EP0425536A1 - Nonglycosylated human interleukin-3 compositions - Google Patents

Nonglycosylated human interleukin-3 compositions

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Publication number
EP0425536A1
EP0425536A1 EP19890907981 EP89907981A EP0425536A1 EP 0425536 A1 EP0425536 A1 EP 0425536A1 EP 19890907981 EP19890907981 EP 19890907981 EP 89907981 A EP89907981 A EP 89907981A EP 0425536 A1 EP0425536 A1 EP 0425536A1
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Prior art keywords
leu
asp
pro
ala
thr
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German (de)
French (fr)
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EP0425536A4 (en
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Dirk M. Anderson
David J. Cosman
Virginia L. Price
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Immunex Corp
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Immunex Corp
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • C07K14/5403IL-3
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00Drugs for disorders of the blood or the extracellular fluid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to lymphokines, and particularly to pharmaceutical compositions comprising nonglycosylated recombinant analog human interleukin-3 (rhuIL-3) proteins having increased biological activity relative to wild-type proteins.
  • rhuIL-3 human interleukin-3
  • CSFs colony-stimulating factors
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Other CSFs include macrophage CSF (M-CSF or CSF-1), which induces the selective proliferation of macrophages, and granulocyte CSF (G-CSF), which induces development of granulocyte progenitors from bone marrow precursors.
  • M-CSF or CSF-1 macrophage CSF
  • G-CSF granulocyte CSF
  • An additional CSF isolated first in murine systems, and more recently from human cell sources, has been designated IL-3 or multi-CSF.
  • 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).
  • compositions having human IL-3 activity can be employed alone or in combination with other lymphokines, e.g., IL-1, IL-2, IL-4, IL-5, IL-6,
  • IL-7 IL-7, G-CSF, GM-CSF, M-CSF or other growth factors, to potentiate immune responsiveness to infectious pathogens, or to assist in reconstituting normal blood cell populations following viral infection or radiation- or chemotherapy-induced hematopoietic cell suppression.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant human IL-3 protein analog, rhuIL-3 (Asp 15 , Asp 70 ) which is derived by yeast expression, wherein the composition in characterized by a biological activity of at least 3.0 x 10 7 units per milligram (U/mg) in a human bone marrow proliferation assay, a binding affinity for human monocyte IL-3 receptors, expressed as an inhibition constant (Ki), of at least 2.0 x 10 10 M" 1 , and an endotoxin content of less than 1 ng per mg protein.
  • Ki inhibition constant
  • BRTEF DESCRIPTION OF THE DRAWINGS Figure 1 indicates the nucleic acid and corresponding a ino acid sequence of a cDNA sequence corresponding to a wild-type human IL-3 allele designated Pro 8 .
  • the N-terminus of the mature protein begins at the alanine (Ala) residue encoded by nucleotides 1-3.
  • Figure 2 illustrates oligonucleotides employed in the site-specific oligonucleotide mutagenesis procedures described in the Examples.
  • 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 the probes and analyzed by restriction endonuclease cleavage, agarose gel electrophoresis, and additional hybridization experiments ("Southern blots") involving the electrophoresed fragments.
  • Southern blots additional hybridization experiments involving the electrophoresed fragments.
  • the hybridizing segment of one huIL-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.
  • Site-specific oligonucleotide mutagenesis was then performed to provide a cDNA encoding an analog huma IL-3 (Pro 8 , Asp 15 , Asp 70 ) lacking N-linked glycosylation sites.
  • This cDNA was employed to construct a yeast expression vector, which was used to transform an appropriate yeast expression strain, which was then grown under fermentation conditions promoting derepression of the yeast promoter.
  • the resulting protein was then purified by a combination of reversed-phase high-performance liquid chromatography and ion-exchange chromatography to provide a purified product.
  • compositions of this invention are characterized by a biological activity of at least 3.0 x 10 7 U/mg in a human bone marrow proliferation assay, and a binding affinity for human monocyte IL-3 receptors, expressed as an inhibition constant, or KT, of at least 2.0 x 10 10 M" 1 .
  • the compositions of this invention exhibit a biological specific activity of at least 4.0 x 10 7 U/mg, and a Ki of at least 4.0 x 10 10 M" 1 .
  • Human interleukin-3 and “huIL-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 identical to the sequence set forth in FIG. 1.
  • substantially identical means that a particular subject amino acid 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, and wherein the subject sequence is at least 90% identical to the reference sequence.
  • rhuIL-3 (Asp 15 , Asp 70 )
  • residue 1 refers to the first amino acid of the mature protein (see description of Figure 1 above).
  • mutant amino acid sequence refers to a polypeptide encoded by a nucleotide sequence intentionally made variant from a native sequence.
  • mutant protein means a protein comprising a mutant amino acid sequence.
  • N-glycosylation site refers to a nucleotide or amino acid sequence which is identical to a wild-type or native form of a gene or protein.
  • activate means 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, essentially free of native endogenous substances and native mammalian-pattern glycosylation.
  • "Derived by yeast expression” means a protein produced by fermentation of a yeast strain transformed with an expression plasmid. Generally, protein expressed in yeast will have a glycosylation pattern distinguishable from that provided by mammalian cell expression.
  • the analog proteins which are the active component of the pharmaceutical compositions of the present invention lack N-linked carbohydrates. As indicated in the following Examples, this modification provides a protein having higher biological activity and increased affinity for human monocyte IL-3 receptors.
  • DNA segment refers to a DNA polymer, in the form of an isolated fragment or 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 to produce the proteins of the presen invention.
  • tissue culture flasks containing 2 x 10 6 cells per ml pre-warmed, pre-gassed serum-fre 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.
  • 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), an 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
  • Fifty ⁇ 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 ma ⁇ row cells, in a volume of 100 ⁇ l, are then added to each well. Plates are incubated for 4 days at 37°C, 5 CO2, in a plastic box containing sterile distilled H2O to prevent desiccation.
  • binding medium means RPMI 1640 with 2.5% (w/v) bovine serum albumin (BSA), 0.2% (w/v) sodium azide, and 20 mM HEPES, pH 7.2.
  • 125 I rhuIL-3 stock [1 x 10 -9 M in binding medium, specific activity 4 - 8 x 10 15 cpm/mmole], prepared as described below -(Example 3), are then added to each well, followed by 50 ⁇ l of human monocytes [harvested from culture by centrifugation, washed twice with RPMI 1640, and resuspended to 4 x 10 7 cells/ml in binding medium].
  • Each plate is then incubated for 1 hour at 37° C on a rotary shaker with vigorous mixing. After incubation, cells and bound 125 I rhuIL-3 are separated from unbound 125 I rhuIL-3 by a phthalate oil centrifugation technique, conducted as follows.
  • Max is the level of binding of 125 I rhuIL-3 in the presence of medium alone
  • Min is the level of binding in the presence of 1.5 ⁇ g/ml unlabeled rhuIL-3
  • Test is the level of binding in the presence of test sample dilutions. Results are analyzed by non-linear least squares fitting Equation (2) below:
  • Equation (1) M (%) is the maximal level of inhibition
  • K (M" 1 ) is the binding constant of 125 I rhuIL-3
  • C (M) is the concentration of 125 I rhuIL-3
  • 1 (M) is the concentration of the test sample
  • Ki (M" 1 ) is the inhibition (binding) constant of the test sample.
  • Ki for test samples are scaled to that of an rhuIL-3 standard by dividing [K ⁇ /Ks] when Ks is the inhibition constant of the standard, to yield a relative binding activity.
  • the bulk product is assayed for the presence of bacterial endotoxins by a limulus amebocyte lysate assay (LAL), using an LAL test kit in accordance with the manufacturer's instructions.
  • LAL limulus amebocyte lysate assay
  • the compositions of this invention contain not greater than 1 ng endotoxin per mg protein.
  • nucleotides 1-3 provide a GCT codon corresponding to the N-terminal alanine of the mature native protein.
  • 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 huIL-3 can be obtained by screening of appropriate cDNA libraries or by assembly of artificially synthesized oligonucleotides.
  • Complete synthetic human IL-3 cDNAs are now articles of commerce (Beckman Specialty Biochemicals, P.O. Box 10015, Palo Alto, CA, USA; British Bio ⁇ technology Limited, Oxford, England).
  • huIL-3 sequences incorporating codons specifying proline or serine at position 8 of the mature sequence can be assembled usin synthetic oligonucleotides and cDNA fragments.
  • Yeast systems are 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 1- 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. /. Biol. Chem.258:261 A (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 o 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 bears the wild-type human GM-CSF gene.
  • pY ⁇ HuGM ATCC 53157
  • Others are known to those skilled in the art
  • the construction of pY ⁇ HuGM is described in EPA 183,350, and pBC102-K22 is described in EPA 243,153, the respective disclosures of which are 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 ⁇ trpl gall adel his2: J17 (ATCC 52683; a hi ⁇ 2 adel trpl met 14 ura3); and IL166-5B (ATCC 46183; £_ hisl t l).
  • 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.
  • Suitable transformation protocols are described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929 (1978), or the lithium acetate method described by Sherman et al., Laboratory Course Manual or Methods in Yeast Genetics, Cold Spring Laboratory, 1986, 121, selecting for Trp + transformants.
  • 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.
  • Recombinant human IL-3 (rhuIL-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., /. Chromatog.296:171 (1984), and Grabstein et al., J. Exp. Med. 163:1405 (1986).
  • yeast-conditioned medium containing rhuIL-3 can be filtered through a
  • purified mixtures of recombinant glycoproteins such as human or murine granulocvte- macrophage colony stimulating factor (GM-CSF) can consist of from 0 to 50% carbohydrate by weight.
  • GM-CSF granulocvte- macrophage colony stimulating factor
  • glycoprotein 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.
  • rhuIL-3 (Asp 15 , Asp 70 ) analog proteins of the present invention which lack N- linked carbohydrates, exhibit 2-4 fold greater biological specific activity and human monocyte IL-3 receptor binding affinity than its counterpart having unmodified N-glycosylation sites, when each protein is expressed in a recombinant yeast system.
  • N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-Ai-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 .
  • the analog proteins of the present invention have an aspartic acid (Asp) residue substituted for each asparagine in the N-glycosylation sites present in the native human IL-3 sequence.
  • Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes a mutein having the desired amino acid insertion, substitution, or deletion.
  • 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 and Walder, Gene 42:133 (1986); Bauer 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.
  • 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
  • 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. Conditions are employed which result in preferential hybridization of the primer to the mutated 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.
  • huIL-3 proteins are expressed as fusion constructs comprising an octapeptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) fused to the N-terminus of the mature huIL-3 protein.
  • the octapeptide 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 can also be specifically cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing if desired.
  • Peripheral blood lymphocytes were isolated from buf ⁇ y coats prepared from whole blood (Portland Red Cross, Portland, Oregon, USA) by Ficoll hypaque density centrifugation. T cells were isolated by resetting with 2-aminoethylthiouronium bromide-treated sheep red blood cells. Cells were cultured in 175 cm 2 flasks at 5 x 10 6 cells/ml for 18 hour in 100 ml RPMI, 10% fetal calf serum, 50 ⁇ M ⁇ -mercaptoethanol, 1% phytohemagglutinin (PHA) and 1 ng/ml phorbol 12-myristate 13-acetate (PMA).
  • PHA phytohemagglutinin
  • PMA phytohemagglutinin
  • RNA was extracted by the guanidinium CsCI method and poly A + RNA prepared by oligo-dT cellulose chromatography (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982).
  • cDNA was prepared from poly A + RNA essentially as described by Gubler and Hoffman, Gene 25:263-269 (1983) 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.
  • constructs were digested with EcoRI to remove all but one copy of the linkers at each end of the cDNA, ligated to EcoRI-cut and dephosphorylate arms of bacteriophage lgtlO (Huynh et al., DNA Cloning: A Practical Approach, Glover, ed., IRL Press, pp. 49-78), and packaged into lambda phage extracts (Stratagene, San Diego, CA, USA) according to the manufacturer's instructions. 500,000 recombinants were plated on E. coli strain C600hf 1" and screened by standard plaque hybridization techniques using the following probes.
  • Two oligonucleotides were synthesized, with sequences complementary to selected 5 and 3' sequences of the huIL-3 gene.
  • the 5' probe complementary to a sequence encoding part of the huIL-3 leader, had the sequence 5'-GAGTTGGAGCAGGAGCAGGAC-3'.
  • the probe corresponding to a region encoding amino acids 123-130 of the mature protein, had the sequence 5'-GATCGCGAGGCTCAAAGTCGT-3'.
  • the method of synthesis was a standard automated triester method substantially similar to that disclosed by Sood et al., Nucl. Acids Res.42- 557 (1977) and Hirose et al., Tet. Lett. 28:2449 (1978).
  • oligonucleotides were deblocked and purified by preparative gel electrophoresis.
  • the oligonucleotides were terminally radiolabeled with 32 P-ATP and T4 polynucleotide kinase using techniques similar to those disclosed by Maniatis et al.
  • the E. col strain used for library screening was C600hfl- (Huynh et al., 1985, supra).
  • the nucleotide sequences of selected clones were determined by the chain termination method. Specifically, partial EcoRI digestion of ⁇ GT10:IL-3 clones 2, 3, 4 and 5 yielded fragments ranging from 850 bp to 1,000 bp in size which were separately subcloned into the EcoRI site of pGEMBLl ⁇ .
  • the inserts of the pGEMBL:rhuIL-3 subclones were sequenced using a universal primer that binds adjacent to the multiple cloning site of pGEMBLl ⁇ , and synthetic oligonucleotide primers derived from the huIL-3 sequence. Restriction mapping indicated the presence of restriction sites corresponding to those previously reported for human IL-3.
  • PCR polymerase chain reaction
  • a yeast expression vector was constructed by digesting pBC102*K22 (ATCC
  • cDNA encoding huIL-3 was excised from a pGEMB l ⁇ cloning vector (see Example A, above) by digestion with Hpal and BamEH, providing a fragment extending from amino acid 14 of mature huIL-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 s-equence encoding a synthetic N-terminal "flag" identification peptide (DYKDDDDK; s»ee above); and (3) a short sequence encoding the N-terminal 14 amino acids of mature huTL-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:
  • Plasmid DNA from an E. coli clone containing the pBC125 vector was isolated and used to transform yeast strain XV2181 for expression of the rhuIL-3 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. These experiments indicated high level expression of the rhuIL-3 protein.
  • Example 1 Modification of N-GIycosylation Sites Encoded by huIL-3 cDNA and Assembly of Expression Vector for rhuIL-3 (Pro 8 -. Asp 5 -. Asp 7 - 0 -)
  • the two asparagine linked glycosylation sites present in the natural protein (Asn 15 an Asn 70 ) were removed by changing the codons at these positions to ones that encode aspartic acid. This prevents N-linked glycosylation (often hyperglycosylation) of the secreted protein by the yeast cells and a more homogeneous product is obtained. These changes were made as described below upon subcloning the huIL-3 cDNA into the yeast expression vector pD Y120.
  • the yeast expression vector pIXY120 is substantially identical to pBC115, described in Example B, above, except that the following synthetic oligonucleotide containing multiple cloning sites was inserted from the Asp718 site (amino acid 79) near the 3' end of the ⁇ -factor signal peptide to the Spel site contained in the 2 ⁇ sequences: Asp718 -iNcoI
  • a 514 bp DNA fragment derived from the single-stranded bacteriophage fl containing the origin of replication and intergenic region was inserted at the Nrul site in the pBR322 DNA sequences.
  • the presence of the f 1 origin of replication allows one to generate single-stranded copies of the vector when transformed into appropriate (male) strains of E. coli and superinfected with bacteriophage f 1. This capability facilitates DNA sequencing of the vector and allows the possibility to do in vitro mutagenesis.
  • the yeast expression vector pIXYl 20 was digested with the restriction enzymes Asp718, which cleaves near the 3' end of the ⁇ -factor leader peptide (nucleotide 237), and BamHl , which cleaves in the polylinker.
  • the large vector fragment was purified and ligated to the following DNA fragments: (1) a huIL-3 cDNA fragment derived from plasmid GEMBL18:huIL-3 from the Cla 1 site (nucleotide 58 of mature huIL-3) to the BamHl site (3' to the huIL-3 cDNA in a polylinker); and (2) a synthetic oligonucleotide linker (oligonucleotide A, Figure 2) regenerating the sequence encoding the C-terminus of the a-factor leader peptide and fusing it in-frame to the octapeptide DYKDDDDK, which is, in turn, fused to the N- terminus of mature rhuIL-3.
  • a synthetic oligonucleotide linker oligonucleotide A, Figure 2
  • This fusion to the rhuIL-3 protein allows detection with antibody specific for the octapeptide and was used initially for monitoring the expression and purification of rhuIL-3.
  • This oligonucleotide also encodes an amino acid change at position 15 (Asn 1 ⁇ Asp 15 ) to alter this N-linked glycosylation site.
  • the underlined nucleotides in FIG. 2 represent changes from the wild type cDNA sequence. Only the A to G and C to T changes at nucleotides 43 and 45, respectively (counting from the codon corresponding to the N-terminal alanine of the mature huIL-3 molecule) result in an amino acid change (Asp 15 ).
  • the other base changes introduce convenient restriction sites (Ahall and PvuII) without altering the amino acid sequence.
  • the resulting plasmid was designated pIXY139 and contains a rhuIL-3 cDNA with one remaining N-linked glycosylation consensus sequence (Asn 70 ).
  • Plasmid pIXY139 was used to perform oligonucleotide-directed mutagenesis to remove the second N-linked glycosylation consensus sequence by changing Asn 70 to Asp 70 .
  • the in vitro mutagenesis was conducted by a method similar to that described by Walder and Walder, supra.
  • the yeast vector, pIXY139 contains the origin of replication for the single- stranded bacteriophage f 1 and is capable of generating single-stranded DNA when present in a suitable (male) strain of E. coli and superinfected with helper phage.
  • Single-stranded DNA was generated by transforming E. coli strain JM107 and superinfecting with helper phage IR1. Single-stranded DNA was isolated and annealed to the mutagenic oligonucleotide B, shown in FIG. 2, which provides a codon switch substituting Asp for Asn at position 70 of mature huIL-3. Annealing and yeast transformation conditions were done as described by Walder and Walder, supra. Yeast transformants were selected by growth on medium lacking tryptophan, pooled, and DNA extracted as described by Holm et al., Gene 42:169 (1986). This DNA, containing a mixture of wild type and mutant plasmid DNA, was used to transform E. coli RR1 to ampicillin resistance.
  • Plasmids comprising DNA encoding huIL-3 Asp 70 were identified by the hybridization to radiolabelled oligonucleotide B under stringent conditions and verified by nucleotide sequencing.
  • the resulting yeast expression plasmid was designated pDCY138, and contained the huIL-3 gene encoding the Asp 15 Asp 70 amino acid changes and the octapeptide DYKDDDDK at the N-terminus.
  • the final yeast expression plasmid is identical to pIXY138 except that it lacks the nucleotide sequences coding for the octapeptide, thus generating mature rhuIL-3 as the product.
  • the final yeast expression plasmid was constructed as described below.
  • the yeast expression vector pIXY120 was cleaved with the restriction enzymes Asp718 and BamHl as described above.
  • the large vector fragment was ligated together with (1) a huIL-3 cDNA fragment derived from plasmid pIXY138 that extended from the AhaH site (which cleaves a nucleotide 19 of mature huIL-3) to the BamHl site 3' to the cDNA, and (2) a synthetic oligonucleotide (oligonucleotide C, Figure 2) regenerating the 3' end of the ⁇ -factor leader peptide from the Asp718 site (the amino acids Pro-Leu-Asp-Lys-Arg) and the N-terminal seven amino acids of huIL-3 to the Ahall site.
  • the resulting plasmid was designated pD Y151.
  • This vector when present in yeast, allows glucose-regulated expression and secretion of rhuTL-3 (Pro 8 , Asp 15 , Asp 70 ).
  • Example 2 Expression of Protein rhuIL-3 (Pro 8 -. Asp-1- 5 -. Asp 7 - 0 -)
  • the host strain XV2181, a diploid S. cerevisiae strain, was formed by mating
  • Yeast containing the expression plasmid pIXY151 are maintained on YNB-trp agar plates stored at 4°C.
  • a preculture is started by inoculating several isolated recombinant yeast colonies into one liter of YNB-ttp medium (6.7 g L Yeast Nitrogen Base, 5 g/L casamino acids, 40 mg/L adenine, 160 mg/L uracil, and 200 mg L tyrosine), and i grown overnight in two 2-liter flasks at 30°C with vigorous shaking.
  • YNB-ttp medium 6.7 g L Yeast Nitrogen Base, 5 g/L casamino acids, 40 mg/L adenine, 160 mg/L uracil, and 200 mg L tyrosine
  • the fermenters (three machines of 10 liter working volume), previously cleaned and sterilized, are filled to 80% of their working capacity with SD-2 medium (4.0 g/L ammonium sulfate, 3.2 g/L monobasic potassium phosphate, 3.0 g/L yeast extract, 1.0 g L citric acid, 0.1 g/L sodium chloride, 5 ml/L 2% calcium chloride, 2.5 ml/L vitamin 101 solution, 0.5 ml L trace elements solution, 0.5 ml/L 20% magnesium sulfate, 2.0 ml/L glucose) and maintained at 30"C with 500-600 rpm agitation and 10-161pm aeration. The inoculum is added.
  • SD-2 medium 4.0 g/L ammonium sulfate, 3.2 g/L monobasic potassium phosphate, 3.0 g/L yeast extract, 1.0 g L citric acid, 0.1 g/L sodium chloride, 5 ml/L 2% calcium chloride, 2.5
  • a nutrient feed of 50% glucose is begun at a rate such that 50 g/L is added over a period of 10-12 hours.
  • the nutrient feed is then shifted to 50% ethanol added at 30-40 ml hr until harvest.
  • Total elapsed time of fermentation is approximately 20 hours, after which optical density (600nm) ranges from 30 to 45.
  • the fermenters are then cooled to 20°C, pH of the yeast beer is adjusted to 8.0 by the addition of 5M NaOH, and the resulting material filtered through a Millipore Pellicon filter system equipped with a 0.45 ⁇ m filter cassette, and collected in a sterile 10 L carboy.
  • the rhuJL-3 contained in the yeast broth is applied to a 5 cm X 30 cm column packed with 15-20 ⁇ m C-4 reversed-phase silica (Vydac, Separations Group, Hesperia, CA, USA) by pumping the filtered yeast broth directly onto the column.
  • the column is equilibrated in 0.1% trifluoroacetic acid in water (Solvent A) prior to application of the yeast broth and is flushed with this solvent following the completion of the application of the broth to the column until the optical absorbance of the effluent approaches base line values.
  • Fractions containing rhuIL-3 from the first step are stored at 4°C in polyethylene bottles until approximately 1 gram total protein is obtained. To the pool is added 2 volumes of 0.1% trifluoroacetic acid in water. This solution is then pumped onto a second 5 cm X 30 cm column packed with 15-20 ⁇ C-18 silica (Vydac, Separations Group, Hesperia, CA, USA) that is equilibrated in Solvent A (0.1% TFA in water). Following application of the material, the column is flushed with Solvent A and then a gradient identical to that described above is established at a rate of change of 1% Solvent B per minute and at a flow rate of 100 ml/min. Fractions are collected every 0.4 minutes 24 minutes into the gradient. Aliquots of the fractions are analyzed for protein content by the fluorescamine assay using BSA as a standard.
  • Peak fractions from the C-18 column are pooled and 1/10 volume of 0.5M ⁇ -alanine pH 3.6 is added. A sample is taken and then the pool is applied to a 10 ml S-Sepharose Column (1 cm X 10 cm, Pharmacia) at 5 ml/minute. After sample application, the column is washed with 50 ml of lOmM Tris, pH 7.4 and the IL-3 is eluted with a linear gradient from lOmM Tris, pH 7.4 to 200mM Tris, pH 7.4 (200 ml total gradient volume).
  • rhuIL-3 Three rhuIL-3 proteins were prepared for assay as follows. First, rhuIL-3 (Pro 8 ) comprising the N-terminal octapeptide DYKDDDDK was expressed using the yeast expression plasmid pBC125 described above. Purification of this fusion protein from the yeast culture broth was accomplished in a single affinity chromatography step using a monoclonal antibody specific for the octapeptide, as described in published PCT Application PCT/US 87/03113. Second, rhuIL-3 (Pro 8 , Asp 15 , Asp 70 ) was prepared by yeast fermentation and was purified from yeast broth by reversed-phase high performance liquid chromatography as described in Example 2, above.
  • a rhuIL-3 containing both the octapeptide and the changes at positions 15 and 70 was prepared by fermentation of yeast comprising expression plasmid pD Y138 described in Example 1, above, and purified by affinity chromatography as described above.
  • Concentrations of the purified rhuIL-3 proteins were determined by amino acid analysis.
  • rhuIL-3 (octapeptide fusion protein) was radiolabeled using the enzymobead radioiodination reagent (BioRad Laboratories, Richmond, CA, USA) essentially as previously described for murine GM-CSF (25). Briefly, 2.5 ⁇ g of IL-3 in 0.1 M glycine HC1, pH 7.4, was brought to a final volume of 50 ⁇ l with 0.2 M sodium phosphate, pH 7.2. Fifty ⁇ l of enzymobead reagent, 20 ⁇ l of 125 I (2 mCi) and 10 ⁇ l of 2.5% ⁇ -D-glucose were added and the mixture incubated at 25°C for 10 min.
  • rhuIL-3 Sodium azide (10 ⁇ l of 50 mM) and sodium metabisulfite (10 ⁇ l of 5 mg/ml) were then added sequentially. After 5 min at 25°C, iodinated rhuIL-3 was separated from free 125 I by chromatography on a 2 ml Sephadex G-25 column equilibrated in RPMI-1640 containing 2% bovine serum albumin, 20 mM Hepes buffer and 0.2% sodium azide, pH 7.2 (binding medium). Fractions containing rhuIL-3 were pooled, diluted to a concentration of 0.5 ⁇ g/ml in the same buffer and stored at 4°C.
  • Bioactivity of 125 I-rhuIL-3 was determined in the human bone marrow proliferation assay described above o samples in which sodium azide had been deleted during the separation step.
  • the specific activities of radiolabeled preparations were estimated to be 3-6 x 10 15 cpm/mmole.
  • the N- terminal octapeptide of the fusion protein contains a tyrosine residue which, in the case of rhuIL-3, is useful in labeling of this molecule with 125 I to high specific activity, since the nativ IL-3 molecule contains only a single tyrosine residue.

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Abstract

On a mis au point une composition pharmaceutique comprenant un analogue de protéine IL-3 humaine recombinante, rhuIL-3(Asp15, Asp70), dérivé par expression à la levure et présentant une activité biologique d'au moins 3,0 x 107 unités par milligramme dans un échantillon de prolifération de moelle osseuse humaine, ainsi qu'une affinité de liaison pour des récepteurs d'IL-3 de monocytes humains, exprimée en tant que constante d'inhibition, d'au moins 2,0 x 1010 M-1, et une teneur en endotoxine inférieure à 1 ng par mg de protéine.A pharmaceutical composition has been developed comprising a recombinant human IL-3 protein analog, rhuIL-3 (Asp15, Asp70), derived by expression with yeast and having a biological activity of at least 3.0 × 10 7 units per milligram in a human bone marrow proliferation sample, as well as a binding affinity for human monocyte IL-3 receptors, expressed as an inhibition constant, of at least 2.0 x 1010 M- 1, and an endotoxin content of less than 1 ng per mg of protein.

Description

TITLE Nonglycosylated Human Interleukin-3 Compositions
BACKGROUND OF THE INVENTION The present invention relates generally to lymphokines, and particularly to pharmaceutical compositions comprising nonglycosylated recombinant analog human interleukin-3 (rhuIL-3) proteins having increased biological activity relative to wild-type proteins.
The differentiation and proliferation of hematopoietic cells is regulated by secreted glycoproteins collectively known as colony-stimulating factors (CSFs). These proteins include granulocyte-macrophage colony-stimulating factor (GM-CSF), which promotes granulocyte and macrophage production from normal bone marrow and which also appears to regulate the activity of mature, differentiated granulocytes and macrophages. Other CSFs include macrophage CSF (M-CSF or CSF-1), which induces the selective proliferation of macrophages, and granulocyte CSF (G-CSF), which induces development of granulocyte progenitors from bone marrow precursors. An additional CSF, isolated first in murine systems, and more recently from human cell sources, has been designated IL-3 or multi-CSF.
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, 20α-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 Yang et al., Cell 47:3 (1986). The deduced amino acid sequence of the human IL-3 homologue was approximately 29% homologous to murine IL-3. Ihle and Weinstein, Adv. Immunol.39: 1 (1986) provide a comprehensive review of publications relating to the biological activities of IL- 3.
Preclinical studies involving IL-3 indicate clinical utility as a therapeutic agent in treatment of various cytopenias. Therapeutic compositions having human IL-3 activity can be employed alone or in combination with other lymphokines, e.g., IL-1, IL-2, IL-4, IL-5, IL-6,
IL-7, G-CSF, GM-CSF, M-CSF or other growth factors, to potentiate immune responsiveness to infectious pathogens, or to assist in reconstituting normal blood cell populations following viral infection or radiation- or chemotherapy-induced hematopoietic cell suppression.
SUMMARY OF THE INVENTION
The present invention provides a pharmaceutical composition comprising a recombinant human IL-3 protein analog, rhuIL-3 (Asp15, Asp70) which is derived by yeast expression, wherein the composition in characterized by a biological activity of at least 3.0 x 107 units per milligram (U/mg) in a human bone marrow proliferation assay, a binding affinity for human monocyte IL-3 receptors, expressed as an inhibition constant (Ki), of at least 2.0 x 1010 M"1, and an endotoxin content of less than 1 ng per mg protein.
BRTEF DESCRIPTION OF THE DRAWINGS Figure 1 indicates the nucleic acid and corresponding a ino acid sequence of a cDNA sequence corresponding to a wild-type human IL-3 allele designated Pro8. The N-terminus of the mature protein begins at the alanine (Ala) residue encoded by nucleotides 1-3.
Figure 2 illustrates oligonucleotides employed in the site-specific oligonucleotide mutagenesis procedures described in the Examples.
DETAILED DESCRIPTION OF THE INVENTION 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 the probes and analyzed by restriction endonuclease cleavage, agarose gel electrophoresis, and additional hybridization experiments ("Southern blots") involving the electrophoresed fragments. After isolating several clones which hybridized to each of the probes, the hybridizing segment of one huIL-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.
Sequencing of several clones isolated from the PBT library indicated the presence in each of a proline residue at position 8 of the mature huIL-3 sequence. Site-specific oligonucleotide mutagenesis was then performed to provide a cDNA encoding an analog huma IL-3 (Pro8, Asp15, Asp70) lacking N-linked glycosylation sites. This cDNA was employed to construct a yeast expression vector, which was used to transform an appropriate yeast expression strain, which was then grown under fermentation conditions promoting derepression of the yeast promoter. The resulting protein was then purified by a combination of reversed-phase high-performance liquid chromatography and ion-exchange chromatography to provide a purified product. Assay of this product using a human bone marrow proliferation assay and human IL-3 receptor binding assay confirmed expression of an analog product suitable for human pharmaceutical use which exhibits higher biological activity than comparabl human IL-3 proteins lacking similar molecular modifications. The compositions of this invention are characterized by a biological activity of at least 3.0 x 107 U/mg in a human bone marrow proliferation assay, and a binding affinity for human monocyte IL-3 receptors, expressed as an inhibition constant, or KT, of at least 2.0 x 1010 M"1. Preferably, the compositions of this invention exhibit a biological specific activity of at least 4.0 x 107 U/mg, and a Ki of at least 4.0 x 1010 M"1.
Definitions "Human interleukin-3" and "huIL-3" refer to a human endogenous secretory protein which induces proliferation of granulocyte, macrophage, and erythrocyte progenitors from populations of multipotent hematopoietic stem cells. As used throughout the specification, the term means a protein having IL-3 biological activity and an amino acid sequence which is substantially identical to the sequence set forth in FIG. 1. "Substantially identical" means that a particular subject amino acid 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, and wherein the subject sequence is at least 90% identical to the reference sequence. Sequences having lesser degrees of identity, comparable bioactivity, and equivalent expression characteristics are considered equivalents. For purposes of determining substantial identity, truncations and additions to the reference sequence are disregarded. The term "rhuIL-3 (Asp15, Asp70)" means a recombinant human interleukin-3 polypeptide having aspartic acid (Asp) residues at positions 15 and 70, wherein residue 1 refers to the first amino acid of the mature protein (see description of Figure 1 above). "Mutant amino acid sequence" refers to a polypeptide encoded by a nucleotide sequence intentionally made variant from a native sequence. "Mutant protein," "analog protein" or "mutein" means a protein comprising a mutant amino acid sequence. "Native sequence" refers to a nucleotide or amino acid sequence which is identical to a wild-type or native form of a gene or protein. The term "N-glycosylation site" is defined below. The term "inactivate," as used in defining particular aspects of the present invention, means to alter an N-glycosylation site to preclude covalent bonding of oligosaccharide moieties to particular amino acid residues.
"Recombinant," as used herein, means that a protein is derived from recombinant microbial (e.g., bacterial or fungal) expression systems, essentially free of native endogenous substances and native mammalian-pattern glycosylation. "Derived by yeast expression" means a protein produced by fermentation of a yeast strain transformed with an expression plasmid. Generally, protein expressed in yeast will have a glycosylation pattern distinguishable from that provided by mammalian cell expression. However, the analog proteins which are the active component of the pharmaceutical compositions of the present invention lack N-linked carbohydrates. As indicated in the following Examples, this modification provides a protein having higher biological activity and increased affinity for human monocyte IL-3 receptors. "DNA segment" refers to a DNA polymer, in the form of an isolated fragment or 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.
Preferably, 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 to produce the proteins of the presen invention.
Assays for rhuIL-3 Biological Activity Assays used to measure rhuIL-3 biological activity are described below. 1. Human Bone Marrow Proliferation Assay Freshly isolated human bone marrow cells are preincubated for 2 hours at 37°C, 5%
CO*2, in tissue culture flasks containing 2 x 106 cells per ml pre-warmed, pre-gassed serum-fre RPMI 1640 medium (Gibco, Chagrin Falls, OH, USA) containing 50 units/ml penicillin, 50 μg/ml streptomycin, and 300 μg ml fresh L-glutamine (hereinafter "assay medium"). After preincubation, nonadherent cells are removed by pipetting the media gently over the surface of the flask. 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), an 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.
Fifty μl assay medium are added to each well of a 96-well flat bottom tissue culture plate. 50 μl 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 104 bone maπrow cells, in a volume of 100 μl, are then added to each well. Plates are incubated for 4 days at 37°C, 5 CO2, in a plastic box containing sterile distilled H2O to prevent desiccation. On day 4, 25 μl o assay medium containing 5% FBS and 80 μCi/ml [3H]-thymidine (80 Ci mmol) are .added to each well and the plate incubated 5 hr at 37βC, 5% CO2, in a plastic box. After incubation, cells are harvested onto glass fiber filters, washed, and tested for incorporated radioactivity using a scintillation counter. Units of huIL-3 activity are calculated by reference to the quanti of huIL-3 which induces 50% of maximal thymidine incorporation. For example, if a 100 μl 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 μl, or 5 μl. The sample would therefore contai 1000 divided by 5, or 200 units per milliliter (U/ml) of huIL-3 activity. 2. Human IL-3 Receptor Binding Assay
In a 96- well round bottom microtiter plate, ten 2-fold dilutions of each sample are prepared in RPMI 1640 medium beginning at 0.075 μg ml (50 μl each). For each assay plate, triplicate 50 μl aliquots of rhuTL-3 standards (1.5 μg/ml) and negative controls (binding medium) are set up. For purposes of this assay, "binding medium" means RPMI 1640 with 2.5% (w/v) bovine serum albumin (BSA), 0.2% (w/v) sodium azide, and 20 mM HEPES, pH 7.2. 50 μl of 125I rhuIL-3 stock [1 x 10-9 M in binding medium, specific activity 4 - 8 x 1015 cpm/mmole], prepared as described below -(Example 3), are then added to each well, followed by 50 μl of human monocytes [harvested from culture by centrifugation, washed twice with RPMI 1640, and resuspended to 4 x 107 cells/ml in binding medium]. Each plate is then incubated for 1 hour at 37° C on a rotary shaker with vigorous mixing. After incubation, cells and bound 125I rhuIL-3 are separated from unbound 125I rhuIL-3 by a phthalate oil centrifugation technique, conducted as follows. First, duplicate 65 μl aliquots are removed from each incubation mixture, and each aliquot is layered onto 200 μl of a cold phthalate oil mixture [1.5 parts dibutyl phthalate, 1 part bis(-2-ethylhexyl)-phthalate (Eastm.an Kodak Co., Rochester, NY, USA)] in 400 μl polyethylene centrifuge tubes and centrifuged for 1 minute in a microcentrifuge at 8°C. The cells plus bound 125I rhuIL-3 sediment through the phthalate oil mixture while the less dense binding medium containing unbound 125I rhuIL-3 is left on its surface. 125I rhuIL-3 bound to cells is measured by cutting the tubes in half and counting top and tip portions for 125I.
The results of the IL-3 receptor binding assay are expressed as inhibition (%) calculated according to Equation (1) below: l ( % ) = 1QQ x (Max - es ) Equation 1 (Max - Min)
where Max is the level of binding of 125I rhuIL-3 in the presence of medium alone, Min is the level of binding in the presence of 1.5 μg/ml unlabeled rhuIL-3 and Test is the level of binding in the presence of test sample dilutions. Results are analyzed by non-linear least squares fitting Equation (2) below:
Equation 2 M (% ) = M • K I
1 + K C + Ki I
to the data generated by Equation (1) where M (%) is the maximal level of inhibition, K (M"1) is the binding constant of 125I rhuIL-3, C (M) is the concentration of 125I rhuIL-3, 1 (M) is the concentration of the test sample and Ki (M"1) is the inhibition (binding) constant of the test sample.
The values of Ki for test samples are scaled to that of an rhuIL-3 standard by dividing [Kτ/Ks] when Ks is the inhibition constant of the standard, to yield a relative binding activity. 3. Limulus Amebocvte Lvsate Assay for Detection of Bacterial Endotoxins
The bulk product is assayed for the presence of bacterial endotoxins by a limulus amebocyte lysate assay (LAL), using an LAL test kit in accordance with the manufacturer's instructions. The compositions of this invention contain not greater than 1 ng endotoxin per mg protein.
The Native huIL-3 Sequence The nucleotide and corresponding amino acid sequences of the huIL-3 cDNA isolated as described below are set forth in FIG. 1. In FIG. 1, nucleotides 1-3 provide a GCT codon corresponding to the N-terminal alanine of the mature native protein. 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 huIL-3 can be obtained by screening of appropriate cDNA libraries or by assembly of artificially synthesized oligonucleotides. Complete synthetic human IL-3 cDNAs are now articles of commerce (Beckman Specialty Biochemicals, P.O. Box 10015, Palo Alto, CA, USA; British Bio¬ technology Limited, Oxford, England). As a matter of choice, huIL-3 sequences incorporating codons specifying proline or serine at position 8 of the mature sequence can be assembled usin synthetic oligonucleotides and cDNA fragments.
Protein Expression in Recombinant Yeast Systems
Yeast systems are 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 (Ap1- gene and origin of replication) and yeast DNA sequences including a glucose-repressible alcohol dehydrogenase 2 (ADH2) promoter. The ADH2 promoter has been described by Russell et al. /. Biol. Chem.258:261 A (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 μ origin of replication. Adjacent to the promoter is the yeast α-factor leader sequence enabling secretion o heterologous proteins from a yeast host. The α-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. 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). Alternative expression vectors are yeast vectors which comprise an α-factor promoter, for example pYαHuGM (ATCC 53157), which bears the wild-type human GM-CSF gene. Others are known to those skilled in the art The construction of pYαHuGM is described in EPA 183,350, and pBC102-K22 is described in EPA 243,153, the respective disclosures of which are incorporated by reference herein.
The choice of appropriate 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 pYαHuGM include strains X2181-1B, available from the Yeast Genetic Stock Center, Berkeley, CA, USA (see below), having the genotype αtrpl gall adel his2: J17 (ATCC 52683; a hi§2 adel trpl met 14 ura3); and IL166-5B (ATCC 46183; £_ hisl t l). 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. Suitable transformation protocols are described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929 (1978), or the lithium acetate method described by Sherman et al., Laboratory Course Manual or Methods in Yeast Genetics, Cold Spring Laboratory, 1986, 121, selecting for Trp+ transformants.
Host strains transformed by vectors comprising the ADH2 or α-factor promoters are grown for expression in a rich medium consisting of 1 % yeast extract, 2% peptone, and 1 % glucose supplemented with 80 μg/ml adenine and 80 μg/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.
Purification of IL-3
Recombinant human IL-3 (rhuIL-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., /. Chromatog.296:171 (1984), and Grabstein et al., J. Exp. Med. 163:1405 (1986). For example, yeast-conditioned medium containing rhuIL-3 can be filtered through a
0.45 μ filter and pumped, at a flow rate of 100 ml/min, onto a 5 cm x 30 cm column packed with 15-20 μm 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. At this time, a gradient of 0.1% trifluoroacetic acid in acetonitrile (Solvent B) can be established that leads from 0 to 100% Solvent B at a rate of change of 2% per minute and at a flow rate of 100 ml/min. At a suitable time following initiation of the gradient, one minute fractions are collected and aliquots of the fractions analyzed for protein content by polyacrylamide gel electrophoresis and fluorescamine protein determination. Additional HPLC steps can be employed if indicated. A complete purification protocol additionally comprising application and elution from S-Sepharose is detailed in Example 2, below. Inactivation of N-glvcosylation Sites Many secreted proteins acquire covalently attached carbohydrate units following translation, frequently in the form of σligosaccharide units linked to asparagine side chains by N-glycosidic bonds. Both the structure and number of oligosaccharide units attached to a particular secreted protein can be highly variable, resulting in a wide range of apparent molecular masses attributable to a single glycoprotein. Recombinant huIL-3 is a secreted glycoprotein of this type. Attempts to express glycoproteins in recombinant systems can be complicated by the heterogeneity attributable to this variable carbohydrate component. For example, purified mixtures of recombinant glycoproteins such as human or murine granulocvte- macrophage colony stimulating factor (GM-CSF) can consist of from 0 to 50% carbohydrate by weight. Miyajima et al., EMBO J.5:1193 (1986) reported expression of a recombinant murine GM-CSF in which N-glycosylation sites had been mutated to preclude glycosylation and reduce heterogeneity of the yeast-expressed product.
The presence of variable quantities of associated carbohydrate in recombinant secreted glycoproteins complicates purification procedures, thereby reducing yield. In addition, should the 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. Further, the rhuIL-3 (Asp15, Asp70) analog proteins of the present invention, which lack N- linked carbohydrates, exhibit 2-4 fold greater biological specific activity and human monocyte IL-3 receptor binding affinity than its counterpart having unmodified N-glycosylation sites, when each protein is expressed in a recombinant yeast system.
N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-Ai-Z, where A1 is any amino acid except Pro, and Z is Ser or Thr. In this sequence, 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 A1 and Z, or an amino acid other than Asn between Asn and A1. The analog proteins of the present invention have an aspartic acid (Asp) residue substituted for each asparagine in the N-glycosylation sites present in the native human IL-3 sequence.
Construction of Analog Sequences and Muteins Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes a mutein having the desired amino acid insertion, substitution, or deletion.
Alternatively, 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 and Walder, Gene 42:133 (1986); Bauer 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.
For either approach, conventional techniques for oligonucleotide synthesis are suitable, for example, the triester synthesis procedures disclosed by Sood et al., Nucl. Acids Res. 4:2557 (1977) and Hirose et al., Tet. Lett. 28:2449 (1978).
In site-specific mutagenesis, 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. If an insertion is desired, the primer will include a new codon, at the appropriate location in the sequence, specifying the amino acid to be inserted. Preferably, the substitute codon, deleted codon, or inserted codon is located at or near the center of the oligonucleotide.
The size of the 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. Thus, oligonucleotides used in accordance with the present invention will usually contain from about 15 to about 25 bases.
In a preferred approach (see Walder and Walder, supra), the resulting oligonucleotide/ss vector hybrid is directly transformed into yeast. Alternatively, a mutagenic primer is hybridized to a gapped duplex having a single-stranded template segment containing the gene to be altered. In the latter case, 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. In both cases, replication of the heteroduplex by the host provides progeny of both strands. In 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. Conditions are employed which result in preferential hybridization of the primer to the mutated 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.
Construction of Fusion Proteins In one embodiment of the present invention, huIL-3 proteins are expressed as fusion constructs comprising an octapeptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) fused to the N-terminus of the mature huIL-3 protein. The octapeptide 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 can also be specifically cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing if desired.
EXPERIMENTAL DISCUSSION Example A. Isolation of cDNA encoding huIL-3
Peripheral blood lymphocytes were isolated from bufϊy coats prepared from whole blood (Portland Red Cross, Portland, Oregon, USA) by Ficoll hypaque density centrifugation. T cells were isolated by resetting with 2-aminoethylthiouronium bromide-treated sheep red blood cells. Cells were cultured in 175 cm2 flasks at 5 x 106 cells/ml for 18 hour in 100 ml RPMI, 10% fetal calf serum, 50 μM β-mercaptoethanol, 1% phytohemagglutinin (PHA) and 1 ng/ml phorbol 12-myristate 13-acetate (PMA). RNA was extracted by the guanidinium CsCI method and poly A+ RNA prepared by oligo-dT cellulose chromatography (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982). cDNA was prepared from poly A+ RNA essentially as described by Gubler and Hoffman, Gene 25:263-269 (1983) 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. These constructs were digested with EcoRI to remove all but one copy of the linkers at each end of the cDNA, ligated to EcoRI-cut and dephosphorylate arms of bacteriophage lgtlO (Huynh et al., DNA Cloning: A Practical Approach, Glover, ed., IRL Press, pp. 49-78), and packaged into lambda phage extracts (Stratagene, San Diego, CA, USA) according to the manufacturer's instructions. 500,000 recombinants were plated on E. coli strain C600hf 1" and screened by standard plaque hybridization techniques using the following probes.
Two oligonucleotides were synthesized, with sequences complementary to selected 5 and 3' sequences of the huIL-3 gene. The 5' probe, complementary to a sequence encoding part of the huIL-3 leader, had the sequence 5'-GAGTTGGAGCAGGAGCAGGAC-3'. The probe, corresponding to a region encoding amino acids 123-130 of the mature protein, had the sequence 5'-GATCGCGAGGCTCAAAGTCGT-3'. The method of synthesis was a standard automated triester method substantially similar to that disclosed by Sood et al., Nucl. Acids Res.42- 557 (1977) and Hirose et al., Tet. Lett. 28:2449 (1978). Following synthesis, oligonucleotides were deblocked and purified by preparative gel electrophoresis. For use as screening probes, the oligonucleotides were terminally radiolabeled with 32P-ATP and T4 polynucleotide kinase using techniques similar to those disclosed by Maniatis et al. The E. col strain used for library screening was C600hfl- (Huynh et al., 1985, supra).
Thirteen positive plaques were purified and re-probed separately with the two hybridization probes. Eleven clones hybridized to both oligonucleotides. The cDNA inserts from several positive recombinant phage were subcloned into a an EcoRI-cut derivative (pGEMBLlδ) of the standard cloning vector pBR322 containing a polylinker having a unique EcoRI site, a BamHl site and numerous other unique restriction sites. An exemplary vector of this type, pEMBL, is described by Dente et al., Nucl. Acids Res. 77:1645-1655 (1983), in which the promoters for SP6 and T7 polymerases flank the multiple cloning sites. The nucleotide sequences of selected clones were determined by the chain termination method. Specifically, partial EcoRI digestion of λGT10:IL-3 clones 2, 3, 4 and 5 yielded fragments ranging from 850 bp to 1,000 bp in size which were separately subcloned into the EcoRI site of pGEMBLlδ. The inserts of the pGEMBL:rhuIL-3 subclones were sequenced using a universal primer that binds adjacent to the multiple cloning site of pGEMBLlδ, and synthetic oligonucleotide primers derived from the huIL-3 sequence. Restriction mapping indicated the presence of restriction sites corresponding to those previously reported for human IL-3. Sequencing of clone 5 revealed the presence of a codon (CCC) encoding proline (Pro) in position 8 of the mature protein. Sequencing corresponding cDNAs isolated from three additional clones (2, 3 and 4) confirmed this variation from the sequence reported by Yang et al., Cell 47:3 (1986). This single T→C change at nucleotide 22 of mature huIL-3 results in a Ser→Pro amino acid substitution at residue 8 of mature huIL-3. Several individuals were screened for Pro8 and Ser8 IL-3 genes in order to (1) determine if huIL-3 containing proline at position 8 represented a naturally occurring huIL-3 gene, and (2) ascertain the relative abundance of Ser8 versus Pro8 huIL-3 genes. The polymerase chain reaction (PCR) described by Mullis et al., Cold Spring Harbor Symposia on Quantitative Biology, Vol. LI:263 (1986) was used to amplify the DNA (from peripheral blood lymphocytes) contained in a portion of the IL-3 gene from thirteen individuals. The amplified DNA sequences, encompassing those that coded for amino acid eight, were hybridized to oligonucleotide probes specific to either Ser8 or Pro8 DNA sequences. This analysis showed that, of thirteen individuals tested, all were positive for huIL-3 with proline at amino acid eight and three were also positive for serine at amino acid eight (thus heterozygotes). The huIL-3 cDNA that was isolated (Pro8) is thus a naturally occurring huIL-3 gene and is the more prevalent of the two alleles.
Example B. Construction of Expression Vector for Expression of rhuIL-3 Fusion Protein Comprising N-terminal Octapeptide
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 oligonucleotide polylinker to the vector fragment:
Asp718 i-NcoI -istul
≤TA£CAGCTAGCTAGCTAGCTCCATGGATCCAGGCCTA
GTCGATCGATCGATCGAGGTACCTAGGTCCGGATGATC
TBamHI Spel The resulting vector was designated pBCl 15. cDNA encoding huIL-3 was excised from a pGEMB lδ cloning vector (see Example A, above) by digestion with Hpal and BamEH, providing a fragment extending from amino acid 14 of mature huIL-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 α-factor leader peptide, beginning with the Asp718 site and terminating in a KEX2 protease recognition site; (2) an 8-codon s-equence encoding a synthetic N-terminal "flag" identification peptide (DYKDDDDK; s»ee above); and (3) a short sequence encoding the N-terminal 14 amino acids of mature huTL-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:
5--GTA CCT TTG GAT AAA AGA GAC TAC AAG GAC GAC GAT GAC AAG- GA AAC CTA TTT TCT CTG ATG TTC CTG CTG CTA CTG TTC- Val Pro Leu Asp Lys Arg Asp Tyr Lys Asp Asp Asp Asp Lys < α-factor leader >|< identification peptide >|
-GCT CCC ATG ACC CAG ACG ACG CCC TTG AAG ACC AGC TGG GTT-3' -CGA GGG TAC TGG GTC TGC TGC AGG AAC TTC TGG TCG ACC CAA Ala Pro Met Thr Gin Thr Thr Pro Leu Lys Thr Ser Trp Val
I huIL-3 >
The foregoing fragment, the Asp718-BamHI vector fragment derived from pBCl 15, and the Hpal-BamHI huIL-3 fragment were ligated together to provide an expression plasmid, designated pBC125. Plasmid DNA from an E. coli clone containing the pBC125 vector was isolated and used to transform yeast strain XV2181 for expression of the rhuIL-3 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. These experiments indicated high level expression of the rhuIL-3 protein.
Example 1: Modification of N-GIycosylation Sites Encoded by huIL-3 cDNA and Assembly of Expression Vector for rhuIL-3 (Pro8-. Asp 5-. Asp7-0-)
The two asparagine linked glycosylation sites present in the natural protein (Asn15 an Asn70) were removed by changing the codons at these positions to ones that encode aspartic acid. This prevents N-linked glycosylation (often hyperglycosylation) of the secreted protein by the yeast cells and a more homogeneous product is obtained. These changes were made as described below upon subcloning the huIL-3 cDNA into the yeast expression vector pD Y120.
The yeast expression vector pIXY120 is substantially identical to pBC115, described in Example B, above, except that the following synthetic oligonucleotide containing multiple cloning sites was inserted from the Asp718 site (amino acid 79) near the 3' end of the α-factor signal peptide to the Spel site contained in the 2μ sequences: Asp718 -iNcoI
GIZ^CTTTGGATAAAAGAGACTACAAGGACGACGATGACAAGAGGCCTCCATGGATCCCCCGGGACA
GAAACCTATTTTCTCTGATGTTCCTGCTGCTACTGTTCTCCGGAGGTACCTAGGGGGCCCTGTGA_I£ TBamHI Spel
In addition, a 514 bp DNA fragment derived from the single-stranded bacteriophage fl containing the origin of replication and intergenic region was inserted at the Nrul site in the pBR322 DNA sequences. The presence of the f 1 origin of replication allows one to generate single-stranded copies of the vector when transformed into appropriate (male) strains of E. coli and superinfected with bacteriophage f 1. This capability facilitates DNA sequencing of the vector and allows the possibility to do in vitro mutagenesis.
The yeast expression vector pIXYl 20 was digested with the restriction enzymes Asp718, which cleaves near the 3' end of the α-factor leader peptide (nucleotide 237), and BamHl , which cleaves in the polylinker. The large vector fragment was purified and ligated to the following DNA fragments: (1) a huIL-3 cDNA fragment derived from plasmid GEMBL18:huIL-3 from the Cla 1 site (nucleotide 58 of mature huIL-3) to the BamHl site (3' to the huIL-3 cDNA in a polylinker); and (2) a synthetic oligonucleotide linker (oligonucleotide A, Figure 2) regenerating the sequence encoding the C-terminus of the a-factor leader peptide and fusing it in-frame to the octapeptide DYKDDDDK, which is, in turn, fused to the N- terminus of mature rhuIL-3. This fusion to the rhuIL-3 protein allows detection with antibody specific for the octapeptide and was used initially for monitoring the expression and purification of rhuIL-3. This oligonucleotide also encodes an amino acid change at position 15 (Asn1 →Asp15) to alter this N-linked glycosylation site. The underlined nucleotides in FIG. 2 represent changes from the wild type cDNA sequence. Only the A to G and C to T changes at nucleotides 43 and 45, respectively (counting from the codon corresponding to the N-terminal alanine of the mature huIL-3 molecule) result in an amino acid change (Asp15). The other base changes introduce convenient restriction sites (Ahall and PvuII) without altering the amino acid sequence. The resulting plasmid was designated pIXY139 and contains a rhuIL-3 cDNA with one remaining N-linked glycosylation consensus sequence (Asn70).
Plasmid pIXY139 was used to perform oligonucleotide-directed mutagenesis to remove the second N-linked glycosylation consensus sequence by changing Asn70 to Asp70. The in vitro mutagenesis was conducted by a method similar to that described by Walder and Walder, supra. The yeast vector, pIXY139, contains the origin of replication for the single- stranded bacteriophage f 1 and is capable of generating single-stranded DNA when present in a suitable (male) strain of E. coli and superinfected with helper phage.
Single-stranded DNA was generated by transforming E. coli strain JM107 and superinfecting with helper phage IR1. Single-stranded DNA was isolated and annealed to the mutagenic oligonucleotide B, shown in FIG. 2, which provides a codon switch substituting Asp for Asn at position 70 of mature huIL-3. Annealing and yeast transformation conditions were done as described by Walder and Walder, supra. Yeast transformants were selected by growth on medium lacking tryptophan, pooled, and DNA extracted as described by Holm et al., Gene 42:169 (1986). This DNA, containing a mixture of wild type and mutant plasmid DNA, was used to transform E. coli RR1 to ampicillin resistance. The resulting colonies were screened by hybridization to radiolabelled oligonucleotide B using standard techniques. Plasmids comprising DNA encoding huIL-3 Asp70 were identified by the hybridization to radiolabelled oligonucleotide B under stringent conditions and verified by nucleotide sequencing.
The resulting yeast expression plasmid was designated pDCY138, and contained the huIL-3 gene encoding the Asp15 Asp70 amino acid changes and the octapeptide DYKDDDDK at the N-terminus. The final yeast expression plasmid is identical to pIXY138 except that it lacks the nucleotide sequences coding for the octapeptide, thus generating mature rhuIL-3 as the product.
The final yeast expression plasmid was constructed as described below. The yeast expression vector pIXY120 was cleaved with the restriction enzymes Asp718 and BamHl as described above. The large vector fragment was ligated together with (1) a huIL-3 cDNA fragment derived from plasmid pIXY138 that extended from the AhaH site (which cleaves a nucleotide 19 of mature huIL-3) to the BamHl site 3' to the cDNA, and (2) a synthetic oligonucleotide (oligonucleotide C, Figure 2) regenerating the 3' end of the α-factor leader peptide from the Asp718 site (the amino acids Pro-Leu-Asp-Lys-Arg) and the N-terminal seven amino acids of huIL-3 to the Ahall site.
The resulting plasmid was designated pD Y151. This vector, when present in yeast, allows glucose-regulated expression and secretion of rhuTL-3 (Pro8, Asp15, Asp70).
Example 2: Expression of Protein rhuIL-3 (Pro8-. Asp-1-5-. Asp7-0-)
The host strain, XV2181, a diploid S. cerevisiae strain, was formed by mating
XV617-1-3B [n, his6. Ieu2-1. trpl-1. ura3. ste5]. obtained from the University of Washington
Dept. of Genetics Yeast Strain Bank, Seattle, WA, USA, and X2181-1B [β, trpl-1. gall, adel. his2], obtained from the Yeast Genetic Stock Center, University of California, Berkeley, CA, USA. The host strain is transformed with the expression plasmid by the method of Sherman et al., supra.
Yeast containing the expression plasmid pIXY151 (see Example 1, above) are maintained on YNB-trp agar plates stored at 4°C. A preculture is started by inoculating several isolated recombinant yeast colonies into one liter of YNB-ttp medium (6.7 g L Yeast Nitrogen Base, 5 g/L casamino acids, 40 mg/L adenine, 160 mg/L uracil, and 200 mg L tyrosine), and i grown overnight in two 2-liter flasks at 30°C with vigorous shaking. By morning the culture i saturated, in stationary phase, at an OD»5oo of 2 to 7.
The fermenters (three machines of 10 liter working volume), previously cleaned and sterilized, are filled to 80% of their working capacity with SD-2 medium (4.0 g/L ammonium sulfate, 3.2 g/L monobasic potassium phosphate, 3.0 g/L yeast extract, 1.0 g L citric acid, 0.1 g/L sodium chloride, 5 ml/L 2% calcium chloride, 2.5 ml/L vitamin 101 solution, 0.5 ml L trace elements solution, 0.5 ml/L 20% magnesium sulfate, 2.0 ml/L glucose) and maintained at 30"C with 500-600 rpm agitation and 10-161pm aeration. The inoculum is added. After two hours of growth a nutrient feed of 50% glucose is begun at a rate such that 50 g/L is added over a period of 10-12 hours. The nutrient feed is then shifted to 50% ethanol added at 30-40 ml hr until harvest.
Total elapsed time of fermentation is approximately 20 hours, after which optical density (600nm) ranges from 30 to 45. The fermenters are then cooled to 20°C, pH of the yeast beer is adjusted to 8.0 by the addition of 5M NaOH, and the resulting material filtered through a Millipore Pellicon filter system equipped with a 0.45 μm filter cassette, and collected in a sterile 10 L carboy.
The rhuJL-3 contained in the yeast broth is applied to a 5 cm X 30 cm column packed with 15-20 μm C-4 reversed-phase silica (Vydac, Separations Group, Hesperia, CA, USA) by pumping the filtered yeast broth directly onto the column. The column is equilibrated in 0.1% trifluoroacetic acid in water (Solvent A) prior to application of the yeast broth and is flushed with this solvent following the completion of the application of the broth to the column until the optical absorbance of the effluent approaches base line values. At this time a gradient of 0.1% trifluoroacetic acid in acetonitrile (Solvent B) is established from 0% B to 100% B at a rate of change of 2% B per minute and at a flow rate of 100 ml/minute. A pause of five minutes when the gradient reaches 30% B is programmed into the gradient controller. Twenty minutes after the initiation of the gradient, one minute fractions are collected. Aliquots of the fractions are analyzed for protein content by a fluorescamine assay using bovine serum albumin (BSA) as a standard. RhuIL-3 (Asp15, Asp70) was found to elute in fractions 8, 9 and 10.
Fractions containing rhuIL-3 from the first step are stored at 4°C in polyethylene bottles until approximately 1 gram total protein is obtained. To the pool is added 2 volumes of 0.1% trifluoroacetic acid in water. This solution is then pumped onto a second 5 cm X 30 cm column packed with 15-20 μ C-18 silica (Vydac, Separations Group, Hesperia, CA, USA) that is equilibrated in Solvent A (0.1% TFA in water). Following application of the material, the column is flushed with Solvent A and then a gradient identical to that described above is established at a rate of change of 1% Solvent B per minute and at a flow rate of 100 ml/min. Fractions are collected every 0.4 minutes 24 minutes into the gradient. Aliquots of the fractions are analyzed for protein content by the fluorescamine assay using BSA as a standard.
Peak fractions from the C-18 column are pooled and 1/10 volume of 0.5M β-alanine pH 3.6 is added. A sample is taken and then the pool is applied to a 10 ml S-Sepharose Column (1 cm X 10 cm, Pharmacia) at 5 ml/minute. After sample application, the column is washed with 50 ml of lOmM Tris, pH 7.4 and the IL-3 is eluted with a linear gradient from lOmM Tris, pH 7.4 to 200mM Tris, pH 7.4 (200 ml total gradient volume). Peak fractions of rhuB -3 are then pooled and dialyzed against lOOmM Tris, pH 7.4, overnight at 4°C, then sterile filtered. Example 3: Comparison of Biological Activities of Analog rhuIL-3 Proteins
Three rhuIL-3 proteins were prepared for assay as follows. First, rhuIL-3 (Pro8) comprising the N-terminal octapeptide DYKDDDDK was expressed using the yeast expression plasmid pBC125 described above. Purification of this fusion protein from the yeast culture broth was accomplished in a single affinity chromatography step using a monoclonal antibody specific for the octapeptide, as described in published PCT Application PCT/US 87/03113. Second, rhuIL-3 (Pro8, Asp15, Asp70) was prepared by yeast fermentation and was purified from yeast broth by reversed-phase high performance liquid chromatography as described in Example 2, above. Third, a rhuIL-3 containing both the octapeptide and the changes at positions 15 and 70, was prepared by fermentation of yeast comprising expression plasmid pD Y138 described in Example 1, above, and purified by affinity chromatography as described above.
Concentrations of the purified rhuIL-3 proteins were determined by amino acid analysis.
Purified rhuIL-3 (octapeptide fusion protein) was radiolabeled using the enzymobead radioiodination reagent (BioRad Laboratories, Richmond, CA, USA) essentially as previously described for murine GM-CSF (25). Briefly, 2.5 μg of IL-3 in 0.1 M glycine HC1, pH 7.4, was brought to a final volume of 50 μl with 0.2 M sodium phosphate, pH 7.2. Fifty μl of enzymobead reagent, 20 μl of 125I (2 mCi) and 10 μl of 2.5% β-D-glucose were added and the mixture incubated at 25°C for 10 min. Sodium azide (10 μl of 50 mM) and sodium metabisulfite (10 μl of 5 mg/ml) were then added sequentially. After 5 min at 25°C, iodinated rhuIL-3 was separated from free 125I by chromatography on a 2 ml Sephadex G-25 column equilibrated in RPMI-1640 containing 2% bovine serum albumin, 20 mM Hepes buffer and 0.2% sodium azide, pH 7.2 (binding medium). Fractions containing rhuIL-3 were pooled, diluted to a concentration of 0.5 μg/ml in the same buffer and stored at 4°C. Bioactivity of 125I-rhuIL-3 was determined in the human bone marrow proliferation assay described above o samples in which sodium azide had been deleted during the separation step. The specific activities of radiolabeled preparations were estimated to be 3-6 x 1015 cpm/mmole. The N- terminal octapeptide of the fusion protein contains a tyrosine residue which, in the case of rhuIL-3, is useful in labeling of this molecule with 125I to high specific activity, since the nativ IL-3 molecule contains only a single tyrosine residue.
The biological activity (units/μg) and binding affinity of the various rhuIL-3 constructs described above were then determined by the human bone marrow proliferation and human monocyte IL-3 receptor binding assays described above. The results are set forth in Table I, below. In Table I, all values are expressed as the averages ± S.D. of samples run in triplicate in two separate assays. TABLE I: Biolo ical Activit an Bindin Affini -
As shown by the data summarized in Table I, determination of both the biological activity and inhibition constants (Ki) for several forms of the human IL-3 molecule showed that addition of the octapeptide DYKDDDDK to the amino terminus does not substantially alter either of these parameters, providing evidence that the fusion protein is a legitimate form of human IL-3 to use for receptor binding studies, as a research reagent, and as a pharmaceutical. Furthermore, changing the asparagines at positions 15 and 70 to aspartic acid, thereby preventing addition of N-linked carbohydrate, results in a significant (2-4 fold) increase in both biological activity and binding affinity of human IL-3.

Claims

CLATMSWhat is claimed is:
1. A pharmaceutical composition comprising a pharmaceutically effective quantity of a recombinant human IL-3 protein analog, rhuIL-3 (Asp15, Asp70), derived by yeast expression, wherein said composition is characterized by:
(a) a biological specific activity of at least 3.0 xlO7 units per milligram in a human bone marrow proliferation assay;
(b) a binding affinity for human monocyte IL-3 receptors, expressed as an inhibition constant, of at least 2.0 x 1010 M*1; and
(c) an endotoxin content of less than 1 ng per mg protein.
2. A composition according to Claim 1, wherein the recombinant human IL-3 protein analog has an amino acid sequence substantially identical to the sequence:
Ala Pro Met Thr Gin Thr Thr Pro Leu Lys Thr Ser Trp Val Asp
Cys Ser Asn Met lie Asp Glu lie lie Thr His Leu Lys Gin Pro
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin
Asp lie Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gin Asp Ala Ser Ala lie Glu
Ser lie Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala
Ala Pro Thr Arg His Pro lie His lie Lys Asp Gly Asp Trp Asn
Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn
Ala Gin Ala Gin Gin Thr Thr Leu Ser Leu Ala lie Phe.
3. A composition according to Claim 2, wherein the recombinant human IL-3 protein has an amino acid sequence identical to such sequence.
4. A composition according to Claim 1, characterized by a biological specific activity of at least 4.0 x 107 U/mg in a human bone marrow proliferation assay, and a binding affinity for human monocyte IL-3 receptors of at least 4.0 x 1010 M"1.
5. A composition according to Claim 1, comprising an N-terminal octapeptide
DYKDDDDK.
6. A composition according to Claim 1 which is formulated with a physiologically acceptable diluent for injection or continuous infusion.
7. A composition according to Claim 5, additionally comprising a pharmaceutically effective quantity of one or more biological response modifiers selected from the group consisting of IL-lα, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-7, GM-CSF, G-CSF, M-CSF, EPO or TNF.
EP19890907981 1988-07-20 1989-06-14 Nonglycosylated human interleukin-3 compositions Withdrawn EP0425536A4 (en)

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