Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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

• the present invention relates to human c-mpl receptor agonists (thrombopoietin) with activity on hematopoietic differentiation and expansion.
• MK Megakaryocyte maturation and platelet production has been long thought to be regulated by lineage specific humoral growth factors in a manner similar to cytokines that induce erythrocyte (erythropoietin) and granulocyte (G-CSF) expansion and maturation. Platelets are responsible for the prevention of bleeding in response to vascular injury. Therefore, platelet production is a vital component of hematopoietic regulation. Patients undergoing chemotherapy or bone marrow transplantation usually experience severely depressed platelet levels (thrombocytopenia) which may result in life threatening bleeding episodes.
• IL-3 IL-6, IL-11, SCF
• Plasma, serum and urine from thrombocytopenic dogs and humans have been found to contain growth factors that have specific megakarypoietic and thrombopoietic activities distinct from all known cytokines. These factors have been termed Meg-CSF, MK-CSF, megakaryocyte growth and development factor (MGDF) , megakaryopoietin, and thrombopoietin but the molecular structure has not been identified until recently.
• Meg-CSF Meg-CSF
• MK-CSF megakaryocyte growth and development factor
• MGDF megakaryopoietin
• thrombopoietin thrombopoietin
• the identification of the thrombopoietic cytokine, c-mpl ligand, originated with the identification of a myeloproliferative leukemia virus (MPLV, Wending et al., Virology 149:242-246, 1986) . Mice infected with this virus gave rise to multi-lineage myeloproliferation. Subsequent studies (Souyri et al. , Cell 63:1137-1147, 1990) demonstrated that the retrovirus encoded an oncogene (v-mpl) that when fused with viral envelope gene gave rise to a membrane anchored protein that resembles the cytoplasmic domain of the hematopoietic growth factor receptor family.
• v-mpl oncogene
• V-mpl was used to probe both human and murine RNA libraries for homologous genes. Clones were identified in both species and termed c- mpl (Vigon et al. , PNAS USA 89:5640-5644, 1992; Vigon et al . , Oncogene 8:2607-2615, 1993).
• C-mpl is a member of the cytokine receptor super-family with regions of homology to mIL-5rc, II_3rc, IL4rc, mEPOrc and mGCSFrc. A chimera of the intracellular domain of c-mpl and the extracellular domain of hIL4rc was transfected into a growth factor dependent cell line (BaF3).
• RT PCR reverse transcriptase polymerase chain reaction
• c-mpl ligand or thrombopoietin is a peptide with a predicted molecular mass of 35,000 kDa.
• the protein has a two domain structure with an amino-terminal domain (153 amino acids) with homology to erythropoietin and a carboxy-terminus rich in serine, threonine and proline residues which also contains several glycosylation sites. There are two potential arginine cleavage sites resulting in two shorter peptides of 25 kDa and 31 kDA forms both of which are biologically active. There is high inter species homology between human, murine, porcine, canine, rat and rabbit c-mpl ligand and most forms are active on all species tested.
• C-mpl ligand has been shown to stimulate the differentiation of CD34+ cells into cells megakaryocyte characteristics.
• CD34+ cells in the presence of c-mpl ligand underwent endomitosis (Kaushansky et al . , Nature 369:568-571, 1994), expressed the megakaryocyte lineage specific cell surface antigen CD41a and had morphology characteristic of megakaryocytes.
• endomitosis Korean et al. , Nature 369:568-571, 1994
• CD41a the megakaryocyte lineage specific cell surface antigen CD41a
• morphology characteristic of megakaryocytes had morphology characteristic of megakaryocytes.
• In vivo administration of c-mpl ligand have given rise to increased circulating platelets in normal mice (Lok et al., Nature 369:565-568, 1994).
• C-mpl deficient mice generated by gene targeting demonstrated a 85% decrease in circulating platelets and megakaryocytes but had normal amounts of other hematopoietic lineages (Gurney et al. , Science 265:1445-1447, 1994). Absolute thrombocytopenia was not observed in these animals indicating that other cytokines may have some activity in expansion of the MK lineage.
• c-mpl ligand is a cytokine with specific activity on the maturation of megakaryocytes and in platelet production.
• Other cytokines have been shown to have activity on megakaryocyte expansion and differentiation, including IL-3, IL-6, IL-11 and c-kit ligand.
• IL-3 IL-3
• IL-6 IL-6
• IL-11 IL-11
• c-mpl ligand may have therapeutic use in restoring circulating platelets to normal amounts in those cases where the number of platelets have been reduced due to diseases or therapeutic treatments such as radiation and/or chemotherapy.
• EP 675,201 Al relates to the c-mpl ligand (Megakaryocyte growth and development factor or MGDF) , allelic variations of c-mpl ligand and c-mpl ligand attached to water soluble polymers such as polyethylene glycol.
• MGDF Megakaryocyte growth and development factor
• WO 95/21920 provides the murine and human c-mpl ligand and polypeptide fragments thereof.
• the proteins are useful for in vivo and ex vivo therapy for stimulating platelet production.
• WO 95/27732 discloses circularly permuted GM-CSF, G- CSF, IL-2 and IL-4 and fusions with Pseudomonas exotoxin.
• the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain.
• linker an additional portion of sequence
• proteins which range in size from 58 to 462 amino acids (Goldenberg & Creighton, J. Mol . Biol . 165:407-413, 1983; Li & Coffino, Mol . Cell . Biol . 13:2377-2383, 1993) .
• the proteins examined have represented a broad range of structural classes, including proteins that contain predominantly ⁇ -helix (interleukin-4; Kreitman et al. , Cytokine 7:311-318, 1995), ⁇ -sheet (interleukin-1; Horlick et al. , Protein Eng.
• the positions of the internal breakpoints used in the studies cited here are found exclusively on the surface of proteins, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle (the variation in the relative distance from the original N- terminus to the breakpoint is ca. 10 to 80% of the total sequence length) .
• the linkers connecting the original N- and C-termini in these studies have ranged from 0 to 9 residues. In one case (Yang & Schachman, Proc. Natl . Acad. Sci . U. S.A . 90:11980-11984, 1993), a portion of sequence has been deleted from the original C-terminal segment, and the connection made from the truncated C-terminus to the original N-terminus.
• the present invention relates to novel c-mpl receptor agonists of the following formulas:
• Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or Met;
• Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met;
• Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu,
• Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or
• N-termmus is ⁇ omed to the C-termmus directly or through a linker capable of joining the N-termmus to the C-termmus and having new C- and N- termini at ammo acids;
• said c-mpl receptor agonist can be immediately preceded by (methionine -1 ), (alanine -1 ) or (methionine -2 , alanine -1 ) .
• Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro,
• Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu,
• Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn;
• N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
• said c-mpl receptor agonist can be immediately preceded by (methionine -1 ), (alanine -1 ) or (methionine -2 , alanine- ) .
• the more preferred breakpoints at which new C-termmus and N-termmus can be made in the amino acid sequence of (SEQ ID NO:2) above are; 80-81, 81-82, 82-83, 83-84, 84-85, 85-86, 86-87, 108-109, 109-110, 110-111, 111-112, 112-113, 113-114, 114-115, 115-116, 116-117, 117-118, 118-119, 119-120, 120-
• the most preferred breakpoints at which new C-termmus and N-termmus can be made in the amino acid sequence of (SEQ ID NO:2) above are; 81-82, 108-109, 115-116, 119-120, 122-123 and 125-126.
• human c-mpl receptor agonists may contain am o acid substitutions, deletions and/or insertions and may also have amino acid deletions at either/or both the N- and C- termini.
• the modified human c-mpl receptor agonists of the present mvention can be represented by the Formula: X 1 - ( L ) a -X 2
• X 1 is a peptide comprising an amino acid sequence corresponding to the sequence of residues n+l through J;
• X 2 is a peptide comprising an amino acid sequence corresponding to the sequence of residues 1 through n; n is an integer ranging from 1 to J-1; and
• L is a linker
• residues of human c-mpl ligand are numbered sequentially 1 through J from the amino to the carboxy terminus.
• a pair of adjacent amino acids within this protein may be numbered n and n+l respectively where n is an integer ranging from 1 to J-1.
• the residue n+l becomes the new N-terminus of the new c- pl receptor agonist and the residue n becomes the new C- terminus of the the new c-mpl receptor agonist.
• the linker (L) joining the N-terminus to the C-terminus is a polypeptide selected from the group consisting of: GlyGlyGlySer (SEQ ID NO:73); GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:74);
• GlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:75); SerGlyGlySerGlyGlySer (SEQ ID NO:76); GluPheGlyAsnMetAla (SEQ ID NO:77); GluPheGlyGlyAsnMetAla (SEQ ID NO:78); GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79); and GlyGlySerAspMetAlaGly (SEQ ID NO:80) .
• the present invention also encompasses recombinant human c-mpl receptor agonists co-administrated with one or more additional colony stimulating factors (CSF) including, cytokines, lymphokines, interleukins, hematopoietic growth factors (herein collectively referred to as "colony stimulating factors”) each of which may act through a different and specific cell receptor to initiate complementary biological activities.
• CSF colony stimulating factors
• co-administrated molecules may be characterized by having the usual activity of both of the peptides or it may be further characterized by having a biological or physiological activity greater than simply the additive function of the presence of human c-mpl receptor agonist or the second colony stimulating factor alone.
• the co ⁇ administration may also unexpectedly provide an enhanced effect on the activity or an activity different from that expected by the presence of human c-mpl receptor agonist or the second colony stimulating factor or human c-mpl ligand variant.
• the co-administration may also have an improved activity profile which may include reduction of undesirable biological activities associated with native human c-mpl ligand or native cytokine.
• in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before infusion into patients.
• Figure 1 schematically illustrates the sequence rearrangement of a protein.
• the N-termmus (N) and the C- term us (C) of the native protem are joined through a lmker, or joined directly.
• the protem is opened at a breakpoint creating a new N-termmus (new N) and a new C- term us (new-C) resultmg in a protein with a new linear am o acid sequence.
• a rearranged molecule may be synthesized de novo as linear molecule and not go through the steps of joining the original N-terminus and the C-termmus and opening of the protem at the breakpoint.
• Figure 2 shows a schematic of Method I, for creating new protems in which the original N-terminus and C-termmus of the native protem are joined with a linker and different N- termmus and C-termmus of the protem are created.
• the sequence rearrangement results in a new gene encoding a protein with a new N-termmus created at ammo acid 97 of the original protein, the original C- termmus (a.a. 174) joined to the ammo acid 11 (a.a. 1- 10 are deleted) through a linker region and a new C-termmus created at amino acid 96 of the original sequence.
• Figure 3 shows a schematic of Method II, for creating new proteins in which the original N-termmus and C-terminus of the native protem are joined without a lmker and different N-terminus and C-termmus of the protein are created.
• the sequence rearrangement results in a new gene encoding a protem with a new N- terminus created at ammo acid 97 of the original protem, the original C-terminus (a.a. 174) joined to the original N- termmus and a new C-termmus created at am o acid 96 of the original sequence.
• Figure 4 shows a schematic of Method III, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created.
• the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C- terminus (a.a. 174) joined to amino acid 1 through a linker region and a new C-terminus created at amino acid 96 of the original sequence.
• Receptor agonists of the present invention may be useful in the treatment of diseases characterized by a decreased levels of megakaryocyte cells of the hematopoietic system.
• a c-mpl receptor agonist may be useful in the treatment or prevention of thrombocytopenia.
• thrombocytopenia Currently the only therapy for thrombocytopenia is platelet transfusions which are costly and carry the significant risks of infection (HIV, HBV) and alloimunization.
• a c-mpl receptor agonist may alleviate or dimmish the need for platelet transfusions. Severe thrombocytopenia may result from genetic defects such as Fanconi ' s Anemia, Wiscott-Aldrich, or May-Hegglm syndromes.
• thrombocytopenia may result from auto- or allo-antibodies as Immune Thrombocytopenida Purpura, Systemic Lupus Erythromatosis, hemolytic anemia, or fetal maternal incompatibility.
• splenomegaly, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, infection or prosthetic heart valves may result in thrombocytopenia.
• Severe thrombocytopenia may also result from chemotherapy and/or radiation therapy or cancer.
• Thrombocytopenia may also result from marrow invasion by carcinoma, lymphoma, leukemia or fibrosis.
• the c-mpl receptor agonists of the present mvention may be useful in the mobilization of hematopoietic progenitors and stem cells into peripheral blood.
• Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation.
• Hematopoietic growth factors including G-CSF and GM-CSF have been shown to enhance the number of circulating progenitors and stem cells in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of plasmaphereses required.
• the c-mpl receptor agonists may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.
• drugs may cause bone marrow suppression or hematopoietic deficiencies.
• examples of such drugs are AZT, DDI, alkylating agents and anti-metabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin, gancyclovir, daunomycin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, analgesics such as aminopyrine and dipyrone, anti convulsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics.
• the c-mpl receptor agonists may be useful in preventing or treating the bone marrow suppression or hematopoietic deficiencies which often occur in patients treated with these drugs.
• Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, e.g., dialysis, c-mpl ligand may be useful in treating such hematopoietic deficiency.
• a novel method for producing the novel family of human c-mpl receptor agonists involves culturing suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of a novel c-mpl receptor agonist polypeptide.
• suitable cells or cell lines may include various strains of E. coli , yeast, mammalian cells, or insect cells may be utilized as host cells in the method of the present invention.
• Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel c-mpl receptor agonists.
• These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention.
• Appropriate vectors which can transform microorganisms capable of expressing the c-mpl receptor agonists include expression vectors comprising nucleotide sequences coding for the c-mpl receptor agonists joined to transcriptional and translational regulatory sequences which are selected according to the host cells used.
• Vectors incorporating modified sequences as described above are mcluded in the present invention and are useful in the production of the c-mpl receptor agonist polypeptides.
• the vector employed in the method also contams selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells.
• Other aspects of the present invention are methods and therapeutic compositions for treating the conditions referred to above.
• Such compositions comprise a therapeutically effective amount of one or more of the c-mpl receptor agonists of the present invention in a mixture with a pharmaceutically acceptable carrier. This composition can be administered either parenterally, intravenously or subcutaneously.
• the therapeutic composition for use m this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution.
• a parenterally acceptable protein solution having due regard to pH, isotonicity, stability and the like, is within the skill of the art.
• the c-mpl receptor agonists of the present invention may be useful in the mobilization of multipotential hematopoietic progenitors m peripheral blood.
• Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation.
• Hematopoietic growth factors including G-CSF and GM-CSF have been shown to enhance the number of circulating progenitors and stem ceils in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of pheresis required.
• the c-mpl receptor agonists may be useful in mobilization of multipotential hematopoietic cells and further enhance the efficacy of peripheral blood cell transplantation.
• the c-mpl receptor agonists of the present invention may also be useful in the ex vivo expansion of multipotential hematopoietic cells.
• Colony stimulating factors such as hIL-3
• CSFs colony stimulating factors
• hIL-3 colony stimulating factors
• myeloid lineage which is comprised of monocytes (macrophages), granulocytes (including neutrophils) and megakaryocytes, is critical in preventing infections and bleeding which can be life-threatening.
• Neutropenia and thrombocytopenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy.
• Bone marrow transplants have been used to treat this patient population.
• several problems are associated with the use of bone marrow to reconstitute a compromised hematopoietic system including: 1) the number of stem cells in bone marrow or other is limited, 2) Graft Versus Host Disease, 3) graft rejection and 4) possible contamination with tumor cells.
• Stem cells make up a very small percentage of the nucleated cells in the bone marrow, spleen and peripheral blood. It is clear that a dose response exits such that a greater number of stem cells will enhance hematopoietic recovery.
• Bone marrow from an allogeneic donor has been used to provide bone marrow for transplant.
• Graft Versus Host Disease and graft rejection limit bone marrow transplantation even in recipients with HLA-matched sibling donors.
• An alternative to allogeneic bone marrow transplants is autologous bone marrow transplants. In autologous bone marrow transplants, some of the patient's own marrow is harvested prior to myeloablative therapy, e.g. high dose chemotherapy, and is transplanted back mto the patient afterwards. Autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection.
• stem cells can be specifically isolated selected based on the presence of specific surface antigen such as CD34+ in order to decrease tumor cell contamination of the marrow graft.
• 5,061,620 relates to compositions comprising human hematopoietic stem cells provided by separatmg the stem cells from dedicated cells.
• 5,199,942 describes a method for autologous hematopoietic cell transplantation comprising: (1) obtaining hematopoietic progenitor cells from a patient; (2) ex-vivo expansion of cells with a growth factor selected from the group consisting of IL-3, flk3 ligand, c-kit ligand, GM CSF, IL-l, GM-CSF/IL-3 fusion protein and combinations thereof; (3) administering cellular preparation to a patient.
• a growth factor selected from the group consisting of IL-3, flk3 ligand, c-kit ligand, GM CSF, IL-l, GM-CSF/IL-3 fusion protein and combinations thereof.
• 5,240,856 relates to a cell separator that includes an apparatus for automatically controlling the cell separation process.
• WO 91/16116 describes devices and methods for selectively isolating and separating target cells from a mixture of cells.
• WO 91/18972 describes methods for in vitro culturing of bone marrow, by incubating suspension of bone marrow cells, using a hollow fiber bioreactor.
• WO 92/18615 relates to a process for maintaining and expanding bone marrow cells, in a culture medium containing specific mixtures of cytokines, for use in transplants.
• WO 93/08268 describes a method for selectively expanding ste cells, comprising the steps of (a) separating CD34+ stem cells from other cells and (b) incubating the separated cells in a selective medium, such that the stem cells are selectively expanded.
• WO 93/18136 describes a process for in vitro support of mammalian cells derived from peripheral blood.
• WO 93/18648 relates to a composition comprising human neutrophil precursor cells with a high content of myeloblasts and promyelocytes for treating genetic or acquired neutropenia.
• WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.
• WO 94/11493 describes a stem cell population that are CD34+ and small in size, which are isolated using a counterflow elutriation method.
• WO 94/27698 relates to a method combining immunoaffinity separation and continuous flow centrifugal separation for the selective separation of a nucleated heterogeneous cell population from a heterogeneous cell mixture.
• WO 94/25848 describes a cell separation apparatus for collection and manipulation of target cells.
• stem cell refers to the multipotential hematopoietic cells as well as early progenitor and precursors cells which can be isolated from bone marrow, spleen or peripheral blood.
• expansion refers to the differentiation and proliferation of the cells.
• the present mvention provides a method for selective ex-vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells, (b) culturing said separated stem cells with a selected medium which contains a c-mpl receptor agonist and optionally a colony stimulating factor; and (c) harvesting said stems cells.
• Stem cells as well as committed progenitor cells destined to become neutrophils, erythrocytes, platelets, etc., may be distinguished from most other cells by the presence or absence of particular progenitor marker antigens, such as CD34, that are present on the surface of these cells and/or by morphological characteristics.
• progenitor marker antigens such as CD34
• the phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-1+ and lin-, but it is to be understood that the present invention is not limited to the expansion of this stem cell population.
• the CD34+ enriched human stem cell fraction can be separated by a number of reported methods, including affmity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+.
• CD34+ progenitors are heterogeneous, and may be divided into several sub-populations characterized by the presence or absence of co-expression of different lineage associated cell surface associated molecules .
• the most immature progenitor cells do not express any known lineage associated markers, such as HLA-DR or CD38, but they may express CD90(thy-l).
• CD33, CD38, CD41, CD71, HLA-DR or c-kit can also be used to selectively isolate hematopoietic progenitors.
• the separated cells can be incubated in selected medium in a culture flask, sterile bag or in hollow fibers.
• Various colony stimulating factors may be utilized in order to selectively expand cells.
• Representative factors that have been utilized for ex-vivo expansion of bone marrow include, c-kit ligand, IL-3 G-CSF, GM-CSF, IL-l, IL-6, IL-11, flt-3 ligand or combinations thereof.
• the proliferation of the stem cells can be monitored by enumerating the number of stem cells and other cells, by standard techniques (e.g.
• hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609, 1993; Kobayashi et al . , Blood 73:1836-1841, 1989) .
• no single factor has been shown to be as effective as the combination of multiple factors.
• the present invention provides methods for ex vivo expansion that utilize c-mpl receptor agonists that are more effective.
• Another aspect of the invention provides methods of sustaining and/or expanding hematopoietic precursor cells which includes inoculating the cells into a culture vessel which contains a culture medium that has been conditioned by exposure to a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a c-mpl receptor agonist of the present invention.
• a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a c-mpl receptor agonist of the present invention.
• hematopoietic progenitor cells are good candidates for ex vivo gene transfection.
• Hematopoietic stem cells cycle at a very low frequency which means that growth factors may be useful to promote gene transduction and thereby enhance the clinical prospects for gene therapy.
• Gene therapy Potential applications include; 1) the treatment of many congenital metabolic disorders and immunodifiencies (Kay and Woo, Trends Genet . 10:253-257, 1994), 2) neurological disorders (Friedmann, Trends Genet . 10:210-214, 1994) , 3) cancer (Culver and Blaese, Trends Genet . 10:174-178, 1994) and 4) infectious diseases (Gilboa and Smith, Trends Genet . 10:139-144, 1994) .
• Viral based vectors include; 1) replication deficient recombinant retrovirus (Boris-Lawrie and Temin, Curr. Opin . Genet . Dev. 3:102-109, 1993; Boris-Lawrie and Temin, Annal . New York Acad. Sci . 716:59-71, 1994; Miller, Current Top. Microbiol . Immunol .
• Non-viral based vectors include protein/DNA complexes (Cristiano et al., PNAS USA . 90:2122-2126, 1993; Curiel et al. , PNAS USA 88:8850- 8854, 1991; Curiel, Annal. Ne York Acad. Sci . 716:36-58, 1994), electroporation and liposome mediated delivery such as cationic liposomes (Farhood et al. , Annal. New York Acad. Sci . 716:23-35, 1994) .
• the present invention provides an improvement to the existing methods of expanding hematopoietic cells, which new genetic material has been introduced, in that it provides methods utilizing c-mpl receptor agonists that have improved biological activity, including an activity not seen by any single colony stimulation factor and/or physical properties.
• a daily regimen may be in the range of 0.2 - 150 ⁇ g/kg of non- glycosylated c-mpl receptor agonists protein per kilogram of body weight. Dosages would be adjusted relative to the activity of a given receptor agonist and it would not be unreasonable to note that dosage regimens may include doses as low as 0.1 microgram and as high as 1 milligram per kilogram of body weight per day.
• c-mpl receptor agonist there may exist specific circumstances where dosages of c-mpl receptor agonist would be adjusted higher or lower than the range of 0.2 - 150 micrograms per kilogram of body weight. These include co-administration with other CSF or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated c-mpl receptor agonists; and various patient-related issues mentioned earlier in this section. As indicated above, the therapeutic method and compositions may also include co-administration with other human factors.
• a non-exclusive list of other appropriate hematopoietins, CSFs and interleukins for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, G-CSF, M-CSF, erythropoietin
• EPO IL-l, IL-4, IL-2, IL-3 , IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13 , IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand, (herein collectively referred to as "colony stimulating factors”), or combinations thereof.
• SCF stem cell factor
• colony stimulating factors also known as steel factor or c-kit ligand
• IL-3 variants taught in WO 94/12639 and WO 94/12638 can be co-administered with the polypeptides of the present invention.
• the c-mpl receptor agonists of the present invention can be co-administered as with another "colony stimulating factor" as discussed above in a fashion taught in WO 95/20976 and WO 95/20977.
• the dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition.
• Progress of the treated patient can be monitored by periodic assessment of the hematological profile, e.g., differential cell count and the like.
• the length of the amino acid sequence of the linker can be selected empirically or with guidance from structural information, or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to 50 A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp & Woods, Mol . Immunol . 20: 483-489, 1983; Kyte & Doolittle, J. Mol . Biol . 157:105- 132, 1992; solvent exposed surface area, Lee & Richards, J. Mol . Biol .
• linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly-Gly-Ser (SEQ ID NO:3) cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used.
• Sequences of c-mpl ligand of foldmg to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from withm the original polypeptide chain while using the linker sequence as described above.
• Ammo and carboxyl termini are selected from withm a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below.
• a novel ammo acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. In many cases the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus.
• those skilled in the art will recognize that selections of termini anywhere withm the region may function, and that these will effectively lead to either deletions or additions to the amino or carboxyl portions of the new sequence.
• Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets, chain reversals and turns, and loops; Kabsch & Sander, Biopolymers 22: 2577-2637, 1983), the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, Ann. .Rev. Biochem . 53:537-572, 1984) and the static and dynamic distribution of conformations along the polypeptide chain (Alber & Mathews, Method ⁇ Enzymol . 154: 511-533, 1987).
• solvent exposure of residues is a site of post- translational attachment of carbohydrate which is necessarily on the surface of the protein.
• methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops.
• Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile & Salvatore, Eur. J. Biochem .
• regions of ammo acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the so-called hydrophobic core of the protem and should also be avoided for selection of ammo and carboxyl termini.
• those regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity are the preferred sites for location of the extremes of the polypeptide chain. Continuous stretches of amino acid sequence that are preferred based on the above criteria are referred to as a breakpoint region.
• Methods for creation of ⁇ enes w th new N-term nus/C-terminus Method I. Creation of genes with new N-terminus/C-terminus which contain a linker region.
• the first primer set (“new start” and “Linker start”) is used to create and amplify, from the gene sequence, the DNA fragment (“Fragment Start”) that contains the sequence encoding the new N-terminal portion of the new protein followed by the linker that connects the C-terminal and N-terminal ends of the original protein.
• the second primer set (“new stop” and “linker stop”) is used to create and amplify, from the gene sequence, the DNA fragment (“Fragment Stop”) that encodes the same linker as used above, followed by the new C-terminal portion of the new protein.
• the “new start” and “new stop” primers are designed to include the appropriate restriction sites which allow cloning of the new gene into expression plasmids.
• Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension for seven minutes.
• a Perkin Elmer GeneAmp PCR Core Reagents kit is used.
• a 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 UM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgC12.
• PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) .
• primers "new start” and “new stop” are added to the annealed fragments to create and amplify the full-length new N- terminus/C-terminus gene.
• Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycle? 94°C denaturation for one minute, 60°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension fov seven minutes.
• a Perkin Elmer GeneAmp PCR Core Reagents kit is used.
• a 100 ul reaction contains 100 pmole of each primer and approximately 0.5 ug of DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2.
• PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
• New N-terminus/C-terminus genes without a linker joining the original N-terminus and C-terminus can be made using two steps of PCR amplification and a blunt end ligation.
• the steps are illustrated in Figure 3.
• the first primer set (“new start” and "P-bl start”) is used to create and amplify, from the original gene sequence, the DNA fragment (“Fragment Start”) that contains the sequence encoding the new N-terminal portion of the new protein.
• the second primer set (“new stop” and "P-bl stop”) is used to create and amplify, from gene sequence, the DNA fragment (“Fragment Stop”) that contains the sequence encoding the new C-terminal portion of the new protein.
• the “new start” and “new stop” primers are designed to include appropriate restriction sites which allow cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for 45 seconds and 72°C extension for 45 seconds. Deep Vent polymerase (New England Biolabs) is used to reduce the occurrence of overhangs in conditions recommended by the manufacturer.
• the "P-bl start” and “P-bl stop” primers are phosphorylated at the 5' end to aid in the subsequent blunt end ligation of "Fragment Start” and “Fragment Stop” to each other.
• a 100 ul reaction contained 150 pmole of each primer and one ug of template DNA; and lx Vent buffer (New England Biolabs), 300 uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit
• PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) .
• PCR reaction products are purified using a Wizard PCR Preps kit (Promega) .
• the primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors.
• "Fragment Start” is designed to create Ncol restriction site
• “Fragment Stop” is designed to create a Hindlll restriction site.
• Restriction digest reactions are purified using a Magic DNA Clean-up System kit (Promega) .
• Fragments Start and Stop are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen) . These fragments are combined with and annealed to the ends of the - 3800 base pair Ncol/Hindlll vector fragment of pMON3934 by heating at 50°C for ten minutes and allowed to slow cool.
• the three fragments are ligated together using T4 DNA ligase (Boehringer Mannheim) .
• the result is a plasmid containing the full-length new N-terminus/C-terminus gene.
• a portion of the ligation reaction is used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD) . Plasmid DNA is purified and sequence confirmed as below.
• New terminus/C-terminus genes can be made based on the method described in R. A. Horlick, et al Protein Eng. 5:427- 431, 1992) . Polymerase chain reaction (PCR) amplification of the new N-terminus/C-terminus genes is performed using a tandemly duplicated template DNA. The steps are illustrated in Figure 4.
• PCR Polymerase chain reaction
• the tandemly-duplicated template DNA is created by cloning and contains two similar, but not necessarily identical, copies of the gene separated by DNA sequence encoding a linker connecting the original C- and N-terminal ends of the two copies of the gene.
• Specific primer sets are used to create and amplify a full-length new N terminus/C- terminus gene from the tandemly-duplicated template DNA. These primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72 n extension for one minute; plus one cycle 72°C extension for seven minutes.
• a Perkin Elmer GeneAmp PCR Core Reagents kit (Perkin Elmer Corporation, Norwalk, CT) is used.
• a 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl 2 .
• PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) . PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
• the construction of the PCR templates involves the steps outlined below;
• Step 1 Reverse transc.rn-r.ase/polvmerase chain reaction
• c-mpl ligand The two forms of c-mpl ligand, one with a deletion of am o acids 112-115 and one without the deletion, can be isolated RT/PCR technology.
• Synthetic primers are designed so that they would anneal to either c-mpl ligand DNA or mRNA (c- mpl ligand sequence based on Genebank accession #L33410 or de Sauvage et al., Nature 369, 1994, pp.533 538) for priming first-strand complementary DNA (cDNA) synthesis.
• the resulting cDNA is used as a template in PCR (Saik , 1985) to generate double-stranded DNA (dsDNA or DNA) which can be used in additional PCR or digested with appropriate restriction enzymes for transfer to E. coli or mammalian expression plasmids.
• RNA for the reveise transcriptase (RT) reaction, human fetal (lot #38130) and adult liver (lot #46018) A+ RNA can be obtained from Clontech (Palo Alto, CA) .
• the RT reactions are carried out using a cDNA CycleTM Kit obtamed from Invitrogen (San Diego, CA) .
• One microgram (ug) of each RNA sample is combmed and denatured at 65°C for 10 mm. in the presence of either random primers, oligo dT primer or a specific 3' anti- sense primer.
• the samples are cooled for 2 min. on ice and spun down for 10 sec. at 10,000 x g.
• RNAse inhibitor, reverse transcriptase buffer, deoxynucleotides, sodium pyrophosphate and reverse transcriptase are added as described by manufacturer, and the 20 microliter reaction is cubated at 42°C tor 1 hr.
• PCR For PCR a specific 5' sense primer and 3' anti-sense primers are added to the RT reactions and the PCR is carried out using reagents from Boehringer Mannheim (Indianapolis, IN) or Perkm-Elmer (Norwalk, CT) as described by the manufacturers using Taq polymerase. The PCR reactions are subjected to 30 cycles of the following; 1 min. @ 94°C, 1 min. @ 58°C, 90 sec. @ 72°C.
• PCR reactions are purified using a WizardTM PCR Preps kit from Promega (Madison, WI) . Briefly, the PCR reactions are added to 100 microliters of Direct Purification buffer, and 1 milliliter (mL) of PCR Preps DNA Purification Resin is added to this mixture. After 1 mmute incubation at 24°C, the supernatant is removed by vacuum filtration through a filtration column. Two mLs of 80% isopropanol is used to wash the resm via vacuum filtration. The column containing the resm is then subjected to centrifugation at 10,000 x g for 30 seconds to remove residual isopropanol.
• WizardTM PCR Preps kit from Promega (Madison, WI) . Briefly, the PCR reactions are added to 100 microliters of Direct Purification buffer, and 1 milliliter (mL) of PCR Preps DNA Purification Resin is added to this mixture. After 1 mmute incubation
• the PCR product is eluted with 50 microliters of 10 mM Tris-Cl, 1 mM EDTA, pH7. , via centrifugation at 10,000 x g for 30 seconds followed by transfer of supernatant to a new tube.
• Step 2 Subcloning the PCR products mto a mammalian expression vector
• the c-mpl ligand PCR products are digested with the appropriate restriction enzymes for ligation to a mammalian expression vector.
• the mammalian expression vector is a derivative of pMON3359 which is a pUCl ⁇ -based vector contammg a mammalian expression cassette.
• the cassette includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 secretion signal sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned mto the pUCl ⁇ polylinker (Hippenmeyer et al., Bio /Technology, 1037-1041, 1993) .
• Restriction enzyme digestions are mcubated for 1 hour at 37°C as described by the manufacturer prior to electrophoresis through a 1% agarose/lx TBE/EtBr gel. Fragments are first visualized by long-wave UV and gel-purified using a Qiaex DNA Extraction kit (Qiagen, Chatsworth, CA) . The DNA fragments are purified from the resin by agarose solublization, addition of a DNA- bindmg resm, and extensive washing of the resm prior to elution with water. The purified DNA products are combined at a molar excess of PCR product to vector fragment and the ligation reactions are carried out according to the manufacturer's recommended conditions for T4 DNA ligase.
• Qiaex DNA Extraction kit Qiagen, Chatsworth, CA
• An E. coli strain is then transformed with the ligation, plated out onto LB-agar plus ampicillin (100 ug/ml) .
• the colonies are screened for presence of a c-mpl ligand gene and DNA is isolated for DNA sequencing to identify both forms c-mpl ligand, one with amino acids 112-115 deleted and one with them present.
• the linkers that join c-mpl (1-153) ligand genes are created by annealing 200 picomoles each of a pair of complementary synthetic oligonucleotides in 5 microliters of ligation buffer (Boehringer-Mannheim #1243 292) .
• Each linker which has flanking EcoRI and Afllll sites, is ligated overnight with a 3.7 Kbp EcoRI/BstXI fragment from a form of c-mpl ligand with amino acids 1-153 (step 2), and a 1 Kbp NcoI/BstXI fragment from the either of the two types of clones in step 2 above, one with and one without a deletion of amino acids 112-115.
• the resulting DNA is used to transform E.
• coli DH5 ⁇ TM cells Transformed cells are selected on LB agar plates containing ampicillin (100 ug/ml). Plasmid DNA is obtained from single colonies of several clones and sequenced to verify correct assembly of the dimer via the linker. The resulting plasmid DNA template can be used in making novel c-mpl ligand molecules via the Horlick method (Prot. Eng. 5:427-433, 1992).
• 5' sense ana 3' anti-sense primers are combined with a dimer template for PCR using reagents from Boehringer Mannheim (Indianapolis, IN) or Perkin-Elmer (Norwalk, CT) as described by the manufacturersusing Taq polymerase.
• the PCR reactions are subjected to 30 cycles of the following; 1 min. G. 94°C, 1 min. @ 58°C, 90 sec. @ 72°C.
• the product (about 480 base pairs) is visualized using a short-wave UV light source.
• the reactions are purified using a WizardTM PCR Preps kit from Promega (Madison, WI) .
• PCR reactions are added to 100 microliters of Direct Purification buffer, and 1 milliliter (mL) of PCR Preps DNA Purification Resin is added to this mixture. After 1 minute incubation at 24°C, the supernatant is removed by vacuum filtration through a filtration column. Two mLs of 80% isopropanol is used to wash the resin via vacuum filtration. The column containing the resin is then subjected to centrifugation at 10,000 x g for 30 seconds to remove residual isopropanol.
• the PCR product is eluted with 50 microliters of 10 mM Tris-Cl, 1 mM EDTA, pH7.4, via centrifugation at 10,000 x g for 30 seconds followed by transfer of supernatant to a new tube.
• novel c-mpl receptor agonists PCR products are digested with the appropriate restriction enzymes for ligation to either mammalian or E. coli expression vectors.
• the mammalian expression vectors are derivatives of pMON3359 which is a pUCl ⁇ -based vector containing a mammalian expression cassette.
• the cassette includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 signal peptide sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned into the pUC18 polylinker [See Hippenmeyer et al. , Bio/Technology, 1993, pp.1037-1041] . Restriction enzyme digestions are incubated for 1 hour at 37°C as described by the manufacturer prior to electrophoresis through a 1% agarose/lx TBE/EtBr gel.
• Fragments are first visualized by long-wave UV and gel- purified using a Qiaex DNA Extraction kit (Qiagen, Chatsworth, CA) .
• the DNA fragments are purified from the resin by agarose solublization, addition of a DNA-binding resin, and extensive washing of the resin prior to elution with water.
• the purified DNA products are combined at a molar excess of PCR product to vector and the ligation reactions are carried out according to the manufacturer's recommended conditions for T4 DNA ligase.
• the £. coli expression vectors that direct high-level production of heterologous proteins in the cytoplasm are derivatives of that described elsewhere (Olins et al., Methods Enzym. , 185:115-119, 1988 and Rangwala et al., Gene, 122: 263-269, 1992).
• the expression cassette consists of the recA promoter and T7 gene 10 ribosome binding site (RBS) as well as an M13 origin of replication or a tandem inverted repeat of a phage P22 gene which acts as a transcription terminator. These cassettes are on a plasmid with the pBR327 origin of replication and encode a gene either for spectinomycin or ampicillin resistance.
• E. coli strains DH5 ⁇ TM (Life Technologies, Gaithersburg,
• E. coli strain MON105 can be obtained from the American Type Culture Collection (ATCC, Rockville, MD) and is the host for expressing alternate forms of c-mpl ligand in the cytoplasm or periplasmic space, respectively, of E. coli .
• MON105 ATCC#55204 F-, lamda-, IN(rrnD, rrE)l, rpoD+, rpoH358 DH5 ⁇ TM: F-, phi80dlacZdeltaMl5, delta (lacZYA-argF)Ul69 , deoR, recAl, endAl, hsdRl7 (rk-,mk+) , phoA, supE441amda-, thi-1, gyrA96, relAl
• TGI delta(lac-pro) , supE, thi-1, hsdD5/F' (traD36, proA+B+, laclq, lacZdeltaMl5)
• DH5 ⁇ TM Subcloning efficiency cells are purchased as competent cells and are ready for transformation using the manufacturer's protocol, while both E. coli strains TGI and MON105 are rendered competent to take up DNA using a CaCl2 method.
• 20 to 50 mLs of cells are grown in LB medium (1% bacto-tryptone, 0.5% bacto-yeast extract, 150 millimolar NaCl) to a density of approximately 1.0 optical density units at 600 nanometers (OD600) as measured by a Baush & Lomb Spectronic spectrophotometer (Rochester, NY) .
• the cells are collected by centrifugation and resuspended in one-fifth culture volume of CaCl2 solution (50 millimolar CaCl2, 10 millimolar Tris-Cl, pH7.4) and are held at 4"C for 30 minutes.
• the cells are again collected by centrifugation and resuspended in one tenth culture volume of CaCl2 solution.
• Ligated DNA is added to 0.2 mL of these cells, and the samples are held at 4'C for 1 hour.
• the samples are shifted to 42"C for two minute ⁇ and 1.0 mL of LB is added prior to shaking the samples at 37"C for one hour.
• Cells from these samples are spread on plates (LB medium plus 1.5% bacto-agar) containing either ampicillin (100 micrograms/mL, ug/mL) when selecting for ampicillin-resistant transformants, or spectinomycin (75 ug/mL) when selecting for spectinomycin- resistant transformants.
• the plates are incubated overnight at 37'C. Single colonies are picked, grown in LB supplemented with appropriate antibiotic for 6-16 hours at 37'C with shaking.
• Colonies are picked and inoculated into LB plus appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL spectinomycin) and are grown at 37°C while shaking. Before harvesting the cultures, 1 ul of cells are analyzed by PCR for the presence of a c-mpl ligand gene. The PCR is carried out using a combination of primers that anneal to the c-mpl ligand gene and/or vector. After the PCR is complete, loading dye is added to the sample followed by electrophoresis as described earlier. A gene has been ligated to the vector when a PCR product of the expected size is observed.
• appropriate antibiotic 100 ug/mL ampicillin or 75 ug/mL spectinomycin
• Plasmid DNA is isolated using the Promega WizardTM Miniprep kit (Madison, WI) or the Qiagen QIAwell Plasmid isolation kits (Chatsworth, CA) . Both kits follow the same general procedure for plasmid DNA isolation. Briefly, cells are pelleted by centrifugation (5000 x g) , plasmid DNA released with sequential NaOH & acid treatment, and cellular debris is removed by centrifugation (10000 x g) . The supernatant (containing the plasmid DNA) is loaded onto a column containing a DNA-binding resin, the column is washed, and plasmid DNA eluted with TE. After screening for the colonies with the plasmid of interest, the E. coli cells are inoculated into 100 mis of LB plus appropriate antibiotic for overnight growth at 37°C in an air incubator while shaking. Plasmid DNA is isolated using the Qiagen Plasmid Midi kit
• Purified recombinant double-stranded DNA is sequenced using the Applied Biosystems Inc. (ABI, Foster City, CA) PRISMTM Ready Reaction DyeDeoxyTM Terminator Sequencing system.
• the ABI system relies on incorporation of four fluorescence labelled dideoxy nucleotides into single- stranded DNA during multiple rounds of amplification. Plasmid DNA and a sequencing primer are added to the reaction mixture (including Taq DNA polmerase, buffer and nucleotides) , which is subjected to 25 cycles of amplification (30 seconds at 96°C, 15 seconds at 50°C, 4 minutes at 60°C) .
• the BHK-21 cell line can be obtained from the ATCC (Rockville, MD) .
• the cells are cultured in Dulbecco's modified Eagle media (DMEM/high-glucose) , supplemented to 2 millimolar (mM) L-glutamine and 10% fetal bovine serum (FBS).
• DMEM/high-glucose Dulbecco's modified Eagle media
• FBS fetal bovine serum
• This formulation is designated BHK growth media.
• Selective media is BHK growth media supplemented with 453 units/ml hygromycin B (Caibiochem, San Diego, CA) .
• the BHK-21 cell line was previously stably transfected with the HSV transactivating protein VPI6, which transactivates the IE110 promoter found on the plasmid pMON3359 (Hippenmeyer et al., Bio/Technology, 1037-1041, 1993).
• the VP16 protein drives expression of genes inserted behind the IE110 promoter.
• BHK-21 cells expressing the transactivating protein VP16 is designated BHK-VP16.
• the plasmid pMONlll ⁇ (Highkin et al.. Poul try Sci . , 70:970-981, 1991) expresses the hygromycin resistance gene from the SV40 promoter.
• a similar plasmid is available from ATCC, pSV2- hph.
• BHK-VP16 cells are seeded into a 60 millimeter (mm) tissue culture dish at 3 X IO 5 cells per dish 24 hours prior to transfection.
• Cells are transfected for 16 hours in 3 mL of "OPTIMEM”TM (Gibco-BRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug hygromycin resistance plasmid, pMONlll ⁇ , and 80 ug of Gibco- BRL "LIPOFECTAMINE”TM per dish.
• the media is subsequently aspirated and replaced with 3 mL of growth media.
• At 48 hours post-transfection media from each dish is collected and assayed for activity (transient conditioned media) .
• the cells are removed from the dish by trypsin-EDTA, diluted 1:10 and transferred to 100 mm tissue culture dishes containing 10 mL of selective media. After approximately 7 days in selective media, resistant cells grow into colonies several millimeters in diameter. The colonies are removed from the dish with filter paper (cut to approximately the same size as the colonies and soaked in trypsin/EDTA) and transferred to individual wells of a 24 well plate containing 1 mL of selective media. After the clones are grown to confluency, the conditioned media is reassayed, and positive clones are expanded into growth media.
• E. coli strain MON105 harboring the plasmid of interest are grown at 37°C in M9 plus casamino acids medium with shaking in a air incubator Model G25 from New Brunswick Scientific (Edison, New Jersey) . Growth is monitored at OD600 until it reaches a value of 1.0 at which time Nalidixic acid (10 milligrams/mL) in 0.1 N NaOH is added to a final concentration of 50 ⁇ g/mL. The cultures are then shaken at 37°C for thr.- ⁇ to four additional hours. A high degree of aeration is maintained throughout culture period in order to achieve maximal production of the desired gene product. The cells are examined under a light microscope for the presence of inclusion bodies (IB) .
• IB inclusion bodies
• One mL aliquots of the culture are removed for analysis of protem content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE (Maniatis et al. Molecular Cloning: A Laboratory Manual, 1982) .
• the first step in purification of the protem is either sonication or homogenization of the cells.
• the cells are resuspended in one-tenth volume (based on culture size) sonication buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA).
• resuspended cells are subjected to several repeated sonication t, irsts using the microtip from a Sonicator cell disrupter, Model W-375 obtamed from Heat Systems-Ultrasonics Inc. (Farmingdale, New York) .
• the extent of sonication is monitored by examining the homogenates under a light microscope. After all of the cells are disrupted, the homogenates are fractionated by centrifugation at 10000 x g for 20 mmutes at 4°C in a JA-20 rotor and J2-21 centrifuge (Beckman, Fullerton, CA) .
• the IBs are released from the cells by lysing the cells in sonication buffer with a Manton-Gaulm homogenizer (Holland) followed by centrifugation as above.
• the IB pellets which are highly enriched for the recombinant protein, are then subjected to another round of sonication and centrifugation as described above.
• the recombinant protein is purified by a variety of standard methods.
• the protein is purified from £. coli contaminants using ion-exhange chomotography, such as Q-sepharose (anion) and S-sepharose (cation) , gel filtration, hydrophobic chromatography or reversed phase HPLC. After dialysis against a low ionic strength buffer, the purified protein is stored frozen or lyophilized.
• RT reaction random primers and oligo dT primer are used to generate cDNA from a combination of human and fetal liver mRNA.
• the RT product serves as the template for PCR with a combination of the primers, Forward primer: c-mplNcol (SEQ ID NO:4) and Reverse primer: Ecompl (SEQ ID NO-.5) .
• the c-mplNcol (SEQ ID NO:4) primer anneals to the c-mpl ligand gene (bases #279-311 based on c-mpl ligand sequence from Genebank accession #L33410 or de Sauvage et al., Nature 369:533-538, 1994) and encodes a ⁇ col restriction enzyme site just 5' to the first mature codon (Ser ⁇ ) .
• the ⁇ col restriction enzyme site codes for methionine and alanine codons prior to ser-1 and includes codon degeneracy for the Ala codon and the first four c-mpl ligand codons (Ser 1 , Pro 2 , Ala 3 , & Pro 4 ) .
• the Ecompl (SEQ ID NO:5) primer anneals to bases #720-737 of c-mpl ligand and encodes a EcoRI recognition site in-frame with the c-mpl ligand gene immediately following Arg 1 ⁇ _ r ⁇ g EcoRI site creates Glu- ⁇ 4 and Phe-*-55 codons following Argl53 _
• the ca. 480 bp PCR product is purified, digested with Ncol and EcoRI and ligated to the NcoI-EcoRl vector fragment of pMON3993 (ca. 4550 bp.) .
• pMON3993 is a derivative of pMON3359.
• the expression cassette in pMON3359 includes a herpes simplex viral promoter IE110 (-800 to +120), an IL-3 signal peptide sequence and a SV40 late poly-adenylation (poly-A) signal which has been subcloned into the pUC18 polylinker (See Hippen eyer et al., Bio/Technology, 1037-1041, 1993).
• the human IL-3 signal peptide sequence which had been subloned as a BamHI fragment into the unique BamHI site between the IE110 promoter and poly-A signal, contains an Ncol site at its 3' end and is then followed by a unique EcoRI site.
• the DNA sequence of the signal peptide is shown below (restriction enzyme sites are indicated above) .
• the ATG (methionine) codon within the Ncol site is in-frame with the initiator ATG of the signal peptide (underlined) ; BamHI
• pMON26458 coding for c-mpl ligand amino acids 1-153, is a result of this cloning.
• the RT reaction from Example 1 serves as the template for PCR with a combination of the following primers; c-mplNcol (SEQ ID NO:4) (forward primer) and c-mplHindlll (SEQ ID NO: 6) (reverse primer) .
• the c- mplNcoI (SEQ ID NO:4) primer is described in Example 1.
• PCR products Two types are generated from the RT cDNA samples, one with a deletion of the codons for amino acids 112-115 and one without the deletion of these codons.
• the c- mpl ligand PCR products (ca. 480 bp) are digested with Ncol and Hindlll restriction enzymes for transfer to a mammalian expression vector, pMON3934, which is a derivative of pMON3359 (see Example 1) .
• pMON3934 is digested with Ncol and Hindlll (ca. 3800 bp) and will accept the PCR products.
• the plasmid, pMON32132 contians the DNA sequence of (SEQ ID NO:82) which encodes for amino acids 1-153 of c-mpl ligand (SEQ ID NO: .4) was a result of this cloning.
• the plasmid, pMON32133 contains the DNA sequence of (SEQ ID NO:83) which encodes amino acids 1-153 of c-mpl ligand with a deletion of codons 112-115 ( ⁇ 112-115) (SEQ ID NO:45) was also a result of this cloning.
• a PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of PMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32133 (containing a deletion of amino acids 112-115) along with the EcoRI/AflHI 5L synthetic oligonucleotide linker 5L-5' (SEQ ID NO:9) and 5L-3 ' (SEQ ID NO:10) .
• the EcoRI end of the linker will ligate to the EcoRI end of PMON26458.
• the Afllll end of the linker will ligate to the Ncol site of pMON32133, and neither restriction site will be retained upon ligation.
• the BstXl sites of pMON26458 and pMON32133 will ligate as well.
• Plasmid, pMON28548, is a result of the cloning and contains the DNA sequence of (SEQ ID NO:38) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker to amino acids 1-153 c-mpl ligand that contains a deletion of amino acids 112-115 (SEQ ID NO:43).
• SEQ ID NO:38 the DNA sequence of (SEQ ID NO:38) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker to amino acids 1-153 c-mpl ligand that contains a deletion of amino acids 112-115 (SEQ ID NO:43).
• a PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132 along with the EcoRI/AflHI 4L synthetic oligonucleotide linker 4L-5' (SEQ ID NO:7) and 4L-3 ' (SEQ ID NO:8) .
• Plasmid, pMON28500 is a result of the cloning and contains the DNA sequence of (SEQ ID NO:39) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyAsnMetAla (SEQ ID NO:77) linker (4L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:46) .
• a PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of PMON26458 to the 1 Kbp NcoI/BstXI fragment from pMON32132 along with the EcoRI/AflHI 5L synthetic oligonucleotide linker 5L-5' (SEQ ID NO:9) and 5L-3 ' (SEQ ID NO:10).
• the EcoRI end of the linker will ligate to the EcoRI end of PMON26458.
• the Afllll end of the linker will ligate to the Ncol site of pMON32132, and neither restriction site will be retained upon ligation.
• PLasmid, pMON28501 is a result of the cloning and contains the DNA sequence of (SEQ ID NO: 40) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:78) linker (5L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:47) .
• a PCR template for generating novel forms of c-mpl ligand is constructed by ligating the 3.7 Kbp BstXI/EcoRI fragment of pMON26458 to the 1 Kbp NcoI/BstXI fragment from PMON32132 along with the EcoRI/AflHI 8L synthetic oligonucleotide linker 8L-5' (SEQ ID NO:ll) and 8L-3 ' (SEQ ID NO:12) .
• the EcoRI end of the linker will ligate to the EcoRI end of PMON26458.
• the Afllll end of the linker will ligate to the Ncol site of pMON32132, and neither restriction site will be retained upon ligation.
• Plasmid, pMON32136 is a result of the cloning which contains the DNA sequence of (SEQ ID NO:41) and encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:79) linker (8L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:48) .
• Novel c-mpl ligand genes are generated using the Horlick method.
• the PCR reaction was carried out using dimer template pMON28501 and one of the sets of synthetic primer sets below (number refers to first amino acid of new molecule) .
• the products that are generated are about 480 bp and are purified via Magic PCR Clean up kits (Promega) .
• Table 4 shows the template, the primer set used in the PCR reaction and the breakpoint for each Example.
• the expression vector, pMON3934 is digested with Ncol and
• Hindlll (ca. 3800 bp) and accepts the PCR products as Ncol- Hindlll or Afllll-Hindlll fragments.
• Table 4 shows the restriction digest of the PCR product and the resulting expression plasmid pMON designation.
• dimer templates pMON28500, pMON32136 and pMON28548 could be used in the PCR reaction as described in Examples 7-18.
• Transfected cell lines Cell lines such as Baf/3 cell line can be transfected with a colony stimulating factor receptor, such as the human IL-3 receptor or human c-mpl receptor, which the cell line does not normally have. These transfected cell lines can be used to determine the activity of the ligand for which the receptor has been transfected into the cell line.
• a colony stimulating factor receptor such as the human IL-3 receptor or human c-mpl receptor
• One such transfected Baf/3 cell line was made by cloning the cDNA encoding c-mpl from a library made from a c- mpl responsive cell line and cloned into the multiple cloning site of the plasmid pcDNA3 (Invitrogen, San Diego Ca. ) .
• Baf/3 cells were transfected with the plasmid via electroporation. The cells were grown under G418 selection in the presence of mouse IL-3 in Wehi conditioned media. Clones were established through limited dilution.
• the BHK expression levels and bioactivity data of some of the c-mpl receptor agonists of the present invention are shown in Table 5.
• the supernatant from the transfected BHK cells was evaluated for expression of the c-mpl receptor agonists by western analysis using an antobody raised against c-mpl ligand. Constructs that expressed at a "++++" level were assayed in the Baf-3/c-mpl cell proliferation assay.
• Bone marrow aspirates are obtained from normal allogeneic marrow donors after informed consent.
• Cells are diluted 1:3 in phosphate buffered saline (PBS, Gibco- BRL) , 30 mL are layered over 15 mL Histopaque-1077 (Sigma) and centrifuged for 30 minutes at 300 RCF. The mononuclear interface layer is collected and washed in PBS.
• CD34+ cells are enriched from the mononuclear cell preparation using an affinity column per manufacturers instructions (CellPro, Inc, Bothell WA) .
• CD34+ cells After enrichment, the purity of CD34+ cells is 70% on average as determined by using flow cytometric analysis using anti CD34 monoclonal antibody conjugated to fluorescein and anti-CD38 conjugated to phycoerythrin (Becton Dickinson, San Jose CA) .
• Cells are resuspended at 40,000 cells/mL in X-Vivo 10 media (Bio-Whittaker, Walkersville, MD) and 1 mL is plated in 12-well tissue culture plates (Costar). Human IL-3 variant, PMON13288, is used at 10 ng/mL or 100 ng/mL.
• Conditioned media from BHK cells transfected with plasmid encoding c-mpl ligand are tested by addition of 100 ⁇ l of supernatant added to 1 mL cultures (approximately a 10% dilution) . Cells are incubated at 37°C for 8-14 days at 5% C02 in a 37°C humidified incubator, b. Cell Harvest and Analysis:
• MK buffer 13.6 mM Sodium Citrate, 1 mM Theophylline, 2.2 ⁇ m PGE1, 11 mM Glucose, 3% w/v BSA, in PBS, pH 7.4,
• MK buffer 13.6 mM Sodium Citrate, 1 mM Theophylline, 2.2 ⁇ m PGE1, 11 mM Glucose, 3% w/v BSA, in PBS, pH 7.4,
• DNA analysis cells are made permeable in MK buffer containing 0.5% Tween 20 (Fisher, Fair Lawn NJ) for 20 minutes on ice followed by fixation in 0.5% Tween-20 and 1% paraformaldehyde (Fisher Chemical) for 30 minutes followed by incubation in Propidium Iodide (Caibiochem , La Jolla Ca) (50 ⁇ g/mL) with RNAase (400 U/mL) in 55% v/v MK buffer (200 mOsm) for 1-2 hours on ice. Cells are analyzed on a FACScan or Vantage flow cytometer (Becton Dickinson, San Jose, CA) .
• CD41a-FlTC Green fluorescence
• PI red fluorescence
• All cells are collected to determine the percent of cells that are CD41+.
• Data analysis is performed using LYSIS software (Becton Dickinson, San Jose, CA) .
• Percent of cells expressing the CD41 antigen is obtained from flow cytometry analysis (Percent) .
• CD34+ enriched population are isolated as described above.
• Cells are suspended at 25,000 cells/mL with/without cytokine(s) in a media consisting of a base Iscoves IMDM media supplemented with 0.3% BSA, 0.4 mg/mL apo-transferrin, 6.67 ⁇ M FeCl2, 25 ⁇ g/mL CaCl2, 25 ⁇ g/mL L asparagine, 500 ⁇ g/mL E-amino-n-caproic acid and Penicillin/Streptomycin.
• thrombin Prior to plating into 35 mm plates, thrombin is added (0.25 units/mL) to initiate clot formation.
• Cells are incubated at 37°C for 13 days at 5% C02 in a 37°C humidified incubator. At the end of the culture period plates are fixed with methanol:acetone (1:3), air dried and stored at -200C until staining.
• a peroxidase immunocytochemistry staining procedure is used
• CFU-MK small colonies, 1-2 foci and less that approx. 25 cells
• BFU MK large, multi-foci colonies with > 25 cells
• mixed colonies mixture of both positive and negative cells
• GTCCTTCACA GCAGACTGAG CCAGTGCCCA
• GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120
• GTCCTTCACA GCAGACTGAG CCAGTGCCCA
• GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120 CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 180
• GTCCTTCACA GCAGACTGAG CCAGTGCCCA
• GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 600
• GTCCTTCACA GCAGACTGAG CCAGTGCCCA
• GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120
• GTCCTTCACA GCAGACTGAG CCAGTGCCCA
• GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 120

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