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WO1991013986A1 - SEQUENCES DE NUCLEOTIDE ET POLYPEPTIDES D'INTERLEUKINE-1α PORCINE - Google Patents

SEQUENCES DE NUCLEOTIDE ET POLYPEPTIDES D'INTERLEUKINE-1α PORCINE Download PDF

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WO1991013986A1
WO1991013986A1 PCT/US1991/001567 US9101567W WO9113986A1 WO 1991013986 A1 WO1991013986 A1 WO 1991013986A1 US 9101567 W US9101567 W US 9101567W WO 9113986 A1 WO9113986 A1 WO 9113986A1
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leu
ser
asp
asn
lys
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Charles R. Maliszewski
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Immunex Corp
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Immunex Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to mammalian cytokines, and particularly to cloning and expression of biologically active mammalian homologues of human IL-l ⁇ , such as porcine interleukin-l ⁇ .
  • Interleukin-1 is the designation given to a family of polypeptides, released by macrophages and certain other cell types in response to immunogenic and traumatic stimulation, which have a primary role in initiating host response to injury and infection. These cytokines have been associated with a complex spectrum of biological activities. For example, IL-1 peptides create a primary immunostimulatory signal capable of inducing thymocyte proliferation via induction of interleukin-2 release, and of stimulating proliferation and maturation of B lymphocytes. In addition, IL-1 has been linked with prostaglandin production and induction of fever, and with promotion of wound healing. Reviews of the literature relating to IL-1 include Oppenheim et al., Immunol. Today 7:45 (1986), and Durum et al., Ann. Rev. Immunol. 3:263 (1985).
  • Human IL-1 activity resides in at least two distantly related proteins, which have been designated E -l ⁇ and IL-l ⁇ (March et al., Nature 315:641 (1985)). Both molecules are normally synthesized as larger precursors having molecular weights of about 30,000 daltons, which are subsequently processed by proteolytic cleavage to yield mature forms having molecular weights of approximately 17,500 daltons. While the precursor of human IL-l ⁇ exhibits IL-1 biological activity, the precursor of human IL- 1 ⁇ is biologically inactive, and must be cleaved to provide a mature version having IL-1 activity.
  • Sow mastitis and agalactia are generally grouped with metritis as mastitis- metritis-agalactia syndrome or MMA.
  • MMA is a poorly understood group of symptoms that generally occur after fallowing (Wagner Endocrine Function in Normal and Agalactic Sows" J. Amin. Sci. 34:270-72 1982).
  • causative etiological agents e.g., E. coli, Klebsiella, etc.
  • mastitis or MMA In cases where mastitis is less severe, bacterial pathogens cannot be isolated although the sow is agalactic. This results in neonatal hypoglycemia and death due to starvation.
  • a porcine IL-l ⁇ may be useful as a prophylaxis against mastitis-causing environmental pathogens, such as the colifor group of pathogens. Sows with coliform mastitis develop a characteristic clinical picture that includes fever, hot mammary glands and leukopenia associated with bacterial endotoxemia (Ross "Klebsiella-induced Mastitis and Efficacy of an Experimental Drug for Control of the Disease" NC127 Annu. Rept. Iowa State Univ. 1975). Similarly to cattle, it is difficult to diagnose subclinical mastitis in swine. Often, the only overt symptom is hypoglycemia in the neonates.
  • porcine vaccine adjuvants to potentiate antibody production in response to vaccine antigens, and also to promote rapid epidermal wound-healing.
  • porcine antiinflammatory agents to control the pig's recovery from traumatic injury. The ability to obtain reliable quantities of a porcine IL-l ⁇ or derivatives thereof can address this need in the art.
  • the present invention is directed to porcine IL-l ⁇ polypeptides and derivatives thereof, DNA segments encoding porcine IL-l ⁇ polypeptides and derivatives thereof, recombinant expression vectors comprising the DNA segments, microbial expression systems comprising the recombinant expression vectors, and processes for making the polypeptides and derivatives thereof using the recombinant expression systems.
  • the present invention was made by cloning the gene encoding porcine IL- l ⁇ (pIL-l ⁇ ), and expressing the isolated cDNA sequence.
  • the deduced mature amino acid sequence of porcine IL-l ⁇ is: Met Ala Lys Val Pro Asp Leu Phe Glu Asp Leu Lys Asn Cys Tyr Ser Glu Asn Glu Glu Tyr Ser Ser Asp He Asp His Leu Ser Leu Asn Gin Lys Ser Phe Tyr Asp Ala Ser Tyr Glu Pro Leu Pro Gly Asp Gly Met Asp Lys Phe Met Pro Leu Ser Thr Ser Lys Thr Ser Lys Thr Ser Arg Leu Asn Phe Lys Asp Ser Val Val Met Ala Ala Ala Asn Gly Lys lie Leu Lys Lys Arg Arg Leu Ser Leu Asn Gin Phe Be Thr Asp Asp Asp Leu Glu Ala He Ala Asn Asp Thr Glu Glu Glu He He Lys Pro Arg Ser Ala Thr Tyr Ser Phe Gin Ser Asn Met Lys Tyr Asn Phe Met Arg Val He Asn His Gin Cys He Leu Asn Asp Ala Arg Asn Gin Ser ⁇
  • native porcine pIL-l ⁇ has 270 amino acids with a predicted molecular weight of 30,788.
  • the expressed pIL-l ⁇ was expressed and secreted by COS-7 cells transf ⁇ cted with a porcine IL-l ⁇ cDNA.
  • An isolated cDNA segment encoding porcine IL-l ⁇ is shown in Figure 1.
  • the invention is directed to functional derivatives of native porcine IL-l ⁇ that exhibit activity characteristics substantially equivalent to native porcine IL-l ⁇ and may be more or less potent than native porcine IL-l ⁇ .
  • Examples of functional derivatives include the mature sequence, containing the C-terminal sequence beginning at about the Ser residue at position 113 or another amino acid within nine amino acids of the 113 position.
  • porcine IL-l ⁇ polypeptides with IL-1 activity includes shortened mature derivatives from about position 127 to the C terminal at position 270 or have the C-terminal shortened up to about 10 amino acids.
  • the mature polypeptide sequence in other species for IL-l ⁇ polypeptides has been shown to begin at either position 113 of position 119. Therefore, the present invention encompasses polypeptide sequences having the full 270 amino acid sequence and truncated versions representing the mature form of the polypeptide from about amino acid 104 to about amino acid 127 at the N-terminal and extending from about amino acid 260 to amino acid 270 at the C-terminal.
  • the present invention further encompasses replicable DNA expression vectors which contain a gene sequence coding for a pIL-l ⁇ polypeptide or a derivative thereof.
  • the invention is also directed to recombinant host cells, such as microorganism strains or cell lines transformed with such vectors, and to the cultures thereof.
  • the invention is directed to compositions comprising the porcine IL-l ⁇ polypeptide of derivatives thereof for parenteral administration.
  • the polypeptides of the present invention are useful for vaccine adjuvants, wound healing, antiinflammatory agents and other activities associated with EL-1 polypeptides. BRTEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 illustrates the nucleotide sequence of a cDNA clone comprising the coding sequence of recombinant porcine IL-l ⁇ .
  • Figure 2 illustrates a comparison of the amino acid sequences of Bovine, Porcine, Rabbit, Human and Murine IL-l ⁇ peptides showing the regions of interspecies homology and the regions where the sequences differ for each species.
  • Figure 3 illustrates a comparison of the biological activity of recombinant porcine IL-l ⁇ compared with other immunologically active polypeptides.
  • a DNA segment encoding porcine IL-l ⁇ was isolated from a cDNA library prepared by reverse transcription of polyadenylated RNA isolated from alveolar macrophages from pigs infected with Ascaris suum.
  • a cDNA fragment corresponding to part of the coding sequence of human IL-l ⁇ was employed to screen the library by conventional DNA hybridization techniques. Clones which hybridized to the probe were analyzed by restriction endonuclease cleavage, agarose gel electrophoresis, and additional hybridization experiments ("Southern blots") involving the electrophoresed fragments.
  • the hybridizing segment of one pIL-l ⁇ clone was subcloned and sequenced by conventional techniques.
  • the coding sequence corresponding to the putative amino acid sequence of mature pIL-l ⁇ , as determined by comparison to the corresponding native human sequence, was inserted into an appropriate expression vector and used to transform a suitable strain of mammalian cells.
  • the recombinant polypeptide, in the supernatant of the transfected mammalian cells grown in culture was tested for H -1 activity in a porcine thymocyte proliferation assay.
  • mammalian cells grown in culture was tested for IL-1 activity in a porcine thymocyte proliferation assay.
  • Porcine interleukin-l ⁇ refers to a polypeptide whose biological properties include induction of porcine thymocyte proliferation via induction of D -2 release, and stimulation of proliferation and maturation of porcine B-lymphocytes, or biological activity substantially the same as native pIL-l ⁇ and normally associated with IL-1.
  • pIL-l ⁇ further denotes porcine fl-l ⁇ or its fragments produced by cell or cell-free culture (or translation) systems in bioactive forms having the capacity to influence wound healing, inflammation and other activities normally associated with an IL-1 polypeptide.
  • the observed biological properties of the human homologue of porcine IL-l ⁇ also include induction of prostaglandin production and provision of a chemotactic signal to fibroblasts.
  • Different alleles ofpIL-l ⁇ may exist in nature. These variations may be characterized by differences in the nucleotide sequence of the structural gene coding for proteins having substantially equivalent biological function. It is possible to produce derivatives having single or multiple amino acid substitutions, deletions, additions, or replacements. All such allelic variations, modifications, and analogs resulting in derivatives of pIL-l ⁇ which retain the biologic properties of native pIL- l ⁇ are included within the scope of this invention.
  • “Mutant amino acid sequence” refers to a polypeptide encoded by a nucleotide sequence intentionally made variant from a native sequence.
  • “Mutant protein” or “mutein” means a protein comprising a mutant amino acid sequence.
  • “Native sequence” refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein.
  • Recombinant refers to a protein derived from recombinant (e.g., microbial or mammalian) expression systems.
  • Microbial refers to bacterial or fungal (e.g., yeast) host cells that are used as a part of the expression system. Protein expressed in bacterial cultures will be free of polysaccharide; protein expressed in yeast will have a glycosylation pattern different from that expressed in mammalian cells.
  • DNA segment refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector.
  • Nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.
  • Recombinant expression vector refers to a plasmid or vector 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, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.
  • transcriptional units intended for use in yeast expression systems include 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.
  • Porcine IL-l ⁇ activity can be monitored by a thymocyte mitogenesis assay, which involves ascertaining the capacity of a sample to induce proliferation of thymocytes from freshly killed pigs.
  • a thymocyte mitogenesis assay involves ascertaining the capacity of a sample to induce proliferation of thymocytes from freshly killed pigs.
  • Ficoll-Hypaque purified porcine thymocytes are dispensed into wells of a flat-bottom microtiter plate (Corning Plastics, Corning, NY, USA) in the presence of a submitogenic concentration of phytohemagglutinin-M (PHA-M) and serial three-fold serial dilutions of samples to be tested for pIL- 1 activity.
  • PHA-M phytohemagglutinin-M
  • Thymocytes are cultured in RPMI 1640 medium containing 50 U/ml penicillin, 50 ⁇ g/ml streptomycin, 2 mM glui___ ⁇ ne, 0.2 mM gentamycin, 10 mM HEPES (N-2-hydroxyethylpiperazine- N'-2-ethanesulf_nic acid) buffer, pH 7.4, 10" 5 M 2-mercaptoethanol, and 10% (v/v) fetal bovine serum. The samples are incubated for 68 hours at 37°C in a hurmdified atmosphere of 5% CO2 in air.
  • RPMI 1640 medium containing 50 U/ml penicillin, 50 ⁇ g/ml streptomycin, 2 mM glui___ ⁇ ne, 0.2 mM gentamycin, 10 mM HEPES (N-2-hydroxyethylpiperazine- N'-2-ethanesulf_nic acid) buffer, pH 7.4, 10" 5 M 2-mer
  • cultures are pulsed for approximately 4 hours with 0.5 microcuries ( ⁇ Ci) of tritiated thymidine ( 3 H-Tdr), and then harvested onto glass fiber filter strips with the aid of a multiple-automated sample harvester. Details of this procedure are provided in U. S. Patent 4,411,992 the disclosure of which is incorporated by reference herein.
  • IL-1 activity is calculated from the linear portion of the 3 H-Tdr incorporation data. Units of D -1 activity are determined as the reciprocal dilution of a sample which generates 50% of maximal thymocyte 3 H-Tdr incorporation, where one unit of activity is provided by a standard solution comprising purified recombinant human IL-l ⁇ at a concentration of 100 ⁇ g/ml.
  • IL-1 activity may be assayed by an IL-1 conversion assay.
  • IL-1 conversion assays are described by Conlon. J. Immunol. 131:1280 (1983) and Lowenthal et al., __ Immunol. 137: 1226 (1986).
  • cells to be induced are first inactivated by treatment with 50 ⁇ g/ml mitomycin C and then incubated in the presence of a suboptimal mitogenic concentration of PHA-M, varying dilutions of sample, and IL-2 dependent cells (e.g., the murine T-cell line CTLL-2 (ATCC TIB 214)). Only the IL-2 dependent cells added to wells previously contacted with IL-1 (thereby inducing IL-2 production by the inactivated cells) should proliferate and incorporate radiolabel.
  • IL-2 dependent cells e.g., the murine T-cell line CTLL-2 (ATCC TIB 214)
  • Conversion assays of this type are generally more rapid and more sensitive than the thymocyte mitogenesis assay.
  • approximately 5 x 10 4 inactivated EL4-6.1 cells are dispensed into wells of a flat-bottom microtiter plate containing serial threefold dilutions of samples to be tested for activity.
  • Cells are cultured in a total volume of 100 microliters of complete Clicks medium containing 50 U/ml penicillin, 50 ⁇ g ml streptomycin, 2 mM glutamine, 0.2 mM gentamycin, 10 mM HEPES buffer, pH 7.4, 10" 5 M 2-mercaptoethanol, and 10% (v/v) fetal bovine serum.
  • Protein concentrations can be determined by any suitable method. However, the Bio-Rad total protein assay (Bio-Rad Laboratories, Richmond, California, USA) is preferred. SDS-PAGE can also be employed to monitor purification progress, substantially as described by Kronheim et al., J. Exp. Med. !6_l:490 (1985), or other suitable techniques. Additional details regarding use of variants of the IL-1 assays described above are disclosed by Conlon, J. Immun. Hi: 1280 (1983) and Kronheim et al., supra. Endotoxin levels in protein compositions can be assayed using a commercial kit, such as one available from Whittaker Bioproducts, Walkersville, Maryland, U.S A.
  • Quantitative Chromogenic LAL QCL-1000 Quantitative Chromogenic LAL QCL-1000 or its equivalent. This method uses a ⁇ Kxiified Limulus amebocyte lysate and synthetic color-producing substrate to detect endotoxin chromogenically. Purified recombinant pIL-l ⁇ is tested for presence of endotoxin at multiple dilutions. The assay is preferably performed shortly following completion of purification and prior to storage at -70°C. To minimize the possibility of bacterial contamination during the purification process itself, sterile buffers should be employed.
  • a cDNA library was prepared by reverse transcription of polyadenylated RNA isolated from alveolar macrophages from pigs infected with Ascaris suwn.
  • the alveolar cells were isolated from pigs after lung lavage.
  • the pigs were first inoculated orally with either 50,000 or 100,000 A. suum eggs and the alveolar cells collected approximately ten or eleven days later.
  • the method used for preparation of infective A. suum eggs is described in Urban et al. "A Rapid Method for Hatching Ascaris suum Eggs in vitro" Proc. Helminthol. Soc. Wash. 48:241:43 (1981).
  • Some of the infected pigs had liver reactions, such as liver f ⁇ brosis, indicating the exposure to A . suum.
  • nucleotide sequence of a cDNA clone isolated from a porcine alveolar macrophage Hbrary is set forth in Figure 1.
  • the initiator methionine is at nucleotide 31 and the stop codon is at nucleotide 841.
  • Figure 2 compares the interspecies amino acid alignment of IL-l ⁇ of the pig, bovine, rabbit, murine and human origins. The regions of amino acid homology are marked. The amino acid sequence of porcine IL-l ⁇ exhibits 90%, 82% and 75% homology with bovine, human and murine IL-l ⁇ , respectively.
  • Porcine IL-l ⁇ can be expressed in bacterial, yeast, mammalian, or other cells under the control of appropriate inducible promoters.
  • Appropriate expression vectors for bacterial use are constructed by inserting the heterologous structural DNA sequence encoding pIL- l ⁇ together with translational initiation and termination signals in operable reading phase with a functional promoter.
  • the vector comprises one or more phenotypic selectable markers and an origin of replication to ensure amplification within the host.
  • the heterologous sequence can be integrated into the vector such that it is translated as a fusion protein, in conjunction with an identification peptide (e.g., DYKDDDDK) or other sequence imparting desired characteristics relating to stabilization or purification of expressed protein.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising sequences derived from the well known cloning vector pBR322 (ATCC 37017).
  • commercial vectors include, for example, pKK223- 3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA). These "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • a particularly useful bacterial expression system employs the phage ⁇ PL promoter and cI857ts thermolabile repressor.
  • Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the ⁇ PL promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082).
  • Other useful promoters for expression in E. coli include the T7 RNA polymerase promoter described by Studier et al., J. Mol. Biol. 189: 113 (1986), the lac promoter described by Lauer, J. Mol. Appl. Genet. 1:139-147 (1981) and available as ATCC 37121, and the tac promoter described by Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, 1982, p 412) and available as ATCC 37138.
  • yeast vectors will include origins of replication and selectable markers permitting transformation of both yeast and E. coli. e.g., the ampicillin resistance gene of R coli and yeast TRP1 gene, and a promoter derived from a highly-expressed yeast gene to induce transcription of a downstream structural sequence.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence causing secretion of translated protein into the extracellular medium.
  • Useful yeast vectors can be assembled using DNA sequences from pBR322 for selection and replication in E. c ⁇ ji (Ap r gene and origin of replication) and yeast DNA sequences including a glucose-repressible alcohol dehydrogenase 2 (ADH2) promoter.
  • ADH2 promoter has been described by Russell et al., J. Biol. Chem. 258:2674 (1982) and Beier et al., Nature 300:724 (1982).
  • Such vectors may also include a yeast TRP1 gene as a selectable marker and the yeast 2 ⁇ origin of replication.
  • the yeast ⁇ -factor leader sequence enabling secretion of heterologous proteins from a yeast host, can be inserted adjacent to the promoter and translation initiation sequence and in phase with the structural gene to be expressed.
  • the ⁇ -factor leader sequence may be modified to contain, near its 3' end, one or more useful restriction sites to facilitate fusion of the leader sequence to foreign genes.
  • Alternative expression vectors are yeast vectors which comprise other promoters, for example, the yeast ⁇ -factor promoter or 3-phosphoglycerate kinase (PGK) promoter.
  • Suitable yeast transformation protocols are known to those of skill in the art; and exemplary technique is described by Hinnen, et al., Proc. Natl. Acad. Sci. USA 7__:1929 (1978), selecting for Trp + transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ⁇ g/ml adenine and 20 ⁇ g ml uracil.
  • Host strains transformed by vectors comprising the ADH2 or ⁇ -factor promoters are grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ⁇ g/ml adenine and 80 ⁇ g/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and frozen or held at 4°C prior to further purification.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23 : 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors may comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the S V40 viral genome for example, S V40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • Microbial Expression and Protein Purification One general purification scheme involves an initial acid extraction from cell pellets, followed by ion exchange chromatography in aqueous media.
  • the ion exchange chromatography may comprise cation exchange chromatography followed by anion exchange chromatography.
  • Suitable cation exchange chromatography media include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.
  • the matrices can be acrylamide, agarose, dextran, cellulose or other ion exchange resins or substrates commonly employed in protein purification.
  • a particularly useful material for cation exchange chromatography of recombinant pLL-l ⁇ (rpIL-l ⁇ ) is Sulphopropyl Sephadex (SPS) C-25 (Pharmacia Fine Chemicals, Uppsala, Sweden).
  • extracts containing rpIL-l ⁇ species are applied at a pH of about 4.0, in a suitable buffer such as sodium citrate.
  • rpIL-l ⁇ is bound by the ion exchanger, and can be eluted by application of a weakly basic eluant, for example, 10 mM Tris- HC1, pH 8.1.
  • Suitable anion exchange chromatography media include various insoluble matrices comprising ⁇ _ei_ ⁇ ylaminoethyl (DEAE) or diethyl-(2- hydroxypropyl)arninoethyl (QAE) groups. DEAE groups are preferred.
  • the matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
  • a useful material for cation exchange chromatography of rpIL- 1 ⁇ is DEAE-Sephacel (Pharmacia) .
  • extracts containing rpIL-l ⁇ are applied at a weakly basic pH.
  • pooled IL-l ⁇ -containing fractions resulting from a previous cation exchange chromatography step (at a pH of about 8.1) can be applied directly in a suitable buffer such as Tris-HCl.
  • rpIL-l ⁇ elutes (in wash fractions) unbound by DEAE Sephacel, while substantially all R cpJi protein contaminants, including pyrogens, are bound.
  • rpIL-l ⁇ can be efficiently produced by growth and derepression of appropriate R coli cells harboring high level thermoinducible expression plasmids.
  • Cells are grown, for example, in a 10 liter fermenter employing conditions of maximum aeration and vigorous agitation.
  • An antifoaming agent (Antifoam A) is preferably employed.
  • the cell mass is initially concentrated by filtration or other means, then centrifuged at 10,000 x g for 10 minutes at 4°C followed by rapid freezing the cell pellet
  • cell pellets are suspended in 30 mM Tris-HCl buffer, pH 8, containing 5 mM EDTA and 1 mM phenyhnethylsulfonyl fluoride (PMSF).
  • the resulting suspension is rapidly frozen in a dry ice/methanol bath and then thawed.
  • 30 mM sodium citrate buffer at pH 4.0, containing 5 mM EDTA and 250 ⁇ g/ml lysozyme is added to the suspensions.
  • cells can be disrupted in pH 4.0 buffers using a cell homogenizer.
  • the resulting acid suspensions are incubated for 60 minutes in a 37°C water bath.
  • the extracts are rapidly frozen in a dry-ice/methanol bath, thawed, and then centrifuged at 4°C for 45 minutes at 38,000 x g. Supernatants are then decanted for use in the next purification step.
  • Extracts containing rpIL-l ⁇ can be applied at pH 4.0 to an SPS C-25 column pretreated with 0.1% Triton X-100 (polyoxyethylene ether, Sigma Chemical Company, St. Louis, Missouri, USA) and 10% fetal calf serum. The column can then be washed with 3 column volumes of 10 mM 2-(N- morpholino)ethanesulfonic acid (MES) buffer, pH 5.0, and protein eluted from the column with 10 mM Tris-HCl, pH 8.1.
  • Triton X-100 polyoxyethylene ether
  • MES 2-(N- morpholino)ethanesulfonic acid
  • Fractions containing pIL-l ⁇ activity from the SPS step can then be combined and applied to columns containing DEAE-Sephacel previously equilibrated with 10 mM Tris-HCl pH 8.1.
  • the DEAE columns are washed with additional starting buffer to elute pIL-l ⁇ which is substantially pure by SDS- PAGE.
  • the foregoing ion exchange chromatography procedures can be repeated to attain further purification, or combined with subsequent size exclusion chromatography or high-performance liquid chromatography (HPLC) steps to attain a final product of high purity.
  • porcine IL-l ⁇ is administered to a mammal for treatment in a manner appropriate to the indication.
  • pIL-l ⁇ administered as a vaccine adjuvant
  • pIL-l ⁇ will be given in conjunction with or shortly following administration of an appropriate vaccine antigen.
  • Administration may be by injection, continuous infusion, sustained release from implants, or other suitable technique.
  • pIL-l ⁇ is administered as an aid to wound healing, it will typically be applied topically to the site of injury, for example, in conjunction with a wound dressing.
  • therapeutic dosages will range from about 0.1 to 1000 ng per kg rpIL-l ⁇ per kg body weight, preferably 1-100 ng/kg.
  • pIL-l ⁇ will be administered in the form of a composition comprising purified protein in conjunction with physiologically acceptable carriers, excipients or diluents.
  • Such compositions will preferably include conventional pharmaceutically acceptable carriers and may include other medicinal agents, carriers, adjuvants, excipients, etc. See,
  • pHL-l ⁇ polypeptides or derivatives thereof may be via any of the conventional accepted routes of administration of IL-l ⁇ , such as topically, parenterally, subcutaneously, or by intravenous administration.
  • routes of administration of IL-l ⁇ such as topically, parenterally, subcutaneously, or by intravenous administration.
  • the following example is offered to illustrate this invention and is not to be construed to limit the scope of the invention.
  • Example 1 This example illustrates a bioassay for porcine IL-l ⁇ to determine the authenticity of the coding sequence for porcine IL-l ⁇ as shown in Figure 1.
  • the Figure 1 cDNA sequence was inserted into a mammalian expression plasmid (CAV) in either the forward or reverse orientation.
  • the plasmid contained a CMV promoter to drive the expression of the inserted cDNA in the CAV plasmid.
  • the plasmid was transfected into COS-7 cells.
  • the transformed COS-7 cells were incubated to allow for recombinant protein production.
  • Supernatant fluids from the transfected cells were collected and assayed for the ability to stimulate the proliferation of porcine thymocytes in the presence of the lectin phytohemagglutinin-M (PHA), as described herein. This is a standard assay for IL-1 activity.
  • PHA phytohemagglutinin-M
  • Purified bovine IL-l ⁇ was included as a positive control.
  • the disclosed pIL-l ⁇ nucleotide sequence encodes a polypeptide having IL-1 activity.

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Abstract

Séquence de nucléotides et séquence d'acides aminés déduites de IL-1α porcine ainsi que ces dérivés. L'invention concerne également un procédé d'utilisation de IL-1α porcine et de ses dérivés.
PCT/US1991/001567 1990-03-13 1991-03-07 SEQUENCES DE NUCLEOTIDE ET POLYPEPTIDES D'INTERLEUKINE-1α PORCINE Ceased WO1991013986A1 (fr)

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Publication number Priority date Publication date Assignee Title
NL9301929A (nl) * 1993-03-29 1994-10-17 Stichting Rega V Z W Recombinant interleukine-1Beta.
US6270758B1 (en) 1998-10-08 2001-08-07 Duke University Substantially non-toxic biologically active mucosal adjuvants in vertebrate subjects
US7041294B2 (en) 1998-10-08 2006-05-09 Duke University Substantially non-toxic biologically active mucosal adjuvants in vertebrate subjects

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