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EP0503050A1 - Facteurs de croissance hybride - Google Patents

Facteurs de croissance hybride

Info

Publication number
EP0503050A1
EP0503050A1 EP91918221A EP91918221A EP0503050A1 EP 0503050 A1 EP0503050 A1 EP 0503050A1 EP 91918221 A EP91918221 A EP 91918221A EP 91918221 A EP91918221 A EP 91918221A EP 0503050 A1 EP0503050 A1 EP 0503050A1
Authority
EP
European Patent Office
Prior art keywords
leu
ala
ser
thr
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Withdrawn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
EP91918221A
Other languages
German (de)
English (en)
Other versions
EP0503050A4 (en
Inventor
Jonathan I. Rosen
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.)
Ortho Pharmaceutical Corp
Original Assignee
Ortho Pharmaceutical Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Ortho Pharmaceutical Corp filed Critical Ortho Pharmaceutical Corp
Publication of EP0503050A1 publication Critical patent/EP0503050A1/fr
Publication of EP0503050A4 publication Critical patent/EP0503050A4/en
Withdrawn legal-status Critical Current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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • a variety of factors can influence the activity of a cell. Frequently a factor exerts its influence by interacting with a receptor on the surface of a cell. After binding to the receptor, the signal which determines the cellular response to the factor can be mediated through a number of different events, including internalization of the factor or alterations of the receptor caused by ligand binding.
  • a number of different factors are involved in the maturation of a pluripotent stem cell into a fully differentiated cell. The activities of these factors during the course of hematopoietic differentiation have resulted in these factors being characterized as early factors or late factors.
  • factors such as interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) are considered early factors, while erythropoietin (Epo), macrophage colony stimulating factor (M-CSF), and granulocyte colony stimulating factor (G-CSF) are considered late factors.
  • IL-3 interleukin-3
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • Epo erythropoietin
  • M-CSF macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • IL-3 and GM-CSF act on pluripotent cells before they become committed to a particular hematopoietic pathway. After the events stimulated by these factors are underway, such lineage restricted cells become receptive to further differentiation mediated by such late factors as Epo, (which leads to the maturation of erythrocytes), G-CSF (which leads cells into the granulocytic pathway), and M-CSF (which leads to the maturation of macrophages) .
  • Epo which leads to the maturation of erythrocytes
  • G-CSF which leads cells into the granulocytic pathway
  • M-CSF which leads to the maturation of macrophages
  • the present invention concerns hybrid molecules comprising early and late differentiation factors produced by genetic manipulation.
  • the local concentration of the late factor is very high at the surface of a cell to which the early factor is bound.
  • binding of late factors to any remaining low-affinity receptors e.g. Epo receptors, could be enhanced, thus reducing the amount of late factor required to stimulate the cell.
  • such early factor may act more specifically to stimulate only the desired lineage, thus reducing any undesirable effects mediated by the early factor.
  • it is considerably easier to produce and administer to a patient a single factor with two activities rather it would to produce and administer two separate factors.
  • Figure 1 shows a Western blot analysis of IL-3/Epo hybrid growth factors in CHO CM.
  • CHO CM was collected from clones 23-10 (IL-3:Epo Flex), 5-4 (IL-3:Epo Short) and 17-3-1 (Epo:IL-3 Short).
  • Hybrid growth factor concentrations were determined by ELISA assay.
  • CM containing 74 ng of IL-3:Epo Flex (having a 23 aa flexible linker (lane 2), 73.5 ng of IL-3:Epo Short (having a short 2 aa linker) (lane 3), 80 ng of Epo:IL-3 Short (having a 3 aa linker) (lane 4 ) were subjected to SDS-PAGE (10-20% gel) electrophoresis and were assayed for Epo by Western blotting with a mouse anti-Epo polyclonal antisera as described in Example 7.
  • FIG. 2 shows AML193 cells proliferate in response to the IL-3 moiety of the hybrid growth factors.
  • AML193 cells were grown to stationary phase and suspended in RPMI-1640 plus 10% FCS and growth factor deprived for 16 hours. The indicated concentrations of growth
  • CM medium conditioned by CHO cells transfected with the vector pEe ⁇
  • Epo rHu Epo
  • IL-3 rHu IL-3
  • IL-3:Epo Flex CHO CM containing IL-
  • Figure 3 shows dose response of IL-3 adapted AML193 cells to the IL-3 moiety of the hybrid growth factors.
  • IL-3 adapted AML 193 cells were grown to stationary phase and suspended in RPMI-1640 plus 10% FCS minus growth factor for 16 hours. Increasing concentrations of IL-3 and fusion proteins were added and the assay was carried out as described in Figure 2 and in Example 7.
  • IL-3:Epo Flex (CHO CM containing IL-3:Epo fusion protein with a 23aa flexible linker);
  • IL- 3:Epo Short (CHO CM containing IL-3:Epo fusion protein with a 2aa linker); Epo:IL-3 Short (CHO CM containing Epo:IL-3 fusion protein with a 3aa linker).
  • FIG 4 shows FDC-Pl/ER cells proliferate in response to the Epo moiety of the hybrid growth factors.
  • FDC-Pl/ER cells were grown to stationary phase and suspended in RPMI-1640 plus 10% FCS without growth factor for 16 hours. The indicated concentrations of growth factors were added for 42 hours followed by a 6 hour pulse of ( H) thymidine as described in Example 7. Columns are labeled as described in Figure 2.
  • WEHI3 CM medium conditioned by murine WEHI3 cells which produce and secrete IL-3).
  • Figure 5 shows dose response of FDC-Pl/ER cells to the Epo moiety of the hybrid growth factors.
  • FDC-Pl/ER cells were grown to stationary phase and suspended in RPMI-1640 plus 10% FCS and deprived of growth factor for 16 hours. Increasing concentrations of Epo and fusion proteins were added and the assay was carried out as described in Example 7.
  • Hybrid growth factors are as designated in Figure 3.
  • Figure 6 shows IL-3 plus Epo responsiveness of IL-3 adapted TF-1 cells. TF-1 cells adapted for growth in IL-3 were grown to stationary phase and suspended in RPMI-1640 plus 10% FCS minus growth factor for 16 hours.
  • Figure 7 shows dose responsiveness of IL-3 adapted TF-1 cells to the hybrid growth factors.
  • TF-1 cells adapted for growth in IL-3 were grown to log phase and suspended in RPMI-1640 plus 10% FCS minus growth factor for 16 hours. Increasing concentrations of hybrid growth factors were added and the cells were incubated for 8 hours. 3
  • Thymidine (1 ⁇ Ci/well) was added and the incubation was continued for 16 hours.
  • A Dose response to hybrid growth factor, concentrations of 0 to 30 fmol/ml.
  • B Represents the same data as in A for concentrations of 0 to 1.875 fmol/ml to emphasize the differences between hybrid factors.
  • Hybrid growth factors are as designated in Figure 3.
  • Figure 8 shows dose responsiveness of GM-CSF adapted TF-1 cells to the hybrid growth factors.
  • TF-1 cells maintained in GM-CSF were grown to log phase and suspended in RPMI-1640 plus 10% FCS minus growth factor for 16 hours. Increasing concentrations of hybrid growth factors were added and the assay was carried out as described above for Figure 5.
  • A Dose response to hybrid growth factor ccncentrations, of 0 to 30 fmol/ml.
  • B Represents the same data as in A for concentrations of 0 to 1.875 fmol/ml to emphasize the differences between hybrid factors.
  • Hybrid growth factors are as designated in Figure 3.
  • FIG 9 shows TF-1 cells proliferate in response to the IL-3 moiety of the IL-3/G-CSF hybrid growth factor.
  • TF-1 cells were grown to stationary phase and suspended in RPMI-1640 plus 10% FCS deprived of growth factor for 16 hours. The indicated concentrations of growth factors were added and the assay was carried out as described in Example 7.
  • Factors are as designated in Figure 2 except, G-CSF (rHu G-CSF); IL-3/G-CSF (CHO CM containing IL-3/G-CSF fusion protein with a lOaa linker).
  • Figure 10 shows NFS-60 cells proliferate in response to the G-CSF moiety of the IL-3/G-CSF hybrid growth factor.
  • NFS-60 cells were grown to stationary phase and suspended in RPMI-1640 plus 10% FCS minus growth factor for 16 hours. The indicated concentrations of growth factors were added and the assay was carried out as described in Example 7. Growth factors are as designated in Figures 2 and 9.
  • Figure 11 shows dose responsiveness of AML193 cells to the IL-3:G-CSF hybrid growth factor.
  • AML193 cells were grown to stationary phase and suspended in RPMI-1640 plus 10% FCS deprived of growth factor for 16 hours. The indicated concentrations of growth factors were added and the assay was carried out as described in Example 7. Growth factors are as designated in Figures 2 and 9.
  • the present invention provides a recombinant hematopoietic molecule comprising at least a portion of a first hematopoietic molecule having early myeloid differentiation activity and at least a portion of a second hematopoietic molecule having late myeloid differentiation activity.
  • This recombinant molecule has early myeloid differentiation activity associated with the first hematopoietic molecule and late myeloid differentiation activity associated with the second hematopoietic molecule.
  • hematopoietic molecule means a molecule which promotes and/or regulates hematopoiesis.
  • Hematopoietic molecules exert such promotional or regulatory activities at different stages during hematopoiesis, such stages being referred to herein as early myeloid differentiation and late myeloid differentiation.
  • early myeloid differentiation activity means the ability to promote the differentiation, self-renewal, or proliferation of pluripotent myeloid cells, i.e., stem cells or colony forming unit, granulocyte-erythrocyte-monocyte-megacaryocyte, cells.
  • late myeloid differentiation activity means the ability to promote the maturation or differentiation of a lineage restricted myeloid cell, i.e., a myeloid precursor cell committed to a specific cell lineage such as erythrocytes, megakaryocytes,. monocytes, neutrophils, eosinophils, and basophils.
  • the first hematopoietic molecule is selected from the group consisting of IL-3 and GM-CSF.
  • the second hemopoietic molecule is selected from the group consisting of Epo, G-CSF, IL-5 and M-CSF.
  • the portion of the first hematopoietic molecule is linked to the portion of the second hematopoietic molecule by an amino acid linker sequence comprising at least two amino acid residues.
  • the recombinant molecule comprises the entire amino acid sequence of human IL-3 (SEQ ID NO: 1). Moreover, the recombinant hematopoietic molecule may preferably comprise a 79 amino acid sequence derived from human IL-3 (SEQ ID NO: 2),i.e. residues 1-79 of SEQ ID NO: 1.
  • the recombinant molecule comprises the entire amino acid sequence of human erythropoietin (SEQ ID NO: 3).
  • the hemopoietic molecule comprises a 155 amino acid sequence derived from human erythropoietin (SEQ ID NO: 4), i.e., residues 7-161 of SEQ ID NO: 3.
  • the recombinant hematopoietic molecule comprises the entire amino acid sequence of human G-CSF (SEQ ID NO: 5).
  • the first hematopoietic molecule is IL-3 and the second hematopoietic molecule is erythropoietin.
  • the first hematopoietic molecule, i.e. IL-3 may comprise the amino portion and the second hematopoietic molecule, i.e. Epo, may comprise the carboxyl portion of the recombinant molecule.
  • the recombinant hematopoietic molecule comprises the amino acid sequence from amino acid 1 to amino acid 302 of SEQ ID NO: 6.
  • the recombinant hematopoietic molecule comprises the amino acid sequence from amino acid 1 to amino acid 321 of SEQ ID NO: 7.
  • the first hematopoietic molecule i.e. IL-3
  • the second hemopoietic molecule i.e. Epo
  • the recombinant molecule comprises the amino acid sequence from amino acid 1 to amino acid 303 of SEQ ID NO: 8.
  • the recombinant molecule comprises the amino acid sequence from amino acid 1 to amino acid 322 of SEQ ID NO: 9.
  • the first hematopoietic molecule is IL-3 and the second hematopoietic molecule is G-CSF.
  • the first hematopoietic molecule comprises the amino portion and the second hematopoietic molecule comprises the carboxyl portion of the recombinant molecule.
  • the recombinant molecule comprises the amino acid sequence from amino acid 1 to amino acid 317 of SEQ ID NO: 10.
  • the subject invention also provides nucleic acid molecules which encode the recombinant hematopoietic molecules of the subject invention.
  • nucleic acid molecules are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.
  • vectors which comprise the nucleic acid molecules of the subject invention are also disclosed.
  • the vector comprises a plasmid.
  • host vector systems for the production of a recombinant hematopoietic molecule of the present invention are provided which comprise a vector of the present invention in a suitable host, preferably a mammalian cell such as a CHO or COS cell.
  • This host vector system may be grown under suitable conditions which permit the expression of the recombinant hematopoietic molecule, which may be recovered by purification techniques known in the art, e.g. ion exchange chromatography, affinity chromatography, and size exclusion chromatography.
  • the present invention further provides pharmaceutical compositions useful for treating patients suffering from anemias of various origins, e.g. renal failure, and AIDS. Moreover, these pharmaceutical compositions are useful for administering to patients for preoperative autologous blood donations, patients receiving or donating bone marrow for transplantation purposes, and patients undergoing cancer chemotherapy. These pharmaceutical compositions comprise effective hematopoiesis-pro oting amounts of a recombinant molecule of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are known in the art and are disclosed in The Pharmacopeia of the United States and the National Formulary. Depending on the specific application contemplated, the pharmaceutical composition may be formulated as a solution, suspension, parenteral preparation, or spray.
  • Parenteral preparations may include a vehicle such as specially distilled, pyrogen-free water, phosphate buffer, or normal saline.
  • Oral and/or transmucosal dosage forms may comprise phospholipids, often in the form of liposomes.
  • Also provided is a method for treating a patient to promote hematopoiesis which comprises administering to the patient an effective hematopoiesis-promoting amount of a pharmaceutical composition of the present invention.
  • hybrid protein genes Genes encoding IL-3 (SEQ ID NO: 16), Epo (SEQ ID NO: 17) and G-CSF (SEQ ID NO: 18) were purchased from British Biotech. Ltd. These genes were utilized to construct three different hybrid hematopoietic proteins, i.e., IL-3:Epo, Epo:IL-3 and IL-3:G-CSF. In these hybrids the first named gene forms the amino portion and the second named gene the carboxyl portion of the hybrid protein.
  • a nucleic acid molecule encoding an IL-3:Epo hybrid growth factor was constructed as follows: CSF, the native leader sequence of IL-3 was synthesized as 4 oligonucleotides (SEQ ID NOS: 19-22; see Table I) which represents both strands of the leader sequence.
  • SEQ ID NOS: 19-22 the native leader sequence of IL-3 was synthesized as 4 oligonucleotides (SEQ ID NOS: 19-22; see Table I) which represents both strands of the leader sequence.
  • the 5' end of the leader (SEQ ID NO: 19) encoded a convenient restriction enzyme overhang (EcoRI), although the EcoRI site was not regenerated, in front of the ATG start codon.
  • the 3' end of the leader included the first several amino acid codons of IL-3 and an Spel overhang so that the annealed leader sequence could be, easily ligated to IL-3, which was altered by British Biotech to include an Spel site.
  • the leader sequence was annealed and ligated to pKS (Stratagene Cloning Systems, Inc., San Diego, CA) cleaved with EcoRI and Spel. The resulting plasmid was designated pKSO.
  • the IL-3 containing pUC18 plasmid obtained from British Biotech was cleaved with Spel and Nhel, then ligated to a linker oligonucleotide (complimentary oligonucleotide SEQ ID NOS: 23 and 24; see Table I) which contained the following three restriction sites: Nhel, Xbal and Ncol. Cleavage was then performed with Spel and Xbal. The resulting 379 base pair fragment was then ligated to PKSO cleaved with
  • the resulting plasmid (pKSOIL-a) contained the IL-3 leader, the IL-3 gene and a small linker fragment.
  • Epo gene was inserted into pEe6 (Celltech, Ltd., Slough, U.K.), a mammalian expression vector which contains the human Cytomegalovirus promoter, a polylinker region and a poly-A addition site in addition to ampicillin resistance and a bacterial origin of replication, by cleaving the Epo containing plasmid obtained from British Biotech with Hindlll and BamHI. Epo was then cleaved with Ncol. The same linker comprising oligonucleotide SEQ ID NOS: 23 and 24 as described earlier was ligated to Epo and then cleaved with Xbal to yield the entire Epo gene.
  • pEe6 Celltech, Ltd., Slough, U.K.
  • PKSOIL-a was cleaved with EcoRV and an Xbal linker was ligated to the blunt ends followed by cleavage with Xbal, which released the IL-3 gene with the leader sequence.
  • Xbal linker was ligated to the blunt ends followed by cleavage with Xbal, which released the IL-3 gene with the leader sequence.
  • Xbal cleaved Peepo to yield a plasmid containing an entire hybrid protein gene (pEepie-a) (see SEQ ID NO: 11 for the structure of the inserted hybrid gene, designated herein IL- 3:Epo Short).
  • the glutamine synthetase (gs) gene was then inserted into the BamHI site of pEepie-a to yield pEepogs-a or pEpogs-b, depending upon the orientation of the gs gene.
  • Glutamine synthetase confers resistance to methionine sulphoximine (MSX) in order to select cells which have taken up the plasmid after transfection.
  • MSX methionine sulphoximine
  • After the plasmid was constructed a large batch was grown, purified by CsCT ultracentrifugation, and used for transfection. At eac ⁇ step in this process all ligation joints between fragments were analyzed by DNA sequence analysis in order to assure that there were no changes that would cause frameshifts and prevent the hybrid gene from being expressed.
  • pEepie-a was cleaved with Nhel and annealed oligonucleotide SEQ ID NOS: 25 and 26 (see Table I) were ligated into the cleaved plasmid.
  • This linker encodes the flexible amino acid sequence Gly Ser Gly Ser Gly Ser (SEQ ID NO: 27).
  • Clones with the insert in the proper orientation were selected by probing colonies with the junction oligonucleotide SEQ ID NO: 28 (see SEQ ID NO: 14 for the structure of the inserted hybrid gene, designated herein IL-3:Epo Flex). The glutamine synthetase gene was then added to the construct as described above.
  • a nucleic acid molecule encoding an IL-3:G-CSF hybrid growth factor was constructed as follows: pUC18 containing G-CSF (British Biotech) was cleaved with Hindlll. A linker composed of an overhanging Xbal site, a NotI site and an overhanging Hindlll site (oligonucleotide SEQ ID NOS: 29 and 30; see Table I) was ligated to the pUC18:G-CSF. This was then cleaved with Xbal and BamHI which released the entire G-CSF gene. The G-CSF fragment was then inserted into Xbal and Bell cleaved pEe6 (pEe6:G-CSF).
  • IL-3 with its signal sequence was removed from the IL-3:Epo plasmid pEepogs-a as an Xbal fragment.
  • This IL-3 fragment was then inserted into Xbal cleaved pEe6-G-CSF.
  • pEGll a plasmid containing the IL-3 gene in the proper orientation was obtained (pEGll), this plasmid encoded a gene capable of expressing IL-3 and G-CSF as a hybrid protein (see SEQ ID NO: 13 for the structure of the inserted hybrid gene, designated herein IL- 3:G-CSF).
  • the gs gene was inserted into this plasmid as described in Example 1 above to yield plasmids pEG13 and pEG14, depending upon the orientation of the gs gene.
  • a nucleic acid molecule encoding an Epo:IL-3 hybrid growth factor was constructed by first synthesizing the native Epo signal sequence as oligonucleotide SEQ ID NOS: 31-36 (see Table I). These were annealed to yield an overhanging 5' Xhol sequence and a 3' PstI sequence. These were then ligated and subcloned as an XhoI/PstI fragment (pEpol). In order to obtain the proper reading frame and signal sequence processing site, the plasmid containing the signal sequence was cleaved with PstI and the 3' overhang left by PstI was enzymatically removed with T4 polymerase. This was then cleaved with BamHI.
  • Epo gene was then amplified by PCR as a fragment with a 5' blunt end using oligonucleotide SEQ ID NO: 37 as a primer and a 3' BamHI end using oligonucleotide SEQ ID NO: 38 as a primer. This fragment was then ligated into pEpol to yield a complete Epo gene with its leader sequence. PCR was used to amplify the Epo gene with its signal sequence as an (5') Xbal and (3') Notl fragment using oligonucleotide SEQ ID NOS: 39 and 40 as primers. This was then digested with Xbal and Notl.
  • IL-3 fragment was amplified by PCR as a (5 Notl and (3') BamHI fragment using oligonucieotide SEQ ID NOS: 41 and 42, followed by digestion with Notl and BamHI. These two fragments were ligated to pEe6 cleaved with Xbal and Bell to yield a full length hybrid gene encoding both Epo and IL-3 (pEG16) (see SEQ ID NO: 12 for the structure of the inserted hybrid gene, designated herein Epo:IL-3 Short).
  • the gs gene was inserted as described in Example 1 above to yield pEG17 and pEG18, depending upon the orientation of the gs gene.
  • a flexible linker is inserted into Epo:IL-3 by cleaving pEG17 or pEGl ⁇ with Notl.
  • Annealed oligonucleotide SEQ ID NOS: 43 and 44 are -then ligated into the cleaved plasmid.
  • Clones with the insert in the proper orientation are selected by probing colonies with a junction oligonucleotide as described above (see SEQ ID NO: 15 for the structure of the inserted hybrid gene.)
  • CTAGCGATCT TTCTAGA (SEQ ID NO: 23) CATGTCTAGA AAGATCG (SEQ ID NO: 24)
  • CTAGTCTCTA GAATGGGGGT CCACGAATGT (SEQ ID NO: 39)
  • the cells were transferred to GMEM-S supplemented with 25 mM MSX after 24 hours. The MSX concentration was subsequently increased to 50 M after one week. Cloning rings were used to subclone MSX resistant colonies and each of these colonies was placed into an individual well of a 24 well plate. Selected clones were incubated in the absence of MSX to insure that the hybrid protein gene was stably integrated. Strongly positive clones were grown in large cultures to provide larger amounts of hybrid proteins for further analysis.
  • IL-3:Epo Short was analyzed by Western blot analysis. The blot was
  • B ⁇ SUtA (5) is a ultipotential hematopoietic progenitor cell line established from nonadherent cell populations removed from continuous B6.S mouse bone marrow culture. This cell line demonstrates absolute dependence upon a source of growth factor(s).
  • Epo a population of the cells synthesize hemoglobin.
  • TF-1 it is a cell line of immature erythroid origin established from a patient with erythroleukemia.
  • the cell line shows complete dependency on GM-CSF or IL-3.
  • Epo sustains short-term growth of TF-1 and will induce hemoglobin synthesis in a very small population of cells (8%).
  • Hemin and w-aminolevulinic acid induce hemoglobin synthesis in most of the cells.
  • B ⁇ SUtA cells are carried in murine IL-3. In each experiment, they are washed thoroughly and set up with growth factors at 10 cells/ml. Cell growth and hemoglobin content were monitored on days 3 and 6 of each experiment. Cells grown in the presence of concentrated (10X) CHO conditioned medium (CM) containing IL-3:Epo Short at a final concentration equivalent to 4.8 units/ml of Epo grew as well as cells grown in an equivalent amount of recombinant human (rHu) Epo.
  • CM CHO conditioned medium
  • the percentage of cells which synthesized hemoglobin in response to the CH0-IL-3:Epo Short CM was always four times that of cells exposed to rHu Epo.
  • B6SUtA cells grown in the presence of rHu IL-3 and rHu Epo grew as well as cells grown in the presence of IL-3:Epo Short and induced hemoglobin synthesis in the same percentage of cells as did rHu Epo.
  • Cells exposed to recombinant murine IL-3 (rMu IL-3) and rHu Epo grew similarly to cells exposed to rMu IL-3 alone and neither effectively induced cells to synthesize hemoglobin.
  • Concentrated control CHO CM did not support the growth of B6SUtA cells nor did it induce hemoglobin synthesis.
  • CHO CM plus rHu Epo supported cell growth and hemoglobinization as well as CH0-IL-3:Epo Short CM.
  • the GM-CSF/IL-3/Epo dependent human TF-1 cell line and the G-CSF dependent murine NFS-60 cell line were grown and maintained as described (7,8,).
  • the GM-CSF dependent human cell line AML 193 (9) was adapted for growth in IL-3 by continuous cultu r e of the cells in RPMI-1640 plus 10% FCS supplemented with rHu IL-3 for 6 weeks.
  • the TF-1 derived cell line, TF-136 was selected by continuous culture of the TF-1 line in RPMI-1640 plus 10% FCS supplemented with 5ng/ml of rHu IL-3 for 6 months, followed by single cell suspension cloning of the resultant IL-3 dependent cells.
  • the Epo dependent murine cell line, FDC-Pl/ER was derived from the IL-3 dependent line, FDC-P1, by introduction of the murine Epo receptor into these cells.
  • FDC-Pl/ER cells are maintained in RPMI-1640 plus 10% FCS supplemented with 1 unit/ml of rHu Epo.
  • Recombinant human Epo was obtained from Ortho Biologicals, Inc (Raritan, NJ).
  • Recombinant human IL-3, rHu G-CSF and rHu GM-CSF were purchased from R & D Systems (Minneapolis, MN).
  • Capture ELISA Assay - ELISA plate was coated with 5 ⁇ g/200 ⁇ l/well of goat anti-human IL-3 (R & D Systems) in PBS at 40°C overnight. Excess antibody was removed by washing with PBS. Blocking was carried out with 300 ⁇ l/well of 1% non-fat milk in PBS for 1 hour at 37°C followed by washing with 0.05% TweenTM in PBS. Samples were then incubated with the IL-3 antibody for 1 hour at 37°C in 0.5% non-fat milk, 0.025% TweenTM. Following extensive washing, the second antibody, a mouse anti-Epo monoclonal (Genzyme, Cambridge, MA ), was added to the plate which was incubated for 1 hour at 37°C.
  • the plate was washed and incubated with conjugate antibody (Goat anti-mouse-horseradish peroxidase) for 30 minutes at 37°C. Color development was carried out with the addition of o-phenylenediamine/HpOp at room temperature (RT) for 30 minutes. The reaction was stopped with IN H SO. and the samples were read at 495 nm.
  • conjugate antibody Goat anti-mouse-horseradish peroxidase
  • Colonies were counted at day 7 for CFU-E and at day 14 for BFU-E under an inverted microscope.
  • the transfer efficiency was monitored by visual examination of the completeness of transfer of prestained molecular weight markers (Bio Rad).
  • the nitrocellulose membrane was incubated in PBS containing 3% BSA for 1 hour at room temperature and subsequently washed in PBS containing 0.5% Tween (PBS-T) for 5 minutes at room temperature.
  • the membrane was probed with primary anti-Epo anti-sera in 3% BSA in PBS. Excess antibody was removed by 3, 5 minute room temperature washes in PBS-T.
  • the nitrocellulose membrane was then probed with a secondary antibody conjugate (Goat anti-Rabbit IgG/ Alkaline Phosphatase, Bio Rad) for 1 hour at room temperature.
  • a secondary antibody conjugate Goat anti-Rabbit IgG/ Alkaline Phosphatase, Bio Rad
  • Hybrid growth factor plasmid amplification Individual transfected CHO cell clones producing significant amounts of the desired hybrid growth factor were identified by ELISA capture assay, Table II. The clones were plated out and placed in medium with increasing concentrations of MSX, ranging between 100 ⁇ M and 500 M. Colonies surviving at the highest concentration of MSX were isolated and grown to confluence. Serum and drug-free medium was then added to the cells and collected after 4 days. At the time of collection fresh serum and drug-free medium was added to the cells. A total of 3 collections were taken. The amount of hybrid growth factor produced in the collections was determined by Epo or G-CSF ELISA assay (Table III) and appropriate collections were pooled. The pooled CM was used as a source of hybrid growth factors in all cellular assays.
  • Epo bioactivitv of the IL-3:Epo and Epo:IL-3 hybrid growth factors was evaluated ( Figure 4).
  • This line derived from FDC-P1 cells expresses the murine Epo receptor (10), and is dependent on either murine IL-3 or Epo (murine and human) for growth ( Figure 4).
  • IL-3 is a species specific growth factor
  • murine IL-3-deoendent cells do not respond to human IL-3 (12). Therefore, when using the FDC-Pl/ER cell line to evaluate functionality, only the activity of the Epo moiety is measured.
  • CM containing rHu Epo and levels of hybrid growth factors sufficient to support maximal proliferation were added to the culture medium.
  • the cells were then pulsed with ( H) thymidine and the radioactivity incorporated into the DNA was used as a measure of cell growth.
  • Cells exposed to CHO CM which did not contain cytokines did not support the proliferation of FDC-Pl/ER cells.
  • Each of the fusion proteins when present in excess stimulated the growth of FDC-Pl/ER cells to the same extent as did rHu Epo. ( Figure 4)
  • Epo linkage of Epo to a second protein does not impair its ability to bind its receptor or transduce a signal. Epo could therefore be useful as a carrier protein which would target a molecule or compound of interest to those cells expressing the Epo receptor.
  • IL-3 plus Epo bioactivitv of the IL-3:Epo and Epo:IL-3 hybrid growth factors were measured.
  • proliferation of a human cell line, TF-1 (7), dependent on IL-3 and Epo for growth was measured. This experiment was done on a cytokine weight basis and the results are represented on a molar basis ( Figure 6).
  • rHu IL-3 (R & D Systems) made in E coli is nonglycosylated.
  • rHu Epo and hybrid growth factors made in CHO cells are glycosylated. Therefore, when equal weights of the growth factors were added to the cell culture medium, approximately twice the number of unglycosylated molecules of IL-3 were added as compared to glycosylated Epo and hybrid growth factor molecules.
  • CHO CM containing rhu IL-3 plus rHu Epo and levels of hybrid growth factors which support suboptimal proliferation of TF-1 cells adapted for growth in IL-3 were added to the culture medium. Cell growth was monitored by radioactivity incorporated into the DNA.
  • Figure 6 The activitjes of IL-3 plus Epo were not synergistic in this cell line, nor were they additive. At these low levels, the activities of the IL-3:Epo Flex and IL-3:Epo Short fusion proteins were comparable to those of a mixture of the two cytokines. Epo:IL-3 Short activity was again reduced in comparison to that of the IL-3:Epo hybrid growth factors and the combination of IL-3 plus Epo. This is likely to be due to decreased IL-3 activity.
  • a Mononuclear human bone marrow cells were used as a target cell population, b BFU-E were counted 14 days after plating. c CFU-E were counted 7 days after plating.
  • IL-3 bioactivitv of the IL-3:G-CSF hybrid growth factor To determine whether the IL-3 moiety of the IL-3:G-CSF hybrid growth factor was functional, its ability to support growth of the IL-3-dependent human cell line TF-1, was evaluated in a dose response experiment (Figure 9). Quantitation of IL-3:G-CSF protein in CHO CM was performed using a G-CSF ELISA assay in which the standard is unglycosylated G-CSF. Since the IL-3:G-CSF fusion protein is glycosylated, measurements are approximate. G-CSF does not support growth of TF-1 cells ( Figure 9), therefore, the only activity measured in this assay system was IL-3.
  • CHO CM containing rhu IL-3, rHu G-CSF, and IL-3:G-CSF hybrid growth factor were added to the culture medium.
  • the radioactivity incorporated into the DNA was used as a measure of cell proliferation.
  • CHO CM did not support growth of TF-1 cells.
  • the mixture of rhu IL-3 plus rHu G-CSF stimulated proliferation to the same extent as did rhu IL-3 alone.
  • the IL-3:G-CSF hybrid growth factor induced a dose response similar to that observed with IL-3.
  • G-CSF bioactivitv of the IL-3:6-CSF hybrid growth factor To evaluate the biological function of the G-CSF moiety of the IL-3-.G-CSF hybrid growth factor, its ability to stimulate proliferation of the murine cell line, NSF-60, was tested. (Figure 10) G-CSF, unlike IL-3 is not species specific, therefore, human G-CSF will actively support growth of murine cells (21). Cells exposed to CHO CM containing no growth factors, supported the proliferation of NSF-60 cells to the same extent as did cells grown in medium alone. The IL-3:G-CSF hybrid growth factor stimulated growth in a dose dependent manner equivalent to that observed with G-CSF.
  • IL-3 plus G-CSF bioactivitv of the IL-3:G-CSF hybrid growth factor The biological function of the IL-3:G-CSF hybrid growth factor was evaluated by its ability to support growth of an IL-3-, G-CSF-dependent human cell line, AML193.
  • CHO CM containing rHu IL-3, rHu G-CSF and IL-3:G-CSF hybrid growth factor were added to the culture medium. Cell proliferation was monitored by incorporation of radioactivity into the DNA. ( Figure 11). Both IL-3 and G-CSF supported growth of this cell line in a dose dependent manner. The two cytokine activities were not synergistic, nor were they additive.
  • the IL-3:G-CSF hybrid growth factor stimulated AML193 proliferation to a greater extent than did the mixture of the two cytokines.
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE N-terminal
  • MOLECULE ' TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • AATTGCCGCC ACC ATG AGC CGC CTG CCC GTC CTG CTC CTG CTC CAA CTC 49 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gin Leu -19 -15 -10
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • AATTGCCGCC ACC ATG AGC CGC CTG CCC GTC CTG CTC CTG CTC CAA CTC 49 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gin Leu -19 -15 -10
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • AATTGCCGCC ACC ATG AGC CGC CTG CCC GTC CTG CTC CTG CTC CAA CTC 49 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gin Leu -19 ' -15 -10
  • GCT GCT CCA CTC CGA ACA ATC ACT GCT GAC ACT TTC CGC AAA CTC TTC 961 Ala Ala Pro Leu Arg Thr He Thr Ala Asp Thr Phe Arg Lys Leu Phe 285 290 295
  • MOLECULE TYPE DNA (genomic) ( i i i ) HYPOTHETICAL: NO ( iv) ANTI -SENSE : NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID N0:29: CTAGAAGCGG CCGCA 15
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:37: GCCCCACCAC GCCTCATCTG T 21
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

Molécules hématopoïétiques recombinées comprenant au moins une partie d'une première molécule hématopoïétique présentant une activité de différenciation myéloïde précoce et au moins une partie d'une seconde molécule hématopoïétique présentant une activité de différenciation myéloïde tardive. L'invention concerne également des molécules d'acide nucléique codant lesdites molécules recombinées, ainsi que des compositions pharmaceutiques comprenant lesdits facteurs recombinés.
EP19910918221 1990-09-28 1991-09-26 Hybrid growth factors Withdrawn EP0503050A4 (en)

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US5718893A (en) * 1984-04-15 1998-02-17 Foster; Preston F. Use of G-CSF to reduce acute rejection
AU651152B2 (en) * 1990-08-29 1994-07-14 Genetics Institute, Llc Multidomain hematopoiesis stimulators
US6057133A (en) * 1992-11-24 2000-05-02 G. D. Searle Multivariant human IL-3 fusion proteins and their recombinant production
US5738849A (en) * 1992-11-24 1998-04-14 G. D. Searle & Co. Interleukin-3 (IL-3) variant fusion proteins, their recombinant production, and therapeutic compositions comprising them
PT670898E (pt) * 1992-11-24 2004-02-27 Searle & Co Polipeptidos de multiplas mutacoes de interleucina-3 (il-3)
US6153183A (en) 1992-11-24 2000-11-28 G. D. Searle & Company Co-administration of interleukin-3 mutant polypeptides with CSF's or cytokines for multi-lineage hematopoietic cell production
US5501962A (en) * 1993-06-21 1996-03-26 G. D. Searle & Co. Interleuken-3 (IL-3) human/murine hybrid polypeptides and recombinant production of the same
WO1995028427A1 (fr) * 1994-04-15 1995-10-26 Imclone Systems Incorporated Lymphokine chimere composee de l'interleukine 3 et d'une muteine de l'interleukine 6
US5536495A (en) * 1994-04-15 1996-07-16 Foster; Preston F. Use of G-CSF to reduce acute rejection
ITFI940106A1 (it) * 1994-05-27 1995-11-27 Menarini Ricerche Sud Spa Molecola ibrida di formula gm-csf-l-epo o epo-l-gm-csf per la stimolaz ione eritropoietica
IT1271688B (it) * 1994-08-04 1997-06-04 Menarini Ricerche Sud Spa Molecole ibride per il trattamento antitumorale loro preparazione e loro uso in composizioni farmaceutiche
AU705064B2 (en) * 1995-04-26 1999-05-13 Kyowa Hakko Kogyo Co. Ltd. Novel polypeptides
US6066318A (en) 1995-10-05 2000-05-23 G.D. Searle & Co. Multi-functional hematopoietic fusion proteins between sequence rearranged C-MPL receptor agonists and other hematopoietic factors
ATE209354T1 (de) * 1996-09-20 2001-12-15 Ortho Mcneil Pharm Inc Methode zur in vitro-bestimmung der bioaktivität von erythropoietin
US6967092B1 (en) 1996-10-25 2005-11-22 Mc Kearn John P Multi-functional chimeric hematopoietic receptor agonists
CZ130199A3 (cs) * 1996-10-25 1999-07-14 G. D. Searle & Co. Cirkulárně permutovaní agonisté receptoru erythropoietinu
KR100497423B1 (ko) * 1996-10-25 2005-07-07 지.디. 썰 엘엘씨 다기능 키메라 조혈 수용체 아고니스트
US6187564B1 (en) * 1997-07-10 2001-02-13 Beth Israel Deaconess Medical Center DNA encoding erythropoietin multimers having modified 5′ and 3′ sequences and its use to prepare EPO therapeutics
US6165476A (en) * 1997-07-10 2000-12-26 Beth Israel Deaconess Medical Center Fusion proteins with an immunoglobulin hinge region linker
ES2208798T3 (es) * 1997-09-01 2004-06-16 Aventis Pharma Deutschland Gmbh Eritropoyetina humana recombinante con un perfil de glicosilacion ventajoso.
AU2866599A (en) * 1998-02-17 1999-08-30 Hyseq, Inc. A novel interleukin-3 and uses thereof
BR9917606A (pt) * 1998-11-06 2002-12-31 Bio Sidus S A Procedimento para a purificação de eritropoetina humana recombinante a partir de sobrenadantes de cultivo de células e eritropoetina humana recombinante obtida com tal procedimento
DE60109625T3 (de) 2000-05-15 2017-08-03 F. Hoffmann-La Roche Ag Flüssige arzneizubereitung enthaltend ein erythropoietin derivat
US7220407B2 (en) 2003-10-27 2007-05-22 Amgen Inc. G-CSF therapy as an adjunct to reperfusion therapy in the treatment of acute myocardial infarction
CA2586365A1 (fr) 2004-11-05 2006-05-26 Northwestern University Utilisation de scf et de scf dans le traitement de l'ischemie cerebrale et des troubles neurologiques
PE20130557A1 (es) 2010-03-04 2013-05-19 Pfenex Inc Metodo para producir proteinas de interferon recombinantes solubles sin desnaturalizacion
EP2552949B1 (fr) 2010-04-01 2016-08-17 Pfenex Inc. Procédés pour la production de g-csf dans une cellule hôte de pseudomonas
TWI502016B (zh) 2012-07-31 2015-10-01 Asahi Kasei E Materials Corp Epoxy resin compositions, epoxy resins and hardened products
LT6161B (lt) 2013-09-27 2015-06-25 Uab Profarma Granuliocitų kolonijas stimuliuojančio faktoriaus sulieti baltymai su kitais augimo faktoriais, optimaliai su kamieninių ląstelių faktoriumi, ir jų gavimo būdas

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WO1990012874A2 (fr) * 1989-04-21 1990-11-01 Genetics Institute, Inc. Variantes de polypeptides par addition de cysteine, et leurs modifications chimiques
WO1991002754A1 (fr) * 1989-08-22 1991-03-07 Immunex Corporation Proteines de fusion comprenant gm-csf et il-3
WO1991007988A1 (fr) * 1989-12-01 1991-06-13 Amgen Inc. Production de megacaryocytes
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See also references of WO9206116A1 *

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ZA917766B (en) 1993-03-29
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CA2069746A1 (fr) 1992-03-29
AU1157695A (en) 1995-04-13

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