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EP0503050A1 - Hybrid growth factors - Google Patents

Hybrid growth factors

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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Withdrawn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)
French (fr)
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
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Filing date
Publication date
Application filed by Ortho Pharmaceutical Corp filed Critical Ortho Pharmaceutical Corp
Publication of EP0503050A1 publication Critical patent/EP0503050A1/en
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Withdrawn legal-status Critical Current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.
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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.Recombinant hematopoietic molecules comprising at least part of a first hematopoietic molecule exhibiting early myeloid differentiation activity and at least part of a second hematopoietic molecule exhibiting late myeloid differentiation activity. The invention also relates to nucleic acid molecules encoding said recombinant molecules, as well as pharmaceutical compositions comprising said recombinant factors.

Description

HYBRID GROWTH FACTORS
BACKGROUND OF THE INVENTION
Within this application several publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the Sequence Listing. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
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. During the course of hematopoietic differentiation, 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. For example, 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.
Based upon studies performed with purified factors and in vitro colony forming unit assays, it appears that both 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) . Experiments described in recent publications (1,2,3) have demonstrated in vitro that early or late factors alone are poor stimuli of colony formation. However, when an early factor such as IL-3 or GM-CSF is combined with a late factor, levels of colony formation equivalent to that seen with conditioned media having full activity is observed. Thus, differentiation appears to be dependent upon the dual activities of early and late factors.
Despite a clear requirement for both IL-3 or GM-CSF and Epo for the formation of erythroid colony forming units, published results indicate that IL-3 can down-modulate high affinity Epo receptors (4). Because the amount of IL-3 required to demonstrate down-modulation of the Epo receptor was higher than that reported by others who demonstrated functional full IL-3 activity in the presence of Epo, it is unclear whether this phenomenon is relevant in vivo.
Previous experiments in animals (22-26) suggest that under conditions of hematopoietic regeneration, optimal expansion of late progenitors could only occur in the presence of an adequate early progenitor pool. This then makes manipulations that result in the expansion of early hematopoietic progenitor pools extremely desirable. IL-3 has been shown to exert a differentiative and proliferative effect on early progenitor cells and at IL-3 concentrations which had little or no effect alone, Epo acted synergistically to induce proliferation and differentiation of erythroid progenitors. (27) By targeting a molecule with both early (IL-3) and late (Epo and G-CSF) activities to early progenitor cells, optimal expansion of a desired lineage should be possible. SUMMARY OF THE INVENTION
The present invention concerns hybrid molecules comprising early and late differentiation factors produced by genetic manipulation. By covalently linking such factors the local concentration of the late factor is very high at the surface of a cell to which the early factor is bound. Additionally, if down-modulation is relevant in vivo, 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. Furthermore, by linking an early factor with a late factor, such early factor may act more specifically to stimulate only the desired lineage, thus reducing any undesirable effects mediated by the early factor. Finally, 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.
BRIEF DESCRIPTION OF THE FIGURES
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. Medium conditioned by CHO cells transfected with the vector pEeβ (lane 5) and rHu Epo 10 ng (lane 6), 20 ng (lane 7), 30 ng (lane 8), 70 ng (lane 9), and 100 ng (lane 10) were included. Molecular size markers in kilodaltons (lane 1).
Figure 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
3 factors were added for 42 hours followed by a 6 hour pulse of ( H) thy idine as described in Example 7. NO GF (no growth factor); CHO
CM (medium conditioned by CHO cells transfected with the vector pEeδ); Epo (rHu Epo); IL-3 (rHu IL-3); 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).
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).
Figure 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. Increasing concentrations of growth factors, 0.75 fmol/ml hybrid growth factors and 0.75 fmol/ml Epo plus 1.5 fmol/ml IL-3 (1), 1.5 fmol/ml hybrid growth factors and 1.5 fmol/ml of Epo plus 3.0 fmol/ml IL-3 (2), 3.0 fmol/ml hybrid growth factors and 3.0 fmol/ml Epo plus 6.0 fmol/ml IL-3 (3), were added and the assay was carried out as described in Example 7. Hybrid growth factors are as designated in Figure 3.
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 ( H) Thymidine (1 μCi/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.
Figure 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.
DETAILED DESCRIPTION OF THE INVENTION
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. Within this application, "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. Also within this application, "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. Moreover, within this application, "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.
In one embodiment of the invention, the first hematopoietic molecule is selected from the group consisting of IL-3 and GM-CSF. In another embodiment of the invention, the second hemopoietic molecule is selected from the group consisting of Epo, G-CSF, IL-5 and M-CSF. In a preferred embodiment of the invention, 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.
Within the context of the present invention, it is understood that variations in proteins and nucleic acids exist among individuals, e.g. amino acid or nucleotide substitutions, deletions, insertions, and degree or location of glycosylation, and that functional derivatives resulting therefrom are included within the scope of the pre'sent invention.
In a preferred embodiment of the invention, 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.
Further still, in yet another preferred embodiment of the invention, the recombinant molecule comprises the entire amino acid sequence of human erythropoietin (SEQ ID NO: 3). In still a further embodiment of the invention, 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.
In another preferred embodiment of the invention, the recombinant hematopoietic molecule comprises the entire amino acid sequence of human G-CSF (SEQ ID NO: 5).
In one embodiment of the invention, 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. Preferably, the recombinant hematopoietic molecule comprises the amino acid sequence from amino acid 1 to amino acid 302 of SEQ ID NO: 6. Also preferably, the recombinant hematopoietic molecule comprises the amino acid sequence from amino acid 1 to amino acid 321 of SEQ ID NO: 7. However, in another embodiment of the invention, the first hematopoietic molecule, i.e. IL-3, may comprise the carboxyl portion and the second hemopoietic molecule, i.e. Epo, may comprise the amino portion of the recombinant molecule. In a preferred embodiment of the invention, the recombinant molecule comprises the amino acid sequence from amino acid 1 to amino acid 303 of SEQ ID NO: 8. In yet another preferred embodiment, the recombinant molecule comprises the amino acid sequence from amino acid 1 to amino acid 322 of SEQ ID NO: 9.
In still a further embodiment of the invention, the first hematopoietic molecule is IL-3 and the second hematopoietic molecule is G-CSF. In one such embodiment, the first hematopoietic molecule comprises the amino portion and the second hematopoietic molecule comprises the carboxyl portion of the recombinant molecule. In yet a more specific embodiment, 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. Examples of such 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.
Moreover, vectors which comprise the nucleic acid molecules of the subject invention are also disclosed. In one embodiment of the invention, the vector comprises a plasmid. Moreover, 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. The recombinant hematopoietic molecules, nucleic acid molecules, pharmaceutical compositions and methods of the present invention will be better understood by reference to the following experiments and examples, which are provided for purposes of illustration and are not to be construed as in any way limiting the scope of the invention, which is defined by the claims appended hereto.
Examples
Construction of the 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.
Example 1
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. In addition, 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 (SEQ ID NO: 21) 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
Spel and Xbal. The resulting plasmid (pKSOIL-a) contained the IL-3 leader, the IL-3 gene and a small linker fragment.
The 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. This was then ligated to Xbal and Bell cleaved pEeδ to yield pEe6 containing the Epo gene (pEepo). 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. This was then ligated to 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. 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.
To construct a nucleic acid molecule encoding an IL-3:Epo hybrid growth factor with a longer linker sequence separating IL-3 and Epo, 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.
Example 2
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. After restriction analysis, 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.
Example 3
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. The 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. At the same time, a purified 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 pEGlδ 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.)
TABLE I OLIGONUCLEOTIDES All oligonucleotides are listed in the 5' to 3' orientation:
AATTGCCGCC ACCATGAGCC GCCTGCCCGT CCTGCTCCT (SEQ ID NO: 19)
GCTCCAACTC CTGGTCCGCC CCGGACTCCA AGCTCCCATG ACCCAGACAA (SEQ ID NO: 20)
CTAGTTGTCT GGGTCATGGG AGCTTGGAGT CCGGGGCGG (SEQ ID NO: 21)
ACCAGGAGTT GGAGCAGGAG CAGGACGGGC AGGCGGCTCAT GGTGGCGGC (SEQ ID NO: 22)
CTAGCGATCT TTCTAGA (SEQ ID NO: 23) CATGTCTAGA AAGATCG (SEQ ID NO: 24)
CTAGAAGCGG CCGCA (SEQ ID NO: 29)
TTCGCCGGCG TTCGA (SEQ ID NO: 30)
TCGAGCCATG GGGGTGCACG AATGTCCT (SEQ ID NO: 31)
GCCTGGCTGT GGCTTCTCCT GTCCCTGCTG TC (SEQ ID NO: 32) GCTCCCTCTG GGCCTCCCAG TCCTGGGCTG CA (SEQ ID NO: 33)
GCCCAGGACT-GGGAGGCCCA GAGGGA (SEQ ID NO: 34)
GCGACAGCAG GGACAGGAGA AGCCACAGCC AGGCAGGACA TT (SEQ ID NO: 35)
CGTGCACCCC CATGGC (SEQ ID NO: 36)
GCCCCACCAC GCCTCATCTG T (SEQ ID NO: 37) GAATTCGGAT CCTTATCATC T (SEQ ID NO: 38)
CTAGTCTCTA GAATGGGGGT CCACGAATGT (SEQ ID NO: 39)
AGCCATGGCG GCCGCTCTGT CCCCTGTCCT (SEQ ID NO: 40)
GACAGAGCGG CCGCCATGGC TCCCATGACC (SEQ ID NO: 41)
GAATTCGGAT CCTTACTAAA AGATCGCTAG (SEQ ID NO: 42) CTAGCGTCCG GAGGCGGTGG CTCGGGCGGT GGCGGCTCGG GTGGCGGCG GCTCTGCG
(SEQ ID NO: 25)
CTAGCGCAGA GCCGCCGCCA CCGCAGCCGC CACCGCCCGA GCCACCGCC TCCGGACG
(SEQ ID NO: 26)
TTGTCGCTAG CGTCCGGAGG C (SEQ ID NO: 28) GGCCGCTTCC GGAGGCGGTG GCTCGGGCGG TGGCGGCTCG GGTGGCGGC GGCTCTGC
(SEQ ID NO: 43)
GGCCGCAGAG CCGCCGCCAC CCGAGCCGCC ACCGCCCGAG CCACCGCCT CCGGCAGC
(SEQ ID NO: 44) Example 4
Transfection of the hybrid gene containing plasmids. All transfections were performed using the Lipofectinς transfection kit (Bethesda Research Labs, Gaithersburg, MD) using 15-30 mg. of purified plasmid DNA (pEepogs-a, pEepogs-b, pEG13, pEG14, pEG17, and pEGlβ). The following alterations were made to the protocol provided by the company: the growth medium in these experiments was GMEM-S and the CH0-K1 cells were incubated in the presence of 10% C02; after addition of the lipofectin:DNA complex, cells were incubated without selection for 24 hours. 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.
Example 5
Assays for hybrid protein production. Cell supernatants from transfected or control cells were assayed using several different assays. In order to demonstrate Epo production, an RIA kit for Epo was used (Incstar Corp., Stillwater, MN). The presence of IL-3 was determined using an ELISA assay in which the capture antibody was a polyclonal goat anti-IL-3 (R&D Systems, Minneapolis, MN) and the probe antibody was a murine anti-IL-3 monoclonal. Goat anti-mouse conjugated to horseradish peroxidase followed by suitable substrate was used to detect the presence of the monoclonal anti-IL-3. A very similar assay was used to demonstrate the presence of the hybrid proteins except that a murine anti-Epo monoclonal or anti-G-CSF monoclonal was used in place of anti-IL-3 monoclonal. Additionally, IL-3:Epo Short was analyzed by Western blot analysis. The blot was
125 probed with antibody to Epo and then with I goat anti-mouse. A single broad band appeared on the autoradiogram with a molecular weight of slightly more than 50,000 daltons.
Example 6
Cellular assays. Epo and/or IL-3 dependent and responsive cell lines were used to test the biological activities of the hybrid proteins. BδSUtA (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). In response to Epo a population of the cells synthesize hemoglobin. Studies of globin expression indicated that the σlobin programs of B6SUtA cells are similar to those of erythroid progenitors at the period of transition from the yolk sac to fetal liver erythropoiesis. TF-1 (6) 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.
Human IL-3 will not bind the murine IL-3 receptor, therefore experiments that were done with B6SUtA cells measured only the functionality of the Epo moiety of the hybrid. BδSUtA 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. 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.
CH0-IL-3:Epo Short CM as well as CHO-rHu IL-3 CM both supported growth of human TF-1 cells. Control CHO CM supported only limited growth of the TF-1 cells.
Discussion
The above-mentioned results demonstrate that a hybrid protein comprising two growth factors can be expressed in mammalian cell culture systems. In vitro assays of IL-3:Epo Short indicate that this hybrid protein has the activities of both IL-3 and Epo. The therapeutic application of such hybrid factors has advantages over using two factors separately simply in terms of patient administration, and moreover since the production, purification and formulation of one factor is less labor intensive than for two separate factors.
Example 7
Factor Dependent Cell Lines and Culture Media - 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 culture 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. (10) 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 μg/200 μl/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 μl/well of 1% non-fat milk in PBS for 1 hour at 37°C followed by washing with 0.05% Tween™ 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% Tween™. 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.
Gene Amplification- CHO cell lines producing significant amounts of the hybrid growth factors were isolated and 10 cells were plated in a 75 mm T-flask in GMEM-S medium containing various concentrations of methionine sulphoximine (MSX), ranging between 100zM and 500Λ. Colonies resistant to the highest MSX concentration (IL-3:Epo Flex 200zM; IL-3:Epo Short 250 M; Epo:IL-3 Short 250zM; IL-3:G-CSF 250μM) were isolated and expanded. Those clones producing the highest levels of hybrid growth factors as determined by Epo or G-CSF ELISA assay (Amgen) were used for subsequent studies. IL-3:Epo Flex (clone 23-10); IL-3:Epo Short (clone 5-4); Epo:IL-3 Short (17-3-1).
Cell Proliferation Assays - Factor dependent cells were grown to stationary phase, washed, and incubated for 16 hours in media plus
10% FCS deprived of growth factor. The cells were plated at a 5 concentration of 2 X 10 cells/ml in a 96 well icrotiter plate (200 μl/well) with and without growth factor. Recombinant human growth factors were diluted into CHO conditioned medium (CM) before addition to cells. Following incubation for 42 hours, ( H) thymidine (1 μCi/ well; New England Nuclear, Boston, MA) was added and the cells were incubated for another 6 hours. The cells were then hypotonically lysed and harvested onto glass fiber filters. The filters were washed with distilled water, dried and counted in liquid scintillation fluid.
Bone Marrow Cultures - Informed consent was obtained prior to aspirating bone marrow from normal volunteers. Aspirated bone marrow was diluted 1:1 in α- medium without nucleosides containing preservative-free sodium heparin. A single cell suspension was prepared, layered, over an equal volume of Ficoll-Hypaque (sp gr 1.077 g/ml) and then centrifuged for 25 minutes at 1,500 rpm at 40°C. The light-density ononuclear cells were collected and washed and diluted
5 to 5 X10 cells/ml with Iscove's modified Dulbecco's medium plus 20% modified FCS (Gibco BRL). Cells (1.25 X 105 /ml) were plated in 0.8% methylcellulose supplemented with various concentrations of rHu Epo, rHu IL-3 and hybrid growth factors. Cultures were incubated for either 7 or 14 days in a humidified atmosphere with 5% CO~ at 37°C.
Colonies were counted at day 7 for CFU-E and at day 14 for BFU-E under an inverted microscope.
Western Blot Analysis - CHO CM containing approximately 75 ng of IL- 3:Epo fusion protein was electrophoresed on a 10-20% gradient SDS PAGE gel (Integrated Separations Systems) under reducing and denaturing conditions. Samples were loaded in 0.0625 M Tris-HCl (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% glycerol and 0.002% bromophenolblue following heat treatment at 100°C for 3 minutes. The proteins were transferred to nitrocellulose (Bio Rad) in 25 mM Tris, 129 mM glycine, pH 8.3, 20% methanol, at 150 V, constant power, for 90 minutes. 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. Excess secondary antibody was removed by two washes with PBS-T as above. Color development was carried out by incubation with color reagents (Bio Rad) in alkaline phosphatase buffer (100 mM Tris HC1, pH 9.5, 100 mM NaCl, 5 mM MgCl2). The reaction was stopped by immersion of the membrane in cold (4°C) distilled H20.
Results and Discussion
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 μM 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.
Conditioned medium from CHO cells transfected with the vector pEeδ.
Concentrations were determined by Epo and G-CSF ELISA Assay.
Detection of hybrid growth factor production. In order to confirm that the IL-3 and Epo detected in the ELISA capture assays were being produced in the form of a fusion protein, Western blot analysis was performed. Conditioned medium from CHO cells transfected with IL- 3:Epo Flex cDNA (Figure 1, lane 2), IL-3:Epo Short cDNA (Figure 1, lane 3) and Epo:IL-3 Short cDNA (Figure 1, lane 4) were probed with mouse anti-Epo polyclonal anti-sera. Immunoreactive material corresponding to a molecular weight of approximately 50,000 daltons, the expected size of the IL-3:Epo and Epo:IL-3 hybrid growth factors, was detected in each sample. Comparison with increasing concentrations of rHu Epo (Figure 1, lanes 6-10) indicated that the antibody used in this analysis recognizes the Epo moiety of the fusion proteins efficiently.
IL-3 bioactivitv of the IL-3:Epo and EDO:IL-3 hybrid growth factors.
To determine whether the IL-3 moiety of the IL-3:Epo and Epo:IL-3 hybrid growth factors was functional, its ability to support growth of the IL-3-dependent human cell line, AML193, was evaluated (Figure 2).
As Epo does not support growth of these cells (Figure 2), only IL-3 activity was measured in this assay system. CHO CM containing rHu IL-
3 and levels of hybrid growth factors sufficient to support maximal proliferation were added to the culture medium. The cells were then 3 pulsed with ( H) thymidine and the radioactivity incorporated into the
DNA was used as a measure of cell growth. Cells exposed to CHO CM containing no growth factors, supported the proliferation of AML193 cells to the same extent as did cells grown in medium alone. Each of the fusion proteins when present in excess, supported the growth of AML193 cells in a manner equivalent to that of rHu IL-3.
The functional activity of the IL-3 portion of the IL-3:Epo and Epo:IL-3 hybrid growth factors was further evaluated by comparing the fusion proteins to rHu IL-3 in dose response experiments (Figure 3). The incorporation of ( H) thymidine into AML193 DNA was again used as a measure of cell proliferation. When IL-3 was located at the N- ter inus of the hybrid growth factor protein (IL-3:Epo), its ability to support AML193 proliferation was equivalent to that of rHu IL-3 (ED50 = 5 fmol/ml). Size (2 aa versus 23 aa) and flexibility of the linker did not greatly impact the function of the IL-3 moiety. However, when IL-3 was located at the C-terminus of the fusion Drotein (Epo:IL-3), its ability to support the growth of AML193 cells was less (ED50 = 200 fmol/ml) than that of rHu IL-3 and the IL-3:Epo hybrid factors. These results suggest that linkage of IL-3 at the N-terminus interferes with function while linkage at the C-terminus does not. It has previously been reported that modification of the C-terminus of murine IL-3 did not interfere with its activity (11). Therefore, it should be possible to target any molecule or compound of interest to cells expressing the IL-3 receptor through linkage to IL-3 at its C- terminus.
Epo bioactivitv of the IL-3:Epo and Epo:IL-3 hybrid growth factors. To determine whether the Epo moiety of the hybrid growth factors was functional, its ability to support the growth of the Epo-dependent murine cell line FDC-Pl/ER, 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). As 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. CHO
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)
The biological function of the Epo portion of the IL-3:Epo and Epo:IL-
3 hybrid growth factors was further analyzed by comparing the fusion proteins to rHu Epo in dose response experiments (Figure 5). The
3 incorporation of ( H) thymidine into FDC-Pl/ER cells was used as a measure of ceil proliferation. Each of the hybrid growth factors was equivalent to rHu Epo in ability to stimulate proliferation of FDC- Pl/ER cells (ED50 = 50 fmol/ml) . Size (2-3 aa versus 23 aa) and flexibility of the linker, as well as the orientation of Epo within the protein (N-terminus versus C-terminus) did not alter function. Evidence exists suggesting that the N-terminus of Epo is not involved in receptor binding as a monoclonal antibody directed toward the N- terminus of Epo does not neutralize its activity (13). The results presented here suggest that 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. In order to study the effects of IL-3 and Epo in combination, 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.
The biological activity of the IL-3:Epo and Epo:IL-3 hybrid growth factors was further evaluated in dose response experiments (Figures 7 & 8). TF-1 cells adapted for optimal growth in IL-3 were exposed to CHO CM containing hybrid growth factors (Figure 7). Each of the fusion proteins when present in excess were able to support growth of the cells to the same extent (Figure 7A). At lower doses, the IL- 3:Epo Flex protein appeared to be slightly more potent than the short linkered hybrids (IL-3:Epo Flex ED50 = 0.37 fmol/ml; IL-3:Epo Short ED50 = 0.75 fmol/ml; Epo:IL-3 Short ED50 = 0.9 fmol/ml) (Figure 7B), This result was more pronounced in experiments done with TF-1 cells adapted for optimal growth in GM-CSF (Figure 8). The IL-3:Epo Flex protein was dramatically more potent than the hybrid growth factors containing short linkers (IL-3:Epo Flex ED50 = 0.07 fmol/ml; IL-3:Epo. Short and Epo:IL-3 Short ED50 = 0.75 fmol/ml (Figure 8B). When present in excess with the GM-CSF adapted TF-1 cells, each of the fusion proteins stimulated cell proliferation to a similar extent (Figure 8A). These results suggest that when IL-3 and Epo are fused, the 23 aa flexible linker allows more efficient receptor interaction than does a short (2-3 aa) linker.
It appears that induction of receptor expression is possible by growing a cell in the presence of a cytokine whose receptor it has the potential to express. An up regulation of Epo receptor expression has been reported in IL-3-dependent cells transferred into medium supplemented with Epo (14). Thus, it is likely that growing TF-1 cells in IL-3 or GM-CSF, preferentially increases the appearance of cell surface IL-3 or GM-CSF receptors. Several research groups (15-20) have observed a subset population of GM-CSF and IL-3 receptors on primary human cells and hematopoietic cell lines capable of binding both GM-CSF and IL-3. It has been suggested that a single accessory molecule preferentially interacts with this subset of GM-CSF/IL-3 receptors allowing the transduction of signal. Our results raise the possibility that GM-CSF could be inducing the expression of an accessory molecule in TF-1 cells which may be important for binding and could possibly link IL-3:Epo signal transduction. This protein could be identical to the GM-CSF/IL-3 receptor accessory protein.
Ervthroid colony formation stimulated bv IL-3:EDO and Epo:IL-3 hybrid growth factors. To assess the biological activity of the fusion proteins on normal hematopoietic progenitor cells, analysis of the formation of erythroid (BFU-E and CFU-E) colonies from nonadherent mononuclear human bone marrow cells was performed. (Table IV) As was observed with the cell lines, the Epo moiety of each of the fusion proteins was equally active (CFU-E formation) on bone marrow progenitor cells. The IL-3:Epo Flex protein was the most active hybrid growth factor while the Epo:IL-3 Short protein was the least. (It stimulated two-thirds the number of BFU-E as did the IL-3:Epo Flex.) These results suggest that the IL-3:Epo Flex fusion protein may have significant clinical benefits where indications for combination therapy with IL-3 and Epo may prove efficacious.
CFU-E
+++
+++
+++
+++ +++
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. REFERENCES
I. Sonoda, et al., Proc. Natl. Acad. Sci. USA, 85:4630-4364 (1988).
2. Migliaccio, et al., Blood, Vol. 72, No. 3, 844-851 (1988).
3. Sieff, et al., Blood, Vol.73, No. 3, 688-693 (1989).
4. Fraser, et al., Exp. Hematol., 16: 769-775 (1988).
5. Enver, et al, Proc. atl. Acad. Sci. USA, 85:9091-9095 (1988).
6. Kitamura, et al., J. Physiol., 140:323-334 (1989).
7. Kitamura, et al., J. Cell. Physiol., 140 : 323-334 (1989).
8. Holmes, et al., Proc. Natl. Acad. Sci. USA, 82 : 6687-6691 (1985).
9. Lange, et al., Blood, 70 : 192-199 (1987).
10. Carroll, et al., J. Biol. Chem., in press (1991).
II. Duπbar, et al., Science, 245 : 1493-1496 (1989).
12. Yang, et al., Cell, 47 : 3-10 (1986).
13. Sue, et al., Proc. Natl. Acad. Sci. USA, 80 : 3651-3655 (1983).
14. Sakaguchi, et al., Biochem. Biophys. Res. Commun., 146 : 7-12 (1987).
15. Park, et al., J. Biol. Chem., 264 : 5420-5427 (1989).
16. Gesner, et al., J. Cell. Physiol., 136 : 493-499 (1988). 17. Elliot, et al., Blood, 74 : 2349-2359 (1989).
18. Lopez, et al., Proc. Natl. Acad. Sci. USA, 86 : 7022-7026 (1989).
19. Budel, et al., Blood, 75 : 1439-1445 (1990).
20. Cannistra, et al., J. Biol. Chem., 265 : 12656-12663 (1990).
21. Souza, et al., Science, 232 : 61-65 (1986).
22. Umemura et al., Proc. Natl. Acad. Sci. USA, 85 : 9278-9282 (1988).
23. Udupa, et al., Blood, 53 : 1164-1171 (1979).
24. Papayannopoulou, et al., J. Clin. Invest., 51 : 1179-1185 (1972).
25. Migliaccio, et al., Blood, 72 : 944-951 (1988).
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Rosen, Jonathan I. (ii) TITLE OF INVENTION: HYBRID GROWTH FACTORS (iii) NUMBER OF SEQUENCES: 44
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Robert L. Minier
(B) STREET: 1 Johnson & Johnson Plaza
(C) CITY: New Brunswick
(D) STATE: New Jersey
(E) COUNTRY: USA
(F) ZIP: 08933
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release *1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: 435
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/589,958
(B) FILING DATE: 28-SEP-1990
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Stark, Michael
(B) REGISTRATION NUMBER: 32,495
(C) REFERENCE/DOCKET NUMBER: BCI-15
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-524-2817
(B) TELEFAX: 908-524-2808
(C) TELEX: 844-481
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 133 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Ala Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15
Ser Asn Met He ASD Glu He He Thr His Leu Lys Gin Pro Pro Leu 20 25 30
Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp He Leu 35 40 45
Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala 50 55 60
Val Lys Ser Leu Gin Asn Ala Ser Ala He Glu Ser He Leu Lys Asn 65 70 75 80
Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro 85 90 95
He His He Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr 100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu 115 120 125
Ser Leu Ala He Phe 130
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: Ala Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15
Ser Asn Met He Asp Glu He He Thr His Leu Lys Gin Pro Pro Leu 20 25 30
Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp He Leu 35 40 45
Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala 50 55 60
Val Lys Ser Leu Gin Asn Ala Ser Ala He Glu Ser He Leu Lys 65 70 75
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE'TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Pro Pro Arg Leu He Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu
1 5 10 15
Leu Glu Ala Lys Glu Ala Glu Asn He Thr Thr Gly Cys Ala Glu His 20 25 30
Cys Ser Leu Asn Glu Asn He Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45
Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp 50 55 60
Gin Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu 65 70 75 80
Leu Val Asn Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp 85 90 95
Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Gin Lys Glu Ala He Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125
Pro Leu Arg Thr He Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140
Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160
Cys Arg Thr Gly Asp Arg 165
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala 1 5 10 15
Glu Asn He Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn 20 25 30
He Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met
35 40 45
Glu Val Gly Gin Gin Ala Val Glu Val TrD Gin Gly Leu Ala Leu Leu 50 55 60
Ser Glu Ala Val Leu Arg Gly Gin Ala Leu Leu Val Asn Ser Ser Gin 65 70 75 80
Pro Trp Glu Pro Leu Gin Leu His Val Asp Lys Ala Val Ser Gly Leu 85 90 95
Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gin Lys Glu Ala 100 105 110 He Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr He Thr 115 120 125
Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg 130 135 140
Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys 145 150 155
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 imino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys 1 5 10 15
Cys Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin 20 25 30
Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 35 40 45
Leu Leu Gly His Ser Leu Gl He Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60
Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser 65 70 75 80
Gly leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser 85 90 95
Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp 100 105 110
Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro 115 120 125
Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140 Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe 145 150 155 160
Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 165 170
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 302 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Ala Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Ser Asn Met He Asp Glu He He Thr His Leu Lys Gin Pro Pro Leu 20 25 30
Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp He Leu 35 40 45
Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala 50 55 60
Val Lys Ser Leu Gin Asn Ala Ser Ala He Glu Ser He Leu Lys Asn 65 70 75 80
Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro 85 90 95
He His He Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr 100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu 115 120 125
Ser Leu Ala He Phe Leu Asp Met Ala Pro Pro Arg Leu He Cys Asp 130 135 140
Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn 145 150 155 160 He Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn He Thr 165 170 175
Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val 180 185 190
Gly Gin Gin Ala Val Glu Val Trp Gin Gly Leu Ala Leu Leu Ser Glu 195 200 205
Ala Val Leu Arg Gly Gin Ala Leu Leu Val Asn Ser Ser Gin Pro Trp 210 215 220
Glu Pro Leu Gin Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser 225 230 235 240
Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gin Lys Glu Ala He Ser 245 250 255
Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr He Thr Ala Asp 260 265 270
Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys 275 280 285
Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg 290 295 300
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 321 amino acids
(B) TYPE: amino acid
(C) -STRANDEDNESS: single
(D) TOPOLOGY: linear
( i i ) MOLECULE TYPE: protein ( i i i ) HYPOTHETICAL: NO ( iv) ANTI -SENSE : NO
(xi ) SEQUENCE DESCRIPTION: SEQ ID N0: 7 :
Ala Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15
Ser Asn Met He Asp Glu He He Thr His Leu Lys Gin Pro Pro Leu 20 25 30
Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp He Leu 35 40 45 Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala 50 55 60
Val Lys Ser Leu Gin Asn Ala Ser Ala He Glu Ser He Leu Lys Asn
65 70 75 80
Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro 85 90 95
He His He Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr 100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu 115 120 125
Ser Leu Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Ala Leu Ala He Phe Leu Asp Met Ala Pro Pro Arg Leu
145 150 155 160
He Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 165 170 175
Ala Glu Asn He Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 180 185 190
Asn He Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 195 200 205
Met Glu Val Gly Gin Gin Ala Val Glu Val Trp Gin Gly Leu Ala Leu 210 215 220
Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu Leu Val Asn Ser Ser 225 230 235 240
Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp Lys Ala Val Ser Gly 245 250 255
Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gin Lys Glu 260 265 270
Ala He Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr He 275 280 285
Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 290 295 300
Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 305 310 315 320
Arg (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 303 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Ala Pro Pro Arg Leu He Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15
Leu Glu Ala Lys Glu Ala Glu Asn He Thr Thr Gly Cys Ala Glu His 20 25 30
Cys Ser Leu Asn Glu Asn He Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45
Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp 50 55 60
Gin Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu 65 70 75 80
Leu Val Asn Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp 85 90 95
Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110
Gly Ala Gin Lys Glu Ala He Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125
Pro Leu Arg Thr He Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140
Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160
Cys Arg Thr Gly Asp Arg Ala Ala Ala Met Ala Pro Met Thr Gin Thr 165 170 175
Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met He Asp Glu 180 185 190 He He Thr His Leu Lys Gin Pro Pro Leu Pro Leu Leu Asp Phe Asn
195 200 205
Asn Leu Asn Gly Glu Asp Gin Asp He Leu Met Glu Asn Asn Leu Arg 210 215 220
Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gin Asn 225 230 235 240
Ala Ser Ala He Glu Ser He Leu Lys Asn Leu Leu Pro Cys Leu Pro 245 250 255
Leu Ala Thr Ala Ala Pro Thr Arg His Pro He His He Lys Asp Gly 260 265 270
Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu
275 280 285
Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu Ser Leu Ala He Phe
290 295 300
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 322 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Ala Pro Pro Arg Leu He Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15
Leu Glu Ala Lys Glu Ala Glu Asn He Thr Thr Gly Cys Ala Glu His 20 25 30
Cys Ser Leu Asn Glu Asn He Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45
Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp 50 55 60
Gin Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp 85 90 95
Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110
Gly Ala Gin Lys Glu Ala He Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125
Pro Leu Arg Thr lie Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140
Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160
Cys Arg Thr Gly Asp Arg Ala Ala Ala Ser Gly Gly Gly Gly Ser Gly 165 170 175
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ala Met Ala Pro Met 180 185 190
Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met 195 200 205
He Asp Glu He He Thr His Leu Lys Gin Pro Pro Leu Pro Leu Leu 210 215 220
Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp He Leu Met Glu Asn 225 230 235 240
Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser 245 250 255
Leu Gin Asn Ala Ser Ala He Glu Ser He Leu Lys Asn Leu Leu Pro 260 265 270
Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro He His He 275 280 285
Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu 290 295 300
Lys Thr Leu Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu Ser Leu Ala 305 310 315 320
He Phe
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 317 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ala Pro Met Thr Gin Tnr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15
Ser Asn Met He Asp Glu He He Thr His Leu Lys Gin Pro Pro Leu 20 25 30
Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu ASD Gin Asp He Leu 35 40 45
Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala 50 55 60
Val Lys Ser Leu Gin Asn Ala Ser Ala He Glu Ser He Leu Lys Asn 65 70 75 80
Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro 85 90 95
He His He Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr 100 105 110
Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu 115 120 125
Ser Leu Ala He Phe Leu Glu Ala Ala Ala Ser Leu Pro Ala Met Thr 130 135 140
Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Cys 145 150 155 160
Leu Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu 165 170 175
Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu 180 185 190
Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser Cys Pro 195 200 205
Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly 210 215 220 Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He Ser Pro 225 230 235 240
Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe 245 250 255
Ala Thr Thr He Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala 260 265 270
Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin 275 280 285
Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu 290 295 300
Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 305 310 315
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 994 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 14..977
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 71..977
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
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
CTG GTC CGC CCC GGA CTC CAA GCT CCC ATG ACC CAG ACA ACT AGT TTG 97 Leu Val Arg Pro Gly Leu Gin Ala Pro Met Thr Gin Thr Thr Ser Leu -5 1 5 AAG ACA AGC TGG GTT AAC TGC TCT AAC ATG ATC GAT GAA ATT ATA ACA 145 Lys Thr Ser Trp Val Asn Cys Ser Asn Met He Asp Glu He He Thr 10 15 20 25
CAC TTA AAC GAG CCA CCT TTG CCT TTG CTG GAC TTC AAC AAC CTC AAT 193 His Leu Asn Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn 30 35 40
GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA AGG CCA AAC 241 Gly Glu Asp Gin Asp He Leu Met Glu Asn Asn Leu Arg Arg Pro Asn 45 50 55
CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAT GCA TCA GCA 289 Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gin Asn Ala Ser Ala 60 65 70
ATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG 337 He Glu Ser He Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr 75 80 85
GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT 385 Ala Ala Pro Thr Arg His Pro He His He Lys Asp Gly Asp Trp Asn 90 95 100 105
GAA TTC CGG AGG AAA CTG ACG TTC TAT CTG AAA ACC CTT GAG AAT GCG 433 Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala 110 115 120
CAG GCT CAA CAG ACG ACT TTG TCG CTA GCG ATC TTT CTA GAC ATG GCC 481 Gin Ala Gin Gin Thr Thr Leu Ser Leu Ala He Phe Leu Asp Met Ala 125 130 135
CCA CCA CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG TAC CTC TTG 529 Pro Pro Arg Leu He Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu 140 145 150
GAG GCC AAG GAG GCC GAG AAT ATC ACG ACG GGC TGT GCT GAA CAC TGC 577 Glu Ala Lys Glu Ala Glu Asn Tie Thr Thr Gly Cys Ala Glu His Cys 155 160 165
AGC TTG AAT GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT AAT TTC TAC 625 Ser Leu Asn Glu Asn He Thr Val Pro Asp Thr Lys Val Asn Phe Tyr 170 175 180 185
GCG TGG AAG AGG ATG GAG GTC GGC CAG CAG GCC GTA GAA GTC TGG CAG 673 Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp Gin 190 195 200
GGC CTG GCC CTG CTG TCG GAA GCT GTC CTG CGG GGC CAG GCC CTG TTG 721 Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu Leu 205 210 215 GTC AAC TCG AGC CAG CCG TGG GAG CCC CTG CAA CTG CAT GTG GAT AAA 769
Val Asn Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp Lys 220 225 230
GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT CGG GCT CTG GGA 817
Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly 235 240 245
GCT CAG AAG GAA GCC ATC TCC CCT CCA GAT GCG GCC TCA GCT GCT CCA 865
Ala Gin Lys Glu Ala He Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro 250 255 260 265
CTC CGA ACA ATC ACT GCT GAC ACT TTC CGC AAA CTC TTC CGA GTC TAC 913
Leu Arg Thr He Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr 270 275 280
TCC AAT TTC CTC CGG GGA AAG CTG AAG CTG TAC ACA GGG GAG GCA TGC 961
Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys
285 290 295
AGG ACA GGG GAC AGA T GATAAGGATC CGAATTC 994
Arg Thr Gly Asp Arg 300
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1015 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 8..998
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 89..998
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TCGAGCC ATG GGG GTG CAC GAA TGT CCT GCC TGG CTG TGG CTT CTC CTG 49 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu -27 -25 -20 -15 TCC CTG CTG TCG CTC CCT CTG GGC CTC CCA GTC CTG GGC GCC CCA CCA 97 Ser Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro -10 -5 1
CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG TAC CTC TTG GAG GCC 145 Arg Leu He Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala 5 10 15
AAG GAG GCC GAG AAT ATC ACG ACG GGC TGT GCT GAA CAC TGC AGC TTG 193 Lys Glu Ala Glu Asn He Thr Thr Gly Cys Ala Glu His Cys Ser Leu 20 25 30 35
AAT GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT AAT TTC TAC GCG TGG 241 Asn Glu Asn He Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp 40 45 50
AAG AGG ATG GAG GTC GGC CAG CAG GCC GTA GAA GTC TGG CAG GGC CTG 289 Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp Gin Gly Leu 55 60 65
GCC CTG CTG TCG GAA GCT GTC CTG CGG GGC CAG GCC CTG TTG GTC AAC 337 Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu Leu Val Asn 70 75 80
TCG AGC CAG CCG TGG GAG CCC CTG CAA CTG CAT GTG GAT AAA GCC GTC 385 Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp Lys Ala Val 85 90 95
AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT CGG GCT CTG GGA GCT CAG 433 Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gin 100 105 110 115
AAG GAA GCC ATC TCC CCT CCA GAT GCG GCC TCA GCT GCT CCA CTC CGA 481 Lys Glu Ala He Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg 120 125 130
ACA ATC ACT GCT GAC ACT TTC CGC AAA CTC TTC CGA GTC TAC TCC AAT 529 Thr He Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn 135 140 145
TTC CTC CGG GGA AAG CTG AAG CTG TAC ACA GGG GAG GCA TGC AGG ACA 577 Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr 150 155 160
GGG GAC AGA GCG GCC GCC ATG GCT CCC ATG ACC CAG ACA ACT AGT TTG 625 Gly Asp Arg Ala Ala Ala Met Ala Pro Met Thr Gin Thr Thr Ser Leu 165 170 175
AAG ACA AGC TGG GTT AAC TGC TCT AAC ATG ATC GAT GAA ATT ATA ACA 673 Lys Thr Ser Trp Val Asn Cys Ser Asn Met He Asp Glu He He Thr 180 185 190 195 CAC TTA AAC GAG CCA CCT TTG CCT TTG CTG GAC TTC AAC AAC CTC AAT 721 His Leu Asn Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn 200 205 210
GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA AGG CCA AAC 769 Gly Glu Asp Gin Asp He Leu Met Glu Asn Asn Leu Arg Arg Pro Asn 215 220 225
CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAT GCA TCA GCA 817 Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gin Asn Ala Ser Ala 230 . 235 240
ATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG 865 He Glu Ser He Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr 245 250 255
GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT 913 Ala Ala Pro Thr Arg His Pro He His He Lys Asp Gly Asp Trp Asn 260 265 270 275
GAA TTC CGG AGG AAA CTG ACG TTC TAT CTG AAA ACC CTT GAG AAT GCG 961 Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala 280 285 290
CAG GCT CAA CAG ACG ACT TTG TCG CTA GCG ATC TTT T AGTAAGGATC 1008 Gin Ala Gin Gin Thr Thr Leu Ser Leu Ala He Phe 295 300
CGAATTC 1015
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1039 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)'ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 14..1021
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 71..1021 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
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
CTG GTC CGC CCC GGA CTC CAA GCT CCC ATG ACC CAG ACA ACT AGT TTG 97 Leu Val Arg Pro Gly Leu Gin Ala Pro Met Thr Gin Thr Thr Ser Leu -5 1 5
AAG ACA AGC TGG GTT AAC TGC TCT AAC ATG ATC GAT GAA ATT ATA ACA 145 Lys Thr Ser Trp Val Asn Cys Ser Asn Met He Asp Glu He He Thr 10 15 20 25
CAC TTA AAC GAG CCA CCT TTG CCT TTG CTG GAC TTC AAC AAC CTC AAT 193 His Leu Asn Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn 30 35 40
GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA AGG CCA AAC 241 Gly Glu Asp Gin Asp He Leu Met Glu Asn Asn Leu Arg Arg Pro Asn 45 50 55
CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAT GCA TCA GCA 289 Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gin Asn Ala Ser Ala 60 65 70
ATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG 337 He Glu Ser He Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr 75 80 85
GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT 385 Ala Ala Pro Thr Arg His Pro He His He Lys Asp Gly Asp Trp Asn 90 95 100 105
GAA TTC CGG AGG AAA CTG ACG TTC TAT CTG AAA ACC CTT GAG AAT GCG 433 Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala 110 115 120
CAG GCT CAA CAG ACG ACT TTG TCG CTA GCG ATC TTT CTA GAA GCG GCC 481 Gin Ala Gin Gin Thr Thr Leu Ser Leu Ala He Phe Leu Glu Ala Ala 125 130 135
GCA AGC TTA CCT GCC ATG ACC CCC CTG GGC CCT GCC AGC TCC CTG CCC 529 Ala Ser Leu Pro Ala Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro 140 145 150
CAG AGC TTC CTG CTC AAG TGC TTA GAG CAA GTG AGG AAG ATC CAG GGC 577 Gin Ser Phe Leu Leu Lys Cys Leu Glu Gin Val Arg Lys He Gin Gly 155 160 165
GAT GGC GCA GCG CTC CAG GAG AAG CTG TGT GCC ACC TAC AAG CTG TGC 625 Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys 170 175 180 185 CAC CCC GAG GAG CTG GTG CTG CTC GGA CAC TCT CTG GGC ATC CCC TGG 673
His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp 190 195 200
GCT CCC CTG AGC TCC TGC CCC AGC CAG GCC CTG CAG CTG GCA GGC TGC 721
Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys 205 210 215
TTG AGC CAA CTC CAT AGC GGC CTT TTC CTC TAC CAG GGG CTC CTG CAG 769
Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin 220 225 230
GCC CTG GAA GGG ATA TCC CCC GAG TTG GGT CCC ACC TTG CAC ACA CTG 817
Ala Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu His Thr Leu 235 240 245
CAG CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GAA 865
Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu
250 255 260 265
GAA CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT GCC ATG CCG 913
Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro 270 275 280
GCC TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG GTC CTG GTT GCT 961
Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala 285 290 295
AGC CAT CTG CAG AGC TTC CTG GAG GTG TCG TAC CGC GTT CTA CGC CAC 1009
Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His 300 305 310
CTT GCG CAG CCC TGATAAGGAT CCGAATTC 1039
Leu Ala Gin Pro 315
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1051 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 14..1033
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 71..1033
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
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
CTG GTC CGC CCC GGA CTC CΛA GCT CCC ATG ACC CAG ACA ACT AGT TTG 97 Leu Val Arg Pro Gly Leu Gin Ala Pro Met Thr Gin Thr Thr Ser Leu -5 1 5
MAG ACA AGC TGG GTT AAC TGC TCT AAC ATG ATC GAT GAA ATT ATA ACA 145 Lys Thr Ser Trp Val Asn Cys Ser Asn Met He Asp Glu He He Thr 10 15 20 25
CAC TTA AAC GAG CCA CCT TTG CCT TTG CTG GAC TTC AAC AAC CTC AAT 193 His Leu Asn Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn 30 35 40
GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA AGG CCA AAC 241 Gly Glu Asp Gin Asp He Leu Met Glu Asn Asn Leu Arg Arg Pro Asn 45 50 55
CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAT GCA TCA GCA 289 Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gin Asn Ala Ser Ala 60 65 70
ATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG 337 He Glu Ser He Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr 75 80 85
GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT 385 Ala Ala Pro Thr Arg His Pro He His He Lys Asp Gly Asp Trp Asn 90 95 100 105
GAA TTC CGG AGG AAA CTG ACG TTC TAT CTG AAA ACC CTT GAG AAT GCG 433 Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala 110 115 120
CAG GCT CAA CAG ACG ACT TTG TCG CTA GCG TCC GGA GGC GGT GGC TCG 481 Gin Ala Gin Gin Thr Thr Leu Ser Leu Ala Ser Gly Gly Gly Gly Ser 125 130 135
GGC GGT GGC GGC TCG GGT GGC GGC GGC TCT GCG CTA GCG ATC TTT CTA 529 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Ala He Phe Leu 140 145 150 GAC ATG GCC CCA CCA CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG 577 Asp Met Ala Pro Pro Arg Leu He Cys Asp Ser Arg Val Leu Glu Arg 155 160 165
TAC CTC TTG GAG GCC AAG GAG GCC GAG AAT ATC ACG ACG GGC TGT GCT 625 Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn He Thr Thr Gly Cys Ala 170 175 180 185
GAA CAC TGC AGC TTG AAT GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT 673 Glu His Cys Ser Leu Asn Glu Asn He Thr Val Pro Asp Thr Lys Val 190 195 200
AAT TTC TAC GCG TGG AAG AGG ATG GAG GTC GGC CAG CAG GCC GTA GAA 721 Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu 205 210 215
GTC TGG CAG GGC CTG GCC CTG CTG TCG GAA GCT GTC CTG CGG GGC CAG 769 Val Trp Gin Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin 220 225 230
GCC CTG TTG GTC AAC TCG AGC CAG CCG TGG GAG CCC CTG CAA CTG CAT 817 Ala Leu Leu Val Asn Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His 235 240 245
GTG GAT AAA GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT CGG 865 Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg 250 255 260 265
GCT CTG GGA GCT CAG AAG GAA GCC ATC TCC CCT CCA GAT GCG GCC TCA 913 Ala Leu Gly Ala Gin Lys Glu Ala He Ser Pro Pro Asp Ala Ala Ser 270 275 280
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
CGA GTC TAC TCC AAT TTC CTC CGG GGA AAG CTG AAG CTG TAC ACA GGG 1009 Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly 300 305 310
GAG GCA TGC AGG ACA GGG GAC AGA TGATAAGGAT CCGAATTC 1051
Glu Ala Cys Arg Thr Gly Asp Arg 315 320
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1072 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic) ( i i i ) HYPOTHETICAL: NO ( iv) ANTI -SENSE : NO
( ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 8..1054
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 89..1054
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
TCGAGCC ATG GGG GTG CAC GAA TGT CCT GCC TGG CTG TGG CTT CTC CTG 49 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu -27 -25 -20 -15
TCC CTG CTG TCG CTC CCT CTG GGC CTC CCA GTC CTG GGC GCC CCA CCA 97 Ser Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro -10 -5 1
CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG TAC CTC TTG GAG GCC 145 Arg Leu He Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala 5 10 15
AAG GAG GCC GAG AAT ATC ACG ACG GGC TGT GCT GAA CAC TGC AGC TTG 193 Lys Glu Ala Glu Asn He Thr Thr Gly Cys Ala Glu His Cys Ser Leu 20 25 30 35
AAT GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT AAT TTC TAC GCG TGG 241 Asn Glu Asn He Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp 40 45 50
AAG AGG ATG GAG GTC GGC CAG CAG GCC GTA GAA GTC TGG CAG GGC CTG 289 Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp Gin Gly Leu 55 60 65
GCC CTG CTG TCG GAA GCT GTC CTG CGG GGC CAG GCC CTG TTG GTC AAC 337 Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu Leu Val Asn 70 75 80
TCG AGC CAG CCG TGG GAG CCC CTG CAA CTG CAT GTG GAT AAA GCC GTC 385 Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp Lys Ala Val 85 90 95
AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT CGG GCT CTG GGA GCT CAG 433 Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gin 100 105 110 115 AAG GAA GCC ATC TCC CCT CCA GAT GCG GCC TCA GCT GCT CCA CTC CGA 481 Lys Glu Ala He Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg 120 125 130
ACA ATC ACT GCT GAC ACT TTC CGC AAA CTC TTC CGA GTC TAC TCC AAT 529 Thr He Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn 135 140 145
TTC CTC CGG GGA AAG CTG AAG CTG TAC ACA GGG GAG GCA TGC AGG ACA 577 Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr 150 155 160
GGG GAC AGA GCG GCC GCC TCC GGA GGC GGT GGC TCG GGC GGT GGC GGC 625 Gly Asp Arg Ala Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 165 170 175
TCG GGT GGC GGC GGC TCT GCG GCC GCC ATG GCT CCC ATG ACC CAG ACA 673 Ser Gly Gly Gly Gly Ser Ala Ala Ala Met Ala Pro Met Thr Gin Thr 180 185 190 195
ACT AGT TTG AAG ACA AGC TGG GTT AAC TGC TCT AAC ATG ATC GAT GAA 721 Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met He Asp Glu 200 205 210
ATT ATA ACA CAC TTA AAC GAG CCA CCT TTG CCT TTG CTG GAC TTC AAC 769 He He Thr His Leu Asn Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn 215 220 225
AAC CTC AAT GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA 817 Asn Leu Asn Gly Glu Asp Gin Asp He Leu Met Glu Asn Asn Leu Arg 230 235 240
AGG CCA AAC CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAT 865 Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gin Asn 245 250 255
GCA TCA GCA ATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC 913 Ala Ser Ala He Glu Ser He Leu Lys Asn Leu Leu Pro Cys Leu Pro 260 265 270 275
CTG GCC ACG GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT 961 Leu Ala Thr Ala Ala Pro Thr Arg His Pro He His He Lys Asp Gly 280 285 290
GAC TGG AAT GAA TTC CGG AGG AAA CTG ACG TTC TAT CTG AAA ACC CTT 1009 Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu 295 300 305
GAG AAT GCG CAG GCT CAA CAG ACG ACT TTG TCG CTA GCG ATC TTT 1054 Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu Ser Leu Ala He Phe 310 315 320
TAGTAAGGAT CCGAATTC 1072 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 429 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 10..411
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 13..411
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
AAGCTTACC ATG GCT CCC ATG ACC CAG ACA ACT AGT TTG AAG ACA AGC 48 Met Ala Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser -1 1 5 10
TGG GTT AAC TGC TCT AAC ATG ATC GAT GAA ATT ATA ACA CAC TTA AAC 96 Trp Val Asn Cys Ser Asn Met He Asp Glu He He Thr His Leu Asn 15 20 25
GAG CCA CCT TTG CCT TTG CTG GAC TTC AAC AAC CTC AAT GGG GAA GAC 144 Glu Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp 30 35 40
CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA AGG CCA AAC CTG GAG GCA 192 Gin Asp He Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala 45 50 55 60
TTC AAC AGG GCT GTC AAG AGT TTA CAG AAT GCA TCA GCA ATT GAG AGC 240 Phe Asn Arg Ala Val Lys Ser Leu Gin Asn Ala Ser Ala He Glu Ser 65 70 75
ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG GCC GCA CCC 288 He Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro 80 85 90
ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT GAA TTC CGG 336 Thr Arg His Pro He His He Lys Asp Gly Asp Trp Asn Glu Phe Arg 95 100 105 AGG AAA CTG ACG TTC TAT CTG AAA ACC CTT GAG AAT GCG CAG GCT CAA 384
Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gin Ala Gin 110 115 120
CAG ACG ACT TTG TCG CTA GCG ATC TTT TAGTAAGGAT CCGAATTC 429
Gin Thr Thr Leu Ser Leu Ala He Phe
125 130
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 532 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 14..514
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 17..514
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
AAGCTTACCT GCC ATG GCC CCA CCA CGC CTC ATC TGT GAC AGC CGA GTC 49 Met Ala Pro Pro Arg Leu He Cys Asp Ser Arg Val -1 1 5 10
CTG GAG AGG TAC CTC TTG GAG GCC AAG GAG GCC GAG AAT ATC ACG ACG 97 Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn He Thr Thr 15 20 25
GGC TGT GCT GAA CAC TGC AGC TTG AAT GAG AAT ATC ACT GTC CCA GAC 145 Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn He Thr Val Pro Asp 30 35 40
ACC AAA GTT AAT TTC TAC GCG TGG AAG AGG ATG GAG GTC GGC CAG CAG 193 Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin 45 50 55
GCC GTA GAA GTC TGG CAG GGC CTG GCC CTG CTG TCG GAA GCT GTC CTG 241 Ala Val Glu Val Trp Gin Gly Leu Ala Leu Leu Ser Glu Ala Val Leu 60 65 70 75 CGG GGC CAG GCC CTG TTG GTC AAC TCG AGC CAG CCG TGG GAG CCC CTG 289 Arg Gly Gin Ala Leu Leu Val Asn Ser Ser Gin Pro Trp Glu Pro Leu 80 85 90
CAA CTG CAT GTG GAT AAA GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT 337 Gin Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr 95 100 105
CTG CTT CGG GCT CTG GGA GCT CAG AAG GAA GCC ATC TCC CCT CCA GAT 385 Leu Leu Arg Ala Leu Gly Ala Gin Lys Glu Ala He Ser Pro Pro Asp 110 115 120
GCG GCC TCA GCT GCT CCA CTC CGA ACA ATC ACT GCT GAC ACT TTC CGC 433 Ala Ala Ser Ala Ala Pro Leu Arg Thr He Thr Ala Asp Thr Phe Arg 125 130 135
AAA CTC TTC CGA GTC TAC TCC AAT TTC CTC CGG GGA AAG CTG AAG CTG 481 Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu 140 145 150 155
TAC ACA GGG GAG GCA TGC AGG ACA GGG GAC AG ATGATAAGGA TCCGAATTC 532 Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 160 165
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 556 base pairs
(B) YPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 14..538
(ix) FEATURE:
(A) NAME/KEY : mat_peptide
(B) LOCATION: 17. .538
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
AAGCTTACCT GCC ATG ACC CCC CTG GGC CCT GCC AGC TCC CTG CCC CAG 49 Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin -1 1 5 10 AGC TTC CTG CTC AAG TGC TTA GAG CAA GTG AGG AAG ATC CAG GGC GAT 97 Ser Phe Leu Leu Lys Cys Leu Glu Gin Val Arg Lys He Gin Gly Asp 15 20 25
GGC GCA GCG CTC CAG GAG AAG CTG TGT GCC ACC TAC AAG CTG TGC CAC 145 Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His 30 35 40
CCC GAG GAG CTG GTG CTG CTC GGA CAC TCT CTG GGC ATC CCC TGG GCT 193 Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala 45 50 55
CCC CTG AGC TCC TGC CCC AGC CAG GCC CTG CAG CTG GCA GGC TGC TTG 241 Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu 60 65 70 75
AGC CAA CTC CAT AGC GGC CTT TTC CTC TAC CAG GGG CTC CTG CAG GCC 289 Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala 80 85 90
CTG GAA GGG ATA TCC CCC GAG TTG GGT CCC ACC TTG CAC ACA CTG CAG 337 Leu Glu Gly He Ser Pro Glu Leu Gly Pro Thr Leu His Thr Leu Gin 95 , 100 105
CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GAA GAA 385 Leu Asp Val Ala Asp Phe Ala Thr Thr He Trp Gin Gin Met Glu Glu 110 115 120
CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT GCC ATG CCG GCC 433 Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala 125 130 135
TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG GTC CTG GTT GCT AGC 481 Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser 140 145 150 155
CAT CTG CAG AGC TTC CTG GAG GTG TCG TAC CGC GTT CTA CGC CAC CTT 529 His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu 160 165 170
GCG CAG CCC TGATAAGGAT CCGAATTC 556
Ala Gin Pro
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19: AATTGCCGCC ACCATGAGCC GCCTGCCCGT CCTGCTCCT 39
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20: GCTCCAACTC CTGGTCCGCC CCGGACTCCA AGCTCCCATG ACCCAGACAA 50
(2) INFORMATION FOR SEQ ID N0:21.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21: CTAGTTGTCT GGGTCATGGG AGCTTGGAGT CCGGGGCGG 39 ( 2 ) INFORMATION FOR SEQ ID NO: 22 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ACCAGGAGTT GGAGCAGGAG CAGGACGGGC AGGCGGCTCA TGGTGGCGGC 50
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23: CTAGCGATCT TTCTAGA 17
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24: CATGTCTAGA AAGATCG 17
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: CTAGCGTCCG GAGGCGGTGG CTCGGGCGGT GGCGGCTCGG GTGGCGGCGG CTCTGCG 57 (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26: CTAGCGCAGA GCCGCCGCCA CCGCAGCCGC CACCGCCCGA GCCACCGCCT CCGGACG 57
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28: TTGTCGCTAG CGTCCGGAGG C 21
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29: CTAGAAGCGG CCGCA 15
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30: TTCGCCGGCG TTCGA 15
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS: (A)-LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31: TCGAGCCATG GGGGTGCACG AATGTCCT 28
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32: GCCTGGCTGT GGCTTCTCCT GTCCCTGCTG TC 32
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid -
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GCTCCCTCTG GGCCTCCCAG TCCTGGGCTG CA 32
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34: GCCCAGGACT GGGAGGCCCA GAGGGA 26 (2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35: GCGACAGCAG GGACAGGAGA AGCCACAGCC AGGCAGGACA TT 42
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36: CGTGCACCCC CATGGC 16
(2) INFORMATION FOR SEQ ID N0-.37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GCCCCACCAC GCCTCATCTG T 21
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38: GAATTCGGAT CCTTATCATC T 21
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39: CTAGTCTCTA GAATGGGGGT CCACGAATGT 30
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40: AGCCATGGCG GCCGCTCTGT CCCCTGTCCT 30
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41: GACAGAGCGG CCGCCATGGC TCCCATGACC 30
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42: GAATTCGGAT CCTTACTAAA AGATCGCTAG 30 (2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43: GGCCGCTTCC GGAGGCGGTG GCTCGGGCGG TGGCGGCTCG GGTGGCGGCG GCTCTGC 57 (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: GGCCGCAGAG CCGCCGCCAC CCGAGCCGCC ACCGCCCGAG CCACCGCCTC CGGCAGC 57

Claims

What is claimed is:
1. 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, said recombinant hematopoietic molecule having early myeloid differentiation activity associated with said first hematopoietic molecule and late myeloid differentiation activity associated with said second hematopoietic molecule.
2. A recombinant hematopoietic molecule of claim 1 wherein the first hematopoietic molecule is selected from the group consisting of IL-3 and GM-CSF.
3. A recombinant hematopoietic molecule of claim 1 wherein the second hematopoietic molecule is selected from the group consisting of Epo, G-CSF, IL-5 and M-CSF.
4. A recombinant hematopoietic molecule of claim 1 wherein the portion of the first hematopoietic molecule is linked to the portion of the second hematopoietic molecule by an amino acid linker sequence of at least two amino acid residues.
5. A recombinant hematopoietic molecule of claim 1 comprising SEQ ID NO: 1.
6. A recombinant hematopoietic molecule of claim 1 comprising an amino acid sequence contained within SEQ ID NO: 2.
7. A recombinant hematopoietic molecule of claim 1 comprising SEQ ID NO: 3.
8. A recombinant hematopoietic molecule of claim 1 comprising an amino acid sequence contained within SEQ ID NO: 4.
9. A recombinant hematopoietic molecule of claim 1 comprising SEQ ID NO: 5.
10. A recombinant hematopoietic molecule of claim 1 wherein the first hematopoietic molecule is IL-3 and the second hematopoietic molecule is Epo.
11. A recombinant hematopoietic molecule of claim 10 wherein the first hematopoietic molecule comprises the amino portion and the second hematopoietic molecule comprises the carboxy portion of the recombinant hematopoietic molecule.
12. A recombinant hematopoietic molecule of claim 11 which comprises SEQ ID NO: 6.
13. A recombinant hematopoietic molecule of claim 11 which comprises SEQ ID NO: 7.
14. A recombinant hematopoietic molecule of claim 10 wherein the first hematopoietic molecule comprises the carboxy portion and the second hematopoietic molecule comprises the amino portion of the recombinant hematopoietic molecule.
15. A recomoinant hematopoietic molecule of claim 14 which comprises SEQ ID NO: 8.
16. A recombinant hematopoietic molecule of claim 14 which comprises SEQ ID NO: 9.
17. A recombinant hematopoietic molecule of c.aim 1 wherein the first hematopoietic molecule is IL-3 and the second hematopoietic molecule is G-CSF
18. A recombinant hematopoietic molecule of claim 17 wherein the first hematopoietic molecule comprises the amino portion and the second hematopoietic molecule comprises the carboxy portion of the recombinant hematopoietic molecule.
19. A recombinant hematopoietic molecule of claim 18 which comprises SEQ ID NO: 10.
20. A nucleic acid molecule which encodes the recombinant hematopoietic molecule of claim 1.
21. An expression vector which comprises the nucleic acid molecule of claim 20.
22. A host cell transformed with the expression vector of claim 19.
23. A host cell of claim 22 which comprises a mammalian cell.
24. A method for producing 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, which comprises culturing a host cell of claim 22 under suitable conditions so as to allow the expression of such recombinant hematopoietic molecule, and recovering such recombinant hematopoietic molecule.
25. A pharmaceutical composition which comprises a recombinant hematopoietic molecule of claim 1 and a pharmaceutically acceptable carrier.
26. A method for promoting hematopoiesis in a patient which comprises administering to such patient a pharmaceutical composition of claim 25.
EP19910918221 1990-09-28 1991-09-26 Hybrid growth factors Withdrawn EP0503050A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US58995890A 1990-09-28 1990-09-28
US589958 1990-09-28

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EP0503050A4 EP0503050A4 (en) 1994-07-06

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EP (1) EP0503050A4 (en)
JP (1) JPH05502463A (en)
AU (2) AU8735991A (en)
CA (1) CA2069746A1 (en)
IE (1) IE913426A1 (en)
PT (1) PT99107A (en)
WO (1) WO1992006116A1 (en)
ZA (1) ZA917766B (en)

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US6057133A (en) * 1992-11-24 2000-05-02 G. D. Searle Multivariant human IL-3 fusion proteins and their recombinant production
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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 (en) * 1994-04-15 1995-10-26 Imclone Systems Incorporated Chimeric interleukin-3/mutein interleukin-6 lymphokine
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IE913426A1 (en) 1992-04-08
AU8735991A (en) 1992-04-28
PT99107A (en) 1992-08-31
EP0503050A4 (en) 1994-07-06
WO1992006116A1 (en) 1992-04-16
ZA917766B (en) 1993-03-29
JPH05502463A (en) 1993-04-28
CA2069746A1 (en) 1992-03-29
AU1157695A (en) 1995-04-13

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