WO1992000319A1 - Proteine de fsc-meg humaine et procedes - Google Patents

Proteine de fsc-meg humaine et procedes Download PDF

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Publication number
WO1992000319A1
WO1992000319A1 PCT/US1991/004698 US9104698W WO9200319A1 WO 1992000319 A1 WO1992000319 A1 WO 1992000319A1 US 9104698 W US9104698 W US 9104698W WO 9200319 A1 WO9200319 A1 WO 9200319A1
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Prior art keywords
csf
protein
hmeg
meg
fraction
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PCT/US1991/004698
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English (en)
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Martin J. Murphy
Ralph E. Parchment
Connie L. Erickson-Miller
Wei Dai
Zhao-Geng Zhang
Lance A. Liotta
Henry Krutzsch
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Hipple Cancer Research Center
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Priority to KR1019920703438A priority Critical patent/KR930701475A/ko
Priority to JP3512921A priority patent/JPH06502621A/ja
Publication of WO1992000319A1 publication Critical patent/WO1992000319A1/fr
Priority to NO92924995A priority patent/NO924995L/no
Priority to FI925983A priority patent/FI925983A/fi

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to an isolated human megakaryocyte-colony stimulating factor protein (hMeg-CSF), to pharmaceutical formulations comprising said factor, and to methods relative to producing, isolating and using the same.
  • hMeg-CSF human megakaryocyte-colony stimulating factor protein
  • the hematopoietic i.e., the blood-forming system
  • the bone marrow is responsible for producing the cellular elements of the blood.
  • Mammalian (including human) blood is composed of minute cellular fragments which are called platelets or thrombocytes, and highly specialized cells called red blood cells (erythrocytes) and white blood cells (leukocytes) all of which are suspended in the plasma. Platelets perform an essential function in the mammalian clotting system: in response to wound-induced chemicals, they cause blood to clot.
  • the red blood cells are responsible for imparting to blood its characteristic deep red color and for transporting oxygen (0 2 ) and other nutrients to the tissues throughout the mammalian body where the 0 2 and other nutrients are exchanged for carbon dioxide (C0 2 ) and waste products.
  • the white blood cells on the other hand are responsible for defending the body against infection. Because the blood in the body is in constant motion, i.e., it is circulating through a closed network of blood vessels, both platelets and blood cells are normally well-suspended in the plasma.
  • the mature red and white blood cells as well as the platelets, i.e., the cellular components of the blood, are formed from primitive undifferentiated precursor cells produced in the mammalian bone marrow. These undifferentiated precursor cells are variously referred to as pluripotent stem cells or progenitor cells. Each stem cell has the potential to differentiate and develop into either mature erythrocytes (red blood cells), or leukocytes (white blood cells) or
  • the stem cells are regarded as primitive pluripotent precursors to the mature erythrocytes, leukocytes and megakaryocytes.
  • hematopoietic system are developed from the primitive undifferentiated stem cells produced in the bone marrow, as illustrated in Fig. 6.
  • Hematopoietins which are also commonly known as blood-cell growth factors, are a specialized group of glycoproteins that promote growth and differentiation of pluripotent stem cells into mature blood cells. See Fig. 6.
  • Colony stimulating factors (CSFs) are a specific class of hematopoietic growth factors or proteins which are believed to be capable of initiating precursor cell proliferation and differentiation into the different types of mature blood cells.
  • CSFs are believed to be responsible for causing primitive undifferentiated precursor cells to commit to, and develop in accordance with, a specific blood cell lineage, i.e., to commit to either the erythrocytic, leukocytic or megakaryocytic lineage.
  • a specific blood cell lineage i.e., to commit to either the erythrocytic, leukocytic or megakaryocytic lineage.
  • the particular type of mature blood cell that results from a primitive undifferentiated precursor cell depends upon the type of hematopoietins the stem cell encounters.
  • erythropoietin causes primitive undifferentiated precursor cells in the bone marrow to commit to the erythrocytic lineage, i.e., to differentiate and mature into erythrocytes, whereas granulocyte/macrophage colony-stimulating factor (GM-CSF) is believed to cause the precursor cells to differentiate and mature into particular types of leukocytes called granulocytes and monocytes.
  • EPO erythropoietin
  • GM-CSF granulocyte/macrophage colony-stimulating factor
  • Platelets are terminal products of megakaryocyte differentiation. Megakaryocytes also originate from primitive undifferentiated precursor cells of the bone marrow, as illustrated in Fig. 6.
  • the earliest recognizable member of the megakaryocyte series developed from stem cells in the bone marrow is the megakaryoblast, which possesses an immature nucleus embedded in a basophilic cytoplasm with a minimum number of granules. Megakaryoblasts and megakaryocytes can be identified by specific cell surface markers; acetylcholinesterase in murine cells and Ilb/IIIb in human cells. Through a complex maturation process, megakaryoblasts mature into
  • megakaryocytes the process involves the formation of a multilobulated polyploid nucleus and distinctive, highly specialized cytoplasmic granules, as illustrated in Fig. 6.
  • the mature megakaryocytes form platelets by pinching off fragments of their cytoplasm and releasing them into the circulating blood, as depicted in Fig. 6, by a process that is not yet well-understood.
  • thrombocytopenia As indicated above, platelets are critical minute cytoplasmic particles which regulate blood clotting. Depletion of the circulating level of platelets, called thrombocytopenia, occurs in various clinical conditions and disorders. Thrombocytopenia is dangerous because patients with this condition are subject to uncontrolled bleeding episodes. If the cause of thrombocytopenia is an external insult or injury (as opposed to a disorder in the production, or maturation of megakaryocytes and platelets) platelet levels are usually restored within a short period of time (about 4-5 days in humans) if the injury or insult (chemical) has been removed. But if a platelet disorder underlies this condition, it persists for as long as the disorder is present, often throughout the patient's life. The only treatment up to the present time has been frequent platelet transfusions with all the attendant dangers that it entails (which range from infection to immune reaction).
  • TPO thrombopoietin
  • TSF thrombopoietic stimulatory factor
  • Meg-CSF megakaryocyte colonystimulating factor
  • megakaryocytes is regulated by thrombopoietin and that in fact the blood concentration of thrombopoietin is in turn influenced by changes in the level of circulating platelets. See, for example. Murphy, M.J., et al., Acta Haematol. JPN. 46(7):
  • IL-3 interleukin-3
  • This protein has a molecular weight of about 14-28,000 daltons and an isoelectric point (Pi) equal to about 4.5-8.0.
  • this IL-3 protein has the ability to stimulate formation of granulocyte, macrophage, erythroid, and megakaryocyte colonies from bone marrow in vitro
  • the human form of the IL-3 protein is species-specific and unable to produce megakaryocyte colonies in an in vitro murine fibrin clot assay.
  • EPO erythropoietin
  • GM-CSF granulocyte/macrophage colony-stimulating factor
  • IL-6 interleukin-6
  • IL-6 has a molecular weight of about 2126,000 daltons and a Pi equal to approximately 6.2-6.4 as determined by chromatofocusing of plasmacytoma growth factor.
  • the IL-6 is reported as having the ability to increase mature megakaryocyte size, the number of megakaryocytes with higher ploidy, and the number of cells in a megakaryocyte colony (all which are maturation-type functions) in vivo, the reported IL-6 lacks the ability to produce megakaryocyte colonies in vitro. See, for example, Lotem J., et al., Blood 14:1545-1551 (1989); Ishibashi T., et al., Proc. Natl. Acad. Sci. 86: 5953-5957 (1989); and Bruno E., et al., Exp. Hematol. 11:1038-1043 (1989).
  • TPO thrombopoietin
  • the TPO protein is said to be an acidic protein having a molecular weight of about 15,000 daltons and a Pi equal to about 4.5.
  • the thrombopoietin protein is stable to 2-mercaptoethanol.
  • This TPO is reported to increase the diameter of megakaryocytes, a maturation-type function, but is unable to stimulate formation of megakaryocytic colonies from bone marrow in vitro. See also, McDonald, T.P., et al., Int. J.
  • MSF megakaryocyte stimulating factor
  • IL-9 interleukin-9
  • IL-9 is reported to have a molecular weight of approximately 20-30,000 daltons or a molecular weight of 32-39,000 daltons based on information for murine p40, the murine homologue of human IL-9, and a Pi equal to approximately 10 based on flow-through on MonoQ Chromatography at pH 9.5, as reported by Uyttenhove, C, et al., Proc. Natl. Acad. Sci. 85:6934-6938 (1988).
  • the IL-9 protein is reported as having the ability to stimulate erythroid colony formation in vitro: see Yang, Y-C, et al., Blood 74 (Suppl. 1) :116a (1989); but no ability to stimulate formation of megakaryocyte colonies in vitro. Donahue, R.E. et al., Blood, 1990, 15:2271-2275.
  • Meg-CSF type proteins For instance, Kawakita, M., et al., Br. J. Haematol. 52: 429-438 (1982), reports a protein having a molecular weight of approximately 155,000 daltons and 76,000 daltons by gel filtration (Sephadex G-200), and 45,000 daltons when the gel filtration is performed with 6 M
  • Meg-CSF protein a Meg-CSF protein
  • the existence, identity, structure and biological activity of this postulated MegCSF protein (or proteins) has up to now remained elusive and controversial. Consequently, there are serious needs in the scientific and medical communities to confirm the existence, identity and activity of a human Meg-CSF protein and to isolate, sequence, and reproduce same for purposes of, among other things, combatting and better understanding the causes of thrombocytopenia, and the mechanism of platelet production.
  • the present invention alleviates the above-mentioned problems and shortcomings of the present state of the art through the discovery of a novel, isolated homogeneous human megakaryocyte-colony stimulating factor (hMeg-CSF) and methods of obtaining same.
  • hMeg-CSF human megakaryocyte-colony stimulating factor
  • the present invention is directed to an isolated, purified human megakaryocyte colony stimulating factor, said factor having the following properties:
  • the present invention is directed to an isolated, purified human megakaryocyte colony stimulating factor preparation comprising at least about 90% protein said preparation being characterized as:
  • hMegCSF having the ability to induce the formation of megakaryocyte colony forming units in a murine fibrin clot assay in vitro.
  • This highly purified but non-homogeneous hMegCSF fraction can be used to elucidate the sequence of hMeg-CSF as well as the mechanism of platelet production.
  • the molecular weight of human Meg-CSF protein species in this virtually pure fraction, when the protein is in glycosylated and sialyated form is within the range of about
  • a further aspect of the present invention is directed to isolated, purified recombinant polypeptides having human megakaryocyte colony stimulating factor activity and to methods for isolating DNA encoding this factor.
  • Another aspect of the present invention is directed to a pharmaceutical formulation for administration to a mammal suffering from a disease related to the production of platelets comprising an isolated, purified human megakaryocyte colony stimulating factor protein, said protein having the following properties:
  • a still further aspect of the present invention is directed to a pharmaceutical formulation for administration to a mammal suffering from a disease related to the production of platelets comprising an isolated, purified polypeptide having human megakaryocyte colony stimulating factor activity and comprising at its amino terminus the amino acid sequence X-AspPro-Val-Glu-Ser-Pro-Val-Pro-Y (wherein X and Y are unspecified amino acid residues).
  • Yet another aspect of the present invention is directed to a method for isolating a human Meg-CSF protein fraction said fraction having a protein content of at least 90% and being free of EPO and GM-CSF activity, said method comprising the steps of:
  • step (e) A variation of the foregoing purification scheme comprising chromatofocusing in step (e) and further comprising step (g) cation exchange HPLC following step (f) yields
  • Meg-CSF which includes either the larger species alone or a combination of the larger and the smaller species ).
  • Figs. 1A and 1B are general outlines of the preferred steps used in the present invention to isolate hMeg-CSF protein in substantially pure, virtually pure, functionally homogeneous and homogeneous form, from urine of aplastic anemia patients.
  • Fig. 2 is a graphic illustration of various hMeg-CSF containing fractions recovered following preparative 5%
  • Fig. 1A polyacrylamide gel electrophoresis of crude urine extract obtained from aplastic anemia patients in accordance with the method of the present invention
  • Fig. 2 also illustrates graphically the number of CFU-Meg and CFU-GM colonies stimulated by the protein fractions.
  • Substantially pure hMeg-CSF protein is in fractions 1-5.
  • Fig. 3 is a graphic illustration of various protein fractions isolated from an isoelectric gel run at a pH range of about 3.5-10 in accordance with the methods of Fig. 1A using the DEAE-Cellulose ion exchange support and the IEF pathways.
  • Fig. 3 also illustrates graphically the amount of protein per fraction, the number of CFU-Meg colonies stimulated by each protein fraction, and the pH of each protein fraction.
  • Virtually pure hMeg-CSF protein is in fraction number 16. Protein fraction number 16 stimulates the formation of the largest number of CFU-Meg colonies in vitro (compared to the other fractions, as illustrated in Fig. 3) and has a pH of between about 7.2-7.4.
  • Fig. 4 is a graphic illustration showing the absorbance at 280 nm, the number of CFU-Meg colonies formed and the pH of groups isolated from the CM-Sepharose ion exchange support and the MonoP chromatofocusing column pathways in accordance with the methods of Fig. 1A.
  • Virtually pure hMegCSF protein is in group number 5.
  • Protein group number 5 stimulates the formation of the largest number of CFU-Meg colonies and has a pH of about 7.0-7.5.
  • the protein fraction under group number 5 has three main peaks of protein.
  • Fig. 5 is a graphic illustration showing the number of CFU-Meg colonies formed, the percent of solvent (acetonitrile) and the absorbance at 280 nm of protein fractions obtained from the CM-Sepharose ion exchange support, the MonoP and the C18 reverse-phase HPLC pathways in accordance with the methods of the instant invention (Fig. 1A).
  • Isolated, hMeg-CSF protein is in fraction numbers 32-34. Protein fraction numbers 32-34 stimulate the formation of the largest number of CFU-Meg colonies and fall between two absorbance peaks as illustrated.
  • Fig. 1A Isolated, hMeg-CSF protein is in fraction numbers 32-34. Protein fraction numbers 32-34 stimulate the formation of the largest number of CFU-Meg colonies and fall between two absorbance peaks as illustrated.
  • FIG. 6 is a pictorial illustration generally depicting the development and differentiation of the various components of the blood from a primitive precursor undifferentiated pluripotent stem cell in accordance with the erythrocytic, leukocytic and megakaryocytic lineages.
  • Fig. 6 has been generally quoted and reproduced from a photograph appearing in the Schering Plough/Sandoz Pharmaceuticals 1990 calendar for background purposes and, more particularly, for illustrating and generally teaching the development, differentiation and production of the highly specialized blood cells and platelets from stem cells in the bone marrow.
  • Fig. 7 is a photograph of a silver stained SDS-PAGE gel of a purified Meg-CSF-containing preparation of the invention and depicts in vertical lane labelled "fraction number 3234", protein bands of isolated functionally homogeneous hMegCSF species, one having a molecular weight of about 24,00035,000 daltons and the other having a molecular weight of about 50,000-70,000 daltons, as determined by 12% analytical SDS-PAGE stained with silver (BioRad, Richmond, CA).
  • the five vertical lanes depicted in this Fig. 7 contain fractions from C18 reverse-phase HPLC generated in accordance with the method of Fig. 1A. Molecular weight markers (kD) are shown to the right of the five fraction lanes.
  • the hMeg-CSF protein of this Fig. 7 is isolated via the CM-Sepharose ion exchange support, the MonoP and the C18 reverse phase HPLC pathways in accordance with the methods of this invention.
  • Figure 8 depicts the results of Meg-CSF transfer to Immobilon PVDF Membrane and in particular the single band obtained upon SDS-PAGE of homogeneous hMeg-CSF (produced according to the Fig. IB scheme) and transfer to an Immobilon PVDF membrane.
  • This single band is the higher m.w. species. The lower m.w. species is not evident.
  • Fig. 9 is a chromatographic profile of Meg-CSF purification on a polyaspartic acid (WCX) HPLC column. Absorbance was monitored at 280 nm with a full scale of 0.1 absor-nadoe units. Flow rate is 1 mL/min. Initial column equilibration is in 0.05 M sodium phosphate, pH 6.0. The gradient of increasing NaCl is signified by the dashed line. 2.5 ml fractions are collected and pooled as follows:
  • Fig. 10A is a photograph of a silver stained SDS-PAGE gel of individual fractions #16-28 from two WCX HPLC columns concentrated by Centricon 10 (Amicon: Danvers, MA). Aliquots of individual fractions were put in SDS-PAGE sample buffer
  • Fig. 10B is a bar diagram of the Meg-CSF activity of other aliquots of the individual fractions 16-30 from the two HPLC WCX columns above which were sterile filtered and assayed for biological activity by murine fibrin clot assay.
  • Fig. 11A is a bar diagram showing the Meg-CSF activity profile of SDS-PAGE elution as a function of gel slice position.
  • This figure reports the biological activity of each slice and its corresponding molecular weight after SDS PAGE under non-reducing conditions.
  • Fig. 11B is the same type of diagram generated after SDS PAGE under reducing conditions. A shift in activity from the high m.w. species to the low m.w. species can be seen, which indicates that the 24-35 kD species may be a fragment or monomer of the 50-70 kD species.
  • Fig. 12 is an autoradiograph of DNA produced by polymerase chain reaction. Placenta genomic DNA was purchased from Clontech Lab, Inc. (CA). One ⁇ g of the DNA was amplified using oligo 1 and 3 according to the protocol provided by
  • Lanes 9 and 10 show the 300 bp and 1.7 kbp fragments re-amplified respectively with oligos 1 and 3.
  • Lane 11 represents the primary amplification product of genomic DNA using oligos 1 and 3.
  • Lane 8, molecular marker (one kb ladder, BRL).
  • “Mammals” is defined herein to mean any organism having a hematopoietic system and susceptible to a disease related to the production of platelets and includes humans.
  • Fully homogeneous human Meg-CSF is defined herein as a hMeg-CSF fraction, although not purified to homogeneity, contains no other detectable hematopoietins (as assessed by standard activity tests: Absence of erythropoietin contamination is assessed by murine spleen cell assay according to Krystal G., Exp. Hematol., 1983, 11:649-660, which can detect as little as 0.05 units/ml of EPO activity; absence of GM-CSF, IL-3 and IL-9 is assayed by M-07-e bioassay according to
  • an hMeg-CSF protein contained in this fraction was used to generate the amino terminal amino acid sequence obtained as shown in Example 5 below, which was identified from the larger species (50-70 kD) even though the smaller species (24-35 kD) was also present in the preparation. Either the smaller species does not have the amino terminal or it is blocked (i.e. not free) or the amount of material available was not sufficient to permit sequencing of the N-terminal from the smaller species.
  • the preparation of Fig. 7 which is "functionally homogeneous" does contain other bands in addition to the two species having hMeg-CSF activity. Therefore, there is no implication here that only the two hMeg-CSF species may be present in a functionally homogeneous preparation.
  • “Homogeneous human Meg-CSF” is defined herein as a polypeptide which has human Meg-CSF activity, migrates as a single band upon electrophoresis in SDS-PAGE gels and after transfer to PVDF Immobilon membrane has a single amino terminal amino acid sequence. While this definition is cast in terms of the higher m.w. species, copresence of the lower m.w. species should not be interpreted as negating homogeneity. The 24-35 kD species has hMeg-CSF activity of its own. This is apparent in Fig. 11B. Moreover, the fact that a preparation containing both species is still “functionally homogeneous" and does not have any other cytokine negates the possibility that the lower m.w.
  • Human Meg-CSF protein (of either or both molecular weights) can be isolated from the urine of patients suffering from aplastic anemia (or another condition, such as bone marrow transplant or thrombocytopenia of a different origin which causes Meg-CSF activity to be present in the urine), but should be purified to homogeneity for therapeutic use.
  • hMeg-CSF derived from natural sources should also be purified to functional homogeneity or homogeneity before it is sequenced or otherwise used in preparation of recombinant or synthetic techniques for producing recombinant hMeg-CSF (e.g., for producing monoclonal antibodies). Because of species crossreactivity between mouse and human, it is anticipated that the hMeg-CSF of the present invention will be useful in treating other mammals, such as pets which are in need of such treatment (e.g., pets undergoing chemotherapy).
  • the hMeg-CSF protein of the instant invention has a Pi of about 7.2-7.4 as determined by isoelectric-focusing and a molecular weight ranging between about 50,000 and 70,000 daltons (or 24,000 and about 35,000 daltons for the smaller species) as determined by SDS-PAGE when the hMeg-CSF protein is in the glycosylated and sialyated form, as illustrated in Fig. 7.
  • carbohydrate residues including the biantennary carbohydrate structures can be cleaved from the hMeg-CSF protein (either species) via appropriate glycosidases, such as endoglycosidase F, endoglycosidase H and N-glycanase.
  • the sialic acid moieties of the hMeg-CSF protein can be removed by treatment with neuraminidase. Even when the carbohydrate and sialic acid moieties are cleaved from the hMeg-CSF protein of the instant invention (both species) to form a naked hMeg-CSF protein, it retains its biological activity in vitro.
  • the novel hMeg-CSF protein of the instant invention which is preferably purified to homogeneity, has the ability to regulate megakaryocytopoiesis and platelet production. More particularly, the novel hMeg-CSF protein of the present invention has the ability to stimulate proliferation of megakaryocytes and production of platelets in vivo, and has the further ability to stimulate proliferation of megakaryocytic lineage type cells, e.g., megakaryocyte-colony forming units into megakaryoblasts, in an in vitro murine mouse megakaryocytecolony forming fibrin clot assay as well as in a serum-free system.
  • megakaryocytic lineage type cells e.g., megakaryocyte-colony forming units into megakaryoblasts
  • each species is able to induce primitive precursor cells in the bone marrow to commit to, and to grow and differentiate in accordance with, the megakaryocytic lineage.
  • the present invention which has eluded the scientific and medical communities heretofore, is predicated upon the confirmed discovery (as well as the isolation, purification and characterization) of a novel human hematopoietin or blood cell growth factor, i.e., a human megakaryocyte colony-stimulating factor, which is believed to be involved in at least the first phase of human megakaryocytopoiesis.
  • the present invention is based upon the discovery of a unique protein produced by humans which is specifically involved in the production of blood platelets.
  • the higher m.w. hMeg-CSF protein of the present invention is a weakly basic, homogeneous protein as judged by isoelectric focusing, SDS-PAGE, chromatofocusing, C18 reversephase and WCX cation exchange HPLC and is characterized as having the single N-terminal amino acid sequence X-Asp-Pro-ValGlu-Ser-Pro-Val-Pro-Y, wherein X and Y represent as yet undetermined amino acid residues.
  • hMeg-CSF protein retains its ability to increase platelet counts and bone marrow megakaryocyte numbers m vivo following amidation or treatment with neuraminidase, it has been found to lose some activity following treatment with 5,5,-dithio bis 2-nitrobenzoic acid (DTNB).
  • DTNB 5,5,-dithio bis 2-nitrobenzoic acid
  • Reduction of the hMeg-CSF of the invention does not by itself cause inactivation although it causes a shift of the bulk of the activity towards the smaller species.
  • reduction by dithiothreitol
  • alkylation with iodoacetamide
  • mercuration with mercury chloride
  • the biological activity of the novel hMeg-CSF protein of the instant invention is believed to be lost when greater than about 30% of the amino acid residues thereof are carbamylated (since carbamylation was not preceded by reduction both species are presumed to have been inactivated).
  • the hMeg-CSF protein of the instant invention is further characterized as a glycoprotein having biantennary carbohydrate structures and beta-galactose residues as the terminal or penultimate sugars.
  • the novel hMeg-CSF of the instant invention is further characterized as containing sialic acid. These characteristics are confirmed by the binding characteristics of the hMeg-CSF protein to an RCA I agarose. Con A Sepharose and Lentil Lectin columns in sialyated and desialyated forms.
  • hMeg- CSF protein activity binds to an RCA I agarose column
  • about 41% hMeg-CSF protein activity binds to a Con A Sepharose column (about 31% activity following elution with about 15 mM alphamethylglucoside or about 10% activity following elution with about 200 mM alpha-methylglucoside)
  • about 45% hMeg-CSF protein activity binds to a Lentil Lectin column. The portion of the binding activity that is attributable to each species has not been determined.
  • hMeg-CSF is obtained, preferably in functionally homogeneous form, from urine of thrombocytopenic patients.
  • Preferred procedures for isolating hMeg-CSF in functionally homogeneous form are generally outlined in Fig. 1A and are typically performed in two phases. In the first phase a protein fraction containing hMeg-CSF but contaminated with EPO and GM-CSF proteins is produced. In the second phase functionally homogeneous hMeg-CSF invention is produced.
  • substantially pure hMeg-CSF protein fraction is produced following the preparative PAGE step of the second phase.
  • substantially pure hMeg-CSF protein fraction is used herein to refer to a protein fraction which is believed to comprise at least about 50% of hMeg-CSF protein and to be essentially free of contaminating EPO and GM-CSF proteins.
  • "Essentially free of contaminating EPO and GM-CSF” is defined herein based on a content of less than 100 units GM- CSF per mg protein and less than 0.5 units EPO per mg protein as determined e.g., by the Krystal assay (Krystal, G. Exp.
  • step D does contain approximately 1-2x10 4 units M-CSF and G-CSF per mg protein.
  • the preparative PAGE step of the second phase of the methodology is responsible for eliminating from the generated substantially pure hMeg-CSF protein fraction a major portion of the contaminating EPO and GM-CSF proteins as well as other contaminants.
  • hMeg-CSF protein fraction is used herein to define a protein fraction which comprises at least about 90% protein and be virtually, if not totally, free of contaminating EPO and GM-CSF proteins. While contaminating EPO and GM-CSF proteins are believed to be totally removed from the virtually pure hMeg-CSF protein fraction, this virtually pure hMeg-CSF protein fraction is not purified to structural homogeneity at this point. A further "functionally homoge- neous" sequenceable hMeg-CSF fraction is produced after reverse phase HPLC (See Fig. 1A).
  • the functionally homogeneous sequenceable hMeg-CSF is eluted from this column with approximately 50% acetonitrile and about 0.1% TFA.
  • This functionally homogeneous hMeg-CBF is at a level of purity which has allowed for accurate N-terminal amino acid sequencing via standard techniques in the sequencing field as shown in Example 5 below.
  • this final functionally homogeneous hMeg-CSF has a specific activity of at least approximately 4 x 10 3 CFU-meg colonies/mg protein (this figures is the total activity of both the smaller and the larger species).
  • hMeg-CSF entirely homogeneous hMeg-CSF is produced (containing either one or both 24-35 and 50-70 kD species).
  • the hMeg-CSF is chromatofocused using a MonoP column (as described above) followed by reverse phase and cation exchange HPLC. It is believed that the isolated hMeg-CSF protein, which is purified to homogeneity, is produced in this embodiment following a reverse phase HPLC and a cation exchange HPLC step of the second phase of the methodology as shown in Fig. 1B.
  • urine is collected from patients with aplastic anemia and concentrated preferably by ultrafiltration.
  • lyophilization and dialysis can be used to concentrate the urine.
  • Use of Amicon YM10 membrane for ultrafiltration is particularly preferred (Amicon, Beverly, MA).
  • Urinary salts are removed by molecular sieve chromatography (using, for Example Sephadex G-50 gel filtration, Pharmacia, Piscataway, NJ).
  • Other molecular sieve columns such as BioGel P-10 (BioRad, Richmond, CA) or Bio Gel P-30 (BioRad) or Sephadex G-25 (Pharmacia, Uppsala, Sweden) can also be used.
  • the pH and salt concentration of the peak protein pooled from the molecular sieve column is adjusted and the material is applied to an anion exchange support such as DEAE cellulose, (Whatman, Clifton, NJ) or DEAE Bio Gel A (Bio Rad, Richmond, CA).
  • An anion exchange support such as DEAE cellulose, (Whatman, Clifton, NJ) or DEAE Bio Gel A (Bio Rad, Richmond, CA).
  • the objective is to remove nonionic (non- binding) matter.
  • non-binding proteins are removed by rinsing with e.g., phosphate buffer but binding proteins, including hMeg-CSF, are eluted with 0.15 M NaCl.
  • the fraction(s) recovered from the separation according to molecular weight can be applied to a cation exchange support using e.g., CM-Sepharose (Pharmacia, Piscataway, NJ) or CM Bio Gel A (Bio Rad, Richmond, CA).
  • CM-Sepharose Pharmacia, Piscataway, NJ
  • CM Bio Gel A Bio Rad, Richmond, CA
  • hMeg-CSF will also bind to these supports under the given conditions and is eluted in a similar fashion.
  • the use of CM-cation exchange has yielded more hMeg protein and is preferred.
  • the active material recovered from either ion exchange support is preferably dialyzed extensively to remove interfering salts, lyophilized, resuspended and ready for further purification steps.
  • These steps include preparative polyacrylamide gel electrophoresis using native (non-detergent) conditions which generates a substantially pure hMeg-CSF protein fraction.
  • This is followed by a step that separates the substantially pure hMeg-CSF protein fraction from residual impurities on the basis of its weakly basic pH to generate a virtually pure hMeg-CSF protein fraction.
  • This step can be performed by either IEF in either liquid or immobilized phases or preferably MonoP.
  • the MonoP chromatography results in recovery of virtually pure hMeg-CSF protein fractions when the pH of the eluting buffer is between 7.0 and 7.5.
  • a virtually pure hMeg-CSF protein fraction is detected in IEF gels at a pH ranging between about pH 7.2 and about pH 7.4.
  • hMeg-CSF protein fraction obtained from either MonoP or IEF or WCX HPLC can be further purified by application to a C18 reverse-phase HPLC (e.g., from Beckman Instruments, Fullerton, CA), as shown generally in Fig. 1A.
  • C8, C4 or C1 reverse phase HPLC columns can be used such as Brownlee RP-4, Brownlee Aquapore RP-300 or
  • Brownlee RP-8 (Rainen, Woburn, MA), Vydec C-4 Protein/Peptide Column, Vydec C-8 (Rainen), Beckman Ultrasphere Octyl Column (Rainen) or a Cl + C4 Column (Pharmacia, Uppsala, Sweden).
  • the hMeg CSF protein is eluted from the C18 column with approximately 50% acetonitrile and about 0.1% TFA (trifluoroacetic acid) to produce functionally homogeneous hMeg-CSF, (i.e., the only hematopoietic activity detected is hMeg-CSF, but it is not constitutively homogeneous).
  • Functionally homogeneous hMeg-CSF protein is at a level of purity which allowed for accurate amino acid sequencing via standard techniques in the sequencing field illustrated in Example 5 below.
  • homogeneous hMeg-CSF is produced.
  • the protein is chromatofocused using a MonoP HR 5/20 column (Pharmacia).
  • the fractions containing hMeg-CSF activity are then further purified by chromatography on reverse phase HPLC using a C18 column followed by polyaspartate WCX cation exchange HPLC.
  • Homogeneous hMeg-CSF protein is obtained after binding to a polyaspartate WCX cation exchange HPLC column and elution with substantially greater than 0.15M NaCl.
  • any other reverse-phase and cation exchange HPLC columns as those skilled in the art will appreciate can be used.
  • the buffers and conditions will vary but they can be ascertained by no more than routine experimentation by those of ordinary skill in the art.
  • a streamlined purification procedure can be used to isolate hMeg-CSF.
  • This procedure leads to the production of homogenous hMeg-CSF but requires much less manipulation of the protein.
  • Aplastic anemia urine concentrate obtained as described is dissolved in 100 ml of 0.8 M urea containing two micrograms/ml of the protease inhibitor leupeptin (Boehringer Mannheim). The material is concentrated on a 10 6 molecular weight cut-off membrane such as Omegacell (Filtron Technology Corporation, Clinton, MA). The material retained on the membrane is discarded and the flow-through is collected and concentrated on a 10 5 cut-off membrane. The flow-through is collected and concentrated on a 10 4 cut-off membrane.
  • hMeg-CSF contained in the 10 4 - 10 5 fraction is further purified by chromatography using a Polyaspartic acid WCX cation exchange column which may be repeated multiple times.
  • hMeg-CSF elutes from the column at greater than 0.5 M NaCl.
  • the material obtained using this streamlined isolation procedure appears to give a much better recovery of the biological activity of the hMeg-CSF protein and can form a single band on SDS-PAGE.
  • the hMeg-CSF of the instant invention can be used in methods to treat mammals, such as to potentiate platelet formation in patients with thrombocytopenia or atherosclerosis, in wound healing, in patients with antibody to platelets, or to make drugs to enhance, alter or possibly decrease platelet function.
  • mammals such as to potentiate platelet formation in patients with thrombocytopenia or atherosclerosis, in wound healing, in patients with antibody to platelets, or to make drugs to enhance, alter or possibly decrease platelet function.
  • structural analogs which are true analogs of hMeg-CSF, can be produced based on a detailed characterization of the binding of hMeg-CSF to its cellular receptor. These analogs would bind tightly to the receptor without activating it and, thus, block the biologic activities of hMeg-CSF.
  • hMeg-CSF may be used in the treatment of diseases characterized by a decrease in the level of hematopoietic cells, particularly those of the megakaryocytic lineage. It may be used directly to stimulate megakaryocyte and platelet production and may indirectly stimulate other hematopoietic lineages.
  • conditions susceptible to treatment with hMeg-CSF protein of the present invention is thrombocytopenia, a reduction in the number of circulating platelets in peripheral blood. Thrombocytopenia may be induced by exposure to certain viruses, drugs or radiation. It is often a side effect of various forms of cancer and/or AIDS therapy, e.g., exposure to chemotherapeutic drugs.
  • hMeg-CSF protein compositions may avoid undesirable side effects caused by treatment with presently available drugs.
  • the amount of hMeg-CSF to be used will, of course, depend upon the severity of the condition being treated, the route of administration chosen, the specific activity of the hMeg-CSF protein and the responsiveness of the individual patients' receptors to the hMeg-CSF protein, and ultimately will be decided by the attending physician or veterinarian.
  • Such amount of hMeg-CSF protein as determined by the attending physician or veterinarian is also referred to herein as a
  • treatment effective or a “therapeutically effective amount”.
  • Typical hMeg-CSF protein treatment effective amounts are contemplated to be in the range of about 0.1-100, preferably about 1-50, and more preferably about 2-15 units of hMeg-CSF protein/kg body weight, for a period of about 3-60 days, and preferably 15-45 days.
  • hMeg-CSF protein fractions for treating mammals including humans and forming medicaments are also contemplated by the instant invention as these hMeg-CSF protein fractions can be rendered pyrogen-free using, for example polymyxin B resins to remove endotoxins (if present) as is well known in the art. Either or both higher and lower m.w. species can be used.
  • hMeg-CSF may enhance hematopoiesis.
  • the present invention may also be employed, alone or in combination with other
  • hematopoietins in the treatment of other blood cell deficiencies or anemia (red cell deficiency). Such therapeutic compositions may also be administered in conjunction with other human factors.
  • a non-exclusive list of other appropriate hematopoietins including CSFs and interleukins for interaction with the hMeg-CSF protein of the present invention includes GM- CSF, G-CSF, M-CSF, erythropoietin (EPO), IL-1, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7 (all available from Amgen, Thousand Oaks, CA), and IL-9 (obtainable as described in Yang, Y-C et al., Blood 74, 1880-1884, 1989).
  • Activins and Inhibins include without limitation Activins and Inhibins (Genentech, South San Francisco, CA), TPO/TSF (obtainable as described in McDonald, T.P. et al., Int. J. Cell Cloning 7:139-155, 1989), IL-11
  • hMeg-CSF protein is in the treatment of patients recovering from chemotherapy, therapeutic radiation, bone marrow transplants, enhancing host blood clotting abilities during surgery and in burn patients.
  • hMeg-CSF may also be employed to develop monoclonal and polyclonal antibodies generated by standard methods for diagnostic, hMeg-CSF gene therapeutic use, or research reagent use (e.g., in the identification of the hMeg-CSF gene and in screening recombinant microorganisms producing hMeg-CSF).
  • yet another aspect of the invention is directed to pharmaceutical formulations or dosage forms for treating the conditions referred to above.
  • Such pharmaceutical formulations comprise a therapeutically effective amount of the hMeg-CSF protein(s) of the present invention in admixture with a pharmaceutically acceptable carrier, such as sterile water, sterile normal saline, dextran, parabens, citrates, stearate calcium or the like.
  • a pharmaceutically acceptable carrier such as sterile water, sterile normal saline, dextran, parabens, citrates, stearate calcium or the like.
  • Such compositions can be systemically administered either parenterally, intravenously or subcutaneously, if appropriate, although oral or inhalable delivery systems suitable for protection of proteins from the gastric environment are also contemplated. Examples are U.S.P. No.
  • the pharmaceutical formulations for use in the present invention are preferably in the form of pyrogen-free, paren terally acceptable aqueous solutions.
  • the preparation of such a parenterally acceptable protein solution having due regard to pH, isotonicity, stability and the like, is well within the skill of the art. While it is possible for the novel hMeg-CSF to be administered as the pure homogeneous protein, functionally homogeneous or virtually pure protein fraction, it is generally preferable to present it as a pharmaceutical formulation or preparation.
  • the formulations of the present invention both for veterinary and for human use, therefore comprise a hMeg-CSF protein, as described above, together with one or more pharmaceutically acceptable carriers or diluents or excipients thereof and optionally other therapeutic ingredients.
  • the carrier(s) must be physiologically "acceptable” in the sense of being compatible with the other ingredients of the formulation, if any, and not deleterious to the recipient thereof.
  • the formulation should not include oxidizing agents and other substances with which peptides are known to be incompatible.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient, e.g., a hMeg-CSF protein, with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.
  • the active ingredient e.g., a hMeg-CSF protein
  • liquid carriers or finely divided solid carriers or both include human serum albumin, dextran-imbedded hMeg-CSF, or other biocompatible polymers degraded in the body.
  • Time- and sustained-release implants could also be used as delivery systems.
  • the new gene therapy technology could also be used to insert the gene(s) for hMeg-CSF into autologous blood cells which are then reinjected in the patient as in vivo production sites. Rosenberg, R.D. et al.
  • Formulations suitable for parenteral administration conveniently comprise sterile aqueous solutions of the active ingredient, e.g., isolated pure homogeneous hMeg-CSF protein, functionally homogeneous or virtually pure hMeg-CSF protein fraction, with solutions which are preferably isotonic with the blood of the recipient.
  • Such formulations may be conveniently prepared by dissolving solid hMeg-CSF proteins in water to produce an aqueous solution, and rendering said solution sterile.
  • the resultant solution may be presented in unit or multi-dose containers, for example sealed ampoules or vials.
  • the pharmaceutical formulations or dosage forms of the present invention need not contain an effective amount of the hMeg-CSF of the present invention as such effective amounts can be achieved by administration of a plurality of such formulations or dosage forms.
  • the present invention also contemplates the use of biologically active fragments of hMeg-CSF.
  • Biologically active fragments (substantially smaller than the entire hMeg-CSF protein molecule) of hMeg-CSF can be obtained for example by limited proteolytic digestion of the hMeg-CSF protein (either purified to homogeneity using techniques described above or purified recombinant protein as described in Example 7 below) using e.g., trypsin, papain, chymotrypsin, V-8 protease, endoproteases or other proteases well-known in the art or other agents such as cyanogen bromide (which cleaves after methionine residues) and testing for hMeg-CSF activity after isolation in, e.g., the in vitro murine fibrin clot assay of Example 3 below.
  • Example 6 limited nuclease digestion of the cDNA using e.g., Bal-31 nuclease, can be performed and the resultant product cloned and expressed as taught in Examples 6-8 below. The protein product can then be tested for activity, leading to the identification of active hMeg-CSF fragments.
  • methods for completing the sequencing of hMeg-CSF and for identifying the gene encoding the hMeg-CSF of the present invention, cloning it and expressing recombinant hMeg-CSF are also disclosed.
  • anemia was collected and mixed with about 50% phenol in ethanol to a final concentration of about 0.1% phenol.
  • the aplastic anemia patients had pancytopenia with hypocellular bone marrow and no signs of systemic or urinary tract infections. Platelet counts were less than about 20, 000/mm 3 , leukocyte counts were less than about 1,500/mm 3 and hemoglobin concentration was maintained around 6g/dl by blood transfusions.
  • the collected urine was filtered and concentrated on a YM10 filter (10,000 molecular weight cut-off, obtained from Amicon, Beverly, MA) by ultrafiltration.
  • the ultrafiltered urine was desalted using a 10 ⁇ 100 cm column of Sephadex G-50 (obtained from Pharmacia, Piscataway, NJ).
  • the initial protein peak (the exclusion effluent) was collected.
  • the G-50 pool was adjusted with about 0.02 M NaH 2 PO 4 and NaCl to pH 4.8-5.0.
  • DEAE-Cellulose (DE-22, obtained from Whatman, Clifton, NJ) equilibrated with about 0.025 M NaH 2 PO 4 was added to the G-50 pool (about 1 g dry powder/1 liter urine) and stirred for about 30 min. The mixture was allowed to settle for about 2 hours at about 4oC and poured into a column with gentle suction. The absorbed protein fraction was eluted with about 0.05 M NaH 2 PO 4 with about 0.15 M NaCl. The eluted sample was lyophilized to dryness and stored at about -70°C.
  • the concentrated, desalted urine from the G-50 column was loaded on a CM-Sepharose Fast Flow column (obtained from Pharmacia) and equilibrated with about 0.01 M phosphate buffer, pH of about 5.5.
  • the bound ionic protein material (including hMeg-CSF protein) was eluted with about 0.05 M Na 2 HPO 4 and about 0.15 M NaCl. The eluted material was then dialyzed and lyophilized.
  • the Crude Urinary Extract obtained above was dissolved in about 30 ml/g and dialyzed for about 2 days against about 4 liters of water (the water was exchanged three times a day), using dialysis tubing, such as Spectra/Por, (Spectrum Medical Industries, Inc., Los Angeles, CA) with a molecular weight cut-off of approximately 10 kDa. After dialysis, the material was lyophilized and stored at about -70 oC.
  • dialysis tubing such as Spectra/Por, (Spectrum Medical Industries, Inc., Los Angeles, CA) with a molecular weight cut-off of approximately 10 kDa.
  • the native preparative gel was about 5% polyacrylamide (about 30% T and about 2.6% C) in about 0.05 M Tris-Hcl (pH 6.8).
  • the 14 ⁇ 16 ⁇ 0.3 cm gel had no stacking gel, and about 0.5 g dry powder of the freshly dialyzed, lyophilized extract was added per gel.
  • the upper and lower reservoir buffer contained about 0.025 M Tris-glycine (pH 8.3).
  • the gel was run at about 50 mA per gel until the front was about 13 cm from the top.
  • the gel was removed from the apparatus and cut into about 1 cm slices which were
  • Fig. 2 illustrates the substantially pure hMeg-CSF protein fractions and biological activity of 1 cm slices of the 5% PAGE, with fraction #1 being at the top of the gel.
  • Isoelectric focusing using a pH range of about 3.5-10, documents that the pl of hMeg-CSF protein in glycosylated and sialyated form was about 7.2-7.4 (Fig. 3).
  • the 115 ⁇ 0.2 ⁇ 25 cm gel is composed of about 5.4% acrylamide, about 0.144% Bis-acrylamide and ampholines (pH equal to about 3.5-10) with the sample as part of the gel composition.
  • a MonoP HR 5/20 column (Pharmacia) was equilibrated with about 0.025 M Triethanolamine (brought to a pH of about 8.3 with acetic acid) at a flow rate of about 1 ml/min.
  • the sample about 70 mg of substantially pure hMeg-CSF protein from the preparative PAGE step, is preferably filtered (0.22 mi- crons) and injected onto the column.
  • Fractions were collected every min for 40 mins.
  • the fractions eluting at 16-20 min contain virtually pure hMeg-CSF protein and had a pH of about 7.0-7.5 (Fig. 4). These fractions were pooled, dialyzed for two days and lyophilized.
  • the hMeg-CSF protein fractions (about 0.5 mg) from the MonoP column were resuspended in about 0.05 M phosphate buffer (pH of about 6.45) and filtered (0.22 microns).
  • the sample was injected into a Polyaspartic Acid WCX HPLC column (The Nest Group, Southboro, MA; 4.6 mm ⁇ 10 cm) and about a 30 minute gradient of about 0-0.5 M NaCl followed by a 5 minute gradient of 0.5-1 M NaCl in about 0.05 M phosphate buffer (pH of about 6.45) was used to elute the hMeg-CSF protein fractions which were present in fractions #13-17 (i.e., at about 0.5-1 M NaCl).
  • the virtually pure hMeg-CSF protein fraction(s) obtained from the MonoP were resuspended in about 0.1% TFA (trifluoroacetic acid) in water. The sample was filtered (0.22 microns) and injected into a C18 reverse-phase HPLC column
  • the hMeg-CSF is purified using steps A-E above.
  • the preparative PAGE step (Step E) is followed by chromatofocusing using a MonoP HR 5/20 column as in step G above.
  • the active fractions from the preparative PAGE step were pooled, dialyzed for at least two days against distilled water, lyophilized, resuspended in 0.025M Triethanolamine, pH 8.3, and filtered
  • the hMeg-CSF protein fractions from the C18 Reverse Phase HPLC column were lyophilized and resuspended in about 0.05 M phosphate buffer (pH of about 6.45) at a concentration of about lmg/ml protein.
  • the sample was injected into a
  • the material eluted from the WCX column was dialyzed, lyophilized and loaded in a single lane of a 12% non-reduced SDS-PAGE gel, electrophoresed, transferred to an Immobilon PVDF membrane (Millipore Corp., Bedford, MA) and stained with
  • Homogeneous hMeg-CSF may be obtained using the following procedure.
  • aplastic anemia urine concentrate 50 liters of aplastic anemia urine concentrate is dissolved in 100 ml of 0.8 M Urea containing two micrograms/ml of the protease inhibitor leupeptin (obtained from Boehringer Mannheim). The material is concentrated on a 10 6 molecular weight cut-off membrane (Omegacell, Filtron Technology Corporation, Clinton, MA). Material retained on the 10 6 membrane was discarded. The flow-through was collected and concentrated on a 10 5 cut-off membrane. The flow-through was collected and concentrated on a 10 4 cut-off membrane. The concentrate in each step was washed with three volumes of 0.8 M Urea plus two micrograms/ml leupeptin.
  • Human megakaryocyte colony-stimulating activity was detected in the 10 5 - 10 6 fraction and in the 10 4 - 10 5 fraction.
  • the 10 5 - 10 6 fraction has also been found to contain erythropoietin and GM-CSF activities.
  • the material contained in the 10 4 - 10 5 fraction was further purified by weak cation exchange HPLC using a polyaspartic acid WCX column (The Nest Group, Southboro, MA) .
  • the 10 4 - 10 5 fraction was adjusted to a pH of 6.0 before injection into the WCX HPLC column.
  • the column (100 X 4.6 mm; 5 microns) was equilibrated with 0.05 M sodium phosphate buffer, pH 6.0. A flow rate of 1.0 ml/min was used.
  • hMeg-CSF activity was detectable in pool A (fraction nos. 1-6), pool D (fractions nos.
  • a pool of material from fraction nos. 16-32 from the WCX HPLC column was electrophoresed on a 12% analytical SDS-PAGE under both reducing (5% 2-mercaptoethanol) and non-reducing conditions.
  • the gel was sliced in 1 and 0.5 cm slices and the protein eluted by two consecutive overnight incubations at 4°C in IMDM medium plus 1% FCS (1 ml/cm gel each).
  • Pre-stained molecular weight standards were run in adjacent lanes to determine the molecular weight.
  • a single band spanning the size range of about 57-66 kD was detected within the expected 50-70 kD range of hMeg-CSF activity, as shown in Fig. 10A.
  • hMeg-CSF was detected under non-reducing conditions both at molecular weights of about 2435 kDa and about 50-70 kDa but most of the activity was associated with the 50-70 kD species. Under reducing conditions (2-mercaptoethanol added) of the same material, hMeg-CSF was also detected at 24-35 kDa and 50-70 kDa as shown in Fig. 11B.
  • Meg-CSF Human urine contains, in addition to Meg-CSF, other cytokines and growth factors including EPO, M-CSF and G-CSF (Das, S.K. et al., Blood 58: 630-641, 1981; Miyake, R. et al., J. Biol. Chem. 252: 55582-5564, 1977; Kohsaki, M. et al., Proc. Natl. Acad. Sci. USA 80:3802-3806, 1983).
  • a systematic exploration of the biological activities present in Meg-CSF preparations from each of the purification steps was performed. The results presented here show that Meg-CSF purified through the MonoP step is virtually pure - the only biological activity detectable by assay being Meg-CSF. The specific activities are set forth in Table I below.
  • AA aplastic anemia
  • urinary extracts and the fraction separated by chromatographic procedures were tested for EPO activity using a murine splenocyte 3 H-thymidine assay for EPO (Krystal, G., Exp. Hematol. 11:649-660. 1983).
  • EPO presents the largest and most vexing contaminant.
  • Meg-CSF Meg-CSF and EPO (which has a Pi of approximately 3.5). It has been determined that as little as 0.7 U/mg EPO is capable of stimulating megakaryocytic colonies in fibrin clot assays. Although 0.3 U/mg EPO does not stimulate
  • the murine fibrin clot culture system was used to detect megakaryocyte progenitors (CFU-Meg) as described in Kuriya, S-I, et al., Exp. Hematol. 15:896-901, 1987.
  • IMDM Dulbecco's medium
  • FCS fetal calf serum
  • FCS HyClone Laboratories, Logan, UT
  • marrow cells final concentration about 5 x 10 5 cells/ml
  • fibrinogen Sigma Chemical Co.
  • thrombin Sigma Chemical Co.
  • fibrin clots were dried and stained for acetylcholmesterase (AChE) activity, as disclosed in Karnov- sky, M., et al., J. Histochem. Cvtochem. 12:219 (1964), and Jackson, C.W., Blood 42:413 (1973). Colonies containing 3 or more AChE-positive cells were counted. The mean and the standard error were determined for triplicate plates.
  • AChE acetylcholmesterase
  • the protein content of the samples was determined and the lyophilized samples were resuspended in Iscove's medium to a concentration of about 1 mg/ml and sterilized by filtration (0.22 microns). The samples were tested at three concentrations: about 6.7, 3.3 and 0.67 micrograms/ml assay mixture. The results are set forth in Table II hereinbelow.
  • One unit is defined as one megakaryocyte colony in themurine fibrin clot assay.
  • the Specific Activity of the DEAE-Cellulose sample (1) and the preparative PAGE sample (2) is reported in Table II as having greater activity than the MonoP and C18 HPLC samples (3 and 4, respectively). This is believed to be due to the contaminating proteins, such as EPO, GM-CSF,etc, in the DEAE-Cellulose and preparative PAGE samples (1 and 2, respectively). 4 ) Sample obtained from the DEAE- 4.0 ⁇ 10 4
  • mice Groups of 5 rats will be injected intraperitoneally with doses of hMeg-CSF that produce about 20-30 colonies per 2xl0 5 cells in the mouse CFU-Meg assay. Injections will be administered for three consecutive days. The animals will then be bled from the tail vein after twenty-four hours and the number and size of platelets determined by Coulter Counter. Controls will consist of animals injected with: 1) supernatants or lysates of cells not transfected in hMeg-CSF cDNA; 2) physiological saline; and/or 3) non-human hMeg-CSF-containing fractions devoid of other cytokines known to stimulate CFU-Meg.
  • the in vivo activity can also be determined by measuring the number of megakaryocyte progenitor cells of mice after treatment with hMeg-CSF.
  • Five mice will be injected with a single dose of purified natural or recombinant hMeg-CSF.
  • the animals will be sacrificed after 2, 4 and 6 days post-inoculation and their spleens and femurs will be removed aseptically.
  • Single cell suspensions will be made by disrupting the spleen and flushing the marrow from the femur.
  • the cells will then be plated in the fibrin clot assay in place of the murine marrow cells and stimulated with pokeweed mitogen spleen conditioned medium (PWM-SCM), a source of Meg-CSF.
  • PWM-SCM pokeweed mitogen spleen conditioned medium
  • Meg-CSF increased the number of murine splenic megakaryocyte progenitors as evidenced by an increase in colonies produced by the spleen cells compared to saline controls. There was no increase in bone marrow megakaryocyte progenitors, perhaps due to the low concentrations of Meg-CSF employed. Therefore, the purified hMeg-CSF of the present invention (both naturally derived and recombinant) is anticipated to increase the number of spleen megakaryocyte progenitors and increase the number of bone marrow progenitor cells in animals receiving the hMeg-CSF of the present invention.
  • hMeg-CSF protein fraction described above was used to determine the putative N-terminal amino acid sequence which may be included in Meg-CSF thus demonstrating that preparations within the present invention are useful to sequence hMeg-CSF.
  • the protein sequence was determined from protein immobilized on PVDF membranes described above after
  • the N-terminal amino acid sequence was X-Asp-Pro-Val-Glu-Ser-Pro-Val-Pro-Y, wherein X and Y are undetermined residues.
  • the species thus partially sequenced is the higher molecular weight (50-70 kD) species.
  • DOS deoxyoligonucleotide sequences
  • Peptides corresponding to N-Terminal Meg-CSF will be synthesized and conjugated to carrier molecules.
  • the polypeptide conjugates will be used to make anti-peptide antibodies which recognize hMeg-CSF using techniques well known by those skilled in the art which would also provide an alternate approach to obtaining Meg-CSF cDNA via immunological screening of an expression cDNA library.
  • Selective amplification and subsequent cloning of specific full-length (or near full length) cDNA can be accomplished by the following steps: (1) First strand synthesis can be initiated by use of oligo-dT primer (which binds to the polyA tail found on eukaryotic mRNAs ) , second strand cDNA synthesis can be accomplished by the Klenow fragment of DNA polymerase 1. (2) PCR utilizing a specific DOS primer and an oligo-dT primer can be used to amplify the cDNA of interest prior to cloning into Ml3 phage or other vectors. (3) A specific cDNA library can be screened with 32 P-labeled DOS primers and positive clones will be characterized by restriction mapping and sequencing.
  • the second strand can then be synthesized in the same manner as above and the double stranded cDNAs can be cloned into an appropriate vector (e.g., the lambda ZAPII vector (Stratagene, La Jolla, CA). Subsequently, DNA can be isolated from the entire lambda ZAPII cDNA library and Meg-CSF specific clones will be amplified by PCR utilizing a specific DOS and a vector-based primer. The amplified products will then be cloned into e.g., an M13 vector (GIBCO/BRL Life Technologies,
  • the primer pair to be used for Meg-CSF mRNA signal amplification will be a first strand oligo-dT 17 "universal" primer which binds to the sense strand, and a second strand specific primer (DOS A) which binds to the antisense strand.
  • the DOS A is a 17-mer which corresponds to all codon combinations derived from the N-terminal amino acid sequence of Meg-CSF, and has the sequence
  • DOSA 1 GAC/T CCN GTN GAA/G TCN CC;
  • DOSA 2 GCT/C CCN GTN GAA G TGC/T CC
  • C/T means C or T
  • N denotes any one of the four bases.
  • DOS B another specific primer
  • Meg-CSF expression is of crucial importance in successfully cloning this factor.
  • T cells and spleen cells (Ogata, K. et al.. Blood 74: (Suppl. 1) :330a. 1989) together with endothelial tissue, placenta, stromal cells and other tissues (e.g., liver) that potentially produce Meg-CSF will be examined for their Meg-CSF mRNA expression according to Cantrell et al., PNAS (USA)
  • RNA will be extracted from different tissues (Chirgwin, J.M. et al., Biochem. 11:5294-99, 1979) and poly A + RNA will then be isolated by two cycles of chromatography on an oligo-dT cellulose column (Sambrook, J. et al.. Molecular
  • the first strand cDNA will be synthesized using Mouse Moloney Leukemia Virus (M-MLV) reverse transcriptase (GIBCO/BRL Life Technologies) with an oligo-dT primer, and the second strand using the Gubler and Hoffman procedure (Gubler, U. et al., Gene 25:263-269, 1983; Dai, W. et al., Biochem., Biophys . Res. Comm. 168: 1-8, 1990). The double stranded cDNAs will then be subjected to 30 cycles of PCR
  • nitrocellulose membranes such as those available from Schleiker and Schul, Keene, NH. After baking under vacuum at 80°C for 2 hrs, the nitrocellulose blot will be probed sequentially with 32 P- labeled DOS A and B. The tissue that gives the strongest specific signal to probe DOS A and B will be used as the primary source for Meg-CSF cDNA cloning.
  • Double stranded cDNA synthesized and amplified as described above will be methylated with Eco RI methylase, blunt-ended by incubation with Klenow enzyme and dNTP, and ligated with Eco RI linkers (GIBCO/BRL Life Technologies).
  • cDNAs After digestion with Eco RI, cDNAs will be separated by size on an agarose gel. The cDNAs larger than the minimal coding sequence will be eluted by electroelution and ligated with the M13 mpl9 phage vector which has been cut with Eco RI and dephosphorylated. The ligated products will be transformed into CaCl 2 treated E. coli JM 101 cells and plated on
  • random primers will be used for the first strand cDNA synthesis. This approach is of particular importance if the size of Meg-CSF mRNA is more than 3 kb.
  • double stranded cDNA will be ligated into the lambda ZAP II vector (Stratagene) to make a complete cDNA library. The entire cDNA library will be amplified once for DNA isolation and the amplified phage library will be then eluted. Recombinant phage DNA will be prepared (Sambrook, J. et al., supra) and used as the starting material for PCR amplification.
  • Meg-CSF specific cDNA cloned in the lambda ZAP II vector will be amplified by 30 cycles of PCR using a specific DOS A primer and a second primer corresponding to the lambda ZAP II phage sequences (containing a Hind III site at the multicloning site) located in the lac Z gene. Following amplification, DNA will be blunt-ended with Klenow enzyme in the presence of dNTP. Blunt-ended DNA will then be cut with Hind III and ligated to a Hind III/Sma I cut M13 phage vector (GIBCO/BRL Life Technologies). The ligation products will be transformed into E.
  • Meg-CSF coli JM101 competent cells and recombinant phages will be screened for Meg-CSF cDNA inserts as described above. Overlapping cDNA inserts from positive clones will be used to construct a full length cDNA clone for Meg-CSF.
  • the peptide should be conjugated as disclosed in Lerner R.A. Nature 299: 592-596, 1982.
  • the peptide may be synthesized on a lysine core as disclosed in Tarn, J.P. Proc. Nat. Acad. Sci. USA 85: 5409-5413, 1988. This antipeptide antibody will provide an alternate method to screen for Meg-CSF cDNA clones.
  • recombinant phages grown on LB plates for 4 hr at 42oC will be overlaid with nitrocellulose filters impregnated with 2 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) (Sigma Chemical Co.). After an additional 4 hr incubation at 37°C, the filters will be removed and probed with antiMeg-CSF serum which will have been preabsorbed with lysate from the E. coli host. The membranes will be rinsed with Tris buffered saline (TBS)/Tween (Huynh, T.V.
  • TBS Tris buffered saline
  • Tween Huynh, T.V.
  • phage clones will be rescued into phagemid (Bluescript, Stratagene, La Jolla, CA) in the presence of "helper" phage R408 according to the protocols provided by the supplier (Stratagene).
  • cDNA inserts will be mapped with restriction enzymes and various restriction fragments will be subcloned into M13, M118 and mp 19 phage vectors and sequenced as outlined above.
  • Meg-CSF cDNA will be expressed immediately using both prokaryotic (e.g.. E. coli) and eukaryotic (Baculovirus) systems (e.g.. Current
  • a chimeric gene with an appropriate "handle” peptide added to the Meg-CSF can be constructed in an appropriate vector using a DNA engineering approach.
  • the resulting vector will be introduced into host cells for expres-r sion of the fusion protein.
  • the fusion protein can be easily identified and purified because of the added "handle” (see below). If the fusion protein interferes with folding and/or biological activity, the peptide "handle" can be removed by specific proteases.
  • EXAMPLE 8 Expression of Meg-CSF in E. coli
  • E. coli Two characteristics of E. coli that make it ideally suited for an expression system for proteins are: (i) its ease of manipulation and (ii) its rapid growth in inexpensive media. Since early studies showed that the carbohydrate moieties of native Meg-CSF are not necessary for its biological activity (Murphy, M.J. et al., Acta Hematol. Jpn. 46: 1380-1396, 1983), E. coli will be the first choice for expression of recombinant Meg-CSF.
  • a prokaryotic alternative is the Baculovirus system which uses eukaryotic machinery for protein expression and post-translational modifications. Both systems are merely mentioned as non-limiting examples of the expression systems that can be used.
  • the two plasmids are maintained within the same E. coli host cell.
  • the expression vector contains P T7 upstream of the Meg-CSF gene.
  • P T7 is the promoter element with which bacteriophage T7 RNA polymerase interacts (Tabor, S., et al., Proc. Natl. Acad. Sci. USA 12:1074-1078, 1985; Ausubel, F.M. et al., (eds)
  • the second plasmid contains the T7 RNA polymerase gene under the control of a heat-inducible E. coli promoter. Upon heat induction, the T7 RNA polymerase is produced which in turn initiates transcription of the expression vector by recognizing the control element of P T7 .
  • the Bluescript plasmid containing the full length Meg-CSF cDNA insert will be linearized at the 5' end with two appropriate restriction enzymes (to cut the vector multicloning and cDNA sites respectively) which remove the cDNA insert from the 5' end up to, or slightly beyond, the Meg-CSF coding region.
  • the amount of insert removed will depend on the availability of a restriction site in the cDNA. If no appropri te site is available, a unidirectional deletion approach with Exo III nuclease will be employed (Sambrook, J. et al., supra). The linearized vector plus most, if not all, of the coding sequence will be eluted from agarose gel with the
  • Plasmid pGPl-2 (obtainable from Dr. Stanley Tabor, Harvard Medical School, Boston, MA) contains a T7 RNA polymerase gene which is silent in host cells grown at 30°C and is induced by raising the temperature to 42°C. Bacteria harboring the pGPl-2 plasmid will be selected on LB/Kanamycin. pT7-7/Meg-CSF plasmid DNA will be transformed into E. coli
  • K38/pGPl-2 cells and transformants will be selected on LB plates containing ampicillin and kanamycin at 30°C. Colonies containing two plasmids will be selected for expression of "handle" peptide/Meg-CSF fusion protein. Fresh LB/ampicillin/Kanamycin medium will be inoculated with a single colony of bacteria containing two plasmids and bacterial cells will be grown at 30 °C to an OD 590 of approximately 0.4. Expression of T7 RNA polymerase, which then activates Meg-CSF fusion gene expression, is induced by raising the temperature to 42°C.
  • the expressed product will be analyzed biochemically by SDS-PAGE and Western blotting and functionally by in vivo and in vitro bioassays for Meg-CSF. Purification of the fusion protein can be achieved by affinity column chromatography using anti ⁇
  • Handle peptide antibody (Immunex Inc., Seattle, WA) or by the procedures described above. Fused "handle” peptide can be removed by the protease enterokinase (which specifically recognizes the "handle” and cleaves at the junction of
  • the E. coli expression system has limitations such as the lack of post-translational modification, protein processing and transfer mechanisms present in eukaryotes. There are also insolubility problems for some over-expressed foreign proteins. Baculoviruses use eukaryotic machinery for expression, and therefore, baculoviral vectors (such as those disclosed in U.S. Patent No. 4,745,051) will be used for Meg-CSF expression to circumvent potential problems. In addition to the advantages of post-translational modification and solubility of most overexpressed foreign protein, baculoviruses are noninfectious to vertebrates (Carbonell, L.F., et al., J. Virol. 56:153-160, 1985). This is particularly important when expressing potentially toxic proteins or oncogene products.
  • Meg-CSF cDNA will be inserted downstream of the viral polyhedrin promoter in an appropriate Baculoviral plasmid vector (e.g., pVL series; Invitrogen, Seattle, WA) as described (Ausubel, F.M. et al., supra) and the Meg-CSF cDNA will be flanked both 5' and 3' by polyhedrin gene-specific sequences.
  • Baculoviral plasmid vector e.g., pVL series; Invitrogen, Seattle, WA
  • the Baculoviral plasmid DNA containing Meg-CSF cDNA will be isolated from bacterial transformants and cotransfected into Sf9 cells (Invitrogen, San Diego, CA) with wild-type viral DNA by the well known CaCl 2 - phosphate co-precipitation procedure (Rosenthal, N. et al..
  • Transfected cells will be maintained in complete medium (Grace's Antheraea medium; GIBCO/BRL Life Technologies) with 10% FBS/50 microg/ml
  • Gentamycin Three to four days after transfection, the culture medium containing both wild type and recombinant (as a result of homologous recombination during or post co-transfection) viruses will be collected. Recombinant viruses will be purified from wild type viruses by several rounds of plaque purification (Ausubel, F.M. et al., supra). The isolated recombinant viral plaques along with agarose plugs will be placed into serum-free medium and stored at 4°C.
  • Sf9 cells will be seeded at a density of 2.5 ⁇ 10 6 /25 cm 2 flask in 5 ml complete medium and maintained at 27°C for about 3 hr to allow cells to attach.
  • Putative recombinant Baculoviruses from individual stocks in serum-free complete medium (5 ⁇ 10 3 particles) are added to the seeded cells.
  • Meg-CSF is a secreted protein and should possess a leader peptide (when no "handle" peptide sequence is fused to the N-terminus) for secretion, culture supernatant (and cell lysates) will be first analyzed for the Meg-CSF antigen and activity as described below.
  • DNA encoding the secretion signal of another protein can be ligated to the Meg-CSF gene and the corresponding signal sequence can be subsequently cleaved.
  • Cells infected with wild type Baculoviruses will be used as a negative control. Recombinant Baculoviral stock which shows Meg-CSF activity and gives high titers will be saved for further studies.
  • Meg-CSF The final purification of recombinant Meg-CSF will be performed using, for example, any or all of the following techniques: gel exclusion chromatography, reverse phase HPLC, ion exchange liquid chromatography or HPLC, hydrophobic interaction chromatography, chromatofocusing, preparative polyacrylamide gel electrophoresis, bioaffinity chromatography (e.g.
  • the glycosylation of native hMeg-CSF may have a significant effect on its behavior, especially on the ion exchange columns. Therefore, it may be necessary to use alternative methods to purify recombinant materials expressed in E. coli, as disclosed in Seetharam, R. et al. (supra), which would be unglycosylated. These include expressing recombinant hMeg-CSF as a fusion protein which can be isolated by an affinity step using available antibodies (Immunex Inc., Seattie, WA) to the "handle" peptide described above or other techniques, as disclosed in Seetharam, R. et al. (supra), such as ultrafiltration, or solution based chromatofocusing described above and apparent to those skilled in the art.
  • the homogeneous hMeg-CSF of the present invention will be employed in experiments using either human or murine bone marrow to elucidate the role of Meg-CSF in
  • megakaryocytopoiesis The following questions will be asked: Does Meg-CSF cause proliferation of bone marrow progenitors so that more megakaryocytes are produced? Does it cause differentiation of progenitors into megakaryocytes, thereby affecting the number of platelets and/or the rate at which they are formed? Does it interact with other cytokines to increase the proliferation and/or differentiation of megakaryocytes?
  • CFU-Meg assays will be added either simultaneously or sequentially with other factors (before or after) to determine which factors promote stem cell differentiation down the megakaryocytic lineage versus those that drive CFU-meg proliferation versus those that increase maturation to platelet formation and release.
  • the number of CFU-Meg that are formed will be determined. It will also be determined whether these are increased over the control cultures. It will also be determined if the number of megakaryocytes are increased over those formed with other cytokines.
  • An alternative to the CFU-Meg assays will be to use liquid cultures of medium and fetal calf serum to grow the bone marrow and count the number of megakaryocytes that are formed.
  • Meg-CSF produced according to the present invention synergizes with other cytokines such as IL-6, GM-CSF, IL-3, EPO, IL-11, SCF, LIF, activins/inhibins, or TPO/TSF to produce even more
  • cytokines such as IL-6, GM-CSF, IL-3, EPO, IL-11, SCF, LIF, activins/inhibins, or TPO/TSF
  • megakaryocytes in the CFU-Meg assay the number of megakaryocyte colonies and/or number of megakaryocyte cells per colony can be measured
  • the maturity of the resulting megakaryocytes by their size, intensity for staining for specific platelet proteins (such as acetylcholmesterase for mice; von Willebrand's factor,
  • gpIIb/IIIa, factor VIII for human megakaryocytes gpIIb/IIIa, factor VIII for human megakaryocytes
  • ploidy measured by specific DNA staining (e.g. Hoescht 33258 stain) and flow cytometry will be determined.
  • Genomic fragments were amplified from a human placenta genomic DNA library according to a protocol provided by the supplier (Perkin Elmer Cetus, Inc, Norwalk, Connecticut ) .
  • One ⁇ g of the DNA was used in a final reaction volume of 100 ⁇ l using primer pairs as specified in Fig. 12.
  • PCR was performed at 94oC for 1 min., 45'C for 2 min. and 72'C for 3 min. for 3 cycles and 94oC for 1 min., 50'C for 2 min. and 72oC for 3 min. for 40 additional cycles.
  • the amplified products were analyzed on a 1% agarose gel as shown in Fig. 12. Using oligos No.
  • agarose gel electrophoresis showed that two specific products were amplified with molecular sizes of 1.65 kbp and 300 bp, as shown in Fig. 12.
  • the two fragments were eluted from the agarose gel and subjected to a second round of PCR amplification using oligos 2 and 3.
  • the larger fragment was amplified as predicted in terms of its specificity and molecular size.
  • the smaller one 300 bp
  • Meg-CSF genes could exist either as alleles or as different genes on the haploid chromosome of which the Meg-CSF gene giving rise to the 300 bp fragment is deprived of at least part of oligo 2 sequences.
  • WO 91/02001 may be directed to a portion of one but not the other;
  • WO 91/02001 is directed to DNA sequences which encode yet another different species of hMeg-CSF.
  • the DNA amplified i.e., the 300 bp fragment
  • the 1.65 bp fragment is anticipated to be useful for identifying the present hMeg-CSF by hybridization under low-stringency conditions.

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Abstract

Facteur de stimulation de colonies de mégacaryocyte humaines isolées (protéine de FSC-Megh) purifié de préférence jusqu'à homogénéité à partir de l'urine de patients souffrant d'anémie aplastique. La nouvelle protéine de FSC-Megh est une protéine de base ayant un pI égal à environ 7,2 à 7,4 déterminé par focalisation isoélectrique, ainsi qu'une masse moléculaire comprise entre environ 29.000 et 34.000 daltons déterminée par SDS-PAGE lorsqu'elle se présente sous forme glycosilée et sialée. La nouvelle protéine de FSC-Megh a le pouvoir d'induire la formation d'unités formant des colonies de mégacaryocyte dans un dosage in-vitro de caillots fibrineux murins, et également le pouvoir de réguler la mégacaryocytopoïese ainsi que la production plaquettaire in-vivo. La protéine de FSC-Megh peut également être caractérisée comme étant une glycoprotéine ayant des structures glucidiques biantennaires, des restes de bêtagalactose comme sucres terminaux ou pénultièmes ainsi que des fractions d'acide sialique. On pense que la protéine de FSC-Megh a une activité spécifique d'au moins environ 4.000 colonies CFU-Meg/Mg de protéine dans un dosage de caillots fibrineux murins. L'invention concerne également des agents pharmaceutiques uniques contenant la protéine de FSC-Megh permettant le traitement d'animaux et de l'homme, ainsi que des procédés d'isolement de la nouvelle protéine de FSC-Megh à divers niveaux de pureté.
PCT/US1991/004698 1990-07-02 1991-07-02 Proteine de fsc-meg humaine et procedes WO1992000319A1 (fr)

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KR1019920703438A KR930701475A (ko) 1990-07-02 1991-07-02 사람의 거핵구-집락 촉진 인자(hMeg-CSF) 및 그것의 생산방법
JP3512921A JPH06502621A (ja) 1990-07-02 1991-07-02 ヒトｍｅｇ―ｃｓｆタンパク質と方法
NO92924995A NO924995L (no) 1990-07-02 1992-12-23 Humant meg-csf-protein og fremgangsmaater
FI925983A FI925983A (fi) 1990-07-02 1992-12-31 Maenniskans meg-csf-protein och foerfaranden

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5260417A (en) * 1989-04-03 1993-11-09 Genetics Institute, Inc. Megakaryocyte growth promoting activity protein
US5766581A (en) * 1994-03-31 1998-06-16 Amgen Inc. Method for treating mammals with monopegylated proteins that stimulates megakaryocyte growth and differentiation
US5795569A (en) * 1994-03-31 1998-08-18 Amgen Inc. Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation
US6433142B1 (en) 1989-08-08 2002-08-13 Genetics Institute, Llc Megakaryocyte stimulating factors

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4894440A (en) * 1986-09-17 1990-01-16 Massachusetts Institute Of Technology Method of isolating megakaryocyte stimulatory factor

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE154611T1 (de) * 1988-09-21 1997-07-15 Meiji Seika Kaisha Neuer megakaryokytischer koloniestimulierender faktor und verfahren zur herstellung
WO1991002001A1 (fr) * 1989-08-08 1991-02-21 Genetics Institute, Inc. Facteur megakaryocytopoïetique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4894440A (en) * 1986-09-17 1990-01-16 Massachusetts Institute Of Technology Method of isolating megakaryocyte stimulatory factor

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 105, issued 1986, M. KAWAKITA et al., "Human Urinary Megakaryocyte colony- and Thrombopoiesis-Stimulating Factor", see page 166, column 1, Abstract No. 105: 109405y. *
CHEMICAL ABSTRACTS, Volume 98, issued 1983, T. MIYAKE et al., "Partial Purification and Biological Properties of Thrombopoietin Extracted from Urine of Aplastic Anemia Patients", see page 63, column 1, Abstract No. 98: 83667v. *
J. Clin. Inc., Volume 75, issued April 1985, HOFFMAN et al., "Purification and Partial Characterization of a Megakaryocyte colony-Stimulating Factor from Human Plasma", pages 1174-1182, see the Abstract and introduction on page 1174. *
J. Clin. Invest., Volume 77, issued June 1986, H.H. YANG et al., "Studies of Human Megakaryocytopoiesis Using an Anti-Megakarocyte Colony-stimulating Factor Antiserum", pages 1873-1880, see the left-hand column on page 1873. *
See also references of EP0540575A4 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5260417A (en) * 1989-04-03 1993-11-09 Genetics Institute, Inc. Megakaryocyte growth promoting activity protein
US6433142B1 (en) 1989-08-08 2002-08-13 Genetics Institute, Llc Megakaryocyte stimulating factors
US7030223B2 (en) 1989-08-08 2006-04-18 Genetics Institute Llc Megakaryocyte stimulating factors
US7361738B2 (en) 1989-08-08 2008-04-22 Genetics Institute, Inc. Megakaryocyte stimulating factors
US5766581A (en) * 1994-03-31 1998-06-16 Amgen Inc. Method for treating mammals with monopegylated proteins that stimulates megakaryocyte growth and differentiation
US5795569A (en) * 1994-03-31 1998-08-18 Amgen Inc. Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation

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EP0540575A4 (en) 1994-06-08
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