, ligation mixtures are used to transform strain 204 of E.coli 1612 (ATCC No. 31, 446) and selected successful variants of ampicillin or tetracycline resistance. Whichever is Labia the 5 transformants are prepared”; then hydrolyzed by the enzyme “endonuclease 0” and/or sequenced by the method of Messing et al., Nucleic Acids] [Res. , 9:309 (1981) or by [Maxam et al, Methods in Enzymology (1980) 65:499]. This invention is in the assay vectors for genotype-0 that are provided for cross-expression of the genotype in senescent cells of DNA encoding for a polypeptide ligand (m). As dale, cross-expression of genotype includes the use of a genotype-expressing vector that is able to efficiently perform 0 replication in a (Jile) cell such that the host cell accumulates many copies of the genotype-expressing vector; In turn, it synthesizes Ble levels of a desired polypeptide encoded by the genotype expression vector.

— 1950 Systems allow transverse expression of the form [Sambrook et al., supra, pp (16.17 -16.22)]; cloned containing a suitable vector for expression of the genotype and a host cell achieved recognition; Also, DNAs encoded by 'positive polypeptide' corresponding to the biological or physiological properties of a polypeptide' can quickly classify such desirable and desirable ones. Therefore, cross-expression systems of form Genetics are useful as 0 ligand “polypeptide” especially in the invention for the purposes of identifying homologues (analogues) and variants (mpl) of the ligand “polypeptide” that have biological activity of (mp1)

Suitable Exemplary Vertebrate Cell Vectors (mpl) (ix): Cell Vectors (mpl) Other methods have been described and vectors; And host cells suitable for adaptation and conditioning with ligand manufacturing 0

Gething et al., Nature].

Mantel et al, Nature, 281: 40-46 (1979)] ¢ [293: 620-623 (1981) and (EP 117, 058) (EP 117, 060) and in European patents no. Levinson et al. ” in particular for the expression of the genotype of mammalian cell culture medium of the group ial) plasmid and US patent (EP 307, 247) [European patent no. pRKS is (mpl) associative ve

PCT [Publication No. WO (91/08291) : Selection and Transformation of Host Cells [(US. Patent No. 5, 258, 287)) D. Selection and Transformation of Host Cells The host cells suitable here for cloning or expressing the genotype of vectors are normally highly nucleated host cells that have been DA, yeast, or prokaryote cells.

Yo — — described by. Suitable eukaryotic cells include eubacteria such as Gram-negative microorganisms (01800-06880376) or Gram-positive (eg E.coli). B types. subtilis “¢ and 5 species (P. aeruginosa or “Salmonella typhimurium” or “Serratia marcescans”). The preferred host of E.coli© is this strain 31.446 E.coli 294 (ATCC No. Although there are other strains that would be suitable for this purpose, see W «E.coli x 1776 (ATCC No. 31, 537) «E.coli B Jia See (ATCC No. 27, 325) 3110. These examples are provided for the purpose of Illustrative only and not for the purpose of limiting and limiting.It is preferable that the host cell secrete minimal amounts of proteolytic enzymes.Alternatively, laboratory methods of cloning (eg PCR or DNA polymerase reactions (os SAY) In addition to 8, Jie eukaryotic microbes, filamentous fungi or yeast are suitable hosts for pi binding vectors (mpl). Saccharomyces cerevisiae or the familiar baker's yeast is the most commonly used of the micro-organisms of the lower eukaryotic host and although there are a number of other genera, species, and strains commonly and usefully available here Jie {[Beach and Nurse, Nature, 290: 140 (1981)] Schizosaccharomycespombe and EP No. 139, 383 (EP published ¥ May AAC)4 and Kluyveromyces hosts published in American Patent No. (4, 943, 529) “(U.S.

Paten No. 0 for example:

17 -

K. lactis (Louvencourt et al., J. Bacteriol., 737 11983]), 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 [1 988]). Candida.

Trichoderma reesia (EP 244,234). Neurospora crassa (Case et awl., Proc. Natl. Acad.

Sci. USA, 76:5259-5263 [1979]) °: and filamentous fungi, such as that, for example.

Neurospora, Penicillium, Tolypocladium (WO 9 1/00357 published 10 January 1991), and

Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.

Commun.. 112:284-289 [1983); Tilburn er a!., Gene, 26:205-221 [1983]; Yelton et al.,

Proc. Natl. Acad. Sci. USA. 81:1470-1474 [1984]) and A. niger (Kelly and 01

Hynes, EMBO j, 4:475-479 [1985] (that is, glycosylated (mpl) host cells derived suitable for assaying the genotype of the host ligand capable of performing LIA processing are multicellular microorganisms. Such cells from, and from the basic principles; the glycosylation is complex and carry out glucosylating activities (producing a compound to work and to operate whether this SHB is high it is eukaryotic any culture medium for a cell culture 10 "vertebrates" or Invertebrate Examples of cells include vertebrate the home medium of a vertebrate culture Insect viral strains or cells of non-invertebrate plants have been identified with many permissive cells and variants and baculoviral bacillus Aedes cells aegypti «Spodoptera frungiperda (caterpillar) Corresponding from the family Mthel

- 118 -

Bombyx «Drosophila 116180025161 (fruitifly) «Aedes albopictus (mosquito) ¢«(mosquito): Tagged. See, for example, the S85 cmori

Neurospora crassa ( Case et.al,. Proc.. Natl. Acad. Sci USA, 76 : 5259 - 5263 [1979], and Filamentous Fungi such as, e.g. Neurospora Luckow et al.. Bio/Technology, 6:47-55

[1988]. Miller et al., Genetic Engineermg, Setlow et al., eds. Vol. 8 (Plenum Publishing ° 1986), pp. 277-279; and Maeda et al, Nature, 315:592-594 [1985] A variety of genetic da slaall transfection viral strains are commonly available, for example the variant (L-1) of "Autographa califonica NPV" and the inherited (Bm-5) of “Bombyx mori NPV” and Jia may use these viruses in the form of the virus used herein according to Spodoptera frugiperda' of the present invention and in particular to transfer the genetic information to cells of 0 culture media can be used For a plant cell culture for each of the “cotton”, corn, and potatoes “potato”; soybean; petunia "American nightshade"; tomato; Tobacco, typically in the form of 'hosts'; The genetic information is transferred to the plant cells by incubating with specific strains taken from the bacterial “Agrobacterium tumefaciens” that had been previously processed to contain the cl (mpl) Atay (DNA) incubation of the homeostatic medium of the ell cell. With “A. tumefaciens” the DNA of the ligand (mpl) is transferred to the host of the plant cell so that it transfers 0 by assimilating the information in the plant cell; and in appropriate conditions; it expresses the genetic formula of the (mpl) ligand (mpl) In addition, regulatory and signaling sequences 0 that are compatible with plant cells are available, such as the catalyst for nopaline synthase.

1018! — Depicker et al., J. and polyadenylation signal sequences.

Mol.

Apple.

Gen.). signal (1982) 561:1. In addition, DNA fragments separated from the region twofold of a gene (T-DNA 780 gene) are capable of activating or increasing transcript levels of genes amenable to expression. The genetic formula of a genetically engineered plant, DNA « European Patent No. 321, 196 (EP 0) published on June 11 (1989 AD). In any case, interest has reached its peak in vertebrate cells, and the spread of Vertebrate cells in tissue culture (in tissue culture media) have been a routine procedure in recent years [Tissue]. [Culture, Academic Press, Kruse and Patterson, editors (1973) Examples of beneficial mammalian host cell lineages 0171 cells of a monkey kidney kidney transformed by COS-7, ATCC” “SV40 (CRL 1651 0) and a human embryonic kidney line (293 cells or 3 and 2 cloned back-to-back on growth in culture or culture medium as suspension” Graham et al., ] “suspension (J.

Gen Virol, 36: 59 (1977), kidney cells of a newborn BHK, ATCC) baby hamster ¢ (CCLI1O) and ovarian cells of a DHFR (CHO, [3 Chinese hamster ovary cells ¢ Urlauband Chasin, Proc.

Natl, Acad.

Sci.

USA, 77: 4216 (1980) and mouse sertoli cells 0 (1980) 243-251:23¢(TM4, Mather, Biol.

Reprod., monkey kidney cells ¢(CV1 ATCC CCL 70) Kidney cells, African green monkey kidney cells DA ¢(VERO-76, ATCC CRL-1 587) cervical carcinoma kidney cells human cervical ¢ (HELA, ATCC CCL2) carcinoma cells, MDOK,) canine kidney cells (ATCC CCL 34, aS cells BRL 3A, ATCC CRL) buffalo rat liver cells 0 1442 (W 138, ATCC CCL 75) human lung cells, human 2S cells (Hep G2, HB 8065) human liver cells, and MMT) mouse mammary tumor ‏

— 111 -

Mather et Led Annals N.Y. Acad. Sci. 383: 44-68] TR1 cells (060562, ATCC CCL 51 (1982)[¢, 5" FS4 Wa MRC cells, human hepatoma cell line (Hep G2) line. It is preferable to transduce The genetic information of the host cells and transformed by the vectors 0 mentioned above in this invention and to be cultured in a normal conventional food culture medium modified to be suitable for inducing promoters or suitable for selecting transformants + or to repeat amplifying the genes encoding the desired sequences.

La genetic form by vector to assimilate the vector Transfection term 0 yu) the host whether or not the host can express any information encoded in this vector. I have known many ways to Jil and to transform the genetic information as it is known by ordinary skilled technicians for example by +0; And the process of formation of pores electrically (electroporation) and in the manner of dale can distinguish transmission and successful genetic transformation is known when there are indications for the expression of the carrier within the host cell. Jl is receptive to DNA where in order for the organism to be in a microorganism DNA transformation means insertion of an exogenous 1 or an extrachromosomal element whether in the form of a chromosomal element replicable el a) and according to the host cell used; The .chromosomal integrant transformation is performed using standard techniques suitable for such cells. It is generally used for treatment

Sambrook — (1.82); As described in section calcium chloride by calcium 0 et al.; This is in prokaryotes or other WALs that have septa walls

111 - — cellular cell-wall . Infection with Agrobacterium tumfaciens is used to transform the cells of a specific globule; As described by [(1983) 315:23 [Show et al., Gene, and International Patent Application No. 89/05859 (WO) published in YA June (a) AAR) in addition to To this the plant can be transformed by infection using ultrasonic treatment 10,850,000” as described in International Patent Application No. 91/00358 (WO) published on January 01 (1991). For mammalian cells, which do not contain such These cell walls, the precipitation method with calcium phosphate [Graham van der Eb, Virology, [Graham van der Eb, Virology], [Graham van der Eb, Virology], is the preferred method. General features of transformations of a host system of a mammalian cell have been described by ‎ Axel in US Patent No. 4,399,216 of August 116, 1987. Typically, the transformations into yeast are carried out according to the method [130:946 Van Solingen et al, J.

Bact. Jey [Hsiao et al, Proc.

Natl, Acad.

Sci (USA), 76: 3829 (1 979)]. nuclear injection » or electroporation or protoplast fusion » fusion with the protoplasmic content of the cell as a biological unit 0 original ». H. Culturing the Host Cells: The Prokaryotic cells used to produce the (mpl) 4a, polypeptide of this invention were cultured in suitable culture media as generally described in Sumbrook et al. The mammalian host cells used to produce the ligand (mpl) of this invention may be cultured in a variety of culture media. Commercially available culture media for this symptom are eg

AY - - ¢ Ham's Flo (Sigma), Minimal Essential Medium (RPMI-1640 (Sigma) « ([MEM]. Sigma), and Dulbeco's Modified Eagles Medium ((DMEM) ] Sigma) is the appropriate culture media in this field to grow host cells.In addition to that, any culture media then described in each of: Ham and Wallace, Meth.

Enz 58.44 [1979], Bames and Sato. Anal Biochem.

Patent No; 4,767,704 ; 4,657,704; 4,657,866; 4,927,762; or 4,560,655; wo, [1980] wo 87/00195; U.S.A.

Patent Re. 30,995; or copending U.S.S.N. 07/592 OR :90/034430 07/592,141 both of which were filed on “Oct (+39 (2) 07/592,141 which are all disclosed 0 and incorporated herein by reference and with the help of Bahaa may be used as culture media (cultivators) Any culture medium from these media can be supplemented if necessary with hormones and/or other growth factors (such as insulin, transferrin , or schiem61 growth factor). epidermal growth ) and/or salts (such as sodium chloride, magnesium calcium, “phosphate” and/or buffer solutions (such as “HEPES” 0 and/or the ) thymidine “adenosine J—is) nucleosides, antibiotics (Ji) antibiotics (Gentamycinw) and trace elements aa (identified as inorganic compounds) usually present in final concentrations in micromolar range limits) and glucose or an equivalent energy source. Other necessary supplement substances may also be included in appropriate concentrations which will be well known 0 a dy skilled in such a field as this art. The conditions of the culture medium (the farm) are ml

117 - The degree of pall pH ¢ (Ph) and the like are similar to those conditions previously used with the selected host cell to express the hereditary trait and Jie these conditions will be quite clear to those of ordinary skilled technicians in such a field . The host cells referred to herein in this text include the cells in culture media 0 of the Led Ws in vitro culture as well as the cells inside a dle animal of the host animal. And the. Detecting Gene Amplification/Expression: The frequency of gene duplication and/or expression of a gene in a sample may be measured directly; For example, WJ by Southern blotting and Northern blotting to determine the amount of “mRNA” copies and measure them [Thomas, Proc.

Natl.

Acad.

Sci.

USA, 77: 5201-5205 (1980), dot blotting (DNA analysis)] with “DNA analysis or in situ hybridization; Using probe No appropriately addressed to a specific card; On the basis of the sequences provided and provided here. Multiple labels probes may be used; Most of them are normally common radioisotopes, especially P and on any (Ja) other techniques may be used. For example, biotin-modified nucleotides are used to be inserted into The polynucleotide then; biotin A forms the site to bind to avidin or

116 - — with antibodies; Which may be tattooed in many ways with a wide variety of; such as aia elements, fluorescers, enzymes » or the like. In the Alay method, antibodies that can distinguish duplexes of a special kind may be used, including DNA-RNA hybrid duplexes, RNA duplexes, DNA duplexes, and DNA-protein duplexes. Antibodies are also identified in turn by marking them with a probe and the pair is attached to one of the surfaces, when the pair is formed on the surface, then the presence of the antibody bound to the double can be detected and identified.In an alternative way, the expression of the genetic character of the gene may be measured by methods of immunological study J— immunological methods Studying the immunohistochemical staining 0 or the staining of different tissue segments and testing all cell culture media or body fluids, in order to quantify the expression of the genetic formula of the gene product directly, and by using the technical methods of this chemical formula Immunohistochemical A cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for product 1 gene conjugate; Whereas, it is usual for the probes to have labels visible and identifiable as the enzyme probes; fluorescent adhesives; luminescent labels; and the like. The dyeing or coloring technique suitable for use in the present invention is described in [Hsu et al, Am, J.

Clean.

Path, [1980] 75:734-738.

#11 — - Antibodies useful in immunohistochemical formulation and/or test sample fluids may be either monoclonal or polyclonal; It may be prepared in any mammal. It is aD that the antibodies against a natural ligand group polypeptide (mpl) or against a {clin peptide} are prepared on the basis of the DNA sequences provided here and described below. g. Purification of mpl ligand Polypeptide (mpl): It is preferable that the ligand (mpl) be recovered from the culture medium as a secreted polypeptide, although it can be recovered from lysis products and cells. 1,588 host when the genotype is expressed directly without a secretory signal.

0 When the genetic form of a ligand (mpl) is expressed in a genetically engineered cell other than that of human origin, the ligand (mpl) is completely clean of proteins or polypeptides of human origin. any way; As usual, there is a need to purify a ligand (mpl) from the proteins or polypeptides of the genetically engineered cell to obtain a homogeneous mpl-ligand preparation. As a first step, culture medium or solubilization products are formed and cell lysis is formed to remove residue

0 cell residue debris 0611. The membrane fragments and the soluble protein fragments are then separated. in an alternative way; A commercially available protein concentration filter (eg “Amicon™,” “Millipore™” or “Pellicon™” brand ultrafiltration units (JU) may be used). The (mpl) ligand may be purified from the GLE soluble portion and from the membrane portion of the cell lysis product, depending on whether it is a (mpl) ligand.

0 is membrane bound or not. after that; The ligand (mpl) is purified from proteinolate and L contaminants

117 - - ALN polypeptide for solubility, by salt separation or forced exchange, or by carrying out chromatographic processes that use several tissue gels. These textile materials include cellulose » dextran » agarose ¢ acrylamide and other common and familiar materials in the field of protein purification. Ideal chromatographic procedures suitable for protein purification include: Immunoaffinity 0 (eg to an anti-hmpl ligand mpl)(((Mab) ligand mpl) Receptor affinity (eg mpl-IgG) or protein A); hydrophobic attraction chromatography (HIC) e.g. butyl sf cether ¢ i.e. phenyl (Toyopearl) lectin chromatography (e.g. Jiu Con A - (lentil-lectin-Sepharose) (Sepharose) size exclusion (ex; 6-75” (Sephadex 0) and exchange columns for each of the anion cation (ex (DEAE) “JL” carboxymethyl sf or ¢(sulfopropyl-cellulose and reverse-phase high performance liquid chromatography (RP-HPLC), Sl) [see eg; (1984) 171:296 [Urdal et al, J.

Chromatog., where two consecutive primary steps of (RP-HPLC) are used to purify engineered human (IL-2). Other optional purification steps 1 include: precipitation of ethanol; and precipitation of ammonium sulfate. SDS-PAGE “preparative chromatofocusing” and the like. Ligament variants (mpl) in which residues have been deleted, inserted ¢, or substituted are restored in the same manner as in the case of the natural 0(m) ligand, taking into account Consider any fundamental changes in properties that are incidentally caused by variability. For example, preparation of ligand fusion (mpl) with another protein or with

117 - — . AT polypeptide such as antigen ie (JE) bacterial or viral; facilitates purification; an immunoaffinity column containing an alias of the antigen in order to adsorb the specific polypeptide. A rabbit polyclonal (anti-mpl) ligand antibody column, Jie Immunodeficiency Column, is used to uptake variant 0 Aad (mpl) ligand with the remaining antibody unit. The (mpl) ligand may be purified by affinity chromatography using (mpl-IgG) conjugated (preferably) to “(Bio-Rad., Richmond CA) Affi —Gel 10 immobilized or similar; by Well known means in Jie in this area of the art.Protease inhibitor a— 3) Jails asia phenyl methyl sulfonyl fluoride (PMSF) Jie may also be involved in inhibiting proteolysis during the 0-purification procedure; Antibiotics may be included to prevent the growth of incidental cross-contaminants. One skilled in such an art is aware that methods of proper purification of the natural (mpl) ligand may require adjustment to account for changes that occur in the characteristic of the (mpl) ligand or its variants according to the expression of the genotype in the culture medium of an engineered cell behind. h. Covalent Modifications of mpl ligano (mpl) Polypeptide 0: Covalent modifications of a polypeptide' (mpl) ligand are included within the scope of this invention. May be done covalently Modification of both natural (mpl) ligand variants and variants of the (mpl) ligand amino acid sequence One type of covalent modification included within the scope of this invention was a portion of the (mpl) ligand that may be suitably prepared © Parts of a ligand variant (mpl) 1 have up to £1 residues of acid

VIA - - formed amino by chemical synthesis or by enzymatic or caspase fragment of the full length of the ligand mpl Lie polypeptide variants. Other types of covalent modifications of the (mpl) ligand or parts thereof have been introduced into the molecule by the interaction of the target amino acid building blocks of the (mpl) ligand or parts thereof with an organic derivatizing agent 0 that is able to interact With select (sub)side chains or with (N-) or carboxylic (-) amino-terminal units. In a normal, familiar way most of the time, “Cysteinyl” building blocks interact with “-haloacetates” (and their corresponding amines); chloroacetic acid Jie or cchloroacetamide to give carboxymethyl or carboxyamidomethyl . It can also derive “cysteinyl” building blocks by 0 reaction with bromotrifluoroacetone ¢ or a-b romo- B- (5 - imidozoyl)propionic acid » or chioroacetyl phosphate » or N- alkylmaleimides “or 3-nitro-2pyridyl disulfide” or 2chioromercuri-4- gb ¢ p-chloromercuribenzoate ol “methyl 2-pyridyl disulfide nitrophenol” or chloro-7-nitrobenzo-2-oxa-1 » or .3-diazole building blocks Histidyl residues are derived by interaction with diethylpyrocarbonate at a pH of 0 "17 m" ranging from (0.0) (V0) given that this factor is of a relatively special quality For the histidyl sub-chain Parabromophenacyl bromide is also useful for Lad and it is preferable that the reaction be carried out in (0.1) mol cacodylate ("1:H" and at a pH = (300) Lysingl residues and residues at the amino terminal react with succinic anhydride 0 or with another carboxylic acid anhydrides. The derivatization with these factors is of

1019 - — charge reversal effect of the lysinyl residues. Other suitable reactants for the derivation of amino group-containing residues include imidoesters such as methyl phosphate pyridoxal ¢ picolinimidate ; chloroborohydride ¢ pyridoxal ¢ 4-pentanedione ¢ methylisourea -0 ¢ trinitrobenzenesulfonic acid,2; And the reaction catalyzed by the enzyme 4ll3 transaminase-catalyzed with .glyoxylate. Arginyl building blocks are modified by reacting with one ligand or several binding reactants, including ninhydrin 1,2-cyclohexanedione ¢ 2,3-butanedione. ¢ “Phenylglyoxal derivation of arginine residues requires that the reaction be carried out under alkaline conditions due to the high pKa of the guanidine functional group. In addition to “ly”, these compounds may interact with 0 groups of the lysine, as well as the arginine epsilon-amino group. labels inside tyrosyl building blocks by reacting with aromatic diazonium compounds or with -tetranitromethane and in a normal and familiar way Sis ' 0 N-acetylimidizole 20110061876 is used to form types of O-acetyl tyrosyl 01 | 806068; and 3-nitro derivatives in the 0 order, then the tyrosyl building blocks of 0 are treated with PT PT to prepare radioactive proteins for use in the radioimmunity assay 1 » and the previously described 1 chloramine method is the appropriate method. And the carboxyl side groups (for desulphation or desoldering) are selectively modified by 0 reaction with (R-N=C=N-R) 5s + in which RR is two alkyl groups

1101 — Two Differents” -cyclohexyl.3-(2-morpholinyl. 4-ethyl)carbodiimide Jie 1 i.e. lethyl-3-(4-azonia- 4-dimethylpentyl)carbodiimide 4 . In addition to J afi ld asparaginyl building blocks « glutaminyl to glutaminyl building blocks » asparaginyl by reacting with -ammonium ions the derivation using Jal go bifunctional agents is useful to perform © cross bonding crosslinking of the ligand (mpl) with a water-insoluble support matrix or with a water-insoluble surface for use in the method for antibody purification of the (anti-mpl) mpl and vice versa . Commonly used Jal gall cross-linking agents include, for example: N N-hydroxysuccinimide ¢1 .1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde esters 0; 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 methyl-3- J—— Je Bai IW J dle [(p.azidophenyl)dithio]propioimidate leads to the production of yall-activate intermediates photoactive Vo, which is capable of cross-linking in the presence of light. in an alternative way; The use of textile matrices that are insoluble in water such as cyanogen bromide - activated carbohydrates and reactive materials Allg are described in US Patent No. (3,969,287)» (3,691,016)» ) (4, 195, 128) (4, 247, 642)” (4, 229, 537) (4, 330, 440) using protein.

111. The amides are recursively removed from both the glutamingl and asparaginyl residues to where they are converted to the corresponding glutamyl and aspartyl residues; Respectively. Deamidated from these building blocks under neutral or basic conditions. The deamidated form of these building blocks falls within the scope of the present invention. Other modifications include the introduction of de gene'/hydroxylation groups (hydroxylation of lysine » proline ¢) and the phosphorylation procedure by phosphorylation of the hydroxyl groups of each of the building blocks seryl or threonyl building blocks; then perform the methylation process (insert [ methylation groups] for the 0-amino 00 groups of both carginine » lysine and histidine side chains [T-E. Creighton, Proteins, Structure and Molecular Properties, 11711. Freeman & Co., “SanFrancisco pp. (79-86) (1983)] and perform the amidation process (the insertion of amidation groups of the carboxyl terminus of the protein 0) Another type of covalent modification of a polypeptide ligand (mpl) is included within the scope of 1 of this invention and includes Altering the pattern of natural glycosylation of the -polypeptide The use of the term “by altering” means the cancellation of one or more of the carbohydrate moieties present in the natural ligand (mpl) and/or the addition of a single site One or more glycosylation sites that are not present in the normal (mpl) ligand. The glycosylation of the polypeptide is typically Ul bonded to a nitrogen (N-linked) or to an oxygen atom (0-0160). indicate associated with a nitrogen atom

17 — (N-linked) to the binding of the carbohydrate moiety to the (sub)side chain of the asparagine residue The asparagine-Xthreonine sequences are “asparagine-X-serine—-U tripeptide ¢ where X is Any amino acid except for proline; They are the sequences characteristic of the enzymatic bonding of carbohydrates with the asparagine sub-chain of that; The presence of Ld© from these chains of tnpeptlde highlights a site amenable to glycosylation. O-linked glycosylation refers to the specific binding of a sugar “N-acetylgalactosamine” or galactose or 6 with 6-hydroxyamino acid commonly known to be a metabolite threonine; Although 5-hydroxyproline or 5-hydroxylysine can be used the addition of glycosylation sites to a ligand polypeptide (mpl) can conveniently be implemented by changing the amino acid sequences so that they contain one or more sequences Of the previously described 6 sequences (for the N-linked glycosylation sites), the change may also be made by adding or substituting one or more residues, serine or threonine. The natural (mpl) ligand sequence lal has 0 glycosylation sites bound to an oxygen atom (‘O-linked’). The level of the ¢ (DNA), in particular by making a mutation of the (DNA) encoding the polypeptide ligand (mpl) according to the previously selected bases so that the codons that will be translated and interpreted are generated by transforming them into the required amino acids. DNA mutation(s) may be performed using the methods previously described by Yo under the heading “Sequence Variants of mpl Ligand (mpl) .

- 117 consists in performing (mpl) on a carbohydrate ligand. There is another way to increase the number of clefts. Such procedures are useful polypeptide with glycosides chemical or enzymatic conjugation of the host cell that It has polypeptide capabilities and is unique in that it does not need to produce L

N-) of "0106000" attached to a glycosylation nitrogen atom of treatment procedure 0010 or attached to an oxygen atom (0-100100). Depending on the pairing pattern used; A sugar may bind to (b) free carboxyl groups; or (b) histidine or arginine groups (sugars linked to) to (b) cysteine free groups such as these Those of the sulfhydryl groups or “hydroxyproline” or of the “compacted” or free serine, such as those of the hydroxyl groups or “tyrosine” or “phenylalanine” such as those of the aromatic residues (d) Building units These methods have been described in Request glutamine for the amide; or (e) tryptophan group 0 and in Dads 11 F (1947) published WO 87/05330. International Patent No. 0 [Aplin and Wriston, CRC Crit. Rev. Biochem, pp. 259-306 [1981] chemically or (mpl) Atay polypeptide contained in carbohydrate removal of polypeptide derivatives may be performed Enzymatic exposure to deglycosylation Requiring chemical treatment to remove it or another equivalent compound This compound leads to ¢ trifluoromethanesulfonic acid L1 acid

N=) The treatment resulted in breaking the bonds of most or all of the sugars and splitting them, except for the sugar bonds, intact. The polypeptide has been left while the ¢ (N-acetylgalactosamine or acetylglucosamine and chemical Hakimuddin et al. by deglycosylation and removal of dae [Edge et al, and by «[Hakimuddin et al. , Arch. Biochem. Biophys. 259: 52 (1987)].

1740 exo-glycosidases; and endo by using a variety of polypeptide enzymes on the -[Thotakura et al., Meth.

Enzymol., 138: 350 (1987) [as described by — at potential 0 sites by using a glycosylation compound aie [Duskin et al, J.

Biol.

Chem., 257: 3105 as described by tunicamycin + protein-N-glycoside linkages by blocking the formation of tunicamycin bonds. A polypeptide ligand is bound by a compound (1982). mpl).

Patent No. (4,640,835) American numbers (4,179,337) or (4,49,689)” or (4,301,144)” or (4,670,417) or (4,791,192) or 0 The linker of the recovered JE to select the mpl variant and requires the need to display a ligand that has immunological and/or biological activity that has been previously identified and defined. MPL can take place in a genetically engineered cell culture medium (a culture) for stability, separating a single variable for the purpose of achieving proteolytic cleavage, or in plasma (for example; in contrast to proteolysis and oxidative stability). Towards oxidation « mpl High affinity or high affinity for a species 1 The change in JU) and the ability to be excreted at high concentrations are measured and estimated, and the like. For example, such as affinity and affinity towards (mpl) The polypeptide ligand is the immunoassay characteristic of a competitive antibody by performing competitive type immunoassay experiments, and other potential modifications of type immunoassay properties are also performed, such as stability testing experiments against antigens. The oxidation of a polypeptide characteristic of a protein or of ©

178 - redox and shorthand; thermal stability ¢ or hydrophobicity or proteolysis; The aforementioned test answers are performed using methods known to be well-knowledgeable in such a field of this art. -117 General methods for the preparation of ligand antibodies (mpl) Preparation of the antibody: (1) Polyclonal antibodies: In general; the formation of polyclonal antibodies against polypeptides lk vd'm parts (mpl) in animals by performing multiple injections subcutaneous (sc) or into the peritoneal (mpl) gvfd'm intraperitoneal (ip) and for an additional adjuvant. That a ligand (mpl) or part thereof containing the target amino acid sequence be conjugated with a protein that is immunogenic in the organisms to be immunized, for example “keyhole limpet hemocyanin —S” or JY serum “albumin” or bovine thyroglobulin; or “soybean trypsin” inhibitor by using a bifunctional agent or derivative agent; 0 eg maleimidobenzoyl sulfosuccinimide ester (bonding through N-hydroxysuccinimide sf “(cysteine)” units (through glytaraldehyde sf “(lysine) residues “ie succinic anhydride” or aR N=C=NR | (SOCL,) the L is the two different alkyl groups.

— 1171 -

The ill gall is immunized against a polypeptide ligand (mpl) or part of a ligand (mpl) or conjugates

immunoglobulins or immunogenic derivatives thereof by mixing 1 mg; to 1 microgram of peptide or

Paired (for rabbits or mice, respectively) with ¥ volumes of additional full auxiliary material

Freund's — complete adjuvant Then inject the solution into the skin at several different sites. And yet

© 1 month ago Animal injections were boosted and fortified with 1 / 1 to 1 / 10 original quantity

of the peptide in Freund's complete adjuvant by subcutaneous injection procedure

in several different locations. After a period ranging from UV to VE days; blood is drawn from

The animals are then examined and the serum tested for the level of concentrations of antibody to the ligand (mpl) being enhanced

Immunity in animals reaches a level sufficient to reach the stable sh plateau level

0 . It is (Jaa) that the immunity in the animal is strengthened by the pairing of the same ligand (mpl), but

In combination with a different protein and/or through the use of a different cross-linker. can be done

Lia makes conjugates in cell culture medium for genetic engineering in the form of protein fusions. It is also

Lad dal so can be used in combination with aggregating agents such as alum to enhance immune response.

: Monoclonal antibodies Gi) 10 Monoclonal antibodies are obtained from many numbers or populations of essentially homologous antibodies; That is, the individual antibodies that make up these sentinel numbers are identical, except for those that are likely to exist naturally resulting from mutations that may be present in small quantities. So; the "monoclonal" modifier refers to the characteristic of the antibody

© A because it is not in the form of a mixture of different antibodies.

177 — a for example; Monoclonal ligand antibodies (mpl) may be made using the hybridoma first ll characterization 495:256 [Kohler & Milstein, Nature, [197]) or they may be made by genetic engineering methods of (DNA) [US Patent No. [U.S.

Patent (No. 4, 816, 567) by Cabilly et al. 0 in the oncogenic hybridization method; An immunized mouse or other suitable diile animal such as a hamster, as described above, is vaccinated to induce lymphocytes that produce or are capable of producing antibodies that will bind specifically to a protein used to induce immunity or the necessary immunity. in an alternative way; Lymphocytes may be inoculated into laboratory test tubes. At that time; Lymphocytes are combined with WDA myeloma cells using an appropriate fusion agent 0; polyethylene glycol jie; This is to form a hybrid and tumor cell. [Godding, Monoclonal Antibodies; Principles and Proctice, pp. (59-103), Academic-Press, (1986)] Hybrid cells are cultured in a tumor form and conditioned for their growth in a suitable culture medium, which is the medium that preferably contains one or more substances that inhibit the growth of non-yo cells. Hybrid and uncombined from the original WA myeloma. For example; If the original myeloma cells of origin lack the enzyme hypoxauthine guanine phosphoribosyl (HPRT | HGPRT) trausferase, the stabilizing medium for oncological hybridization will typically include caminopterin hypoxauthine and thymidine (HAT), which is They are substances that inhibit the growth of HGPRT-deficient cells

— YVA —

Preferred myeloma cells are those (ALL WAY) integrating eli and support a high level of affinity-specific antibody genotype expression by selected antibody-producing cells; they are sensitive to a medium such as medium (0187). Among these (LDA) The preferred myeloma cell lineage is a marine myeloma cell line;

mClK derived from mouse tumors (MPC-11) (MOPC-21) available at the Salk Institute Cell Distribution Center in San Digo, California; US

USA ¢ (California, U.S.A.) and cells (SP-2) (the American Culture Collection Rockville, Marryland, U.S.A.) ©170. ADL has also been described as a human myeloma cell and a heterozygous mouse-human myeloma cell lineage (moace - human hetevomy).

[Kozbor, J.

Immunol, 133: 3001 When Human Monoclone Antibodies Are Produced 0 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications,

-pp. 51-63, Marcel Dekker, Inc., New York, (1 987] The culture medium in which the oncogenic hybrid cells grow is tested for the production of monoclonal antibodies directed against the ligand (mpl). vo The binding of monoclonal antibodies produced by hybrid tumor cells by immunoprecipitation or by cin vitvo binding test such as radioimmunoassay (RIA) or radioimmunoassay

J The binding affinity of a monoclonal antibody can be determined by the v procedure. The “Scatchard” analysis of L [(1980) 107: 220] [Munson & Pollard, Anal.

Biochem.,

1794 - After the oncological hybrid cells are identified and distinguished, which produce antibodies of a special quality; And / or Ad) and / or activity and / or effectiveness required and desired (Cele), the products may be duplicated, subcloned by identifying and developing mitigation procedures by standard methods (Goding, supra) including appropriate homeostatic media For this purpose, for example on Dulbeceo's Modified Eagle's Medium or RPMI- medium 1640. In addition; Hybrid cells may develop tumors in the body of the organism, Jie, ascites tumors in an animal. It's appropriate; That the monoclonal antibodies secreted by the replicated subclones be separated from the culture medium; or from ascites fluid eli uu” or from one of the serums by carrying out immunoglobulin “purification” procedures such as, for example, protein A-Sepharose; Or by hydroxylapatite “chromatography” or by “gel electrophoresis” or “dialysis” or affinity chromatography, the DNA encoding monoclonal antibodies of the present invention is quickly separated and 1 of its sequences are determined This is done using conventional procedures (for example, by using oligonucleotide probes that are able to perform specific binding to genes encoding murine heavy and light chains (antibodies). Hybridoma cells are used as a preferred source of such DNA. Once the separation has been performed, the DNA may be placed into vectors expressing the genotype, which are then transfected into host cells such as simian COS cells, or hamster ovary cells.

186 = - Chinese (CHO) or IS tumor [elds] that do not somehow produce the immunoglobulin protein immunoglobulin to elicit monoclonal antibody manufacture in genetically engineered host cells. DNA may also be modified, for example by substitution of the coding sequence for human heavy and light chain constant domains instead of the different murine sequences © Fig. 6851 :81 Proc.

Nat.

Acad.

Sci. Mullah [Cabilly et al, supra; Marrison et. (1984) or by covalent binding to all or part of the sequence encoding an immunoglobulin, for all or part of the sequence encoding a polypeptide other than immunoglobulin. Typically, Jia is replaced by This polypeptide is non-immunoglobulin 0 of the constant domains of the antibody of the invention or is substituted for the variable domains of one site bound with 0 of the antibody of the invention to be synthesized or to form a bivalent scrambled antibody (with genetic substitution ("chimeric" shall have a locus associated with an antigen of specific quality for the associative group (mpl) and another site associated with an antigen of specific quality for .antigen ve may be Lad Preparation of chimeric or hybridized antibodies in laboratory test tubes using known methods in synthetic protein chemistry including cross-linking agents For example, immunotoxins may be constructed using a disulfide exchange reaction i.e. disulfide or by forming a thioether bond Examples of suitable chemicals in this place for this purpose include methyl-4- ¢ iminothiolate -mercaptobutyrimidate 0

— YAY - for diagnostic applications; The antibodies of the invention will typically be identified and labeled with a detectable and identifiable moiety. A detectable slit can be any slit that is SUE to produce a detectable and identifiable signal, either directly or indirectly. For example; The detectable moiety may be a 0° radioisotope such as CH, SC 0, 8, or 1, or be a fluorescent substance or a chemiluminescent compound » such as rhodamine » fluorescein isothiocyanate » or luciferin and isotope-characterized probes (da dial such as EP DT or ©*'; or CH or be an enzyme; such as salkaline phosphatase, i.e., beta-galactosidase, i.e., 'horseradish peroxidase' 0 Any of the methods known in this field of art can be used to separately conjugate the antibody to a detectable and identifiable moiety, including those described. By: [Hunter, et al., Nature, 144: 945 (1962)] ¢ [David, et al., Biochemistry, 13: 1014 (1974)] ¢ [Pain, et al, J. Immunol. , 40: 219 (1981)] ¢ [Nygren, J. Histochem.

Cytochem. 30: 407 (1982)]. Vo Antibodies of the present invention may be used in any of the methods of testing “competitive binding assays” known Jia competitive binding assays; direct and indirect sandwich assays

Zola, indirect and immunoprecipitation assays. And methods of testing by immunoprecipitation

187 - Mononclonal Antibodies: A Manual of Techniques, pp. (147-158) (CRC Press, Inc.) 1987. Competitive ligation assay experiments depend on the ability of a specific standard (which may be a ligand (mpl) or an immunoglobulin elie Ja thereof) to compete with the analyte of a sample

0 The test (which is the "1M" ligand) Lad relates to binding with a specific amount of antibody. The amount of ligand (mpl) contained in the test die is inversely proportional to the amount of bil that becomes bound; In general, antibodies are insoluble before and after competition; This is because it may be appropriate to separate the antibody-bound standard and analyte from the standard and analyte that remain unbound.

0 Sandwich assays involve the use of two opposite objects; Each is able to bind to a different immune system; or in the epitope or antigen space; Or the protein (the “m(«”) ligand to be detected and identified. In the sandwich analysis, the test sample is bound by the first antibody i on a solid suppor; then the second antibody binds to the product of dial thus forms three insoluble complexes. David & Greene US.

Patent

No 4,376,110\e The second antibody may be tagged with a probe using a detectable and identifiable moiety (direct sandwich method assay experiments) or it may be measured and quantified using an antibody to immunoglobulin that is tagged with a detectable and identifiable moiety (experiments). indirect sandwich method test). For example; One variant of the sandwich method is an analytical assay (ELISA) in which the detectable moiety is an enzyme.

For example, the enzyme horseradish peroxidase

18# - - Z (ii) Humanized and human antibodies It has been known in such art and know so well the ways in which non-human antibodies are rendered human antibodies. In general; The antibody that is intended to be made human has a building block of one or more amino acids inserted into it from a non-human. In lla) these non-human M0 amino acid residues are referred to as "import" residues which are typically taken from an "external" variable domain. Humanization can be carried out primarily by following Winter's method and with the help of [Winter and [Riechmann et al, Nature, 322: ¢co-workers Jones et al., Nature, 321: 522-525 (1986)] [Verhoeyen et al, Science, 239: 1534-1536 (1988)] £323-327 (1988)] by substituting 1 for the two rodent CDRs or CDR sequences for their corresponding sequences of the human antibody. accordingly; Such humanized antibodies are chimeric antibodies "that is, they have a hereditary Ja" et al, supra ("atta0"); in which less than one healthy human variable domain replaced by the corresponding sequence of non-human species has been essentially formed. and in practice; Humanized antibodies are typically human antibodies in which some of the 001 residues and possibly some of the FR residues are replaced by residues from similar sites found in rodent antibodies. The choice of human variable domains; Both light and heavy ones and intended to be used to make antibodies into human antibodies are important to reduce antigenicity 0 and according to the appropriate best-adjusted method 88-51";

164 for known human variable field sequences. clin The human sequence that is closest to the rodent sequence is accepted as the human framework (FR) of the humanized antibody [(19993) 2296:151 [Chothia and Lesk, J. ¢[Sims] et al., J.

Immunol.

Biol, 196:901 (1987)] Another method uses a system-specific structure derived from the overall accurate statistic of all human antibodies to a specific subset of light chains or heavy chains. The same system structure may also be used for several different antibodies made into human antibodies [Carter et al, Proc.

Natl.

Acad.

Sci.

USA, 89: 4285 (1992)] ¢ [Presta et al., Immnol., -])1993( 2623 : 151 0 It is important, in addition to the foregoing, that the antibodies intended to be converted into human ini so that it retains a high affinity for 0 and other preferred biological properties.To further this goal according to a preferred method, human-transformed antibodies are prepared by the process of analyzing the original sequences and multiple conceptual human-transformed products using commonly available three-dimensional models. 1 Computer software is available which illustrates and presents the common structures three-dimensional boundaries of the selected candidate immunoglobulin sequences. Examination and study of these presentations allows analysis of the potential role of the residues In the functionality of the immunoglobulin sequence, which is a candidate for this work, i.e. the analysis of the residues that affect the ability of the diseased immunoglobulin to bind its antigen 0. In this way, the selection of (FR) residues can be carried out. And make it unite from

a YA — the extraneous overall statistical exact sequence (from an external domain); In order to achieve the desired and desirable antibody specific characteristics such as increased affinity for the target antigen(s), In general; Residues (CDR) are directly and primarily involved in affecting antigen binding. For additional details, see © US Patent Application Serial No. (934/07, 373) filed on August 1, 1997 ), which partly shares with the American patent application serial No. (715/07, 272) filed in VE in June of the year (9911 AD).

in an alternative way; It is now possible to produce transgenic animals (such as oo) iil that, when vaccinated, are able to produce a repertoire of 0 human antibodies in the absence of endogenous immunoglobulin production and growth. . For example (Jal), it has been described that homozygous deletion of the ALE antibody chain linking to the (J) gene in a mouse strain with a mutated chimeric ID {ys) leads to complete and complete inactivation. for endogenous antibody production and growth. Transfer of the human strain's immunoglobulin gene sequence system into such a mouse ADL would result in the production of antibody

Vo is a human antibody when it is stimulated by an antigen. See for example; Jakobovits et al., Proc.

Natl.

Acad.

Sci.

USA. 90:2551-255 [1993]; Jakobovits et al., Nature, 362:255-258 [1993]; Bruggermann et al.. Year in Immuno., 7:33 [1993].

— 17 -

Human antibodies can also be produced in phage-labeled presentation kits (Hoogenboom and Winter, J.

Mol.

Biol. 227, 381 [1991]; Marks et al., J.

Mol.

Biol. 222,

581 [1991]).

(iv) © Bispecific antibodies:

Bispecific antibodies are monoclonal; preferably human or humanized antibodies that have at least two different binding types. The workings of bispecific antibodies are known in such art.

0 in normal conventional form; The production of genetically engineered bispecific bodies is based on the expression of the common genotype of the ALE chain - light chain pairs of immunoglobulin ¢ where the two heavy chains have different qualities [Millstein and Cuello, .Nature, 305: 537 -539 (1983)] Due to the random assortment of the heavy and light chains of immunoglobulin, these hybrid tumors

Hybridomas 0 ('quadroma') produce a potential mixture of (Vr) different antibody molecules; of which only one has the correct bispecific structure. The process of purifying the correct molecule; which is usually performed By carrying out the steps of affinity and affinity chromatography, it is troublesome and cumbersome to the point of cle and the yield of the product is low. Similar procedures have also been revealed in the International Patent Application (PCT) No. 93/08829 (Wo

YAY - - (published May 1, 1999 AD); As disclosed in [Traunecker et al, EMBO, ](1991) 10:3655-3659. According to different and preferred methods in the “JST” image, variable domains of the antibody are combined with the desired binding types (“antibody” binding sites). antigen”) with sequences of a stable M domain of immunoglobulin. It is preferable to combine with a constant domain of the ALE chain of immunoglobulin that includes at least part of the hinge (the connecting part ); of the two regions (of both groups) (CH2 0113. It is preferable to have the first ALE chain-binding region (CHI) containing the necessary light chain binding site present in at least one of the fusions. Jay) the nucleic acids (DNAs) encoded 0 fusions the immunoglobulin heavy chain “immunoglobulin” and as desired Lad can be inserted into the light chain of the immunoglobulinde lil in separate vectors expressing the genotype; Vectors are cotransfected into & yids of a suitable host microorganism. This provides great flexibility in adjusting the mutual ratios of the ADEN polypeptide moieties in models when there are unequal ratios of the three polypeptide 0 chains used in a composition that yields optimal yields. It is anyway; Sequences encoding two polypeptide chains of all three polypeptide chains may be inserted into a single genotype-expressing vector when the expression of the genotype of at least two peptide chains is equal to the ratios resulting from high yields or when These proportions are not particularly significant. In a preferred embodiment of this method; The bispecific antibodies consist of a heavy chain hybridized to an immunoglobulin with a primary binding type in one arm; A hybrid pair of the ALE Series Light Series L

YAN — immunoglobulin (with a second binding type provided) in the other arm. It was found that this asymmetric Jot structure dissociates the desired and desirable bispecific complex from the unwanted immunoglobulin chain conjugates; Since the immunoglobulin light chain is present in only one half of the bispecific molecule the method provides facilitation of separation. This method has been revealed in a patent application that has not yet been decided and bears a serial number (931/07, 811); Filed VV August 39)(a) (EP No. “656064”). For more details regarding the production of bi-specialized bodies; See for example; -[Suresh et al, Methods in Enzymology, 121: 210 (1986)] (v) Heteroconjugate antibodies heteroconjugated.

0 The heterogeneously bound Loaf antibodies fall within the scope of the present invention. Heteroconjugated antibodies consist of two covalently linked antibodies. These antibodies, for example, have been proposed to target cells of the immune system to convert to unwanted LA (US Patent No. 4, 676, 980 (US.

Patent No. and for the treatment of HIV infection [PCT No.

WO 91/00360(WO 92/00373) and EP No. 03089[(EP). Heteroconjugated antibodies may be made heterologously using suitable cross-linking methods. Suitable cross-linking agents are well known in Such art has been disclosed in US Patent No. 4, 676, 980 (U.S.

Patent No. spans a number of the many technical methods of cross-linking (cross-linking).

- 1/9 —

Therapeutic Use of the mpl ligand - MK WA megakaryocytopoietic, which is referred to here as an inducing protein to build cells, in the preparation of a sterile pharmaceutical preparation or in a formula (TPO) to build platelets 0 or generate platelets in a sterile pharmaceutical MK LAN for excitation and induction thrombocytopenia in patients with increased platelet insufficiency or damage. The lack or lack of poor sequestration »; or to platelet aplasia of the bone marrow associated with thrombocytopenia (eg, slow-growing tissue hypoplasia as a result of chemotherapy or bone marrow transplantation) it may be treated as aplastic anemia) ie can be used to treat disorders; Loaf efficacious using compounds of this invention as well as thrombocytopenia disseminated intravascular coagulation (DIC) diffuse angiogenesis Jala induced thrombosis B (317) (ITP) (including immune tnrombocytopenia specific blood Idiopathic thrombocytopenia 0¢, Jia congenital at birth; thrombotic thrombocytopenia In addition, such megakaryocytopoietic proteins may be useful in the treatment of myeloproliferative HLS as well as in the treatment of thrombocytosis© resulting from inflammatory conditions. and iron deficiency.

Yq. -- The preferred uses of the MK proliferation-inducing protein or the clot-forming or platelet-producing protein (TPO) of this invention are in conjunction with myelotoxic chemotherapy; myeloablative chemotherapy; where platelet oat is attributed to bone marrow failure 0 and there are still other disorders that are useful to be treated using the MK cells of this invention; Where such disorders include deficiency or damage to blood platelets resulting from taking drugs; Poisoning or activation on artificial surfaces. in these cases; Existing compounds may be used to induce the isolation or shedding of new platelets that have not yet been damaged or undamaged. For more on the Get a larger, more complete list of useful applications in this area; See 0 (0); As well as the references made and to (2) and, especially the sections from the “Background” supra

La la £3 of the present invention in the form of MK may be used to induce proliferation of proteins for other cells of cells; or with hematogens; with cytokines singly or in combination with 1 interleukin; or with sai factors or with antibodies; This is in the treatment of the aforementioned disorders and pathological conditions. So; The present compounds may be used in combination with Protein 351 or another peptide with thrombopoietic or platelet-forming activity including: IL-1 1-087 “LIF” GM-CSF “G-CSF 11-3 ; Erythrocyte component <erythropoietin (EPO)

191- The proteins inducing proliferation of MK cells of the present invention are prepared in the form of a mixture with a pharmaceutically acceptable carrier. The combination medicine can be given by injection into a vein, taken through the nose, or through the lung. The formulation can also be administered parenterally or by subcutaneous injection if desired. and when taken 0 regularly; The therapeutic composition shall be pyrogen-free and in the form of an acceptable solution for injection of an appropriate pH; With an “isotonic” concentration and a high degree of stability. Such conditions are known to those skilled and experienced in Jie in this field of this art. In short; Dosage compositions of the compounds of the present invention for the purposes of storage or hydrolysis are prepared by mixing the compound of the required degree of purity with carrier materials; Or excipients, or stabilizers, all of which are physiologically acceptable. Jia these materials are non-toxic to patients receiving treatment and this is for doses and concentrations A is used; and comprising buffer solutions such as phosphate salts, citrate, cacetate and other organic acid salts; It also includes antioxidants such as “ascorbic acid” and peptides with low molecular weights (less than about ten eo building units) such as cpolyarginine, as well as proteins; serum gelatin i « albumin; Immunoglobulins ¢ and also include hydrophilic polymers polyvinylpyrrolidinone J—is ¢ and also contain amino acids such as glycine, glutamic acid, or aspartic acid, or arginine; It also includes disaccharides “monosaccharides ¢ as well as [La] carbohydrates Y. Others including cellulose or “ili arr glucose” or mannose “or derivatives” as well as factors chelating agents (EDTA) Jia chelating agents as well as alcohols

Vay - - sugary mannitol Jie sugar alcohols or sobitol ; So too are counterions or reverse ions such as ssodium / or non-ionic 5 surfactants Je material bearing the brand polyethyleneglycol si Pluronics™ “Tween™”. (10) milligrams to (004) milligrams of a compound or mixture of the protein that induces the proliferation of MK cells in the form of free acid or sal free base in the form of a pharmaceutically acceptable salt, with a carrier; carrier; palatability; binder; sale preservative 068080078 stabilizer; flavoring, etc., all physiologically acceptable as required or required by acceptable pharmaceutical practice. The quantity of the active ingredient in such formulations is such that the appropriate dose is present within the range indicated. Sterile formulations for injection purposes may be formed or formed in accordance with customary conventional pharmaceutical practices. For example, it may be desirable to Dissolving the active compound or making a suspension of it in a carrier such as water or in a naturally occurring (naturally) vegetable oil such as sesame oil; or peanut oil; or cottonseed oil 0 ; or to be done in Jil synthetic fatty ethyl oleate Jie; or something like that. Buffer solutions can be combined; preservatives; and antioxidant agents (substances) according to acceptable pharmaceutical practices. Suitable examples of sustained-release formulations include semipermeable matrices dite of solid hydrophobic polymers | 70 water containing the polypeptide + which are the tissues that are in

Yay - - image of textured materials; Such as thin films or microcapsules. Examples of intercellular tissues from which the active substance shy is released include WJ Ad Jw Le] hydrogels (poly(2-hydroxyethylmethacrylate) as described by [Langer et al, J.

Biomed. H. [(1981) (167-277) 15: Matter.

Res., ¢ ](1982) (96-105) : 12 Mek 1 ¢ [Langer, Chem, or [poly(vinylalcohol) as including se)je] polylactides (US.

Patent No. 3, 773, 919) and European Patent No. 58, 481 (EP) and copolymers of “L-glutamic acid and Sidman et al,] gamma ethyl-L-glutamate ethylene-vinyl acetate” [Biopolymers, 22: (547-556) (1983) that are not biologically or chemically soluble 0’) (Langer et al, supra) and copolymers of lactic and acidglycolic acid that are biologically or chemically soluble Such as those branded “Lupron Depot™” (injectable microspheres composed of copolymers of lactic acid and “(leuprolide acetate”) glycolic acid (poly-D (-)- 3-hydroxybutyric acid (EP No. 133, 988)

1 Although polymers such as ethylene-vinyl acetate and copolymers of 80116 and glycolic acid are able to release molecules over class (001), there are hydrogels that release proteins over extended periods of time. When the encapsulated proteins remain in the body for a period of time, they may lose their nature or may accumulate as a result of exposure to moisture at a temperature (27 "C); Lam 5033) to their loss of biological activity with

x. Possible changes in the production of immunogens. Rationale strategies for protein stabilization can be devised depending on which mechanism is involved eg;

- Vag is an aggregation if the mechanism of intermolecular aggregation (58-9) is detected by an internal change 01901506; Stabilization may be achieved by adjusting lyophilizing solutions and freeze-drying sulfhydryl residues of the 8 acidic residues; with control of moisture content using appropriate adjuvants; With the development of the polymer matrix formulations of the tissue material for a specific polymer that slowly releases the active substance on MK WAY, it also includes formulations of the protein inducing proliferation. Fat bodies containing the cell proliferation-inducing protein 106 by methods known in [Epstein et al, Proc. Natl. Acad. Sci. ¢ (DE 3, 218, 121) Same as German Patent No. aa [Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-¢ USA, 82: 3688-3692 (1985)]0

EP) «(EP 88, 046) «(EP 36, 676) «(EP 52, 322) and EP No. 4034 (1980)]

Japanese patent application 83-) (EP 142, 641) (143, 949 and American Patent No. 4, 485, 045); (4, 544, 545) and European Patent No. 118008 unilamellar (In a normal way, the lipid bodies are of the monolayer type (EP 102, 324) (the content of which is 208870375 (Ar) to ) 001) The small thin (ranging from about ys al aly adjusting the selected part pig ¢ cholestoro lipid greater than about (770) mol of the ideal. The specialist doctor in this regard, taking into account several factors known to modify the action and effects of drugs, and these factors include the degree of severity of the disease being treated and its type; the weight and gender of the patient being treated, his diet and the period - ©

1018 - — time of drug administration, route of administration ¢, other drugs taken, and other relevant clinical factors. Typically; The daily dose rate regimen administered will be within the range of 1,001 - 1.1 μg/kg body weight. It is preferred that the dose be within a range of 11 µg/kg to 0 (00) µg/kg of body weight. It is more desirable that the dose be within a range of 1 µg/kg. kg to µg/kg/day Optionally, the dose range will be the same for other cytokines, in particular (GM-CSF) (G-CSF) (EPO) and effective doses may be set Uf Ladle by methods performed in laboratory experiments in test tubes or in vivo.

0 Examples without subtracting more from (Coal) It is believed that any ordinary practitioner with skill and experience in such a field of this art can use the foregoing description and illustrative examples; and to carry out the work of the present invention and implement and use it to the fullest extent Therefore, the following examples of work qualitatively illustrate the preferred embodiments of the present invention;

yo is intended to be in some way a limitation of the remainder of the invention; Rather, it has been presented as an example but not limited to.

Va — - Example No. (1) Partial purification of the ligand (mpl) in pigs. Plasma of low platelets was collected from pigs with normal or inactive anemia 16. Pigs were rendered inactive by exposing them to radiation. irradiation by (900 cGy)© of the total radiation exposed to the body using a linear accelerator (£) million electron volts (AMEV) supported pigs that were irradiated by intramuscular injection with cefazolin for a period of (6) to (A) days, then her total blood was removed after being subjected to hepannized total anesthesia, then subjected to a centrifugal force at a rate of 18060 p for a period of (01) cis to obtain platelet-weak plasma It has been found that the excitatory activity of the MK cell reaches its peak after (6) days of exposure to radiation. By adding NaCl at a concentration; molar; Then stirred for 70 minutes at room temperature. Then the precipitate is removed by centrifugation at a rotational speed of 7800 rpm in a device of the type bearing the brand “Sorvall RC 3B™” and then the supernatant is loaded onto a 100 ml daw column. (Phenyl-Toyopearl) was neutralized in 0 mM of .11800 containing ; Molar [2180]. Then the column was washed with this buffer solution until the absorbance became (Aggy > 1,00) and then an elution wash was performed in 0:011. Then the filtered protein peak was eluted with 0:011 diluted to where the electrical conductivity became conductivity 15 mS) then loading was carried out over a (Blue- Sepharose) 00 column with a capacity of 7560 liters equilibrated in PBS and thus; the column was washed by ©

vay - - volumes of column volume in PBS and 10 mmol 108007 pH = 4 containing ? Molar of urea, then proteins were eluted from the column using 01 mM MNaPO, pH = 7.4) containing ¥ MM Veurea M of NaCl. Peak filtered protein elutriated using 1 ¢ octyl glucoside(n-octyl -D-glucopyranoside) (7+, + ) 0 mM in both EDTA and Pefabloc, brand (™) (Bochinger Mannheim) then loaded directly over (with,0) linked hierarchically [(1989) 525-531: 337 Capon DJ. et al, Nature and (Pierce) mpl-lgG Ultralink columns (see below) Lad after).Then a column — (004-186) with a capacity of ?ml was lifted and removed after the sample was loaded; then the column (mpl-IgG) with a capacity of 0ml was washed by 01. Volumes of column volume from both (PBS) and each of (PBS) contained ?molar of “NaCl”, then eluting was performed using 11molar of glycine hydrochlorcle (pH) = 1,75). The parts were assembled into one tenth of a volume of 1 molar Tris-HCl at pH 11M - 8.60. (ad)ela elution of mpl-affinity column 1 was analyzed by using (Novex gel 771 — £) SDS-PAGA under reducing conditions; Or stop having too many proteins (Fig. (*)). The proteins that were highly stained with silver or with a very high density were analyzed as having molecular weights (Mr) of (15000); (28,000); (Faas) (18000); (VE nn) to designate what these proteins are; These proteins were filtered out from the gel as described in Example No. (1) below.

198 — Ultra link Affinity Columns 0.1 to 0.1 mg of Simpl-IgG 004-186 combined with © grams of (Pierce) Ultralink resin as described la according to the manufacturer's instructions. Construction and Expression of mpl- (mpl-IgG) ©1860 “0” to “491”) and the (Fc) region of human “861” in 797 cells. The cDNA fragment encoding amino acids “0” to “91?” of ligand mpl was obtained. Cua macrocytes by (PCR) derived from the cDNA array of pure (CMK) cells morphed into a 0 -_ sequence. end of tip 5 and insertion of site (Bst Eli) at end of tip 3. This Lud fragment was then cloned before the IgGl Fe-encoded region in a Bluescript vector between the two sites (Bst Eli) ¢ ( Clal is located in the DNA coding for the exocellular domain of the mpl ligand. Fc) frame with extracellular domain of the ligand count (mpl) A subcloning of the structure was performed in JU bound by the Lad Pris- ligand between the two sites (its©); (Xbal) then transferred by infection into human genetic kidney cells by the method of calcium phosphate. Cells were selected at (+) mg/ml (6418) and individual clones were isolated or separated. Expression of the genotype (mpl-IgG) was determined from the glje clones using the ELISA method. The specificity of human (Fc) is for cloning

1099 - - the best expressing the genotype Expression level of the genotype at a rate of (1) to (Y) mg / ml of (mpl-IgG) LDA Expression of the genotype of ( Ba/F3 mpl P ai) (cdna) corresponding to the entire coding region of the human (mpl P) ligand was cloned into 0 ligand (PrkS-tkneo) which was thus linearized with (Notl) were then transfected into the IL-3 cell line (Ba/F3) by forming TY “1” electrodeposits (cell; 0 uV). And after three days have passed; The test was initiated in the presence of (Y) μg/mL of (6418). Cells were selected as groups of clones or as individual clones obtained by limiting or restriction dilution in 0 dishes - each with an A cavity. Selected or selected cells were maintained in RPMI containing (0 (1) (1) FBS “mg/mL of (6418); (01) mmol of glutamine” (01)). mmol of 0 ¢ (HEPES) μg/mL from (m060-508). The expression of the ligand genotype (mpl P) present in the selected clones was determined by dal gy analysis ( FACS) using a rabbit anti-ligand polyclonal (mpl P) antibody.

(Ba/F3 mpl) ligand test 0 of (mpl) to determine the presence of ligand L(Y) as shown in Figure No. (mpl) A ligand test was performed for 4 hours at a density of Intracellular IL-3 (mpl P Ba/F3) Many sources; The cells were euthanized in an atmosphere of humidified oP dioxide at a land temperature of (0 x 01) carbon dioxide at a concentration of 5 and air. After euthanizing the 1-3 cells, the cells were placed in culture media in microliters of culture medium, 100 cells per 00,000 lumen with a density of AT in 2 “dishes each”.

_ You in cell culture medium fash 1 hour in YE with or without diluted samples where the culture was done for a period free of blood serum; The content of (RPMI) 71 microliters of culture medium was then added to each of these cavities in the hours from (CH - µ©l (thymidine 1) on the last six to the last eight of the process After that, a toll was collected and then washed several times with water. (GF/C) cells were counted for 40 cavities of 0 filter dishes and filters were counted in the presence of 4 μL of a fluorescent fluid acting as Flash (Packard Top Counter ™) in a counter of the type bearing the trademark (Microscint 20 Example No. (?) thigly purified porcine mpl ligand of high purity mpl ligand Gel elution protocol Gel elutriation system 0 purified alimentarily (mpl) vid'm Equal amounts of 2XLaemmli and buffer solution "mpl-IgG" elutriated were mixed From the “+” column at room temperature and without reducing agent, then as quickly as possible load the resulting mixture onto a *©07 gel. The sample has not been heated. (7701) with a concentration ranging from (?)) to polyacrylamide for resistance; the buffer solution of the sample was passed without a ligand down an adjacent lane. The defrosting was done - 1 hour_approx. It was (Y,Y0) for volt 1 Fo to 1 m at TE at a temperature of all at room temperature. After that, the running buffer was removed, the temperature of the working buffer solution was removed. from the gel box; Then remove the plate from one side of the gel

11- — Then make a replica (imprint) of the gel on nitro cellulise as follows: then wet a piece of "nitro cellulose" with hia ¢ water and then carefully place it on the surface of the exposed gel in order to eliminate Rising air bubbles. Then put reference marks on the nitro cellulise and on the plate of the mount in order to reposition the exact copy correctly after the dye and after about two minutes have passed; 0 nitro cellulise has been carefully removed; The gel was then wrapped in a flexible wrap in plastic wrap and placed in a refrigerator or cooler. The nitro cellulise was stained with Biorad's gald total prorein stain by first shaking it in (© *) 01 ml at a concentration (0.1/) of 20 Tween + 0, + M Tris- HCI +,) + 0.5 M NaCl 0.1 at pH = pH 7.9 over a period of time (0£) minutes; then this was followed by washing with pure water (¥) times by (01) .. 0 ml each time (*01 ml) over a period of (0) minutes. Then cad dyes were added and left to develop until bands or standard colored spots appeared in the compared Ase. And it became visible.Then, the replica (print) was rinsed with water, then it was placed on top of the plastic wrap over the gel and very carefully aligned with the reference marks.The positions of the Novex comparison sample were marked on top of the gel dish, then the lines were placed To indicate the places of the cuts, Aes 1 removed the nitro cellulise and the plastic wrap, then cut the gel along the lines shown with the blade of a sharp blade. After the etl pall was removed the remaining gel was made with silver. The places of the compared sample and the places of the cut were measured. The partial weights corresponding to the cutting positions were determined through the comparison sample (08). The (VY) gel strips were placed inside the cells in model 422 electroeluters 510:80 elliptical filters. In cells, specific membrane coverings have been used to weigh on

- 117 -

The molecule coming out of (K) 2 should not exceed (12K) [i.e. from 0001(HOY).] The buffer solution was (+0) mM ammonium bicarbonate +

9058 (approximately pH = 7.8). Also, (1) liter of the solution was cooled

the regulator in for an hour in a cool room temperature of ; M to oT before it is done

0 usage. Then elutriation of the gel strips was carried out at fma Vv cell rate (initially 0 volts) and in a cold room with a temperature ranging from −°C to +°C. The sequencing took about; hours. shrewdness The cells were carefully removed and the liquid above the ferrite was removed

+ using a pipette. after that; The after-filter has been removed and any other liquid present

Cover over the membrane using a pipette. The liquid Aj was then drawn as a pall into the membrane cover

0 using a pipette or pipetman and then kept. Then 50 µL of complete and equal volumes of pure water were placed in the hood; then it was shaken; It was then removed until the SDS crystals had completely dissolved. The lye was collected together with the liquid held before. lhave

The volume of the total eluate sample was in the range of (Fr) (000) μL per gel slide. The samples were placed in a 10 mm dialysis tube: 50600800 of limited type B.

1 12-14 These tubes have been soaked for several hours in pure water. The dialysis was carried out overnight at a temperature of 4°C to 6°C against Tov ml of phosphate-saturated saline (PBS is approximately mmol in potassium) for each samples. The solution was replaced Then the dialysis process continued for (0.3) hours. (pany) The samples were removed from the dialysis bags and placed in small centrifuge tubes mlcrofuged. © The tubes were placed on ice for (1) hour; then centrifuged by centrifugal force at a rotational speed of “141” rpm (10001) revolutions per minute for “minutes” then the floats were carefully removed from the

117 - The (505) that was deposited. after that; The supernatants were placed on ice for approximately over an hour and then centrifuged again for (£) minutes. Thereafter, the supernatants were diluted in phosphate-buffered saline; the result was then used for an activity and potency test. They were frozen and frozen. The remaining samples at a temperature (- (pV) m_Example No. (¥) Performing the process of sequencing the flour (mpl) gvfd'm of Porcine mpl Ligand Microsequencing, then concentration (Y,1) ml of the fraction Fraction No. 1 taken from the affinity column (mpl-IgG) on a Microcon-10 01 with the brand name (Amicon™). To prevent the ligand (mpl) from adsorbing to the microcon; eluate sl Gal) by 80571; then © 0 µL of solution 701 505 was added to fraction No. 1. Subsequently, twice as much volume of 0 µL of sample buffer (do 01) was added to fraction No. 1 XY) after the microconcentration became μl of Yo and then the volume of SH£0 μl was loaded on a single pass with a ratio of 4 to 771-(Novex) acrylamide gel, then gelation was performed following the system Novex After that, the gel was neutralized for (0) minutes; before conducting electrosorption electroblotting of 0 in 0 mmol buffer solution of 3-(cyclohexylamino)-1-propanesulfonic cacid (CAPS) (pH = 1011); containing + 7) methanol electrophoresis was carried out on (Millipore) Immobilon-PSQ membranes for £0 min at 148 mA constant current in a Bio Rad Trans-Blot cell (battery). © . then stain the film (PVDF) with Coomasie Blue R-250 70.1 dissolved in t+ 1 acetic acid 700 1 methanol 00 for 1 min; Then the dye was removed for ¥ - ¥ min using

Yet _ — 0 acetic nm dissolved in 79 0 methanol The only proteins visible in the molecular weight range were: 1" in a region between (ga (Youve) VA) proteins with Mr with values of 011 kDa. The bands slices (kDa (YY YA os) were subjected to protein sequence work. SDoma) or sequence work was carried out automatically on model (470A) of the device Applied Biosystem sequencer equipped with a “PTH” analyzer unit on the same operating line. The sequencer has been modified to inject from 7/80 to 790 of the sample [Rodriguez: 350: 17-225 (17-225) 70] of the sample. , J.

Chromatogr, Acetone (by -VY μmol/L) was added from solvent “A” to achieve UV absorbance equilibrium. The electrolytically adsorbed proteins were placed sequentially into a 0 printer cartridge 3101 and integration of the Peaks was then performed using Justice Innovation software using Nelson Analytical 970 interfaces. Sequence interpretation has been performed on a 5900 [Henzel et al., J.

Chromatogr., 404: (4 1-52) (1987)] “VAX sequences at the amino terminus of a protein (N ~terminal) (using a monoletter for the amino acid with unconfirmed residues in parentheses) is also indicated as the amount of substance that Obtained (in brackets) 1 _ in Table No. (Y).

#7015 _ — table (Y) of the N —terminal amino-terminal sequences of ligand mpl kDa 1.8 pmol (SEQIDNO:30) 30 : 1 ° "1 Yo Yo Yo (SPAPPA) (CDPRLLNKLLRDD (H/S)VLH (GR L kDa 0.5 pmol (SEQIDNO:31) 28 (S)PAPPAXDPRLLNKLLRDDHVLHGR kDa 0.5 pmol (SEQIDNO:32) 18-22 XPAPPAXDPRLX(N)(K) Example No. (2) Testing a liquid suspension proliferating in MK cells generated with Liquid Suspension Megakaryocytopoiesis Assay, then diluting human peripheral stem cells (PSC) (obtained with patient consent) with an orbital (0) times to medium from (Gibeo) “IMDM” and then subjected to centrifugal force for (10) minutes at room temperature at (2 Ava) rotational force. Then a suspension of mole precipitate was reconstituted in the ( IMDM) 5 and applied as a layer over 0 of the compound 17/01 GES, “Percoll™” g/l (Pharmacia) and then coated with (8 800%) for 0 minutes. Then the mononuclear cells were withdrawn a light density at the interface and then washed Ob ya with medium (IMDM) and placed on plate surfaces at a rate ranging from (Ys XV) to (10 x X) cells/mL in medium (IMDM) Content On “FBS” Ys (until final volume is “0” ml) in Vida wl culture medium groups a cavity of commercial (Costar) was added (APP) or ligand (mpl) depletea depletea of

11 - - (APP) to 01, then the culture media (farms) were left to grow for a period of 11 days to Lap VE in a wet nursery at a temperature of (77 +) and in an atmosphere of (70). Carbon dioxide (60:) and air.The culture media were also left to grow in the presence of (APP 700) with 16 μg of (mpl-IgG) added on the first, second and fourth day. of 0 mpl ligand by APP (je) through affinity column 0 mpl-IgG to quantify platelet-forming MK cells in liquid suspension culture media; a modification (Solberg et al. al.) with the use of a radiolabeled mouse antibody (IgG) monoclone (HP1-1D) of pais (GPILIIL) from Dr.

Nichols, Mayo Clinic and then radioactive tattoos of 100 micrograms of (HP1-1D) [see Grant, B. et al, [Blood 69: (1334-1339) (1987) 0 with:10" of saturated Nal'™ using (Biorad, Richmond, CA) Enzymobeads as described in accordance with the manufacturer's instructions. Radioactive (HPI-ID) has been stored at temperature = 17 C in (PBS) containing 0 7 octyl-glucoside The typical specific activities were Le: specific activities ranging from (01 XY) to (TV eX Y) / microgram (more than 1425 precipitation by trichloroacetic acid 0 with a concentration of 0.0 Y) (7) The liquid suspension culture media (cultivators) were prepared and prepared in three triplicates for each experimental point. After a light ranging from 11 days to VE days in the culture medium (the culture)”; the 1 ml cultures were transferred into (eppendorf) tubes of 0.5 da capacity, then centrifuged by (x Ae) p ) for 10 minutes at room temperature; then the resulting cell precipitate © was resuspended in 100 μl of PBS containing 70.07 UY (EDTA) of serum

Bovine calf serum LLY! Then 10 ng of (1]110110"!) dissolved in ov 1 μl of buffer were added to the suspended cells and then incubated for 10 minutes at room temperature with occasional shaking and stirring. Cells were collected by centrifugal force (g X Ave) for 10 minutes at room temperature, then washed with Ge using buffer to perform the test.Irradiation was counted for 1 minute on a gamma counter Lela (Packard type) Non-specific radiation was determined by the addition of 1 μg of non-radioactive (HP1-1D) and for 1 4883 01 before (HP1-1D) was added Then the specific radioactive binding was set as the total binding (1101-10”*) minus that binding in the case of an excess of non-radioactive (HP1-ID). 0 _ Example No. (5) prefixes Oligonucleotide PCR Primers Oligonucleotide PCR Based on the amino acid sequences at the amino-terminal protein and the values obtained from proteins with k Da 28 k Da 30 k Da 18-22 degenerate oligonucleotides were determined For use as primers for the PCR reaction (see yo Table 4). Two sets of primers were manufactured; One of them is mer 01 positive sense of the group encoding the amino acid residues ¥ - mpl 1A and the second is anti-sense mer 71 antl-sense of a group complementary to the sequences encoding amino acids (mpl 2) 7 4 -18

YA - Table No. (2) Paddy oligonucleotide groups Soluble by 01448 times 3 mpl 1:5 CCN GCN CCN CCN GCN TGY GA Soluble by 44 mp12:5' NCC RTG NAR NACRTGRTCRTC 3' 3 3a 01 Porcine genomic DNA isolated from porcine peripheral blood lymphocytes was used as a base for the 008 construct. The reaction contained 1 μL of A + μg of acid. Porcine genomic DNA in 0 mM —Tris M HCL (pH 005 mM (AY) 1601, mM:1480, 100 μg/ml BSA £9 44 μM ANTP , and 1 micromolar of each group of primers, Vos, a unit of the Taq polymerase enzyme, fads denaturation at 86 degrees Asada minutes, followed by Yo, a cycle of £0 seconds each at 14 °C and 1 minute at 00 °C and 1 minute at VY C. The final cycle was allowed to be extended for 10 minutes at 7 °C.0 PCR products were separated by electrophoresis on polyacrylamide gel 11 and were Showing it for visibility by staining it with ethidium bromide It is estimated that if the amino acid sequence on the amino terminus of the protein is encoded by a single exon, the correct PCR product is expected to be 14 bp sac ld and the third part is extracted DNA of this size from the gel and then cloned into -(promega) pGEMT, then clarified with Vall, clones in List No. ( ).

(5) Table of porcine genomic number with length of 1 base pair DNA fragments from

GemT3 S'CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC

TGCCTCGTGA 3GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG

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

ACTGGTGCAA GTCGTGCCG (SEQ ID NO: 38) gemT7 5'CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC

TGCTTCGTGA 30010010046 GCCGTACACT GGGGGCTGAG GATRATTITG

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

GCTGGTGCAG GTAGTGCCG (SEQ ID NO: 40) gemT9P R LL NK L L R (SEQID NO: 32) 5'CCAGCACCGCCGGCATGTGACCCCCGACTCCTAAATAAACTGCTTCGTGACG 3'

GGTCGTGGCGGCCGTACACTGGGGGCTGAGGATTTATTTGACGAAGCACTGC

ATCATGTCTATCAGGT 3' (SEQ ID NO: 41)

TAGTACAGATAGTGCCA 5' (SEQ ID NO: 42)

701 - The position of the POR prefixes is indicated by the underlined rules. These results verify the N-terminal sequence obtained for amino acids 4 - 17 for the 01 kDa, YA -18 kDa and YY -18 kDa proteins and show that these sequences are encoded by single exon of porcine DNA. 0 Example No. (1):

Human mpl Ligand Gene

Based on the results of example (0). A 45-mer ¢ deoxyoligonucleotide construct called PRY was designed to browse the genomic library. The p-45 sequence had the following:

5 GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG 3

YA: (sequence ID number 0) kinase was used to assay Ty stained with m2 (stm)-(m) and this oligonucleotide enzyme induces slack hybridization conditions + gem 12 in a human genomic A human genome was collected and analyzed by plot maps with plaque (see Example No.). Positive clones were captured, the plaque was purified, and clone No. was selected for additional analysis. southern blotting analysis

1 A 7,4 lf base clone of BamHI-Xbal fragments hybridized to:45-006 was transformed into .p.Bluescript SK. DNA sequencing of this clone was determined using an oligonucleotide. Lala with the DNA sequence of a pig mpl ligand as a primer. The obtained sequence proved that the DNA encoding the human-like ligament of the pig mpl was then led je. And then the cutoff position is detected

7110 - - b for 2003 in the sequence, allowing us to isolate a fragment of 180 pairs of EcoRI-Xbal sxc from Y,A base from Bamltl-Xbal, as well as re-cloning them in -pBluescript SK. The sequence of the two sequences was studied for this part. The human DNA sequence and inferred amino acid sequence are shown in Figure (4) (sequence ID numbers ' and 4). Predicted locations 0 of 1010008 in the genomic sequence are also indicated by arrows; It specifies the putative exon (© exon). eu predicted amino acid sequence tests that the first amino acid of the mature mpl ligand is senne ; This was also determined from direct analysis of amino acid identity. Immediately prior to this codon, the predicted amino acid sequence is highly suggestive of a sequence that plays a signaling role in mature mpl ligand secretion. It is possible that a coding region of the signal sequence does not communicate with an intron at the A nucleotide position. The exon appears to end at 197 nucleotides. So that exon encodes a sequence ?; Amino acid; 1% of them are likely to be part of a signal sequence and 17 of them are part of the mature human mpl ligand. Example No. (v): cDNA 0 of a Full Length Human mpl Ligand cDNA Based on the “human” exon sequence (Example 1) two non-dissolving oligonucleotides were synthesized degenerate corresponding to the v and © ends of ¥oexon (Table + ( .

711 - - Table No. (7) Non-dissolving Oligonucleotid Primers for 008 L. in Human cDNA Human cDNA Non-degenerate PCR Oligonucleotid Primers (No. ia) Forward prefix: GCT AGC TCT AGA AAT TGC TCC TCG TGG '5 sequence : 5 ) | TCATGC TTC T3' (reverse prefix ID: '3 CAG TCT GCC 616 AAG GAC ATG G 5 sequence : 44 ) These two primers were used in DNA—S308 reactions wall PCR data from different human DNA kits or 1 ng of cDNA Quick Clone (Clonetech) from different tissues using the conditions described in Example 8. The size was 0 expected. For the correct PCR product Yeo pair bp sac ld and after analyzing the PCR products on 11 7 polyacrylamide gel ¢ a DNA molecule of the expected size was detected in the cDNA groups prepared from the kidney of an adult animal 797 fetal kidney cell DNA y prepared from roasted fetal liver (Clonetech cat. # 7171-1). IX) - 0 5 with the same 45mer oligonucleotide used in the human genomic assembly test. The oligonucleotide was tattooed with (y?P)~ATP using polynucleotide kinase Ty.

710” - 0 and the batch was tested under loose-breeding conditions. The filtrate was pre-hybridized for 2 hours and then hybridized using the probe along Jl at EY °C in 100 10xDenhardt's y SXSSC 5 formamide 5 0 + , + sodium phosphate (m pH ( f ) + sodium pyrophosphate 5 +© μg/ml DNA from Cla sally© sonicated salmon sperm for 11 hours. (registered tag) overnight.Positive clones were captured;plaque purified;the insert ligand size was determined by PCR using an oligonucleotide to surround flanking BamH1-Xbal clone in cat.#6475- 1) ADR; 010061600). A © microliter from the phage virus stock was used as an information source. The first sal was 0 for a duration of 0 minutes at 1 m and was followed by a cycle of Vo (1 minute at m, 1 minute at 0 m, 0 min at VY m). The final extension was for 11 minutes at 77 °C, and clone No. FL2b had an insert of 0.8 kilobase and was selected for analysis. el Install Clonetech.

ACDR; & pDR2 cloning and Expression System) pDR2 se Dll 42 m (Library Protocol Handbook, Content Jala virus arms ¢ACDR, phage as Vo described by Manufacturer Clonetech.

CDR, & pDR2 cloning and Expression) (System Library Protocol Handbook, p29-30). The plasmid — restriction analyzes of Pdr2-FL2b using BamHI and Xbal indicated the presence of an endogenous 380111 cutoff site in The inserter at the +0 site. Digestion of plasmid B 380111-58 cleaved the inserter into two parts, one with M0 +1000 bases and one with 1,10000 bases. The template is derived from the work of the pDR2-FL2b plasmid with three different classes of information bases. The DNA sequence of a double-stranded plasmid was studied using a method.

71 - Automated fluorescent DNA sequencing (Applied Biosystems, Foster AB1373 automated fluorescent City.

California) using standard protocols for dye-stained dideoxy nucleoside terminators (triphosphate trimers) and normal synthetic walking primers (Sanger et al, Proc.

Natl Academy.

Sci.

USA, 74:5463-5467 [1977]: smith et al., Nature, 0 ) [1986] 321:674-676). The PCR-increased fragments were directly sequenced from 0 using the 1373 AB sequencing medium using normal primers and dye termination reactions. A single stranded database was produced using the M13 Janus vector (DNASTAR.

Inc., Madison, Wisconsin) (Burland et al., Nucl.

Acids Res. , 21:3385-3390 BamH1-Xbal (1.15 kb) and BamHI (0.65kb) )[1993]« Le—1je was reported on pDR2- plasmid FL2b 0 and the ends were filled with polymerase 14 DNA in the presence of deoxynucleotldes and then cloned into the -MI13 Janus locus—! Smal sequencing was performed using standard protocols for 1,113 universal primers, reverse primers, mobile primers, and dye terminators. Manual sequencing reactions were performed on M13 DNA single sequences using mobile primers and terminal termination chemistry with a standard Proc.

Natl.

Acad.

Sci. has Sanger et (USA, 74:5463-5467 [1977] 0 with 337 at a-dATP-site and coenzyme 86 (trademark of Biochemical Corporation USA.; Cleveland; Ohio). United (States Biochemical Corp., Cleveland.

Ohio and the DNA sequence sequence was assembled using -Sequencher V2.1b12 (Gene Codes Corporation, Ann Arbor, Michigan) and the nucleotide was provided and the inferred sequences AML are shown in Figure (1) The sequence: 1

#1 - - Example No. (A) Isolation of the Human mpl Ligand (TPO) Gene (TPO) J je Human mpl ligand (TPO) gene clones were cloned gene by reviewing the assay of a genome set in 1-08012 with the previously described 5 nucleotide oligo-probe under relaxed conditions (see example oF or inducing extreme conditions with a molecular homologue to the 7 half-terminus of the human cDNA codon for the mpl ligand). From the cut position of BamHI to terminal 0 (v), two overlapping synonymous A-shells spanning Wo spanning a thousand bases were isolated. The sequence of the two synonymous fragments (Eco R15 BamHi) containing along TPO The structure of the human gene includes exons 1 within 7,000 bases of genomic DNA (Fig. generated for mammalian genes. (Shopiro, .M.

B.. nucleus.

Acids Res. 15:7155 [1987]) 1 exon and Y exon contain the untranslated sequence on the © side and the first four amino acids of the signal peptide . The remainder of the secretion signal and the first YI amino acids of the mature protein are encoded within the exon.” The complete (20-) carboxyl domain is encoded as well as the part of the untranslated 1¥ plus about 0 amino acids from the space preceding erythropoietin all located in exon Jala 1. The four related amino acids are The omission noted within Hml-2 (Htpo-2) is located at the © end of exon 1.

711 - - Example No. (3) Transient genetic expression of a human mpl ligand (hML) to clone the full-length insertion contained in 0012-1126 The plasmid is digested with Xbal to the end; Then digested with fragment B, BamH1. The DNA fragment equivalent to the 1,8k0 base helix was purified by gel and cloned into PrkS (PrkS-hmpl1) (refer to US Patent No. 7 for the formation of PrkS under Direct early promoter control of cytomegalovirus Then prepare DNA from PrkS-hmpll formed by PEG method and transfected the gene into YAY human embryonic kidney cell placed in Dulbecco's modified Eagle's medium (DMEM) was added to a mixture of nutrients 7-12 and 70 mmol Hepes (pH 7.4) 0 and 1072 dae fetal bovine serum, and the genes were transferred to the cells in a manner calcium 6 mm This has been described in: Gorman, C. [1985] in DNA Cloning: A Practical Approach (Glover.

D. 1 ed.) Vol.

Ill.pp. 143-190, IRL Press.

Washington, D. ©(0© 1 hour after gene transfer, the supernatant of infected cells was tested for yo on proliferative activity (see Example No. 1). The supernatant was not given from YAY pRK-transfected cell only did not induce Ba/F3 WIA or Ba/F3-mpl cells (Fig. effect on Ba/F3 cells but dramatically inhibited the proliferation of Ba/F3-mpl WDA (Fig. No. (NY) indicating that the aforementioned cDNA is a functionally active human mpl ligand.

711 - Example No. (Yo): Homologous forms of the human mpl ligand 63012 and 211.3 3 -hML4 In order to determine the alternatively spliced forms of 1141, the corresponding primers ball was built For each end of the hML coding sequence these primers were used in RT-PCR 0 repetition of an adult human liver RNA increase. additionally; Selected regions of flanker flankers were generated with internal primers of interest (see what Gh and Silas forms were used. Direct arrangement of the ends of the POR product revealed a single sequence exactly equivalent to the DNA sequence isolated from the human fetal liver genome (See Fig. No. (1) [Sequence ID No.: 1]. On the other hand, a region close to the © end of the EPO-space (in the center of the PCR product) showed a complex sequence pattern 0 suggestive The presence of different possible conjugation isoforms in that region.To isolate these different conjugation isoforms, the primers listed in Table (7) that flank the region of interest in PCR were used as a cDNA database from human adult AS. 7 PCR primers of human ML isoforms (sequencing ID: £0) | phmplicdna 361: STGTGGACTHAGCTTGGGAGAATGS' (series ID: £7) pbx4.£2: SGGTCCAGGGACCTGGAQGTTTG3' replicated clone PCR products blunt in 3. Sequencing of individual ye subclones revealed the presence of at least ML homozygotes.

YYA - - (hMLs; The sequences of the four human mpl ligand isoforms shown from longest (hML) to shortest (hML-4) are listed in (Fig. 11 [sequence ID numbers: >, 4, 5, and 10]). No. (11): Creation and episodic genotypes of homologous forms of the human mpl ligand and the actual variants hML3 5 hML2 and (154 -hML(R153A, sale) reconstituted homologues hML2, 1113, and hML(R153A, R154A) substitutional derived variants of hML using a genetically engineered 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 Snmsky & T.J White Editors. In all structures the "overlapping" prefixes used are shown in Table No. Sadly (A) "overlapping" is illustrated in Table No. (3)

114 - Table No. (4) Outer Bushings (Series ID No.: 597) 012 5'ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G3' (Series ID No.: HMPLL-R: (8A) 500 AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA3' Table No. (1) Overlapping Bushings hML-2: MLd4.F: CTC CTT GGA ACC CAG GGC AGG ACC 3 | (£9) (No. identity

MLa4.R 5001 CCT GCC CTG GGT TCC AAG GAG 3' | hML-3: (0) : (sequence ID number) hML#116+: 5'CTG CTC CGA GGA AAG GAC TTC TGG ATT 3 (oF : (sequence ID number) NEXT-16: SAAT CCA GAA GTC CTT TCC TCG GAG CAG 3' hML(R153A.R154A): (oF : (sequence ID number)

RR-KO-F: 5'CCC TCT GCG TCG CGG CGG CCC CAC

OCA C 3' (of : (sequence ID number

RR-KO-R: 5010 GGT GGG GCC GCC GCG ACG CAG

AGGG 3'

YY. - - All replications were performed by PCR using a pfu DNA polymerase enzyme cloned (Stratagene) under the following conditions. Denaturation of the initial information base Alta ic was done for 1 min and this was followed by a “0 cycle of 1 min at 14°C and 1 min at 1,052°00 min at 77°C. The final cycle was allowed to extend for 10 minutes at a VY and the final PCR 0 product was digested using Clat-Xbal gel, then gel purified, cloned into pRKStkneo., and genes transferred into a YAY cell using different constructs. As described previously, the supernatant was tested using the Ba/F3-mpl proliferation assay. The hML-2 eds and hML-3 did not show any significant activity in this assay in terms of dls. The hML activity was (R153A, R154A) similar to AML activity, which indicates that processing at this site is a double base locus not necessary for activity 0 (see fig. 11). Example No. (VY) CDNA mML-3 3 mML-2 3 mML murine mpl-ligand:- ~:icDNA mML J je DNA 44 5a corresponding to the full coding region of the human mpl-ligand was obtained by PCR 40, gel-purified and stained with primer random sic in the presence of 0-1877* and (0-001). This probe was used to test 01 clones of a cDNA pool of mouse livers from 1.6110 (Clontech Cat # ML 3001 a) Duplicate filters were crossed. filters in 7Yo SDS 70.1 5 Denhardt's X 01 and SSC X © formamide 3 0 +, + molar sodium phosphate (sodium pyrophosphate pH 71, 1.0 pH, 100 µg/mL DNA + Salmon sperm treated with ultrasound all night in the presence of the probe. rinse was rinsed

7710 - -— Filters in XY 556 and then washed once in 0, + X 550 and 1 X SDS at “EY” temperature. Hybridized phage plaques were purified and phage cloned partially inserts cDNA at cut-off position 1 of the Bluescript-SK plasmid. The “LD” clone with an insert of 1.5 thousand bases was selected for further analysis, and the sequences of both strands were analyzed as Cag. 0 previously for human ML 0008. The nucleotide and amino acid sequences deduced from clone 110 are shown in Figure VE (sequential ID numbers 1 and 11). The mature ML of that clone is 71 amino acid long residues and is identified as mMLay (mML-2, for reasons described below). Similar to EPO 0 for those JML's on the other hand, when the inferred amino acid sequences of 7 were aligned for both human and mouse ¢ it was found that mouse lla was characterized by a loss of tetrapeptide deletion between the constituent units of the acids amino -111; 11 in humans that corresponds to a missing VY nucleotide after nucleotide position 114 that is seen in DNA in both human (see above) and pig (see below). Accordingly; Additional clones were tested to detect any possible UML Vo homologues. One of the "17" clones had a 0.4 k base insert with an inferred sequence of 715 amino acids containing the "missing" LPLQ tetrapeptide. It is believed that this form is ML Fari full length and is referred to by the name Talt or Wadmlal 0h. The nucleotide and amino acid sequence for .20 are shown in the form of the number (V1) (sequence ID numbers: 11 and 71). Finally; The knowledge and sequence of the "12" clone was isolated and sequenced. This clone has a deletion of the 111 nucleotides corresponding to hML3 00 and is therefore called 0201-3. A comparison of the inferred amino acid sequences for these two homologues is shown in Figure No. (16).

7717 - - Genetic expression of genetically engineered .01/1 Expnession of recombinaut mML :- Then preparation of expression vectors for mouse ML, mainly as described in Example No. (4). The clones encoding mML-2.5 mML were partially cloned from pRKStkeo and a mammalian expression vector providing expression under the control of promoter 7 and polyadenylation signal 0 9940 of -SV40 and the information was temporarily reduced. Genetic expression of the resulting expressive mMLpRK 5tkeo and 101/12015051160 in YAY cells using the calcium phosphate method. After the accidental transfer of genetic information, the media were conditioned for ∼ days. Cells were maintained in high glucose DMEM media supplemented with + 7) Jac fetal calf serum (0) fetl calf serum (0) - Gol expression in mouse mpl (mmpl). In —:Ba/F3 cells, stable cell lineages genetically expressing Compl were obtained via is genetic information to “mmpl pRKStkeo as described mainly for human mpl in Example No. JS ago) brief; Expressive vector 760107 600165100 (01 μg; (linearized jas) transfected containing the complete coding sequence of mouse mpl was transcribed by Skoda, R.C. et., et at., EMBO 1.12:2645-2653(1993) 1 o to 3/80 cells by electrophoresis (0 '01 x cell; You v; uF do.) This was followed by selection of a neomycin resistance polis of [ana Y ml G418]. Expression was evaluated. Genetic transmission of mpl by flow cytometry analysis using mp-1-1gG antisera anti-mouse-rabbit antimurine Ba/F3 cells were maintained in medium 1640/2014 cells WEHI-3B as a source of IL-3 + supernatants from 717 cells to which genetic information was transfected accidentally from mML were evaluated.

and mML-2 in Ba/F3 Wa transposons from hmpl 9 mmpl as described in Example No. (Example No. (VF) cDNA for pML-2 5 Pml from Porcine mpl ligand: -m and then porcine ML cDNA Je (PML) by .RACE PCR in brief; then designing a short 07 primer and two specific primers based on the exon sequence The porcine ML gene encoding the amino-terminus of ML purified from aplastic porcine serum and then obtaining cDNA prepared from different porcine aplastic tissues increased by replication. IVEY base pair in the kidney and re-cloned 0 5900010060. He studied the sequence of different clones and found that they encode a mpl ligand of a mature pig (does not contain a complete secretion signal). “#1 Amino Acid (2:10M) has the sequence shown as a number (VA) (Seq ID 115) Method: - 0 Isolation of the M and Gakhlan gene: - Genomic clones of the porcine ML gene were isolated Screening a pig genome in (Clontech Inc) EMBL3 using 1845. The review was mainly as described in Example No. (7). The different clones were isolated and the sequence of the exon encoding the identical amino acid sequence as that obtained from purified ML was studied. 00178 was obtained from

porcine ML using a modification of the RACE PCR protocol. Two specific ML primers were designed based on the ML gene sequence from a pig. Polyadenylated mRNA was isolated from the kidney of aplastic pigs primarily as described previously. cDNA was prepared by reverse transcription 6 using primer 38001. (BamdT : 5 GACTCGAGGATCCATCGATTTTTTTTTTTITTTTTTS 0° (sequence ID: 00) wave against the polyadenosine tail of Khanted. An initial YA replication repeat cycle was performed. cycle at 2°40 for 10 sec; OA um for 1 sec and 77”C for 0 sec) using special primer h-forward-1 for —ML (h-forward- 1: 5° GCTAGCTCTAGAAATTGCTCCTCGTGGICATGCTTCT 3 0 01 (La sequence number: 1) and the prefix BAMAD (BAMAD:5'GACTCGAGGATCCATCG3 0 (Sequence ID number B) (in 001 dels) 04 mM !160 0.59 mM MgCl, 10 mM “Tris JY sa pH” and 71 mM “NTPs” with 00 units/mL polymerase enzyme ([Perkin Elmer Inc.] Amplitaq, the PCR product was then digested with 1, extracted using (1:1) phenol-chloroform, precipitated with ethanol, and bound with 11 mg of SK-vector.

YYo — (Stratagene Inc.) Bluescript vector that was cut using 1 .Kpnl and after incubating for 2 hours at room temperature then adding one quarter of the bound mixture directly to a second cycle of the Yy PCR cycle as described. previously) using a second primer specific to forward-1 primer -ML (forward-I: 5' GCTAGCTCTAGAAGCCCGGCTCCTCCTGCCTG 3") ° (sequence ID number: (OV and oligonucleotide ) Ts associated with sequence Adjacent to the multiple cloning region Jalanegion transporter Bluescript SK oligonucleotide (CGAAATTAACCCTCACTAAAG 3 7 ' 5) 0 (sequence ID number (0A): Then digest the obtained PCR product Using 1 and 11e0, the Lf ja clone was re-subcloned in Bluescript SK, and the sequences of different clones resulting from independent PCR reactions were studied.Once again, there is a second form, Sand 0141-2, that encodes a protein missing ; amino acid residues 0 (A) “© amino acid residue)” is defined (see Figure YY [sequence ID number: 17]). Comparison of pML-2 amino acid sequences shows 5 pML The latter formulation is identical except that the tetrapeptide 01.0 corresponding to residues 11-111 has been completely canceled (see figure YY [sequence ID numbers VA and 1?]). All four cancellations occurred

The amino acid observed in cDNA in mouse, human, and porcine ML is in exactly the same position within the predicted proteins. Example No. (f) - CMK assay to induce thrombopoietin (TPO) induction for genetic expression of platelet antigen GPIIb Illa. CMK cells were preserved in 1640 medium ( (Sigma Rempl) supplemented with 7) fetal bovine serum and 10 mM glutamine in preparation for the test “Cells are harvested, washed, and resuspended at ©” X 01 cells/mL in serum-free GIf medium supplemented with © mg bovine bovine insulin, 1107 mg/l apo-transferrin, and 1” trace minerals, in a flat-bottomed dish of 976 eyes, standard or experimental TPO samples were added to each eye at an appropriate dilution. In volumes of 100 μl, 100 μl of 016 cell suspension was added to each eye kidney and the plates were incubated at “FV” at 70 CO, as incubation medium for fA for 1 hour. After incubation, the plates were rotated at a speed of 1,000 rpm at 0°C for five minutes. The AUD fragments were excluded and 100 μL of 212 antibody monoclonal Ia monoclonal 00115 conjugated with 7716 was added to each eye. After incubation at m for 1 hour, the unbound antibody was removed and 100 μL of BSA-PBS 7 + wash was added to each eye. The lye step (7 358-085) was repeated three times. The cells were then analyzed on a FASCAN using one parameter analysis _ to measure 0 relative fluorescence intensity.

Example No. (10) Test 1 for Thrombopoietin (TPO) by measuring the mitotic activity of DAMI cells in small calibrated dishes from 976 eyes. Then 111 cells were preserved in 701 + IMDM horse serum (H: 6:5) horse serum E plus 01 mM glutamine, 100 ng/mL 6 Penicillin and 1 µg/mL streptomycin © In preparation for the test, cells are harvested, washed and resuspended in cell/mL ella in a IMDM +) 7 HP Serum. In a round-bottom dish with an AT well, 100 μl of standard or experimental TPO samples were added to the DAME cell suspension and the cells were then incubated for $A h at sa YY Incubation apparatus with 70 .CO, and after incubation; The plates are rotated in the Sorvall 6000 B centrifuge at 0,001 rpm for o minutes at ; M. The supernatants are discarded and the washing step is repeated with 10,000 µl of alg BSA 70.1 — PBS LA fixed by adding 00 µl ice-cold ethanol frozen PBS-0 and reconstituted Suspension by aspiration After incubation at °C for 11 minutes, the plates are rotated at 70 005 rpm for 10 minutes and 101 μL of RNAse 0 at a concentration of 1 mg/mL is added. Contains 0.1 mg/ml propidium iodide Tween -20 7+, 0 to each eye After 1 hour of incubation at YY Changes in DNA content are measured by flow cytometry Flow cytometry, the number of chromosomes (Polyploldy) was calculated and quantified as follows:

YYA - - Normalized Polyploldy Number Ratio (NPR) = Da ) in >,6 + /M 96 cells in (M + Go< with TPO a Tre RT (7 cells in Z0 M+ 7 cells in (M+ Gx) in the comparison sample Example No. (01): Thrombopoietin test in the laboratory (Rebound Assay test in platelets of 0 mice): Laboratory Test to Determine 3g as a Standard for Platelet Production A. 057816 mice (obtained from Charles River) were intraperitoneally (IP) injected with 1 mL of goat serum containing anti-mouse platelets. 1 (amps) on day 1 to induce platelet reduction -thrombocytopenia on days © and;0 mice were given factor or PBS as a sample compared to two intraperitoneal injections. On day 7; © is injected Microcurrie obituary of 11827750,11827750 in 1 mL of saline intravenously and measured the inclusion ratio of 8" in platelets from the parenteral dose and circuit in blood samples taken from control and treated mice. A platelet count was performed. And white blood cells at the same time in blood taken from a cavity 0 retro-orbital sinus oe Example No. (for 1 7): KIRA ELISA for (TPO) Thrombopoietin by measuring the phosphorylation of the mpl- receptor Rse-gD receptor has a genetic Jada chimeric. A human mpl receptor was detected Vigenet al, PNAS, USA,—:4ddaul (1992) 5640-5644: 89: A receptor with a genetic mixture that includes the extracellular space was made.

extracellular domain (ECD) of the mpl receptor, transmembrane domain ™, and intracellular domain (ICD) of 10720-10728 (269):14( Mark et al, J. of Biol. Chem: RSE ([1994] using a peptide tattooed in the carboxyl-terminal flag “ih” (ie Rse.gD) for use in the KIRA ELISA described herein. See fig. (+7) and (17) 0 for the graphic description of the test. al., Protein Engineering 3(6): ) Herpes simplex virus

[1990] 547-553) The purified stock was adjusted to be ¥ [alae ml in Js las buffered saline 0 b “(PBS) phosphate pH 7.4 and a fraction of 1 was stored mL at 1 °C 5(b) Anti-phosphotyrosme antlboav preparation * - 4610 monoclonal anti-phosphotyrosine was purchased from (Lake Placid, NY) UBL and processed blotinylated using biotin-N-hydroxysuccinamide 00 long chain (Biot in -X- NHS, Research Organics, Cleveland,). (OH (c) ligand dnagil. :- Then prepare mpl day, by the techniques of genetic engineering then described herein.The purified mpl ligand was stored at 1 °C or as a stock solution +

- YY. -:186.81 (preparation of DNA (4) construction of the coding sequence, ole stranded, standard double synthetic oligonucleotides, a human was used, and Rse —) 640-- (C - stranded) for ten amino acids at the end The antibody 586 and another amino acid epitope codon containing the YY epitope add the final sequence to the structural part of the fused gene. (V+) Table No. provides stop signal © (01) table human number. Rse double-stranded synthetic fragment of a fusion gene (ID number coding strand coding strand (0% : sequence | 51 1GCAGCAAGGGCTACTGCCACACTCGAGCTGCGCAG

ATGCTAGCCTCAAGATGGCTG

ATCCAAATCGATTCCGCGGCAAAGATCTTCCGGTCCTGTA

GAAGCT-3' (ID number) noncoding (anti-sense) strand noncoding string (V+: HE 1 5 A GCTTCTACAGGACCGGAAGATCTTTGCCGCGGAA

TCGATITrGGATCAGCCA

TCTTGAGGCTAGCATCTGCGCAGCTCGAGTGTGGCAGTAG

CCCTTGCTGCA-3'

771 - - Synthetic DNA was linked with the coding for 1 amino acid of cDNA - 880 of human Rse at the position Pst] starting from nucleotide 744 of human Rse cDNA sequence published in:- (Mark et al., Journal of Biological Chemistry 269(14):1072010728 [1994]) and Hindill loci in the polylinker of the expression vector pSVI17.IDLL (see fig. A-L 7 0 no. Sequence ID: (YY) to produce the expression plasmid expression vector pSV17.1D.LL .pSV.ID Rse.qD In short, the expression plasmid includes a dicistronic primer containing a sequence encoded for DHFR is bounded on the one hand by © a granting piece and on the other by © a place to receive a donated piece in cintron cut and glued places, followed by a sequence encoding the Rse.gD The complete message J shall (non-spliced ) contains DHFR as the first open reading frame 01 thus producing DHFR (yg) and allowing selection of stable mutants. (e) Preparation of DNA 'mpl-Rse.gD The expression plasmid pSV.ID.Rse was developed. The resulting gD as described above to produce a plasmid 6 containing the coding sequence for the ECD of a human mpl (1-491 amino acids) incorporated into the transmembrane space and intracellular space. For Rse.gD vo (amino acids 474 - 111). Synthetic oligonucleotides were used to conjugate the human mpl extracellular space protein primer sequence to coding sequence protein 1856 in a two-step PCR cloning reaction as described by 26166-26171:267 Market al, J.

Biol.

Chem (1992) and the primers used for the first PCR reaction were 141:- (5-TCTCGCTACCGTTTACAG-3)

171 - - (sequence ID number: 61) and 102:- (5'-CAGGTACCCACCAGGCGGTCTCGGT-3 7 (sequence ID number: TY) with cDNA database for Ris mpl (5-GGGCCATGACACTGTCAA-3') (sequence ID: ?1) and Ry -GACCGCCACCGAGACCGCCTGGTGGGTACCTGTGGTCCTT-3 7 (5) 0 (sequence ID: ?1) with database cDNA of .Rse and the Pyyll-Smal fragment of that fusion splice was used to form a full-length chimeric fragment complex receiver. (5) Cell transformation: Electroporation of 00120110 cells (BrA) EP No. ye 307247 published Vo March) 1989 using 05171040846 which was made linear at a unique Notl position in the main part of the plasmid and ethanol was deposited after extraction of phenol/chloroform It was resuspended in 01 μl .EDTA - Tris 0 / 1 and thus completed

770 - 01 µg of DNA was incubated with 3" 0012 0110 cell in 1 mL of ice-cold PBS for 0 min before electrophoresis at 50860 V 1" uf and cells were returned to ice for Yo. minutes before placing them in dishes in a non-selective medium, and after Y¢ an hour, then ol Ag in a nucleoside-free medium to select clones + stable DHFR. In —:KIRA ELISA, clones expressing MPL/Rse.gD were identified by western-blotting method on whole cell extract after being separated by SDS-PAGE using antibody 5836, which detects the presence of the adhesive epitope. gD fd) Media: -0 then grow F12/DMEM cells with a ratio of +0:+© Gibco/BRL, Life Technologies, Grand Island, (NY). The medium was supplemented with FBS. 70 mM filter (Hyclone, Logan, Ulab) diafiltered You mM Y s HEPES mM 'L-glutamine (i) KIRA ELISA:— DP12CHO cells transformed from 1 Y) mpl-Rse.gD in each eye) from the eyes of the \o flat-bottomed culture dish consisting of an eye in Yoo μl volume medium and were cultured overnight at 17 °C in 786 . CO, and the next morning; The supernatants in the eyes were filtered out and the plates were lined lightly on blotting paper. Then 50 μL of media containing LJ experimental samples, You, 8, .71, YY, YA +, 7.15", 0, or 0 ng/ ml of mpl ligand into each eye. Cells were stimulated at “FY” for ¾ min. The floating fractions were aly filtered again by stacking the plates lightly on a sheet of paper

blotter. To dissolve cells and make ALS soluble receptors. 100 μl of solvent was added to each eye. The lysed buffer consisted of Yor mM NaCl JY ga containing 5906 mM (Gibco) HEPES, 0 Triton-X 100 (Gibco), and 0 1 [klu 01 thimerosal] Biochemicals, Aurora , OH) aprotinin 1017 and 1 mM (AEBSF, ICN Biochemicals) 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride 0 and 50 μM (ICN Biochemicals) leupeptin and 1 mM sodium solution.” (Nazvo,, Sigma Chemicals Co, St Louismo) orthovanadate, pH 7.0. The dishes were then gently shaken on a dish shaker (Bellco.

Instruments, Vineland.

NJ) for 10 minutes at room temperature. Lay. A was dissolving; The ELISA titration plate (Nunc Maxisorp, Inter Med, Denamark) that is coated overnight at ; m with anti-Gd monoclonal antibody 6 (0 μg/mL in ©mM carbonate as pH 01.1 (9.1) μl/eye), filtered, placed on blotting paper and blocked with 0 µL/eye of PBS Block Buffer containing 0.05 D BSA 7 (Intergen Company, Purchase, NY) and 70.01 thimerosal] for 10 minutes at room temperature with After 10 minutes, the anti-60586 coated plate was washed 7 times with a PBS wash buffer containing 10.06 Tween-20 and 1000 thimerosal using a Scan Washer 300 Skatron automatic dishwasher. Instruments.

Inc. (Sterling, VA) Lysis product containing 80 1001/3886 from cell culture with microcalibration eyes ©AO (μl/eye) was transferred to an ELISA (pe coated with anti-Gd5B6). sealed, and incubated for 2 hours at room temperature with gentle shaking.mpl-Rse.gD was removed by washing with buffer and 1,100 μL of biotinylated 4610 (anti-phosphotyrosine) diluted At a ratio of 1:1,800,060 in a diluent

YYo _ — PBS) containing 750.5 Tween-20 750.06 3 BSA 5 © mM +1*YSEDTA 1 thimerosal (¢ means 0% ng/mL added to each eye. After Incubation for 2 hours at room temperature The plate was washed and 100 μL of horseradish peroxidase enzyme streptavidin-conjugated (HRPO) (Zymed Laboratories, 5. San Francisco, CA) © diluted 0:000 was added. 1 with buffer diluent into each well.The plate was incubated for 1 min at room temperature with light shaking.The free avidin-conjugate was washed out and 100 µL of substrate 5 (33S) was added. Then prepare fresh [TMB] tetramethyl benzidine) a two-component substrate kit ¢ Kirkegaard and Perry, Gaithersburg «MD» to each eye. The reaction was continued for 0,01 minutes; Then the color development was stopped by adding 100 μL/eye of 0.11:00 at a concentration of 1 M. Absorbance was read at £00 nm with a reference wavelength of 1560 nm (ABSusois0) using a dish reader (Molecular Devices, Palo, Alto, CA) Vmax controlled with a 650 Macintoch Centris. (trademark (Apple Computer, Cupertino, CA) and computer software (BioMetallics, Inc, Princeton, NJ) Delta Soft. 0 Standard curve plotted for dpl2.trkAB cells or CD cells using 001, So 15, 17, CVA, 1.19, EA, +, or 0 ng/mL of ligand presented as TPO ng ml/ng Against the mean of ABS, sosso t sd using Delta Soft software. The concentrations of the samples were obtained by interpolation of absorbance on the standard curve and were expressed in terms of TPO activity in units of ng / Jo. It was found that The mpl ligand is able to activate the chimeric transgenic mpl-Rse-gD receptor in a concentration-dependent and ligand-specific manner. (shown) or 7001 Plasma (not shown), allowing for ease of use for easy examination of patient and ph samples.

7031 - - Standard Curve for TPO;3; Product B Yay Standard Curve TTPO, YT SAA TT B 0, 7 Usa A 8 b 7 Both and | Hu Ser 10056 a 0, 2 w v, o w and y 1< 7 1 7 7 f* gh } aa ER Ys vo 3 [a 1 * 1 0 TPO concentration (nanograms/mL) EC50 EC50 TPO formulas Cells) Molarity Molecular Ratio pM 4 7 ng/mL (332) (293) Hu TPO pM AY) 4 ng/mL (332) 293 Mu TPO -0 ng/mL (293) 153 Hu TPO 4 ng/mL Hu TPO 155 (E. coli) sili “AYA pM Y),A g/mL | Hu TPO 153met E. coli) Example No. A) \ ( : ELISA Based on a Receiver — Receptor Based ELISA for (TPO) Thrombopoietin (TPO) ELISA plates were coated with F (ab), from human rabbit anti-(Fe)186 in buffered carbonate and then in L at pH 2 gene q 0 1 pH and at $C overnight.

YY - — 0 bovine serum albumin in PBS at room temperature for 1 hour. Fermentation product containing the genetically assembled receptor was added; mpl-lgG was attached to the occlusal and incubated for 2 hours. Two times more dilutions (02.79 - Yo ng/Ja) were added from standard serial dilutions (D: 100 produced in 747 cells at a concentration determined by quantitative analysis of amino acids) in 74, 0 bovine serum albumin and 2070.05-tween were transferred to plates and incubated for 2 hours.TPO bound was detected using A-iia protein and biotinylated co-JY antibodies against F-10 produced in Escherichia coli. E.coli (1 hour incubation); this was followed by streptavidin-peroxidase (1 minute incubation) and tetramethyl benzidine 3,3,5,5 as substrate #0 #6.dad was read. Absorbance at a wavelength of £04 nm The plates were washed between steps For data analysis, the curves were fitted using Kaleidagraph {aul 5; four-parameter curve fitting program day variables} sample concentrations were calculated. Standard curve Jha No. (4 1 vo) Genetic expression and purification of TPO from YAY cell:- (1) Preparation of cellular JIL YAY expressive Expression Vectors:- Then obtaining a corresponding cDNA For each interest open reading frame — for a total open reading frame TPO by PCR using the following oligonucleotides Labs Primers:—

0-77/8 Table No. (11) 797 PCR primer Clg FLF: 5 ATC GAT ATC GAT CAG CCA GAC ACC | (sequence ID number (to: CCG GCC AG 3 AGC TCT AGA CAG GGA AGG GAG 067: 5L| (sequence ID number: (EA) CTG TAC ATG AGA 3' Then using PRKS5-hmpl1 (which is then described in Example 9) as a model for the reaction in the presence of an enzyme Polymerase for (Stratagene) pfu DNA and denaturation was JY denaturation for 17 minutes at 3 5 degrees followed by a cycle of (sale) construct (1 minute at ¢) (a 1 minute They are at 00 a and 1 minute at 777 C).The final extension was for 11 minutes at a VY and the PCR product was purified and 1 cloned between the xbal 5 Clal restriction sites of the pRKS5tkneo plasmid. It is a pRKS-derived vector modified for the expression of a resistance-0 gene under the control of a thymidine kinase promoter to yield the PRKS5tkneo-transporter.

ORF A second structure corresponding to a homologous epo space was constructed in the same way, but using 0.081 as a forward prefix and the following reverse prefix:- Arg STOP.Xba: 5 TCT AGA TCT AGA TCA CCT GAC GCA GAG GGT GGA CC 3 (sequence ID number: 1%) The final structure is called .pRKS-thnoeoEPO-D and both sequences are verified as described in Example # (VY)

Yrs - - (¥) transferring the genetic information to human embryonic kidney cells using the 0800 method, as described in Example No. (9): - After transferring the infection by 8 ¥ hours, the selection of neomycin-resistant clones began in the presence of 4 mg / ml 8. After that, with a period ranging from 01 to Vo days, individual clones were transferred to the same AT dishes and left to grow until perfect confluency. The expression of ML153 or 141,332 was evaluated in the conditioned media of those clones. Using the Ba/F3-mpl reproducibility test (LS) this is described in Example #1. (Y) Purification —:thML332 293-rthMI332 conditioned medium was placed in the (pharmacia) column Blue- Sepharose was then saturated with 0l01110:8160© in 0mM sodium phosphate at pH 2.4 pH sala (buffer A) and the column was washed sequentially with ten column sizes; Each pan was composed of regulator A and a buffer A containing urea with a concentration of ¥ M. The (aad) was then sequentially washed using regulator (A) containing urea at a concentration of 1 M NaCls. at a concentration of 1 molar. The Blue-Sepharose eluatet was immediately then placed into a WGA-Sepharose 10 column saturated in buffer A. Then, the WGA- Sepharose column was washed using ten column sizes of buffer A containing urea 7 M and 11801 1 M and washed sequentially with the same buffer containing 3S yu N-acetyl-D-glucosamine 0 ,+ Molar. The WGA-Sepharose wash-out media was placed on a (Synchrom, Inc.) C4-HPLC column neutralized in 760.1 TFA and then the C-HPLC column was washed with intermittently graded amounts of Yo (Yo — Jia) propanol #0 min —¥o 19). It was found that thMLy;, is being washed into the realm

‎YE. _ — propanol 1 . —YA while grading. rhML;3, purified by 01M 5105-0, is transmitted broadly in the region of 8+-80 kDa and 1022 for the gel (see fig. (Vo)£) Purified and resorbed:- The conditioned medium was fractionated with 293-thML The media was resolved on Blue-Sepharose as described in Womtal, and the sequential filter fluid exiting the Blue-Sepharose was directly placed in the mpl-affinity column as described above. The f5 outflow from the mpl-affinity column was purified to homogeneity using C4- column cycle HPLC under the same conditions as described for .thMLys; and by SDS-PAGE the thML ps analysis showed Two major bonds and two minor bands, two major and two minor bands, 0, with a molecular weight: 14, in the range of 70001 - 18001 (see Figure No. (Yo) Example No. HY) Genetic expression and purification of TPO From Expression and Purification of TPO from CHO —:CHO -1 Description of expressive vectors for CHO yo The expression vectors used in the electroporation protocols that will be described later have the following names:- pSV15.ID.

LL MLORF (Full Length OR, B8770); 5 pSV1S.ID.LLMLEPO-D (truncated or -(hTPO,s;

141- The relevant properties of these plasmids are shown in the form of the number (Y¥) and (?1). Y - Preparation of CHO expressive vectors:— The cDNA corresponding to the hTPO-total open reading frame was obtained by PCR reaction 0 using oligonucleotide primers shown in Table (VY) Table No. (VY) PCR Primers of Expressive Vector CHO FL 2 S'ATC GAT ATC GAT AGC CAG ACA CCC 014 | Sequence ID: (8Y CGG CCA G 3' ORE S41 5 AGT CGA CGT CGA CGT CGG CAG TGT | (Sequence ID: 67) CTG AGA ACC 3' then al using 1 PRK S-hmpl (which was then described in Example 7 and q) as a model for the reaction in the presence of (Stratagene) pfu DNA polymerase. The first 0 denaturation lasted for 1 min at a q¢ and followed. Yo cycle of iterations (1 min at €2°, 1 min at #dam and 1 min at 77°).The final extension was continued for 11 min at VY 2° and the PCR product was purified and cloned between loci sall 5 Clal cutoff of plasmid 05715.10 to obtain the vector .pSVIS.ID.LLMLORF A second construct corresponding to the homo-domain EPO was constructed in the same reverse and forward primer method but using 018.117 as the following forward primer:- primer

EPOD.Sal 5' AGT CGA CGT CGA CTC ACC TGA CGC AGA GGG TGG ACC 3) 68 : (sequence ID number o) The final structure is called pSVISID.LLMLEPO-D and the sequence of both structures has been verified as described This is in Example (V) 5 (2). Basically, the coding sequence of the full-length truncated ligand ld is inserted into the multiple cloning site of the CHO expressive vector represented by pSVISID. .LL vector contains enhancer/early promoter 51740, a modified splice unit containing DHFR cDNA/mouse, and a multiplex locus to insert the gene of interest (in this case the described TPO sequences), a polyadenylation signal of SV40, an origin of replication, and a B-lactamase gene for plasmid selection and virulence in bacteria.” —How to Create Cell Lineages CHO constant expressing TPOss, 100 human genetically engineered recombinant a- Description of cell line 1 of mother (parent cell line 0110). The CHO host cell strain (fi wala ovary Chinese Hamster Ovary) used to express the TPO molecules then described herein is known as CHO-DP12 (see EP 247 EP307). Posted on March 11 (Azar) 4 1 ( and then

Selection of the dal cell lineage as a clonally selected from cells of the parent transduced lineage (al) CHO-K1 DUX-B11 (DHFR-) - Obtained from Dr.

Frank Lee of Stanford University with permission from Dr.L.Chasin (Dr.L.Chasin) used Jib for preproinsulin to obtain progenitors with low insulin requirements. These cells are also DHFR deficient, and progenitors can be selected in the presence of the transporter homologues. DHFR CDNA was supplemented by complementation in MA medium of nucleoside additions (hypoxanthine “glycine) and 0 (thymidine) supplements. In general, the aforementioned selection system was used for the stable expression of CHO Wa strains b- Genetic cutting method ( Transfection method (electroporation): Lisa, 10, and 7100 cell strains were produced by transfecting genetic information into 0 109012 cells by electronic transmission; see, for example, Andreason, G.L.J.

Tiss.

Cult) (Meth., 15.56 (1993) using pSV15.ID.LLMLEPO-si pSV15.ID.LLMLORF plasmids 0 converted to the linear form, respectively. ¥ enzyme reaction mixtures were formed to cut 5 at 01 µg 5 YO µg and © 1 µg of vector with enzyme 1 by standard molecular biology - methods 10. The said cut-1 locus is present only once in the vector in the linearization region On the ¥ side and outside the TPO ligand cloning units (see Figure No. (YY), the reactions were prepared with a volume of 100 μL for overnight incubation at 0°C, and the next day, phenol mixtures were extracted. 0 ) - chloroform - isoamyl alcohol ?: 1) once and the precipitate was placed with ethnol on dry ice for approximately one hour.Then the precipitate was collected after centrifugation for 1 minute and then dried The linearized DNA was resuspended in 00 μL of medium

Ham's DMEM-F12 in a 1:1 ratio plus standard antibiotics and ? mM glutamine. WA 7012 grown in suspension was collected and washed once in the medium described for DNA resuspension. Cells are finally suspended in the same medium at a concentration of “01 cells in each Vou oo”. 0 μl cell volumes (VO μl) as well as all linearized DNA mixture were incubated with each other at room temperature for 1 hour and then transferred to the BRL electropermeability chamber and each reaction mixture was then subjected to To conduct the electrical transmittance in a standard BRL electrical transmittance tester at YOu volts set at low YY capacitance microfarads (uF) and after conducting the electrical transmittance the cells were allowed to remain in the device Bad 0 minutes and then They were placed on ice for another 10 minutes.The cells tested for electrical permeability were transferred to 01 mm cell culture dishes containing ¾ ml of standardized complete growth medium for CHO cells (high glucose 014534-12 ratio; 10 00 without glycine plus 1 GHT and ?5 mM glutamine 70 fetal calf serum) and cultured overnight in a cell culture incubator at CO; salaction and screening On day J, the cells were stained with trypsin to separate them from the dishes by standard methods and transferred to YOu mm tissue culture dishes containing Ham's S.DMEM-selective DHFR medium. 2 medium 1:1 previously described plus either MDLs 77 or 16 fetal calf serum 40 but free of HTPoxauthine glycine and APSIN; This is the selective DHFR 0 medium we use.) Cells were then placed from each 0 mm Te dish

Plates * / 0 millimeter mm, and then the cells were incubated for a period ranging from 01 to 116 days (with one medium change) at 7.7” m / 00.75 until clones appeared and reached sizes that could be transferred to dishes AT eye. and within a period of 4 to 0 days »; Cell strains were transferred to 16-well dishes using sterile yellow pipette tips and then adjusted to a volume of 50 ml. Cells were allowed to grow to perfection com (typically from ¥ - 0 days) then the trays were trypsinized and two replicated copies of the original tray were reproduced. Two of these clones were stored short-term under freezing with dilution of JS cells sampled in 00 μl of 5 01 in DMSO Samples of 0 ali conditioned medium, free from serum, were analyzed from eyes pooled in The third tray for transgenic expression of TPO via a BaF cell-based activity assay. The clones with the highest expression based on that assay were activated after storage and placed in two 7 101 mm collection vials for transfer to a group Cell culture to prepare them for suspension, re-assay, and storage banking. D- Amplification Protocol: Thus, many groups of cell strains with higher concentrations than Transmission Group 10, which were previously described, were placed through a system Enlarge methotrexate to produce higher concentration clones. Clones of CHO cells were then extended and placed in 0 cm dishes with four concentrations of methotrexate (i.e., 84 nM, 100 nM, 0000 nM, and 1005 nM) at two or three cell numbers (01) and 80 010 x and 10 cells/dish). fis cells were then incubated at 7 °C / 70 CO, until the clones are formed and are able to transfer to dishes with 476 wells for the additional test.

- +7147 higher concentrations of methotrexate (i.e. 0 nM, 8060 nM, 1000 nM)

and 1101 nanomolar) and as previously explained, resistant clones are allowed to form

establish, then transfer to 976 plates, and then test it.

; - Cultivation of CHO 435 cell lines expressing: 170 and: 170 human genetically engineered

,. recombinant 0

Banked cells are activated and the number of cells is increased by standard culture methods in empty medium

of serum or containing serum. and after increasing to sufficient WIA concentration; Cells are washed to remove

remaining cell culture media. The cells are then cultured by any standard method including:

batch, fed-batch, or continuous culture 0 at NYO 40 "C; neutral pH".

The dissolved 0p content is not less than 70 so that the secreted TPO is mainly accumulated. It is then separated

Cell culture fluid is obtained from the cells by a mechanical means, Jie, centrifugation.

© - Human TPO Remediation Produced by Genetic Engineering from Fluids Culture ~:CHO

Harvested cell culture fluid is placed directly and then collected (HCCF) in 0 column (pharmacia) Blue sepharose 6 Fast Flow saturated with Na Phosphate at a concentration of 0.11

Molar pH NaCly oY, € Ph at a concentration of 106 molar at a ratio of about 001 pH

107 per liter of resin with a linear flow rate of approximately 01 mL/

hour/cm'. Then the column was washed with an amount ranging from “ to © of the volume of the column of the material

This is followed by washing with an amount ranging from ©” to © of the column size of Na Phosphate © at a concentration of 100 M at a pH of 21 7.4 and finally urea at a concentration of ? Molar. And then it is done

541 - Filtering of Lal TPO using from “ to © column volume of Na Phosphate at a concentration of 100 M; At a pH of 1.4 pH and 3S urea ¥ molar NaCly at a concentration of 1 molar. The amount of Blue Sepharose containing TPO is then placed in a column of Wheat Germ Lectin (pharmacia) Sepharose 6MB 0 saturated in Na Phosphate at a concentration of 800 M at a pH of 4 and 8N. at a concentration of ¥ mol NaCly at a concentration of 1 M in a ratio of 8 to 11 ml of 0001 Blue Sepharose per ml of resin at a flow rate of approximately 50 ml/h/You and then washed column with a quantity ranging from * to ? of the column volume of an equivalent structured substance. The TPO was then filtered using an amount of ? To © from the column volume of 0 sodium phosphate at a concentration of 00 M at pH 4 and 68 at a concentration of ¥ M and N-acetyl-D-glucosamine at a concentration of v.0 M. The Wheat Germ Lectin pool is adjusted to a final concentration of 1004 pH and 1,16 (TFA) trifluroacetic acid. The resulting pool is placed in a Jalas (Vydoc214TP1022)C4 reverse phase column in 70:1 Cig 70.0 pH. 5 TFA at load ranges from 0 - approximately 1.7 to +10 mg protein per mL of resin at a flow rate of 07 mL/h/cm'. The protein is sequentially filtered in linearly graduated amounts in two stages of 80010116”#containing 228-741 and 7505 Ci. The first stage consists of linearly graduated amounts of zero to 0 2661001016 in 11 minutes. The second stage consists of linear graded quantities from Fr to

YEA - — acetonitrile 11 in 0 minutes. The TPO is sequentially filtered using approximately +70 acetonitrile. The group was formed on an SDS-PAGE basis and the group was then diluted with a volume of 8.61 M sodium phosphate at pH 7.4 NaCly 8 M on an AmiconYM membrane or similar multifilter 0 membrane having Cut - Off separation limit at a molecular weight of 00001 to va *" Daltons. alg Immediately after that the binary filtrate is treated which is then obtained or further concentrated by multiple filtration. The binary filtration is set Diafiltrate the concentrate to a final concentration of 6201 Tween-80. All or part of the binary filtrate/concentrate equal to ?to 70 of the calculated column volume of 0 is then placed in a (pharmacia) Sephacryl 5-300 column. HR equivalent in Na Phosphate at a concentration of 6501 M at a pH of NaCly ¢ pH at a concentration of 1216 M and Tween-80 at a concentration of 10000 and purified by chromatography at a flow rate of 11 ml/h/cm. The TPO-containing fractions that are free from clumps and Ala proteolysis products are aggregated on a 505-5 basis.The resulting aggregate is filtered onto a YY filter, + Millex-Gv 10 µA or similar that; It is stored at a temperature ranging from ? To AA Example No. (1): Transformation and Induction of TPO Protein Synthesis in E. coli: 1- Creating expression vectors 0 in Escherichia E. coli:

All pMP21 plasmids and pMP202 5 pMPS7 5 pMP4L pMP151 plasmids are engineered to express the first 05 amino acids of TPO right downstream from a small lead guide that changes between all constructs. Leader functions primarily provide a high level of translation initiation and fast filtering. Plasmids Pmp210-1,18,21-,22~©,24-,25- are engineered to express the first amino acid Yor of TPO after initiation of methionine and differ only in the 0 codon used for the + first amino acid. of TPO while the pMP251 plasmid is a derivative of pMP210-1 in which the carboxy terminus of 0 is extended by ¥ an amino acid. JS of these plasmids will produce high levels of expression. The intracellular interior of TPO in Escherichia coli upon tryptophan stimulation. : Yansura, 10. G. et. al Methods in Enzymology ( Goeddel, D.

V., Ed.) 185:54-60, Academic Press, San Diego [1990). The pMP172 5 pMP1 plasmids are intermediates in the formation of the aforementioned TPO-expressing plasmids in the intracellular sphere. —:pMP1 plasmids )( 0 The pMP1 plasmids are vectors for secretion of Yoo, the primary amino acid of TPO, formed by linking 6 DNA molecules together as shown in figure (YT). The first of these is transporter 1 from which the Malul-BamH1 minor 0 has been removed and 001021 is considered to be the derivative of 1 swelling C.

N.et.

Al, Gene 55:189196 [1987])_ the human growth hormone gene 0 was replaced by the phoA gene from E. coli and the

Yo. —— the position of the 1001 geometric segments in the coding sequence relative to the signal sequence 8711 at amino acid 1?”-XY Then pre-join the next two segments of a base pair from the Hinti-Pstl segment of pRKS DNA -hmpl (Example 3) which encodes amino acids in TPO 19 to 10' oo and the synthesized segment of DNA encoding amino acids 1 - 18 : - 5'- CGCGTATGCCAGCCCGGCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTA AACTGCTTCGTG ATACGGTCGGGCCGAGGAGGACGAACACTGGAGGCTCAGGAGTCAITIGACG AAGC ACTGA-5' (sequence ID number (V8): Y 0 (sequence ID number (v 0) using ligase enzyme 14-01 18 and cut off a second using 0 The fourth was 1091 base pairs of pstl-Haelll segments of PrkShmpll encoding amino acids;10-101 of TPO and the last was 7; a base pair of segments of segments 5011-71 of pdh108 containing lambda eo termination of reproduction as previously described:- (Scholtissek, 5. et al., NAR 15: 3185 [1987])

- 01 -

(2<) The Pmp21 Plasmid The pMP21 Plasmid is engineered to express the first yoo amino acid of TPO with the help of a 1” amino acid leader segment that comprises part of the STI signaling sequence. It was formed by joining ¥ DNA segments together as shown in Figure (TE) the first ie oo is the pVEG31 vector in which the Xbal-Sphl small segment is removed.

1 is a derivative of pHGH207-1 (de Boer, H.

A.et. al. in Promoter Structure and Function (Rodriguez.

R. 1. and Chamberlain, M.

J., Ed), 462, Praeger, New York [1982]) in which the human growth hormone gene is replaced by a gene for vascular endothelial growth factor (the corresponding vector fragment can be obtained mentioned from the last plasmid). The second segment in the splicing was a double-stranded synthetic DNA with the following sequence:- CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAA 5. TTAATACTTTTTCTTATAGCGTAAAGAAGAATTGC-S've (sequence identifier number (VY): (sequence identifier number) VY: the last fragment was a 0 Y base pair of the Milul-Sphl fragments of pMP1 encoding 100 amino acids of TPO

75017 - c) Plasmid 110151m:- Plasmid 0000151 is engineered to express the first Y00 amino acid of TPO after leader 5: containing 1 amino acid for homology of signal 9111 and A 85 And the position of cutting the Xa factor as shown in the figure (Yo), and then the installation of pMP151 by linking three DNA parts with each other. The younger Xbal-Sphl was described previously, and the second was a structural DNA duplex with the following sequence:- 0. CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACAT 5 CGAAG 6106105 TTAATACTTTTTCTTAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTTC 01 CAGC AT-5 (String ID : (vv (Ve: mall id) the latter was a Bgll-Sphl molecule with 0174 base pairs of 0081011 encoding for Voi 11 amino acids in -TPO and the pMP11 plasmid is identical to pMP1 except for a few codonal changes 8 in signal sequence 5111 (that part can be obtained from pMP 1 .

YoY - — pMP202 Plasmid (=) pMP202 plasmid 55: very similar to expression vector 0000151 except that the factor Xa cut-off position in the leader is replaced by the thrombin cut-off position as shown in fig. oF) pMP202 was created by joining together three DNA fragments. The first, Leia, is the previously described 01785031 o from which the Xbal-Sphl molecule has been eluted. The second represents a structural DNA duplex having the following sequence:- CTAGAATTATGAAAAAGAATATCG CArrTCATCACCATCACCATCACCATCA 5. CATCGAACCACGTAGCC TTAATACTTTTTCTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGC TTGGTGCAT-5 0 (sequence ID number: (Vo) The sequence identity: , ) 077 The final fragment was a Bgll-Shpl molecule with 01164 base pairs from the plasmid pMP11 described previously.

(e) —:Pmp172 plasmid vo The Pmpl72 plasmid is a secretion vector for YoY the first amino acid of TPO and is an intermediate for the construction of pMP210 as shown in FIG. (FV) pMP172 was prepared by ligation of three clea DNA segments, the first of which was the pLS32lamB vector, from which segment 260621-71 was removed. The second was an EcoRI-Hagl fragment with 157 base pairs

Yo¢ — from the previously described 1m plasmid. The last fragment was a double-stranded DNA with the following sequence: - SUTCCACCCTCTGCGTCAGGT | (VY ID No.: Amal GGAGACGCAGTCCATCGA-S' | (VY ID No.: (VA) Plasmid 110210AD:-

The pMP210 plasmid is engineered to express the 1st amino acid YoY in TPO after translational initiation methionine. Basically, this plasmid was made as a bank of plasmlds. The first six codons were placed at 0 randomly in the third position of each codon and constructed as shown in the figure (VA) for By linking three DNA molecules, the first of which is vector 0115031, which was described previously and which was removed

0 - Xbal-Sphl minor part of it. The second was the double structural DNA shown Lid follows and processed (Klenow) DNA polymerasel — Y followed by a flash using Xbal and Amnctl and encoding methionine start 0 and the first six codons in TPO .

5. GCAGCAGTTCGAATTATGTCNCCNGCNCCNCCNGC (ID No. 1 consecutive NTGTGACCTCCGAACACTGGAGGCT: (V4)

ie No. GTTCTCAGTAAACAAGAGTCATTTGACGAAGCACTGA (A+: 45d | GGGTACAGGAAG-5

— Yoo —— The third was a Hintl-Sphl molecule with +A9 base pair of acid-encoding pMP172

TPO at 19-107 amino from the pMP210 plasmid bank of approximately 700 clones was retransferred into LB dishes with highly concentrated ykdm ffh]zhj hgjv[lm (04 µg/mL) for clone selection. tetracycline. translational initiation clones 0 (Yansura, D. G. et. al., Methods: A Companion to Methods in Enzymology4: 151158

[1992]). Among the eight clones that took place in tetracycline dishes; Five of the preferred TPO-expressing ones were subjected to a DNA sequencing procedure, and the results are shown in the form of No. (19) (No. 1 Sequence ID 77, 7 Cy 7, 6 XY, nor 7 Ag). ¥) pMP41 Plasmid (g) The pMP4 1 plasmid is engineered to express the 1st amino acid Yoo native in TPO fused to a leader consisting of 1 amino acid of signal sequence 5111 followed by a cutoff locus The .Xa factor and then the creation of the plasmid as shown in the form of a number ($) binds three pieces of DNA with 1 to each other. The first of them is the pVEG3T vector, which was previously described. The Xbal-molecular was removed. Sphi from it.The second was the following double structural DNA:-

5. CTAGAATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC | Sequence ID: (AY TTAATACTTTTTCTTATAGCGTAAATAGCTTCCAGCAT-S' | Sequence ID (A vl) The last segment to bind was a Bgll-Sphl molecule with 1 + 53 > base of plasmid 1 whose (h) —:pMP57 plasmid © plasmid 01057 expresses the first amino acid Yoo in TPO after the amino acid leader of signal sequence 5111 and the base duplex site Lgs-Arg dibasic site. The aforementioned double base position provides a means to remove the leader using a protease enzyme. This plasmid was constructed as shown in figure (£1) by linking three pieces of DNA together. The first, Leia, is the 1031m vector that previously described from which a small Xbal-Sphl molecule was extracted.a The second was the following structural duplex DNA: CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTA .5 | (Ls no. AACGTAGCC | sequence: (AY) TTAATACTTTTTCTTAGCGTAAATAGCTTCCAGCAT- 5' | consecutive number i (Af:

581—The last fragment of the ligand was a Ball-Sphl molecule with 014 014 base pairs of the previously described pMP11 plasmid. (i) —:plasmid pMP251 The pMP251 plasmid is a derivative of pMP210-1 that includes two additional 0 amino acids of TPO on the carboxy terminal end. As shown in Figure No. (7;); Then the assembly of the plasmid by linking two pieces of DNA (the first of which is pMP21, which was described previously, from which the Xbal-Apal part was removed. The second part of the linkage was an Xbal-Apal part with 01 Base pair of 0100210-1.” - Transformation and Induction of E. coli with known vectors —:coli with TPO expression vectors TPO E. coli 44C6 (w3110 topaz rpoHts ion3 clpP galE) using the 06012 heat shock method (Mandel.

M. et al.. J.

Mol.

Biol., 53:159-162, [1970]). Transformed cells were first grown at FY 113 media containing 1 µg/ml carbenicillin Ov until the optical density reached Tov. ) nanometers (nm) of culture to approx. ml carbenicillin and after growing with ventilation at 10°C for an hour, indole-3-acrylic acid was added to a final concentration of ov.

— 0 C — µg/0 Je The culture was then allowed to continue to grow at ¥ 0 °C with aeration for another 0 hours at which point the cells were harvested by centrifugation. Example No. (Y) X: Production of biologically active TPO (1-153) (Met) in Escherichia coli (E.coli) The following procedures can be used to produce biologically active (1-153) TPO (Met Refold the 0 in a similar way to process other TPO shapes Lay including the extended images at the N end Cy Soluble—:non-soluble E. coli cells expressing TPO-1-153 (Met) encoded by the plasmid Pmp210-10 were prepared as described above. Approximately 100 g of cells were resuspended in 1 liter (100 volumes) of cell disruption buffer 0 mM Tris; © mM EDTA pH (A Ph). with a Polytron homogenate and LAD was centrifuged at X©0081 p for 0’ min. The washed cell pellet was resuspended again in 1 L shredded buffer LOAN with the homogenate Polytron homogenizer 1 and the molar suspension is passed through an LH Cell Disruption ad uy (Inc, LH-Inceitech) or through a microfluidizer Lik (Microfluidics International) Microfividizer according to the manufacturer's instructions. The suspension is then centrifuged at g 5 0 00001 for 6 ? minute; Then it was re-suspended and centrifuged again to obtain a precipitate with a brittle structure. The washed precipitate is used immediately and can be stored frozen at -71a degree

— 4e - b) solubility and purification (1-153) (Met 0 monomeric): The resulting pellet is resuspended in high volumes by weight of Yo mM Tris pH A pH with > -A YO 5 mM guanidine (dithiothreitol) DTT and stirred for 1 to ¥ hours; or cll length at 78° to make TPO 0 soluble Higher concentrations of urea are beneficial (1-molar Lad) but generally result in a lower yield compared to guanidine. After conversion to soluble material (Olas), the solution is centrifuged at 00060 X p for 1? min to produce a sale supernatant containing monomeric Jaw TPO 0 protein. The fraction is then purified. The supernatant was chromatographically placed on a 200 X 1 Pharmacia (Pharmacia) Superdex +" (0 pm) gel filter column at a flow rate of ? ml / i, and the protein was sequentially filtered using 1 mM Yo of Na phosphate ». pH using 01 mM aggregation of DTT fractions containing monomeric denaturedJie TPO protein from a separating column between 100 and 6000 ml.The TPO protein is then purified on a phase column (01 XY) preparative Cy reversed phase column cm (VYDAC) and the sample is placed at 6 Vo ml/min in a column saturated with +7 (trifluoroacetic acid) TFA using .acetonitrile 7 1 and the protein eluted using linear gradient amounts of 711 - 71 (acetonitrile in <1 min). acetonitrile + 7.0 approx. This material is used for refolding to obtain a different form of biologically active TPO.

C- Production of biologically active TPO (1-153 —: (Met) approximately yo mg of protein monomeric TPO reduced and reduced in 560 ml) 7 acetonitrile 70+ / TFA In “01 mL of buffered refolding material ideally contains the following reagents:- 1 mM Tris ¥,¢ M NaCl © 7 mM EDTA CHAPS detergent glycerol 7 Yo 0 © mM glutathione oxidized glutathione 1 mM glutathione reduced glutathione pH adjusted to AY after mixing; The refolding (all sale) sale organization is slightly stirred at a degree; or for a duration of VY SA-hour to obtain refolding results as high as possible for the intact disulfide-bonded form 1 of TPO (see below). Then the acidified solution was acidified with TFA final concentration of 7857 and the product was filtered through a filter from 0.406 to 7 μm Na and then ali .alg .acetonitrile aaa 1 / 01 was added after that the solution was pumped

71711 - - directly to a reverse phase column and the refolded purified TPO elution (1-153 (Met) is filtered using the same gradient program as above. The refolded purified TPO elution is filtered using acetonitrile 50 Under these conditions, unsuitable bonded B-6 variants of TPO are pre-filtered. Final purified TPO (1-153 (Met! SDS gel and analysis using reverse phase chromatography © For animal studies, the purified material was dialyzed in physiologically compatible buffers. mMolar acetate; pH © or 0 mM Na succinate « hydrate alginate pH 0.0 or 0 mM phosphate 118 pH (0.4 pH) contains Tween -80 mM 1180 and 7001 151 on 0 half in a 38/79 sample (the half-maximum induction process TPO is activated due to the high efficiency of dled it is possible to obtain a substance (pg/ml) at about ?pg/ml maximal stimulation detergents and biological detergents using different conditions for the buffers in most cases; Aull is done on the one hand. redox conditions, reducing and oxidizing substances for manufacturing processes (001). Obtaining a small amount of suitably folded material (less than at least 15 and most commercial 701; a refolding yield of 701 is desirable). More than 70 0 favorites from © to

CHAPS 3 different dodecyl-beta-maltoside and Triton X-100 detergents Ahrigo 1 — 1 Zwittergent, Tween - 80, Tween - 20, sarkosyl, SDS 3 CHAPSO to determine its efficiency to support high refolding production. Among those cleansers; The © family has been found to be generally useful in the refolding reaction to determine agglomeration (CHAPSO 5 CHAPS) only CHAPS

unwanted protein and disulfide formation. CHAPS levels greater than 71 were most beneficial. Sodium chloride was required for better productivity; at optimum levels between 1 molar and 51 molar. The presence of 1 mM EDTA limits the amount of metal-catalyzed oxidation (aggregation) observed with some formulations. Glycerol concentrations greater than 711 lead to Achieving sale conditions) optimal folding.In order to obtain maximum productivity, it was necessary to have oxidized and reduced glutathione or oxidized and reduced cysteine as a redox reagent pair and in general A higher yield was observed when the molecular ratio of the oxidizing reagent was equal to or greater than the number of reducing agents for the reducer and oxidant pair. Organic solvents (for example, ethanol, acetonitrile, and methanol) in concentrations of Sve -01 lower. High levels of organic solvents led to an increase in the quantities of unfavorably refolded shapes. Tris and phosphate as buffer solutions pH 0 buffer buffers are generally useful for incubation at 0°C and also produce higher levels of unsuitably refolded TPO. A refolding yield of 46-710 (depending on the amount of reduced TPO A Tdi 0 used in the refolding reaction (hl) is typical for the preparation of TPO purified during the first ,© step. Substance can be obtained Effective in the case of less pure formulations (on Jal dpe ©) immediately after the Superdex 200 column or after extraction of the primary fractions

refractile body extraction ) although the yield is lower due to large-scale precipitation and intrusion of non-TPO proteins during the ale process. Cysteine Residues ¢ It is possible to generate three different forms of disulfide for this protein. No. (): Disulfides compounds between building blocks 1 - cysteine and 5-7 Copy No. (7): Disulfides compounds between building blocks cysteine - 7 and 7 - During the first examination to determine cases of refolding; separate units peaks and different containing TPO protein upon reverse phase chromatographic purification, Ben. Only one of these isolates contained clear biological activity when studied using the BaF test; therefore, reconstitution conditions were made The folds are proportional to those yielding such a transcript.Under these conditions, the misfolded transcripts are less than -01 70 of the total product L of the total product of the TPO monomer - and the disulfide in the active TPO Biologically determined is — 8 and -¥ by mass spectrometry 0 and protein sequencing (i.e. 1 das). Fragments of different ©, -column favorites (0 - 01 nmol) were combined using trypsin (molecular YO:1 ratio between trypsin and protein). The mixture was analyzed by matrixassisted laser desorption mass spectrometry before and after DTT reduction and after reduction; The corresponding masses of most trypsin peptides were revealed

114 - - large tryptic peptides of TPO and in unreduced samples; Some of those blocks were absent, while others were still present. The cluster of new peaks corresponding primarily to the sum of individual tryptic peptides was contained in the disulfide pair and thus it was possible to locate a clear JS disulfide of bioactive TPO engineered and refolded to be 1 4- 0-0 and - ?. This is consistent with the known disulfide motif of the related erythropoietin.

D Biological activity of genetically engineered refolded TPO (1-153 —::Met) The refolded and purified TPO (1-153 (Met) has activity in each of the in vitro tests and the in vivo organism in the test 80/15 half of the maximum induction value for thymidine inclusion was achieved (J incorporation 1/F 80 cells at Su YF g pg/mL (, 0 picomolar). ELISA

0 based on the ampl receptor, the half-maximal activity occurs at 0.9 ng/mL (VY) picomolar: and in normal and myelosuppressed animals caused by wal X rays approximately cnear-lethal X-radiation (Met! 1-153) TPO was most effective (efficacy was observed at doses as low as 1 ng/mouse) to induce new platelet production. Example No. (¥"):

-: 15. Bioactive Escherichia coli nah’ TPO production Other different forms of 1 purified 8. coli Three bioactive forms produced in Escherichia coli and refolded into active form are provided below Biologically. From the signal sequence derived from bacterium 5171 in the field MLF-13 fusion of residues when (1)

The N-terminal of TPO (residual units (Yoo - 1).

The resulting sequence is:- Where the leader homologue is underlined and represents C...C from Cys” to Cys! And that different form was fitted to provide tyrosine for radio-iodination expansion To TPO for m future and biological studies. (¥) HSMLF-7 residues from sequence STII and A histidine residues are fused; sequence 1561 is the factor Xa cutting enzyme site fused to the N-terminal domain (Yeo residues). -1) for TPO, and the sequence is represented in:- Where the leader sequence was underlined and represents CnC from Cys to Cys. This different form can be processed when purified and refolded using an enzymatic factor Xa, which It will cut it after the arginine residue of sequence 1261 resulting in a TPO of a different form than Yoo residue in length with a natural serine amino acid at the N-terminal + (©) T-H8MLF is prepared This is also described above for the different form (oY) except that the thrombin-sensitive Allie 0 1508 is combined with the N-terminal domain of .TPO. The resulting sequence is:

Where the leader sequence was underlined and represents C...C from Cys' to Cys'! This different form can be treated after purification and refolding with the enzyme thrombin to generate a different form of TPO with an end Natural N with a length of 105 residues. The different isoforms in E.coli Most of the isoforms are found in refractile bodies, as observed in example (YY) for TPO (1-153©146).Similar steps were performed For the treatment, dissolution and purification of the various forms of monomeric TPO as described in Example No. (YY) similar refolding conditions were used as those used for (Met! 1-153) TPO with an AS yield of (Tr to 0 20 and after sale) folding the different forms of TPO were purified using reverse phase chromatography 0,0 in 70.1 TFA using graded amounts of acetonitrile as previously described: All forms of TPO (in their unproteolyzed form) are biologically active as assessed by a 138/7 test with half maximum activities of 1 - 0 picomolar. (B) Processing of hydrolytic protease of different forms (1), (1), to generate TPO (1-155) with a -N-terminus: a true terminal 0 leader that can be cut enzymatically by reducing the peptide using TPO. The variations (7) and (©) are designed by

Ally after refolding the TPO of the N amino acid residue at the end of the variants (7) and (©) as previously described; Each was subjected to enzyme digestion from the reverse-phase step,© by passing an acetonitrile stream for each form; The ake has been removed

711 - - calm of nitrogen in solution. The two variants were then treated with either factor Xa or thrombin, as described below. For the different form (7) of “TPO”, a Tris buffer, Tns buffer, was added at a concentration of 1 M; pH A pH ; to a solution free of acetonitrile to a final concentration of 0 mM ov ily and adjusted to pH 11 M at A value if necessary; NaCl and CaCl, 11 molar and 1 millimolar, respectively, were added. Factor Xa (New England Biolabs) was added to obtain a molecular ratio of about 1:5 to 100:1 between the enzyme and the desired form of TPO and the sample was incubated at room temperature for 1 - 7 hours to achieve a maximum cleavage of 0 as assessed by separation change with SDS magazines showing loss of leader AE. and then; The reaction mixture was purified using reverse phase chromatography©, using the same stepping volumes and conditions previously described for the purification of different folded conformations. The non-fractured heteromorph 8 was separated from the cleaved heteromorph (7) under these conditions. It turned out that the amino acid at terminus 17 is a “SPAPP,” indicating that the removal of the leader sequence at the vo N-terminus was successful. Factor Xa also generates various amounts of endogenous sequence within the TPO space, and cleavage was observed after the arginine residue at position number (VIA), which generates an additional sequence at the N-terminus that is TTAHKDP (ID number). Succession: (AA) on non-reducing lats; a single band at approximately 117001 D was observed for the cleaved form of factor Xa and on reducing gels two bonds with a molecular weight of approximately 1711 to 001 were observed Dalton" is consistent with the cutoff at .111 arginine and this observation also confirms that

The two parts of the molecule are held together by a disulfide bond between cysteine residues one and four; This was also inferred from the tryptic digestion experiments that were described previously. in the BaF biological test; The salvaged variant of 100-1 (700) after removing the leader sequence at tip 17 and by internal slicing had a half-maximal activity value ranging from 0 7 to 17 picomolar. The intact variant with the leader sequence had a half-maximum activity ranging from NY ¢ picomolar .picomolar For the different form ( ); the buffer used for digestion consisted of 0 μm Tris A pH, 77 CHAPS, 71 mol NaCl, #5 mM SEDTA Human or bovine thrombin (Calbiochem) at a ratio of 1:78 to OR:1 by weight between the enzyme 0 and the protein of the different form of TPO, and the digestion process was carried out at room temperature for a period of ? 1 h. Digestion efficacy was evaluated by Gellat SDS as previously described for the Xa Jalal cleavage reaction) Overall; more than 7550 cleavage for the leader sequence were achieved in that time. The resulting TPO was purified on Reverse phase columns Cy as described by Alle and it turns out that it contains ve on the N terminus required to study the amino acid sequence Only very small amounts (less than 5/) of the internal cleavage were obtained at the same arginine -threonine day) y as observed lle with coefficient Xa, and the resulting TPO protein had high biological activity with half the maximum responses in the Ba/F test at a protein concentration of 07-54 picomolar. In an ELISA based on the mpl receptor this protein had a half-maximal response at 1-Ye?; ng/ml purified protein -171 (740 picomolar) while the healthy variant containing Allie Leader was the lowest protein in both tests with a value ranging from 0 to 01

1195 - - times more than in animal studies; The selected sectioned protein was dialyzed by HPLC chromatography in physiologically acceptable buffers using NaCl at a concentration of 101 mM, Tween 80 at a concentration of 70.01, and acetate 00-LanH80 at a concentration of 01 mM, pH 0, 0 or sodium phosphate 01 mM + pH sodium phosphate § ©,0 pH m 01 mM; pH11M 7.4. The protein purified by HPLC and SDS-gelt was stable for several weeks while stored at ;m; It stimulates platelet production at doses as low as Fe ng / Js 0. Example No. (Ye) Synthetic mpl ligand: - Although the human mpl ligand ( WML) is usually synthesized using genetic engineering methods. Lind can be built by enzymatic ligation of structural peptide fragments using methods described later. Structural production 0 of Hml allows the incorporation of amino acids Mutant unnatural amino acids or synthetic functionalities such as polyethylene glycol and previously +0. Mutant subtilisin BPN enzyme (S22IC/P225A) subtiligase a2 3 “serine protease” was engineered to be Binding of peptide esters efficiently in aqueous medium (1991[4151-4159:30] (Abrahmsen et al., Biochem). It has now been shown that structural peptides can be enzymatically bound in a long, enzymatically active Ly chain peptides. and proteins such as the enzyme (Jackson et al., Science [1994]) ribonuclease A. This technique, which will be described later in more detail, enables the chemical construction of long proteins that previously could only be manufactured using DNA technology and genetic engineering. A general strategy for the synthesis of 1111.153 using the enzyme subtiligase is illustrated (Chart 1).

An unprotected peptide le 01 of a kidney corresponding to the C-terminal molecule of the protein, to which an ester peptide, the C-terminal activated, is added and protected at the terminal = 17, along with the enzyme subtiligase. when the reaction is complete; The product is isolated using HPLC in the Se phase, the protective group is removed from the oN-terminal, the Jl peptide part is attached and the 0 protection is removed, and the process is repeated using successive peptides until a full-length protein is obtained. The process is similar to the solid phase methodology in that the peptide activated at the C-terminal and protected at the N-terminal is attached to the N-terminal protected from the previous peptide and is synthesized. protein in the translocation direction. on the second hand; Because each coupling leads to an addition of up to 0 © 00 residues and the products are isolated after every day, longer proteins of higher purity can be built with a reasonable yield. Scheme No. (1): Method of constructing hML using the enzyme subtiligase and 00- 51-0601108 + R-NH-Peptide,-CO-R' Subtiligase (1 R-NH-Peptide, -CO-NH-Peptide,-CO, Zn/CHZCO,H )2 } H.N-Peptide,-CO-NH-Peptide4-CO, 3jrepeat 1 +2 H N-Peptide;-CO -NH-Peptide,-CO-NH-Peptide,-CO, 0 0 NH = — I~ oY wh NH;8 b 0 CY - 5 N.#© (CH NH, ‏

- 1 No - Based on our knowledge of the location of the subtiligase enzyme specificity of the sequence, and in addition to our knowledge of the amino acid sequence of the biologically active epo pad of 101, 1101153 was divided into seven Stas with a length of Yo -18 amino acids. Quadruple peptides were constructed to locate suitable ligands for the Yo-18 parts. Donor and nucleophile peptides at a concentration of 10 mM were dissolved in tricine at a concentration of 100 mM (pH (V,A pH) at a temperature of YY 2). An enzyme was added. ligase to a final concentration of 0.01 μM from a feedstock of 0.6 mg/mL (about μM Vo) and binding was continued overnight.The yield depends on the percentage of binding versus hydrolysis of the peptides donor hydrolysis ligation mu 9 glc-K-) Donor guild nucleophile set 116 : _ Ligation Hydroly NH; — Nucleophile (NH, % Yosis +A ay SRLS HVLH ( (Y £/vY) 1) Conformance ID: (Ad (Sequence ID No. “og) 4) HSRL SHVL (YF /1Y) 2 5 (Sequence ID No.: (3) (String ID No.: (AY VA YY SLGE AVDF (£V [£1) ¥ (Series ID number) (AY: | (Series ID number) (V6: or LLEG AVIL (Ve 9) ¥ 7 (No. Sequence ID: 0 (4) (Sequence ID number 1 2) LGQL LSSL (3+ JA) ¢m (Sequence ID number (AY): (Sequence ID number: (3A)

YVY - — EE (Sequence ID No.: 34) 1 (Sequential ID No.: (Vor) What (Sequence ID No.: 011) | (Sequential ID No. (Vo: (ID number 1) 017 : AJ (ID number (Vers Amal) (ID number of the sequence 1) Vf: (ID number of the sequence (Yeo:) (ID number of the sequence (Ve: | ) Sequence ID No. (Voy: (Sequence ID No. (V0 A)) | (Sequence ID No.: 0016) Based on these experiments, they should bind to the binding peptides shown in Table VE of Septley gas enzyme A suitable protecting group for the 17-terminus of each ester donor peptide was needed to prevent self-ligation An isonicotinyl protecting group—:(iNOC) group (Veber et al., J.

Org.

Chem., 42: 3286-3289 [1977]) ° because it is soluble in water; They can be included in the final step in solid phase peptide synthesis and are stable to the anhydrous HF used to deprotect and cut peptides from solid phase resin. in addition to; They can be removed from the peptide after each ligation under mild reducing conditions (20/011:00(217) to provide free N0 ends for the next ligation. A glycolate-lysyl-amide (gic-K-NH2) ester was used To activate the C-terminal based on previous experiments that showed that this is efficiently acylated using a separate gas enzyme ([1991], 4151-4159: 30: 30). (Abrahmsen et al, Biochen,

The amide-activated peptides 81-1-2106 were protected with P110 using standard solid phase methods as outlined in (scheme?). The peptides are chain-linked until a complete protein is produced, and the final product is refolded in the lab. Based on analogy with EPO; It is believed that disulfide pairs are formed between residues of (V) cysteine 0 5 (101) and between (YA) and -(0A). Slightly reduced under oxygen atmosphere for several hours. The refolded material can then be purified using HPLC and the fractions containing the active protein are collected and lyophilized. Alternatively, disulfide compounds can be differentially protected to control cascade oxidation between specific disulfide pairs. It will ensure the protection of cysteines (¥) (Yor) vs using the (YA) oxidized acetamidomethyl (acm) groups and (8/*). The acm groups can then be removed and the (V0) residues oxidized. ) 5 (V) Conversely, the (0A) 5 (YA) residues can be protected with acetamidomethyl (acm) and oxidized if consecutive oxidation is required for proper folding. Optionally, (YA) can be substituted cysteines and (4*) with another natural or unnatural residue other than Cys to ensure proper oxidation of (V) cysteines

A and (111).

16 — - Table No. (V€) Peptide fragments used for the total synthesis of L.1-141 using Septley Gas enzyme. Fragments in a sequence: 1 ( sequence ID: 0011) iNOC-HN-SPAPPACDLRVLSKLLRDSHVL-glc-K-NH, (1-22) ¥ ( sequence ID: 011) iNOC- HN-HSRLSQCPEVHPLPTPVLLPAVDF-glc-K-NH, (23-46) Y ( Sequence ID No.: 011) iNOC-HN-SLGEWKTQMEETKAQDILGAVTL-glc-K-NH, (47-69) ¢ ) No. Sequence ID: 011) iNOC-HN-LLEGVMAARGQLGPTCLSSLL-gle-K-NH, (70-90) © ( Sequence ID: 011) iNOC-HN-GQLSGQVRLLLGALQS-gle-K-NH; (90-106) ( Series ID: 0011) iNOC-HN-LLGTQLPPQGRTTAHKDPNAIF-glc-K-NH, (107-128) 1 ( Series ID: (VY) HoN-LSFQHLLRGKVRFLMLVGGSTLCVR -CO, (129-153) peptides are bound at tricine dp YO at a concentration of 100 mM A pH (freshly prepared and degassed by vacuum filtration through #0 filter Typically, the © terminal portion is dissolved in a sale organization peptide (at a concentration of ¥ = © m mM) and a 10x stock solution of enzyme subtiligase 1 (mg/mL in 001) is added mM tricine; pH (A pH) to reach a final enzyme concentration of © micromolar. An excess amount of 0 - ©molar of the activated donor peptide gle-K-NH, is then added. As a dissolved solid, the mixture is allowed to remain at a temperature of #7 “C. The mixture is monitored

YVo - — Bond formation by reverse-phase Cig column HPLC analysis (graded amounts of 0/007 with +7 TFA). The binding products are purified by preparative HPLC and lyophilized. Lyophilized Isonicotinyl (INOC) deprotection was performed by mixing 48 activated zinc powder with HCI with 6 M protected in acetic © .acid. The zine powder was removed by filtration and evaporated. acetic acid in a monogenic medium (.under vacuum) and the (Sl) peptide can be used directly in the next linkage and the process is repeated. 1 can be combined with procedures similar to those previously described for DOH: 14 structural or genetic engineering to produce . The constructive 1101 has many advantages compared to the output of genetic engineering. Unnatural 0 side chains can be introduced to improve potency or specificity. Polymer functionalities such as polyethylene glycol can be included to improve the effect period. For example, “Say” attaches polyethylene glycol to the lysine residues of the individual 58 molecules (Table 0?) before or after performing one or more binding steps. Peptides sensitive to the enzyme Protease bonds can be removed. or altered to improve stability in vivo nvivo Vo In addition, heavy atom derivatives can be synthesized to aid in structure determination.

Building Solid Phase Particles for Segment Ligation: (Y) Scheme No. -: Solid Phase Synthesis of Peptide Fragments for Segment Ligation

CeLS 1

NE) a wy (i) a

R 0 R i 1 2 0 9 R L, ¢ a a a (automated synthesis)

R 0 R = 3 0 ©0 Secret: . ide. NH NH 1 e, f (cleavage) oe (Hoy PE) c) 0 R 0 R 4 0 0 0

CY oA wy (Peptide) ~y NH A oy N he NH, 8! 5 0 Lilithic) 0 8 ° C NH, to make up the core

Isonicotiny! (iINOC) glycolate-lysyl-amide (gle-K-NH,) +,1Y) (MBHA) Lysyl-paramethylbenzhydrylamine (1) The resin was stirred (0 eq.) and °) bromoacetic acid with (Advanced Chem Tech /g; (b) The resin is washed thoroughly with sodium bicarbonate by stirring with (Bachem « 3 eq single)¥ eq. Boc-protected um for #01h at Y¢ for (DMF) dimethylformamide eq) in 1) 01 views (7). The input resin, glycolate-phenylalanyl-amide-resin, is washed on dichloromethane (5 mrsat) ¥) DMF using (Y) amino acetylated on it.

YVYV - -— (CHLCL) )¥ times) and the product can be stored at room temperature for several months 0 The resin (V) can then be packaged in an automated peptide synthesizer (Applied Biosystems 430A) and peptides are elongated using standard solid phase procedures (*). © (c) The 17-0-302 group is deparaffinized using a solution of trifluoroacetic acid 40 in .CH,(Cl,d). Amino acids are activated after protection with benzotriazol-1 N-a-Boc -yl -oxy-tris-(dimethylamino) phosphonium hexafluorophosphate (Bop) ¢ equivalent) and 01 (NMM) N-methylmorpholine equivalent) in DMA and is 1 read 1 sad - 7 hr. (e) The final N-a-Boc Ae gana (TFA)/:011:01 is removed to yield (;) and the isonicotinyl (INOC) protecting group is introduced as previously described (?;) by means of Il with 4-isonicotinyl-2-4-dinitrophenyl carbonate (substitutes) 5 NMM (1 equivalent) in DMA at 75 °C for 4 7 hours. Vo (and gives cutting and deprotection of the atu peptide by treating with anhydrous HF (8 (ethylmethyl sufide 70) [anisole] at zero degrees um for an hour; gives an activated peptide b glycolate-lys-amide protected with INOC (©) purified by HPLC on a reverse-phase 018 column (graded amounts of CH3CN

YVA - - Alig (TFA 71,1 HO) Verifying the identity of all materials by mass spectrometry Mass -spectometry Enables adding SUPPLEMENTAL ENABLEMENT The present invention is able to fulfill the objective as stated in the claims In accordance with the previous complete description and the available references in a simplified form as well as the starting materials. USA (ATCC) » American Type Culture Collection o Escherichia coli ATCC » Escherichia coli 1.1.8, RT-011103-0351; Collection No. (CRL 69575) filed on YE Feb(halal) 1344 ATCC 1 Plasmid 0 01 L051715.10.11.11.01, Lot No. 75958 “CRL Deposited Dec. Y; 144¢6E Cells ML 1/50MCB “CHO DP-12 (No. 1594) ¢ (ATCC Group No. CRL deasall (11770 in + december)” N98 This deposit is made in accordance with the provisions of the Budapest Treaty on the International Treaty 0 Deposit of Microorganisms Deposit of Microorganisms for the purposes of patent procedures and the provisions under these laws (Budapest Treaty) and this guarantees the maintenance of a viable live culture for a period of 71 years from the date of filing. The living organisms are provided by ATCC under the terms of the Budapest Treaty and the subject matter of the agreement between da Application 5 ATCC guarantees unrestricted availability upon issuance of the relevant US patent. don't prefix

- 1795 -

The availability of the deposited breed as a license to practice the invention as a breach of the rights secured under the authority of any

government in accordance with its patent laws.

WHILE THE INVENTION IS NECESSARY DESCRIBED IN CONNECTION WITH PREFERRED EMPLOYMENTS AND SPECIFIC RUNNING EXAMPLES; You will

shall become any of the persons of ordinary experience and after reviewing the foregoing full description; M is able to bring about different changes as well as equivalent substitutions and alternatives in the research topic mentioned here in

This document. Accordingly; Say the practice of the invention in ways different from those specifically described

here in this document.

All references cited in this document are expressly included by reference.

Claims (1)

Why 78 Protection_elements 1 1 - mpl ligand polypeptide Ade is obtained by the Jari Y process at:- 3 0 screening a human genomic library p using an oligonucleotide based on The genomic sequence based on the genomic sequence ° shown in Figure (8) for this purpose is to isolate genomic DNA 1 containing a sequence encoding the exon of a mpl ligand shown in Figure No. ( 3) for this purpose with the remaining exons of gene +5 A (b) inserting DNA into an expression vector and (c) q inserting the transfecting vector of an animal cell Shi mammalion cells Ye and expressing gene ¢ and 11 (3) extraction of polypeptide recovering ligand 1(” from \Y cell culture medium. Polypeptide — ¥ 1 of a mpl ligand obtained by a process that includes:- genmic DNA Je { Y from a genome libray and hybridises 3 under low stringency conditions with the sequence the following DNA:- GCCGTGAAGGACGTGGTCGTCACGAAGCAGTTATTTAGGAGTCG 3' t 5° which contains the encoding exons of the ligand polypeptide “mpl ligand 1 7 b) inserting the DNA into a readable vector expression vector ¢ and A transfecting vector of a mammalian mammalion cells -YAY- 1 and expressing the gene; And Yo d) Extraction of a polypeptide recovering mpl ligand from 11 cell culture medium. The polypeptide - ¥ 1 of the mpl ligand is obtained by a process including:- Y () Identification of 060085 suitable cellular source of RNA for a human mpl v ligand and preparation of one or more cDNA libraries of said RNA and 8 (9) Hybridisation - screening test for group or groups 1 using oligonucleotide based on coding sequence 7 shown in Figure No. (4) in this regard to identify and isolate A6 A clone containing a day) coding sequence “mpl ligand 1 and vector encoding encoded in an expression vector DNA inserting (c) insertion 01 into mammalian cells expression suitable for expression 0 11 and “mammalion cells 7 (a) (d) insertion of the transfecting vector into a mammalian cell ¢/mammalion cells and expression of the gene; and Yo (e) Extraction of a polypeptide recovering mpl ligand from VY cell culture medium. 1; - Polypeptide of mpl ligand as obtained by a process including:- mRNA from cDNA library or libraries or de gana collections prepared (0 ¥ YAY — 3 eextracted “human kiden cells” and (b) perform hybridisation - screening of the collection or 8 collections library or libraries using oligonucleotide based on 1 coding sequence The coding sequence shown in Figure No. (1) in particular to identify and isolate a clone that contains A coding sequence with a “mpl ligand” and 9 (©) input DNA transfecting encoded by coding in an expressive vector Vector Vo 0 _ is suitable for expression in WIS mammalian mammalion cells « and 1 (d) Transfecting a mammalian cell mammalion cells with the vector and expressing the gene expressing gene ¢ and YY(e) polypeptide recovering mpl ligand from cell culture medium \¢ 1 - mpl ligand complex isolated and essentially homogenous substantially homogeneous isolated Y is characterized by the following:- The 0 ligand induces the inclusion of nucleotides «incorporation» (3H thymidine)labelled ¢ in the DNA in 1/3 cells dependent o on IL-3 transfected with human mpl P inserted “1 (b) ligand induces 4S incorporation of platelets 7 in circulating blood in the reverse response test for platelet rebound assay A; (z) 9 The ligand of the ligand is stable at a pH of SDS 7.5 pH at a concentration of Ye 9660.1 and at a concentration of ¥ molar 21/100168 — YAY - “glycoprotein is a ligand ligand 8 11 is a polypeptide of the amino terminal sequence the amino terminal sequence (0) \Y VY is:- P” SPAPPACDPRLLNKLLRDDHVLHGR or ,. SPAPPACDLRVLSKLLRDSHVLHSRL Yo polypeptide - 1 1 of an isolated human mpl ligand comprising a Y sequence amino acid sequence and is biologically active l .biologically active apkastic Lait Obtained by regurgitation from plasma mpl ligand 1 - 1 hydrophobic interaction 4a by affinity chromatography plasma Y immobilised dye oa « chromatography ¥ And the “mpl - affinity chromatography and chromatography with affinity” ¢ “apparent molecular weight mentioned has an apparent molecular weight mpl ligand ligand 0 “reducing conditions Jb reduction conditions in SDS-PAGE as well as detection by analysis 1 kDa 00 or kD kDa YA or kD kDa YY TVA in bounds from v kD A polypeptide - A \mpl ligand isolated nucleic acid encoded by a nucleic acid has sequence 6 hybridizes under moderate conditions 3a all moderately 1 with uncleic acid nucleic acid has a homology of 119 nucleotides to 14%? JSAP number (1) (sequence ID number: ; or the sequence ID number YAS —— sequence 0 : *). 1 4 - mpl ligand isolated Wh isolated to any of the preceding claims Y is biologically active -mpl ligand day polypeptide — 0 1 has at least 96700 saturated percentage shares identity Y of the identity of the identical sequence with the polypeptide according to any of the v protection elements from No. (1) to (A) polypeptide - 1 ligature mpl ligand is an allele or a variant Y of a polypeptide according to any of claims No. (1) to (A) or and part of said mpl ligand or allele or variant ¢ that shares at least one biological feature with -mpl ligand mpl ligand day -1 1 according to Claim No. (AL Cua) 11 Possible Y locus modification or more to cut with a protease enzyme cleavage site in order to make it resistant to cut -resistant to cleavage 1 lish mpl ligand day - 1 1 of protection element number (1) 'where the mentioned potential fission site is at the amino acid position 104 -110 and/or “Yo” © 746 for the bonding compound mpl as described in Figure (16) —_ Qim — 1 4 - mpl ligand according to Claim No. (VF) where Y 10 cleavage site at position Jai YL 104 YoY is disabled .substitution 1 101 1 - mpl ligand according to Claim No. (41), where Y is substituted for the residues Arg-Arg residues at ?1-; 10 building blocks residues science. 110 - A variant of the mpl ligand according to claim no. (11); comprising Y fusion 3 with a heterogeneous polypeptide . 17 1- mpl ligand according to claim No. (1) or claim No. (V1) comprising fusion with a residue residues one or more amino acids at the amino terminus and .amino - and/or carboxy terminus YA 1 - mpl ligand of claim No. (VV) having methionine Y at the N-terminal polypeptide — 3 1 of the mpl ligand according to any of the claims from No. (1) Y to No. (+) and from (A) to (VA) where the mpl ligand is extracted from 1 human strains. human species polypeptide - 0 1 of the mpl ligand according to any of the claims No. 0 (7) to No. 0 and (7) to 0” (YA) wherein the mpl ligand is extracted from 3 non-human strains .non -human species polypeptide - 1 "1 mpl ligand according to which of the above claims 7 did not enter the .unglycosylated YY group 1 - mpl ligand according to For any of the previous claims, Jas yall b non-proteinaceous polymer 1 ©?- mpl ligand according to claim number (YY) where the said polymer Y is For polyethylene glycol, polypropylene, or polyoxyalkylene 1; (YY) YO 1 An isolated nucleic acid molecule is selected Y from :- 7 0 cDNA clone as then obtained from (ii) 3 hill in Claim ¢ (©) or step (ii) in claim no. (;); and 0 (b) DNA sequence capable of hybridizing under stringent conditions 1 to a clone From item (7) and (z) Y a different genetic form of any of the DNA sequences (00) A and (B) which encodes a polypeptide that has a biological characteristic such as polypeptide q for a natural mpl ligand. YAY - — 1 1 - An isolated nucleic acid with a sequence that hybridizes under moderately stringent conditions to a nucleic acid with a sequence of nucleotide 1956-1194 as in the form of the number ¢ (5) (sequence ID number: 6 or sequence ID number 01) and encodes ° .mpl ligand VV ligand 1 - acid S35 isolated Gb isolated nucleic acid of protection element No. (1) Y hybridizes by hybridizing to 118 nucleotides and then to 197 nucleotides as in Figure L No. (5) (sequence ID number : ¢ or sequence ID number : 0) and encodes a mpl ligand or fragment thereof that shares a single biological © feature on BY with the mpl ligand genomic DNA - YA 1 or cDNA equivalent to at least a portion of the mpl ligand Y gene that hybridizes in |b highly stringent conditions 3 using ror nalural nucleic acid molecule has sequence ¢ encoding a ligand “mpl ligand where mpl ligand is as 0 § specifying it in any of the claims from number (1) to (MN) 1 4 — $95(Gb nucleic acid) of any L(Y) (YA)Y claimant operably linked .promoter . YAA — - 0 1 - vector encoding expressive vector containing nucleic acid Y according to any of the claims number (YE) through (79) actively binds to 3 control sequences Accepted control seauences recognized host cell. ©11 - a host cell transformed with DNA according to any of the Y protection elements from number (Y¢) to number (0 *) to be able to express mucleic 7-DNA acid to produce a polypeptide for ligand .mpl © 1 - Process involving expression of engineered nucleic acid recombinant Ld, nucleic acid Y according to any of the claims of (YE) no. (Ve) 1 host cell suitable for the production of polypeptide of mpl ligand 1 *© - process according to claim no. (PY) in which polypeptide Y of mpl ligand is extracted ligand from the host cell or host cell culture medium 7 .1 0© - a process of claim No. (FY) or claim No. (FF) in which it is 7 The host cell is a progeny cell line .CHO cell Yo 1 - process involving expression in cell gene of ligand 40 1 natural 100860005 under the control of a promoter [promoter] enhancer 1 or suppressor or exogenous “transcription modulatory element ¢ which is inserted into the cell genome e is nearby and in a favorable orientation to affect DNA transcription encoding polypeptide 1 of .mpl ligand 1 “© - a composition comprising a mpl ligand according to any of the claims of No. (1) Y to (YF) or as obtained by a process pursuant to any of the claims from ?No. (FY) to (YE) and a pharmaceutically acceptable carrier ¢. FV 1 - A process involving the use of a polypeptide of a mpl ligand pursuant to any of “claims No. (1) through No. (17) or obtained by the process pursuant to any of the 3 claims of No. (VE) (VY) in preparation of jlie 0 medicament ¢ FA) - formulation according to claim No. (M1) may be obtained by process according to " - of claim No. (VV) Which in addition to the above includes an effective ladle and effective amount of agent selected from cytokines, colony stimulating factors and interleukin FA. - Composition according to Claim No. o FA) where the agent is chosen from LIF IL-25 IL-1 5 Epos M-CSF 5s GM-CSF y G-CSF5 "and 3C11A IL-55 IL-75 JL-115 IL-9 5 IL-8 5 Y — Yq. _ nucleic acid test sample an amlifying method for supplying -6 1 a nucleic acid priming nucleic acid polymerase reaction involving the activation Y corresponding to at least part of the nucleic acid using a nucleic acid polymerase and (YA) according to cDNA or genomic claim number 1 ¢1; - a method for determining the presence of a polypeptide of a mpl ligand involving hybridizing a nucleic acid according to of claim number (YA) to ¥ nucleic acid of a test sample and determination of the presence of nucleic acid of the polypeptide of the -mpl ligand ¢1"; — process involving the use of the polypeptide of the Lik ligand A ligand of any of the (1) to (7) protectors or obtained by the lik process for any of the 7 protectors (YY) to (VE) in a specific antibody preparation ¢ for ligand mpl 1; - a process according to Claim No. (aly dua ¢) ey preparation of a monoclonal antibody clone Y 1 £4 - an antibody that is Obtained by the process pursuant to Claim No (47). (17) to No. (1) M “according to any of the claims from No. 0.” Yay - - 1?? - An antibody according to protection element No. (£0), which is a monoclonal antibody Y - monoclonal antibody tv 1 - a hybrid cell line that produces the antibody according to 1 mpl ligand as originally described herein. 1 4 - isolated nucleic acid molecule s ze buy encoding 6 of the mpl ligand or of the mpl ligand gene as originally described in this document. As described in Principally Y of this document. It is also primarily described in this document. duals 443 - oY 1 is a host cell transformed with a nucleic acid encoding the polypeptide encoding Y of ligand 01t as primarily described herein. yay — - oF 1 - a process involving the expression of expression of a genetically engineered nucleic acid Y recombinant in a host cell suitable for the production of a polypeptide for an emp ligand It is also primarily described in this document. of 1 - Method of providing amlifying “using the polymerase chain reaction 1 ¢ of a nucleic acid test sample that is thought to contain 1 nucleic acid encoding a mpl ligand 0 of 3 mpl ligand gene as originally described in this document. 1 00 - Pharmaceutical composition comprising a polypeptide of the ligand “mpl Y” as stated in this document. 1 0% - A spevific antibody specific for the empl ligand as originally described 0 herein. polypeptide - ov 1 has the following amino acid sequence: - SPAPPACDPRLLNKLLRDDHVLHGR v or SPAPPACDLRVLSKLLRDSHVLHSRIL £ + DNA - oA 1 isolate Wh polypeptide encoding ily isolated of protector number 0 (OV).