Classifications

• • 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.
• CSFs 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.
• granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies, respectively
• 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.
• 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 Al 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.
• 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 chai .
• proteins which range in size from 58 to 462 amino acids (Goldenberg & Creighton, J. Mol . Biol . 165:407-413, 1983; Li & Coffino, Mol . Cell . Biol . 13:2377-2383, 1993) .
• the proteins examined have represented a broad range of structural classes, including proteins that contain predominantly ⁇ -helix (interleukin-4; Kreitman et al., Cytokine 7:311-318, 1995) , ⁇ -sheet (interleukin-1; Horlick et al. , Protein Eng .
• sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease Tl, Bacillus ⁇ -glucanase, interleukin-l ⁇ , ⁇ -spectrin SH3 domain, pepsinogen, interleukin-4).
• 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) .
• the SH3 domain of ⁇ -spectrin choosing new termini at locations that corresponded to ⁇ - 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.
• Novel hematopoietic proteins of this invention are represented by the formulas:
• Ri and R2 are independently selected from the group consisting of;
• Xaa at position 1 is Thr, Ser, Arg, Tyr or Gly;
• Xaa at position 2 is Pro or Leu
• Xaa at position 3 is Leu, Arg, Tyr or Ser;
• Xaa at position 13 is Phe, Ser, His, Thr or Pro
• Xaa at position 16 is Lys, Pro, Ser, Thr or His
• Xaa at position 17 is Cys, Ser, Gly, Ala, lie, Tyr or Arg;
• Xaa at position 18 is Leu, Thr, Pro, His, lie or Cys;
• Xaa at position 22 is Arg, Tyr, Ser, Thr or Ala;
• Xaa at position 24 is lie, Pro, Tyr or Leu;
• Xaa at position 27 is Asp, or Gly;
• Xaa at position 30 is Ala, lie, Leu or Gly;
• Xaa at position 34 is Lys or Ser
• Xaa at position 36 is Cys or Ser
• Xaa at position 42 is Cys or Ser
• Xaa at position 43 is His, Thr, Gly, Val, Lys, Trp, Ala, Arg, Cys, or Leu;
• Xaa at position 44 is Pro, Gly, Arg, Asp, Val, Ala, His, Trp, Gin, or Thr;
• Xaa at position 46 is Glu, Arg, Phe, Arg, lie or Ala;
• Xaa at position 47 is Leu or Thr;
• Xaa at position 49 is Leu, Phe, Arg or Ser;
• Xaa at position 50 is Leu, lie, His, Pro or Tyr;
• Xaa at position 54 is Leu or His
• Xaa at position 64 is Cys or Ser; Xaa at position 67 is Gin, Lys, Leu or Cys;
• Xaa at position 70 is Gin, Pro, Leu, Arg or Ser;
• Xaa at position 74 is Cys or Ser
• Xaa at position 104 is Asp, Gly or Val
• Xaa at position 108 is Leu, Ala, Val, Arg, Trp, Gin or Gly;
• Xaa at position 115 is Thr, His, Leu or Ala;
• Xaa at position 120 is Gin, Gly, Arg, Lys or His
• Xaa at position 123 is Glu, Arg, Phe or Thr
• Xaa at position 144 is Phe, His, Arg, Pro, Leu, Gin or Glu;
• Xaa at position 146 is Arg or Gin
• Xaa at position 147 is Arg or Gin
• Xaa at position 156 is His, Gly or Ser;
• Xaa at position 159 is Ser, Arg, Thr, Tyr, Val or Gly;
• Xaa at position 162 is Glu, Leu, Gly or Trp;
• Xaa at position 163 is Val, Gly, Arg or Ala; Xaa at position 169 is Arg, Ser, Leu, Arg or Cys;
• Xaa at position 170 is His, Arg or Ser;
• 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;
• Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gin, or Arg;
• Xaa at position 18 is Asn, His, Leu, lie, Phe, Arg, or Gin;
• Xaa at position 19 is Met, Phe, lie, Arg, Gly, Ala, or Cys;
• Xaa at position 20 is lie, Cys, Gin, Glu, Arg, Pro, or Ala;
• Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gin, Asn, Thr, Ser or Val;
• Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gin, Leu, Val or Gly;
• Xaa at position 23 is He, Val, Ala, Gly, Trp, Lys, Phe, Leu, Ser, or Arg;
• Xaa at position 24 is He, Gly, Val, Arg, Ser, Phe, or Leu;
• Xaa at position 25 is Thr, His, Gly, Gin, Arg, Pro, or Ala;
• Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;
• Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;
• Xaa at position 28 is Lys, Arg, Leu, Gin, Gly, Pro, Val or Trp;
• Xaa at position 29 is Gin, Asn, Leu, Pro, Arg, or Val;
• Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gin, Ser, Leu, or Lys;
• Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gin;
• Xaa at position 32 is Leu, Val, Arg, Gin, Asn, Gly, Ala, or Glu;
• Xaa at position 33 is Pro, Leu, Gin, Ala, Thr, or Glu;
• Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gin, Thr,
• Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gin, or Val
• Xaa at position 36 is Asp, Leu, or Val
• Xaa at position 37 is Phe, Ser, Pro, Trp, or He;
• Xaa at position 38 is Asn, or Ala;
• Xaa at position 40 is Leu, Trp, or Arg;
• Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro
• Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, He, Met or Ala
• Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys,
• Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gin, Ala or Pro;
• Xaa at position 45 is Gin, Pro, Phe, Val, Met, Leu, Thr, Lys,
• Trp Asp, Asn, Arg, Ser, Ala, He, Glu or His
• Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gin, Lys, His, Ala, Tyr, He, Val or Gly
• Xaa at position 47 is He, Gly, Val, Ser, Arg, Pro, or His;
• Xaa at position 48 is Leu, Ser, Cys, Arg, He, His, Phe, Glu,
• Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;
• Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, He, Val, His, Phe, Met or Gin;
• Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His
• Xaa at position 52 is Asn, H s, Arg, Leu, Gly, Ser, or Thr
• Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met
• Xaa at position 54 is Arg, Asp, He, Ser, Val, Thr, Gin, Asn, Lys, His, Ala or Leu;
• Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;
• Xaa at position 56 is Pro, Gly, Cys, Ser, Gin, Glu, Arg, His,
• Xaa at position 57 is Asn or Gly
• Xaa at position 58 is Leu, Ser, Asp, Arg, Gin, Val, or Cys
• Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg;
• Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;
• Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;
• Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or lie;
• Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;
• Xaa at positj-on 64 is Ala, Asn, Pro, Ser, or Lys;
• Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;
• Xaa at position 66 is Lys, He, Arg, Val, Asn, Glu, or Ser;
• Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, He, Pro, or His;
• Xaa at position 68 is Leu, Val, Trp, Ser, He, Phe, Thr, or His;
• Xaa at position 69 is Gin, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;
• Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;
• Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gin, Trp, or Asn;
• Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;
• Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;
• Xaa at position 74 is He, Met, Thr, Pro, Arg, Gly, Ala;
• Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gin, or Leu;
• Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;
• Xaa at position 77 is He, Ser, Arg, Thr, or Leu;
• Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;
• Xaa at position 79 is Lys, Thr, Asn, Met, Arg, He, Gly, or Asp;
• Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;
• Xaa at position 81 is Leu, Gin, Gly, Ala, Trp, Arg, Val, or Lys,-
• Xaa at position 82 is Leu, Gin, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, He, Met or Val;
• Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;
• Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 85 is Leu, Asn, Val, or Gin;
• Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;
• Xaa at position 87 is Leu, Ser, Trp, or Gly;
• Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;
• Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;
• Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, He, or Met;
• Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;
• Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, He or
• Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg
• Xaa at position 94 is Arg, He, Ser, Glu, Leu, Val, Gin, Lys, His, Ala, or Pro
• Xaa at position 95 is His, Gin, Pro, Arg, Val, Leu, Gly, Thr, Asn,
• Xaa at position 96 is Pro, Lys, Tyr, Gly, He, or Thr;
• Xaa at position 97 is He, Val, Lys, Ala, or Asn;
• Xaa at position 98 is His, He, Asn, Leu, Asp, Ala, Thr,
• Xaa at position 99 is He, Leu, Arg, Asp, Val, Pro, Gin, Gly, Ser, Phe, or His
• Xaa at position 100 is Lys, Tyr, Leu, His, Arg, He, Ser, Gin, or Pro
• Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val,
• Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro
• Xaa at position 103 is Asp, or Ser
• Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gin, Lys, Ala, Phe, or Gly
• Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gin, Tyr,
• Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, He, Gly, or Pro
• Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, He, Gin, His, Ser, Ala or Pro
• Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly
• Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gin, His, Glu, Ser, or Trp
• Xaa at position 111 is Leu, He, Arg, Asp, or Met
• Xaa at position 112 is Thr, Val, Gin, Tyr, Glu, His, Ser, or Phe;
• Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp,
• Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;
• Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Tr , or Met;
• Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu,
• Xaa at position 117 is Thr, Ser, Asn, He, Trp, Lys, or Pro;
• Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;
• Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;
• Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gin;
• Xaa at position 121 is Ala, Ser, He, Asn, Pro, Lys, Asp, or Gly;
• Xaa at position 122 is Gin, Ser, Met, Trp, Arg, Phe, Pro, His, He, Tyr, or Cys;
• Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;
• ammo acids can be deleted from the N-termmus and/ or from 1 to 15 amino acids can be deleted from the C-termmus ; 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 ;
• N-terminus is joined to the C-termmus directly or through a linker (L2) capable of joining the N-terminus to the C-termmus and having new C- and N-terrami at ammo acids;
• Xaa at position 112 is deleted or Leu, Ala, Val, lie, Pro, Phe, Trp, or Met
• Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, lie, Trp, or Met
• Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu, lie, Trp, or Met
• Xaa at position 115 is deleted or Gin, Gly, Ser, Thr, Tyr, or Asn; 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;
• Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gin, or Arg
• Xaa at position 18 is Asn, His, Leu, He, Phe, Arg, or Gin;
• Xaa at position 19 is Met, Phe, He, Arg, Gly, Ala, or Cys;
• Xaa at position 20 is He, Cys, Gin, Glu, Arg, Pro, or Ala;
• Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gin, Asn,
• Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gin, Leu, Val or Gly;
• Xaa at position 23 is He, Val, Ala, Gly, Trp, Lys, Phe,
• Xaa at position 24 is He, Gly, Val, Arg, Ser, Phe, or Leu
• Xaa at position 25 is Thr, His, Gly, Gin, Arg, Pro, or Ala
• Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp
• Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala
• Xaa at position 28 is Lys, Arg, Leu, Gin, Gly, Pro, Val or Trp
• Xaa at position 29 is Gin, Asn, Leu, Pro, Arg, or Val
• Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gin, Ser, Leu, or Lys
• Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gin
• Xaa at position 32 is Leu, Val, Arg, Gin, Asn, Gly,
• Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gin, or Val
• Xaa at position 36 is Asp, Leu, or Val
• Xaa at position 37 is Phe, Ser, Pro, Trp, or lie
• Xaa at position 38 is Asn, or Ala
• Xaa at position 40 is Leu, Trp, or Arg;
• Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu,
• Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gin, Arg, Thr, Gly or Ser;
• Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp,
• Xaa at position 45 is Gin, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, A"p, Asn, Arg, Ser, Ala, He, Glu or His
• Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gin, Lys, His, Ala, Tyr, He, Val or Gly
• Xaa at position 47 is He, Gly, Val, Ser, Arg, Pro, or His
• Xaa at position 48 is Leu, Ser, Cys, Arg, He, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn
• Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp
• Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser,
• Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His
• Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr
• Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met
• Xaa at position 54 is Arg, Asp, He, Ser, Val, Thr, Gin, Asn,
• Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly
• Xaa at position 56 is Pro, Gly, Cys, Ser, Gin, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys
• Xaa at position 57 is Asn or Gly;
• Xaa at position 58 is Leu, Ser, Asp, Arg, Gin, Val, or Cys;
• Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg;
• Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;
• Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;
• Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or lie;
• Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;
• Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;
• Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;
• Xaa at position 66 is Lys, He, Arg, Val, Asn, Glu, or Ser;
• Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, He, Pro, or His;
• Xaa at position 68 is Leu, Val, Trp, Ser, He, Phe, Thr, or His;
• Xaa at position 69 is Gin, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;
• Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala
• Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gin, Trp, or Asn;
• Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;
• Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;
• Xaa at position 74 is He, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gin, or Leu;
• Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;
• Xaa at position 77 is He, Ser, Arg, Thr, or Leu;
• Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;
• Xaa at position 79 is Lys, Thr, Asn, Met, Arg, He, Gly, or Asp;
• Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;
• Xaa at position 81 is Leu, Gin, Gly, Ala, Trp, Arg, Val, or Lys;
• Xaa at position 82 is Leu, Gin, Lys, Trp, Arg, Asp, Glu, Asn,
• Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;
• Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;
• Xaa at position 85 is Leu, Asn, Val, or Gin;
• Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;
• Xaa at position 87 is Leu, Ser, Trp, or Gly
• Xaa at position 88 is Ala, Lys, Arg, Val, or Trp
• Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser
• Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, He, or Met
• Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His
• Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, He or Leu;
• Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;
• Xaa at position 94 is Arg, He, Ser, Glu, Leu, Val, Gin, Lys, His,
• Xaa at position 95 is His, Gin, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, He, or Tyr;
• Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gin, Tyr, Leu, Lys, He, Asp, or His;
• Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, He, Gly, or Pro;
• Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, He, Gin, His, Ser,
• Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly
• Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gin, His, Glu, Ser, or Trp
• Xaa at position 111 is Leu, He, Arg, Asp, or Met
• Xaa at position 112 is Thr, Val, Gin, Tyr, Glu, His, Ser, or Phe
• Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, He, Val or Asn
• Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;
• Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr,
• Trp Trp, or Met
• Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gin, or lie;
• Xaa at position 117 is Thr, Ser, Asn, He, Trp, Lys, or Pro;
• Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;
• Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;
• Xaa at posit.on 120 is Asn, Ala, Pro, Leu, His, Val, or Gin;
• Xaa at position 121 is Ala, Ser, He, Asn, Pro, Lys, Asp, or Gly;
• Xaa at position 122 is Gin, Ser, Met, Trp, Arg, Phe, Pro, His,
• Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;
• ammo acids can be deleted from the N-terminus and/or from 1 to 15 ammo acids can be deleted from the C-terminus; and wherein from 1 to 44 of the ammo acids designated by Xaa are different from the corresponding amino acids of native (1-133) human ⁇ nterleukin-3 ; or
• Li is a linker capable of linking Ri to R2;
• Ri or R2 is selected from the polypeptide of formula (I) , (II), or (III);
• said hematopoietic protein can optionally be immediately preceded by (methionme-!) , (alanme- ⁇ -) or (meth ⁇ onine ⁇ 2, .
• the more preferred breakpoints at which new C- termmus and N-termmus can be made m the polypeptide ( I 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.
• 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; i and L 2 are optional peptide spacers: n is an integer ranging from 1 to J-l; 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
• T ⁇ and T 2 are proteins.
• 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 __! 6
• 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 vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before infusion into patients.
• Figure 1 schematically illustrates the sequence rearrangement of a protein.
• the N-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.
• 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 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.
• regulators with hematopoietic proli erative activity There are currently at least 20 known regulators with hematopoietic proli erative 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.
• 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.
• 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)
• EPO erythropoietin
• ⁇ -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 (gpl30) (Taga et al. , Cell
• 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:
• Rl-Li-R2 R2-L1-R1, R1-R2, and R2"Rl
• R2 is preferably a colony stimulating factor with a different but complementary activity than Ri.
• complementary activity is meant activity which enhances or changes the response to another cell modulator.
• the Ri 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.
• Li represents a chemical bond or polypeptide segment to which both Ri and R2 are joined in frame, most commonly Li is a linear peptide to which Rl and R2 are joined by amide bonds linking the carboxy terminus of Ri to the amino terminus or Li and carboxy terminus of Li to the amino terminus of R2.
• joinined in frame is meant that there is no translation termination or disruption between the reading frames of the DNA encoding Ri and R2.
• 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 (SCF) also known as steel fa tor or c-kit ligand.
• CSFs colony stimulating factors
• SCF stem cell factor
• Ri 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
• 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 (Li) 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 Ri and R2 such that Ri and R2 could interact simultaneously with their corresponding receptors on a single cell.
• 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 Li linkers of the present invention include sequences selected from the group of formulas: (Gly 3 Ser) n (SEQ ID NO:4), (Gly 4 Ser) n (SEQ ID N0.5),
• 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: GlyGlyGlySerGlyGlyGlySerGlyGlyGlyGlySerGluGlyGlyGlySerGlu GlyGlyGlySerGluGlyGlyGlySerGluGlyGlyGlySerGlyGlyGlyGlySer (SEQ ID NO:9) .
• 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, chy osin, 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:
• 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.
• the length of the amino acid sequence of the linker L2 to be used in Ri and/or R2 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 conformation of R-"- or R 2 (conformationally flexible; Karplus & Schulz, Naturwissenschaften 72:212-213, 1985) .
• 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.
• Sequences of Ri and R2 capable of folding to biologically active states can be prepared by appropriate selection c l 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.
• 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.
• 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)
• 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.
• Non-covalent Multifunctional hematopoietic growth factors 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.
• 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.
• Rl and R2 are as is defined above.
• Domains Ci and C2 are either identical or non-identical chemical structures, typically roteinaceous, which can form a non-covalent, specific association. Complexes between Ci and C2 result in a one-to-one stoichiometric relationship between Ri 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 Ri and Ci, and R2 and C2, respectively. As indicated in the formulae, the domains Ci and C2 can be present either at the N-terminus or C-terminus of their corresponding hematopoietic growth factor (R) .
• R hematopoietic growth factor
• Multimerization domains 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 336:646-65 , 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 interacting with identical domains.
• the leucine zipper domain of Fos interacts with the Jun leucine zipper domain, but does not interact with other Fos leucine zipper domains.
• 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 enabj rapid assay and facile purification of the multi-functional hematopoietic receptor agonist protein by a specific monoclonal antibody.
• mutant amino acid sequence 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 mutein ⁇ .
• Biological activity of the multi-functional hematopoietj c 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 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.
• 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.
• 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 tor 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.
• 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) is also included in the present invention.
• 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.
• 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 x - 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
• 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 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.
• 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 expression for 24 - 72 hours after transfection of the cells or after establishment of stable cell lines following selection for antibiotic resistance.
• 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.
• 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 xeferences cited therein.
• 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,
• 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 strong Baculovirus promoter such as the polyhedron promoter
• the plasmid pVL1392 obtained from Invitrogen Corp., San Diego, California
• 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.
• diseases 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.
• 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.
• splenomegaly, disseminated intravascular coagulation, thrombotic thro bocytopenic 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 such as hIL-3
• 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.
• CSFs colony stimulating factors
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 proliferati n 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.
• 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 CD90(thy-l) .
• 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.
• hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609 [1993], Kobayashi et al. , Blood 73:1836-1841 [1989]) .
• no single factor has been shown to be as effective as the combination of multiple factors.
• the present invention provides methods for ex vivo expansion that utilize 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.
• 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 .
• 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 stimulation factor.
• 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.
• Immunodeficiencies 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.
• 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.
• 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.
• multi-functional hematopoietic receptor agonist 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.
• CSFs colony stimulating factors
• cytokines cytokines
• lymphokines hematopoietic growth factors and interleukins
• interleukins for simultaneous or serial co- administration with the polypeptides of the present invention
• 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 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)l, rpoD+, rpoH358
• DH5 ⁇ TM F-, phi80dlacZdeltaM15, delta (lacZYA-argF)U169, deoR, recAl, endAl, hsdRl7 (rk- ,mk+) , phoA, supE441amda-, thi-1, gyrA96, relAl TGI: delta (lac-pro) , supE, thi-1, hsdD5/F' (traD36, proA+B+, laclq, lacZdeltaMl5) JM101 ATCC#33876: delta (pro lac), supE, thi , F' (tr ⁇ aD36, 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 CaCl2 solution (50 mM CaCl2, 10 mM Tris- Cl, pH7.4) and are held at 4'C for 30 minutes.
• the cells are again collected by centrifugation and resuspended in one-tenth culture volume of CaCl2 solution.
• Ligated DNA is added to 0.2 mL of these cells, and the samples are held at 4"C for 30-60 minutes.
• the samples are shifted to 42 'C for two minutes and 1.0 mL of LB is added prior to shaking the samples at 37"C for one hour.
• Cells from these samples are spread on plates (LB medium plus 1.5% bacto-agar) containing either ampicillin (100 micrograms/mL, ug/mL) when selecting for ampicillin-resistant transformants, or spectinomycin (75 ug/mL) when selecting for spectinomycin-resistant transformants.
• ampicillin 100 micrograms/mL, ug/mL
• spectinomycin 75 ug/mL
• 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) .
• 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.
• 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 lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2.
• PCR reactions are 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”.
• 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 lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2.
• PCR reactions are purified using a Wizard PCR Preps kit (Promega) .
• New N-terminus/C-terminus genes without a linker joining the original N-terminus and C-termmus can be made using two steps of PCR amplification and a blunt end ligation.
• the steps are illustrated in Figure 3.
• 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 conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for 45 seconds and 72°C extension for 45 seconds. Deep Vent polymerase (New England Biolabs) is used to reduce the occurrence of overhangs in conditions recommended by the manufacturer.
• the "P-bl start” and “P-bl stop” primers are phosphorylated at the 5' end to aid in the subsequent blunt end ligation of "Fragment Start” and “Fragment Stop” to each other.
• a 100 ul reaction contained 150 pmole of each primer and one ug of template DNA; and lx Vent buffer (New England Biolabs), 300 uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit 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 Ncol restriction site
• “Fragment Stop” is designed to create a Hindlll restriction site.
• Restriction digest reactions are purified using a Magic DNA Clean-up System kit (Promega) . Fragments Start and Stop are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen) . These fragments are combined with and annealed to the ends of the - 3800 base pair Ncol/Hindlll vector fragment of pMON3934 by heating at 50°C for ten minutes and allowed to slow cool. The three fragments are ligated together using T4 DNA ligase (Boehringer Mannheim) . The result is a plasmid containing the full-length new N-terminus/C-terminus gene. A portion of the ligation reaction is used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD) . Plasmid
• DNA is purified and sequence confirmed as below.
• New 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.
• PCR Polymerase chain reaction
• 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 lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units A pliTaq 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. coli. 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 mis 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 0 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 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-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 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) .
• 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) .
• DMEM/high-glucose Dulbecco's modified Eagle media
• FBS fetal bovine serum
• This formulation is designated BHK growth media.
• Selective media is BHK growth media supplemented with 453 units/mL hygromycin B (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 pMONlll ⁇ 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, pMONlll ⁇ , 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 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.
• 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 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.
• sonication buffer 10 mM 2-amino-2- (hydroxymethyl) 1,
• 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.
• DTT dithiothreitol
• 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.
• Denatured proteins generally elute later in the gradient than the refolded proteins. Purification:
• 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.
• HOAc acetic acid
• 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 x 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,
• 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, 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 myelogenou ⁇ 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 .
• 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 lxlO 5 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 x 105 to 5 x 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.
• 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 x 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 (negative ccntrol) . AML 193 1.3 cell suspensions prepared as above are added to each well by pipetting 50 ⁇ l (2.5 x 10 4 cells) into each well. Tissue culture plates are incubated at 37°C with 5% C02 in humidified air for 3 days.
• 0.5 ⁇ ci 3 ⁇ -thymidine (2 Ci/mM, New England Nuclear, Boston, MA) is added in 50 ⁇ l of tissue culture medium. Cultures are incubated at 37°C with 5% C02 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.
• scintillation fluid Scintiverse II, Fisher Scientific, St. Louis, MO or BetaPlate Scintillation Fluid, Pharmacia LKB, Gaithersburg, MD
• 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 ( ⁇ H-thymidine incorporation) induced by graded concentrations of interleukin-3 or multi-functional hematopoietic receptor agonist. Typically, concentration ranges from 0.05 pM - 10 ⁇ 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) .
• h-IL3 100 U/mL
• 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 x 10 ⁇ 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 x 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 x 10 ⁇ 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% C0 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% C0 2 and 37° C for four hours. The plate is harvested and counted on a Betaplate counter.
• NNN 3H/well
• 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 Phy ⁇ iol 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
• One such transfected Baf/3 cell line was made by cloning the cDNA encoding c-mpl from a library made from a c-mpl responsive cell line and cloned into the multiple cloning site of the plasmid pcDNA3 (Invitrogen, San Diego Ca.) .
• Baf/3 cells were transfected with the plasmid via electroporation. The cells were grown under G418 selection in the presence of mouse IL-3 in Wehi conditioned media. Clones were established through limited dilution.
• the 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 r.,__rrow 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 L 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) .
• CD34+ cells 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 ⁇ l of supernatant added to 1 mL cultures (approximately a 10% dilution) . Cells were incubated at 37°C for 8-14 days at 5% C02 in a 37°C humidified incubator.
• MK buffer 13.6 mM sodium citrate, 1 mM theophylline, 2.2 ⁇ PGEl, 11 mM glucose, 3% w/v BSA, in PBS, pH 7.4,
• MK buffer 13.6 mM sodium citrate, 1 mM theophylline, 2.2 ⁇ PGEl, 11 mM glucose, 3% w/v BSA, in PBS, pH 7.4,
• CD41a-FITC Green fluorescence
• PI red fluorescence
• 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) .
• 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 FeCl2, 25 ⁇ g/mL CaCl2, 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 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 Neurogen
• 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.
• CFU-GM granulocyte / macrophage colonies
• HPP-CFC high proliferative potential colony forming cell
• 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 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 are prepared with 1x104 cells in 1ml of 0.9% methycellulose containing medium without additional growth factors (Methocult H4230 from Stem Cell Technologies, Vancouver, BC. ) .
• 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.
• N A,C,G or T
• GLYXAl GTAGAGGGCG GTGGAGGCTC C (SEQ ID NO:74)
• GLYXA2 CCGGGGAGCC TCCACCGCCC TCTAC (SEQ ID NO:75)
• lGGGSfor TTCTACGCCA CCTTGCGCAG CCCGGCGGCG
• GCTCTGACAT GTCTACACCA TTG
• lGGGSrev CAATGGTGTA GACATGTCAG AGCCGCCGCC GGGCTGCGCA AGGTGGCGTA GAA (SEQ ID NO:77)
• AAAGAATCTC ATAAATCTCC AAACATGGCT ACCCAGGGTG CCATGCCGGC 501 CTTCGCCTCT GCTTTCCAGC GCCGGGCAGG AGGGGTCCTG GTTGCTAGCC
• CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC 501 CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT
• CTGTGTGCCA CCTACAAGCT GTGCCACCCC GAGGAGCTGG TGCTGCTCGG

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