WO1988005469A1 - Proteines humaines il-3 - Google Patents

Proteines humaines il-3 Download PDF

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Publication number
WO1988005469A1
WO1988005469A1 PCT/US1988/000011 US8800011W WO8805469A1 WO 1988005469 A1 WO1988005469 A1 WO 1988005469A1 US 8800011 W US8800011 W US 8800011W WO 8805469 A1 WO8805469 A1 WO 8805469A1
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Prior art keywords
hil
yeast
protein
sequence
amino acid
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PCT/US1988/000011
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English (en)
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Dirk M. Anderson
David J. Cosman
Virginia L. Price
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Immunex Corporation
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Publication of WO1988005469A1 publication Critical patent/WO1988005469A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5403—IL-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • 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

Definitions

  • the present invention relates generally to colony stimulating factors (CSFs), and particularly to purified recombinant human interleukin-3 (IL-3) protein compositions.
  • CSFs colony stimulating factors
  • IL-3 human interleukin-3
  • CSFs colony stimulating factors
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • M-CSF or CSF-1 macrophage CSF
  • BPA burst promoting activity
  • Murine IL-3 was originally identified by Ihle et al., J. Immunol. 126:2184 (1981) as a factor which induced expression of a T cell associated enzyme, 20 ⁇ -hydroxysteroid dehydrogenase. The factor was purified to homogeneity and shown to regulate the growth and differentiation of numerous subclasses of early hematopoietic and lymphoid progenitor cells. cDNA clones corresponding to murine IL-3 were first isolated by Fung et al., Nature 307:233 (1984) and Yokota et al., Proc. Natl. Acad. Sci. USA 81:1070 (1984). Gibbon and human genomic DNA homologues of the murine IL-3 sequence were disclosed by
  • the present invention concerns recombinant human IL-3 proteins which are expressed and secreted in high yields in microbial systems.
  • the invention also concerns recombinant expression vectors comprising nucleotide sequences encoding the proteins, related microbial expression systems, and processes for making the proteins using the microbial expression systems.
  • the present invention relates generally to colony stimulating factors (CSFs), and particularly to purified recombinant human interleukin-3 (IL-3) protein compositions.
  • CSFs colony stimulating factors
  • IL-3 human interleukin-3
  • CSFs colony stimulating factors
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • M-CSF or CSF-1 macrophage CSF
  • BPA burst promoting activity
  • FIG. 1 indicates the nucleotide sequence and corresponding amino acid sequence of a human IL-3 protein having a proline residue at position 8 of the mature polypeptide (hIL-3[P8]).
  • a DNA segment encoding human IL-3 was isolated from a cDNA library prepared by reverse transcription of polyadenylated RNA isolated from human peripheral blood T-lymphocytes (PBT). Synthetic oligonucleotide probes having sequence homology to N-terminal and C-terminal regions of the native human genomic DNA sequence were employed to screen the library by conventional DNA hybridization techniques. DNA was isolated from those clones which hybridized to thei probes and analyzed by restriction endonuclease cleavage, agarose gel electrophoresis, and additional hybridization experiments ("Southern blots") involving the electrophoresed fragments.
  • PBT peripheral blood T-lymphocytes
  • the hybridizing segment of one hIL-3 clone was subcloned and sequenced by conventional techniques.
  • the segment was also transcribed into RNA in vitro, and the resulting message capped, polyadenylated, and injected into Xenopus oocytes.
  • the resulting oocyte translation products were then tested for the capacity to induce hematopoietic cell proliferation in a human bone marrow assay, described below.
  • Human interleukin-3 and “hIL-3” refer to a human endogenous secretory protein which induces proliferation of granulocyte, macrophage, and erythrocyte progenitors from populations of multipotent hematopoietic stem cells.
  • the term means a protein having IL-3 biological activity and an amino acid sequence which is substantially homologous to the sequence set forth in FIG. 1.
  • substantially homologous which can refer both to nucleic acid and amino acid sequences, means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which do not result in an adverse functional dissimilarity between reference and subject sequences.
  • sequences having greater than 90 percent homology, equivalent biological activity, and equivalent expression characteristics are considered substantially homologous. Sequences having lesser degrees of homology, comparable bioactivity, and equivalent expression characteristics are considered equivalents.
  • “Mutant amino acid sequence” refers to a polypeptide encoded by a nucleotide sequence intentionally made variant from a native sequence.
  • “Mutant protein” or “mutein” means a protein comprising a mutant amino acid sequence.
  • “Native sequence” refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein.
  • the terms “KEX2 protease recognition site” and “N-glycosylation site” are defined below.
  • activate means to alter a selected KEX2 protease recognition site to retard or prevent cleavage by the KEX2 protease of Saccharomyces cerevisiae, or to alter an N-glycosylation site to preclude covalent bonding of oligosaccharide moieties to particular amino acid residues.
  • Recombinant means that a protein is derived from recombinant microbial (e.g., bacterial or fungal) expression systems. As a product, this defines a human protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.
  • Protein expressed in bacterial cultures will be free of polysaccharide; protein expressed in yeast will have a glycosylation pattern different from that expressed in mammalian cells.
  • "Crude yeast-conditioned culture supernatant” refers to media withdrawn from yeast cultures which has not been subjected to concentration or purification procedures.
  • "Purified”, as used in the context of this disclosure, refers to a recombinant protein in the form of a protein composition having a specific activity in a human bone marrow proliferation assay of at least 1 x 10 6 units/mg. The efficiency of the microbial expression systems disclosed herein permit production of sufficient quantities of human IL-3 to permit quantitative purification. Specific activities in the range 10 7 to 10 8 units/mg are contemplated as projected final product criteria.
  • DNA segment refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector.
  • Nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.
  • Recombinant expression vector refers to a plasmid comprising a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, and (2) a structural or coding sequence which is transcribed into mRNA and translated into protein.
  • the transcriptional unit includes a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • Recombinant expression system means a combination of an expression vector and a suitable host microorganism. Yeast expression systems, preferably those employing Saccharomyces cerevisiae, are employed in production of the proteins of the present invention.
  • Freshly isolated human bone marrow cells are preincubated for 2 hours at 37°C, 5% CO 2 , in tissue culture flasks containing 2 x 10 6 cells per ml pre-warmed, pre-gassed serum-free RPMI 1640 medium (Gibco, Chagrin Falls, OH, USA) containing 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, and 300 ⁇ g/ml fresh L-glutamine (hereinafter "assay medium”). After preincubation, nonadherent cells are removed by pipetting the media gently over the surface of the flask.
  • tissue culture flasks containing 2 x 10 6 cells per ml pre-warmed, pre-gassed serum-free RPMI 1640 medium (Gibco, Chagrin Falls, OH, USA) containing 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, and 300 ⁇ g/ml fresh L-glutamine (hereinafter "assay medium
  • Nonadherent cells are collected by centrifugation at 1000 rpm for 10 minutes at 4°C, resuspended in a small volume of assay medium containing 10% fetal bovine serum (FBS), and counted using Trypan blue for viability and Turks stain for recovery of white cells. Cells are kept at about 4°C in assay medium containing 10% FBS until added to assay plates.
  • FBS fetal bovine serum
  • 50 ⁇ l assay medium are added to each well of a 96 well flat bottom tissue culture plate. 50 ⁇ l of sample diluted in assay medium are added to the first well of each row, and serial dilutions are made across each, row in the usual manner. 1.25 x 10 4 bone marrow cells, in a volume of 100 ⁇ l, are then added to each well. Plates are incubated for 4 days at 37oC, 5% CO 2 , in a plastic box containing steriledistilled H 2 O to prevent desiccation.
  • hIL-3 activity are calculated by reference to the quantity of hIL-3 which induces 50% of maximal thymidine incorporation. For example, if a 100 ⁇ l sample generates one-half maximal thymidine incorporation at a dilution of 1:20, one unit is defined as the activity contained in 1/20 of 100 ⁇ l, or 5 ⁇ l.
  • the sample would therefore contain 1000 divided by 5, or 200 units per milliliter (U/ml) of hIL-3 activity.
  • An incubation medium is prepared by mixing seven parts nutrient medium
  • ⁇ -minimum essential medium [ ⁇ -minimum essential medium ( ⁇ MEM) supplemented with vitamins, 28.5% FBS, 0.7 x 10 4 M 2-mercaptoethanol, 0.12 mg/ml asparagine, 0.7 mg/ml glutamine, 150 U/ml penicillin G, and 150 U/ml streptomycin] and three parts agar suspension, and held at 37°C.
  • Percoll treated bone marrow cells are warmed to 37°C and added to the incubation medium to provide a final concentration of approximately 1 x 10 5 cells/ml.
  • the resulting mixture is kept at 37°C while dispensing 250 ⁇ l aliquots into each well. Plates are held at about 23°C until the agar solidifies, then incubated at 37°C in plastic boxes containing distilled water to prevent desiccation.
  • Colonies having 50 or more cells each are counted on days 7 or 10 and 14. Earlier counts are better for granulocyte colonies, while later counts are better for macrophage and mixed colonies.
  • hIL-3 activity expressed in colony forming units per milliliter ("CFU/ml"), is defined as that sample dilution providing one-half of the maximum colonies formed by 1 x 10 5 bone marrow cells, multiplied by the number ⁇ f colonies observed in the half maximal case.
  • Cell types in colonies are determined by staining individual cells with a stain consisting of
  • nucleotide and deduced amino acid sequences of the hIL-3 cDNA isolated as described below are set forth in FIG. 1.
  • nucleotides are numbered beginning with the GCT codon corresponding to the N-terminal alanine of the mature native protein. Similarly, amino acids are numbered from this alanine residue.
  • the native polypeptide includes a leader sequence which is cleaved upon secretion to provide mature protein.
  • a recombinant DNA segment encoding the amino acid sequence of hIL-3 can be obtained by screening of appropriate cDNA libraries or by assembly of artificially synthesized oligonucleotides.
  • hIL-3 sequences incorporating codons specifying proline or serine at position 8 of the mature sequence can be assembled.
  • Yeast systems may be used for expression of the recombinant proteins of this invention.
  • Preferred expression vectors can be derived from pBC102.K22 (ATCC 67,255) which contains DNA sequences from pBR322 for selection and replication in E. coli (Ap r gene and origin of replication) and yeast DNA sequences including a glucose-repressible alcohol dehydrogenase 2 (ADH2) promoter.
  • ADH2 promoter has been described by Russell et al., J. Biol. Chem. 258:2674 (1982) and Beier et al., Nature 300:724 (1982).
  • Plasmid pBC102-K22 also includes a Trpl gene as a selectable marker and the yeast 2 ⁇ origin of replication. Adjacent to the promoter is the yeast ⁇ -factor leader sequence enabling secretion of heterologous proteins from a yeast host.
  • the ⁇ -factor leader sequence is modified to contain, near its 3' end, an Asp718 (Kpnl and Asp718 are isoschizomers) restriction site to facilitate fusion of this sequence to foreign genes.
  • Asp718 Kpnl and Asp718 are isoschizomers restriction site to facilitate fusion of this sequence to foreign genes.
  • a sequence coding for the Glu-Ala-Glu-Ala amino acids was omitted to allow efficient processing of secreted protein, as described by Brake et al., Proc. Natl. Acad. Sci. USA 81:4642 (1984).
  • yeast vectors which comprise an ⁇ -factor promoter, for example pY ⁇ HuGM (ATCC 53157), which hears the wild-type human GM-CSF gene. Others are known to those skilled in the art. The construction of pY ⁇ HuGM is described in published European Patent Application No. 183,350 (8530682.7), the disclosure of which is incorporated by reference herein.
  • yeast strains for transformation will be determined by the nature of the selectable markers and other features of the vector.
  • Appropriate S. cerevisiae strains for transformation by expression vectors derived from pBC102.K22 or pY ⁇ HuGM include strains X2181-1B, available from the Yeast Genetic Stock Center, Berkeley, CA, USA [see below], having the genotype ⁇ trp1 gal1 ade1 his2; J17 (ATCC 52683; ⁇ his2 ade1 trp1 met14 ura3); and IL166-5B (ATCC 46183; ⁇ his1 trp1).
  • a particularly preferred expression strain for use with pBC102-K22, XV2181 is a diploid formed by mating two haploid strains, X2181-1B, available from the Yeast Genetic Stock Center, Department of Biophysics and Medical Physics, University of California, Berkeley, CA 94702, USA; and XV617-1-3B, available from the Department of Genetics, University of Washington, Seattle, WA 98105, USA, or Immunex Corporation, 51 University Street, Seattle, WA 98101, USA.
  • a suitable transformation protocol is that described by Hinnen, et al., Proc. Natl. Acad. Sci.
  • Trp + transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ⁇ g/ml adenine and 20 ⁇ g/ml uracil.
  • Host strains transformed by vectors comprising the ADH2 or ⁇ -factor promoters are grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ⁇ g/ml adenine and 80 ⁇ g/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and frozen or held at 4°C prior to further purification.
  • IL-3 Recombinant human IL-3 resulting from fermentation of yeast strains can be purified by single or sequential reversed-phase HPLC Steps on a preparative HPLC column, by methods analogous to those described by Urdal et al., J. Chromatog. 296:171 (1984), and Grabstein et al., J. Exp. Med. 163:1405 (1986).
  • yeast-conditioned medium containing hIL-3 can be filtered through a 0.45 ⁇ filter and pumped, at a flow rate of 100 ml/min, onto a 5 cm x 30 cm column packed with 10-20 y reversed phase silica (Vydac, The Separations Group, Hesperia, CA, USA).
  • the column can be equilibrated in 0.1% trifluoroacetic acid in water (Solvent A) prior to the application of the yeast-conditioned medium and then flushed with this solvent following application of the medium to the column until the optical absorbance at 280 nm of the effluent approaches baseline values.
  • fractions containing hIL-3 can be pooled, concentrated, and reapplied to a similar HPLC column in 0.9 M acetic acid plus pyridine to pH 4.0, for an additional elution step mediated by gradient of 0.9 M acetic acid, pyridine (pH 4.5) and 60% n-propanol. Fractions eluting from the column can be analyzed for protein concentration by fluorescamine analysis, and hIL-3 activity determined by appropriate assay. Additional HPLC steps can be employed if indicated.
  • the native hIL-3 protein includes an Arg-Arg pairing at position 52 and an Arg-Arg-Lys triplet beginning at position 106, both of which are susceptible to cleavage by the KEX2 protease of Saccharomyces cerevisiae.
  • Site-specific mutagenesis procedures can be employed to inactivate KEX2 protease processing sites by deleting, adding, or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites. The resulting muteins are less susceptible to cleavage by the KEX2 protease at locations other than the yeast ⁇ -factor leader sequence, where cleavage upon secretion is intended.
  • purified mixtures of recombinant glycoproteins such as human or murine granulocyte-macrophage colony stimulating factor (GM-CSF) can consist of from 0 to 50% carbohydrate by weight.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the presence of variable quantities of associated carbohydrate in recombinant secreted glycoproteins complicates purification procedures, thereby reducing yield.
  • glycoprotein be employed as a therapeutic agent, a possibility exists that recipients will develop allergic reactions to the yeast carbohydrate moieties, requiring therapy to be discontinued. For these reasons, biologically active, homogeneous analogs of immunoregulatory glycoproteins having reduced carbohydrate are desirable for therapeutic use.
  • Functional mutant analogs of hIL-3 having inactivated N-glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques as described below. These analog protei an be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems.
  • the present invention contemplates analog forms of human IL-3 comprising an amino acid sequence homologous to the native sequence of hIL-3, but comprising at least one amino acid substitution, deletion, or insertion inactivating at least one N-glycosylation site.
  • N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-A 1 -Z, where A 1 is any amino acid except Pro, and Z is Ser or Thr.
  • asparagine provides a side chain amino group for covalent attachment of carbohydrate.
  • Such a site can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z amino acid between A 1 and Z, or an amino acid other than Asn between Asn and A 1 .
  • substitutions are made conservatively; i.e., the most preferred substitute amino acids are those having physicochemical characteristics resembling those of the residue to be replaced.
  • the potential effect of the deletion or insertion upon biological activity should be considered.
  • an analog hIL-3 lacking N-glycosylation sites is a protein having a mutant amino acid sequence which is substantially homologous to the native sequence set forth in FIG. 1, wherein at least one occurrence Asn-A 1 -Z in the native sequence has been replaced in the mutant sequence by Asn-A 2 -Y or X-A 2 -A 3 , where A 1 , A 2 , and A 3 are the same or different and can be any amino acid, X is any amino acid not Asn; Y is any amino acid not Z; and Z is Ser or Thr.
  • all occurrences of Asn-A 1 -Z in the native sequence are replaced in the mutant sequence by Asn-A 2 -Y or X-A 2 -A 3 .
  • N-glycosylation sites the first being the triplet AsnCysSer beginning at residue 15, and the second being AsnAlaSer beginning at residue 68.
  • Appropriately conservative substitute amino acids for Asn include Asp, Gln, Glu, Ala, Gly, Ser, and Thr, of which Asp, Gin, and Glu are preferred. Where Z is Ser, appropriate substitutes are Met, Leu, Ile, Val, Asp, Gin, Glu, or Asn; of which Met, Leu, lie, and Val are preferred. Other conservative amino acid substitutions could be made to provide protein lacking N-glycosylation sites.
  • oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Walder et al., Gene 42:133 (1986); Bauer et al., Gene 37:73 (1985); Craik, Biotechniques, January 1985, 12-19; Smith et al., Genetic Engineering: Principles and Methods (Plenum Press, 1981); and U. S. Patent 4,518,584 disclose suitable techniques, and are incorporated by reference herein.
  • a strand of the gene to be altered is cloned into an M13 single-stranded phage or other appropriate vector to provide single-stranded (ss) DNA comprising either the sense or antisense strand corresponding to the gene to be altered.
  • This DNA is then hybridized to an oligonucleotide primer complementary to the sequence surrounding the codon to be altered, but comprising a codon (or an antisense codon complementary to such codon) specifying the new amino acid at the point where substitution is to be effected. If a deletion is desired, the primer will lack the particular codon specifying the amino acid to be deleted, while maintaining the correct reading frame.
  • the primer will include a new codon, at the appropriate location in the sequence, specifying the amino acid to be inserted.
  • the substitute codon, deleted codon, or inserted codon is located at or near the center of the oligonucleotide.
  • oligonucleotide primer employed is determined by the need to optimize stable, unique hybridization at the mutation site with the 5' and 3' extensions being of sufficient length to avoid editing of the mutation by exonucleases.
  • oligonucleotides used in accordance with the present invention will usually contain from about 15 to about 25 bases. Oligonucleotides of greater size are not needed.
  • the resulting oligonucleotide/ss vector hybrid is directly transformed into yeast.
  • a mutagenic primer is hybridized to a gapped duplex having a single-stranded template segment containing the gene to be altered.
  • the primer is extended along the template strand by reaction with DNA polymerase I (Klenow fragment), T4 DNA polymerase, or other suitable DNA polymerase, providing a resulting double stranded DNA which is circularized and used to transfect a suitable host strain.
  • DNA polymerase I Klenow fragment
  • T4 DNA polymerase or other suitable DNA polymerase
  • replication of the heteroduplex by the host provides progeny of both strands.
  • E. coli transfected cells are plated to provide colonies, which are screened using a labeled oligonucleotide corresponding to that used in the mutagenesis procedure. If yeast are transformed directly, transformants are pooled, DNA isolated and transformed into E. coli. The resulting colonies are screened by hybridization.
  • DNA but not to the progeny of the parent strand DNA containing the mutated gene is then isolated and spliced into a suitable expression vector, and the vector used to transform a host strain. The host strain is then grown in culture to provide the analog protein.
  • the amino acid sequence of mature hIL-3 is linked to a yeast ⁇ -factor leader sequence via an N-terminal fusion construct comprising a nucleotide encoding the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Lys (DYKDDDDK).
  • DYKDDDDK Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
  • the latter sequence is highly antigenic and provides an epitope reversibly bound by specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein.
  • This sequence is also specifically cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing. Fusion proteins capped with this peptide are also resistant to intracellular degradation prior to secretion.
  • Two oligonucleotides were synthesized, with sequences complementary to selected 5' and 3' sequences of the hIL-3 gene.
  • a cDNA library was constructed by reverse transcription of polyadenylated mRNA isolated from total RNA extracted from human peripheral blood T lymphocytes (PBT) stimulated with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA).
  • PBT peripheral blood T lymphocytes
  • PMA phytohemagglutinin
  • PMA phorbol 12-myristate 13-acetate
  • the cDNA was rendered double-stranded using DNA polymerase I, blunt-ended with T4 DNA polymerase, methylated with EcoRI methylase to protect EcoRI cleavage sites within the cDNA, and ligated to EcoRI linkers.
  • the resulting constructs were digested with EcoRI to remove all but one copy of the linkers at each end of the cDNA, and ligated to EcoRI-cut and dephosphorylated arms of bacteriophage ⁇ gt10 (Huynh et al., DNA Cloning: A Practical Approach, Glover, ed., IRL Press, pp. 49-78).
  • the ligated DNA was packaged into phage particles to generate a library of recombinants. 500,000 recombinants were plated on E. coli strain C600hf1- and screened by standard plaque hybridization techniques. Eleven clones were isolated from the library which hybridized to both probes.
  • a yeast expression vector was constructed by digesting pBC102-K22 (ATCC 67,255) with Asp718 and Spel, removing a fragment comprising the mature sequence of human G-CSF, and ligating the following olig to the vector fragment:
  • the resulting vector was designated pBC115.
  • cDNA encoding hIL-3 was excised from the pGEMBL cloning vector by digestion with Hpal and BamHI, providing a fragment extending from amino acid 14 of mature hIL-3 to a site downstream of the coding sequence.
  • Oligonucleotides were synthesized and assembled to provide a fragment encoding (1) the C-terminal 5 amino acids of the yeast ⁇ -factor leader peptide, beginning with the Asp718 site and terminating in a KEX2 protease recognition site; (2) an 8 codon sequence encoding a synthetic N-terminal "flag" identification peptide (DYKDDDDK; see above); and (3) a short sequence encoding the N-terminal 14 amino acids of mature hIL-3 protein up to and including an Hpal blunt end.
  • the sequence of this 84 base pair Kpnl-Hpal fragment which was constructed from 4 oligomers of approximately 40 nucleotides each, is set forth below:
  • yeast strain XV2181 for expression of the hIL-3 gene product.
  • the transformed yeast strain was grown in shake flask culture under conditions promoting derepression of the ADH2 promoter.
  • Yeast-conditioned supernatants were collected by centrifugation and assayed for bone marrow proliferation-inducing activity.

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Abstract

Protéines humaines IL-3 (hIL-3) purifiées recombinantes, protéines analogues mutantes à activité biologique de hIL-3, vecteurs d'expression recombinants comprenant des séquences de nucléotides codant hIL-3, systèmes d'expression microbiens comprenant des organismes hôtes appropriés transformés par les vecteurs d'expression recombinants susmentionnés, et procédés correspondants.
PCT/US1988/000011 1987-01-20 1988-01-11 Proteines humaines il-3 WO1988005469A1 (fr)

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990010705A2 (fr) * 1989-03-15 1990-09-20 Gist-Brocades N.V. Production et purification d'interleukine-3 humaine de recombinaison et de ses muteines
WO1991000350A1 (fr) * 1989-06-30 1991-01-10 Immunex Corporation Proteines analogues d'interleucine-3 humaine non glycosylatees
US5328988A (en) * 1987-10-26 1994-07-12 Immunex Corporation Interleukin-7
EP0691403A1 (fr) * 1986-07-14 1996-01-10 Genetics Institute, Inc. Famille de facteurs de croissance hématopoiétique de type IP3 d'origine primate
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
US5516512A (en) * 1989-08-14 1996-05-14 Gist-Brocades, N.V. N- and C-terminal truncation and deletion mutants of human interleukin-3
US5604116A (en) * 1992-11-24 1997-02-18 G. D. Searle & Co. Interleukin-3 (IL-3) multiple mutation polypeptides, recombinant production of the same, and corresponding therapeutic methods
EP0810285A2 (fr) 1989-08-14 1997-12-03 Gist-Brocades B.V. Mutants de l'interleukine-3 humaine
US5705149A (en) * 1987-10-26 1998-01-06 Sterling Winthrop Inc. Use of interleukin-7 to stimulate proliferation of hematopoietic cell precursors
US5738849A (en) * 1992-11-24 1998-04-14 G. D. Searle & Co. Interleukin-3 (IL-3) variant fusion proteins, their recombinant production, and therapeutic compositions comprising them
US5772992A (en) * 1992-11-24 1998-06-30 G.D. Searle & Co. Compositions for co-administration of interleukin-3 mutants and other cytokines and hematopoietic factors
WO1999041382A2 (fr) * 1998-02-17 1999-08-19 Hyseq, Inc. Nouvelle interleukine 3 et ses utilisations
US6017523A (en) * 1995-06-06 2000-01-25 G.D. Searle & Co. Therapeutic methods employing mutant human interleukin-3 (IL-3) polypeptides
US6022535A (en) * 1993-11-22 2000-02-08 G. D. Searle & Company Treatment of hematopoietic disorders with fusion proteins comprising multiply mutated interleukin-3 (IL-3) polypeptides and second growth factors
US6060595A (en) * 1996-09-03 2000-05-09 The General Hospital Corporation Inhibition of viral replication
US6060047A (en) * 1992-11-24 2000-05-09 G. D. Searle & Co. Co-administration of interleukin-3 mutant polypeptides with CSF's for multi-lineage hematopoietic cell production
US6103699A (en) * 1996-06-07 2000-08-15 Immunotech Developments Inc. Peptide, a method for its preparation and a pharmaceutical composition containing the peptide
US6361977B1 (en) 1992-11-24 2002-03-26 S. Christopher Bauer Methods of using multivariant IL-3 hematopoiesis fusion protein
US6361976B1 (en) 1992-11-24 2002-03-26 S. Christopher Bauer Co-administration of interleukin-3 mutant polypeptides with CSF'S for multi-lineage hematopoietic cell production
US6403076B1 (en) 1992-11-24 2002-06-11 S. Christopher Bauer Compositions for increasing hematopoiesis with interleukin-3 mutants
US6413509B1 (en) 1992-11-24 2002-07-02 S. Christopher Bauer Methods of ex-vivo expansion of hematopoietic cells using interleukin-3 mutant polypeptides with other hematopoietic growth factors
US7091319B1 (en) 1992-11-24 2006-08-15 Bauer S Christopher IL-3 variant hematopoiesis fusion protein

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EP0691403A1 (fr) * 1986-07-14 1996-01-10 Genetics Institute, Inc. Famille de facteurs de croissance hématopoiétique de type IP3 d'origine primate
US6156301A (en) * 1987-10-26 2000-12-05 Sanofi-Synthelabo Use of interleukin-7 to stimulate proliferation of hematopoietic cell precursors
US5714585A (en) * 1987-10-26 1998-02-03 Sterling Winthrop, Inc. Antibodies that are immunoreactive with interleukin-7
US5705149A (en) * 1987-10-26 1998-01-06 Sterling Winthrop Inc. Use of interleukin-7 to stimulate proliferation of hematopoietic cell precursors
US5965122A (en) * 1987-10-26 1999-10-12 Sanofi Use of interleukin-7 to stimulate proliferation of hematopoietic cell precursors
US5328988A (en) * 1987-10-26 1994-07-12 Immunex Corporation Interleukin-7
EP0390252A3 (fr) * 1989-03-15 1991-06-19 Gist-Brocades N.V. Purification de l'interleukine-3 recombinante humaine
WO1990010705A2 (fr) * 1989-03-15 1990-09-20 Gist-Brocades N.V. Production et purification d'interleukine-3 humaine de recombinaison et de ses muteines
WO1990010705A3 (fr) * 1989-03-15 1990-11-01 Gist Brocades Nv Production et purification d'interleukine-3 humaine de recombinaison et de ses muteines
EP0390252A2 (fr) * 1989-03-15 1990-10-03 Gist-Brocades N.V. Purification de l'interleukine-3 recombinante humaine
WO1991000350A1 (fr) * 1989-06-30 1991-01-10 Immunex Corporation Proteines analogues d'interleucine-3 humaine non glycosylatees
US5516512A (en) * 1989-08-14 1996-05-14 Gist-Brocades, N.V. N- and C-terminal truncation and deletion mutants of human interleukin-3
US6500417B1 (en) 1989-08-14 2002-12-31 Dsm N.V. Mutants of human interleukin-3
EP0810285A3 (fr) * 1989-08-14 1998-07-08 Gist-Brocades B.V. Mutants de l'interleukine-3 humaine
EP0810285A2 (fr) 1989-08-14 1997-12-03 Gist-Brocades B.V. Mutants de l'interleukine-3 humaine
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
US6403076B1 (en) 1992-11-24 2002-06-11 S. Christopher Bauer Compositions for increasing hematopoiesis with interleukin-3 mutants
US5738849A (en) * 1992-11-24 1998-04-14 G. D. Searle & Co. Interleukin-3 (IL-3) variant fusion proteins, their recombinant production, and therapeutic compositions comprising them
US5817486A (en) * 1992-11-24 1998-10-06 G. D. Searle & Co. Recombinant human interleukin-3 (IL-3) multiple mutation polypeptides
US5858347A (en) * 1992-11-24 1999-01-12 G. D. Searle & Co. Therapeutic methods using fusion proteins between interleukin-3 (IL-3) variants and other hematopoietic factors
US7091319B1 (en) 1992-11-24 2006-08-15 Bauer S Christopher IL-3 variant hematopoiesis fusion protein
US5677149A (en) * 1992-11-24 1997-10-14 G.D. Searle & Co., Interleukin-3 (IL-3) mutant polypeptides and their recombinant production
US5997857A (en) * 1992-11-24 1999-12-07 G. D. Searle & Co. Co-administration of interleukin-3 mutants with colony stimulating factors
US6479261B1 (en) 1992-11-24 2002-11-12 Pharmacia Corporation Methods of using interleukin-3 (IL-3) mutant polypeptides for ex-vivo expansion of hematopoietic stem cells
US6458931B1 (en) 1992-11-24 2002-10-01 S. Christopher Bauer Interleukin-3 (IL-3) multiple mutation polypeptides
US6440407B1 (en) 1992-11-24 2002-08-27 G. D. Searle Methods of ex-vivo expansion of hematopoietic cells using interleukin-3 (IL-3) multiple mutation polypeptides
US6030812A (en) * 1992-11-24 2000-02-29 G. D. Searle & Company Fusion proteins comprising multiply mutated interleukin-3 (IL-3) polypeptides and second growth factors
US6051217A (en) * 1992-11-24 2000-04-18 G. D. Searle & Co. Therapeutic uses of interleukin-3 (IL-3) multiple mutation polypeptides
US6057133A (en) * 1992-11-24 2000-05-02 G. D. Searle Multivariant human IL-3 fusion proteins and their recombinant production
US6413509B1 (en) 1992-11-24 2002-07-02 S. Christopher Bauer Methods of ex-vivo expansion of hematopoietic cells using interleukin-3 mutant polypeptides with other hematopoietic growth factors
US6060047A (en) * 1992-11-24 2000-05-09 G. D. Searle & Co. Co-administration of interleukin-3 mutant polypeptides with CSF's for multi-lineage hematopoietic cell production
US6074639A (en) * 1992-11-24 2000-06-13 G. D. Searle & Co. Ex vivo expansion of hematopoietic cells using interleukin-3 (IL-3) variant fusion proteins
US6093395A (en) * 1992-11-24 2000-07-25 G. D. Searle & Co. Co-administration of interleukin-3 mutant polypeptides with CSF's for multi-lineage hematopoietic cell production
US5772992A (en) * 1992-11-24 1998-06-30 G.D. Searle & Co. Compositions for co-administration of interleukin-3 mutants and other cytokines and hematopoietic factors
US6132991A (en) * 1992-11-24 2000-10-17 G. D. Searle & Co. Human interleukin-3 (IL-3) variant fusion proteins
US5604116A (en) * 1992-11-24 1997-02-18 G. D. Searle & Co. Interleukin-3 (IL-3) multiple mutation polypeptides, recombinant production of the same, and corresponding therapeutic methods
US6379662B1 (en) 1992-11-24 2002-04-30 Mckearn John P. Co-administration of interleukin-3 mutant polypeptides with CSF's for multi-lineage hematopoietic cell production
US6361977B1 (en) 1992-11-24 2002-03-26 S. Christopher Bauer Methods of using multivariant IL-3 hematopoiesis fusion protein
US6361976B1 (en) 1992-11-24 2002-03-26 S. Christopher Bauer Co-administration of interleukin-3 mutant polypeptides with CSF'S for multi-lineage hematopoietic cell production
US5543141A (en) * 1993-06-21 1996-08-06 G.D. Searle & Co. Therapeutic methods using interleukin-3 (IL-3) human/murine hybrid polypeptides
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
US6022535A (en) * 1993-11-22 2000-02-08 G. D. Searle & Company Treatment of hematopoietic disorders with fusion proteins comprising multiply mutated interleukin-3 (IL-3) polypeptides and second growth factors
US6017523A (en) * 1995-06-06 2000-01-25 G.D. Searle & Co. Therapeutic methods employing mutant human interleukin-3 (IL-3) polypeptides
US6410515B1 (en) 1995-06-07 2002-06-25 Immunotech Developments Inc. Peptide, a method for its preparation and a pharmaceutical composition containing the peptide
US6103699A (en) * 1996-06-07 2000-08-15 Immunotech Developments Inc. Peptide, a method for its preparation and a pharmaceutical composition containing the peptide
US6060595A (en) * 1996-09-03 2000-05-09 The General Hospital Corporation Inhibition of viral replication
WO1999041382A3 (fr) * 1998-02-17 1999-12-16 Hyseq Inc Nouvelle interleukine 3 et ses utilisations
WO1999041382A2 (fr) * 1998-02-17 1999-08-19 Hyseq, Inc. Nouvelle interleukine 3 et ses utilisations

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EP0342206A4 (en) 1990-09-19
EP0342206A1 (fr) 1989-11-23
JPH02501925A (ja) 1990-06-28
AU1496688A (en) 1988-08-10

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