WO1997012985A2 - Multi-functional hematopoietic receptor agonists - Google Patents

Multi-functional hematopoietic receptor agonists Download PDF

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Publication number
WO1997012985A2
WO1997012985A2 PCT/US1996/015774 US9615774W WO9712985A2 WO 1997012985 A2 WO1997012985 A2 WO 1997012985A2 US 9615774 W US9615774 W US 9615774W WO 9712985 A2 WO9712985 A2 WO 9712985A2
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WIPO (PCT)
Prior art keywords
terminus
amino acids
cells
amino acid
acid sequence
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PCT/US1996/015774
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French (fr)
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WO1997012985A9 (en
WO1997012985A3 (en
Inventor
Yiqing Feng
Nicholas R. Staten
Charles M. Baum
Neena L. Summers
Maire H. Caparon
S. C. Bauer
Linda Zurfluh
John P. Mckearn
Barbara Kure Klein
Stephen C. Lee
Charles A. Mcwherter
Judith G. Giri
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G.D. Searle & Co.
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Priority to NZ320978A priority Critical patent/NZ320978A/en
Application filed by G.D. Searle & Co. filed Critical G.D. Searle & Co.
Priority to EP96936114A priority patent/EP0854928A2/en
Priority to IL12383296A priority patent/IL123832A0/en
Priority to JP9514385A priority patent/JPH11510062A/en
Priority to AU73844/96A priority patent/AU705083B2/en
Priority to PL96326072A priority patent/PL184424B1/en
Priority to BRPI9610977A priority patent/BRPI9610977A2/en
Priority to US08/835,162 priority patent/US6066318A/en
Publication of WO1997012985A2 publication Critical patent/WO1997012985A2/en
Publication of WO1997012985A9 publication Critical patent/WO1997012985A9/en
Publication of WO1997012985A3 publication Critical patent/WO1997012985A3/en
Priority to NO981500A priority patent/NO981500L/en
Priority to US10/695,584 priority patent/US20040171115A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/524—Thrombopoietin, i.e. C-MPL ligand
    • 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]
    • 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
    • C07K14/54—Interleukins [IL]
    • C07K14/5403—IL-3
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates to multi-functional hematopoietic receptor agonists.
  • Colony stimulating factors which stimulate the differentiation and/or proliferation of bone marrow cells have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells.
  • CSFs in both human and murine systems have been identified and distinguished according to their activities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies,
  • GM-CSF and interleukin-3 have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies.
  • IL-3 also stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies.
  • U.S. 4,877,729 and U.S. 4,959,455 disclose human IL-3 and gibbon IL-3 cDNAs and the protein sequences for which they code.
  • the hIL-3 disclosed has serine rather than proline at position 8 in the protein sequence.
  • WO 88/00598 discloses gibbon- and human-like IL-3.
  • the hIL-3 contains a Ser 8 -> Pro 8 replacement. Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites.
  • U.S. 4,810,643 discloses the DNA sequence encoding human G-CSF.
  • WO 91/02754 discloses a fusion protein comprised of GM-CSF and IL-3 which has increased biological activity
  • WO 92/04455 discloses fusion proteins composed of IL-3 fused to a lymphokine selected from the group consisting of IL-3, IL-6, IL-7, IL-9, IL-11, EPO and G-CSF.
  • WO 95/21197 and WO 95/21254 disclose fusion proteins capable of broad multi-functional hematopoietic properties.
  • GB 2,285,446 relates to the c-mpl ligand
  • thrombopoietin and various forms of thrombopoietin which are shown to influence the replication, differentiation and maturation of megakaryocytes and megakaryocytes progenitors which may be used for the treatment of thrombocytopenia.
  • EP 675,201 A1 relates to the c-mpl ligand
  • 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.
  • 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
  • sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease T1, Bacillus ⁇ -glucanase, interleukin-1 ⁇ , ⁇ -spectrin SH3 domain, pepsinogen, interleukin-4).
  • basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease T1 Bacillus ⁇ -glucanase, interleukin-1 ⁇ , ⁇ -spectrin SH3 domain, pepsinogen, interleukin-4.
  • an unexpected improvement over some properties of the natural sequence was observed, e.g., the solubility and refolding rate for rearranged ⁇ -spectrin SH3 domain sequences, and the receptor affinity and anti-tumor activity of transposed interleukin-4—Pseudomonas exotoxin fusion molecule (Kreitman et al.
  • Novel hematopoietic proteins of this invention are represented by the formulas:
  • N-terminus 1-5 from the C-terminus can be deleted; and wherein the 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;
  • mterleukin-3 and wherein the N-terminus is joined to the C-terminus directly or through a linker (L 2 ) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
  • N-terminus is joined to the C-terminus directly or through a linker (L 2 ) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
  • breakpoints at which new C-terminus and N-terminus can be made in the polypeptide (I) above are; 38-39, 48-49, 96-97, 125-126, 132-133 and 141-142.
  • breakpoints at which new C-terminus and N-terminus can be made in the polypeptide (II) above are; 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 66-67, 67-68, 68-69, 69-70, 70-71, 84-85, 85-86, 86-87, 87-88, 88-89, 89-90, 90-91, 98-99, 99-100, 100-101 and 101-102.
  • breakpoints at which new C-terminus and N-terminus can be made in the polypeptide (II) above are; 34-35, 69-70 and 90-91.
  • breakpoints at which new C-terminus and N-terminus can be made in the polypeptide (III) above or the amino acid sequence of (SEQ ID NO:256) 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, 120-121, 121-122, 122-123, 123-124, 124-125, 125-126 and 126-127.
  • the most preferred breakpoints at which new C-terminus and N-terminus can be made in the polypeptide (III) above or the amino acid sequence of (SEQ ID NO:256) are; 81-82, 108-109, 115-116, 119-120, 122-123 and 125-126.
  • the multifunctional receptor agonist of the present invention can also be represented by the following formula:
  • X 1 is a peptide comprising an amino acid sequence corresponding to the sequence of residues n+1 through J of the original protein having amino acids residues numbered sequentially 1 through J with an amino terminus at residue 1;
  • L is an optional linker
  • X 2 is a peptide comprising an amino acid sequence of residues 1 through n of the original protein
  • Y 2 is a peptide comprising an amino acid sequence of residues 1 through n of the original protein
  • L 1 and L 2 are optional peptide spacers:
  • n is an integer ranging from 1 to J-1;
  • b, c, and d are each independently 0 or 1;
  • a and e are either 0 or 1, provided that both a and e cannot both be 0;
  • T 1 and T 2 are proteins.
  • the present invention relates to
  • the invention also relates to
  • compositions containing the multi-functional hematopoietic receptor agonists and methods for using the multi-functional hematopoietic receptor agonists.
  • in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before infusion into patients.
  • N N-terminus
  • C C-terminus
  • the protein is opened at a breakpoint creating a new N-terminus (new N) and a new C-terminus (new-C) resulting in a protein with a new linear amino acid sequence.
  • a rearranged molecule may be
  • FIG. 2 shows a schematic of Method I, 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 the amino acid 11 (a.a. 1- 10 are deleted) through a linker region and a new C-terminus 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-terminus and C-terminus of the native protein are joined without 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 the original N- terminus and a new C-terminus created at amino 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.
  • 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.
  • the present invention encompasses multi-functional hematopoietic receptor agonists formed from covalently linked polypeptides, each of which may act through a
  • Hematopoiesis requires a complex series of cellular events in which stem cells generate continuously into large populations of maturing cells in all major lineages.
  • regulators with hematopoietic proliferative activity.
  • Most of these proliferative regulators can only stimulate one or another type of colony formation in vitro, the precise pattern of colony formation stimulated by each regulator is quite distinctive. No two regulators stimulate exactly the same pattern of colony formation, as evaluated by colony numbers or, more importantly, by the lineage and maturation pattern of the cells making up the developing colonies.
  • Proliferative responses can most readily be analyzed in simplified in vitro culture systems. Three quite different parameters can be distinguished: alteration in colony size, alteration in colony numbers and cell lineage.
  • Two or more factors may act on the progenitor cell, inducing the formation of larger number of progeny thereby increasing the colony size.
  • Two or more factors may allow increased number of progenitor cells to proliferate either because distinct subsets of progenitors cells exist that respond exclusively to one factor or because some progenitors require stimulation by two or more factors before being able to respond.
  • Activation of additional receptors on a cell by the use of two or more factors is likely to enhance the mitotic signal because of coalescence of initially differing signal pathways into a common final pathway reaching the nucleus (Metcalf, Nature 339:27, 1989). Other mechanisms could explain synergy.
  • one signaling pathway is limited by an intermediate activation of an additional signaling pathway which is caused by a second factor, then this may result in a super additive response.
  • activation of one receptor type can induce an enhanced expression of other receptors (Metcalf, Blood 82:3515-3523, 1993).
  • Two or more factors may result in a different pattern of cell lineages than from a single factor.
  • the use of multi-functional hematopoietic receptor agonists may have a potential clinical advantage resulting from a proliferative response that is not possible by any single factor.
  • the receptors of hematopoietic and other growth factors can be grouped into two distinct families of related
  • tyrosine kinase receptors including those for epidermal growth factor, M-CSF (Sherr, Blood 75:1, 1990) and SCF (Yarden et al., EMBO J. 6:3341, 1987): and (2)
  • hematopoietic receptors not containing a tyrosine kinase domain, but exhibiting obvious homology in their
  • EPO erythropoietin
  • IL-6 (Harada et al., PNAS USA 87:857, 1990), IL-5 (Takaki et al., EMBO J. 9:4367, 1990), IL-6
  • ⁇ -chains for GM-CSF, IL-3 and IL-5 share the same ⁇ -chain (Kitamura et al., Cell 66:1165, 1991), Takaki et al., EMBO J. 10:2833-8, 1991) and receptor complexes for IL-6, LIF and IL-11 share a common ⁇ -chain (gp130) (Taga et al., Cell
  • hematopoietic factor may also have a potential advantage by reducing the demands placed on factor-producing cells and their induction systems. If there are limitations in the ability of a cell to produce a factor, then by lowering the required
  • concentrations of each of the factors, and using them in combination may usefully reduce demands on the factor- producing cells.
  • the use of a multiply acting hematopoietic factor may lower the amount of the factors that would be needed, probably reducing the likelihood of adverse side- effects.
  • Novel compounds of this invention are represented by a formula selected from the group consisting of:
  • R 2 is preferably a colony stimulating factor with a different but complementary activity than R 1 .
  • R 1 polypeptide is joined either directly or through a linker segment to the R 2 polypeptide.
  • the term “directly” defines multi-functional hematopoietic receptor agonists in which the polypeptides are joined without a peptide linker.
  • L 1 represents a chemical bond or polypeptide segment to which both R 1 and R 2 are joined in frame, most commonly L 1 is a linear peptide to which R 1 and R 2 are joined by amide bonds linking the carboxy terminus of R 1 to the amino terminus or L 1 and carboxy terminus of L 1 to the amino terminus of R 2 .
  • joinined in frame is meant that there is no translation termination or disruption between the reading frames of the DNA encoding R 1 and R 2 .
  • colony stimulating factors are cytokines
  • lymphokines, interleukins, hematopoietic growth factors which can be joined to (I), (II) or (III) include GM-CSF, G-CSF, c-mpl ligand (also known as TPO or MGDF), M-CSF, erythropoietin (EPO), IL-1, 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 also known as steel factor or c-kit ligand. Additionally, this
  • R 1 or R 2 is an hIL-3 variant, c-mpl ligand variant, or G-CSF variant.
  • a "hIL-3 variant” is defined as a hIL-3 molecule which has amino acid substitutions and/or portions of hIL-3 deleted as disclosed in WO 94/12638, WO 94/12639 and WO 95/00646, as well as other variants known in the art.
  • a "c-mpl ligand variant” is defined an c-mpl ligand molecule which has amino acid substitutions and/or portions of c-mpl ligand deleted, disclosed in United States Application Serial Number
  • G- CSF variant is defined an G-CSF molecule which has amino acid substitutions and/or portions of G-CSF deleted, as disclosed herein, as well as other variants known in the art.
  • the linking group (L 1 ) is generally a polypeptide of between 1 and 500 amino acids in length.
  • the linkers joining the two molecules are preferably designed to (1) allow the two molecules to fold and act independently of each other, (2) not have a propensity for developing an ordered
  • surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence.
  • Other neutral amino acids such as Thr and Ala, may also be used in the linker sequence. Additional amino acids may also be included in the linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the multi-functional
  • Preferred L 1 linkers of the present invention include sequences selected from the group of formulas:
  • a highly-flexible linker is the glycine and serine-rich spacer region present within the pill protein of the filamentous bacteriophages, e.g.
  • This region provides a long, flexible spacer region between two domains of the pill surface protein.
  • the spacer region consists of the amino acid sequence:
  • the present invention also includes linkers in which an endopeptidase recognition sequence is included.
  • a cleavage site may be valuable to separate the individual components of the multi-functional hematopoietic receptor agonist to determine if they are properly folded and active in vitro.
  • various endopeptidases include, but are not limited to, plasmin, enterokinase, kallikrein, urokinase, tissue plasminogen activator, clostripain, chymosin, collagenase, Russell's viper venom protease, postproline cleavage enzyme, V8 protease, Thrombin and factor Xa.
  • Peptide linker segments from the hinge region of heavy chain immunoglobulms IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptides.
  • linkers of the present invention include sequences derived from murine IgG gamma 2b hinge region in which the cysteines have been changed to serines. These linkers may also include an endopeptidase cleavage site. Examples of such linkers include the following sequences: IleSerGluProSerGlyProIleSerThrlleAsnProSerProProSerLys GluSerHisLysSerPro (SEQ ID NO:10) and
  • the present invention is, however, not limited by the form, size or number of linker sequences employed and the only requirement of the linker is that functionally it does not interfere with the folding and function of the linker
  • the length of the amino acid sequence of the linker L 2 to be used in R 1 and/or R 2 can be selected empirically or with guidance from structural information, or by using a combination of the two approaches.
  • 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; solvent exposed surface area, Lee & Richards, J. Mol . Biol . 55:379-400, 1971) and the ability to adopt the necessary conformation with out deranging the
  • 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:12) cassette approach mentioned above; or optionally a
  • the parental amino acid sequence is inspected to classify regions according to whether or not they are integral to the maintenance of secondary and tertiary structure.
  • the occurrence of sequences within regions that are known to be involved in periodic secondary structure are regions that should be avoided.
  • regions of amino 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 protein and should also be avoided for selection of amino and carboxyl termini.
  • Non-covalent Multifunctional hematopoietic growth factors 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.
  • R 1 -C 1 + R 2 -C 2 or C 1 -R 1 + C 2 -R 2 ; C 1 -R 1 + R 2 -C 2 ; or C 1 -R 1 + R 2 -C 2 .
  • R 1 and R 2 are as is defined above. Domains C 1 and C 2 are either identical or non-identical chemical structures, typically proteinaceous, which can form a non-covalent, specific association. Complexes between C 1 and C 2 result in a one-to-one stoichiometric relationship between R 1 and R 2 for each complex. Examples of domains which associate are "leucine zipper" domains of transcription factors,
  • C 1 and C 2 include those derived from the bZIP family of proteins (Abel et al., Nature 341:24-25, 1989; Landshulz et al., Science 240:1759-1764, 1988; Pu et al., Nuc. Acid Res . 21:4348-4355, 1993; Kozarides et al., Nature 336:646-651, 1988), as well as multimerization domains of the helix-loop-helix family of proteins (Abel et al., Nature 341:24-25, 1989; Murre et al., Cell 56:777-783, 1989;
  • Preferred multi-functional hematopoietic receptor agonists of the present invention include colony stimulating factors dimerized by virtue of their incorporation as translational multi-functional hematopoietic receptor agonists with the leucine zipper dimerization domains of the bZIP family proteins Fos and Jun.
  • the leucine zipper domain of Jun is capable of
  • the leucine zipper domain of Fos interacts with the Jun leucine zipper domain, but does not interact with other Fos leucine zipper domains. Mixtures of Fos and Jun predominantly result in formation of Fos-Jun heterodimers. Consequently, when joined to colony stimulating factors, the Jun domain can be used to direct the formation of either homo- or heterodimers. Preferential formation of heterodimers can be achieved if one of the colony stimulating factor partners is engineered to possess the Jun leucine zipper domain while the other is engineered to possess the Fos zipper.
  • Additional peptide sequences may also be added to facilitate purification or identification of multi- functional hematopoietic receptor agonist proteins (e.g., poly-His).
  • a highly antigenic peptide may also be added that would enabjv rapid assay and facile purification of the multi-functional hematopoietic receptor agonist protein by a specific monoclonal antibody.
  • variant protein refers to a polypeptide having an amino acid sequence which varies from a native sequence due to amino acid deletions
  • nucleotide sequence intentionally made variant from a native sequence.
  • “Native sequence” refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein.
  • Hematopoietic growth factors can be characterized by their ability to stimulate colony formation by human hematopoietic progenitor cells.
  • the colonies formed include erythroid, granulocyte, megakaryocyte, granulocytic
  • hematopoietic growth factors have demonstrated the ability to restore bone marrow function and peripheral blood cell populations to therapeutically beneficial levels in studies performed initially in primates and subsequently in humans. Many or all of these biological activities of hematopoietic growth factors involve signal transduction and high affinity receptor binding. Multi-functional hematopoietic receptor agonists of the present invention may exhibit useful
  • properties such as having similar or greater biological activity when compared to a single factor or by having improved half-life or decreased adverse side effects, or a combination of these properties.
  • Multi-functional hematopoietic receptor agonists which have little or no agonist activity maybe useful as
  • antagonists as antigens for the production of antibodies for use in immunology or immunotherapy, as genetic probes or as intermediates used to construct other useful hIL-3 muteins.
  • hematopoietic receptor agonist proteins of the present invention can be determined by DNA synthesis in factor- dependent cell lines or by counting the colony forming units in an in vitro bone marrow assay.
  • the multi-functional hematopoietic receptor agonists of the present invention may have an improved therapeutic profile as compared to single acting hematopoietic agonists.
  • some multi-functional hematopoietic receptor agonists of the present invention may have a similar or more potent growth factor activity relative to other hematopoietic agonists without having a similar or
  • the present invention also includes the DNA sequences which code for the multi-functional hematopoietic receptor agonist proteins, DNA sequences which are
  • agonists of the invention only due to the degeneracy of the genetic code.
  • oligonucleotide intermediates used to construct the mutant DNAs and the polypeptides coded for by these
  • cassette mutagenesis (Wells et al., Gene 34:315-323, 1985) in which a portion of the coding sequence in a plasmid is replaced with synthetic oligonucleotides that encode the desired amino acid substitutions in a portion of the gene between two restriction sites.
  • Pairs of complementary synthetic oligonucleotides encoding the desired gene can be made and annealed to each other.
  • the DNA sequence of the oligonucleotide would encode sequence for amino acids of desired gene with the exception of those substituted and/or deleted from the sequence.
  • Plasmid DNA can be treated with the chosen restriction endonucleases then ligated to the annealed oligonucleotides.
  • the ligated mixtures can be used to transform competent JM101 cells to resistance to an appropriate antibiotic.
  • Single colonies can be picked and the plasmid DNA examined by restriction analysis and/or DNA sequencing to identify plasmids with the desired genes.
  • stimulating factor may be accomplished by the use of intermediate vectors.
  • one gene can be cloned directly into a vector containing the other gene.
  • Linkers and adapters can be used for joining the DNA sequences, as well as replacing lost sequences, where a restriction site was internal to the region of interest.
  • genetic material DNA encoding one polypeptide, peptide linker, and the other polypeptide is inserted into a suitable expression vector which is used to transform bacteria, yeast, insect cells or mammalian cells.
  • the transformed organism is grown and the protein isolated by standard techniques.
  • the resulting product is therefore a new protein which has a colony stimulating factor joined by a linker region to a second colony stimulating factor.
  • Another aspect of the present invention provides plasmid DNA vectors for use in the expression of these novel multi-functional hematopoietic 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 multi-functional hematopoietic receptor agonists include expression vectors comprising nucleotide sequences coding for the multi-functional hematopoietic 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 included in the present invention and are useful in the production of the multi-functional hematopoietic receptor agonist polypeptides.
  • the vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and which are capable of directing the replication and expression thereof in selected host cells.
  • a method for producing the novel multi-functional hematopoietic 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 multi-functional hematopoietic receptor agonist.
  • suitable cells or cell lines may be bacterial cells.
  • E. col i the various strains of E. col i are well-known as host cells in the field of biotechnology. Examples of such strains include E. coli strains JM..01 (Yanish-Perron et al . Gene 33: 103-119, 1985) and MON105 (Obukowicz et al., Applied Environmental
  • the gene encoding the multi-functional hematopoietic receptor agonists of the present invention may also be constructed such that at the 5' end of the gene codons are added to encode Met -Ala -1 - or Met at the N-terminus of the protein.
  • the N termini of proteins made in the cytoplasm of E. coli are affected by post-translational processing by methionine aminopeptidase (Ben Bassat et al., J. Bac. 169:751-757, 1987) and possibly by other peptidases so that upon expression the methionine is cleaved off the N-terminus.
  • the multi-functional hematopoietic receptor agonists of the present invention may include multi-functional hematopoietic receptor agonist polypeptides having Met -1 , Ala -1 or Met -2 -Ala -1 at the N-terminus. These mutant multi-functional hematopoietic receptor agonists may also be expressed in E. coli by fusing a secretion signal peptide to the N-terminus. This signal peptide is cleaved from the polypeptide as part of the secretion process.
  • mammalian cells such as Chinese hamster ovary cells (CHO).
  • CHO Chinese hamster ovary cells
  • An expression vector is constructed in which a strong promoter capable of
  • plasmids such as pcDNA I/Neo, pRc/RSV, and pRc/CMV (obtained from Invitrogen Corp., San Diego, California) can be used.
  • the eukaryotic secretion signal peptide coding region can be from the gene itself or it can be from another secreted mammalian protein (Bayne, M. L. et al., Proc. Natl . Acad. Sci . USA 84: 2638-2642, 1987).
  • the vector DNA is transfected into mammalian cells.
  • Such cells can be, for example, the COS7, HeLa, BHK, CHO, or mouse L lines.
  • the cells can be cultured, for example, in DMEM media (JRH Scientific).
  • the polypeptide secreted into the media can be recovered by standard biochemical approaches following transient
  • Another suitable mammalian cell line is the monkey COS-1 cell line.
  • a similarly useful mammalian cell line is the CV-1 cell line.
  • insect cells may be utilized as host cells in the method of the present invention. See, e.g., Miller et al., Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein. In addition, general methods for expression of foreign genes in insect cells using Baculovirus vectors are described in: Summers, M. D. and Smith, G. E., 1987) - A manual of methods for
  • An expression vector is constructed comprising a Baculovirus transfer vector, in which a strong Baculovirus promoter (such as the polyhedron promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally joined to the coding region for the multi- functional hematopoietic receptor agonist polypeptide.
  • a Baculovirus transfer vector in which a strong Baculovirus promoter (such as the polyhedron promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally joined to the coding region for the multi- functional hematopoietic receptor agonist polypeptide.
  • a strong Baculovirus promoter such as the polyhedron promoter
  • the vector carrying the gene encoding the multi-functional hematopoietic receptor agonist polypeptide two micrograms of this DNA is co-transfected with one microgram of Baculovirus DNA (see Summers & Smith, 1987) into insect cells, strain SF9. Pure recombinant Baculovirus carrying the multi-functional hematopoietic receptor agonist is used to infect cells cultured, for example, in Excell 401 serum-free medium (JRH Biosciences, Lenexa, Kansas). The multi-functional hematopoietic receptor agonist secreted into the medium can be recovered by standard biochemical approaches.
  • Supernatants from mammalian or insect cells expressing the multi-functional hematopoietic receptor agonist protein can be first concentrated using any of a number of commercial concentration units.
  • the multi-functional hematopoietic receptor agonists of the present invention may be useful in the treatment of diseases characterized by decreased levels of either
  • leukopenia a reduction in the number of circulating leukocytes (white cells) in the peripheral blood. Leukopenia may be induced by exposure to certain viruses or to radiation. It is often a side effect of various forms of cancer therapy, e.g., exposure to chemotherapeutic drugs, radiation and of infection or hemorrhage. Therapeutic treatment of
  • leukopenia with these multi-functional hematopoietic receptor agonists of the present invention may avoid
  • the multi-functional hematopoietic receptor agonists of the present invention may be useful in the treatment of neutropenia and, for example, in the treatment of such conditions as aplastic anemia, cyclic neutropenia,
  • idiopathic neutropenia Chediak-Higashi syndrome, systemic lupus erythematosus (SLE), leukemia, myelodysplastic syndrome and myelofibrosis.
  • the multi-functional hematopoietic receptor agonist of the present invention may be useful in the treatment or prevention of thrombocytopenia.
  • thrombocytopenia Currently the only therapy for thrombocytopenia is platelet transfusion which are costly and carry the significant risks of infection (HIV, HBV) and alloimunization.
  • the multi-functional hematopoietic receptor agonist may alleviate or diminish the need for platelet transfusion.
  • Severe thrombocytopenia may result from genetic defects such as Fanconi's Anemia, Wiscott-Aldrich, or May Hegglin syndromes. Acquired thrombocytopenia may result from auto- or allo-antibodies as in Immune
  • Thrombocytopenia Purpura Systemic Lupus Erythromatosis, hemolytic anemia, or fetal maternal incompatibility.
  • 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 multi-functional hematopoietic receptor agonists of the present invention may be useful in the mobilization of hematopoietic progenitors and stem cells in 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
  • the multi-functional hematopoietic receptor agonist may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.
  • the multi-functional hematopoietic receptor agonists of the present invention may also be useful in the ex vivo expansion of hematopoietic progenitors and stem cells.
  • Colony stimulating factors such as hIL-3
  • CSFs Colony stimulating factors
  • the 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
  • Bone marrow transplants have been used to treat this patient population.
  • 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, spleen, or peripheral blood 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 exists such that a greater number of stem cells will enhance hematopoietic recovery. Therefore, the in vitro expansion of stem cells should enhance hematopoietic recovery and patient survival.
  • autologous bone marrow transplants An alternative to allogeneic bone marrow transplants is autologous bone marrow transplants.
  • 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 into the patient afterwards.
  • autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection.
  • transplants still present problems in terms of the limited number of stems cells in the marrow and possible
  • stem cells contamination with tumor cells.
  • the limited number of stem cells may be overcome by ex-vivo expansion of the stem cells.
  • stem cells can be specifically isolated, based on the presence of specific surface antigens such as CD34+ in order to decrease tumor cell contamination of the marrow graft.
  • compositions comprising human
  • hematopoietic stem cells provided by separating 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
  • IL-3 consisting of IL-3, flt3 ligand, c-kit ligand, GM-CSF, IL-1, GM-CSF/IL-3 fusion protein and combinations thereof; (3) administering cellular preparation to a patient.
  • 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 stem 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 are 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 totipotent hematopoietic stem cells as well as early precursors and progenitor cells which can be isolated from bone marrow, spleen or peripheral blood.
  • expansion refers to the differentiation and
  • the present invention 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 selective media which contains multi-functional
  • stems cells as well as committed
  • 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 affinity 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
  • Other surface antigens such as 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-1, IL-6, IL-11, flt-3 ligand or combinations thereof.
  • the proliferation of the stem cells can be
  • stem cells and other cells monitored by enumerating the number of stem cells and other cells, by standard techniques (e.g. hemacytometer, CFU, LTCIC) or by flow cytometry prior and subsequent to
  • 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]). However, 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 multi-functional hematopoietic receptor agonists that are more effective than a single factor alone.
  • 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 multi-functional hematopoietic 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 multi-functional hematopoietic 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.
  • Potential applications of gene therapy (review Crystal, Science 270:404-410
  • 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 . Mi crobi ol . Immunol . 158:1-24 [1992]) and replication-deficient recombinant adenovirus (Berkner, BioTechniques 6:616-629 [1988], Berkner, Current Top .
  • 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 . New York Acad. Sci .
  • 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 multi-functional hematopoietic receptor agonist proteins that have improved biological activity, including an activity not seen by any single colony
  • hematopoietic deficiencies examples include AZT, DDI, alkylating agents and anti-metabolites used in
  • antibiotics such as chloramphenicol
  • phenothiazones 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 multi-functional hematopoietic receptor agonists of the present invention 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.
  • the multi-functional hematopoietic receptor agonists of the present invention may be useful in treating such hematopoietic deficiencies.
  • the treatment of hematopoietic deficiency may include administration of a pharmaceutical composition containing the multi-functional hematopoietic receptor agonists to a patient.
  • the multi-functional hematopoietic receptor agonists of the present invention may also be useful for the activation and amplification of hematopoietic precursor cells by treating these cells in vitro with the multi- functional hematopoietic receptor agonist proteins of the present invention prior to injecting the cells into a patient.
  • arthritis may also be beneficially affected by treatment with the multi-functional hematopoietic receptor agonists of the present invention.
  • Immunodeficiencies may be the result of viral infections, e.g., HTLVI, HTLVII, HTLVIII, severe exposure to radiation, cancer therapy or the result of other medical treatment.
  • the multi-functional hematopoietic receptor agonists of the present invention may also be employed, alone or in combination with other colony
  • polypeptides are the in vivo and ex vivo treatment of patients recovering from bone marrow transplants, and in the development of monoclonal and polyclonal antibodies
  • compositions for treating the conditions referred to above.
  • Such compositions comprise a
  • the therapeutic composition for use in this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • the preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.
  • a daily regimen may be in the range of 0.2 - 150 ⁇ g/kg of multi-functional hematopoietic receptor agonist protein per kilogram of body weight. Dosages would be adjusted relative to the activity of a given multi-functional hematopoietic receptor agonist protein 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. In addition, there may exist specific circumstances where dosages of multi-functional
  • hematopoietic 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 colony stimulating factors or IL-3 variants or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated multi-functional hematopoietic receptor agonist protein; and various patient- related issues mentioned earlier in this section.
  • the therapeutic method and compositions may also include co-administration with other human factors.
  • CSFs colony stimulating factors
  • cytokines cytokines
  • lymphokines hematopoietic growth factors
  • interleukins for simultaneous or serial co- administration with the polypeptides of the present
  • GM-CSF GM-CSF
  • G-CSF G-CSF
  • c-mpl ligand also known as TPO or MGDF
  • M-CSF M-CSF
  • EPO erythropoietin
  • IL-1 IL-4
  • SCF stem cell factor
  • 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.
  • E. coli strains such as DH5 ⁇ TM (Life Technologies, Gaithersburg, MD) and TGI (Amersham Corp., Arlington
  • E. coli strains such as JM101 (Yanisch-Perron, et al., Gene, 33: 103-119, 1985) and MON105
  • MON105 ATCC#55204 F-, lambda-,IN(rrnD, rrE)1, rpoD+, rpoH358
  • DH5 ⁇ TM F-, phi80dlacZdeltaM15, delta (lacZYA-argF) U169, deoR, recA1, endA1, hsdR17 (rk-,mk+), phoA, supE441amda-, thi-1, gyrA96, relA1 TGI: delta (lac-pro), supE, thi-1, hsdD5/F' (traD36, proA+B+, laclq, lacZdeltaM15)
  • JM101 ATCC#33876 delta (pro lac), supE, thi , F'(traD36, proA+B+, laclq, lacZdeltaM15)
  • 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 CaCl 2 method.
  • 20 to 50 mL of cells are grown in LB medium (1% bacto-tryptone, 0.5% bacto-yeast extract, 150 mM NaCl) to a density of approximately 1.0 optical density unit 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 CaCl 2 solution (50 mM CaCl 2 , 10 mM Tris-Cl, pH7.4) and are held at 4oC for 30 minutes. The cells are again collected by centrifugation and resuspended in one-tenth culture volume of CaCl 2 solution. Ligated DNA is added to 0.2 mL of these cells, and the samples are held at 4oC for 30-60 minutes. The samples are shifted to 42oC for two minutes and 1.0 mL of LB is added prior to shaking the samples at 37oC for one hour.
  • CaCl 2 solution 50 mM CaCl 2 , 10 mM Tris-Cl, pH7.4
  • Ligated DNA is added to 0.2 mL of these cells, and the samples are held at 4oC for 30-60 minutes.
  • the samples are shifted to 42oC for two minutes
  • 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
  • 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.
  • appropriate antibiotic 100 ug/mL ampicillin or 75 ug/mL spectinomycin
  • Methods for creation of genes with new N-terminus/C-terminus Method I. Creation of genes with new N-terminus/C-terminus which contain a linker region (L 2 ).
  • the first primer set (“new start” and “linker 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 followed by the linker (L 2 ) 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 original gene sequence, the DNA fragment ("Fragment
  • 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 denaturaticr 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 1x 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 performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT). "Fragment Start” and “Fragment Stop”, which have complementary sequence in the linker region and the coding sequence for the two amino acids on both sides of the linker, are joined together in a third PCR step to make the full-length gene encoding the new protein.
  • fragments "Fragment Start” and “Fragment 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 in equimolar quantities, heated at 70°C for ten minutes and slow cooled to allow annealing through their shared sequence in "linker start” and “linker stop”.
  • 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.
  • 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 1x 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).
  • Method II Creation of genes with new N-terminus/C-terminus without a linker region.
  • 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
  • the 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 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
  • a 100 ul reaction contained 150 pmole of each primer and one ug of template DNA; and 1x Vent buffer (New England Biolabs), 300 uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit Deep Vent polymerase.
  • 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 NcoI restriction site
  • “Fragment Stop” is designed to create a HindIII 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 NcoI/HindIII 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
  • New N-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)
  • tandemly-duplicated template DNA is created by cloning and contains two 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
  • 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 (Perkin Elmer Corporation, Norwalk, CT) is used.
  • a 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and 1x 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 new N-terminus/C-terminus gene is digested with restriction endonucleases to create ends that are compatible to insertion into an expression vector containing another colony stimulating factor gene.
  • This expression vector is likewise digested with restriction endonucleases to form compatible ends.
  • the gene and the vector DNAs are combined and ligated using T4 DNA ligase. A portion of the ligation reaction is used to transform E.
  • Plasmid DNA is purified and sequenced to confirm the correct insert. The correct clones are grown for protein expression.
  • Plasmid DNA can be isolated by a number of different methods and using commercially available kits known to those skilled in the art. A few such methods are shown herein. Plasmid DNA is isolated using the Promega WizardTM Miniprep kit (Madison, WI), the Qiagen QIAwell Plasmid isolation kits (Chatsworth, CA) or Qiagen Plasmid Midi kit. These 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 50-100 mls of LB plus appropriate antibiotic for overnight growth at 37°C in an air incubator while shaking.
  • the purified plasmid DNA is used for DNA sequencing, further restriction enzyme digestion, additional subcloning of DNA fragments and transfection into mammalian, E. coli or other cells.
  • plasmid DNA is resuspended in dH 2 O and quantitated by measuring the absorbance at 260/280 nm in a Bausch and Lomb Spectronic 601 UV spectrometer. DNA samples are sequenced using ABI PRISMTM DyeDeoxyTM terminator sequencing chemistry (Applied Biosystems Division of Perkin Elmer Corporation, Lincoln City, CA) kits (Part Number
  • Sequencing reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) following the recommended amplification conditions. Samples are purified to remove excess dye terminators with Centri-SepTM spin columns (Princeton
  • Fluorescent dye labeled sequencing reactions are resuspended in deionized formamide, and sequenced on denaturing 4.75% polyacrylamide- 8M urea gels using an ABI Model 373A automated DNA
  • the BriK-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 mM (mM) L-glutamine and 10% fetal bovine serum (FBS). This formulation is designated BHK growth media. Selective media is BHK growth media supplemented with 453 units/mL
  • DMEM/high-glucose Dulbecco's modified Eagle media
  • FBS fetal bovine serum
  • hygromycin B Calbiochem, San Diego, CA.
  • the BHK-21 cell line was previously stably transfected with the HSV
  • transactivating protein VP16 which transactivates the IE110 promoter found on the plasmid pMON3359 (See Hippenmeyer et al., Bio /Technology, pp.1037-1041, 1993).
  • the VP16 protein drives expression of genes inserted behind the IE110 promoter.
  • BHK-21 cells expressing the transactivating protein VP16 are designated BHK-VP16.
  • the plasmid pMON1118 (See 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 10 ⁇ 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, pMON1ll ⁇ , and 80 ug of Gibco-BRL "LIPOFECTAMINE”TM per dish.
  • the media is subsequently aspirated and replaced with 3 mL of growth media.
  • 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
  • the conditioned media is re-assayed, and positive clones are expanded into growth media.
  • E. coli strain MON105 or JM101 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 three 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 protein content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE (see Maniatis et al . Molecular Cloning: A Laboratory Manual, 1982).
  • the culture is centrifuged (5000 x g) to pellet the cells. inclusion Body preparation.
  • the IB pellet is resuspended in 10 mL of 50 mM Tris-HCl, pH 9.5, 8 M urea and 5 mM dithiothreitol (DTT) and stirred at room temperature
  • the extraction solution is transferred to a beaker containing 70 mL of 5 mM Tris-HCl, pH 9.5 and 2.3 M urea and gently stirred while exposed to air at 4°C for 18 to 48 hours to allow the proteins to refold.
  • Refolding is monitored by analysis on a Vydac (Hesperia, Ca.) C18 reversed phase high pressure liquid chromatography (RP-HPLC) column (0.46x25 cm).
  • RP-HPLC reversed phase high pressure liquid chromatography
  • a linear gradient of 40% to 65% acetonitrile, containing 0.1% trifluoroacetic acid (TFA) is employed to monitor the refold. This gradient is developed over 30 minutes at a flow rate of 1.5 mL per minute.
  • contaminating E. coli proteins are removed by acid precipitation.
  • the pH of the refold solution is titrated to between pH 5.0 and pH 5.2 using 15% (v/v) acetic acid (HOAc). This solution is stirred at 4°C for 2 hours and then centrifuged for 20 minutes at 12,000 x g to pellet any insoluble protein.
  • the supernatant from the acid precipitation step is dialyzed using a Spectra/Por 3 membrane with a molecular weight cut off (MWCO) of 3,500 daltons.
  • the dialysis is against 2 changes of 4 liters (a 50-fold excess) of 10 mM Tris-HCl, pH 8.0 for a total of 18 hours. Dialysis lowers the sample conductivity and removes urea prior to DEAE chromatography.
  • the sample is then centrifuged (20 minutes at 12,000 x g) to pellet any insoluble protein following dialysis.
  • a Bio-Rad Bio-Scale DEAE2 column (7 ⁇ 52 mm) is used for ion exchange chromatography.
  • the column is equilibrated in a buffer containing 10 mM Tris-HCl, pH 8.0, and a 0-to- 500 mM sodium chloride (NaCl) gradient, in equilibration buffer, over 45 column volumes is used to elute the protein.
  • a flow rate of 1.0 mL per minute is used throughout the run.
  • Column fractions (2.0 mL per fraction) are collected across the gradient and analyzed by RP HPLC on a Vydac (Hesperia,
  • sample is sterile filtered using a 0.22 ⁇ m syringe filter ( ⁇ star LB syringe filter, Costar, Cambridge, Ma.), and stored at 4°C.
  • the folded proteins can be affinity purified using affinity reagents such as mAbs or receptor subunits attached to a suitable matrix.
  • affinity reagents such as mAbs or receptor subunits attached to a suitable matrix.
  • purification can be accomplished using any of a variety of chromatographic methods such as: ion exchange, gel filtration or hydrophobic chromatography or reversed phase HPLC.
  • the purified protein is analyzed by RP-HPLC,
  • AML Proliferation Assay for Bioactive Human Interleukin-3 The factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). This cell line, established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM-CSF supplemented medium (Lange, B., et al., Blood 70: 192, 1987; Valtieri, M., et al., J. Immunol . 138:4042, 1987). The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D., et al., J.
  • a cell line variant was used, AML 193 1.3, which was adapted for long term growth in IL-3 by washing out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours.
  • the cells are then replated at 1 ⁇ 10 5 cells/well in a 24 well plate in media containing 100 U/mL IL-3. It took
  • AML 193 1.3 cells are washed 6 times in cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, NY) by centrifuging cell suspensions at 250 x g for 10 minutes followed by decantation of the supernatant. Pelleted cells are resuspended in HBSS and the procedure is repeated until six wash cycles are completed. Cells washed six times by this procedure are resuspended in tissue culture medium at a density ranging from 2 ⁇ 10 5 to 5 ⁇ 10 5 viable cells/mL. This medium is prepared by supplementing Iscove's modified Dulbecco's Medium (IMDM, Hazelton, Lenexa, KS) with albumin, transferrir, lipids and 2-mercaptoethanol.
  • IMDM Iscove's modified Dulbecco's Medium
  • Bovine albumin (Boehringer-Mannheim, Indianapolis, IN) is added at 500 ⁇ g/mL; human transferrin (Boehringer-Mannheim, Indianapolis, IN) is added at 100 ⁇ g/mL; soybean lipid (Boehringer-Mannheim, Indianapolis, IN) is added at 50 ⁇ g/mL; and 2- mercaptoethanol (Sigma, St. Louis, MO) is added at 5 ⁇ 10 -5 M.
  • Serial dilutions of human interleukin-3 or multi- functional hematopoietic receptor agonist proteins are made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 ⁇ l of medium containing interleukin-3 or multi-functional hematopoietic receptor agonist proteins once serial dilutions are completed.
  • Control wells contained tissue culture medium alone
  • AML 193 1.3 cell suspensions prepared as above are added to each well by pipetting 50 ⁇ l (2.5 ⁇ 10 4 cells) into each well. Tissue culture plates are incubated at 37°C with 5% CO 2 in humidified air for 3 days. On day 3, 0.5 ⁇ ci 3 H-thymidine (2 Ci/mM, New England
  • Beta emissions of samples from individual tissue culture wells are counted in a LKB BetaPlate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, MD) and data is expressed as counts per minute of 3 H-thymidine incorporated into cells from each tissue culture well.
  • hematopoietic receptor agonist hematopoietic receptor agonist. Typically, concentration ranges from 0.05 pM - 10 5 pM are quantitated in these assays.
  • This EC50 value is also equivalent to 1 unit of bioactivity. Every assay is performed with native interleukin-3 as a reference standard so that relative activity levels could be assigned.
  • the multi-functional hematopoietic receptor agonist proteins were tested in a concentration range of 2000 pM to 0.06 pM titrated in serial 2 fold dilutions.
  • the proliferation assay was performed with the multi-functional hematopoietic receptor agonist plus and minus neutralizing monoclonal antibodies to the hIL-3 receptor agonist portion.
  • a fusion molecule with the factor Xa cleavage site was cleaved then purified and the halves of the molecule were assayed for proliferative activity.
  • the c-mpl ligand proliferative activity can be assayed using a subclone of the pluripotential human cell line TF1 (Kitamura et al., J. Cell Physiol 140:323-334. [1989]). TF1 cells are maintained in h-IL3 (100 U/mL). To establish a sub-clone responsive to c-mpl ligand, cells are maintained in passage media containing 10% supernatant from BHK cells transfected with the gene expressing the 1-153 form of c-mpl ligand (pMON26448). Most of the cells die, but a subset of cells survive.
  • a c-mpl ligand responsive clone is selected, and these cells are split into passage media to a density of 0.3 ⁇ 10 6 cells/mL the day prior to at-say set-up.
  • Passage media for these cells is the following: RPMI 1640 (Gibco), 10% FBS (Harlan, Lot #91206), 10% c-mpl ligand supernatant from transfected BHK cells, 1 mM sodium pyruvate (Gibco), 2 mM glutamine (Gibco), and 100 ug/mL penicillin-streptomycin (Gibco).
  • ATL medium consists of the following:IMDM (Gibco), 500 ug/mL of bovine serum albumin, 100 ug/mL of human transferrin, 50 ug/mL soybean lipids, 4 ⁇ 10-8M beta-mercaptoethanol and 2 mL of A9909 (Sigma, antibiotic solution) per 1000 mL of ATL.
  • Cells are diluted in assay media to a final density of 0.25 ⁇ 10 6 cells/mL in a 96-well low evaporation plate (Costar) to a final volume of 50 ul.
  • Transient supernatants (conditioned media) from transfected clones are added at a volume of 50 ul as duplicate samples at a final concentration of 50% and diluted three-fold to a final dilution of 1.8%.
  • 0.0014ng/mL is included as a positive control. Plates are incubated at 5% CO 2 and 37° C. At day six of culture, the plate is pulsed with 0.5 Ci of 3H/well (NEN) in a volume of 20 ul/well and allowed to incubate at 5% CO 2 and 37° C for four hours. The plate is harvested and counted on a
  • in vitro cell based proliferation assays Other in vitro cell based assays, known to those skilled in the art, may also be useful to determine the activity of the multi-functional hematopoietic receptor agonists depending on the factors that comprise the molecule in a similar manner as described in the AML 193.1.3 cell proliferation assay. The following are examples of other useful assays.
  • TF1 proliferation assay TF1 is a pluripotential human cell line (Kitamura et al., J. Cell Physiol 140:323-334. [1989]) that responds to hIL-3.
  • 32D proliferation assay 32D is a murine IL-3 dependent cell line which does not respond to human IL-3 but does respond to human G-CSF which is not species restricted.
  • Baf/3 proliferation assay Baf/3 is a murine IL-3 dependent cell line which does not respond to human IL-3 or human c-mpl ligand but does respond to human G-CSF which is not species restricted.
  • T1165 proliferation assay T1165 cells are a IL-6 dependent murine cell line (Nordan et al., 1986) which respond to IL-6 and IL-11.
  • Human Plasma Clot meg-CSF Assay Used to assay megakaryocyte colony formation activity (Mazur et al., 1981).
  • Cell lines such as the murine Baf/3 cell line can be transfected with a colony stimulating factor receptor, such as the human G-CSF receptor or human c-mpl receptor, which the cell line does not 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 G-CSF receptor or human c-mpl receptor
  • 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 human G-CSF receptor can be transfected into the Baf/3 cell line and used to determine the bioactivity of the multi-functional hematopoietic receptor agoinsts.
  • Bone marrow aspirates (15-20 mL) were obtained from normal allogeneic marrow donors after informed consent.
  • CD34+ cells were prepared using an affinity column per manufacturers instructions (CellPro, Inc, Bothell WA). After enrichment, the purity of CD34+ cells was 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).
  • hematopoietic receptor agonists were tested by addition of 100 ⁇ l of supernatant added to 1 mL cultures (approximately a 10% dilution). Cells were incubated at 37°C for 8-14 days at 5% CO 2 in a 37°C humidified incubator. b. Cell Harvest and Analysis:
  • CD34+ enriched population were isolated as described above.
  • Cells were suspended at 25,000 cells/mL with or without cytokine(s) in a media consisting of a base Iscoves IMDM media supplemented with 0.3% BSA, 0.4mg/mL apo-transferrin, 6.67 ⁇ M FeCl 2 , 25 ⁇ g/mL CaCl 2 , 25 ⁇ g/mL L-asparagine, 500 ⁇ g/mL ⁇ -amino-n-caproic acid and penicillin/streptomycin. Prior to plating into 35mm plates, thrombin was added (0.25
  • This assay reflects the ability of colony stimulating factors to stimulate normal bone marrow cells to produce different types of hematopoietic colonies in vi tro (Bradley et al., Aust . Exp Biol . Sci . 44:287-300, 1966), Pluznik et al., J. Cell Comp . Physio 66:319-324, 1965).
  • CD34+ cells are counted and CD34+ cells are selected using the Ceprate LC (CD34) Kit (CellPro Co., Bothel, WA) column. This fractionation is performed since all stem and progenitor cells within the bone marrow display CD34 surface antigen. Cultures are set up in triplicate with a final volume of 1.0 mL in a 35 X 10 mm petri dish (Nunc#174926). Culture medium is purchased from Terry Fox Labs. (HCC-4230 medium (Terry Fox Labs, Vancouver, B.C., Canada) and erythropoietin (Amgen, Thousand Oaks, CA.) is added to the culture media. 3,000-10,000 CD34+ cells are added per dish.
  • Recombinant hIL-3, GM-CSF, c-mpl ligand and multi-functional hematopoietic receptor agonist are supplied in house.
  • G-CSF Neuropogen
  • Cultures are resuspended using a 3cc syringe and 1.0 mL is dispensed per dish. Control
  • Bone marrow cells are traditionally used for in vitro assays of hematopoietic colony stimulating factor (CSF) activity.
  • CSF colony stimulating factor
  • human bone marrow is not always available, and there is considerable variability between donors.
  • Umbilical cord blood is comparable to bone marrow as a source of hematopoietic stem cells and progenitors (Broxmeyer et al., PNAS USA 89:4109-113, 1992; Mayani et al., Blood 81:3252-3258, 1993). In contrast to bone marrow, cord blood is more readily available on a regular basis.
  • There is also a potential to reduce assay variability by pooling cells obtained fresh from several donors, or to create a bank of cryopreserved cells for this purpose. By modifying the culture conditions, and/or analyzing for lineage specific markers, it is be possible to assay specifically for granulocyte / macrophage colonies (CFU-GM), for
  • Mononuclear cells are isolated from cord blood within 24 hr. of collection, using a standard density gradient (1.077 g/mL Histopaque). Cord blood MNC have been further enriched for stem cells and progenitors by several
  • a new mammalian expression vector was constructed to accept NcoI-HindIII or AflIII-HindIII gene fragments in-frame and 3' to the hIL-3 receptor agonist pMON13146 (WO 94/12638) gene and a mouse IgG2b linker fragment.
  • the single AflIII site was removed from pMON3934, which is a derivative of pMON3359.
  • pMON3359 is a pUC18-based vector containing a mammalian expression cassette.
  • the cassette includes a herpes simplex viral promoter IE110 (-800 to +120) followed by a modified human IL-3 signal peptide sequence and an 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).
  • IE110 herpes simplex viral promoter
  • poly-A SV40 late poly-adenylation
  • the modified human IL-3 signal sequence which facilitates secretion of gene products outside of the cell, is flanked by a BamHI site on the 5' end and a unique NcoI site on the 3' end.
  • a unique HindIII site is 3' to the NcoI site and 5' to the poly-A sequence.
  • the DNA sequence encoding the signal peptide is shown below (restriction enzyme sites are indicated above).
  • the ATG (methionine) codon within the NcoI site is in-frame with the initiator ATG of the signal peptide (underlined);
  • the single AflIII site was removed from pMON3934 by
  • digested DNA fragment was purified via Magic PCR Clean up kit (Promega) and ligated with T4 DNA ligase. The ligation reaction was transformed into DH5 ⁇ TM and the cells were plated onto LB-agar plus ampicillin. Individual colonies were screened for the loss of the AflIII site by restriction analysis with AflIII and HindIII which results in a single fragment if the AflIII site was removed. The resulting plasmid was designated pMON30275.
  • the NcoI-HindIII fragment (ca. 425 bp) from pMON30245 was ligated to the NcoI-HindIII fragment (ca. 3800 bp) of the pMON30275.
  • pMON30245 (WO 94/12638) contains the gene coding for hIL-3 receptor agonist pMON13416 joined to a mouse lgG2b hinge fragment. Immediately 3' to the lgG2b hinge and 5' to the HindIII site is an AflIII site.
  • Genes can be cloned into the AflIII-HindIII sites as NcoI-HindIII or AflIII-HindIII fragments in frame with the hIL-3 variant pMON13416/IgG2b hinge to create novel chimeras.
  • the NcoI site and the AflIII site have compatible overhangs and will ligate but both recognition sites are lost.
  • EXAMPLE 2 Construction of an intermediate plasmid containing one copy of the c-mol ligand (1-153) gene of the dimer template in order to generate a plasmid DNA with the coding sequence of c-mpl (1-153) ligand followed by a unique EcoRI restriction site, the gene is isolated via reverse
  • RNA transcriptase/polymerase chain reaction RT/PCR.
  • Human fetal (lot #38130) and adult liver (lot #46018) A+ RNA are obtained from Clontech (Palo Alto, CA) for source of c-mpl ligand messenger RNA (mRNA).
  • mRNA messenger RNA
  • RNA sequencing reactions are carried out using a cDNA CycleTM Kit obtained from Invitrogen (San Diego, CA).
  • a cDNA CycleTM Kit obtained from Invitrogen (San Diego, CA).
  • 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-mplNcoI (SEQ ID NO:13) and Reverse primer: Ecompl.
  • the c-mplNcoI primer anneals to the c-mpl ligand gene (bases #279-311 based on c-mpl ligand sequence from Gene bank accession #L33410 or de Sauvage et al., Nature 369: 533-538 (1994)) and encodes a NcoI restriction enzyme site immediately 5' to the first codon (Ser+1) of c-mpl ligand.
  • the NcoI The NcoI
  • 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 codons (Ser, Pro, Ala, & Pro) of c-mpl ligand.
  • the Ecompl primer anneals to bases #720- 737 of c-mpl ligand and encodes an EcoRI site (GAATTC) in- frame with the c-mpl ligand gene immediately following Arg- 153.
  • the EcoRI site creates Glu and Phe codons following Arg-153.
  • the ca. 480 bp PCR product was purified, digested with NcoI and EcoRI and ligated to the NcoI-EcoRI vector fragment of pMON3993 (ca.
  • pMON3993 was a derivative of pMON3359 (described in Example 1).
  • the human IL-3 signal peptide sequence which had been subcloned as a BamHI fragment into the unique BamHI site between the IE110 promoter and poly-A signal, contains an NcoI site at its 3' end and is followed by a unique EcoRI site.
  • the RT reaction from Example 2 serves as the template for PCR with a combination of the following primers; c-mplNcoI (SEQ ID NO:13) (forward primer) and c-mplHindIII (SEQ ID NO:15) (reverse primer).
  • the c-mplNcoI (SEQ ID NO:13) primer is described in Example 2.
  • the c-mplHindIII (SEQ ID NO:15) primer which anneals to bases #716-737 of c-mpl ligand, adds both a termination codon and a HindIII restriction enzyme site immediately following the final codon, Arg 153 .
  • 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 NcoI and HindIII restriction enzymes for transfer to a mammalian expression vector, pMON3934.
  • pMON3934 is digested with NcoI and HindIII (ca. 3800 bp) and will accept the PCR products.
  • 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/AflIII 5L synthetic oligonucleotide linker 5L-5' (SEQ ID NO:18) and 5L-3' (SEQ ID NO:19).
  • the EcoRI end of the linker will ligate to the EcoRI end of pMON26458.
  • the AflIII end of the linker will ligate to the NcoI site of pMON32133, and neither restriction site will be retained upon ligation.
  • Plasmid, pMON28548, is a result of the cloning and contains the DNA sequence of (SEQ ID NO:80) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:222) linker to amino acids 1-153 c-mpl ligand that contains a deletion of amino acids 112-115 (SEQ ID NO:162).
  • 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/Af1III 4L synthetic
  • oligonucleotide linker 4L-5' (SEQ ID NO:16) and 4L-3' (SEQ ID NO:17).
  • the EcoRI end of the linker will ligate to the EcoRI end of pMON26458.
  • the AflIII end of the linker will ligate to the NcoI site of pMON32132, and neither restriction site will be retained upon ligation.
  • the BstXI sites of pMON26458 and pMON32132 will ligate as well.
  • the plasmid, pMON28500 is a result of the cloning and contains the DNA sequence of (SEQ ID NO:82) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyAsnMetAla (SEQ ID NO:223) linker (4L) to amino acids 1-153 c-mpl ligand (SEQ ID NO:82) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyAsnMetAla (SEQ ID NO:223) linker (4L) to amino acids 1-153 c-mpl ligand
  • 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/AflIII 5L synthetic
  • oligonucleotide linker 5L-5' SEQ ID NO:18
  • 5L-3' SEQ ID NO:19
  • pMON28501 is a result of the cloning and contains the DNA sequence of (SEQ ID NO: 82) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:222) linker (5D to amino acids 1-153 c-mpl ligand (SEQ ID NO:164).
  • 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 pMON32134 along with the EcoRI/AflIII 8L synthetic
  • oligonucleotide linker 8L-5' SEQ ID NO:20
  • 8L-3' SEQ ID NO:21
  • the EcoRI end of the linker will ligate to the EcoRI end of pMON26458.
  • the AflIII end of the linker will ligate to the NcoI site of pMON32134, and neither restriction site will be retained upon ligation.
  • pMON26458 and pMON32134 will ligate as well.
  • Plasmid, pMON28502 is a result of the cloning which contains the DNA sequence of (SEQ ID NO:83) and encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:224) linker (8D to amino acids 1-153 c-mpl ligand (SEQ ID NO:165).
  • the PCR reactions were carried out using dimer templates, pMONs 28500, 28501, 28502 or 28548 and one of the sets of synthetic primer sets below (The first number refers to the position of the first amino acid in the original sequence.
  • the 31-5' and 31-3' refers to the 5' and 3' oligo primers, receptively, for the sequence beginning at the codon corresponding to residue 31 of the original sequence.).
  • the templates and oligonucleotide sets used in the PCR reactions are shown in Table 4.
  • the products that were generated were about 480 bp and were purified via Magic PCR Clean up kits (Promega).
  • the c-mpl receptor agonist gene PCR products were digested with NcoI and HindIII or AflIII and HindIII restriction enzymes (ca. 470 bp) for transfer to a mammalian expression vector.
  • the expression vector, pMON30304 was digested with NcoI and HindIII (ca. 4200 bp) and accepts the PCR products as NcoI-HindIII or AflIII-HindIII fragments.
  • the restriction digest of the PCR product and the resulting plasmids are shown in Table 4.
  • Plasmid pACYC177 (Chang, A.C.Y. and Cohen, S.N. J. Bacteriol .
  • DNA was digested with restriction enzymes HindIII and BamHI, resulting in a 3092 base pair HindIII, BamHI fragment.
  • Plasmid, pMON13037 (WO 95/21254), DNA was digested with BglII and Fspi, resulting in a 616 base pair BglII, Fspi fragment.
  • a second sample of plasmid, pMON13037, DNA was digested with NcoI and HindIII,
  • Plasmid, pMON15960, DNA was digested with
  • restriction enzyme SmaI used as template in a PCR reaction using synthetic DNA oligonucleotides 38 stop (SEQ ID NO:65) and 39 start (SEQ ID NO:64) as primers, resulting in the amplification of a DNA fragment of 576 base pairs.
  • the amplified fragment was digested with restriction enzymes HindIII and NcoI, resulting in a HindIII, NcoI fragment of 558 base pairs.
  • Plasmid, pMON13181 DNA was digested with HindIII and AflIII, resulting in a HindIII, AflIII fragment of 4068 base pairs. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis, and sequenced to confirm the correct insert.
  • the plasmid, pMON15981 contains the" DNA sequence of (SEQ ID NO:155) which encodes the following amino acid sequence:
  • Plasmid, pMON15960, DNA was digested with
  • restriction enzyme SmaI and used as template in a PCR reaction using synthetic DNA oligonucleotides 96 stop (SEQ ID NO:67) and 97 start (SEQ ID NO:66) as primers, resulting in the amplification of a DNA fragment of 576 base pairs.
  • the amplified fragment was digested with restriction enzymes HindIII and NcoI, resulting in a HindIII, NcoI fragment of 558 base pairs.
  • Plasmid, pMON13181 DNA was digested with HindIII and AflIII, resulting in a HindIII, AflIII fragment of 4068 base pairs.
  • the restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101.
  • Plasmid DNA was isolated, analyzed by restriction analysis, and sequenced to confirm the correct insert.
  • the plasmid, pMON15982 contains the DNA sequence of (SEQ ID NO:157) which encodes the following amino acid sequence:
  • Plasmid, pMON15960, DNA was digested with
  • restriction enzyme SmaI and used as template in a PCR reaction using synthetic DNA oligonucleotides 142 stop (SEQ ID NO:73) and 141 start (SEQ ID NO:72) as primers, resulting in the amplification of a DNA fragment of 576 base pairs.
  • the amplified fragment was digested with restriction enzymes HindIII and NcoI, resulting in a HindIII, NcoI fragment of 558 base pairs.
  • Plasmid, pMON13181 DNA was digested with HindIII and AflIII, resulting in a HindIII, AflIII fragment of 4068 base pairs.
  • the restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101.
  • Plasmid DNA was isolated, analyzed by restriction analysis, and sequenced to confirm the correct insert.
  • the plasmid, pMON15965 contains the DNA sequence of (SEQ ID NO:157) which encodes the following amino acid sequence:
  • Plasmid, pMON15960, DNA was digested with
  • fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101.
  • Plasmid DNA was isolated, analyzed by restriction analysis, and sequenced to confirm the correct insert.
  • the plasmid, pMON15966 contains the DNA sequence of (SEQ ID NO:158) which encodes the following amino acid sequence:
  • Plasmid, pMON15960, DNA was digested with
  • restriction enzyme SmaI and used as template in a PCR reaction using synthetic DNA oligonucleotides 132 stop (SEQ ID NO:71) and 133 start (SEQ ID NO:70) as primers, resulting in the amplification of a DNA fragment of 576 base pairs.
  • the amplified fragment was digested with restriction enzymes HindIII and NcoI, resulting in a HindIII, NcoI fragment of 558 base pairs.
  • Plasmid, pMON13181 DNA was digested with HindIII and AflIII, resulting in a HindIII, AflIII fragment of 4068 base pairs.
  • the restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101.
  • Plasmid DNA was isolated, analyzed by restriction analysis, and sequenced to confirm the correct insert.
  • the plasmid, pMON15967 contains the DNA sequence of (SEQ ID NO: 159) which encodes the following amino acid sequence:
  • pMON13180 an intermediate plasmid used for constructing plasmids that contain DNA sequence encoding multi-functional hematopoietic receptor agonists.
  • Plasmid, pMON13046 (WO 95/21254), DNA was digested with restriction endonucleases XmaI and SnaBI, resulting in a 4018 base pair vector fragment.
  • the 4018 base pair XmaI- SnaBI fragment was purified using a Magic DNA Clean-up
  • oligonucleotides glyxal (SEQ ID NO:74) and glyxa2 (SEQ ID NO:75), were designed to remove sequence encoding a factor Xa cleavage site. When properly assembled these oligonucleotides also result in XmaI and SnaBI ends.
  • the primers, Glyxal and glyxa2 were annealed in annealing buffer (20mM Tris-HCl pH7.5, 10 mM MgCl 2 , 50 mM NaCl) by heating at 70°C for ten minutes and allowed to slow cool.
  • the 4018 base pair XmaI-SnaBI fragment from pMON13046 was ligated with the assembled oligonucleotides using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells (Life Technologies Inc.
  • the resulting plasmid was designated pMON13180 and contains the DNA sequence of (SEQ ID NO:**).
  • pMON13181 an intermediate plasmid used for constructing plasmids that contain DNA sequences encoding multi-functional hematopoietic receptor agonists.
  • Plasmid, pMON13047 (WO 95/21254), DNA was digested with restriction endonucleases XmaI and SnaBI, resulting in a 4063 base pair vector fragment.
  • the 4063 base pair XmaI- SnaBI fragment was purified using a Magic DNA Clean-up
  • glyxal SEQ ID NO:74
  • glyxa2 SEQ ID NO:75
  • glyxal and glyxa2 were annealed in annealing buffer by heating at 70°C for ten minutes and allowed to slow cool.
  • the 4063 base pair XmaI-SnaBI fragment from pMON13047 was ligated with the assembled oligonucleotides using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells (Life).
  • Plasmid DNA was isolated from the transformants and analyzed using a PCR based assay. Plasmid DNA from selected transformants was sequenced to confirm the correct insertion of the oligonucleotides. The resulting plasmid was designated pMON13181 and contains the DNA sequence of (SEQ ID NO:**)
  • the new N-terminus/C-terminus gene in pMON13182 was created using Method I as described in Materials and
  • Fragment Start was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 39 start (SEQ ID NO:64) and L-11 start (SEQ ID NO:60).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence ii, pMON13037 using the primer set, 38 stop (SEQ ID NO:65) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the intermediate plasmid, pMON13180 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
  • plasmid contains the DNA sequence of
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 38 stop (SEQ ID NO:65) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the intermediate plasmid, pMON13181 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison,
  • E. coli strain JM101 was transformed with pMON13183 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13183 contains the DNA sequence of (SEQ ID NO:95) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13184 was created using Method I as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 97 start (SEQ ID NO:66) and L-11 start (SEQ ID NO:60).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:67) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the intermediate plasmid, pMON13180 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison,
  • plasmid was designated pMON13184.
  • E. coli strain JM101 was transformed with pMON13184 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13184 contains the DNA sequence of (SEQ ID NO:96) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13185 was created using Method I as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 97 start (SEQ ID NO:66) and L-11 start (SEQ ID NO:60).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:67 and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the intermediate plasmid, pMON13181 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
  • plasmid was designated pMON13185.
  • E. coli strain JM101 was transformed with pMON13185 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13185 contains the DNA sequence of
  • the new N-terminus/C-terminus gene in pMON13186 was created using Method I as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 126 start (SEQ ID NO:68) and L-11 start (SEQ ID NO:60).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:69) and L-11 stop (SEQ ID NO:61).
  • the full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and amplified from the annealed Fragments Start and Stop using 126 start and 125 stop.
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the intermediate plasmid, pMON13180 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
  • E. coli strain JM101 was transformed with pMON13186 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13186 contains the DNA sequence of (SEQ ID NO:98) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13187 was created using Method I as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 126 start (SEQ ID NO:68) and L-11 start (SEQ ID NO:60).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:69) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the intermediate plasmid, pMON13181 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
  • E. coli strain JM101 was transformed with pMON13187 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13187 contains the DNA sequence of (SEQ ID NO:99) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13188 was created using Method I as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 133 start (SEQ ID NO:70) and L-11 start (SEQ ID NO:60).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO:71) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the intermediate plasmid, pMON13180 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison,
  • E. coli strain JM101 was transformed with pMON13188 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13188 contains the DNA sequence of (SEQ ID NO:100) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13189 was created using Method I as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 133 start (SEQ ID NO:70) and L-11 start (SEQ ID NO:60).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO:71) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the intermediate plasmid, pMON13181 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
  • plasmid was designated pMON13189.
  • E. coli strain JM101 was transformed with pMON13189 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13189 contains the DNA sequence of (SEQ ID NO:101) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13190 was created using Method I as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 142 start (SEQ ID NO:72) and L-11 start (SEQ ID NO:60).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 141 stop (SEQ ID NO:73) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the intermediate plasmid, pMON13180 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison,
  • E. coli strain JM101 was transformed with pMON13190 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13190 contains the DNA sequence of (SEQ ID NO:102) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13191 was created using Method I as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 142 start (SEQ ID NO:72) and L-11 start (SEQ ID NO:60).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 141 stop (SEQ ID NO:73) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the intermediate plasmid, pMON13181 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI).
  • the purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
  • plasmid was designated pMON13191.
  • E. coli strain JM101 was transformed with pMON13191 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13191 contains the DNA sequence of (SEQ ID NO:103) which encodes the following amino acid sequence:
  • Fragment Start was created and amplified from G- CSF sequence in pMON13037 using the primer set, 39 start (SEQ ID NO:64) and P-bl start (SEQ ID NO:62).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 38 stop (SEQ ID NO:65) and P-bl stop (SEQ ID NO:63).
  • Fragment Start was digested with restriction endonuclease NcoI
  • Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
  • the intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser 17 gene and was digested with restriction endonucleases NcoI and
  • HindIII The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N- terminus/C-terminus G-CSF Ser 17 gene was isolated using Geneclean (Bio101, Vista, CA).
  • the intermediate plasmid, pMON13180 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells
  • Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene.
  • the resulting plasmid was designated pMON13192.
  • E. coli strain JM101 was transformed with pMON13192 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13192, contains the DNA sequence of
  • Fragment Start was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 39 start (SEQ ID NO:64) and P-bl start (SEQ ID NO:62).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 38 stop (SEQ ID NO:65) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
  • the intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser 17 gene and was digested with restriction endonucleases NcoI and
  • HindIII The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N- terminus/C-terminus G-CSF Ser 17 gene was isolated using Geneclean (Bio101, Vista, CA).
  • the intermediate plasmid, pMON13181 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells
  • Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene.
  • the resulting plasmid was designated pMON13193.
  • E. coli strain JM101 was transformed with pMON13193 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13193 contains the DNA sequence of (SEQ ID NO:105) encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON25190 was created using Method II as described in Materials and
  • Fragment Start was created and amplified from G- CSF sequence in pMON13037 using the primer set, 97 start
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:67) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was
  • digested with restriction endonuclease HindIII After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
  • the intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser 17 gene and was digested with restriction endonucleases NcoI and
  • bacteria were selected on ampicillin-containing plates.
  • Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene.
  • the resulting plasmid was designated pMON25190.
  • E. coli strain JM101 was transformed with pMON25190 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON25190 contains the DNA sequence of (SEQ ID NO:106) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON25191 was created using Method II as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 97 start (SEQ ID NO:66) and P-bl start (SEQ ID NO:62).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:98) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
  • the intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser 17 gene and was digested with restriction endonucleases NcoI and HindIII.
  • the digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N-terminus/C-terminus G-CSF Ser 17 gene was isolated using Geneclean (Bio101, Vista, CA).
  • the intermediate plasmid, pMON13181 was digested with restriction endonucleases
  • bacteria were selected on ampicillin-containing plates.
  • Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene.
  • the resulting plasmid was designated pMON25191.
  • E. col i strain JM101 was transformed with pMON25191 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON25191 contains the DNA sequence of (SEQ ID NO:107) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13194 was created using Method II as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 126 start (SEQ ID NO:68) and P-bl start (SEQ ID NO:62).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:67) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
  • the intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser 17 gene and was digested with restriction endonucleases NcoI and
  • HindIII The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N- terminus/C-terminus G-CSF Ser 17 gene was isolated using Geneclean (Bio101, Vista, CA).
  • the intermediate plasmid, pMON13180 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells
  • Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene.
  • the resulting plasmid was designated pMON13194.
  • E. coli strain JM101 was transformed with pMON13194 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13194 contains the DNA sequence of (SEQ ID NO:108) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13195 was created using Method II as described in Materials and Methods. Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 126 start (SEQ ID NO:68) and P-bl start (SEQ ID NO:62).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:69) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
  • the intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser 17 gene and was digested with restriction endonucleases NcoI and
  • Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene.
  • the resulting plasmid was designated pMON13195.
  • E. coli strain JM101 was transformed with pMON13195 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13195 contains the DNA sequence of (SEQ ID NO:109) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13196 was created using Method II as described in Materials and
  • Fragment Start was created and amplified from G- CSF sequence in pMON13037 using the primer set, 133 start
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO:71) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
  • the intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser 17 gene and was digested with restriction endonucleases NcoI and
  • Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene.
  • the resulting plasmid was designated pMON13196.
  • E. coli strain JM101 was transformed with pMON13196 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13196 contains the DNA sequence of
  • the new N-terminus/C-terminus gene in pMON13197 was created using Method II as described in Materials and
  • Fragment Start was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 133 start (SEQ ID NO:70) and P-bl start (SEQ ID NO:62).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO:71) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
  • the intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser 17 gene and was digested with restriction endonucleases NcoI and
  • HindIII The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N- terminus/C-terminus G-CSF Ser 17 gene was isolated using Geneclean (Bio101, Vista, CA).
  • the intermediate plasmid, pMON13181 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells
  • Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene.
  • the resulting plasmid was designated pMON13197.
  • E. coli strain JM101 was transformed with pMON13197 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13197 contains the DNA sequence of (SEQ ID NO:111) which encodes the following amino acid sequence:
  • Fragment Start was created and amplified from G-CSF sequence in pMON13037 using the primer set, 142 start (SEQ ID NO:72) and P-bl start (SEQ ID NO:62).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 141 stop (SEQ ID NO:73) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was
  • digested with restriction endonuclease HindIII After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
  • the intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser 17 gene and was digested with restriction endonucleases NcoI and
  • HindIII The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N- terminus/C-terminus G-CSF Ser 17 gene was isolated using Geneclean (Bio101, Vista, CA).
  • the intermediate plasmid, pMON13180 was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells
  • Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene.
  • the resulting plasmid was designated pMON13198.
  • E. coli strain JM101 was transformed with pMON13198 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13198 contains the DNA sequence of (SEQ ID NO:112) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON13199 was created using Method II as described in Materials and
  • Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 142 Start (SEQ ID NO:72) and P-bl start (SEQ ID NO:62).
  • Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 141 stop (SEQ ID NO:73) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI. and Fragment Stop was digested with restriction endonuclease HindIII.
  • digested Fragments Start and Stop were combir.-d with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
  • the intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser 17 gene and was digested with restriction endonucleases NcoI and
  • Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene.
  • the resulting plasmid was designated pMON13199.
  • E. coli strain JM101 was transformed with pMON13199 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON13199 contains the DNA sequence of (SEQ ID NO:113) which encodes the following amino acid sequence:
  • the three DNAs are: 1) pMON13046, containing hIL-3 receptor agonist pMON13416, digested with BstEII and SnaBI; 2 ) the annealed complimentary pair of synthetic oligonucleotides, L1syn. for (SEQ ID NO:48) and L1syn.
  • rev SEQ ID NO:49
  • DNA had been grown in the dam- cells, DM1 (Life Technologies)) and SnaBI.
  • the digested DNAs were resolved on a 0.9% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101).
  • Miniprep DNA was isolated from the transformants, and the transformants were screened using a PCR based assay.
  • Plasmid DNA from selected transformants was sequenced to obtain the correct template.
  • the resulting plasmid was designated syntan1 and contains the DNA sequence of (SEQ ID NO:84).
  • the three DNAs are: 1) pMON13046, containing hIL-3 receptor agonist pMON13416, digested with BstEII and SnaBI; 2) the annealed complimentary pair of synthetic oligonucleotides, L3syn.for (SEQ ID NO:50) and L3syn.rev (SEQ ID NO:51), which contain sequence encoding the linker that connects the C-terminal and N-terminal ends of the original protein and a small amount of surrounding pMON13416 sequence and which when properly assembled result in BstEII and SnaBI ends; and 3) a portion of hIL-3 receptor agonist pMON13416 digested from pMON13046 with Clal (DNA had been grown in the dam- cells, DM1 (Life Technologies)) and SnaBI.
  • the digested DNAs were resolved on a 0.9% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101).
  • E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD). Miniprep DNA was isolated from the transformants, and the transformants were screened using a PCR based assay. Plasmid DNA from selected transformants was sequenced to obtain the correct template. The resulting plasmid was designated syntan3 and contains the DNA sequence of (SEQ ID NO:85).
  • the new N-terminus/C-terminus gene in pMON31104 was created using Method III as described in Materials and
  • the full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntanl, using the primer set 35 start (SEQ ID NO:52) and 34 rev (SEQ ID NO:53).
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI.
  • the digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA).
  • the purified digested DNA fragment was ligated into the expression vector,pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN).
  • the pMON13189 DNA had been previously digested with NcoI and
  • SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD).
  • Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31104. E. coli strain JM101 was transformed with pMON31104 for protein expression and protein isolation from inclusion bodies. The plasmid, pMON31104, contains the DNA sequence of (SEQ ID NO:86) which encodes the following amino acid sequence:
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI.
  • the digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA).
  • the purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN).
  • pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD).
  • Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31105.
  • E. coli strain JM101 was transformed with pMON31105 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON31105 contains the DNA sequence of (SEQ ID NO: 1
  • the new N-terminus/C-terminus gene in pMON31106 was created using Method III as described in Materials and
  • the full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntanl, using the primer set 91 start (SEQ ID NO:56) and 90 rev (SEQ ID NO:57).
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI.
  • the digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean
  • the purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN).
  • the pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide.
  • a portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD).
  • Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31106. E. coli strain JM101 was transformed with pMON31106 for protein expression and protein isolation from inclusion bodies. The plasmid, pMON31106, contains the DNA sequence of (SEQ ID NO:80) which encodes the protein with the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON31107 was created using Method III as described in Materials and Methods.
  • the full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntanl, using the primer set 101 start (SEQ ID NO:58) and 100 rev (SEQ ID NO:59).
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI.
  • the digested was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA).
  • the purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN).
  • T4 DNA ligase Boehringer Mannheim, Indianapolis, IN.
  • pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31107.
  • E. coli strain JM101 was transformed with pMON31107 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON31107 contains the DNA sequence of (SEQ ID NO:89) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON31108 was created using Method III as described in Materials and Methods.
  • the full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntan3, using the primer set 35 start (SEQ ID NO:52) and 34 rev (SEQ ID NO:53).
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI.
  • the digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA).
  • the purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN).
  • the pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a
  • Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31108. E. coli strain JM101 was transformed with pMON31108 for protein expression and protein isolation from inclusion bodies. The plasmid, pMON31108, contains the DNA sequence of (SEQ ID NO:90) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON31109 was created using Method III as described in Materials and Methods.
  • the full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntan3, using the primer set 70 start (SEQ ID NO:54) and 69 rev (SEQ ID NO:55).
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI.
  • the digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA).
  • the purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN).
  • the pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide.
  • a portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31109.
  • E. coli strain JM101 was transformed with pMON31109 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON31109 contains the DNA sequence of (SEQ ID NO:91) which encodes the following amino acid sequence:
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI.
  • the digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA).
  • the purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN).
  • the pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31110.
  • E. coli strain JM101 was transformed with pMON31110 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON31110 contains the DNA sequence of (SEQ ID NO:92) which encodes the following amino acid sequence:
  • the new N-terminus/C-terminus gene in pMON31111 was created using Method III as described in Materials and
  • the full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntan3, using the primer set 101 start (SEQ ID NO:58) and 100 rev (SEQ ID NO:59).
  • the resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI.
  • the digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA).
  • the purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN).
  • pMON13189 DNA had been previously digested with NcoI and
  • SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD).
  • Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31111.
  • E. coli strain JM101 was transformed with pMON31111 for protein expression and protein isolation from inclusion bodies.
  • the plasmid, pMON31111 contains the DNA sequence of (SEQ ID NO:93) which encodes the following amino acid sequence:
  • pMON31112 a plasmid containing DNA sequence encoding a multi-functional hematopoietic receptor agonist which activates the hIL-3 receptor and G-CSF
  • Plasmid, pMON13189 DNA was digested with
  • restriction enzymes NcoI and XmaI resulting in an NcoI, XmaI vector fragment that was isolated and purified from a 0.8% agarose gel.
  • the DNA from a second plasmid, pMON13222 (WO 94/12639, US serial # 08/411,796) was digested with NcoI and EcoRI resulting in a 281 base pair NcoI, EcoRI fragment. This fragment was isolated and purified from a 1.0% agarose gel.
  • SYNNOXAl.REQ SEQ ID NO:240
  • SYNNOXA2.REQ SEQ ID NO:241
  • SEQ ID NO:241 Two oligonucleotides SYNNOXAl.REQ (SEQ ID NO:240) and SYNNOXA2.REQ (SEQ ID NO:241) were annealed and ligated with the 281 base pair DNA fragment from pMON13222 to the DNA vector fragment from pMON13189. A portion of the ligation mixture was then transformed into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis to show the presence of an EcoRV fragment, and sequenced to confirm the correct inserts.
  • the plasmid, pMON31112 contains the DNA sequence of (SEQ ID NO:114) which encodes the following amino acid sequence:
  • pMON31113 a plasmid containing DNA sequence encoding a multi-functional hematopoietic receptor agonist which activates the hIL-3 receptor and G-CSF receptor.
  • Plasmid, pMON13197 DNA was digested with restriction enzymes NcoI and XmaI resulting in an NcoI, XmaI vector fragment that was isolated and purified from a 0.8% agarose gel.
  • the DNA from a second plasmid, pMON13239 (WO 94/12639, US serial # 08/411,796) was digested with NcoI and EcoRI resulting in a 281 base pair NcoI, EcoRI fragment. This fragment was isolated and purified from a 1.0% agarose gel.
  • SYNNOXA2.REQ (SEQ ID NO:241) were annealed and ligated with the 281 base pair DNA fragment from pMON13239 to the DNA vector fragment from pMON13197. A portion of the ligation mixture was then transformed into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis to show the presence of an EcoRV fragment, and sequenced to confirm the correct inserts. The plasmid, pMON31113, contains the DNA sequence of (SEQ ID NO:115) which encodes the following amino acid sequence:
  • pMON31114 Construction of pMON31114, a plasmid containing DNA sequence encoding a multi-functional hematopoietic receptor agonist which activates the hIL-3 receptor and G-CSF receptor.
  • Plasmid, pMON13189 DNA was digested with restriction enzymes NcoI and XmaI resulting in an NcoI, XmaI vector fragment that was isolated and purified from a 0.8% agarose gel.
  • the DNA from a second plasmid, pMON13239 (WO 94/12639, US serial # 08/411,796), was digested with NcoI and EcoRI resulting in a 281 base pair NcoI, EcoRI fragment. This fragment was isolated and purified from a 1.0% agarose gel.
  • SYNNOXA2.REQ (SEQ ID NO:241) were annealed and ligated with the 281 base pair DNA fragment from pMON13239 to the DNA vector fragment from pMON13189. A portion of the ligation mixture was then transformed into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis to show the presence of an EcoRV fragment, and sequenced to confirm the correct inserts.
  • the plasmid, pMON31114 contains the DNA sequence of
  • pMON31115 a plasmid containing DNA sequence encoding a multi-functional hematopoietic receptor agonist which activates the hIL-3 receptor and G-CSF receptor.
  • Plasmid, pMON13197 DNA was digested with restriction enzymes NcoI and XmaI resulting in an NcoI, XmaI vector fragment that was isolated and purified from a 0.8% agarose gel.
  • the DNA from a second plasmid, pMON13222, was digested with NcoI and EcoRI resulting in a 281 base pair NcoI, EcoRI fragment. This fragment was isolated and purified from a 1.0% agarose gel.
  • SYNNOXAl.REQ SEQ ID NO:240
  • SYNNOXA2.REQ SEQ ID NO:241
  • SEQ ID NO:241 Two oligonucleotides SYNNOXAl.REQ (SEQ ID NO:240) and SYNNOXA2.REQ (SEQ ID NO:241) were annealed and ligated with the 281 base pair DNA fragment from pMON13222 to the DNA vector fragment from pMON13197. A portion of the ligation mixture was then transformed into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis to show the presence of an EcoRV fragment, and sequenced to confirm the correct inserts.
  • the plasmid, pMON31115 contains the DNA sequence of (SEQ ID NO:117) which encodes the following amino acid sequence:
  • the protein concentration of the multi-functional hematopoietic receptor agonist protein can be determined using a sandwich ELISA based on an affinity purified
  • the bioactivity of the multi-functional hematopoietic receptor agonist can be determined in a number of in vitro assays.
  • a multi-functional hematopoietic receptor agonist which binds the hIL-3 receptor and G-CSF receptor can be assayed in cell proliferation assays using cell lines expressing the hIL-3 and/or G-CSF receptors.
  • One such assay is the AML-193 cell proliferation assay.
  • AML-193 cells respond to IL-3 and G-CSF which allows for the combined bioactivity of the IL-3/G-CSF multi-functional hematopoietic receptor agonist to be determined.
  • Another such assay is the TFl cell proliferation assay.
  • M-NFS-60 ATCC. CRL 1838
  • 32D which are murine IL-3 dependent cell line
  • the activity of IL-3 is species specific whereas G-CSF is not, therefore the
  • bioactivity of the G-CSF component of the IL-3 /G-CSF multi- functional hematopoietic receptor agonist can be determined independently.
  • Cell lines such as BHK or murine Baf/3, which do not express the receptor for a given ligand can be transfected with a plasmid containing a gene encoding the desired receptor.
  • An example of such a cell line is BaF3 transfected with the hG-CSF receptor (BaF3/hG-CSF).
  • the activity of the multi-functional hematopoietic receptor agonist in these cell lines can be compared with hIL-3 or G- CSF alone or together.
  • the bioactivity of examples of multi- functional hematopoietic receptor agonists of the present invention assayed in the BaF3/hG-CSF cell proliferation and TFl cell proliferation assays is shown in Table 5 and Table 6.
  • the bioactivity of the multi-functional hematopoietic receptor agonist is expressed as relative activity compared with a standard protein pMON13056 (WO 95/21254).
  • the bioactivity of examples of multi-functional hematopoietic receptor agonists of the present invention assayed in the BaF3/c-mpl cell proliferation and TF1 cell proliferation assays is shown in Table 7 and Table 8.
  • the methylcellulose assay can be used to determine the effect of the multi-functional hematopoietic receptor agonists on the expansion of the hematopoietic progenitor cells and the pattern of the different types of hematopoietic colonies in vitro .
  • the methylcellulose assay can provide an estimate of precursor frequency since one measures the frequency of progenitors per 100,000 input cells. Long term, stromal dependent cultures have been used to delineate primitive hematopoietic progenitors and stem cells.
  • This assay can be used to determine whether the multi-functional hematopoietic receptor agonist stimulates the expansion of very primitive progenitors and/or stem cells.
  • limiting dilution cultures can be performed which will indicate the frequency of primitive progenitors stimulated by the multi-functional hematopoietic receptor agonist.
  • G-CSF variants which contain single or multiple amino acid substitutions were made using PCR mutagenesis
  • substitutions can be made one at a time or in combination with other amino acid substitutions, and/or deletions, and/or insertions and/or extensions.
  • the plasmid DNA can be transfected into an appropriate mammalian cell, insect cell or bacterial strain such as E. coli for production.
  • G-CSF which are active, include substitutions at positions 1 (Thr to Ser, Arg or Gly, 2 (Pro to Leu), 3 (Leu to Arg or Ser) and 17 (Cys to Ser) and deletions of amino acids 1-11 (Kuga et al.
  • G-CSF amino acid substitution variants can be used as the template to create the G-CSF receptor agonists in which a new N-terminus and new C-terminys are created. Examples of G-CSF amino acid substitution variants are shown in Table 9. EXAMPLE 89
  • the G-CSF amino acid substitution variants can be assayed for cell proliferation activity using the Baf/3 cell line transtected with the human G-CSF receptor.
  • bioactvity of examples of G-CSF amino acid substitution variants is shown in Table 9 relative to native human G-CSF.
  • a "+” indicates a comparable activity to native and a "-” indicates significantly reduced or no measurable activity.

Abstract

Disclosed are novel multi-functional hematopoietic receptor agonist proteins, DNAs which encode the multi-functional hematopoietic receptor agonists proteins, methods of making the multi-functional hematopoietic receptor agonists proteins and methods of using the multi-functional hematopoietic receptor agonists proteins.

Description

MULTI-FUNCTIONAL HEMATOPOIETIC RECEPTOR AGONISTS
The present application claims priority under 35 USC §119 (e) of United States provisional application Serial No. 60/004,834 filed October 05, 1995.
Field of the Invention
The present invention relates to multi-functional hematopoietic receptor agonists.
Background of the Invention
Colony stimulating factors (CSFs) which stimulate the differentiation and/or proliferation of bone marrow cells have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells. CSFs in both human and murine systems have been identified and distinguished according to their activities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies,
respectively, while GM-CSF and interleukin-3 (IL-3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies. IL-3 also stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies.
U.S. 4,877,729 and U.S. 4,959,455 disclose human IL-3 and gibbon IL-3 cDNAs and the protein sequences for which they code. The hIL-3 disclosed has serine rather than proline at position 8 in the protein sequence.
International Patent Application (PCT) WO 88/00598 discloses gibbon- and human-like IL-3. The hIL-3 contains a Ser8 -> Pro8 replacement. Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites. U.S. 4,810,643 discloses the DNA sequence encoding human G-CSF.
WO 91/02754 discloses a fusion protein comprised of GM-CSF and IL-3 which has increased biological activity
compared to GM-CSF or IL-3 alone. Also disclosed are
nonglycosylated IL-3 and GM-CSF analog proteins as
components of the multi-functional hematopoietic receptor agonist.
WO 92/04455 discloses fusion proteins composed of IL-3 fused to a lymphokine selected from the group consisting of IL-3, IL-6, IL-7, IL-9, IL-11, EPO and G-CSF.
WO 95/21197 and WO 95/21254 disclose fusion proteins capable of broad multi-functional hematopoietic properties.
GB 2,285,446 relates to the c-mpl ligand
(thrombopoietin) and various forms of thrombopoietin which are shown to influence the replication, differentiation and maturation of megakaryocytes and megakaryocytes progenitors which may be used for the treatment of thrombocytopenia.
EP 675,201 A1 relates to the c-mpl ligand
(Megakaryocyte growth and development factor (MGDF) , allelic variations of c-mpl ligand and c-mpl ligand attached to water soluble polymers such as polyethylene glycol.
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.
Rearrangement of Protein Sequences In evolution, rearrangements of DNA sequences serve an important role in generating a diversity of protein
structure and function. Gene duplication and exon shuffling provide an important mechanism to rapidly generate diversity and thereby provide organisms with a competitive advantage, especially since the basal mutation rate is low (Doolittle, Protein Science 1:191-200, 1992).
The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The approach used in creating new sequences resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al., Proc. Natl . Acad. Sci . U. S. A . 76:3218-3222, 1979;
Teather & Erfle, J. Bacteriol . 172: 3837-3841, 1990;
Schimming et al., Eur. J. Biochem. 204: 13-19, 1992;
Yamiuchi and Minamikawa, FEBS Let t . 260:127-130, 1991;
MacGregor et al., FEBS Lett . 378:263-266). The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton ( J. Mol . Biol . 165:407-413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point 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.
This approach has been applied to 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 α-helix (interleukin-4; Kreitman et al., Cytokine 7:311-318, 1995), β-sheet
(interleukin-1; Horlick et al., Protein Eng. 5:427-431, 1992), or mixtures of the two (yeast phosphoribosyl
anthranilate isomerase; Luger et al., Science 243:206-210, 1989). Broad categories of protein function are represented in these sequence reorganization studies:
Enzymes
Figure imgf000006_0001
Figure imgf000007_0001
Enzyme Inhibitor
Figure imgf000007_0002
Cytokines
Figure imgf000007_0003
Tyrosine Kinase Recognition Domain
Figure imgf000007_0004
Transmembrane Protein
Figure imgf000007_0005
Chimeric Protein
Figure imgf000008_0001
The results of these studies have been highly variable. In many cases substantially lower activity, solubility or thermodynamic stability were observed ( E. coli dihydrofolate reductase, aspartate transcarbamoylase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3-phosphate
dehydrogenase, ornithine decarboxylase, omp A, yeast
phosphoglycerate dehydrogenase). In other cases, the
sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease T1, Bacillus β-glucanase, interleukin-1β, α-spectrin SH3 domain, pepsinogen, interleukin-4). In exceptional cases, an unexpected improvement over some properties of the natural sequence was observed, e.g., the solubility and refolding rate for rearranged α-spectrin SH3 domain sequences, and the receptor affinity and anti-tumor activity of transposed interleukin-4—Pseudomonas exotoxin fusion molecule (Kreitman et al., Proc . Natl . Acad . Sci . U. S. A . 91:6889-6893, 1994; Kreitman et al., Cancer Res . 55:3357-3363, 1995).
The primary motivation for these types of studies has been to study the role of short-range and long-range interactions in protein folding and stability. Sequence rearrangements of this type convert a subset of interactions that are long-range in the original sequence into short- range interactions in the new sequence, and vice versa. The fact that many of these sequence rearrangements are able to attain a conformation with at least some activity is persuasive evidence that protein folding occurs by multiple folding pathways (Viguera, et al., J. Mol . Biol . 247:670- 681, 1995). In the case of the SH3 domain of α-spectrin, choosing new termini at locations that corresponded to β- hairpin turns resulted in proteins with slightly less stability, but which were nevertheless able to fold.
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. Flexible hydrophilic residues such as Gly and Ser are frequently used in the linkers. Viguera, et al. (J. Mol . Biol . 247:670-681, 1995) compared joining the original N- and C- termini with 3- or 4-residue linkers; the 3 -residue linker was less
thermodynamically stable. Protasova et al. (Protein Eng. 7:1373-1377, 1994) used 3- or 5-residue linkers in
connecting the original N-termini of E. coli dihydrofolate reductase; only the 3-residue linker produced protein in good yield.
Summary of the Invention
Novel hematopoietic proteins of this invention are represented by the formulas:
R1-L1-R2, R2-L1-R1, R1-R2, or R2-R1 wherein R1 and R2 are independently selected from the group consisting of;
(I) A polypeptide comprising; a modified human G-CSF amino acid sequence of the formula:
Figure imgf000010_0001
Figure imgf000011_0001
wherein
Figure imgf000011_0002
wherein optionally 1-11 amino acids from the N-terminus and
1-5 from the C-terminus can be deleted; and wherein the 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;
Figure imgf000012_0001
(II) A polypeptide comprising; a modified hIL-3 amino acid sequence of the formula:
Figure imgf000012_0002
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0002
wherein optionally from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from 0 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human
mterleukin-3; and wherein the N-terminus is joined to the C-terminus directly or through a linker (L2) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
Figure imgf000017_0001
or
(III) A polypeptide comprising; a modified human c-mpl ligand amino acid sequence of the formula:
Figure imgf000018_0001
Figure imgf000019_0001
wherein the N-terminus is joined to the C-terminus directly or through a linker (L2) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
Figure imgf000020_0001
or (IV) A polypeptide comprising; a modified hIL-3 amino acid sequence of the formula:
Figure imgf000020_0002
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
above are; 38-39, 39-40, 40-41, 41-42, 48-49, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 64-65, 65-66, 66-67, 67-68, 68-69, 69-70, 96-97, 125-126, 126-127, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 136-137, 137-138, 138-139, 139-140, 140-141 and 141-142.
The most preferred breakpoints at which new C-terminus and N-terminus can be made in the polypeptide (I) above are; 38-39, 48-49, 96-97, 125-126, 132-133 and 141-142.
The more preferred breakpoints at which new C-terminus and N-terminus can be made in the polypeptide (II) above are; 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 66-67, 67-68, 68-69, 69-70, 70-71, 84-85, 85-86, 86-87, 87-88, 88-89, 89-90, 90-91, 98-99, 99-100, 100-101 and 101-102.
The most preferred breakpoints at which new C-terminus and N-terminus can be made in the polypeptide (II) above are; 34-35, 69-70 and 90-91.
The more preferred breakpoints at which new C-terminus and N-terminus can be made in the polypeptide (III) above or the amino acid sequence of (SEQ ID NO:256) 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, 120-121, 121-122, 122-123, 123-124, 124-125, 125-126 and 126-127.
The most preferred breakpoints at which new C-terminus and N-terminus can be made in the polypeptide (III) above or the amino acid sequence of (SEQ ID NO:256) are; 81-82, 108-109, 115-116, 119-120, 122-123 and 125-126. The multifunctional receptor agonist of the present invention can also be represented by the following formula:
Figure imgf000027_0001
in which:
X1 is a peptide comprising an amino acid sequence corresponding to the sequence of residues n+1 through J of the original protein having amino acids residues numbered sequentially 1 through J with an amino terminus at residue 1;
L is an optional linker;
X2 is a peptide comprising an amino acid sequence of residues 1 through n of the original protein;
Y1 is a peptide comprising an amino acid sequence corresponding to the sequence of residues n=1 through K of the original protein having amino acids residues numbered sequentially 1 through K with an amino terminus at residue
1;
Y2 is a peptide comprising an amino acid sequence of residues 1 through n of the original protein;
L1 and L2 are optional peptide spacers:
n is an integer ranging from 1 to J-1;
b, c, and d are each independently 0 or 1;
a and e are either 0 or 1, provided that both a and e cannot both be 0; and
T1 and T2 are proteins.
Additionally, the present invention relates to
recombinant expression vectors comprising nucleotide sequences encoding the multi-functional hematopoietic receptor agonists, related microbial expression systems, and processes for making the multi-functional hematopoietic receptor agonists. The invention also relates to
pharmaceutical compositions containing the multi-functional hematopoietic receptor agonists, and methods for using the multi-functional hematopoietic receptor agonists.
In addition to the use of the multi-functional hematopoietic receptor agonists of the present invention in vivo, it is envisioned that in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before infusion into patients.
Brief Description of the Figures
Figure 1 schematically illustrates the sequence
rearrangement of a protein. The N-terminus (N) and the C-terminus (C) of the native protein are joined through a linker, or joined directly. The protein is opened at a breakpoint creating a new N-terminus (new N) and a new C-terminus (new-C) resulting in a protein with a new linear amino 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-terminus and opening of the protein at the breakpoint. Figure 2 shows a schematic of Method I, 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. In the example shown 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 the amino acid 11 (a.a. 1- 10 are deleted) through a linker region and a new C-terminus 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-terminus and C-terminus of the native protein are joined without a linker and different N-terminus and C-terminus of the protein are created. In the example shown 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 the original N- terminus and a new C-terminus created at amino 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. In the example shown 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.
Detailed Description of the Invention
The present invention encompasses multi-functional hematopoietic receptor agonists formed from covalently linked polypeptides, each of which may act through a
different and specific cell receptor to initiate
complementary biological activities. Hematopoiesis requires a complex series of cellular events in which stem cells generate continuously into large populations of maturing cells in all major lineages. There are currently at least 20 known regulators with hematopoietic proliferative activity. Most of these proliferative regulators can only stimulate one or another type of colony formation in vitro, the precise pattern of colony formation stimulated by each regulator is quite distinctive. No two regulators stimulate exactly the same pattern of colony formation, as evaluated by colony numbers or, more importantly, by the lineage and maturation pattern of the cells making up the developing colonies. Proliferative responses can most readily be analyzed in simplified in vitro culture systems. Three quite different parameters can be distinguished: alteration in colony size, alteration in colony numbers and cell lineage. Two or more factors may act on the progenitor cell, inducing the formation of larger number of progeny thereby increasing the colony size. Two or more factors may allow increased number of progenitor cells to proliferate either because distinct subsets of progenitors cells exist that respond exclusively to one factor or because some progenitors require stimulation by two or more factors before being able to respond. Activation of additional receptors on a cell by the use of two or more factors is likely to enhance the mitotic signal because of coalescence of initially differing signal pathways into a common final pathway reaching the nucleus (Metcalf, Nature 339:27, 1989). Other mechanisms could explain synergy. For example, if one signaling pathway is limited by an intermediate activation of an additional signaling pathway which is caused by a second factor, then this may result in a super additive response. In some cases, activation of one receptor type can induce an enhanced expression of other receptors (Metcalf, Blood 82:3515-3523, 1993). Two or more factors may result in a different pattern of cell lineages than from a single factor. The use of multi-functional hematopoietic receptor agonists may have a potential clinical advantage resulting from a proliferative response that is not possible by any single factor.
The receptors of hematopoietic and other growth factors can be grouped into two distinct families of related
proteins: (1) tyrosine kinase receptors, including those for epidermal growth factor, M-CSF (Sherr, Blood 75:1, 1990) and SCF (Yarden et al., EMBO J. 6:3341, 1987): and (2)
hematopoietic receptors, not containing a tyrosine kinase domain, but exhibiting obvious homology in their
extracellular domain (Bazan, PNAS USA 87:6934-6938, 1990). Included in this latter group are erythropoietin (EPO)
(D 'Andrea et al., Cell 57:277, 1989), GM-CSF (Gearing et al., EMBO J. 8:3667, 1989), IL-3 (Kitamura et al., Cell 66:1165, 1991), G-CSF (Fukunaga et al., J. Bio . Chem .
265:14008-15, 1990), IL-4 (Harada et al., PNAS USA 87:857, 1990), IL-5 (Takaki et al., EMBO J. 9:4367, 1990), IL-6
(Yamasaki et al., Science 241:825, 1988), IL-7 (Goodwin et al., Cell 60:941-51, 1990), LIF (Gearing et al., EMBO J. 10:2839, 1991) and IL-2 (Cosman et al., Mol-Immunol. 23: 935-94, 1986). Most of the latter group of receptors exists in a high-affinity form as heterodimers. After ligand binding, the specific α-chains become associated with at least one other receptor chain (β-chain, γ-chain). Many of these factors share a common receptor subunit. The α-chains for GM-CSF, IL-3 and IL-5 share the same β-chain (Kitamura et al., Cell 66:1165, 1991), Takaki et al., EMBO J. 10:2833-8, 1991) and receptor complexes for IL-6, LIF and IL-11 share a common β-chain (gp130) (Taga et al., Cell
58:573-81, 1989; Gearing et al., Science 255:1434-7, 1992). The receptor complexes of IL-2, IL-4, IL-7, IL-9 and IL-15 share a common γ-chain (Kondo et al., Science 262:1874,
1993; Russell et al., Sci ence 266: 1042-1045, 1993;
Noguchi et al., Science 262:1877, 1993; Giri et al., EMBO J. 13:2822-2830, 1994).
The use of a multiply acting hematopoietic factor may also have a potential advantage by reducing the demands placed on factor-producing cells and their induction systems. If there are limitations in the ability of a cell to produce a factor, then by lowering the required
concentrations of each of the factors, and using them in combination may usefully reduce demands on the factor- producing cells. The use of a multiply acting hematopoietic factor may lower the amount of the factors that would be needed, probably reducing the likelihood of adverse side- effects.
Novel compounds of this invention are represented by a formula selected from the group consisting of:
R1-L1-R2, R2-L1-R1, R1-R2, and R2-R1 Where R1 and R2 are as defined above.
R2 is preferably a colony stimulating factor with a different but complementary activity than R1. By
complementary activity is meant activity which enhances or changes the response to another cell modulator. The R1 polypeptide is joined either directly or through a linker segment to the R2 polypeptide. The term "directly" defines multi-functional hematopoietic receptor agonists in which the polypeptides are joined without a peptide linker. Thus L1 represents a chemical bond or polypeptide segment to which both R1 and R2 are joined in frame, most commonly L1 is a linear peptide to which R1 and R2 are joined by amide bonds linking the carboxy terminus of R1 to the amino terminus or L1 and carboxy terminus of L1 to the amino terminus of R2. By "joined in frame" is meant that there is no translation termination or disruption between the reading frames of the DNA encoding R1 and R2.
A non-exclusive list of other growth factors, i.e.
colony stimulating factors (CSFs), are cytokines,
lymphokines, interleukins, hematopoietic growth factors which can be joined to (I), (II) or (III) include GM-CSF, G-CSF, c-mpl ligand (also known as TPO or MGDF), M-CSF, erythropoietin (EPO), IL-1, 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. Additionally, this
invention encompasses the use of modified R1 or R2 molecules or mutated or modified DNA sequences encoding these R1 or R2 molecules. The present invention also includes multifunctional hematopoietic receptor agonists in which R1 or R2 is an hIL-3 variant, c-mpl ligand variant, or G-CSF variant. A "hIL-3 variant" is defined as a hIL-3 molecule which has amino acid substitutions and/or portions of hIL-3 deleted as disclosed in WO 94/12638, WO 94/12639 and WO 95/00646, as well as other variants known in the art. A "c-mpl ligand variant" is defined an c-mpl ligand molecule which has amino acid substitutions and/or portions of c-mpl ligand deleted, disclosed in United States Application Serial Number
08/383,035 as well as other variants known in the art. A "G- CSF variant" is defined an G-CSF molecule which has amino acid substitutions and/or portions of G-CSF deleted, as disclosed herein, as well as other variants known in the art. The linking group (L1) is generally a polypeptide of between 1 and 500 amino acids in length. The linkers joining the two molecules are preferably designed to (1) allow the two molecules to fold and act independently of each other, (2) not have a propensity for developing an ordered
secondary structure which could interfere with the
functional domains of the two proteins, (3) have minimal hydrophobic characteristics which could interact with the functional protein domains and (4) provide steric separation of R1 and R2 such that R1 and R2 could interact
simultaneously with their corresponding receptors on a single cell. Typically surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence. Other neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Additional amino acids may also be included in the linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the multi-functional
hematopoietic receptor agonists.
Preferred L1 linkers of the present invention include sequences selected from the group of formulas:
(Gly3Ser)n (SEQ ID NO:4), (Gly4Ser)n (SEQ ID NO.5),
(Gly5Ser)n (SEQ ID NO:6), (GlynSer)n (SEQ ID NO:7) or
(AlaGlySer)n (SEQ ID NO:8).
One example of a highly-flexible linker is the glycine and serine-rich spacer region present within the pill protein of the filamentous bacteriophages, e.g.
bacteriophages M13 or fd (Schaller et al., PNAS USA 72: 737- 741, 1975). This region provides a long, flexible spacer region between two domains of the pill surface protein. The spacer region consists of the amino acid sequence:
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGluGlyGlyGlySerGlu GlyGlyGlySerGluGlyGlyGlySerGluGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:9).
The present invention also includes linkers in which an endopeptidase recognition sequence is included. Such a cleavage site may be valuable to separate the individual components of the multi-functional hematopoietic receptor agonist to determine if they are properly folded and active in vitro. Examples of various endopeptidases include, but are not limited to, plasmin, enterokinase, kallikrein, urokinase, tissue plasminogen activator, clostripain, chymosin, collagenase, Russell's viper venom protease, postproline cleavage enzyme, V8 protease, Thrombin and factor Xa.
Peptide linker segments from the hinge region of heavy chain immunoglobulms IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptides.
Especially useful are those hinge regions where the
cysteines are replaced with serines. Preferred linkers of the present invention include sequences derived from murine IgG gamma 2b hinge region in which the cysteines have been changed to serines. These linkers may also include an endopeptidase cleavage site. Examples of such linkers include the following sequences: IleSerGluProSerGlyProIleSerThrlleAsnProSerProProSerLys GluSerHisLysSerPro (SEQ ID NO:10) and
IleGluGlyArglleSerGluProSerGlyProIleSerThrlleAsnProSer ProProSerLysGluSerHisLysSerPro (SEQ ID NO:11).
The present invention is, however, not limited by the form, size or number of linker sequences employed and the only requirement of the linker is that functionally it does not interfere with the folding and function of the
individual molecules of the multi-functional hematopoietic receptor agonist. Determination of the Linker L2. The length of the amino acid sequence of the linker L2 to be used in R1 and/or R2 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; solvent exposed surface area, Lee & Richards, J. Mol . Biol . 55:379-400, 1971) and the ability to adopt the necessary conformation with out deranging the
conformation of R1 or R2 (conformationally flexible; Karplus & Schulz, Naturwissenschaften 72:212-213, 1985). Assuming an average of translation of 2.0 to 3.8 A per residue, this would mean the length to test would be between 0 to 30 residues, with 0 to 15 residues being the preferred range. Exemplary of such an empirical series would be to construct linkers using a cassette sequence such as Gly-Gly-Gly-Ser (SEQ ID NO:12) repeated n times, where n is 1, 2, 3 or 4.
Those skilled in the art will recognize that there are many such sequences that vary in length or composition that can serve as linkers with the primary consideration being that they be neither excessively long nor short (cf., Sandhu, Critical Rev. Biotech . 12: 437-462, 1992); if they are too long, entropy effects will likely destabilize the three- dimensional fold, and may also make folding kinetically impractical, and if they are too short, they will likely destabilize the molecule because of torsional or steric strain. Those skilled in the analysis of protein structural information will recognize that using the distance between the chain ends, defined as the distance between the c-alpha carbons, can be used to define the length of the sequence to be used, or at least to limit the number of possibilities that must be tested in an empirical selection of linkers. They will also recognize that it is sometimes the case that the positions of the ends of the polypeptide chain are illdefined in structural models derived from x-ray diffraction or nuclear magnetic resonance spectroscopy data, and that when true, this situation will therefore need to be taken into account in order to properly estimate the length of the linker required. From those residues whose positions are well defined are selected two residues that are close in sequence to the chain ends, and the distance between their c-alpha carbons is used to calculate an approximate length for a linker between them. Using the calculated length as a guide, linkers with a range of number of residues
(calculated using 2 to 3.8A per residue) are then selected. These 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:12) cassette approach mentioned above; or optionally a
combination of the original sequence and new sequence having the appropriate total length may be used.
Determination of the Amino and Carboxyl Termini of R1 and R2
Sequences of R1 and R2 capable of folding to
biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending
(carboxyl terminus) positions from within the original polypeptide chain while using the linker sequence L2 as described above. Amino and carboxyl termini are selected from within a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below. A novel amino 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. However, those skilled in the art will recognize that selections of termini anywhere within 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.
It is a central tenet of molecular biology that the primary amino acid sequence of a protein dictates folding to the three-dimensional structure necessary for expression of its biological function. Methods are known to those skilled in the art to obtain and interpret three-dimensional structural information using x-ray diffraction of single protein crystals or nuclear magnetic resonance spectroscopy of protein solutions. 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, Methods Enzymol . 154: 511-533, 1987). In some cases additional information is known about solvent exposure of residues; one example is a site of post-translational attachment of carbohydrate which is necessarily on the surface of the protein. When
experimental structural information is not available, or is not feasible to obtain, 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 . 218:603-621, 1993)
Thus using either the experimentally derived structural information or predictive methods (e.g., Srinivisan & Rose Proteins : Struct . , Funct . & Genetics, 22: 81-99, 1995) the parental amino acid sequence is inspected to classify regions according to whether or not they are integral to the maintenance of secondary and tertiary structure. The occurrence of sequences within regions that are known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets) are regions that should be avoided. Similarly, regions of amino 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 protein and should also be avoided for selection of amino and carboxyl termini. In contrast, 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. Non-covalent Multifunctional hematopoietic growth factors
An alternative method for connecting two hematopoietic growth factors is by means of a non-covalent interaction. Such complexed proteins can be described by one of the formulae:
R1-C1 + R2-C2; or C1-R1 + C2-R2; C1-R1 + R2-C2; or C1-R1 + R2-C2.
R1 and R2 are as is defined above. Domains C1 and C2 are either identical or non-identical chemical structures, typically proteinaceous, which can form a non-covalent, specific association. Complexes between C1 and C2 result in a one-to-one stoichiometric relationship between R1 and R2 for each complex. Examples of domains which associate are "leucine zipper" domains of transcription factors,
dimerization domains of bacterial transcription repressors and immunoglobulin constant domains. Covalent bonds link R1 and C1, and R2 and C2, respectively. As indicated in the formulae, the domains C1 and C2 can be present either at the N-terminus or C-terminus of their corresponding
hematopoietic growth factor (R). These multimerization domains (C1 and C2) include those derived from the bZIP family of proteins (Abel et al., Nature 341:24-25, 1989; Landshulz et al., Science 240:1759-1764, 1988; Pu et al., Nuc. Acid Res . 21:4348-4355, 1993; Kozarides et al., Nature 336:646-651, 1988), as well as multimerization domains of the helix-loop-helix family of proteins (Abel et al., Nature 341:24-25, 1989; Murre et al., Cell 56:777-783, 1989;
Tapscott et al., Science 242:405-411, 1988; Fisher et al., Genes & Dev. 5:2342-2352, 1991). Preferred multi-functional hematopoietic receptor agonists of the present invention include colony stimulating factors dimerized by virtue of their incorporation as translational multi-functional hematopoietic receptor agonists with the leucine zipper dimerization domains of the bZIP family proteins Fos and Jun. The leucine zipper domain of Jun is capable of
interacting with identical domains. On the other hand, the leucine zipper domain of Fos interacts with the Jun leucine zipper domain, but does not interact with other Fos leucine zipper domains. Mixtures of Fos and Jun predominantly result in formation of Fos-Jun heterodimers. Consequently, when joined to colony stimulating factors, the Jun domain can be used to direct the formation of either homo- or heterodimers. Preferential formation of heterodimers can be achieved if one of the colony stimulating factor partners is engineered to possess the Jun leucine zipper domain while the other is engineered to possess the Fos zipper.
Additional peptide sequences may also be added to facilitate purification or identification of multi- functional hematopoietic receptor agonist proteins (e.g., poly-His). A highly antigenic peptide may also be added that would enabjv rapid assay and facile purification of the multi-functional hematopoietic receptor agonist protein by a specific monoclonal antibody.
"Mutant amino acid sequence," "mutant protein",
"variant protein", "mutein", or "mutant polypeptide" refers to a polypeptide having an amino acid sequence which varies from a native sequence due to amino acid deletions,
substitutions, or both, or is encoded by a nucleotide sequence intentionally made variant from a native sequence.. "Native sequence" refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein.
Hematopoietic growth factors can be characterized by their ability to stimulate colony formation by human hematopoietic progenitor cells. The colonies formed include erythroid, granulocyte, megakaryocyte, granulocytic
macrophages and mixtures thereof. Many of the hematopoietic growth factors have demonstrated the ability to restore bone marrow function and peripheral blood cell populations to therapeutically beneficial levels in studies performed initially in primates and subsequently in humans. Many or all of these biological activities of hematopoietic growth factors involve signal transduction and high affinity receptor binding. Multi-functional hematopoietic receptor agonists of the present invention may exhibit useful
properties such as having similar or greater biological activity when compared to a single factor or by having improved half-life or decreased adverse side effects, or a combination of these properties.
Multi-functional hematopoietic receptor agonists which have little or no agonist activity maybe useful as
antagonists, as antigens for the production of antibodies for use in immunology or immunotherapy, as genetic probes or as intermediates used to construct other useful hIL-3 muteins.
Biological activity of the multi-functional
hematopoietic receptor agonist proteins of the present invention can be determined by DNA synthesis in factor- dependent cell lines or by counting the colony forming units in an in vitro bone marrow assay. The multi-functional hematopoietic receptor agonists of the present invention may have an improved therapeutic profile as compared to single acting hematopoietic agonists. For example, some multi-functional hematopoietic receptor agonists of the present invention may have a similar or more potent growth factor activity relative to other hematopoietic agonists without having a similar or
corresponding increase in side-effects.
The present invention also includes the DNA sequences which code for the multi-functional hematopoietic receptor agonist proteins, DNA sequences which are
substantially similar and perform substantially the same function, and DNA sequences which differ from the DNAs encoding the multi-functional hematopoietic receptor
agonists of the invention only due to the degeneracy of the genetic code. Also included in the present invention are the oligonucleotide intermediates used to construct the mutant DNAs and the polypeptides coded for by these
oligonucleotides. Genetic engineering techniques now standard in the art (United States Patent 4,935,233 and Sambrook et al.,
"Molecular Cloning A Laboratory Manual", Cold Spring Harbor Laboratory, 1989) may be used in the construction of the DNA sequences of the present invention. One such method is cassette mutagenesis (Wells et al., Gene 34:315-323, 1985) in which a portion of the coding sequence in a plasmid is replaced with synthetic oligonucleotides that encode the desired amino acid substitutions in a portion of the gene between two restriction sites.
Pairs of complementary synthetic oligonucleotides encoding the desired gene can be made and annealed to each other. The DNA sequence of the oligonucleotide would encode sequence for amino acids of desired gene with the exception of those substituted and/or deleted from the sequence.
Plasmid DNA can be treated with the chosen restriction endonucleases then ligated to the annealed oligonucleotides. The ligated mixtures can be used to transform competent JM101 cells to resistance to an appropriate antibiotic.
Single colonies can be picked and the plasmid DNA examined by restriction analysis and/or DNA sequencing to identify plasmids with the desired genes.
Cloning of the DNA sequences of the novel
multifunctional hematopoietic agonists wherein at least one of the with the DNA sequence of the other colony
stimulating factor may be accomplished by the use of intermediate vectors. Alternatively one gene can be cloned directly into a vector containing the other gene. Linkers and adapters can be used for joining the DNA sequences, as well as replacing lost sequences, where a restriction site was internal to the region of interest. Thus genetic material (DNA) encoding one polypeptide, peptide linker, and the other polypeptide is inserted into a suitable expression vector which is used to transform bacteria, yeast, insect cells or mammalian cells. The transformed organism is grown and the protein isolated by standard techniques. The resulting product is therefore a new protein which has a colony stimulating factor joined by a linker region to a second colony stimulating factor.
Another aspect of the present invention provides plasmid DNA vectors for use in the expression of these novel multi-functional hematopoietic 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 multi-functional hematopoietic receptor agonists include expression vectors comprising nucleotide sequences coding for the multi-functional hematopoietic 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 included in the present invention and are useful in the production of the multi-functional hematopoietic receptor agonist polypeptides. The vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and which are capable of directing the replication and expression thereof in selected host cells.
As another aspect of the present invention, there is provided a method for producing the novel multi-functional hematopoietic receptor agonists. The method of the present invention involves culturing suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of a novel multi-functional hematopoietic receptor agonist. Suitable cells or cell lines may be bacterial cells. For example, the various strains of E. col i are well-known as host cells in the field of biotechnology. Examples of such strains include E. coli strains JM..01 (Yanish-Perron et al . Gene 33: 103-119, 1985) and MON105 (Obukowicz et al., Applied Environmental
Microbiology 58: 1511-1523, 1992). Also included in the present invention is the expression of the multi-functional hematopoietic receptor agonist protein utilizing a
chromosomal expression vector for E. coli based on the bacteriophage Mu (Weinberg et al., Gene 126: 25-33, 1993). Various strains of B. subtilis may also be employed in this method. Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. When
expressed in the E. coli cytoplasm, the gene encoding the multi-functional hematopoietic receptor agonists of the present invention may also be constructed such that at the 5' end of the gene codons are added to encode Met -Ala-1- or Met at the N-terminus of the protein. The N termini of proteins made in the cytoplasm of E. coli are affected by post-translational processing by methionine aminopeptidase (Ben Bassat et al., J. Bac. 169:751-757, 1987) and possibly by other peptidases so that upon expression the methionine is cleaved off the N-terminus. The multi-functional hematopoietic receptor agonists of the present invention may include multi-functional hematopoietic receptor agonist polypeptides having Met-1, Ala-1 or Met-2-Ala-1 at the N-terminus. These mutant multi-functional hematopoietic receptor agonists may also be expressed in E. coli by fusing a secretion signal peptide to the N-terminus. This signal peptide is cleaved from the polypeptide as part of the secretion process.
Also suitable for use in the present invention are mammalian cells, such as Chinese hamster ovary cells (CHO). General methods for expression of foreign genes in mammalian cells are reviewed in Kaufman, R. J., 1987) Genetic
Engineering, Principles and Methods, Vol. 9, J. K. Setlow, editor, Plenum Press, New York. An expression vector is constructed in which a strong promoter capable of
functioning in mammalian cells drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally joined to the coding region for the multi¬functional hematopoietic receptor agonist. For example, plasmids such as pcDNA I/Neo, pRc/RSV, and pRc/CMV (obtained from Invitrogen Corp., San Diego, California) can be used.
The eukaryotic secretion signal peptide coding region can be from the gene itself or it can be from another secreted mammalian protein (Bayne, M. L. et al., Proc. Natl . Acad. Sci . USA 84: 2638-2642, 1987). After construction of the vector containing the gene, the vector DNA is transfected into mammalian cells. Such cells can be, for example, the COS7, HeLa, BHK, CHO, or mouse L lines. The cells can be cultured, for example, in DMEM media (JRH Scientific). The polypeptide secreted into the media can be recovered by standard biochemical approaches following transient
expression for 24 - 72 hours after transfection of the cells or after establishment of stable cell lines following selection for antibiotic resistance. The selection of suitable mammalian host cells and methods for
transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625,
1981), or alternatively, Kaufman et al, Mol . Cell . Biol . , 5(7):1750-1759, 1985) or Howley et al., U.S. Pat. No.
4,419,446. Another suitable mammalian cell line is the monkey COS-1 cell line. A similarly useful mammalian cell line is the CV-1 cell line.
Where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g., Miller et al., Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein. In addition, general methods for expression of foreign genes in insect cells using Baculovirus vectors are described in: Summers, M. D. and Smith, G. E., 1987) - A manual of methods for
Baculovirus vectors and insect cell culture procedures,
Texas Agricultural Experiment Station Bulletin No. 1555. An expression vector is constructed comprising a Baculovirus transfer vector, in which a strong Baculovirus promoter (such as the polyhedron promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally joined to the coding region for the multi- functional hematopoietic receptor agonist polypeptide. For example, the plasmid pVL1392 (obtained from Invitrogen
Corp., San Diego, California) can be used. After
construction of the vector carrying the gene encoding the multi-functional hematopoietic receptor agonist polypeptide, two micrograms of this DNA is co-transfected with one microgram of Baculovirus DNA (see Summers & Smith, 1987) into insect cells, strain SF9. Pure recombinant Baculovirus carrying the multi-functional hematopoietic receptor agonist is used to infect cells cultured, for example, in Excell 401 serum-free medium (JRH Biosciences, Lenexa, Kansas). The multi-functional hematopoietic receptor agonist secreted into the medium can be recovered by standard biochemical approaches. Supernatants from mammalian or insect cells expressing the multi-functional hematopoietic receptor agonist protein can be first concentrated using any of a number of commercial concentration units. The multi-functional hematopoietic receptor agonists of the present invention may be useful in the treatment of diseases characterized by decreased levels of either
myeloid, erythroid, lymphoid, or megakaryocyte cells of the hematopoietic system or combinations thereof. In addition, they may be used to activate mature myeloid and/or lymphoid cells. Among conditions susceptible to treatment with the polypeptides of the present invention is leukopenia, a reduction in the number of circulating leukocytes (white cells) in the peripheral blood. Leukopenia may be induced by exposure to certain viruses or to radiation. It is often a side effect of various forms of cancer therapy, e.g., exposure to chemotherapeutic drugs, radiation and of infection or hemorrhage. Therapeutic treatment of
leukopenia with these multi-functional hematopoietic receptor agonists of the present invention may avoid
undesirable side effects caused by treatment with presently available drugs.
The multi-functional hematopoietic receptor agonists of the present invention may be useful in the treatment of neutropenia and, for example, in the treatment of such conditions as aplastic anemia, cyclic neutropenia,
idiopathic neutropenia, Chediak-Higashi syndrome, systemic lupus erythematosus (SLE), leukemia, myelodysplastic syndrome and myelofibrosis.
The multi-functional hematopoietic receptor agonist of the present invention may be useful in the treatment or prevention of thrombocytopenia. Currently the only therapy for thrombocytopenia is platelet transfusion which are costly and carry the significant risks of infection (HIV, HBV) and alloimunization. The multi-functional hematopoietic receptor agonist may alleviate or diminish the need for platelet transfusion. Severe thrombocytopenia may result from genetic defects such as Fanconi's Anemia, Wiscott-Aldrich, or May Hegglin syndromes. Acquired thrombocytopenia may result from auto- or allo-antibodies as in Immune
Thrombocytopenia Purpura, Systemic Lupus Erythromatosis, hemolytic anemia, or fetal maternal incompatibility. In addition, 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 multi-functional hematopoietic receptor agonists of the present invention may be useful in the mobilization of hematopoietic progenitors and stem cells in 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 pheresis required. The multi-functional hematopoietic receptor agonist may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.
The multi-functional hematopoietic receptor agonists of the present invention may also be useful in the ex vivo expansion of hematopoietic progenitors and stem cells.
Colony stimulating factors (CSFs), such as hIL-3, have been administered alone, co-administered with other CSFs, or in combination with bone marrow transplants subsequent to high dose chemotherapy to treat the neutropenia and
thrombocytopenia which are often the result of such
treatment. However the period of severe neutropenia and thrombocytopenia may not be totally eliminated. The 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. However, 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, spleen, or peripheral blood 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 exists such that a greater number of stem cells will enhance hematopoietic recovery. Therefore, the in vitro expansion of stem cells should enhance hematopoietic recovery and patient survival. Bone marrow from an
allogeneic donor has been used to provide bone marrow for transplant. However, 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 into the patient afterwards. Autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection. However, autologous bone marrow
transplants still present problems in terms of the limited number of stems cells in the marrow and possible
contamination with tumor cells. The limited number of stem cells may be overcome by ex-vivo expansion of the stem cells. In addition, stem cells can be specifically isolated, based on the presence of specific surface antigens such as CD34+ in order to decrease tumor cell contamination of the marrow graft.
The following patents contain further details on separating stem cells, CD34+ cells, culturing the cells with hematopoietic factors, the use of the cells for the
treatment of patients with hematopoietic disorders and the use of hematopoietic factors for cell expansion and gene therapy.
5,061,620 relates to compositions comprising human
hematopoietic stem cells provided by separating 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, flt3 ligand, c-kit ligand, GM-CSF, IL-1, GM-CSF/IL-3 fusion protein and combinations thereof; (3) administering cellular preparation to a patient.
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 stem 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.
The long term culturing of highly enriched CD34+ precursors of hematopoietic progenitor cells from human bone marrow in cultures containing IL-1a, IL-3, IL-6 or GM-CSF is discussed in Brandt et al J. Clin . Invest . 86:932-941, 1990).
One aspect of the present invention provides a method for selective ex-vivo expansion of stem cells. The term "stem cell" refers to the totipotent hematopoietic stem cells as well as early precursors and progenitor cells which can be isolated from bone marrow, spleen or peripheral blood. The term "expansion" refers to the differentiation and
proliferation of the cells. The present invention 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 selective media which contains multi-functional
hematopoietic receptor agonist protein(s) 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. 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 affinity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+. Further, physical separation methods such as counterflow elutriation may be used to enrich hematopoietic progenitors. The 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-1). Other surface antigens such as 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-1, 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. hemacytometer, CFU, LTCIC) or by flow cytometry prior and subsequent to
incubation.
Several methods for ex-vivo expansion of stem cells have been reported utilizing a number of selection methods and expansion using various colony stimulating factors including c-kit ligand (Brandt et al., Blood 83:1507-1514 [1994], McKenna et al., Bl ood 86:3413-3420 [1995]), IL-3 (Brandt et al., Blood 83:1507-1514 [1994], Sato et al., Blood 82:3600-3609 [1993]), G-CSF (Sato et al., Blood
82:3600-3609 [1993]), GM-CSF (Sato et al., Blood 82:3600- 3609 [1993]), IL-1 (Muench et al., Blood 81:3463-3473
[1993]), IL-6 (Sato et al., Blood 82:3600-3609 [1993]), IL- 11 (Lemoli et al., Exp . Hem. 21:1668-1672 [1993], Sato et al.. Blood 82:3600-3609 [1993]), flt-3 ligand (McKenna et al., Blood 86:3413 3420 [1995]) and/or combinations thereof (Brandt et al., Blood 83:1507 1514 [1994], Haylock et al., Blood 80:1405-1412 [1992], Koller et al., Biotechnology 11:358-363 [1993], (Lemoli et al., Exp . Hem . 21:1668-1672 [1993]), McKenna et al., Blood 86:3413-3420 [1995], Muench et al., Blood 81:3463-3473 [1993], Patchen et al.,
Biotherapy 7:13-26 [1994], Sato et al., Blood 82:3600-3609 [1993], Smith et al., Exp . Hem. 21:870-877 [1993], Steen et al., Stem Cells 12:214-224 [1994], Tsujino et al., Exp . Hem . 21:1379-1386 [1993]). Among the individual colony
stimulating factors, 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]). However, 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 multi-functional hematopoietic receptor agonists that are more effective than a single factor alone.
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 multi-functional hematopoietic receptor agonist of the present invention.
Another projected clinical use of growth factors has been in the in vitro activation of hematopoietic progenitors and stem cells for gene therapy. Due to the long life-span of hematopoietic progenitor cells and the distribution of their daughter cells throughout the entire body,
hematopoietic progenitor cells are good candidates for ex vivo gene transfection. In order to have the gene of
interest incorporated into the genome of the hematopoietic progenitor or stem cell one needs to stimulate cell division and DNA replication. 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. Potential applications of gene therapy (review Crystal, Science 270:404-410
[1995]) include; 1) the treatment of many congenital
metabolic disorders and immunodeficiencies (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]).
There are a variety of methods, known to those with skill in the art, for introducing genetic material into a host cell. A number of vectors, both viral and non-viral have been developed for transferring therapeutic genes into primary cells. 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 . Mi crobi ol . Immunol . 158:1-24 [1992]) and replication-deficient recombinant adenovirus (Berkner, BioTechniques 6:616-629 [1988], Berkner, Current Top .
Microbiol . Immunol . 158:39-66 [1992], Brody and Crystal, Annal . New York Acad. Sci . 716:90-103 [1994]). 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 . New 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 multi-functional hematopoietic receptor agonist proteins that have improved biological activity, including an activity not seen by any single colony
stimulation factor.
Many 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 multi-functional hematopoietic receptor agonists of the present invention 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. The multi-functional hematopoietic receptor agonists of the present invention may be useful in treating such hematopoietic deficiencies.
The treatment of hematopoietic deficiency may include administration of a pharmaceutical composition containing the multi-functional hematopoietic receptor agonists to a patient. The multi-functional hematopoietic receptor agonists of the present invention may also be useful for the activation and amplification of hematopoietic precursor cells by treating these cells in vitro with the multi- functional hematopoietic receptor agonist proteins of the present invention prior to injecting the cells into a patient.
Various immunodeficiencies, e.g., in T and/or B lymphocytes, or immune disorders, e.g., rheumatoid
arthritis, may also be beneficially affected by treatment with the multi-functional hematopoietic receptor agonists of the present invention. Immunodeficiencies may be the result of viral infections, e.g., HTLVI, HTLVII, HTLVIII, severe exposure to radiation, cancer therapy or the result of other medical treatment. The multi-functional hematopoietic receptor agonists of the present invention may also be employed, alone or in combination with other colony
stimulating factors, in the treatment of other blood cell deficiencies, including thrombocytopenia (platelet
deficiency), or anemia. Other uses for these novel
polypeptides are the in vivo and ex vivo treatment of patients recovering from bone marrow transplants, and in the development of monoclonal and polyclonal antibodies
generated by standard methods for diagnostic or therapeutic use.
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 multi-functional hematopoietic receptor agonists of the present invention in a mixture with a pharmaceutically acceptable carrier. This composition can be administered either parenterally, intravenously or subcutaneously . When administered, the therapeutic composition for use in this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.
The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g., the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, a daily regimen may be in the range of 0.2 - 150 μg/kg of multi-functional hematopoietic receptor agonist protein per kilogram of body weight. Dosages would be adjusted relative to the activity of a given multi-functional hematopoietic receptor agonist protein 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. In addition, there may exist specific circumstances where dosages of multi-functional
hematopoietic 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 colony stimulating factors or IL-3 variants or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated multi-functional hematopoietic receptor agonist protein; 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 colony stimulating factors (CSFs), cytokines, lymphokines, hematopoietic growth factors and interleukins for simultaneous or serial co- administration with the polypeptides of the present
invention includes GM-CSF, G-CSF, c-mpl ligand (also known as TPO or MGDF) , M-CSF, erythropoietin (EPO), IL-1, 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, IL-16, LIF, flt3/flk2 ligand, B-cell growth factor, B-cell differentiation factor and eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, or combinations thereof. 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.
MATERIALS AND METHODS Unless noted otherwise, all specialty chemicals were obtained from Sigma, Co. (St. Louis, MO). Restriction endonucleases and T4 DNA ligase were obtained from New
England Biolabs (Beverly, MA) or Boehringer Mannheim
(Indianapolis, IN).
Transformation of E. coli strains
E. coli strains, such as DH5α™ (Life Technologies, Gaithersburg, MD) and TGI (Amersham Corp., Arlington
Heights, IL) are used for transformation of ligation reactions and are the source of plasmid DNA for transfecting mammalian cells. E. coli strains, such as JM101 (Yanisch-Perron, et al., Gene, 33: 103-119, 1985) and MON105
(Obukowicz, et al., Appl . and Envir . Micr . , 58: 1511-1523, 1992) can be used for expressing the multi-functional hematopoietic receptor agonist of the present invention in the cytoplasm or periplasmic space.
MON105 ATCC#55204: F-, lambda-,IN(rrnD, rrE)1, rpoD+, rpoH358
DH5α™: F-, phi80dlacZdeltaM15, delta (lacZYA-argF) U169, deoR, recA1, endA1, hsdR17 (rk-,mk+), phoA, supE441amda-, thi-1, gyrA96, relA1 TGI: delta (lac-pro), supE, thi-1, hsdD5/F' (traD36, proA+B+, laclq, lacZdeltaM15)
JM101 ATCC#33876: delta (pro lac), supE, thi , F'(traD36, proA+B+, laclq, lacZdeltaM15)
DH5α™ 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. Typically, 20 to 50 mL of cells are grown in LB medium (1% bacto-tryptone, 0.5% bacto-yeast extract, 150 mM NaCl) to a density of approximately 1.0 optical density unit 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 mM CaCl2, 10 mM Tris-Cl, pH7.4) and are held at 4ºC 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 4ºC for 30-60 minutes. The samples are shifted to 42ºC for two minutes and 1.0 mL of LB is added prior to shaking the samples at 37ºC 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 37ºC.
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. Methods for creation of genes with new N-terminus/C-terminus Method I. Creation of genes with new N-terminus/C-terminus which contain a linker region (L2).
Genes with new N-terminus/C-terminus which contain a linker region (L2) separating the original C-terminus and N-terminus can be made essentially following the method described in L. S. Mullins, et al J. Am . Chem . Soc . 116, 5529-5533, 1994). Multiple steps of polymerase chain reaction (PCR) amplifications are used to rearrange the DNA sequence encoding the primary amino acid sequence of the protein. The steps are illustrated in Figure 2.
In the first step, the first primer set ("new start" and "linker 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 followed by the linker (L2) that connects the C-terminal and N-terminal ends of the original protein. In the second step, the second primer set ("new stop" and "linker stop") is used to create and amplify, from the original 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 denaturaticr 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 1x 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 performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT). "Fragment Start" and "Fragment Stop", which have complementary sequence in the linker region and the coding sequence for the two amino acids on both sides of the linker, are joined together in a third PCR step to make the full-length gene encoding the new protein. The DNA
fragments "Fragment Start" and "Fragment 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 in equimolar quantities, heated at 70°C for ten minutes and slow cooled to allow annealing through their shared sequence in "linker start" and "linker stop". In the third PCR step, 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 cycles 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 for 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 1x 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).
Method II. Creation of genes with new N-terminus/C-terminus without a linker region. 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. In the first step, the 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. In the second step, the 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 1x Vent buffer (New England Biolabs), 300 uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit Deep Vent polymerase. 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. Typically "Fragment Start" is designed to create NcoI restriction site , and "Fragment Stop" is designed to create a HindIII 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 NcoI/HindIII 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 DH5α cells (Life Technologies, Gaithersburg, MD). Plasmid
DNA is purified and sequence confirmed as below. Method III. Creation of new N-terminus/C-terminus genes by tandem-duplication method
New N-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 3. The tandemly-duplicated template DNA is created by cloning and contains two 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°C 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 1x 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 performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT). PCR reactions are purified using a Wizard PCR Preps kit (Promega).
Cloning of new N-terminus/C-terminus genes into multi-functional receptor agonist expression vectors. The new N-terminus/C-terminus gene is digested with restriction endonucleases to create ends that are compatible to insertion into an expression vector containing another colony stimulating factor gene. This expression vector is likewise digested with restriction endonucleases to form compatible ends. After purification, the gene and the vector DNAs are combined and ligated using T4 DNA ligase. A portion of the ligation reaction is used to transform E.
coli. Plasmid DNA is purified and sequenced to confirm the correct insert. The correct clones are grown for protein expression.
DNA isolation and characterization Plasmid DNA can be isolated by a number of different methods and using commercially available kits known to those skilled in the art. A few such methods are shown herein. Plasmid DNA is isolated using the Promega Wizard™ Miniprep kit (Madison, WI), the Qiagen QIAwell Plasmid isolation kits (Chatsworth, CA) or Qiagen Plasmid Midi kit. These 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 50-100 mls of LB plus appropriate antibiotic for overnight growth at 37°C in an air incubator while shaking. The purified plasmid DNA is used for DNA sequencing, further restriction enzyme digestion, additional subcloning of DNA fragments and transfection into mammalian, E. coli or other cells.
Sequence confirmation.
Purified plasmid DNA is resuspended in dH2O and quantitated by measuring the absorbance at 260/280 nm in a Bausch and Lomb Spectronic 601 UV spectrometer. DNA samples are sequenced using ABI PRISM™ DyeDeoxy™ terminator sequencing chemistry (Applied Biosystems Division of Perkin Elmer Corporation, Lincoln City, CA) kits (Part Number
401388 or 402078) according to the manufacturers suggested protocol usually modified by the addition of 5% DMSO to the sequencing mixture. Sequencing reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) following the recommended amplification conditions. Samples are purified to remove excess dye terminators with Centri-Sep™ spin columns (Princeton
Separations, Adelphia, NJ) and lyophilized. Fluorescent dye labeled sequencing reactions are resuspended in deionized formamide, and sequenced on denaturing 4.75% polyacrylamide- 8M urea gels using an ABI Model 373A automated DNA
sequencer. Overlapping DNA sequence fragments are analyzed and assembled into master DNA contigs using Sequencher v2.1 DNA analysis software (Gene Codes Corporation, Ann Arbor, MI). Expression of multi-functional receptor agonists in
mammalian cells Mammalian Cell Transfection/Production of Conditioned Media
The BriK-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 mM (mM) L-glutamine and 10% fetal bovine serum (FBS). This formulation is designated BHK growth media. Selective media is BHK growth media supplemented with 453 units/mL
hygromycin B (Calbiochem, San Diego, CA). The BHK-21 cell line was previously stably transfected with the HSV
transactivating protein VP16, which transactivates the IE110 promoter found on the plasmid pMON3359 (See Hippenmeyer et al., Bio /Technology, pp.1037-1041, 1993). The VP16 protein drives expression of genes inserted behind the IE110 promoter. BHK-21 cells expressing the transactivating protein VP16 are designated BHK-VP16. The plasmid pMON1118 (See 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 10^ cells per dish 24 hours prior to transfection. Cells are transfected for 16 hours in 3 mL of "OPTIMEM"™ (Gibco-BRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug hygromycin resistance plasmid, pMON1llδ, and 80 ug of Gibco-BRL "LIPOFECTAMINE"™ 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 confluence, the conditioned media is re-assayed, and positive clones are expanded into growth media.
Expression of multi-functional receptor agonists in E. col i
E. coli strain MON105 or JM101 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 μg/mL. The cultures are then shaken at 37°C for three 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). One mL aliquots of the culture are removed for analysis of protein content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE (see Maniatis et al . Molecular Cloning: A Laboratory Manual, 1982). The culture is centrifuged (5000 x g) to pellet the cells. inclusion Body preparation. Extraction, Refolding, Dialysis, DEAE Chromatography, and Characterization of the multi- functional hematopoietic receptor agonists which accumulate as inclusion bodies in E. coli . Isolation of Inclusion Bodies: The cell pellet from a 330 mL E. coli culture is resuspended in 15 mL of sonication buffer (10 mM 2-amino-2-(hydroxymethyl) 1,3-propanediol hydrochloride (Tris-HCl), pH 8.0 + 1 mM ethylenediaminetetraacetic acid (EDTA). These resuspended cells are sonicated using the microtip probe of a Sonicator Cell Disruptor (Model W-375, Heat Systems-Ultrasonics, Inc., Farmingdale, New York). Three rounds of sonication in sonication buffer followed by centrifugation are employed to disrupt the cells and wash the inclusion bodies (IB). The first round of sonication is a 3 minute burst followed by a 1 minute burst, and the final two rounds of sonication are for 1 minute each. Extraction and refolding of proteins from inclusion body pellets:
Following the final centrifugation step, the IB pellet is resuspended in 10 mL of 50 mM Tris-HCl, pH 9.5, 8 M urea and 5 mM dithiothreitol (DTT) and stirred at room
temperature for approximately 45 minutes to allow for denaturation of the expressed protein.
The extraction solution is transferred to a beaker containing 70 mL of 5 mM Tris-HCl, pH 9.5 and 2.3 M urea and gently stirred while exposed to air at 4°C for 18 to 48 hours to allow the proteins to refold. Refolding is monitored by analysis on a Vydac (Hesperia, Ca.) C18 reversed phase high pressure liquid chromatography (RP-HPLC) column (0.46x25 cm). A linear gradient of 40% to 65% acetonitrile, containing 0.1% trifluoroacetic acid (TFA), is employed to monitor the refold. This gradient is developed over 30 minutes at a flow rate of 1.5 mL per minute.
Denatured proteins generally elute later in the gradient than the refolded proteins. Purification:
Following the refold, contaminating E. coli proteins are removed by acid precipitation. The pH of the refold solution is titrated to between pH 5.0 and pH 5.2 using 15% (v/v) acetic acid (HOAc). This solution is stirred at 4°C for 2 hours and then centrifuged for 20 minutes at 12,000 x g to pellet any insoluble protein.
The supernatant from the acid precipitation step is dialyzed using a Spectra/Por 3 membrane with a molecular weight cut off (MWCO) of 3,500 daltons. The dialysis is against 2 changes of 4 liters (a 50-fold excess) of 10 mM Tris-HCl, pH 8.0 for a total of 18 hours. Dialysis lowers the sample conductivity and removes urea prior to DEAE chromatography. The sample is then centrifuged (20 minutes at 12,000 x g) to pellet any insoluble protein following dialysis.
A Bio-Rad Bio-Scale DEAE2 column (7 × 52 mm) is used for ion exchange chromatography. The column is equilibrated in a buffer containing 10 mM Tris-HCl, pH 8.0, and a 0-to- 500 mM sodium chloride (NaCl) gradient, in equilibration buffer, over 45 column volumes is used to elute the protein. A flow rate of 1.0 mL per minute is used throughout the run. Column fractions (2.0 mL per fraction) are collected across the gradient and analyzed by RP HPLC on a Vydac (Hesperia,
Ca.) C18 column (0.46 × 25 cm). A linear gradient of 40% to 65% acetonitrile, containing 0.1% trifluoroacetic acid
(TFA), is employed. This gradient is developed over 30 minutes at a flow rate of 1.5 mL per minute. Pooled fractions are then dialyzed against 2 changes of 4 liters (50-to-500-fold excess) of 10 mM ammonium acetate (NH4Ac), pH 4.0 for a total of 18 hours. Dialysis is performed using a Spectra/Por 3 membrane with a MWCO of 3,500 daltons.
Finally, the sample is sterile filtered using a 0.22μm syringe filter (μstar LB syringe filter, Costar, Cambridge, Ma.), and stored at 4°C.
In some cases the folded proteins can be affinity purified using affinity reagents such as mAbs or receptor subunits attached to a suitable matrix. Alternatively, (or in addition) purification can be accomplished using any of a variety of chromatographic methods such as: ion exchange, gel filtration or hydrophobic chromatography or reversed phase HPLC. These and other protein purification methods are described in detail in Methods in Enzymology, Volume 182 'Guide to Protein Purification' edited by Murray Deutscher, Academic Press, San Diego, CA (1990). Protein Characterization:
The purified protein is analyzed by RP-HPLC,
electrospray mass spectrometry, and SDS-PAGE. The protein quantitation is done by amino acid composition, RP-HPLC, and Bradford protein determination. In some cases tryptic peptide mapping is performed in conjunction with
electrospray mass spectrometry to confirm the identity of the protein. AML Proliferation Assay for Bioactive Human Interleukin-3 The factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). This cell line, established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM-CSF supplemented medium (Lange, B., et al., Blood 70: 192, 1987; Valtieri, M., et al., J. Immunol . 138:4042, 1987). The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D., et al., J. Immunol . 139: 348, 1987). A cell line variant was used, AML 193 1.3, which was adapted for long term growth in IL-3 by washing out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours.
The cells are then replated at 1×105 cells/well in a 24 well plate in media containing 100 U/mL IL-3. It took
approximately 2 months for the cells to grow rapidly in IL-3. These cells are maintained as AML 193 1.3 thereafter by supplementing tissue culture medium (see below) with human IL-3.
AML 193 1.3 cells are washed 6 times in cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, NY) by centrifuging cell suspensions at 250 x g for 10 minutes followed by decantation of the supernatant. Pelleted cells are resuspended in HBSS and the procedure is repeated until six wash cycles are completed. Cells washed six times by this procedure are resuspended in tissue culture medium at a density ranging from 2 × 105 to 5 × 105 viable cells/mL. This medium is prepared by supplementing Iscove's modified Dulbecco's Medium (IMDM, Hazelton, Lenexa, KS) with albumin, transferrir, lipids and 2-mercaptoethanol. Bovine albumin (Boehringer-Mannheim, Indianapolis, IN) is added at 500 μg/mL; human transferrin (Boehringer-Mannheim, Indianapolis, IN) is added at 100 μg/mL; soybean lipid (Boehringer-Mannheim, Indianapolis, IN) is added at 50 μg/mL; and 2- mercaptoethanol (Sigma, St. Louis, MO) is added at 5 × 10-5 M.
Serial dilutions of human interleukin-3 or multi- functional hematopoietic receptor agonist proteins are made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 μl of medium containing interleukin-3 or multi-functional hematopoietic receptor agonist proteins once serial dilutions are completed.
Control wells contained tissue culture medium alone
(negative ccntrol). AML 193 1.3 cell suspensions prepared as above are added to each well by pipetting 50 μl (2.5 × 104 cells) into each well. Tissue culture plates are incubated at 37°C with 5% CO2 in humidified air for 3 days. On day 3, 0.5 μci 3H-thymidine (2 Ci/mM, New England
Nuclear, Boston, MA) is added in 50 μl of tissue culture medium. Cultures are incubated at 37°C with 5% CO2 in humidified air for 18-24 hours. Cellular DNA is harvested onto glass filter mats (Pharmacia LKB, Gaithersburg, MD) using a TOMTEC cell harvester (TOMTEC, Orange, CT) which utilized a water wash cycle followed by a 70% ethanol wash cycle. Filter mats are allowed to air dry and then placed into sample bags to which scintillation fluid (Scintiverse II, Fisher Scientific, St. Louis, MO or BetaPlate
Scintillation Fluid, Pharmacia LKB, Gaithersburg, MD) is added. Beta emissions of samples from individual tissue culture wells are counted in a LKB BetaPlate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, MD) and data is expressed as counts per minute of 3H-thymidine incorporated into cells from each tissue culture well.
Activity of each human interleukin-3 preparation or multi-functional hematopoietic receptor agonist protein
preparation is quantitated by measuring cell proliferation (3H-thymidine incorporation) induced by graded
concentrations of interleukin-3 or multi-functional
hematopoietic receptor agonist. Typically, concentration ranges from 0.05 pM - 105 pM are quantitated in these assays. Activity is determined by measuring the dose of interleukin-3 or multi-functional hematopoietic receptor agonist protein which provides 50% of maximal proliferation (EC50 = 0.5 × (maximum average counts per minute of 3H- thymidine incorporated per well among triplicate cultures of all concentrations of interleukin-3 tested - background proliferation measured by 3H-thymidine incorporation
observed in triplicate cultures lacking interleukin-3).
This EC50 value is also equivalent to 1 unit of bioactivity. Every assay is performed with native interleukin-3 as a reference standard so that relative activity levels could be assigned.
Typically, the multi-functional hematopoietic receptor agonist proteins were tested in a concentration range of 2000 pM to 0.06 pM titrated in serial 2 fold dilutions.
Activity for each sample was determined by the
concentration which gave 50% of the maximal response by fitting a four-parameter logistic model to the data. It was observed that the upper plateau (maximal response) for the sample and the standard with which it was compared did not differ. Therefore relative potency calculation for each sample was determined from EC50 estimations for the sample and the standard as indicated above. AML 193.1.3 cells proliferate in response to hIL-3, hGM-CSF and hG-CSF.
Therefore the following additional assays were performed for some samples to demonstrate that the G-CSF receptor agonist portion of the multi-functional hematopoietic receptor agonist proteins was active. The proliferation assay was performed with the multi-functional hematopoietic receptor agonist plus and minus neutralizing monoclonal antibodies to the hIL-3 receptor agonist portion. In addition, a fusion molecule with the factor Xa cleavage site was cleaved then purified and the halves of the molecule were assayed for proliferative activity. These experiments showed that both components of the multi-functional hematopoietic receptor agonist proteins were active.
TF1 c-mpl ligand dependent proliferation assay
The c-mpl ligand proliferative activity can be assayed using a subclone of the pluripotential human cell line TF1 (Kitamura et al., J. Cell Physiol 140:323-334. [1989]). TF1 cells are maintained in h-IL3 (100 U/mL). To establish a sub-clone responsive to c-mpl ligand, cells are maintained in passage media containing 10% supernatant from BHK cells transfected with the gene expressing the 1-153 form of c-mpl ligand (pMON26448). Most of the cells die, but a subset of cells survive. After dilution cloning, a c-mpl ligand responsive clone is selected, and these cells are split into passage media to a density of 0.3 × 106 cells/mL the day prior to at-say set-up. Passage media for these cells is the following: RPMI 1640 (Gibco), 10% FBS (Harlan, Lot #91206), 10% c-mpl ligand supernatant from transfected BHK cells, 1 mM sodium pyruvate (Gibco), 2 mM glutamine (Gibco), and 100 ug/mL penicillin-streptomycin (Gibco). The next day, cells are harvested and washed twice in RPMI or IMDM media with a final wash in the ATL, or assay media. ATL medium consists of the following:IMDM (Gibco), 500 ug/mL of bovine serum albumin, 100 ug/mL of human transferrin, 50 ug/mL soybean lipids, 4 × 10-8M beta-mercaptoethanol and 2 mL of A9909 (Sigma, antibiotic solution) per 1000 mL of ATL. Cells are diluted in assay media to a final density of 0.25 × 106 cells/mL in a 96-well low evaporation plate (Costar) to a final volume of 50 ul. Transient supernatants (conditioned media) from transfected clones are added at a volume of 50 ul as duplicate samples at a final concentration of 50% and diluted three-fold to a final dilution of 1.8%. Triplicate samples of a dose curve of IL-3 variant pMON13288 starting at 1 ng/mL and diluted using three-fold dilutions to
0.0014ng/mL is included as a positive control. Plates are incubated at 5% CO2 and 37° C. At day six of culture, the plate is pulsed with 0.5 Ci of 3H/well (NEN) in a volume of 20 ul/well and allowed to incubate at 5% CO2 and 37° C for four hours. The plate is harvested and counted on a
Betaplate counter.
Other in vitro cell based proliferation assays Other in vitro cell based assays, known to those skilled in the art, may also be useful to determine the activity of the multi-functional hematopoietic receptor agonists depending on the factors that comprise the molecule in a similar manner as described in the AML 193.1.3 cell proliferation assay. The following are examples of other useful assays.
TF1 proliferation assay: TF1 is a pluripotential human cell line (Kitamura et al., J. Cell Physiol 140:323-334. [1989]) that responds to hIL-3.
32D proliferation assay: 32D is a murine IL-3 dependent cell line which does not respond to human IL-3 but does respond to human G-CSF which is not species restricted.
Baf/3 proliferation assay: Baf/3 is a murine IL-3 dependent cell line which does not respond to human IL-3 or human c-mpl ligand but does respond to human G-CSF which is not species restricted.
T1165 proliferation assay: T1165 cells are a IL-6 dependent murine cell line (Nordan et al., 1986) which respond to IL-6 and IL-11. Human Plasma Clot meg-CSF Assay: Used to assay megakaryocyte colony formation activity (Mazur et al., 1981).
Transfected cell lines:
Cell lines such as the murine Baf/3 cell line can be transfected with a colony stimulating factor receptor, such as the human G-CSF receptor or human c-mpl receptor, which the cell line does not 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.
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.
In a similar manner the human G-CSF receptor can be transfected into the Baf/3 cell line and used to determine the bioactivity of the multi-functional hematopoietic receptor agoinsts.
Analysis of c-mpl ligand proliferative activity Methods
1. Bone marrow proliferation assay
a. CD34+ Cell Purification: Bone marrow aspirates (15-20 mL) were obtained from normal allogeneic marrow donors after informed consent.
Cells were diluted 1:3 in phosphate buffered saline (PBS, Gibco-BRL), 30 mL were layered over 15 mL Histopaque-1077 (Sigma) and centrifuged for 30 minutes at 300 RCF. The mononuclear interface layer was collected and washed in PBS. CD34+ cells were enriched from the mononuclear cell
preparation using an affinity column per manufacturers instructions (CellPro, Inc, Bothell WA). After enrichment, the purity of CD34+ cells was 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 were resuspended at 40,000 cells/mL in X-Vivo 10 media (Bio-Whittaker, Walkersville, MD) and 1 mL was plated in 12-well tissue culture plates (Costar). The growth factor rhIL-3 was added at 100 ng/mL (pMON5873) was added to some wells. hIL3 variants were used at 10 ng/mL to 100 ng/mL. Conditioned media from BHK cells transfected with plasmid encoding c-mpl ligand or multi-functional
hematopoietic receptor agonists were tested by addition of 100 μl of supernatant added to 1 mL cultures (approximately a 10% dilution). Cells were incubated at 37°C for 8-14 days at 5% CO2 in a 37°C humidified incubator. b. Cell Harvest and Analysis:
At the end of the culture period a total cell count was obtained for each condition. For fluorescence analysis and ploidy determination cells were washed in megakaryocyte buffer (MK buffer, 13.6 mM sodium citrate, 1 mM
theophylline, 2.2 μm PGEl , 11 mM glucose, 3% w/v BSA, in PBS, pH 7.4,) (Tomer et al., Blood 70: 1735-1742, 1987) resuspended in 500 μl of MK buffer containing anti-CD41a FITC antibody (1:200, AMAC, Westbrook, ME) and washed in MK buffer. For DNA analysis cells were permeablized in MK buffer containing 0.5% Tween 20 (Fisher, Fair Lawn NJ)for 20 min. on ice followed by fixation in 0.5% Tween-20 and 1% paraformaldehyde (Fisher Chemical) for 30 minutes followed by incubation in propidium iodide (Calbiochem , La Jolla Ca) (50 μg/mL) with RNA-ase (400 U/mL) in 55% v/v MK buffer (200mOsm) for 1-2 hours on ice. Cells were analyzed on a FACScan or Vantage flow cytometer (Becton Dickinson, San Jose, CA). Green fluorescence (CD41a-FITC) was collected along with linear and log signals for red fluorescence (PI) to determine DNA ploidy. All cells were collected to determine the percent of cells that were CD41+. Data analysis was performed using software by LYSIS (Becton Dickinson, San Jose, CA). Percent of cells expressing the CD41 antigen was obtained from flow cytometry
analysis (Percent). Absolute (Abs) number of CD41+ cells/mL was calculated by: (Abs) = (Cell Count)* (Percent)/100. 2. Megakaryocyte fibrin clot assay.
CD34+ enriched population were isolated as described above. Cells were suspended at 25,000 cells/mL with or without cytokine(s) in a media consisting of a base Iscoves IMDM media supplemented with 0.3% BSA, 0.4mg/mL apo-transferrin, 6.67μM FeCl2, 25μg/mL CaCl2, 25μg/mL L-asparagine, 500μg/mL ε-amino-n-caproic acid and penicillin/streptomycin. Prior to plating into 35mm plates, thrombin was added (0.25
Units/mL) to initiate clot formation. Cells were incubated at 37°C for 13 days at 5% CO2 in a 37°C humidified
incubator. At the end of the culture period plates were fixed with methanol:acetone (1:3), air dried and stored at -200C until staining. A peroxidase immunocytochemistry staining
procedure was used (Zymed, Histostain-SP. San Francisco, CA) using a cocktail of primary monoclonal antibodies consisting of anti-CD41a, CD42 and CD61. Colonies were counted after staining and classified as negative, CFU-MK (small colonies, 1-2 foci and less that approx. 25 cells), BFU-MK (large, multi-foci colonies with > 25 cells) or mixed colonies (mixture of both positive and negative cells.
Methylcellulose Assay
This assay reflects the ability of colony stimulating factors to stimulate normal bone marrow cells to produce different types of hematopoietic colonies in vi tro (Bradley et al., Aust . Exp Biol . Sci . 44:287-300, 1966), Pluznik et al., J. Cell Comp . Physio 66:319-324, 1965).
Methods
Approximately 30 mL of fresh, normal, healthy bone marrow aspirate are obtained from individuals following informed consent. Under sterile conditions samples are diluted 1:5 with a IX PBS (#14040.059 Life Technologies, Gaithersburg, MD. ) solution in a 50 mL conical tube (#25339-50 Corning, Corning MD). Ficoll (Histopaque 1077 Sigma H-8889) is layered under the diluted sample and centrifuged, 300 x g for 30 min. The mononuclear cell band is removed and washed two times in IX PBS and once with 1% BSA PBS (CellPro Co., Bothel, WA). Mononuclear cells are counted and CD34+ cells are selected using the Ceprate LC (CD34) Kit (CellPro Co., Bothel, WA) column. This fractionation is performed since all stem and progenitor cells within the bone marrow display CD34 surface antigen. Cultures are set up in triplicate with a final volume of 1.0 mL in a 35 X 10 mm petri dish (Nunc#174926). Culture medium is purchased from Terry Fox Labs. (HCC-4230 medium (Terry Fox Labs, Vancouver, B.C., Canada) and erythropoietin (Amgen, Thousand Oaks, CA.) is added to the culture media. 3,000-10,000 CD34+ cells are added per dish. Recombinant IL- 3, purified from mammalian cells or E. coli , and multi- functional hematopoietic receptor agonist proteins, in conditioned media from transfected mammalian cells or purified from conditioned media from transfected mammalian cells or E. coli , are added to give final concentrations ranging from .001 nM to 10 nM. Recombinant hIL-3, GM-CSF, c-mpl ligand and multi-functional hematopoietic receptor agonist are supplied in house. G-CSF (Neupogen) is from Amgen (Thousand Oaks Calf.). Cultures are resuspended using a 3cc syringe and 1.0 mL is dispensed per dish. Control
(baseline response) cultures received no colony stimulating factors. Positive control cultures received conditioned media (PHA stimulated human cells: Terry Fox Lab. H2400). Cultures are incubated at 37°C, 5% CO2 in humidified air. Hematopoietic colonies which are defined as greater than 50 cells are counted on the day of peak response (days 10-11) using a Nikon inverted phase microscope with a 40x objective combination. Groups of cells containing fewer than 50 cells are referred to as clusters. Alternatively colonies can be identified by spreading the colonies on a slide and stained or they can be picked, resuspended and spun onto cytospin slides for staining.
Human Cord Blood Hemopoietic Growth Factor Assays
Bone marrow cells are traditionally used for in vitro assays of hematopoietic colony stimulating factor (CSF) activity. However, human bone marrow is not always available, and there is considerable variability between donors. Umbilical cord blood is comparable to bone marrow as a source of hematopoietic stem cells and progenitors (Broxmeyer et al., PNAS USA 89:4109-113, 1992; Mayani et al., Blood 81:3252-3258, 1993). In contrast to bone marrow, cord blood is more readily available on a regular basis. There is also a potential to reduce assay variability by pooling cells obtained fresh from several donors, or to create a bank of cryopreserved cells for this purpose. By modifying the culture conditions, and/or analyzing for lineage specific markers, it is be possible to assay specifically for granulocyte / macrophage colonies (CFU-GM), for
megakaryocyte CSF activity, or for high proliferative potential colony forming cell (HPP-CFC) activity.
Methods
Mononuclear cells (MNC) are isolated from cord blood within 24 hr. of collection, using a standard density gradient (1.077 g/mL Histopaque). Cord blood MNC have been further enriched for stem cells and progenitors by several
procedures, including immunomagnetic selection for CD14-, CD34+ cells; panning for SBA-, CD34+ fraction using coated flasks from Applied Immune Science (Santa Clara, CA); and CD34+ selection using a CellPro (Bothell, WA) avidin column. Either freshly isolated or cryopreserved CD34+ cell enriched fractions are used for the assay. Duplicate cultures for each serial dilution of sample (concentration range from 1 pM to 1204 pM) are prepared with 1×104 cells in 1ml of 0.9% methycellulose containing medium without additional growth factors (Methocult H4230 from Stem Cell Technologies,
Vancouver, BC.). In some experiments, Methocult H4330 containing erythropoietin (EPO) was used instead of
Methocult H4230, or Stem Cell Factor (SCF), 50 ng/mL
(Biosource International, Camarillo, CA) was added. After culturing for 7-9 days, colonies containing >30 cells are counted. In order to rule out subjective bias in scoring, assays are scored blind.
Additional details about recombinant DNA methods which may be used to create the variants, express them in bacteria, mammalian cells or insect cells, purification and refold of the desired proteins and assays for determining the bioactvity of the proteins may be found in co-filed Applications WO 95/00646, WO 94/12639, WO 94/12638, WO 95/20976, WO 95/21197, WO 95/20977, WO 95/21254 and US 08/383,035 which are hereby incorporated by reference in their entirety.
Further details known to those skilled in the art may be found in T. Maniatis, et al., Molecular Cloning. A
Laboratory Manual, Cold Spring Harbor Laboratory, 1982) and references cited therein, incorporated herein by reference; and in J. Sambrook, et al., Molecular Cloning, A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, 1989) and references cited therein, incorporated herein by reference.
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The following examples will illustrate the invention in greater detail although it will be understood that the invention is not limited to these specific examples.
EXAMPLE 1
Construction of parental BHK expression vector
A. Removal of AflIII site from mammalian expression plasmid.
A new mammalian expression vector was constructed to accept NcoI-HindIII or AflIII-HindIII gene fragments in-frame and 3' to the hIL-3 receptor agonist pMON13146 (WO 94/12638) gene and a mouse IgG2b linker fragment. First, the single AflIII site was removed from pMON3934, which is a derivative of pMON3359. pMON3359 is a pUC18-based vector containing a mammalian expression cassette. The cassette includes a herpes simplex viral promoter IE110 (-800 to +120) followed by a modified human IL-3 signal peptide sequence and an 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).
The modified human IL-3 signal sequence, which facilitates secretion of gene products outside of the cell, is flanked by a BamHI site on the 5' end and a unique NcoI site on the 3' end. A unique HindIII site is 3' to the NcoI site and 5' to the poly-A sequence. The DNA sequence encoding the signal peptide is shown below (restriction enzyme sites are indicated above). The ATG (methionine) codon within the NcoI site is in-frame with the initiator ATG of the signal peptide (underlined);
Figure imgf000159_0001
The single AflIII site was removed from pMON3934 by
digestion with AflIII followed by filling in the overhangs by addition of a DNA polymerase and nucleotides. The
digested DNA fragment was purified via Magic PCR Clean up kit (Promega) and ligated with T4 DNA ligase. The ligation reaction was transformed into DH5α™ and the cells were plated onto LB-agar plus ampicillin. Individual colonies were screened for the loss of the AflIII site by restriction analysis with AflIII and HindIII which results in a single fragment if the AflIII site was removed. The resulting plasmid was designated pMON30275.
B. Transfer of hIL-3 receptor agonist pMON13416/IgG2b cassette into pMON30275.
The NcoI-HindIII fragment (ca. 425 bp) from pMON30245 was ligated to the NcoI-HindIII fragment (ca. 3800 bp) of the pMON30275. pMON30245 (WO 94/12638) contains the gene coding for hIL-3 receptor agonist pMON13416 joined to a mouse lgG2b hinge fragment. Immediately 3' to the lgG2b hinge and 5' to the HindIII site is an AflIII site. Genes can be cloned into the AflIII-HindIII sites as NcoI-HindIII or AflIII-HindIII fragments in frame with the hIL-3 variant pMON13416/IgG2b hinge to create novel chimeras. The NcoI site and the AflIII site have compatible overhangs and will ligate but both recognition sites are lost. The plasmid, pMON30304 containing the DNA sequence of (SEQ ID NO:78), coding for hIL-3 variant pMON13416 joined with a mouse IgG2b hinge region, was a result of this cloning.
EXAMPLE 2 Construction of an intermediate plasmid containing one copy of the c-mol ligand (1-153) gene of the dimer template in order to generate a plasmid DNA with the coding sequence of c-mpl (1-153) ligand followed by a unique EcoRI restriction site, the gene is isolated via reverse
transcriptase/polymerase chain reaction (RT/PCR). Human fetal (lot #38130) and adult liver (lot #46018) A+ RNA are obtained from Clontech (Palo Alto, CA) for source of c-mpl ligand messenger RNA (mRNA). The first strand cDNA
reactions are carried out using a cDNA Cycle™ Kit obtained from Invitrogen (San Diego, CA). In the RT reaction, random primers and oligo dT primer are used to generate cDNA from a combination of human and fetal liver mRNA. For
amplification of c-mpl ligand gene fragment encoding amino acids 1-153, the RT product serves as the template for PCR with a combination of the primers, Forward primer: c-mplNcoI (SEQ ID NO:13) and Reverse primer: Ecompl. The c-mplNcoI primer anneals to the c-mpl ligand gene (bases #279-311 based on c-mpl ligand sequence from Gene bank accession #L33410 or de Sauvage et al., Nature 369: 533-538 (1994)) and encodes a NcoI restriction enzyme site immediately 5' to the first codon (Ser+1) of c-mpl ligand. The NcoI
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 codons (Ser, Pro, Ala, & Pro) of c-mpl ligand. The Ecompl primer anneals to bases #720- 737 of c-mpl ligand and encodes an EcoRI site (GAATTC) in- frame with the c-mpl ligand gene immediately following Arg- 153. The EcoRI site creates Glu and Phe codons following Arg-153. The ca. 480 bp PCR product was purified, digested with NcoI and EcoRI and ligated to the NcoI-EcoRI vector fragment of pMON3993 (ca. 4550 bp.). pMON3993 was a derivative of pMON3359 (described in Example 1). The human IL-3 signal peptide sequence, which had been subcloned as a BamHI fragment into the unique BamHI site between the IE110 promoter and poly-A signal, contains an NcoI site at its 3' end and is followed by a unique EcoRI site. The plasmid, pMON26458 containing the DNA sequence of (SEQ ID NO:79), coding for c-mpl ligand amino acids 1-153 (SEQ ID NO.161), was the result of this cloning.
EXAMPLE 3
Construction of the parental plasmids containing the second genes of the dimer templates For amplification of c-mpl ligand gene fragments starting at amino acid 1 (Ser) with a termination codon following amino acid 153 (Arg), the RT reaction from Example 2 serves as the template for PCR with a combination of the following primers; c-mplNcoI (SEQ ID NO:13) (forward primer) and c-mplHindIII (SEQ ID NO:15) (reverse primer). The c-mplNcoI (SEQ ID NO:13) primer is described in Example 2. The c-mplHindIII (SEQ ID NO:15) primer, which anneals to bases #716-737 of c-mpl ligand, adds both a termination codon and a HindIII restriction enzyme site immediately following the final codon, Arg153.
Two types of PCR products 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 NcoI and HindIII restriction enzymes for transfer to a mammalian expression vector, pMON3934. pMON3934 is digested with NcoI and HindIII (ca. 3800 bp) and will accept the PCR products.
Plasmid, pMON32132 (SEQ ID NO:249), coding for c-mpl ligand amino acids 1-153 (SEQ ID NO:252) was a result of this cloning. Plasmid, pMON32134 (SEQ ID NO:250), coding for c-mpl ligand amino acids 1-153 (SEQ ID NO:253) was a result of this cloning. Plasmid, pMON32133 (SEQ ID NO:251), coding for c-mpl ligand amino acids 1-153 with a deletion of codons 112-115 (Δ112-115) (SEQ ID NO:254) was also a result of this cloning.
EXAMPLE 4
Generation of PCR dimer template 5L with a Δ112-115 deletion in the second c-mol ligand gene
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/AflIII 5L synthetic oligonucleotide linker 5L-5' (SEQ ID NO:18) and 5L-3' (SEQ ID NO:19).
The EcoRI end of the linker will ligate to the EcoRI end of pMON26458. The AflIII end of the linker will ligate to the NcoI site of pMON32133, and neither restriction site will be retained upon ligation. The BstXI 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:80) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:222) linker to amino acids 1-153 c-mpl ligand that contains a deletion of amino acids 112-115 (SEQ ID NO:162).
EXAMPLE 5
Generation of PCR dimer template 4L
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/Af1III 4L synthetic
oligonucleotide linker 4L-5' (SEQ ID NO:16) and 4L-3' (SEQ ID NO:17).
The EcoRI end of the linker will ligate to the EcoRI end of pMON26458. The AflIII end of the linker will ligate to the NcoI site of pMON32132, and neither restriction site will be retained upon ligation. The BstXI sites of pMON26458 and pMON32132 will ligate as well. The plasmid, pMON28500, is a result of the cloning and contains the DNA sequence of (SEQ ID NO:82) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyAsnMetAla (SEQ ID NO:223) linker (4L) to amino acids 1-153 c-mpl ligand (SEQ ID
NO:163).
EXAMPLE 6
Generation of PCR dimer template 5L 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/AflIII 5L synthetic
oligonucleotide linker 5L-5' (SEQ ID NO:18) and 5L-3' (SEQ ID NO:19).
The EcoRI end of the linker will ligate to the EcoRI end of pMON26458. The AflIII end of the linker will ligate to the NcoI site of pMON32132, and neither restriction site will be retained upon ligation. The BstXI sites of pMON26458 and pMON32132 will ligate as well. Plasmid, pMON28501 is a result of the cloning and contains the DNA sequence of (SEQ ID NO: 82) which encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnMetAla (SEQ ID NO:222) linker (5D to amino acids 1-153 c-mpl ligand (SEQ ID NO:164). EXAMPLE 7
Generation of PCR dimer templates 8L
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 pMON32134 along with the EcoRI/AflIII 8L synthetic
oligonucleotide linker 8L-5' (SEQ ID NO:20) and 8L-3' (SEQ ID NO:21).
The EcoRI end of the linker will ligate to the EcoRI end of pMON26458. The AflIII end of the linker will ligate to the NcoI site of pMON32134, and neither restriction site will be retained upon ligation. The BstXI sites of
pMON26458 and pMON32134 will ligate as well. Plasmid, pMON28502 is a result of the cloning which contains the DNA sequence of (SEQ ID NO:83) and encodes amino acids 1-153 c-mpl ligand fused via a GluPheGlyGlyAsnGlyGlyAsnMetAla (SEQ ID NO:224) linker (8D to amino acids 1-153 c-mpl ligand (SEQ ID NO:165).
EXAMPLES 8-44
Generation of novel c-mpl ligand genes with new N-terminus and C-terminus
A. PCR generation of genes encoding novel c-mpl ligand receptor agonists.
Genes encoding novel c-mpl ligand receptor agonists were generated using Method III (Horlick et al., Prot. Eng.
5:427-433, 1992 ). The PCR reactions were carried out using dimer templates, pMONs 28500, 28501, 28502 or 28548 and one of the sets of synthetic primer sets below (The first number refers to the position of the first amino acid in the original sequence. For example, the 31-5' and 31-3' refers to the 5' and 3' oligo primers, receptively, for the sequence beginning at the codon corresponding to residue 31 of the original sequence.).
31-5' (SEQ ID NO:22) and 31-3' (SEQ ID NO:23), 35-5' (SEQ ID NO:24) and 35-3' (SEQ ID NO:25), 39-5' (SEQ ID NO:26) and 39-3' (SEQ ID NO:27), 43-5' (SEQ ID NO:28) and 43-3' (SEQ ID NO:29), 45-5' (SEQ ID NO:30) and 45-3' (SEQ ID NO:31), 49-5' (SEQ ID NO:32) and 49-3' (SEQ ID NO:33), 82-5' (SEQ ID
NO:34) and 82-3' (SEQ ID NO:35), 109-5' (SEQ ID NO:36) and 109-3' (SEQ ID NO:37), 115-5' (SEQ ID NO:38) and 115-3' (SEQ ID NO:39), 120-5' (SEQ ID NO:40) and 120-3' (SEQ ID NO:41), 123-5' (SEQ ID NO:42) and 123-3' (SEQ ID NO:43), 126-5' (SEQ ID NO:44) and 126-3' (SEQ ID NO:45).
The templates and oligonucleotide sets used in the PCR reactions are shown in Table 4. The products that were generated were about 480 bp and were purified via Magic PCR Clean up kits (Promega).
B. Subcloning of novel c-mpl receptor agonist gene products into mammalian expression vector for generation of chimeras
The c-mpl receptor agonist gene PCR products were digested with NcoI and HindIII or AflIII and HindIII restriction enzymes (ca. 470 bp) for transfer to a mammalian expression vector. The expression vector, pMON30304, was digested with NcoI and HindIII (ca. 4200 bp) and accepts the PCR products as NcoI-HindIII or AflIII-HindIII fragments. The restriction digest of the PCR product and the resulting plasmids are shown in Table 4.
Figure imgf000166_0001
Figure imgf000167_0001
EXAMPLE 45
Construction of pMON15960
Construction of pMON15960, an intermediate plasmid used for constructing plasmids containing DNA sequences encoding G-CSF Ser17 with a new N-terminus and C-terminus. Plasmid pACYC177 (Chang, A.C.Y. and Cohen, S.N. J. Bacteriol .
134:1141-1156, 1978) DNA was digested with restriction enzymes HindIII and BamHI, resulting in a 3092 base pair HindIII, BamHI fragment. Plasmid, pMON13037 (WO 95/21254), DNA was digested with BglII and Fspi, resulting in a 616 base pair BglII, Fspi fragment. A second sample of plasmid, pMON13037, DNA was digested with NcoI and HindIII,
resulting in a 556 base pair NcoI, HindIII fragment. The synthetic DNA oligonucleotides IGGGSfor (SEQ ID NO:76) and lGGGSrev (SEQ ID NO:77) were annealed to each other, and then digested with AflIII and Fspi, resulting in a 21 base pair AflIII, FspI fragment. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and analyzed by restriction analysis to confirm the correct insert.
EXAMPLE 46
Construction of pMON15981
Construction of pMON15981, a plasmid containing DNA
sequences encoding a multi-functional hematopoietic receptor agonist. Plasmid, pMON15960, DNA was digested with
restriction enzyme SmaI and used as template in a PCR reaction using synthetic DNA oligonucleotides 38 stop (SEQ ID NO:65) and 39 start (SEQ ID NO:64) as primers, resulting in the amplification of a DNA fragment of 576 base pairs.
The amplified fragment was digested with restriction enzymes HindIII and NcoI, resulting in a HindIII, NcoI fragment of 558 base pairs. Plasmid, pMON13181, DNA was digested with HindIII and AflIII, resulting in a HindIII, AflIII fragment of 4068 base pairs. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis, and sequenced to confirm the correct insert. The plasmid, pMON15981, contains the" DNA sequence of (SEQ ID NO:155) which encodes the following amino acid sequence:
Figure imgf000168_0001
Figure imgf000169_0002
EXAMPLE 47
Construction of pMON15982
Construction of pMON15982, a plasmid containing DNA
sequences encoding a multi-functional hematopoietic receptor agonist. Plasmid, pMON15960, DNA was digested with
restriction enzyme SmaI and used as template in a PCR reaction using synthetic DNA oligonucleotides 96 stop (SEQ ID NO:67) and 97 start (SEQ ID NO:66) as primers, resulting in the amplification of a DNA fragment of 576 base pairs. The amplified fragment was digested with restriction enzymes HindIII and NcoI, resulting in a HindIII, NcoI fragment of 558 base pairs. Plasmid, pMON13181, DNA was digested with HindIII and AflIII, resulting in a HindIII, AflIII fragment of 4068 base pairs. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis, and sequenced to confirm the correct insert. The plasmid, pMON15982, contains the DNA sequence of (SEQ ID NO:157) which encodes the following amino acid sequence:
Figure imgf000169_0001
Figure imgf000170_0002
EXAMPLE 48
Construction of pMON15965
Construction of pMON15965, a plasmid containing DNA
sequences encoding a multi-functional hematopoietic receptor agonist. Plasmid, pMON15960, DNA was digested with
restriction enzyme SmaI and used as template in a PCR reaction using synthetic DNA oligonucleotides 142 stop (SEQ ID NO:73) and 141 start (SEQ ID NO:72) as primers, resulting in the amplification of a DNA fragment of 576 base pairs. The amplified fragment was digested with restriction enzymes HindIII and NcoI, resulting in a HindIII, NcoI fragment of 558 base pairs. Plasmid, pMON13181, DNA was digested with HindIII and AflIII, resulting in a HindIII, AflIII fragment of 4068 base pairs. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis, and sequenced to confirm the correct insert. The plasmid, pMON15965, contains the DNA sequence of (SEQ ID NO:157) which encodes the following amino acid sequence:
Figure imgf000170_0001
Figure imgf000171_0001
EXAMPLE 49
Construction of pMON15966 Construction of pMON15966, a plasmid containing DNA
sequences encoding a multi-functional hematopoietic receptor agonist. Plasmid, pMON15960, DNA was digested with
restriction enzyme SmaI and used as template in a PCR reaction using synthetic DNA oligonucleotides 126 stop (SEQ ID NO:68) and 125 start (SEQ ID NO:69) as primers,
resulting in the amplification of a DNA fragment of 576 base pairs. The amplified fragment was digested with restriction enzymes HindIII and NcoI, resulting in a HindIII, NcoI fragment of 558 base pairs. Plasmid, pMON13181, DNA was digested with HindIII and AflIII, resulting in a HindIII, AflIII fragment of 4068 base pairs. The restriction
fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101.
Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis, and sequenced to confirm the correct insert. The plasmid, pMON15966, contains the DNA sequence of (SEQ ID NO:158) which encodes the following amino acid sequence:
Figure imgf000171_0002
Figure imgf000172_0001
EXAMPLE 50
Construction of pMON15967
Construction of pMON15967, a plasmid containing DNA
sequences encoding a multi-functional hematopoietic receptor agonist. Plasmid, pMON15960, DNA was digested with
restriction enzyme SmaI and used as template in a PCR reaction using synthetic DNA oligonucleotides 132 stop (SEQ ID NO:71) and 133 start (SEQ ID NO:70) as primers, resulting in the amplification of a DNA fragment of 576 base pairs. The amplified fragment was digested with restriction enzymes HindIII and NcoI, resulting in a HindIII, NcoI fragment of 558 base pairs. Plasmid, pMON13181, DNA was digested with HindIII and AflIII, resulting in a HindIII, AflIII fragment of 4068 base pairs. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis, and sequenced to confirm the correct insert. The plasmid, pMON15967, contains the DNA sequence of (SEQ ID NO: 159) which encodes the following amino acid sequence:
Figure imgf000172_0002
Figure imgf000173_0001
EXAMPLE 51
Construction of pMON13180, an intermediate plasmid used for constructing plasmids that contain DNA sequence encoding multi-functional hematopoietic receptor agonists.
Plasmid, pMON13046 (WO 95/21254), DNA was digested with restriction endonucleases XmaI and SnaBI, resulting in a 4018 base pair vector fragment. The 4018 base pair XmaI- SnaBI fragment was purified using a Magic DNA Clean-up
System kit (Promega, Madison, WI) in which the 25 base pair XmaI-SnaBl insert fragment is not retained. The
complimentary pair of synthetic oligonucleotides, glyxal (SEQ ID NO:74) and glyxa2 (SEQ ID NO:75), were designed to remove sequence encoding a factor Xa cleavage site. When properly assembled these oligonucleotides also result in XmaI and SnaBI ends. The primers, Glyxal and glyxa2, were annealed in annealing buffer (20mM Tris-HCl pH7.5, 10 mM MgCl2, 50 mM NaCl) by heating at 70°C for ten minutes and allowed to slow cool. The 4018 base pair XmaI-SnaBI fragment from pMON13046 was ligated with the assembled oligonucleotides using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life
Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated from the transformants and analyzed using a PCR based assay. Plasmid DNA from selected transformants was sequenced to confirm the correct insertion of the
oligonucleotides. The resulting plasmid was designated pMON13180 and contains the DNA sequence of (SEQ ID NO:**).
EXAMPLE 52
Construction of pMON13181, an intermediate plasmid used for constructing plasmids that contain DNA sequences encoding multi-functional hematopoietic receptor agonists.
Plasmid, pMON13047 (WO 95/21254), DNA was digested with restriction endonucleases XmaI and SnaBI, resulting in a 4063 base pair vector fragment. The 4063 base pair XmaI- SnaBI fragment was purified using a Magic DNA Clean-up
System kit (Promega, Madison, WI) in which the 25 base pair XmaI-SnaBI insert fragment is not retained. The
complimentary pair of synthetic oligonucleotides, glyxal (SEQ ID NO:74) and glyxa2 (SEQ ID NO:75), were designed to remove sequence encoding the factor Xa cleavage site. When properly assembled these oligonucleotides also result in XmaI and SnaBI ends. Glyxal and glyxa2 were annealed in annealing buffer by heating at 70°C for ten minutes and allowed to slow cool. The 4063 base pair XmaI-SnaBI fragment from pMON13047 was ligated with the assembled oligonucleotides using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life
Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated from the transformants and analyzed using a PCR based assay. Plasmid DNA from selected transformants was sequenced to confirm the correct insertion of the oligonucleotides. The resulting plasmid was designated pMON13181 and contains the DNA sequence of (SEQ ID NO:**)
EXAMPLE 53
Construction of pMON13182
The new N-terminus/C-terminus gene in pMON13182 was created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 39 start (SEQ ID NO:64) and L-11 start (SEQ ID NO:60).
Fragment Stop was created and amplified from G-CSF Ser17 sequence ii, pMON13037 using the primer set, 38 stop (SEQ ID NO:65) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using primers 39 start and 38 stop.
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The intermediate plasmid, pMON13180, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life
Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON13182. E. coli strain JM101 was transformed with pMON13182 for protein expression and protein isolation from inclusion bodies. The plasmid, pMON13182, contains the DNA sequence of
(SEQ ID NO:94) which encodes the following amino acid sequence:
Figure imgf000176_0001
EXAMPLE 54
Construction of pMON13183 The new N-terminus/C-terminus gene in pMON13183 was created using Method I as described in Materials and Methods. "Fragment Start" was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 39 start (SEQ ID NO:64) and L-11 start (SEQ ID NO:60).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 38 stop (SEQ ID NO:65) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using 39 start and 38 stop.
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The intermediate plasmid, pMON13181, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison,
WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life
Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON13183.
E. coli strain JM101 was transformed with pMON13183 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13183, contains the DNA sequence of (SEQ ID NO:95) which encodes the following amino acid sequence:
Figure imgf000177_0001
Figure imgf000178_0001
EXAMPLE 55 Construction of pMON13184
The new N-terminus/C-terminus gene in pMON13184 was created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 97 start (SEQ ID NO:66) and L-11 start (SEQ ID NO:60).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:67) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using 97 start and 96 stop.
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The intermediate plasmid, pMON13180, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison,
WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life
Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON13184.
E. coli strain JM101 was transformed with pMON13184 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13184, contains the DNA sequence of (SEQ ID NO:96) which encodes the following amino acid sequence:
Figure imgf000179_0001
EXAMPLE 56 Construction of pMON13185
The new N-terminus/C-terminus gene in pMON13185 was created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 97 start (SEQ ID NO:66) and L-11 start (SEQ ID NO:60).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:67 and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using 97 start and 96 stop.
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The intermediate plasmid, pMON13181, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life
Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON13185.
E. coli strain JM101 was transformed with pMON13185 for protein expression and protein isolation from inclusion bodies. The plasmid, pMON13185, contains the DNA sequence of
(SEQ ID NO:67) which encodes the following amino acid sequence:
Figure imgf000180_0001
Figure imgf000181_0001
EXAMPLE 57 Construction of pMON13186
The new N-terminus/C-terminus gene in pMON13186 was created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 126 start (SEQ ID NO:68) and L-11 start (SEQ ID NO:60).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:69) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and amplified from the annealed Fragments Start and Stop using 126 start and 125 stop.
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The intermediate plasmid, pMON13180, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON13186.
E. coli strain JM101 was transformed with pMON13186 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13186, contains the DNA sequence of (SEQ ID NO:98) which encodes the following amino acid sequence:
Figure imgf000182_0001
EXAMPLE 58 Construction of pMON13187
The new N-terminus/C-terminus gene in pMON13187 was created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 126 start (SEQ ID NO:68) and L-11 start (SEQ ID NO:60). Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:69) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using 126 start and 125 stop.
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The intermediate plasmid, pMON13181, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life
Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON13187.
E. coli strain JM101 was transformed with pMON13187 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13187, contains the DNA sequence of (SEQ ID NO:99) which encodes the following amino acid sequence:
Figure imgf000183_0001
Figure imgf000184_0001
EXAMPLE 59
Construction of pMON13188
The new N-terminus/C-terminus gene in pMON13188 was created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 133 start (SEQ ID NO:70) and L-11 start (SEQ ID NO:60).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO:71) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using 133 start and 132 stop.
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The intermediate plasmid, pMON13180, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison,
WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON13188.
E. coli strain JM101 was transformed with pMON13188 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13188, contains the DNA sequence of (SEQ ID NO:100) which encodes the following amino acid sequence:
Figure imgf000185_0001
EXAMPLE 60
Construction of pMON13189
The new N-terminus/C-terminus gene in pMON13189 was created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 133 start (SEQ ID NO:70) and L-11 start (SEQ ID NO:60). Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO:71) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using 133 start and 132 stop.
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The intermediate plasmid, pMON13181, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life
Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON13189.
E. coli strain JM101 was transformed with pMON13189 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13189, contains the DNA sequence of (SEQ ID NO:101) which encodes the following amino acid sequence:
Figure imgf000186_0001
Figure imgf000187_0001
EXAMPLE 61 Construction of pMON13190
The new N-terminus/C-terminus gene in pMON13190 was created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 142 start (SEQ ID NO:72) and L-11 start (SEQ ID NO:60).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 141 stop (SEQ ID NO:73) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using 142 start and 141 stop.
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The intermediate plasmid, pMON13180, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison,
WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON13190.
E. coli strain JM101 was transformed with pMON13190 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13190, contains the DNA sequence of (SEQ ID NO:102) which encodes the following amino acid sequence:
Figure imgf000188_0001
EXAMPLE 62
Construction of pMON13191
The new N-terminus/C-terminus gene in pMON13191 was created using Method I as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 142 start (SEQ ID NO:72) and L-11 start (SEQ ID NO:60).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 141 stop (SEQ ID NO:73) and L-11 stop (SEQ ID NO:61). The full-length new N terminus/C-terminus G-CSF Ser17 gene was created and
amplified from the annealed Fragments Start and Stop using 142 start and 141 stop.
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The intermediate plasmid, pMON13181, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim,
Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life
Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON13191.
E. coli strain JM101 was transformed with pMON13191 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13191, contains the DNA sequence of (SEQ ID NO:103) which encodes the following amino acid sequence:
Figure imgf000189_0001
Figure imgf000190_0001
EXAMPLE 63
Construction of pMON13192 The new N-terminus/C-terminus gene in pMON13192 was created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF sequence in pMON13037 using the primer set, 39 start (SEQ ID NO:64) and P-bl start (SEQ ID NO:62). Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 38 stop (SEQ ID NO:65) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
The intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser17 gene and was digested with restriction endonucleases NcoI and
HindIII. The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N- terminus/C-terminus G-CSF Ser17 gene was isolated using Geneclean (Bio101, Vista, CA). The intermediate plasmid, pMON13180, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells
(Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates.
Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene. The resulting plasmid was designated pMON13192.
E. coli strain JM101 was transformed with pMON13192 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13192, contains the DNA sequence of
(SEQ ID NO:104) which encodes the following amino acid sequence:
Figure imgf000191_0001
EXAMPLE 64
Construction of pMON13193 The new N-terminus/C-terminus gene in pMON13193 was created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 39 start (SEQ ID NO:64) and P-bl start (SEQ ID NO:62).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 38 stop (SEQ ID NO:65) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
The intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser17 gene and was digested with restriction endonucleases NcoI and
HindIII. The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N- terminus/C-terminus G-CSF Ser17 gene was isolated using Geneclean (Bio101, Vista, CA). The intermediate plasmid, pMON13181, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells
(Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates.
Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene. The resulting plasmid was designated pMON13193.
E. coli strain JM101 was transformed with pMON13193 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13193, contains the DNA sequence of (SEQ ID NO:105) encodes the following amino acid sequence:
Figure imgf000193_0001
EXAMPLE 65 Construction of pMON25190
The new N-terminus/C-terminus gene in pMON25190 was created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF sequence in pMON13037 using the primer set, 97 start
(SEQ ID NO:66) and P-bl start (SEQ ID NO:62). Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:67) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was
digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
The intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser17 gene and was digested with restriction endonucleases NcoI and
HindIII. The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N-terminus/C-terminus G-CSF Ser17 gene was isolated using Geneclean (Bio101, Vista, CA). The intermediate plasmid, pMON13180, was digested with restriction endonucleases
HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells
(Life Technologies, Gaithersburg, MD). Transformmnt
bacteria were selected on ampicillin-containing plates.
Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene. The resulting plasmid was designated pMON25190.
E. coli strain JM101 was transformed with pMON25190 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON25190, contains the DNA sequence of (SEQ ID NO:106) which encodes the following amino acid sequence:
Figure imgf000194_0001
Figure imgf000195_0001
EXAMPLE 66 Construction of pMON25191
The new N-terminus/C-terminus gene in pMON25191 was created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 97 start (SEQ ID NO:66) and P-bl start (SEQ ID NO:62).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:98) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
The intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser17 gene and was digested with restriction endonucleases NcoI and HindIII. The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N-terminus/C-terminus G-CSF Ser17 gene was isolated using Geneclean (Bio101, Vista, CA). The intermediate plasmid, pMON13181, was digested with restriction endonucleases
HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells
(Life Technologies, Gaithersburg, MD). Transformant
bacteria were selected on ampicillin-containing plates.
Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene. The resulting plasmid was designated pMON25191.
E. col i strain JM101 was transformed with pMON25191 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON25191, contains the DNA sequence of (SEQ ID NO:107) which encodes the following amino acid sequence:
Figure imgf000196_0001
Figure imgf000197_0001
EXAMPLE 67 Construction of pMON13194
The new N-terminus/C-terminus gene in pMON13194 was created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 126 start (SEQ ID NO:68) and P-bl start (SEQ ID NO:62).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:67) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
The intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser17 gene and was digested with restriction endonucleases NcoI and
HindIII. The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N- terminus/C-terminus G-CSF Ser17 gene was isolated using Geneclean (Bio101, Vista, CA). The intermediate plasmid, pMON13180, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells
(Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates.
Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene. The resulting plasmid was designated pMON13194.
E. coli strain JM101 was transformed with pMON13194 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13194, contains the DNA sequence of (SEQ ID NO:108) which encodes the following amino acid sequence:
Figure imgf000198_0001
EXAMPLE 68 Construction of pMON13195
The new N-terminus/C-terminus gene in pMON13195 was created using Method II as described in Materials and Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 126 start (SEQ ID NO:68) and P-bl start (SEQ ID NO:62).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:69) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
The intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser17 gene and was digested with restriction endonucleases NcoI and
HindIII. The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N-terminus/C-terminus G-CSF Ser17 gene was isolated using Geneclean (Bio101, Vista, CA). The intermediate plasmid, pMON13181, vas digested with restriction endonucleases
HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer
Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells
(Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates.
Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene. The resulting plasmid was designated pMON13195.
E. coli strain JM101 was transformed with pMON13195 for protein expression and protein isolation from inclusion bodies. The plasmid, pMON13195, contains the DNA sequence of (SEQ ID NO:109) which encodes the following amino acid sequence:
Figure imgf000200_0001
EXAMPLE 69 Construction of pMON13196
The new N-terminus/C-terminus gene in pMON13196 was created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF sequence in pMON13037 using the primer set, 133 start
(SEQ ID NO:70) and P-bl start (SEQ ID NO:62). Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO:71) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
The intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser17 gene and was digested with restriction endonucleases NcoI and
HindIII. The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N-terminus/C-terminus G-CSF Ser17 gene was isolated using Geneclean (Bio101, Vista, CA). The intermediate plasmid, pMON13180, was digested with restriction endonucleases
HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells
(Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates.
Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene. The resulting plasmid was designated pMON13196.
E. coli strain JM101 was transformed with pMON13196 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13196, contains the DNA sequence of
(SEQ ID NO:110) which encodes the following amino acid sequence:
Figure imgf000201_0001
Figure imgf000202_0001
EXAMPLE 70
Construction of pMON13197
The new N-terminus/C-terminus gene in pMON13197 was created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 133 start (SEQ ID NO:70) and P-bl start (SEQ ID NO:62).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO:71) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
The intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser17 gene and was digested with restriction endonucleases NcoI and
HindIII. The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N- terminus/C-terminus G-CSF Ser17 gene was isolated using Geneclean (Bio101, Vista, CA). The intermediate plasmid, pMON13181, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells
(Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates.
Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene. The resulting plasmid was designated pMON13197.
E. coli strain JM101 was transformed with pMON13197 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13197, contains the DNA sequence of (SEQ ID NO:111) which encodes the following amino acid sequence:
Figure imgf000203_0001
EXAMPLE 71
Construction of pMON13198 The new N-terminus/C-terminus gene in pMON13198 was created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G-CSF sequence in pMON13037 using the primer set, 142 start (SEQ ID NO:72) and P-bl start (SEQ ID NO:62). Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 141 stop (SEQ ID NO:73) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was
digested with restriction endonuclease HindIII. After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
The intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser17 gene and was digested with restriction endonucleases NcoI and
HindIII. The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N- terminus/C-terminus G-CSF Ser17 gene was isolated using Geneclean (Bio101, Vista, CA). The intermediate plasmid, pMON13180, was digested with restriction endonucleases HindIII and AflIII, resulting in a 4023 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells
(Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates.
Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene. The resulting plasmid was designated pMON13198.
E. coli strain JM101 was transformed with pMON13198 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13198, contains the DNA sequence of (SEQ ID NO:112) which encodes the following amino acid sequence:
Figure imgf000205_0001
EXAMPLE 72
Construction of pMON13199
The new N-terminus/C-terminus gene in pMON13199 was created using Method II as described in Materials and
Methods. Fragment Start was created and amplified from G- CSF Ser17 sequence in pMON13037 using the primer set, 142 Start (SEQ ID NO:72) and P-bl start (SEQ ID NO:62).
Fragment Stop was created and amplified from G-CSF Ser17 sequence in pMON13037 using the primer set, 141 stop (SEQ ID NO:73) and P-bl stop (SEQ ID NO:63). Fragment Start was digested with restriction endonuclease NcoI. and Fragment Stop was digested with restriction endonuclease HindIII.
After purification, the digested Fragments Start and Stop were combir.-d with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.
The intermediate plasmid described above contained the full length new N-terminus/C-terminus G-CSF Ser17 gene and was digested with restriction endonucleases NcoI and
HindIII. The digested DNA was resolved on a 1% TAE gel, stained with ethidium bromide and the full-length new N-terminus/C-terminus G-CSF Ser17 gene was isolated using Geneclean (Bio101, Vista, CA). The intermediate plasmid, pMON13181, was digested with restriction endonucleases
HindIII and AflIII, resulting in a 4068 base pair vector fragment, and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The purified restriction fragments were combined and ligated using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). A portion of the ligation reaction was used to transform E. coli strain DH5α cells
(Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates.
Plasmid DNA was isolated and sequenced to confirm the correct insertion of the new gene. The resulting plasmid was designated pMON13199.
E. coli strain JM101 was transformed with pMON13199 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON13199, contains the DNA sequence of (SEQ ID NO:113) which encodes the following amino acid sequence:
Figure imgf000206_0001
Figure imgf000207_0001
EXAMPLE 73 Construction of tandemly-duplicated plasmid template,
Syntan1
To create the tandemly-duplicated hIL-3 receptor agonist pMON13416 template, Syntanl, three DNAs were joined by means of ligation using T4 DNA ligase (Boehringer
Mannheim). The three DNAs are: 1) pMON13046, containing hIL-3 receptor agonist pMON13416, digested with BstEII and SnaBI; 2 ) the annealed complimentary pair of synthetic oligonucleotides, L1syn. for (SEQ ID NO:48) and L1syn. rev (SEQ ID NO:49), which contain sequence encoding the linker that connects the C-terminal and N-terminal ends of the original protein and a small amount of surrounding pMON13416 sequence and which when properly assembled result in BstEII and Clal ends; and 3) a portion of hIL-3 receptor agonist pMON13416 digested from pMON13046 with Clal (DNA had been grown in the dam- cells, DM1 (Life Technologies)) and SnaBI. The digested DNAs were resolved on a 0.9% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101).
A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg,
MD). Miniprep DNA was isolated from the transformants, and the transformants were screened using a PCR based assay.
Plasmid DNA from selected transformants was sequenced to obtain the correct template. The resulting plasmid was designated syntan1 and contains the DNA sequence of (SEQ ID NO:84).
EXAMPLE 74 Construction of tandemly-duplicated template, syntan3.
To create the tandemly-duplicated hIL-3 receptor agonist pMON13416 template, syntan3, three DNAs were joined by means or ligation using T4 DNA ligase (Boehringer
Mannheim). The three DNAs are: 1) pMON13046, containing hIL-3 receptor agonist pMON13416, digested with BstEII and SnaBI; 2) the annealed complimentary pair of synthetic oligonucleotides, L3syn.for (SEQ ID NO:50) and L3syn.rev (SEQ ID NO:51), which contain sequence encoding the linker that connects the C-terminal and N-terminal ends of the original protein and a small amount of surrounding pMON13416 sequence and which when properly assembled result in BstEII and SnaBI ends; and 3) a portion of hIL-3 receptor agonist pMON13416 digested from pMON13046 with Clal (DNA had been grown in the dam- cells, DM1 (Life Technologies)) and SnaBI. The digested DNAs were resolved on a 0.9% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101).
A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD). Miniprep DNA was isolated from the transformants, and the transformants were screened using a PCR based assay. Plasmid DNA from selected transformants was sequenced to obtain the correct template. The resulting plasmid was designated syntan3 and contains the DNA sequence of (SEQ ID NO:85).
EXAMPLE 75 Construction of pMON31104
The new N-terminus/C-terminus gene in pMON31104 was created using Method III as described in Materials and
Methods. The full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntanl, using the primer set 35 start (SEQ ID NO:52) and 34 rev (SEQ ID NO:53).
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI. The digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA). The purified digested DNA fragment was ligated into the expression vector,pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). The pMON13189 DNA had been previously digested with NcoI and
SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD).
Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31104. E. coli strain JM101 was transformed with pMON31104 for protein expression and protein isolation from inclusion bodies. The plasmid, pMON31104, contains the DNA sequence of (SEQ ID NO:86) which encodes the following amino acid sequence:
Figure imgf000210_0001
EXAMPLE 76
Construction of pMON31105 The new N-terminus/C-terminus gene in pMON31105 was created using Method III as described in Materials and Methods. The full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntanl, using the primer set 70 start (SEQ ID NO:54) and 69 rev (SEQ ID NO:55).
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI. The digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA). The purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). The
pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD).
Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31105.
E. coli strain JM101 was transformed with pMON31105 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON31105, contains the DNA sequence of (SEQ ID
NO:87) which encodes the protein with the following amino acid sequence:
Figure imgf000211_0001
Figure imgf000212_0001
EXAMPLE 77 Construction of pMON31106
The new N-terminus/C-terminus gene in pMON31106 was created using Method III as described in Materials and
Methods. The full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntanl, using the primer set 91 start (SEQ ID NO:56) and 90 rev (SEQ ID NO:57).
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI. The digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean
(Bio101, Vista, CA). The purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). The pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD).
Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31106. E. coli strain JM101 was transformed with pMON31106 for protein expression and protein isolation from inclusion bodies. The plasmid, pMON31106, contains the DNA sequence of (SEQ ID NO:80) which encodes the protein with the following amino acid sequence:
Figure imgf000213_0001
EXAMPLE 78 Construction of pMON31107
The new N-terminus/C-terminus gene in pMON31107 was created using Method III as described in Materials and Methods. The full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntanl, using the primer set 101 start (SEQ ID NO:58) and 100 rev (SEQ ID NO:59). The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI. The digested The DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA). The purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). The
pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31107.
E. coli strain JM101 was transformed with pMON31107 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON31107, contains the DNA sequence of (SEQ ID NO:89) which encodes the following amino acid sequence:
Figure imgf000214_0001
Figure imgf000215_0001
EXAMPLE 79
Construction of pMON31108
The new N-terminus/C-terminus gene in pMON31108 was created using Method III as described in Materials and Methods. The full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntan3, using the primer set 35 start (SEQ ID NO:52) and 34 rev (SEQ ID NO:53).
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI. The digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA). The purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). The pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a
0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD).
Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31108. E. coli strain JM101 was transformed with pMON31108 for protein expression and protein isolation from inclusion bodies. The plasmid, pMON31108, contains the DNA sequence of (SEQ ID NO:90) which encodes the following amino acid sequence:
Figure imgf000216_0001
EXAMPLE 80
Construction of pMON31109
The new N-terminus/C-terminus gene in pMON31109 was created using Method III as described in Materials and Methods. The full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntan3, using the primer set 70 start (SEQ ID NO:54) and 69 rev (SEQ ID NO:55). The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI. The digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA). The purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). The pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31109.
E. coli strain JM101 was transformed with pMON31109 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON31109, contains the DNA sequence of (SEQ ID NO:91) which encodes the following amino acid sequence:
Figure imgf000217_0001
Figure imgf000218_0001
EXAMPLE 81 Construction of pMON31110 The new N-terminus/C-terminus gene in pMON31110 was created using Method III as described in Materials and
Methods. The full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntan3, using the primer set 91 start (SEQ ID NO:56) and 90 rev (SEQ ID NO:57).
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI. The digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA). The purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). The pMON13189 DNA had been previously digested with NcoI and SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31110.
E. coli strain JM101 was transformed with pMON31110 for protein expression and protein isolation from inclusion bodies.
The plasmid, pMON31110, contains the DNA sequence of (SEQ ID NO:92) which encodes the following amino acid sequence:
Figure imgf000219_0001
EXAMPLE 82
Construction of pMON31111
The new N-terminus/C-terminus gene in pMON31111 was created using Method III as described in Materials and
Methods. The full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMON13416 was created and amplified from the intermediate plasmid, Syntan3, using the primer set 101 start (SEQ ID NO:58) and 100 rev (SEQ ID NO:59).
The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and SnaBI. The digested DNA fragment was resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (Bio101, Vista, CA). The purified digested DNA fragment was ligated into the expression vector pMON13189, using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN). The
pMON13189 DNA had been previously digested with NcoI and
SnaBI to remove the hIL3 receptor agonist pMON13416 coding sequence and the 4254 base pair vector fragment was isolated using Geneclean (Bio101, Vista, CA) after resolution on a 0.8% TAE gel and staining with ethidium bromide. A portion of the ligation reaction was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, MD).
Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON31111.
E. coli strain JM101 was transformed with pMON31111 for protein expression and protein isolation from inclusion bodies. The plasmid, pMON31111, contains the DNA sequence of (SEQ ID NO:93) which encodes the following amino acid sequence:
Figure imgf000220_0001
Figure imgf000221_0001
EXAMPLE 83 Construction of pMON31112
Construction of pMON31112, a plasmid containing DNA sequence encoding a multi-functional hematopoietic receptor agonist which activates the hIL-3 receptor and G-CSF
receptor. Plasmid, pMON13189 DNA was digested with
restriction enzymes NcoI and XmaI resulting in an NcoI, XmaI vector fragment that was isolated and purified from a 0.8% agarose gel. The DNA from a second plasmid, pMON13222 (WO 94/12639, US serial # 08/411,796) was digested with NcoI and EcoRI resulting in a 281 base pair NcoI, EcoRI fragment. This fragment was isolated and purified from a 1.0% agarose gel. Two oligonucleotides SYNNOXAl.REQ (SEQ ID NO:240) and SYNNOXA2.REQ (SEQ ID NO:241) were annealed and ligated with the 281 base pair DNA fragment from pMON13222 to the DNA vector fragment from pMON13189. A portion of the ligation mixture was then transformed into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis to show the presence of an EcoRV fragment, and sequenced to confirm the correct inserts. The plasmid, pMON31112, contains the DNA sequence of (SEQ ID NO:114) which encodes the following amino acid sequence:
Figure imgf000222_0001
Construction of pMON31113
Construction of pMON31113, a plasmid containing DNA sequence encoding a multi-functional hematopoietic receptor agonist which activates the hIL-3 receptor and G-CSF receptor.
Plasmid, pMON13197 DNA was digested with restriction enzymes NcoI and XmaI resulting in an NcoI, XmaI vector fragment that was isolated and purified from a 0.8% agarose gel. The DNA from a second plasmid, pMON13239 (WO 94/12639, US serial # 08/411,796) was digested with NcoI and EcoRI resulting in a 281 base pair NcoI, EcoRI fragment. This fragment was isolated and purified from a 1.0% agarose gel. Two
oligonucleotides SYNNOXA1.REQ (SEQ ID NO:240) and
SYNNOXA2.REQ (SEQ ID NO:241) were annealed and ligated with the 281 base pair DNA fragment from pMON13239 to the DNA vector fragment from pMON13197. A portion of the ligation mixture was then transformed into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis to show the presence of an EcoRV fragment, and sequenced to confirm the correct inserts. The plasmid, pMON31113, contains the DNA sequence of (SEQ ID NO:115) which encodes the following amino acid sequence:
Figure imgf000223_0001
EXAMPLE 85
Construction of pMON31114 Construction of pMON31114, a plasmid containing DNA sequence encoding a multi-functional hematopoietic receptor agonist which activates the hIL-3 receptor and G-CSF receptor.
Plasmid, pMON13189 DNA was digested with restriction enzymes NcoI and XmaI resulting in an NcoI, XmaI vector fragment that was isolated and purified from a 0.8% agarose gel. The DNA from a second plasmid, pMON13239 (WO 94/12639, US serial # 08/411,796), was digested with NcoI and EcoRI resulting in a 281 base pair NcoI, EcoRI fragment. This fragment was isolated and purified from a 1.0% agarose gel. Two
oligonucleotides SYNNOXA1.REQ (SEQ ID NO:240) and
SYNNOXA2.REQ (SEQ ID NO:241) were annealed and ligated with the 281 base pair DNA fragment from pMON13239 to the DNA vector fragment from pMON13189. A portion of the ligation mixture was then transformed into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis to show the presence of an EcoRV fragment, and sequenced to confirm the correct inserts.
The plasmid, pMON31114, contains the DNA sequence of
(SEQ ID NO:116) which encodes the following amino acid sequence:
Figure imgf000224_0001
EXAMPLE 86 Construction of pMON31115
Construction of pMON31115, a plasmid containing DNA sequence encoding a multi-functional hematopoietic receptor agonist which activates the hIL-3 receptor and G-CSF receptor.
Plasmid, pMON13197 DNA was digested with restriction enzymes NcoI and XmaI resulting in an NcoI, XmaI vector fragment that was isolated and purified from a 0.8% agarose gel. The DNA from a second plasmid, pMON13222, was digested with NcoI and EcoRI resulting in a 281 base pair NcoI, EcoRI fragment. This fragment was isolated and purified from a 1.0% agarose gel. Two oligonucleotides SYNNOXAl.REQ (SEQ ID NO:240) and SYNNOXA2.REQ (SEQ ID NO:241) were annealed and ligated with the 281 base pair DNA fragment from pMON13222 to the DNA vector fragment from pMON13197. A portion of the ligation mixture was then transformed into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated, analyzed by restriction analysis to show the presence of an EcoRV fragment, and sequenced to confirm the correct inserts.
The plasmid, pMON31115, contains the DNA sequence of (SEQ ID NO:117) which encodes the following amino acid sequence:
Figure imgf000225_0001
EXAMPLE 87 Determination of the in vitro activity of multi-functional hematopoietic receptor agonist proteins
The protein concentration of the multi-functional hematopoietic receptor agonist protein can be determined using a sandwich ELISA based on an affinity purified
polyclonal antibody. Alternatively the protein concentration can be determined by amino acid composition analysis. The bioactivity of the multi-functional hematopoietic receptor agonist can be determined in a number of in vitro assays. For example a multi-functional hematopoietic receptor agonist which binds the hIL-3 receptor and G-CSF receptor can be assayed in cell proliferation assays using cell lines expressing the hIL-3 and/or G-CSF receptors. One such assay is the AML-193 cell proliferation assay. AML-193 cells respond to IL-3 and G-CSF which allows for the combined bioactivity of the IL-3/G-CSF multi-functional hematopoietic receptor agonist to be determined. Another such assay is the TFl cell proliferation assay.
In addition other factor dependent cell lines, such as
M-NFS-60 (ATCC. CRL 1838) or 32D which are murine IL-3 dependent cell line, may be used. The activity of IL-3 is species specific whereas G-CSF is not, therefore the
bioactivity of the G-CSF component of the IL-3 /G-CSF multi- functional hematopoietic receptor agonist can be determined independently. Cell lines, such as BHK or murine Baf/3, which do not express the receptor for a given ligand can be transfected with a plasmid containing a gene encoding the desired receptor. An example of such a cell line is BaF3 transfected with the hG-CSF receptor (BaF3/hG-CSF). The activity of the multi-functional hematopoietic receptor agonist in these cell lines can be compared with hIL-3 or G- CSF alone or together. The bioactivity of examples of multi- functional hematopoietic receptor agonists of the present invention assayed in the BaF3/hG-CSF cell proliferation and TFl cell proliferation assays is shown in Table 5 and Table 6. The bioactivity of the multi-functional hematopoietic receptor agonist is expressed as relative activity compared with a standard protein pMON13056 (WO 95/21254). The bioactivity of examples of multi-functional hematopoietic receptor agonists of the present invention assayed in the BaF3/c-mpl cell proliferation and TF1 cell proliferation assays is shown in Table 7 and Table 8.
Figure imgf000228_0001
Figure imgf000229_0001
Figure imgf000230_0001
In a similar manner other factor dependent cell lines known to those skilled in the art can be used to measure the bioactivity of the desired multi-functional hematopoietic receptor agonist. The methylcellulose assay can be used to determine the effect of the multi-functional hematopoietic receptor agonists on the expansion of the hematopoietic progenitor cells and the pattern of the different types of hematopoietic colonies in vitro . The methylcellulose assay can provide an estimate of precursor frequency since one measures the frequency of progenitors per 100,000 input cells. Long term, stromal dependent cultures have been used to delineate primitive hematopoietic progenitors and stem cells. This assay can be used to determine whether the multi-functional hematopoietic receptor agonist stimulates the expansion of very primitive progenitors and/or stem cells. In addition, limiting dilution cultures can be performed which will indicate the frequency of primitive progenitors stimulated by the multi-functional hematopoietic receptor agonist.
Figure imgf000232_0001
Figure imgf000233_0001
EXAMPLE 88
G-CSF variants which contain single or multiple amino acid substitutions were made using PCR mutagenesis
techniques as described in WO 94/12639 and WO 94/12638. These and other variants (i.e. amino acid substitutions, insertions or deletions and N-terminal or C-terminal
extensions) could also be made, by one skilled in the art, using a variety of other methods including synthetic gene assembly or site-directed mutagenesis (see Taylor et al., Nucl . Acids Res . , 13: 7864-8785 [1985]; Kunkel et al., Proc . Natl . Acad. Sci . USA, 82: 488-492 [1985]; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, [1989], (WO 94/12639) and (WO 94/12638)). These substitutions can be made one at a time or in combination with other amino acid substitutions, and/or deletions, and/or insertions and/or extensions. After sequence verification of the changes, the plasmid DNA can be transfected into an appropriate mammalian cell, insect cell or bacterial strain such as E. coli for production. Known variants of G-CSF, which are active, include substitutions at positions 1 (Thr to Ser, Arg or Gly, 2 (Pro to Leu), 3 (Leu to Arg or Ser) and 17 (Cys to Ser) and deletions of amino acids 1-11 (Kuga et al.
Biochemicla and Biophysical Research Comm . 159:103-111 (1989)). These G-CSF amino acid substitution variants can be used as the template to create the G-CSF receptor agonists in which a new N-terminus and new C-terminys are created. Examples of G-CSF amino acid substitution variants are shown in Table 9. EXAMPLE 89
Bioactivity determination of G-CSF amino acid substitution variants
The G-CSF amino acid substitution variants can be assayed for cell proliferation activity using the Baf/3 cell line transtected with the human G-CSF receptor. The
bioactvity of examples of G-CSF amino acid substitution variants is shown in Table 9 relative to native human G-CSF. A "+" indicates a comparable activity to native and a "-" indicates significantly reduced or no measurable activity.
Figure imgf000235_0001
Figure imgf000236_0001
Figure imgf000237_0001
Figure imgf000238_0001
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
More details concerning the molecular biology
techniques, protein purification and bioassays can be found in WO 94/12639, WO 94/12638, WO 95/20976, WO 95/21197, WO
95/20977, WO 95/21254, are hereby incorporated by reference in their entirety.
All references, patents or applications cited herein are incorporated by reference in their entirety as if written herein.
Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the
invention. It is intended that all such other examples be included within the scope of the appended claims.
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Figure imgf000443_0001
Figure imgf000444_0001
Figure imgf000445_0001
Figure imgf000446_0001
Figure imgf000447_0001
Figure imgf000448_0001
Figure imgf000449_0001
Figure imgf000450_0002
Figure imgf000450_0001
Figure imgf000451_0001
Figure imgf000452_0001
Figure imgf000453_0001
Figure imgf000454_0001
Figure imgf000455_0001
Figure imgf000456_0001
Figure imgf000457_0001
Figure imgf000458_0001
Figure imgf000459_0001
Figure imgf000460_0001
Figure imgf000461_0001
Figure imgf000462_0001
Figure imgf000463_0001
Figure imgf000464_0001
Figure imgf000465_0001
Figure imgf000466_0001
Figure imgf000467_0001
Figure imgf000468_0001
Figure imgf000469_0001
Figure imgf000470_0001
Figure imgf000471_0001
Figure imgf000472_0001
Figure imgf000473_0001
Figure imgf000474_0001
Figure imgf000475_0001
Figure imgf000476_0001
Figure imgf000477_0001
Figure imgf000478_0001
Figure imgf000479_0001
Figure imgf000480_0001
Figure imgf000481_0001
Figure imgf000482_0001
Figure imgf000483_0001
Figure imgf000484_0001
Figure imgf000485_0001
Figure imgf000486_0001
Figure imgf000487_0001
Figure imgf000488_0001
Figure imgf000489_0001
Figure imgf000490_0001
Figure imgf000491_0001
Figure imgf000492_0001
Figure imgf000493_0001
Figure imgf000494_0001
Figure imgf000495_0001
Figure imgf000496_0001
Figure imgf000497_0001
Figure imgf000498_0001
Figure imgf000499_0001
Figure imgf000500_0001
Figure imgf000501_0001
Figure imgf000502_0001
Figure imgf000503_0001
Figure imgf000504_0001
Figure imgf000505_0001
Figure imgf000506_0001
Figure imgf000507_0001
Figure imgf000508_0001

Claims

WHAT IS CLAIMED IS:
1. A hematopoietic protein comprising; an amino acid sequence of the formula:
R1-L1-R2, R2-L1-R1, R1-R2, or R2-R1 wherein R1 and R2 are independently selected from the group consisting of;
(I) A polypeptide comprising; a modified human G-CSF amino acid sequence of the formula:
Figure imgf000509_0001
Figure imgf000510_0001
wherein
Figure imgf000510_0002
wherein optionally 1-11 amino acids from the N-terminus and 1-5 from the C-terminus can optionally be deleted from said modified human G-CSF amino acid sequence; and wherein the 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;
Figure imgf000511_0001
(II) A polypeptide comprising; a modified human IL-3 amino acid sequence of the formula:
Figure imgf000511_0002
Figure imgf000512_0001
;
Figure imgf000513_0001
Figure imgf000514_0001
Figure imgf000515_0001
Figure imgf000516_0001
wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of said modified human IL-3 amino acid sequence; wherein from 0 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human
interleukin-3; and wherein the N-terminus is joined to the C-terminus directly or through a linker (L2), capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
Figure imgf000516_0002
Figure imgf000517_0001
Figure imgf000518_0001
wherein the N-terminus is joined to the C-terminus directly or through a linker (L2) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
Figure imgf000518_0002
(IV) A polypeptide comprising; a modified human IL-3 amino acid sequence of the formula:
Figure imgf000518_0003
Figure imgf000519_0001
Figure imgf000520_0001
Figure imgf000521_0001
Figure imgf000522_0001
Figure imgf000523_0001
wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of said modified human IL-3 amino acid sequence; and wherein from 1 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human
interleukin-3; and (V) a colony stimulating factor; and wherein L1 is a linker capable of linking R1 to R2; with the proviso that at least R1 or R2 is selected from the polypeptide of formula (I) , (II), or (III); and said hematopoietic protein can optionally be
immediately preceded by (methionine-1), (alanine-1) or (methionine-2, alanine-1).
2. A hematopoietic protein comprising; an amino acid sequence of the formula:
R1-L1-R2, R2-L1-R1, R1-R2, or R2-R1 wherein R1 and R2 are independently selected from the group consisting of;
(I) A polypeptide comprising; a modified human G-CSF amino acid sequence of the formula:
Figure imgf000524_0001
Figure imgf000525_0001
wherein optionally 1-11 amino acids from the N-terminus and 1-5 from the C-terminus can be deleted from said modified human G-CSF amino acid sequence; and wherein the 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;
Figure imgf000526_0001
(II) A polypeptide comprising; a modified human IL-3 amino acid sequence of the formula:
Figure imgf000526_0002
Figure imgf000527_0001
Figure imgf000528_0001
Figure imgf000529_0001
Figure imgf000530_0001
Figure imgf000531_0002
wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of said modified human IL-3 amino acid sequence; and wherein from 0 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human
interleukin-3; and wherein the N-terminus is joined to the C-terminus directly or through a linker (L2) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
Figure imgf000531_0001
Figure imgf000532_0001
Figure imgf000533_0001
wherein the N-terminus is joined to the C-terminus directly or through a linker (L2) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
Figure imgf000533_0002
(IV) A polypeptide comprising; a modified human IL-3 amino acid sequence of the formula:
Figure imgf000533_0003
Figure imgf000534_0001
Figure imgf000535_0001
Figure imgf000536_0001
Figure imgf000537_0001
Figure imgf000538_0001
wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of said modified human IL-3 amino acid sequence; and wherein from 1 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human
interleukin-3; and
(V) a colony stimulating factor; and wherein L1 is a linker capable of linking R1 to R2; with the proviso that at least R1 or R2 is selected from the polypeptide of formula (I) , (II), or (III); and said hematopoietic protein can optionally be
immediately preceded by (methionine-1), (alanme-1) or (methionine-2, alanme-1).
3. The hematopoietic protein as recited in claim 1 wherein the polypeptide of (IV) is selected from the from the group consisting of;
Figure imgf000539_0001
Figure imgf000540_0002
4. The hematopoietic protein as recited in claim 2 wherein the polypeptide of (IV) is selected from the from the group consisting of;
Figure imgf000540_0001
Figure imgf000541_0002
5. A hematopoietic protein comprising; an amino acid sequence of the formula:
R1-L1-R2, R2-L1-R1, R1-R2, or R2-R1 wherein R1 is a polypeptide comprising; a modified human G-CSF amino acid sequence of the formula:
Figure imgf000541_0001
Figure imgf000542_0001
Figure imgf000543_0002
wherein optionally 1-11 amino acids from the N-terminus and
1-5 from the C-terminus can be deleted from said modified human G-CSF amino acid sequence; and wherein the 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;
Figure imgf000543_0001
wherein R2 is a polypeptide comprising; a modified human IL-3 amino acid sequence of the formula:
Figure imgf000544_0001
Figure imgf000545_0001
Figure imgf000546_0001
Figure imgf000547_0001
Figure imgf000548_0001
wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of said modified human interleukin-3 amino acid sequence; and wherein from 0 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human mterleukin-3; and wherein the N-terminus is joined to the C-terminus directly or through a linker (L2) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
Figure imgf000549_0001
wherein L1 is a linker capable of linking R1 to R2; and said hematopoietic protein can optionally be
immediately preceded by (methionine-1), (alanine-1) or (methionine-2, alanine-1).
6. A hematopoietic protein comprising; an amino acid sequence of the formula:
R1-L1-R2, R2-L1-R1, R1-R2, or R2-R1 wherein R1 is a polypeptide comprising; a modified human G-CSF amino acid sequence of the formula:
Figure imgf000549_0002
Figure imgf000550_0001
Figure imgf000551_0001
wherein optionally 1-11 amino acids from the N-terminus and 1-5 from the C-terminus can be deleted from said modified human G-CSF amino acid sequence; and wherein the 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;
Figure imgf000551_0002
Figure imgf000552_0002
R2 is a polypeptide comprising; a modified human IL-3 amino acid sequence of the formula:
Figure imgf000552_0001
Figure imgf000553_0001
Figure imgf000554_0001
Figure imgf000555_0001
Figure imgf000556_0001
wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus; and wherein from 1 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; wherein L1 is a linker capable of linking R1 to R2; and additionally said hematopoietic protein can be
immediately preceded by (methionine-1), (alanine-1) or (methionine-2, alanine-1).
7. A hematopoietic protein comprising; an amino acid sequence of the formula:
R1-L1-R2, R2-L1-R1, R1-R2, or R2-R1 wherein R1 is a polypeptide comprising; a modified human G-CSF amino acid sequence of the formula:
Figure imgf000557_0001
Figure imgf000558_0001
Figure imgf000559_0001
wherein optionally 1-11 amino acids from the N-terminus and
1-5 from the C-terminus can be deleted from said modified human G-CSF amino acid sequence; and wherein the 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;
Figure imgf000559_0002
R2 is a polypeptide comprising; a modified human c-mpl ligand amino acid sequence of the formula:
Figure imgf000559_0003
Figure imgf000560_0001
wherein the N-terminus is joined to the C-terminus directly or through a linker (L2) capable of joining the N-terminus to the C-terminus and having new C- and N-termmi at amino acids;
Figure imgf000560_0002
Figure imgf000561_0001
wherein L1 is a linker capable of linking R1 to R2; and additionally said hematopoietic protein can be
immediately preceded by (methionine-1), (alanine-1) or (methionine-2, alanine-1).
8. A hematopoietic protein comprising; an amino acid sequence of the formula:
R1-L1-R2, R2-L1-R1, R1-R2, or R2-R1 wherein R1 is a polypeptide comprising; a modified human c-mpl ligand amino acid sequence of the formula:
Figure imgf000561_0002
Figure imgf000562_0002
wherein the N-terminus is joined to the C-terminus directly or through a linker (L2) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
Figure imgf000562_0001
Figure imgf000563_0002
wherein R2 is a polypeptide comprising; a modified human IL-3 amino acid sequence of the formula:
Figure imgf000563_0001
Figure imgf000564_0001
Figure imgf000565_0001
Figure imgf000566_0001
Figure imgf000567_0001
wherein from 1 to 14 amino acids can optionally be deleted from the N-terminus and/or from 1 to 15 amino acids can optionally be deleted from the C-terminus of said modified human interleukin-3 amino acid sequence; and wherein from 1 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; wherein L1 is a linker capable of linking R1 to R2; and said hematopoietic protein can optionally be
immediately preceded by (methionine-1), (alanine-1) or
(methionine-2, alanine-1).
9. The hematopoeitic protein of claim 6 or 8 wherein R2 is selected from the group consisting of;
Figure imgf000568_0001
Figure imgf000569_0002
10. A hematopoietic protein comprising; an amino acid sequence of the formula:
R1-L1-R2, R2-L1-R1, R1-R2, or R2-R1 wherein R1 is a polypeptide comprising; a modified human c-mpl ligand amino acid sequence of the formula:
Figure imgf000569_0001
,
Figure imgf000570_0002
wherein the N-terminus is joined to the C-terminus directly or through a linker (L2) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
Figure imgf000570_0001
Figure imgf000571_0001
wherein R2 is G-CSF or G-CSF Ser17 ; wherein L1 is a linker capable of linking R1 to R2; and said hematopoietic protein can optionally be
immediately preceded by (methionine-1), (alanine-1) or (methionine-2, alanine-1).
11. The hematopoietic protein as recited in claim 1, 2, 3, 4, 5, 6, 7, 8, or 10 wherein said linker (L2) is selected from the group consisting of;
Figure imgf000571_0002
12. The hematopoietic protein as recited in claim 9 wherein said linker (L2) is selected from the group
consisting of;
Figure imgf000571_0003
Figure imgf000572_0002
13. The hematopoietic protein as recited in claim 1 wherein said protein is selected from the group consisting of;
Figure imgf000572_0001
Figure imgf000573_0001
Figure imgf000574_0001
Figure imgf000575_0001
Figure imgf000577_0001
Figure imgf000578_0001
Figure imgf000579_0001
Figure imgf000580_0001
Figure imgf000581_0001
Figure imgf000582_0001
Figure imgf000583_0001
Figure imgf000584_0001
Figure imgf000585_0001
Figure imgf000586_0001
Figure imgf000587_0001
Figure imgf000588_0001
Figure imgf000589_0001
Figure imgf000590_0001
.
14. The hematopoietic protein of claim 1, 2, 3, 4, 5, 6, 7, 8, 10 or 11 wherein said colony stimulating factor is selected from the group consisting of GM-CSF, G-CSF, G-CSF Ser17, c-mpl ligand (TPO), M-CSF, erythropoietin (EPO), IL-1, 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).
15. The hematopoietic protein of claim 14 wherein
said colony stimulating factor is selected from the group
consisting of G-CSF, G-CSF Ser17 and c-mpl ligand (TPO).
16. A nucleic acid molecule encoding said hematopoietic protein of claim 1.
17. A nucleic acid molecule encoding said hematopoietic protein of claim 2.
18. A nucleic acid molecule encoding said hematopoietic protein of claim 3.
19. A nucleic acid molecule encoding said hematopoietic protein of claim 4.
20. A nucleic acid molecule encoding said hematopoietic protein of claim 5.
21. A nucleic acid molecule encoding said hematopoietic protein of claim 6.
22. A nucleic acid molecule encoding said hematopoietic protein of claim 7.
23. A nucleic acid molecule encoding said hematopoietic protein of claim 8.
24. A nucleic acid molecule encoding said hematopoietic protein of claim 9.
25. A nucleic acid molecule encoding said hematopoietic protein of claim 10.
26. A nucleic acid molecule encoding said hematopoietic protein of claim 11.
27. A nucleic acid molecule encoding said hematopoietic protein of claim 12.
28. A nucleic acid molecule encoding said hematopoietic protein of claim 13.
29. A nucleic acid molecule encoding said hematopoietic protein of claim 14.
30. A nucleic acid molecule encoding said hematopoietic protein of claim 15.
31. The nucleic acid molecule according to claim 27 selected from group consisting of;
Figure imgf000592_0001
Figure imgf000593_0001
Figure imgf000594_0001
Figure imgf000595_0001
Figure imgf000596_0001
Figure imgf000597_0001
Figure imgf000598_0001
Figure imgf000599_0001
Figure imgf000600_0001
Figure imgf000601_0001
Figure imgf000602_0001
Figure imgf000603_0001
Figure imgf000605_0001
Figure imgf000606_0001
Figure imgf000607_0001
Figure imgf000608_0001
Figure imgf000609_0001
Figure imgf000610_0001
32. A method of producing a hematopoietic protein
comprising: growing under suitable nutrient conditions, a
host cell transformed or transfected with a replicable
vector comprising a nucleic acid molecule of claim 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 in a manner allowing expression of said hematopoietic protein and recovering said hematopoietic protein.
33. A pharmaceutical composition comprising; the
hematopoietic protein according to claim 1, 2, 3, 4, 5, 6,
7, 8, 10, 11, 12, 13 or 14 and a pharmaceutically acceptable carrier.
34. A method of stimulating the production of
hematopoietic cells in a patient comprising the step of;
administering an effective amount of the hematopoietic
protein as recited in claim 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13 or 14 to said patient.
35. A method of stimulating the production of hematopoietic cells in a patient comprising the step of; administering an effective amount of the hematopoietic protein as recited in claim 9 to said patient.
36. 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 culture medium comprising; the hematopoietic protein of claim 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13 or 14; and
(c) harvesting said cultured cells.
37. 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 culture medium comprising; the hematopoietic protein of claim 9; and
(c) harvesting said cultured cells.
38. A method for treatment of a patient having a
hematopoietic disorder, comprising the steps of;
(a) removing stem cells;
(b) separating stem cells from other cells;
(c) culturing said separated stem cells with a selected culture medium comprising; the hematopoietic protein of
claim 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13 or 14;
(d) harvesting said cultured cells; and
(e) transplanting said cultured cells into said
patient.
39. A method for treatment of a patient having a
hematopoietic disorder, comprising the steps of;
(a) removing stem cells;
(b) separating stem cells from other cells;
(c) culturing said separated stem cells with a selected culture medium comprising; the hematopoietic protein of
claim 9;
(d) harvesting said cultured cells; and
(e) transplanting said cultured cells into said
patient.
40. A method of human gene therapy, comprising the steps of;
(a) removing stem cells from a patient;
(b) separating said stem cells from other cells;
(c) culturing said separated stem cells with a selected culture medium comprising; the hematopoietic protein of claim 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13 or 14;
(d) introducing DNA into said cultured cells;
(e) harvesting said transduced cells; and
(f) transplanting said transduced cells into said patient.
41. A method of human gene therapy, comprising the steps of;
(a) removing stem cells from a patient;
(b) separating said stem cells from other cells;
(c) culturing said separated stem cells with a selected culture medium comprising; the hematopoietic protein of claim 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13 or 14;
(d) introducing DNA into said cultured cells;
(e) harvesting said transduced cells; and
(f) transplanting said transduced cells into said patient.
PCT/US1996/015774 1995-10-05 1996-10-04 Multi-functional hematopoietic receptor agonists WO1997012985A2 (en)

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IL12383296A IL123832A0 (en) 1995-10-05 1996-10-04 Multi-functional hematopoietic receptor antagonists
JP9514385A JPH11510062A (en) 1995-10-05 1996-10-04 Multifunctional hematopoietic receptor agonist
AU73844/96A AU705083B2 (en) 1995-10-05 1996-10-04 Multi-functional hematopoietic receptor agonists
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BRPI9610977A BRPI9610977A2 (en) 1995-10-05 1996-10-04 multifunctional hematopoietic receptor agonists
US08/835,162 US6066318A (en) 1995-10-05 1997-04-04 Multi-functional hematopoietic fusion proteins between sequence rearranged C-MPL receptor agonists and other hematopoietic factors
NO981500A NO981500L (en) 1995-10-05 1998-04-02 Multi-functional hematopoietic receptor antagonists
US10/695,584 US20040171115A1 (en) 1995-10-05 2003-10-27 Multi-functional hematopoietic fusion proteins between sequence rearranged G-CSF receptor agonists and other hematopoietic factors

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CN1124348C (en) 2003-10-15
CA2234061A1 (en) 1997-04-10
CN1204369A (en) 1999-01-06
CZ96598A3 (en) 1998-09-16
BRPI9610977A2 (en) 2019-09-17
EP0854928A2 (en) 1998-07-29
AU705083B2 (en) 1999-05-13
KR19990064068A (en) 1999-07-26
WO1997012985A3 (en) 1997-08-07
CN1590407A (en) 2005-03-09
NO981500D0 (en) 1998-04-02
IL123832A0 (en) 1998-10-30
PL326072A1 (en) 1998-08-17
JPH11510062A (en) 1999-09-07
KR100456212B1 (en) 2005-01-15
CZ295518B6 (en) 2005-08-17
NZ320978A (en) 2001-03-30
NO981500L (en) 1998-05-20
MX9802730A (en) 1998-09-30
AU7384496A (en) 1997-04-28
PL184424B1 (en) 2002-10-31

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