EP0859844A1 - Agonistes du recepteur de l'il-3 - Google Patents

Agonistes du recepteur de l'il-3

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Publication number
EP0859844A1
EP0859844A1 EP96936194A EP96936194A EP0859844A1 EP 0859844 A1 EP0859844 A1 EP 0859844A1 EP 96936194 A EP96936194 A EP 96936194A EP 96936194 A EP96936194 A EP 96936194A EP 0859844 A1 EP0859844 A1 EP 0859844A1
Authority
EP
European Patent Office
Prior art keywords
leu
xaa
ala
ser
pro
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96936194A
Other languages
German (de)
English (en)
Inventor
Maire H. Caparon
S. C. Bauer
Linda L. Zurfluh
Yiging Feng
John P. Mckearn
Charles M. Baum
Charles A. Mcwherter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GD Searle LLC
Original Assignee
GD Searle LLC
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Filing date
Publication date
Application filed by GD Searle LLC filed Critical GD Searle LLC
Publication of EP0859844A1 publication Critical patent/EP0859844A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5403—IL-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • the present invention relates to receptor agonists of human interleukin-3 (hIL-3) .
  • hIL-3 agonists retain one or more activities of native hlL-3 and may also show improved hematopoietic cell-stimulating activity and/or an improved activity profile which may include reduction of undesirable biological activities associated with native hlL-3 and/or have improved physical properties which may include increased solubility, stability and refold efficiency.
  • 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
  • macrophage-CSF M-CSF
  • GM-CSF and interleukin-3 IL-3
  • IL-3 also stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies.
  • I -3 Because of its ability to stimulate the proliferation of a number of different cell types and to support the growth and proliferation of progenitor cells, I -3 has potential for therapeutic use in restoring hematopoietic cells to normal amounts in those cases where the number of cells has been reduced due to diseases or to therapeutic treatments such as radiation and chemotherapy.
  • Interleukin-3 is a hematopoietic growth factor which has the property of being able to promote the survival, growth and differentiation of hematopoietic cells.
  • IL-3 biological properties of IL-3 are the ability (a) to support the growth and differentiation of progenitor cells committed to all, or virtually all, blood cell lineages; (b) to interact with early multipotential stem cells; (c) to sustain the growth of pluripotent precursor cells; (d) to stimulate proliferation of chronic myelogenous leukemia (CML) cells; (e) to stimulate proliferation of mast cells, eosinophils and basophils; (f) to stimulate DNA synthesis by human acute myelogenous leukemia (AM ) cells; (g) to prime cells for production of leukotrienes and histamines; (h) to induce leukocyte chemotaxis; and (i) to induce cell surface molecules needed for leukocyte adhesion.
  • Mature human interleukin-3 (hI -3) consists
  • Murine IL-3 (m.IL-3) was first identified by Ihle, et al., J. IMMUNOL. 12ii.:2184, 1981) as a factor which induced expression of a T cell associated enzyme, 20-hydroxysteroid dehydrogenase. The factor was purified to homogeneity and shown to regulate the growth and differentiation of numerous subclasses of early hematopoietic and lymphoid progenitor cells. In 1984, cDNA clones coding for murine IL-3 were isolated (Fung, et al., NATURE 101:233, 1984) and Yokota, et al., PROC. NATL. ACAD. SCI. USA £1:1070, 1984) .
  • the gibbon IL-3 sequence was obtained using a gibbon cDNA expression library.
  • the gibbon IL-3 sequence was then used as a probe against a human genomic library to obtain a human IL-3 sequence.
  • 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.
  • PCT International Patent Application
  • WO 88/00598 discloses gibbon- and human-like IL-3.
  • the hIL-3 contains a Ser8 -> Pro ⁇ 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.
  • EP-A-0275598 (WO 88/04691) illustrates that Ala* can be deleted while retaining biological activity.
  • Some mutant hIL-3 sequences are provided, e.g., two double mutants, Ala 1 -> Asp 1 , Trpl3 -> Arg3-3 (pGB/lL-302) and Ala 1 -> Asp 1 , Met3
  • WO 88/05469 describes how deglycosylation mutants can be obtained and suggests mutants of Arg54Arg55 and ArgiO ⁇ ArgiOSLys 11 ⁇ might avoid proteolysis upon expression in Saccharomvces cerevisiae by KEX2 protease. No mutated proteins are disclosed. Glycosylation and the KEX2 protease activity are only important, in this context, upon expression in yeast. WO 88/06161 mentions various mutants which theoretically may be confor ationally and antigenically neutral. The only actually performed mutations are Met 2 -> He2 and Ilel31 -> Leu!31. it is not disclosed whether the contemplated neutralities were obtained for these two mutations.
  • WO 91/00350 discloses nonglycosylated hIL-3 analog proteins, for example, hIL-3 (Pro8Asp!5Asp70) , Met3 hIL-3 (Pro8Asp 1 5Asp70) ; ⁇ hr4 hIL-3 (Pro8Asp 1 5Asp70)and Thr ⁇ hIL-3 (Pro8Asp 15 Asp 7 0) . It is said that these protein compositions do not exhibit certain adverse side effects associated with native hIL-3 such as urticaria resulting from infiltration of mast cells and lymphocytes into the dermis.
  • the disclosed analog hIL-3 proteins may have N termini at Met3 , ⁇ hr4, or Thr ⁇ .
  • WO 91/12874 discloses cysteine added variants (CAVs) of IL-3 which have at least one Cys residue substituted for a naturally occurring amino acid residue.
  • WO 94/12639 discloses novel variants of IL-3 which contain one to three amino acid substitutions and optionally deletions from the N-terminus and C-terminus which have increased potency and- improved therapeutic window.
  • WO 94/12638 discloses novel variants of IL-3 which contain from four to forty-four amino acid substitutions and optionally deletions from the N-terminus and C-terminus which have increased potency and improved therapeutic window.
  • WO 95/27732 describes, but does not show that the molecule has biological activity, a circularly permuted G-CSF ligand with a breakpoint at positions 68/69 creating a circularly permuted G-CSF ligand with a new N-terminus at the original position 69 of G-CSF and a new C-terminus at the original position 68 of G-CSF.
  • WO 95/27732 also discloses circularly permuted GM-CSF, IL-2 and IL-4.
  • the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain.
  • linker an additional portion of sequence
  • proteins which range in size from 58 to 462 amino acids (Goldenberg & Creighton, J. Mol . Biol . 165:407-413, 1983; Li & Coffino, Mol . Cell . Biol . 13:2377-2383, 1993) .
  • the proteins examined have represented a broad range of structural classes, including proteins that contain predominantly ⁇ -helix (interleukin-4; Kreitman et al. , CytoJcine 7:311-318, 1995), ⁇ -sheet
  • sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease Tl, Bacillus ⁇ - glucanase, interleuki -l ⁇ , ⁇ -spectrin SH3 domain, pepsinogen, interleukin-4) .
  • the positions of the internal breakpoints used in the studies cited here are found exclusively on the surface of proteins, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle (the variation in the relative distance from the original N- terminus to the breakpoint is ca. 10 to 80% of the total sequence length) .
  • the linkers connecting the original N- and C-termini in these studies have ranged from 0 to 9 residues. In one case (Yang & Schachman, Proc . Natl . Acad. Sci . U. S. A . 90:11980-11984, 1993), a portion of sequence has been deleted from the original C-terminal segment, and the connection made from the truncated C-terminus to the original N-terminus.
  • the present invention relates to novel recombinant human interleukin-3 (hIL-3) receptor agonists. These hIL-3 receptor agonists may also contain amino acid substitutions and/or deletions at either/or both the N- and C- termini.
  • the invention also relates to pharmaceutical compositions containing the hlL-3 receptor agonists, and methods for using the receptor agonists.
  • the present invention relates to DNA coding for the receptor agonists, and recombinant expression vectors comprising nucleotide sequences encoding the hIL-3 receptor agonists, related microbial expression systems, and processes for making the hIL-3 receptor agonists using the microbial expression systems.
  • These hIL-3 receptor agonists may have biological activities similar to or better than hIL-3 and, in some cases, may also have an improved side effect profile, i.e., some receptor agonists may have a better therapeutic index than native hlL-3.
  • the present invention also provides molecules which may function as IL-3 antagonists or as discrete antigenic fragments for the production of antibodies useful in immunoassay and immunotherapy protocols.
  • Antagonists of hIL-3 would be particularly useful in blocking the growth of certain cancer cells like AML, CML and certain types of B lymphoid cancers.
  • Other conditions where antagonists would be useful include those in which certain blood cells are produced at abnormally high numbers or are being activated by endogenous ligands.
  • Antagonists would effectively compete for ligands, presumably naturally occurring hemopoietins including and not limited to IL-3, GM-CSF and IL-5, which might trigger or augment the growth of cancer cells by virtue of their ability to bind to the IL-3 receptor complex while intrinsic activation properties of the ligand are diminished.
  • IL-3, GM-CSF and/or IL-5 also play a role in certain asthmatic responses.
  • An antagonist of the IL-3 receptor may have utility in this disease by blocking receptor-mediated activation and recruitment of inflammatory cells.
  • modified human interleukin-3 receptor agonists of the present invention can be represented by the Formula:
  • L is a linker
  • the constituent amino acids residues of human IL-3 are numbered sequentially 1 through J from the amino to the carboxyl terminus.
  • a pair of adjacent amino acids within this protein may be numbered n and n+1 respectively where n is an integer ranging from 1 to J-1.
  • the residue n+1 becomes the new N-terminus of the new interleukin-3 receptor agonist and the residue n becomes the new C-terminus of the new interleukin-3 receptor agonist.
  • a preferred embodiment of the present invention are modified human interleukin-3 receptor agonists of the Formula:
  • 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 lie, Val, Ala, Gly, Trp, Lys, Phe, Leu, Ser, or Arg
  • Xaa at position 24 is lie, 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 Glir, 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, Arg, Ala, Phe, lie or Met
  • 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, Val, Glu, Phe, Tyr, He, Met or Ala
  • 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, Asp, Asn, Arg, Ser, Ala, He, Glu or His;
  • Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gin,
  • 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, 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, 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 s 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, 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 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, Ala, or Pro;
  • Xaa at position 95 is His, Gin, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe
  • Xaa at position 99 is He, Leu, Arg, Asp, Val, Pro, Gin,
  • 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, Tyr, Glu, Asn, Ser, Ala, Gly, He, Leu, or Gin;
  • 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, 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, 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,
  • 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,
  • Trp Trp, 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,
  • Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;
  • amino acids designated by Xaa are different from the corresponding amino acids of native (1- 133) human interleukin-3 ; wherein optionally from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus;
  • N-terminus is joined to the C-terminus directly or through a linker (L) capable of joining the N-terminus to the C-terminus and having new C- and N-terminus at amino acids;
  • modified human interleukin-3 receptor agonist can be immediately preceded by (methionine -1 ) ,
  • the more preferred breakpoints at which new C-terminus and N-terminus can be made 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 are; 34-35, 69-70 and 90-91.
  • linker (L) joining the N-terminus to the C-terminus is a polypeptide selected from the group consisting of:
  • GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:34); SerGlyGlySerGlyGlySer (SEQ ID NO:35) ;
  • 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 relates to novel receptor agonists of human interleukin-3 (hIL-3) in which the sequence has been rearranged to have a new N-terminus and a new C-terminus, and which may also have amino acid substitutions and/or insertions and/or deletions, and which have substantially the same structure and substantially the same biological activity.
  • the novel receptor agonists of human interleukin-3 (hIL-3) may also contain the amino acid substitutions of naturally occurring variants of hIL-3 polypeptide amino acids (for example, the allele in which proline rather than serine is at position 8 in the hIL-3 polypeptide sequence) or the IL-3 variants described in WO 94/12639, WO 94/12638, WO
  • hIL-3 receptor agonists which are modified post-translationally (e.g. glycosylation) .
  • the present invention also includes the DNA sequences which code for the receptor agonists, DNA sequences which are substantially similar and perform substantially the same function, and DNA sequences which differ from the DNAs encoding the receptor agonists of the invention only due to the degeneracy of the genetic code. Also included in the present invention are the DNA sequences coding for the receptor agonists of the present invention; the oligonucleotide intermediates used to construct the receptor agonists DNAs; and the polypeptides coded for by these oligonucleotides. These polypeptides may be useful as antagonists or as antigenic fragments for the production of antibodies useful in immunoassay and immunotherapy protocols.
  • Human IL-3 can be characterized by its ability to stimulate colony formation by human hematopoietic progenitor cells. The colonies formed include erythroid, granulocyte, megakaryocyte, granulocytic macrophages and mixtures thereof. Human IL-3 has demonstrated an 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 (Gillio, A. P., et al., 1990; Ganser, A, et al., 1990; Falk, S., et al., 1991) .
  • hIL-3 Additional activities include the ability to stimulate leukocyte migration and chemotaxis; the ability to prime human leukocytes to produce high levels of inflammatory mediators like leukotrienes and histamine; the ability to induce cell surface expression of molecules needed for leukocyte adhesion; and the ability to trigger dermal inflammatory responses and fever. Many or all of these biological activities of hlL-3 involve signal transduction and high affinity receptor binding. Receptor agonists of the present invention may exhibit useful properties such as having similar or greater biological activity when compared to native hIL-3 or by having improved half-life or decreased adverse side effects or physical properties, or a combination of these properties. They may also be useful as antagonists.
  • hIL-3 mutant polypeptides which have little or no activity when compared to native hIL-3 may still be 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 receptor agonists. Since hIL-3 functions by binding to its receptor(s) and triggering second messages resulting in competent signal transduction, hIL-3 receptor agonists of this invention may be useful in helping to determine which specific amino acid sequences are responsible for these activities.
  • novel hIL-3 receptor agonists of the present invention will preferably have at least one biological property of human IL-3 or of an IL-3-like growth factor and may have more than one IL-3-like biological property, or an improved property, or a reduction in an undesirable biological property of human IL-3.
  • an IL-3-like biological property is the stimulation of granulocytic type colonies, megakaryocytic type colonies, monocyte/macrophage type colonies, and erythroid bursts.
  • IL-3-like properties are the interaction with early multipotential stem cells, the sustaining of the growth of pluripotent precursor cells, the ability to stimulate chronic myelogenous leukemia (CML) cell proliferation, the stimulation of proliferation of mast cells, the ability to support the growth of various factor- dependent cell lines, and the ability to trigger immature bone marrow cell progenitors.
  • CML chronic myelogenous leukemia
  • Other biological properties of IL-3 have been disclosed in the art. Human IL-3 also has some biological activities which may in some cases be undesirable, for example the ability to stimulate leukotriene release and the ability to stimulate increased histamine synthesis in spleen and bone marrow cultures and in vivo.
  • Some receptor agonists of the present invention may also exhibit an improved side effect profile. For example, they may exhibit a decrease in leukotriene release or histamine release when compared to native hIL-3 or (15-125) hlL-3.
  • Such hIL-3 or hIL-3-like biological properties may include one or more of the following biological characteristics and in vivo and in vitro activities.
  • Biological activity of hIL-3 and hIL-3 receptor agonists of the present invention is determined by DNA synthesis by human acute myelogenous leukemia cells (AML) .
  • the factor- dependent cell line AML 193 was adapted for use in testing biological activity.
  • “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.
  • hlL-3 receptor agonists of the present invention particularly those which retain activity similar to or better than that of native hIL-3, is that it may be possible to use a smaller amount of the biologically active receptor agonists to produce the desired therapeutic effect. This may make it possible to reduce the number of treatments necessary to produce the desired therapeutic effect. The use of smaller amounts may also reduce the possibility of any potential antigenic effects or other possible undesirable side effects. For example, if a desired therapeutic effect can be achieved with a smaller amount of polypeptide it may be possible to reduce or eliminate side effects associated with the administration of native IL-3 such as the stimulation of leukotriene and/or histamine release.
  • hIL-3 receptor agonists of the present invention may also be useful in the activation of stem cells or progenitors which have low receptor numbers.
  • Pharmaceutical compositions containing hIL-3 receptor agonists of the present invention can be administered parenterally, intravenously, or subcutaneously.
  • the modified hIL-3 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 modified hIL-3 receptor agonist may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.
  • the modified hIL-3 receptor agonists of the present invention may also be useful in the ex vivo expansion of hematopoietic progenitors and stem cells.
  • Colony stimulating factors CSFs
  • CSFs Colony stimulating factors
  • hlL-3 Colony stimulating factors
  • the myeloid lineage which is comprised of monocytes (macrophages), granulocytes (including neutrophils) and megakaryocytes, is critical in preventing infections and bleeding which can be life-threatening.
  • Neutropenia and thrombocytopenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy.
  • Bone marrow transplants have been used to treat this patient population.
  • several problems are associated with the use of bone marrow to reconstitute a compromised hematopoietic system including: 1) the number of stem cells in bone marrow, 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.
  • 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 proliferation of the cells.
  • the present invention provides a method for selective ex-vivo expansion of stem cells, comprising the steps of: (a) separating stem cells from other cells, (b) culturing the separated stem cells with a selective culture medium which contains hIL-3 receptor agonist (s) of the present invention and (c) harvesting the cultured 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 modified hIL-3 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,
  • 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 . Microbiol . Immunol .
  • Non-viral based vectors include protein/DNA complexes (Cristiano et al., PNAS USA. 90:2122-2126, 1993; Curiel et al. , PNAS USA 88:8850-8854, 1991; Curiel, Annal . New York Acad. Sci .
  • the present invention provides an improvement to the existing methods of expanding hematopoietic cells, into which new genetic material has been introduced, in that it provides methods utilizing hIL-3 receptor agonists that have improved biological activity, including an activity not seen by any single colony stimulation factor and/or physical properties. Many drugs may cause bone marrow suppression or hematopoietic deficiencies.
  • drugs examples include 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-convuIsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics.
  • the modified hIL-3 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 modified hIL-3 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 modified hIL-3 receptor agonists to a patient.
  • the modified hIL-3 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 modified hlL-3 receptor agonist proteins of the present invention prior to injecting the cells into a patient.
  • immunodeficiencies e.g., in T and/or B lymphocytes, or immune disorders, e.g., rheumatoid arthritis, may also be beneficially affected by treatment with the modified hlL-3 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 modified hIL-3 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 modified hIL-3 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.
  • Another aspect is to provide DNA sequence that encode for the novel hIL-3 receptor agonists and DNA sequences which are the same except for the degeneracy of the genetic code. It is well known to those skilled in the art that the DNA sequence can be altered to remove "rare" codons, modified to alter the GC content .of the DNA sequences, modified to increase RNA message stability and modified at the N-terminus to improve expression in a particular host cell.
  • Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel hlL-3 receptor agonists.
  • These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention.
  • Appropriate vectors which can transform microorganisms capable of expressing the hIL-3 receptor agonists include expression vectors comprising nucleotide sequences coding for the hIL-3 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 hIL-3 receptor agonists.
  • the vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells.
  • a method for producing the novel modified hIL-3 receptor agonists involves culturing the suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of a novel modified hlL-3 receptor agonist.
  • suitable cells or cell lines may be bacterial cells.
  • E. coli the various strains of E. coli are well- known as host cells in the field of biotechnology. Examples of such strains include E. coli strains JM101 (Yanish-Perron et al. Gene 33:103-119, 1985) and MON105 (Obukowicz et al.. Applied Environmental Microbiology 58:1511-1523, 1992) .
  • the expression of the modified hlL-3 receptor agonist protein utilizing a chromosomal expression vector for E. coli based on the bacteriophage Mu (Weinberg et al., Gene 126:25-33, 1993).
  • a chromosomal expression vector for E. coli based on the bacteriophage Mu
  • 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.
  • the gene encoding the modified hIL-3 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 - or Met at the N-terminus of the protein.
  • modified hIL-3 receptor agonists of the present invention may include modified hIL-3 receptor agonist polypeptides
  • mutant modified hIL-3 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 modified hIL-3 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 D ⁇ A 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 references cited therein. In addition, general methods for expression of foreign genes in insect cells using Baculovirus vectors are described in: Summers, M. D. and Smith, G. E., 1987) - A manual of methods for Baculovirus vectors and insect cell culture procedures, Texas Agricultural Experiment Station Bulletin No. 1555.
  • An expression vector is constructed comprising a Baculovirus transfer vector, in which a strong Baculovirus promoter (such as the polyhedron promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally joined to the coding region for the modified hIL-3 receptor agonist polypeptide.
  • a strong Baculovirus promoter such as the polyhedron promoter
  • the plasmid pVLl392 obtained from Invitrogen Corp., San Diego, California
  • two micrograms of this DNA is co- transfected with one microgram of Baculovirus DNA (see Summers & Smith, 1987) into insect cells, strain SF9. Pure recombinant Baculovirus carrying the modified hIL-3 receptor agonist gene is used to infect cells cultured, for example, in Excell 401 serum-free medium (JRH Biosciences, Lenexa,
  • the modified hIL-3 receptor agonist secreted into the medium can be recovered by standard biochemical approaches.
  • Supernatants from mammalian or insect cells expressing the modified hIL-3 receptor agonist protein can be first concentrated using any of a number of commercial concentration units.
  • a daily regimen may be in the range of 0.2 - 150 ⁇ g/kg of non- glycosylated IL-3 protein per kilogram of body weight.
  • This dosage regimen is referenced to a standard level of biological activity which recognizes that native IL-3 generally possesses an EC50 at or about 10 picoMolar to 100 picoMolar in the AML proliferation assay described herein. Therefore, dosages would be adjusted relative to the activity of a given mutein vs.
  • dosage regimens may include doses as low as 0.1 microgram and as high as 1 milligram per kilogram of body weight per day.
  • dosages of IL-3 receptor agonist would be adjusted higher or lower than the range of 10 - 200 micrograms per kilogram of body weight.
  • these include co-administration with other CSF or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated IL-3 receptor agonists; and various patient-related issues mentioned earlier in this section.
  • the therapeutic method and compositions may also include co-administration with other human factors.
  • colony stimulating factors for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, G- CSF, c-mpl ligand (also known as TPO or MGDF) , M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3 , IL-5, IL 6, IL- 7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand, or combinations thereof.
  • SCF stem cell factor
  • the length of the amino acid sequence of the linker can be selected empirically or with guidance from structural information, or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to 50 A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp & Woods, Mol . Immunol . 20: 483-489,1983, Kyte & Doolittle, J. Mol . Biol . 157:105- 132, 1982); solvent exposed surface area, Lee & Richards, J. Mol . Biol .
  • linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic .as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly-Gly-Ser (SEQ ID NO:2) cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used.
  • Sequences capable of folding to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from within the original polypeptide chain while using the linker sequence 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.
  • Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets, chain reversals and turns, and loops; Kabsch & Sander, Biopolymers 22: 2577-2637, 1983), the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, Ann . .Rev. Biochem . 53:537-572, 1984) and the static and dynamic distribution of conformations along the polypeptide chain (Alber & Mathews, Methods Enzymol . 154: 511-533, 1987) .
  • solvent exposure of residues is a site of post- translational attachment of carbohydrate which is necessarily on the surface of the protein.
  • methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops.
  • Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile & Salvatore, Eur. J. Biochem.
  • regions of 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.
  • 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 modified hlL-3 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-, phi80dlacZdeltaMl5, delta(lacZYA-argF)Ul69, deoR, recAl, endAl, hsdRl7 (rk-,mk+) , phoA, supE441amda-, thi-1, gyrA96, relAl
  • TGI delta (lac-pro) , supE, thi-1, hsdD5/F AtraD36, proA+B+, laclq, lacZdeltaMl5)
  • JM101 ATCC#33876 delta (pro lac), supE , thi , F' (traD36, proA+B+, laclq, lacZdeltaMl5)
  • DH5 ⁇ TM Subcloning efficiency cells are purchased as competent cells and are ready for transformation using the manufacturer's protocol, while both E. coli strains TGI and MON105 are rendered competent to take up DNA using a CaCl2 method.
  • 20 to 50 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. The plates are incubated overnight at 37'C.
  • Colonies are picked and inoculated into LB plus appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL spectinomycin) and are grown at 37°C while shaking.
  • appropriate antibiotic 100 ug/mL ampicillin or 75 ug/mL spectinomycin
  • Method I Creation of genes with new N-terminus/C-terminus which contain a linker region.
  • the 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 that connects the C-terminal and N-terminal ends of the original protein.
  • the primer set (“new stop” and “linker stop”) is used to create and amplify, from the original gene sequence, the DNA fragment (“Fragment Stop”) that encodes the same linker as used above, followed by the new C-terminal portion of the new protein.
  • the “new start” and “new stop” primers are designed to include the appropriate restriction sites which allow cloning of the new gene into expression plasmids.
  • Typical PCR conditions are one cycle 95°C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50°C annealing for one minute and 72°C extension for one minute; plus one cycle 72°C extension for seven minutes.
  • a Perkin Elmer GeneAmp PCR Core Reagents kit is used.
  • a 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl2.
  • PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) .
  • primers "new start” and “new stop” are added to the annealed fragments to create and amplify the full-length new N- terminus/C-terminus gene.
  • Typical PCR conditions are one cycle 95°C melting for two minutes; 25 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-terminus can be made using two steps of PCR amplification and a blunt end ligation. The steps are illustrated in Figure 2.
  • 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 the original 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 AmpliTaq DNA polymerase and 2 mM MgCl 2 .
  • PCR reactions are performed in a
  • the new N-terminus/C-terminus gene is digested with restriction endonucleases to create ends that are compatible to insertion into an expression vector.
  • 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 QIAwe11 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.
  • the E. coli cells of selected transformants are inoculated into 50-100 mLs of LB plus appropriate antibiotic for overnight growth at 37°C in an air incubator while shaking.
  • the purified plasmid DNA is used for DNA sequencing, further restriction enzyme digestion, additional subcloning of DNA fragments and transfection into mammalian, E. coli or other cells.
  • Purified plasmid DNA is resuspended in dl ⁇ O and quantitated by measuring the absorbance at 260/280 nm in a Bausch and Lomb Spectronic 601 UV spectrometer.
  • DNA samples are sequenced using ABI PRISMTM DyeDeoxyTM terminator sequencing chemistry (Applied Biosystems Division of Perkin Elmer Corporation, Lincoln City, CA) kits (Part Number 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 Separations, Adelphia, NJ) and lyophilized. Fluorescent dye labeled sequencing reactions are resuspended in deionized formamide, and sequenced on denaturing 4.75% polyacrylamide-8M urea gels using an ABI Model 373A automated DNA sequencer. Overlapping DNA sequence fragments are analyzed and assembled into master DNA contigs using Sequencher v2.1 DNA analysis software (Gene Codes Corporation, Ann Arbor, MI) .
  • the BHK-21 cell line can be obtained from the ATCC (Rockville, MD) .
  • the cells are cultured in Dulbecco's modified Eagle media (DMEM/high-glucose) , supplemented to 2 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., Poultry 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 5 cells per dish 24 hours prior to transfection.
  • Cells are transfected for 16 hours in 3 mL of "OPTIMEM”TM (Gibco-BRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug hygromycin resistance plasmid, pMONlll ⁇ , and 80 ug of Gibco- BRL "LIPOFECTAMINE”TM per dish.
  • the media is subsequently aspirated and replaced with 3 mL of growth media.
  • 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 Chromato ⁇ raphv. and Characterization of the modified hIL-3 receptor agonists which accumulate as inclusion bodies in E. coli .
  • 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) .
  • sonication buffer 10 mM 2-amino-2- (hydroxymethyl) 1,3-propanediol hydrochloride (Tris-HCl) , pH 8.0 + 1 mM ethylenediaminetetraacetic acid (EDTA) .
  • Tris-HCl 2-amino-2- (hydroxymethyl) 1,3-propanediol hydrochloride
  • EDTA ethylenediaminetetraacetic acid
  • These resuspended cells are sonicated using the microtip probe of a Sonicator Cell Disruptor (Model W-375, Heat Systems- Ultrasonics
  • 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.
  • 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, Ca.) C18 column (0.46 x 25 cm) .
  • a linear gradient of 40% to 65% acetonitrile, containing 0.1% trifluoroacetic acid (TFA) is employed.
  • 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.
  • the factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) .
  • This cell line established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM-CSF supplemented medium (Lange, B., et al. , Blood 70: 192, 1987) Valtieri, M. , et al., J. Immunol . 138:4042, 1987).
  • the ability of AML . 19-3 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D., et al., J. Immunol . 139: 348, 1987).
  • a cell line variant was used, AML 193 1.3, which was adapted for long term growth in IL-3 by washing out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours. The cells are then replated at 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 10 ⁇ to 5 x 10 ⁇ viable cells/mL. This medium is prepared by supplementing Iscove's modified Dulbecco's Medium (IMDM, Hazelton, Lenexa, KS) with albumin, transferrin, 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 modified hlL- 3 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 modified hIL-3 receptor agonist proteins once serial dilutions are completed. Control wells contained tissue culture medium alone (negative control) . AML 193 1.3 cell suspensions prepared as above are added to each well by pipetting 50 ⁇ l (2.5 x 10 cells) into each well. Tissue cu ture plates are incubated at 37°C with 5% C02 in humidified air for 3 days.
  • 0.5 ⁇ ci 3 H- 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
  • Beta emissions of samples from individual tissue culture wells are counted in a LKB BetaPlate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, MD) and data is expressed as counts per minute of 3 ⁇ -thymidine incorporated into cells from each tissue culture well.
  • Activity of each human interleukin-3 preparation or modified hIL-3 receptor agonist proteins preparation is quantitated by measuring cell proliferation (3H-thymidine incorporation) induced by graded concentrations of interleukin-3 or modified hIL-3 receptor agonist. Typically, concentration ranges from 0.05 pM - 105 M 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 modified hIL-3 receptor agonist proteins are tested in a concentration range of 2000 pM to 0.06 pM titrated in serial 2 fold dilutions.
  • Activity for each sample is determined by the concentration which gave 50% of the maximal response by fitting a four-parameter logistic model to the data. It was observed that the upper plateau (maximal response) for the sample and the standard with which it was compared did not differ. Therefore relative potency calculation for each sample is determined from EC50 estimations for the sample and the standard as indicated above.
  • 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.,
  • 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 modified hIL-3 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 modified hIL-3 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 baseline response
  • Positive control cultures received conditioned media (PHA stimulated human cells: Terry Fox Lab. H2400) . Cultures are incubated at 37°C, 5% C02 in humidified air.
  • Hematopoietic colonies which are defined as greater than 50 cells are counted on the day of peak response (days 10-11) using a Nikon inverted phase microscope with a 40x objective combination. Groups of cells containing fewer than 50 cells are referred to as clusters. Alternatively colonies can be identified by spreading the colonies on a slide and stained or they can be picked, resuspended and spun onto cytospin slides for staining.
  • 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) .
  • cord blood is more readily available on a regular basis.
  • 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 ⁇ BA-, CD34+ fraction using coated flasks from Applied Immune Science (Santa Clara, CA) ; and CD34+ selection using a CellPro (Bothell, WA) avidin column. Either freshly isolated or cryopreserved CD34+ cell enriched fractions are used for the assay.
  • Duplicate cultures for each serial dilution of sample (concentration range from 1 pM to 1204 pM) are prepared with 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) is used instead of Methocult H4230, or Stem Cell Factor (SCF) , 50 ng/mL (Biosource O 97/12979
  • Ala lie Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser Ala Thr Ala
  • Miniprep DNA was isolated from the transformants, and the transformants were screened using a PCR based assay. Plasmid DNA from selected transformants was sequenced to obtain the correct template. The resulting plasmid was designated syntanl.
  • syntan3 SEQ ID N0:16
  • T4 DNA ligase Boehringer Mannheim
  • the three DNAs are: 1) pMON13046, containing hIL-3 receptor agonist pMONl3416, digested with BstEII and SnaBI; 2) the annealed complimentary pair of synthetic oligonucleotides, L3syn.for (SEQ ID NO:5) and L3syn.rev (SEQ ID NO:6), which contain sequence encoding the linker that connects the C-terminal and N-terminal ends of the original protein and a small amount of surrounding pMONl3416 sequence and which when properly assembled result in BstEII and SnaBI ends; and 3) a portion of hIL-3 receptor agonist pMONl3416 digested from pMONl3046 with Clal (DNA had been grown in the dam- cells, DM1 (Life Technologies) and SnaBI. The digested DNAs were resolved on a 0.9% TAE gel, stained with ethidium bromide and isolated using Geneclean (BiolOl) .
  • Miniprep DNA was isolated from the transformants, and the transformants were screened using a PCR based assay. Plasmid DNA from selected transformants was sequenced to obtain the correct template. The resulting plasmid was designated syntan3.
  • the new N-terminus/C-terminus gene in pMON31155 is created using Method III as described in Materials and Methods.
  • the full length new N-terminus/C-terminus gene of hIL-3 receptor agonist pMONl3416 is created and amplified from the intermediate plasmid, Syntanl, using the primer set 35start (SEQ ID NO:7) and 34stop (SEQ ID NO:8) .
  • the resulting DNA fragment which contains the new gene is digested with restriction endonucleases Ncol and Hindlll.
  • the digested DNA fragment is resolved on a 1% TAE gel, stained with ethidium bromide and isolated using Geneclean (BiolOl, Vista, CA) .
  • the purified digested DNA fragment is ligated into an expression vector using T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN) .
  • the expression vector DNA is digested with Ncol and Hindlll .
  • a portion of the ligation reaction is used to transform E. coli strain DH5 ⁇ cells (Life Technologies, Gaithersburg, MD) .
  • Transformant bacteria are selected on ampicillm-containing plates. Plasmid DNA is isolated and sequenced to confirm the correct insert. The resulting plasmid is designated
  • E. coli strain JM101 was transformed with pMON31155 for protein expression and protein isolation from inclusion bodies .
  • Examples 4-10 are constructed in a similar manner as 70 described in Example 3 using the primers indicated in Table 4.
  • the resulting proteins have new N-terminus and C-terminus as indicated in Table 4.
  • NAME G. D. Searle & Co.
  • Trp Pro, Ser, Ala, His, Asp, Asn, Gin, Leu, Val or Gly;

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Abstract

L'invention porte sur de nouvelles protéines agonistes du récepteur de l'IL-3, sur de l'ADN codant pour des protéines agonistes du récepteur hématopoïétique IL-3, ainsi que sur des procédés d'obtention et d'utilisation desdites protéines.
EP96936194A 1995-10-05 1996-10-04 Agonistes du recepteur de l'il-3 Withdrawn EP0859844A1 (fr)

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US483595P 1995-10-05 1995-10-05
PCT/US1996/015941 WO1997012979A1 (fr) 1995-10-05 1996-10-04 Agonistes du recepteur de l'il-3
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EP1073755A2 (fr) * 1998-04-28 2001-02-07 Axys Pharmaceuticals, Inc. Nouvelle serine protease apte au clivage selectif de la proteine de liaison du facteur de croissance insuline-like

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US6057133A (en) * 1992-11-24 2000-05-02 G. D. Searle Multivariant human IL-3 fusion proteins and their recombinant production

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