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
  • C07K14/475—Growth factors; Growth regulators
• • 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
  • A61P7/06—Antianaemics
• • 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 chimeric proteins or multi-functional hematopoietic receptor agonists, comprising a human flt3 agonist.
• These chimera proteins retain one or more activities of native flt3 ligand and the other component of the chimera protein.
• the chimera protein may also show improved hematopoietic cell-stimulating activity or an activity not seen for flt3 ligand and the other factor when co-administered.
• the chimera protein may also show an improved activity profile which may include reduction of undesirable biological activities associated with native flt3 ligand 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.
• Colony stimulating factors 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 while GM-CSF and interl,eu in-3 (I -3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies.
• G- CSF granulocyte-CSF
• M-CSF macrophage-CSF
• I -3 interl,eu in-3
• Certain factors such as flt3 ligand are able to predominately affect stem cells.
• Tyrosine kinase receptors are growth factor receptors that regulate the proliferation and differentiation of a number of cell. Certain tyrosine kinase receptors function within the hematopoietic system. Flt3 (Roseate et al . , Oncogene, 6:1641-1650, 1991) and flk-2 (Matthews et al . , Cell , 65:1143-1152, 1991) are forms of a tyrosine kinase receptor that is related to c-fms and c-kit receptors. The flk-2 and flt3 receptors are similar in amino acid sequence and vary at two amino acid residues in the extracellular domain and diverge in a 31 amino acid segment located near the C-terminus.
• Flt3 ligand is a hematopoietic growth factor which has the property of being able to regulate the growth and differentiation of hematopoietic progenitor and stem cells. Because of its ability to support the growth and proliferation of progenitor cells, flt3 receptor agonists have potential for therapeutic use in treating hematopoietic disorders such as aplastic anemia and myelodysplastic syndromes. Additionally, flt3 receptor agonists will be useful 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.
• WO 94/28391 discloses the native flt3 ligand protein sequence and a cDNA sequence encoding the flt3 ligand, methods of expressing flt3 ligand in a host cell transfected with the cDNA and methods of treating patients with a hematopoietic disorder using flt3 ligand.
• US Patent No. 5,554,512 is directed to human flt3 ligand as an isolated protein, DNA encoding the flt3 ligand, host cells transfected with cDNAs encoding flt3 ligand and methods for treating patients with flt3 ligand.
• WO 94/26891 provides mammalian flt3 ligands, including an isolate that has an insertion of 29 amino acids, and fragments there of.
• the human blood-forming (hematopoietic) system replaces a variety of white blood cells (including neutrophils, macrophages, and basophils/mast cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes/platelets) .
• white blood cells including neutrophils, macrophages, and basophils/mast cells
• red blood cells erythrocytes
• clot-forming cells megakaryocytes/platelets
• U.S. Patent 4,810,643 relates to DNA and methods of making G-CSF and Cys to Ser substitution variants of G-CSF.
• Kuga et al . (Biochem . + Biophys . Res . Comm . 159:103-111, 1988) made a series of G-CSF variants to partially define the structure-function relationship.
• Kuga et al . found that internal and C-terminal deletions abolished activity, while N-terminal deletions of up to 11 amino acids and amino acid substitutions at positions 1, 2 and 3 were active.
• Erythropoietin is a naturally-occurring glycoprotein hormone with a molecular weight that was first reported to be approximately 39,000 daltons (T. Miyaki et al . , J. Biol . Chem . 252:5558-5564 (1977)).
• the mature hormone is 166 amino acids long and the "prepro" form of the hormone, with its leader peptide, is 193 amino acids long (F. Lin, U.S. Patent No. 4,703,008).
• the mature hormone has a molecular weight, calculated from its amino acid sequence, of 18,399 daltons (K. Jacobs et al . , Nature 313:806-810 (1985); J. K. Browne et al . , Cold Spring Harbor Symp . Quant . Biol . 5:1693-702 (1986).
• the first mutant erythropoietins (i.e., erythropoietin analogs), prepared by making amino acid substitutions and deletions, have demonstrated reduced or unimproved activity.
• replacement of the tyrosine residues at positions 15, 40 and 145 with phenylalanine residues, replacement of the cysteine residue at position 7 with an histidine, substitution of the proline at position 2 with an asparagine, deletion of residues 2-6, deletion of residues 163- 166, and deletion of residues 27-55 does not result in an apparent increase in biological activity.
• the Cys 7 -to-His 7 mutation eliminates biological activity.
• Oligonucleotide-directed mutagenesis of erythropoietin glycosylation sites has effectively probed the function of glycosylation but has failed, as yet, to provide insight into an effective strategy for significantly improving the characteristics of the hormone for therapeutic applications.
• a series of single amino acid substitution or deletion mutants have been constructed of erythropoietin, involving amino acid residues 15, 24, 49, 76, 78, 83, 143, 145, 160, 162, 163, 164, 165 and 166.
• the mutants have been administered to animals while monitoring hemoglobin, hematocrit and reticulocyte levels (EP No. 0 409 113) .
• the human erythropoietin molecule contains two disulfide bridges, one linking the cysteine residues at positions 7 and 161, and a second connecting cysteines at positions 29 and 33 (P.H. Lai et al . , J. Biol . Chem . 261:3116-3121 (1986)). Oligonucleotide- directed mutagenesis has been used to probe the function of the disulfide bridge linking cysteines 29 and 33 in human erythropoietin. The cysteine at position 33 has been converted to a proline residue, which, mimics the structure of murine erythropoietin at this residue. The resulting mutant has greatly reduced in vi tro activity.
• EPO muteins modified EPO proteins
• WO 91/05867 discloses analogs of human erythropoietin having a greater number of sites for carbohydrate attachment than human erythropoietin, such as EPO (Asn 69 ), EPO (As ' n 125 , Ser 127 ), EPO (Thr 125 ), and EPO (Pro 124 , Thr 125 ).
• WO 94 /24160 discloses erythropoietin muteins which have enhanced activity, specifically amino acid substitutions at positions 20, 49, 73, 140, 143, 146, 147 and 154.
• WO 94/25055 discloses erythropoietin analogs, including EPO (X 33 , Cys 139 , des-Arg 166 ) and EPO
• Stem cell factor has the ability to stimulate growth of early hematopoietic progenitors which are capable of maturing to erythroid, megakaryocyte, granulocyte, lymphocyte and macrophage cells. Stem cell factor treatment of mammals results in absolute increases in hematopoietic cells of both the myeloid and lymphoid cells.
• EP 0 423 980 discloses novel stem cell factor (SCF) polypeptides including SCF 1"148 , SCF 1"157 , SCF 1"160 , SCF 1"161 , SCF 1"162 , SCF 1"164 , SCF 1"165 , SCF 1"183 , SCF 1"185 , SCF 1"
• SCF stem cell factor
• 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 -> Pro8 replacement.
• 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
• 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 present invention encompasses recombinant chimeric proteins comprising a flt3 agonist and another factor.
• the other factor may be a colony stimulating factor (CSF) , cytokine, lymphokine, interleukin, hematopoietic growth factor which include but are not limited to GM-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 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, stem cell growth factor (SCGF) (Hiraoka,
• the chimera proteins can also be co-administered or sequentially administered with one or more additional colony stimulating factor (s) , cytokine (s), lymphokine ( s ) , interleukin (s) , hematopoietic growth factor (s) which include but are not limited to GM- CSF, c-mpl ligand (also known as TPO or MGDF) , M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2 , IL-3 , IL-5.
• colony stimulating factor s
• cytokine cytokine
• lymphokine s
• s interleukin
• hematopoietic growth factor hematopoietic growth factor
• EPO erythropoietin
• hematopoietic growth factors IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, SCGF and SDF-1 (herein collectively referred to "hematopoietic growth factors".
• SCF stem cell factor
• SCGF also known as steel factor or c-kit ligand
• SDF-1 also known as steel factor or c-kit ligand
• the chimeric protein may also provide an enhanced effect on the activity or an activity different from that expected by the presence of the flt3 ligand or the second colony stimulating factor.
• the chimeric protein may also have an improved activity profile which may include reduction of undesirable biological activities associated with native human flt3.
• the present invention encompasses multifunctional hematopoietic receptor agonists or chimeric proteins 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 proliferative activity There are currently at least 20 known regulators with hematopoietic proliferative activity. Most of these proliferative regulators can only stimulate one or another type of colony formation in vitro, the precise pattern of colony formation stimulated by each regulator is quite distinctive.
• No two regulators stimulate exactly the same pattern of colony formation, as evaluated by colony numbers or, more importantly, by the lineage and maturation pattern of the cells making up the developing colonies.
• Proliferative responses can most readily be analyzed in simplified in vitro culture systems. Three quite different parameters can be distinguished: alteration in colony size, alteration in colony numbers and cell lineage. Two or more factors may act on the progenitor cell, inducing the formation of larger number of progeny thereby increasing the colony size. Two or more factors may allow increased number of progenitor cells to proliferate either because distinct subsets of progenitors cells exist that respond exclusively to one factor or because some progenitors require stimulation by two or more factors before being able to respond.
• Activation of additional receptors on a cell by the use of two or more factors is likely to enhance the mitotic signal because of coalescence of initially differing signal pathways into a common final pathway reaching the nucleus (Metcalf, Nature 339:27, 1989).
• Other mechanisms could explain synergy. For example, if one signaling pathway is limited by an intermediate activation of an additional signaling pathway which is caused by a second factor, then this may result in a super additive response.
• 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.
• hematopoietic receptor agonists may have a potential clinical advantage resulting from a proliferative response that is not possible by any single factor.
• the receptors of hematopoietic and other growth factors can be grouped into two distinct families of related proteins: (1) tyrosine kinase receptors, including those for epidermal growth factor, M-CSF (Sherr, Blood 75:1, 1990) and SCF (Yarden et al . , EMBO J.
• EPO erythropoietin
• GM-CSF Gearing et al . , EMBO J. 8:3667, 1989
• IL-3 Kitamura et al . , Cell 66:1165, 1991
• G-CSF Fukunaga et al . , J " . Bio . Chem . 265:14008-15, 1990
• IL-4 Harada et al . , PNAS USA 87:857, 1990
• IL-5 Takaki et al . , EMBO J. 9:4367, 1990
• IL-6 Yamamoto et al., Science 241:825, 1988
• IL-7 Goodwin et al .
• the use of a multiply acting hematopoietic factor may also have a potential advantage by reducing the demands placed on factor-producing cells and their induction systems. If there are limitations in the ability of a cell to produce a factor, then by lowering the required concentrations of each of the factors, and using them in combination may usefully reduce demands on the factor-producing cells .
• the use of a multiply acting hematopoietic factor may lower the amount of the factors that would be needed, probably reducing the likelihood of adverse side- effects.
• Novel compounds of this invention are represented by a formula selected from the group consisting of:
• Ri is a flt3 agonist and R2 is a hematopoietic growth factor.
• R2 is a hematopoietic growth 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
• 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 of Li and carboxy terminus of Li to the amino terminus of R2.
• joined in frame is meant that there is no translation termination or disruption between the reading frames of the DNA encoding Ri and R2 -
• CSFs colony stimulating factors
• cytokines are cytokines, lymphokines, interleukins, hematopoietic growth factors which can be joined to Ri
• Ri 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 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand IL- 12.
• SCF stem cell factor
• Ri or R2 molecules or mutated or modified DNA sequences encoding these Ri or R2 molecules .
• a "c-mpl ligand variant” is defined an c-mpl ligand molecule which has amino acid substitutions and/or portions of c-mpl ligand deleted, disclosed in United States Application Serial Number 08/383,035 as well as other variants known in the art.
• a "G-CSF variant” is defined an G-CSF molecule which has amino acid substitutions and/or portions of G-CSF deleted, as disclosed herein, as well as other variants known in the art.
• R2 is G-CSF, GM-CSF, c-mpl ligand or EPO.
• 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.
• Thr and Ala may also be used in the linker sequence. Additional amino acids may also be included in the 41inkers 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:
• 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 :
• the present invention also includes linkers in which an endopeptidase recognition sequence is included. Such a cleavage site may be valuable to separate the individual components of the multifunctional hematopoietic receptor agonist to determine if they are properly folded and active in vitro.
• endopeptidases include, but are not limited to, plasmin, enterokinase, kallikrein, urokinase, tissue plasminogen activator, clostripain, chymosin, collagenase, Russell's viper venom protease, postproline cleavage enzyme, V8 protease, Thrombin and factor Xa.
• Peptide linker segments from the hinge region of heavy chain immunoglobulins 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 (Bell et al . US Patent 4,936,233). These linkers may also include an endopeptidase cleavage site. Examples of such linkers include the following sequences:
• ProProSerLysGluSerHisLysSerPro (SEQ ID NO : 8 ) (collectively referred to herein as "IgG2b" linkers) .
• 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.
• Hematopoietic growth factors can be characterized by their ability to stimulate colony formation by human hematopoietic progenitor cells.
• the colonies formed include erythroid, granulocyte, megakaryocyte, granulocytic macrophages and mixtures thereof.
• Many of the hematopoietic growth factors have demonstrated the ability to restore bone marrow function and peripheral blood cell populations to therapeutically beneficial levels in studies performed initially in primates and subsequently in humans . Many or all of these biological activities of hematopoietic growth factors involve signal transduction and high affinity receptor binding.
• Multi-functional hematopoietic receptor agonists of the present invention may exhibit useful properties such as having similar or greater biological activity when compared to a single factor or by having improved half-life or decreased adverse side effects, or a combination of these properties .
• Multi-functional hematopoietic receptor agonists which have little or no agonist activity maybe useful as antagonists, as antigens for the production of antibodies for use in immunology or immunotherapy, as genetic probes or as intermediates used to construct other useful hIL-3 muteins.
• 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 multifunctional 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 .
• Pairs of complementary synthetic oligonucleotides encoding the desired gene can be made and annealed to each other.
• the DNA sequence of the oligonucleotide would encode sequence for amino acids of desired gene with the exception of those substituted and/or deleted from the sequence.
• Plasmid DNA can be treated with the chosen restriction endonucleases then ligated to the annealed oligonucleotides.
• the ligated mixtures can be used to transform competent bacterial cells such as E. coli strain JM101 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 hematopoietic growth 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.
• DNA genetic material
• 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 hematopoietic growth factor joined by a linker region to a second colony stimulating factor.
• Another aspect of the present invention provides plasmid DNA vectors for use in the expression of these novel multi-functional hematopoietic receptor agonists.
• These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention.
• Appropriate vectors which can transform microorganisms capable of expressing the multi-functional hematopoietic receptor agonists include expression vectors comprising nucleotide sequences coding for the multifunctional 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 multifunctional hematopoietic receptor agonist polypeptides.
• the vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and which are capable of directing the replication and expression thereof in selected host cells .
• a method for producing the novel multi-functional hematopoietic receptor agonists involves culturing suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of a novel multi-functional hematopoietic receptor agonist.
• suitable cells or cell lines may be bacterial cells.
• E. 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 .
• 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).
• 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 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
• the multi functional hematopoietic receptor agonists of the present invention may include multi-functional hematopoietic receptor agonist polypeptides having
• 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 C0S7 , 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.
• general methods for expression of foreign genes in insect cells using Baculovirus vectors are described in: Summers, M. D. and Smith, G. E., 1987) - A manual of methods for Baculovirus vectors and insect cell culture procedures, Texas Agricultural Experiment Station Bulletin No. 1555.
• An expression vector is constructed comprising a Baculovirus transfer vector, in which a strong Baculovirus promoter (such as the polyhedron promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally joined to the coding region for the multi-functional hematopoietic receptor agonist polypeptide.
• a strong Baculovirus promoter such as the polyhedron promoter
• the plasmid pVL1392 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 multi-functional hematopoietic receptor agonist is used to infect cells cultured, for example, in Excell 401 serum-free medium (JRH Biosciences, Lenexa, Kansas) .
• the multi-functional hematopoietic receptor agonist secreted into the medium can be recovered by standard biochemical approaches .
• Supernatants from mammalian or insect cells expressing the multifunctional hematopoietic receptor agonist protein can be first concentrated using any of a number of commercial concentration units.
• the multi-functional hematopoietic receptor agonists of the present invention may be useful in the treatment of diseases characterized by decreased levels of either myeloid, erythroid, lymphoid, or megakaryocyte cells of the hematopoietic system or combinations thereof. In addition, they may be used to activate mature myeloid and/or lymphoid cells.
• 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,
• thrombocytopenia Systemic Lupus Erythromatosis , hemolytic anemia, or fetal maternal incompatibility.
• splenomegaly, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, infection or prosthetic heart valves may result in thrombocytopenia.
• Severe thrombocytopenia may also result from chemotherapy and/or radiation therapy or cancer.
• Thrombocytopenia may also result from marrow invasion by carcinoma, lymphoma, leukemia or fibrosis .
• the multi-functional hematopoietic receptor agonists of the present invention may be useful in the mobilization of hematopoietic progenitors and stem cells in peripheral blood.
• Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation.
• Hematopoietic growth factors including G-CSF and GM-CSF have been shown to enhance the number of circulating progenitors and stem cells in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of pheresis required.
• the multi-functional hematopoietic receptor agonist may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.
• the multi-functional hematopoietic receptor agonists of the present invention may also be useful in the ex vivo expansion of hematopoietic progenitors and stem cells.
• Colony stimulating factors (CSFs) such as hIL-3
• CSFs Colony stimulating factors
• hIL-3 Colony stimulating factors
• 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.
• 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 chimera 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 chimera 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 yeloblasts 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 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, flt3 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.
• GM-CSF (Sato et al . , Blood 82:3600-3609 [1993] ) IL-1 (Muench et al . , Blood 81:3463-3473 [1993] ) IL-6 (Sato et al . , Blood 82:3600-3609 [1993] ) IL-11 (Lemoli et al . , Exp . Hem. 21:1668-1672 [1993], Sato et al . , Blood 82:3600-3609 [1993]), flt3 ligand (McKenna et al .
• 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.
• Gene therapy Potential applications include; 1) the treatment of many congenital metabolic disorders and immunodeficiencies (Kay and Woo, Trends Genet . 10:253-257 [1994]), 2) neurological disorders (Friedmann, Trends Genet . 10:210-214 [1994]), 3) cancer (Culver and Blaese, Trends Genet . 10:174-178 [1994]) and 4) infectious diseases (Gilboa and Smith, Trends Genet . 10:139-144 [1994] ) .
• 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 . 716:36-58 [1994]), electroporation and liposome mediated delivery such as cationic liposomes (Farhood et al., Annal. New York Acad. Sci . 716:23-35 [1994]).
• the present invention provides an improvement to the existing methods of expanding hematopoietic cells, which new genetic material has been introduced, in that it provides methods utilizing multi-functional hematopoietic receptor agonist proteins that have improved biological activity, including an activity not seen by any single colony stimulation factor.
• 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.
• 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
• 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.
• 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.
• compositions for treating the conditions referred to above 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.
• 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.
• 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.
• multifunctional hematopoietic receptor agonist there may exist specific circumstances where dosages of multifunctional 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 hematopoietic growth 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 or sequential administration other hematopoietic growth factors.
• a non-exclusive list of other appropriate hematopoietic growth factors, colony stimulating factors (CSFs), cytokines, lymphokines, and interleukins for simultaneous or serial co- administration with the chimeric proteins of the present invention includes GM-CSF, G-CSF, G-CSF, G-CSF
• c-mpl ligand also known as TPO or MGDF
• M- CSF erythropoietin
• EPO erythropoietin
• IL-1 also known as TPO or MGDF
• EPO erythropoietin
• IL-4 also known as TPO or MGDF
• EPO erythropoietin
• IL-4 also known as TPO or MGDF
• EPO erythropoietin
• IL-1 also known as TPO or MGDF
• EPO erythropoietin
• IL-1 also known as TPO or MGDF
• EPO erythropoietin
• IL-1 also known as TPO or MGDF
• EPO erythropoietin
• 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.
• 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.
• Biological activity of the multi-functional hematopoietic receptor agonist proteins of the present invention can be determined by DNA synthesis in factor-dependent cell lines or by counting the colony forming units in an in vitro bone marrow assay.
• the chimeric proteins may be assayed by a number of in vitro and in vivo models known to those skilled in the art. Examples of such assays include put are not limited to:
• 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) .
• samples are diluted 1:5 with a IX PBS (#14040.059 Life Technologies, Gaithersburg, MD. ) solution in a 50 mL conical tube (#25339-50 Corning, Corning MD) .
• Ficoll Histopaque 1077 Sigma H-8889 is layered under the diluted sample and centrifuged, 300 x g for 30 min. The mononuclear cell band is removed and washed two times in IX PBS and once with 1% BSA PBS (Cell ro Co., Bothel, WA) .
• 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 multifunctional 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 multifunctional 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 baseline response
• cultures received no colony stimulating factors .
• Positive control cultures received conditioned media (PHA stimulated human cells: Terry Fox Lab. H2400). Cultures are incubated at 37 °C, 5% 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). 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.
• Methocult H4330 containing erythropoietin (EPO) was used instead of Methocult H4230, or Stem Cell Factor (SCF) , 50 ng/mL (Biosource International, Camarillo,
• CA CA
• the factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) .
• This cell line established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM-CSF supplemented medium (Lange, B., et al . , Blood 70: 192, 1987; Valtieri, M., et al., J. Immunol . 138:4042, 1987).
• the ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D. , et al., J. Immunol . 139: 348, 1987).
• a cell line variant was used, AML 193 1.3, which was adapted for long term growth in IL-3 by washing out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours.
• the cells are
• 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
• 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
• albumin transferrin
• lipids transferrin
• 2- mercaptoethanol 2- mercaptoethanol
• Serial dilutions of human interleukin-3 or multi-functional chimeric 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 chimeric hematopoietic 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
• Activity of each human interleukin-3 preparation or multi-functional chimeric hematopoietic receptor agonist protein preparation is quantitated by measuring cell proliferation ' ( 3 H-thymidine incorporation) induced by graded concentrations of interleukin-3 or multi- functional chimeric hematopoietic receptor agonist.
• concentration ranges from 0.05 pM - 10 pM are quantitated in these assays .
• This EC 50 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 chimeric hematopoietic receptor agonist proteins were tested in a concentration range of 2000 pM to 0.06 pM titrated in serial 2 fold dilutions.
• the c-mpl ligand proliferative activity can be assayed using a subclone of the pluripotential human cell line TFl (Kitamura et al . , J. Cell Physiol
• TFl 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. After dilution cloning, a c-mpl ligand responsive clone is selected, and these cells are split into passage media to a density of 0.3 x 10 cells/mL the day prior to assay 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) .
• RPMI 1640 Gibco
• FBS Hard, Lot #91206
• 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 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%.
• MUTZ-2 Cell Proliferation Assay A cell line such as MUTZ-2, which is a human myeloid leukemia cell line (German Collection of Microorganisms and Cell Cultures, DSM ACC 271), can be used to determine the cell proliferative activity of flt3 receptor agonists. MUTZ-2 cultures are maintained with recombinant native flt3 ligand (20- lOOng/mL) in the growth medium. Eighteen hours prior to assay set-up, MUTZ-2 cells are washed in IMDM medium (Gibco) three times and are resuspended in IMDM medium alone at a concentration of 0.5-0.7 x
• the starved MUTZ-2 cells are washed two times in IMDM medium and resuspended in 50 ⁇ l assay media. 50 ⁇ l of cells are added to each well resulting in a final concentration of 0.25 x 10E6cells/mL. Assay plates containing cells are incubated at 37°C and 5%C0 2 for
• Each well is then pulsed with l ⁇ Ci/well of tritiated thymidine in a volume of 20 ⁇ l for four hours. Plates are then harvested and counted.
• in vitro cell based proliferation assays Other in vitro cell based assays, known to those skilled in the art, may also be useful to determine the activity of the multi-functional chimeric 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.
• TFl proliferation assay TFl is a pluripotential human cell line (Kitamura et al . , J. Cell Physiol 140:323-334. [1989]) that responds to hIL-3.
• 32D proliferation assay 32D is a murine IL-3 dependent cell line which does not respond to human IL-3 but does respond to human G-CSF which is not species restricted.
• Baf/3 proliferation assay Baf/3 is a murine IL-3 dependent cell line which does not respond to human
• IL-3 human flt3 ligand 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 hematopoietic growth 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 hematopoietic growth 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 chimeric hematopoietic receptor agonists.
• Bone marrow aspirates (15-20 mL) were obtained from normal allogeneic marrow donors after informed consent.
• Cells were diluted 1:3 in phosphate buffered saline (PBS, Gibco-BRL) , 30 mL were layered over 15 mL Histopaque-1077 (Sigma) and centrifuged for 30 minutes at 300 RCF .
• the mononuclear interface layer was collected and washed in PBS.
• CD34+ cells were enriched from the mononuclear cell preparation using an affinity column per manufacturers instructions (CellPro, Inc., Bothell WA) .
• 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 chimeric 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% C0 2 in a 37°C humidified incubator.
• MK buffer 13.6 mM sodium citrate, 1 mM theophylline, 2.2 ⁇ m PGE1, 11 mM glucose, 3% w/v BSA, in PBS, pH 7.4,
• MK buffer 13.6 mM sodium citrate, 1 mM theophylline, 2.2 ⁇ m PGE1, 11 mM glucose, 3% w/v BSA, in PBS, pH 7.4,
• CD41a-FITC Green fluorescence
• PI red fluorescence
• 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,
• EXAMPLES 1 & 2 Construction of an expression vectors, pMON32364 and pMON32377, comprising a DNA sequence encoding a multi-functional receptor agonist comprising an IL-3 (15-125) variant joined via the IgG2b linker to a flt-3 (1-134) ligand and an IL-3 (15-125) variant joined via the IgG2b linker to a flt-3 (1-139) ligand, respectively.
• the ligation mixtures were used to transform competent DH5a cells (Gibco BRL cat #18265-017) following the manufacturer's recommended protocol, and vector DNA was isolated from ampicillin-resistant colonies.
• the DNA sequence of resulting genes (SEQ ID NO: 21 and SEQ ID NO: 22 respectively) were determined by automated fluorescent DNA sequencing on an ABI 373/377 DNA sequencer (Perkin Elmer ABI) using Sequencher (Gene Codes) software.
• the resulting vectors, pMON32364 and pMON32377 encodes the proteins of SEQ ID NO: 42 and SEQ ID NO: 43 respectively.
• an expression vectors PMON30247 and pMON30246, comprising a DNA sequence encoding a multi-functional receptor agonist comprising an IL-3 (15-125) variant joined via the IgG2b linker to a flt-3 (1-134) ligand and an IL-3 (15-125) variant joined via the GlySer linker to a flt-3 (1-134) ligand, respectively.
• an expression vectors PMON30249 and pMON30248, comprising a DNA sequence encoding a multi-functional receptor agonist comprising an IL-3 (15-125) variant joined via the IgG2b linker to a flt-3 (1-139) ligand and an IL-3 (15-125) variant joined via the GlySer linker to a flt-3 (1-139) ligand, respectively.
• Plasmids, PMON30248 and pMON30249, were constructed by cloning the gel purified Ncol/Afllll restriction fragment from pMON30244 (GlySer linker) and pMON30245 (IgG2B linker) respectively, into vector, pMON30238, digested with Ncol (which contains hFlt3L 1-139) as described in Examples 1 & 2.
• the DNA sequence of resulting genes, SEQ ID NO: 15 and SEQ ID NO: 16 encodes the proteins of SEQ ID NO: 36 and SEQ ID NO: 37, respectively.
• PMON32392 and pMON32393, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-134) ligand joined via the IgG2b linker to an IL-3 (15-125) variant and a flt-3 (1-139) ligand joined via the IgG2b linker to an IL-3 (15-125) variant, respectively.
• Plasmids, pMON32392 and pMON32393, were constructed by Polymerase Chain Reaction (PCR) methods.
• Plasmid, pMON30237 and pMON30238, DNA was used as the template in the PCR reaction with primer pairs N-term SEQ ID NO:29/134rev SEQ ID NO:30 and N-term SEQ ID NO:29/139rev SEQ ID NO:31, respectively, to introduce an in-frame SnaBI restriction site at the C-terminus.
• Standard PCR reaction mixtures were set up using an Invitrogen PCR Optimizer kit (Invitrogen) .
• Amplification cycle conditions were as follows: seven cycles of 94°C, one minute, 65°C, two minutes, and 72°C 2 1/2 minutes; followed by ten cycles of 94°C, one minute, 70°C, two minutes, and 72 °C 2 1/2 minutes.
• the product of the PCR reactions were purified using the Wizard PCR Purification kit (Promega) , and eluted in 50 ⁇ l dH 2 0. 20 ⁇ l of each purified PCR product were digested in 50 ⁇ l reaction mixture volumes with 10U each of Ncol and SnaBI for 90 minutes at 37°C.
• One ⁇ g of vector, PMON26431 (derivative of pMONl3061 - W095/21254), was digested with 7.5U each of Ncol and SnaBI in a 20 ⁇ l reaction volume for 90 minutes at 37°C, followed by the addition of 1U shrimp alkaline phosphatase.
• the reaction was incubated an additional 10 minutes at 37°C, and both inserts and vector were gel purified as described previously. Ligation times and temperatures were modified to include incubation at 16°C for 3 hour, followed by 2 hour at ambient temperature.
• Plasmid, pMON30328 comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-134) ligand joined via the IgG2b linker to a G-CSF receptor agonist.
• Plasmid, pMON30328 was constructed by subcloning the gel-purified Ncol/Hindlll restriction fragment from pMON30237 into plasmid, pMON30309 (derivative of pMONl3149 - W095/21254), digested with Afllll/Hindlll (contains G-CSF/IgG2b-Afllll/Hindlll) as described in Examples 1 & 2.
• the DNA sequence of resulting gene, SEQ ID NO: 50 encodes the protein of SEQ ID NO: 60.
• EXAMPLE 10 Construction of an expression vector, pMON30329, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a G- CSF receptor agonist joined via the IgG2b linker to a flt3 (1-139) ligand. Plasmid, pMON30329, was constructed by subcloning the gel-purified Ncol/Hindlll restriction fragment from pMON30238 into plasmid pMON30309 digested with Afllll/Hindlll (which contains G-CSF/IgG2b-
• Plasmid, pMON32175, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a G-CSF receptor agonist.
• Plasmid, pMON32175, was constructed by subcloning the gel-purified NcoI/SnaBI restriction fragment from pMON32393 into pMON26430 (derivative of pMON13060 - W095/21254) digested with NcoI/SnaBI as described in Examples 1 & 2.
• the DNA sequence of resulting gene, SEQ ID NO: 19, encodes the protein of SEQ ID NO: 40.
• an expression vector comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a G-CSF receptor agonist.
• pMON32191 was assembled by subcloning the gel-purified NcoI/SnaBI restriction fragment from pMON32393 SEQ ID NO: 58 into plasmid pMON31123 digested with NcoI/SnaBI (which contains the GlySer/G-CSF moiety) as described in Examples 1 & 2.
• the DNA sequence of resulting gene, SEQ ID NO: 20, encodes the protein of SEQ ID NO: 41.
• Plasmid, pMON35767 comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a G-CSF receptor agonist.
• Plasmid, pMON35767 was constructed by subcloning the gel-purified Ncol/Hindlll restriction fragment from pMON32191 SEQ ID NO: 20 into the BHK expression vector PMON3934, which is a derivative of pMON3359.
• pMON3359 is a pUC18-based vector containing a mammalian expression cassette.
• the cassette includes a herpes simplex viral promoter IE110 (- 800 to +120) followed by a modified human IL-3 signal peptide sequence and an SV40 late polyadenylation (poly-A) signal which has been subcloned into the pUC18 polylinker (See Hippenmeyer et al . , Bio/Technology, 1993, pp.1037- 1041) .
• Plasmid, pMON32173, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a flt3 (1-139) ligand.
• Plasmid, pMON32173, was constructed by subcloning the gel-purified ⁇ 130bp NcoI/SacI restriction fragment from pMON32342 SEQ ID NO: 52 and the ⁇ 290bp SacI/SnaBI restriction fragment from pMON32393 into plasmid pMON30329 digested with NcoI/SnaBI as described in Examples 1 & 2.
• the DNA sequence of resulting gene, SEQ ID NO: 18, encodes the protein of SEQ ID NO: 39.
• an expression vector comprising a DNA sequence encoding a multi-functional receptor agonist comprising a c-mpl (1-153) ligand joined via the IgG2b linker to a flt3 (1-139) ligand.
• Plasmid, pMON45419 was constructed by subcloning the NcoI/SnaBI restriction fragment from pMON26474 (derivative of pMON26472 - W095/21254) into plasmid, PMON32173 SEQ ID NO:56, digested with NcoI/SnaBI as described in Examples 1 & 2.
• the DNA sequence of resulting gene, SEQ ID NO: 25, encodes the protein of SEQ ID NO: 46.
• pMON45420 comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a c-mpl (1-153) ligand.
• Plasmid, pMON45420 (derivative of pMON26471 - W095/21254) was assembled by subcloning the
• NcoI/SnaBI restriction fragment from pMON32191 into plasmid, pMON26473, digested with NcoI/SnaBI as described in Examples 1 & 2.
• the DNA sequence of resulting gene, SEQ ID NO: 26, encodes the protein of SEQ ID NO: 47.
• Plasmid pMON46408 which encodes a multi-functional receptor agonist comprising EPO joined via a Gly Ser linker to a flt3 (1-139) ligand
• Plasmid pMON46408 was constructed in a two step cloning procedure. First, an intermediate plasmid, pMON46406, was constructed. This plasmid encodes the human EPO sequence joined to a GlySer linker sequence containing the restriction enzyme sites Afllll and Hindlll. The following three DNA fragments were ligated together to form plasmid pMON46406: 1. A 480 bp NcoI-StuI fragment encoding EPO except for the terminal 6 amino acids
• the ligation mixture was used to transform competent MON105 cells and transformants were selected on LB Amp plates . Colonies were picked and analyzed by DNA sequencing analysis to identify a correct clone. A correct clone was assigned pMON46406.
• plasmid pMON46406 was digested with Afllll and Hindlll and the vector portion was purified. This was ligated with the 423 bp Ncol-Hindlll fragment of plasmid PMON32342 SEQ ID NO: 52, which encodes the flt-3 (1-139) ligand. The ligated ligation mixture was used to transform competent MON105 cells and transformants were selected on LB Amp plates. Colonies were picked and analyzed by DNA sequencing analysis to identify a correct clone. A correct clone was assigned pMON46408. The DNA sequence of resulting gene, SEQ ID NO: 28, encodes the protein of SEQ ID NO: 49.
• EXAMPLE 18 Determination of bioactivity of selected chimera proteins Selected chimeras of the present invention were assayed in a Baf3 cell line transfected with the flt3/flk2 receptor (Baf3/flt3) to determine flt3 ligand bioactivity.
• MOLECULE TYPE peptide ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 1 :
• AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCATGGATC 360
• Lys Ser Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu
• Phe Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr 225 230 235 240
• AAAGAATCTC ATAAATCTCC AAACATGGCT ACCCAGGACT GCTCCTTCCA ACACAGCCCC 660
• CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG 780 GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG 340
• AAAGAATCTC ATAAATCTCC AAACATGGCC ACCCAGGACT GCTCCTTCCA ACACAGCCCC 660
• AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCATGGATC 360
• CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG 660 CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT 720
• AAAGAATCTC ATAAATCTCC AAACATGGCT AACTGCTCTA TAATGATCGA TGAAATTATA 540
• AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360 ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420
• GTCCTTCACA GCAGACTGAG CCAGTGCCCA
• GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 600
• CAAGATTACC CAGTCACCGT GGCCTCCAAC CTGCAGGACG AGGAGCTCTG CGGGGGCCTC 720
• GCCCCACCAC GCCTCATCTG TGACAGCCGA GTCCTGGAGA GGTACCTCTT GGAGGCCAAG 60

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Hematology (AREA)
• Biochemistry (AREA)
• Genetics & Genomics (AREA)
• Gastroenterology & Hepatology (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Zoology (AREA)
• Toxicology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Diabetes (AREA)
• Biophysics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Priority Applications (7)

Applications Claiming Priority (2)

Publications (1)

Family

ID=25273300

Family Applications (1)

Country Status (8)

Cited By (5)

Citations (3)

• 1998
  • 1998-04-10 JP JP54422398A patent/JP2001527396A/ja not_active Abandoned
  • 1998-04-10 EP EP98915573A patent/EP0973900A1/en not_active Withdrawn
  • 1998-04-10 AU AU69721/98A patent/AU751498B2/en not_active Ceased
  • 1998-04-10 BR BR9808514-0A patent/BR9808514A/pt not_active Application Discontinuation
  • 1998-04-10 PL PL98336159A patent/PL336159A1/xx unknown
  • 1998-04-10 CA CA002284127A patent/CA2284127A1/en not_active Abandoned
  • 1998-04-10 NZ NZ337911A patent/NZ337911A/en unknown
  • 1998-04-10 WO PCT/US1998/007511 patent/WO1998046750A1/en not_active Application Discontinuation

Patent Citations (3)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events