EP0544719A1 - Cytokine production - Google Patents

Cytokine production

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Publication number
EP0544719A1
EP0544719A1 EP91914480A EP91914480A EP0544719A1 EP 0544719 A1 EP0544719 A1 EP 0544719A1 EP 91914480 A EP91914480 A EP 91914480A EP 91914480 A EP91914480 A EP 91914480A EP 0544719 A1 EP0544719 A1 EP 0544719A1
Authority
EP
European Patent Office
Prior art keywords
cytokine
ovine
dna sequence
approximately
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91914480A
Other languages
German (de)
French (fr)
Other versions
EP0544719A4 (en
Inventor
Malcolm Roy Brandon
Arna Elizabeth Andrews
Andrew Donald Nash
Elsa Nicole Theresia Meeusen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Melbourne
Australian Wool Research and Promotion Organization AWRAP
Original Assignee
University of Melbourne
Australian Wool Research and Promotion Organization AWRAP
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Melbourne, Australian Wool Research and Promotion Organization AWRAP filed Critical University of Melbourne
Publication of EP0544719A1 publication Critical patent/EP0544719A1/en
Publication of EP0544719A4 publication Critical patent/EP0544719A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to cytokine production, particularly ovine cytokine production and to pharmaceutical compositions including ovine cytokines.
  • Cytokines are important polypeptides which display significant immuno-regulatory and inflammatory activities in animals. Whilst the ovine immune system has been extensively utilised in lymphocyte recirculation studies and as a large animal model for the study of immune responses to infectious diseases, little is known about either the production of cytokines by ovine leukocytes or the regulation of cellular function by cytokines.
  • IL-1 Interleukin-1
  • Tumor Necrosis Factor ⁇ Tumor Necrosis Factor ⁇
  • IL-1 The pleiotropic bioactivities of these two cytokines are in many instances overlapping.
  • the bioactivities ascribed to IL-1 are produced by two molecules (IL-l ⁇ and IL-l ⁇ ) encoded for by two distinct genes. The amino acid sequence of these two molecules is only 29% homologous, nevertheless, they bind to the same receptor and exert the same biological activity.
  • IL-1 As a mediator of the inflammatory response IL-1 induces secretion of acute phase proteins, stimulates release of prostaglandin E2 and proteolytic enzymes, and is chemotactic for neutrophils.
  • TNF ⁇ As a product of activated macrophages, TNF ⁇ also plays an important role in the inflammatory response. Bioactivities that overlap with IL-1 include mitogenicity for non-immune cells such as fibroblasts and endothelial cells and induction of secretion of prostaglandins, proteolytic enzymes and possibly acute phase proteins. TNF ⁇ serum levels are associated with onset of septic shock and, in chronic conditions, cachexia. Other important cytokines include Interleukin-6
  • IL-6 and the Interleukin-2 (IL-2).
  • interleukin-2 The production of interleukin-2 and expression of its high affinity receptor is essential for T cell proliferation and differentiation in the development of an immune response.
  • Interleukin-2 is produced by activated T cells. It induces the expression of a 55 kD Receptor protein ( ⁇ chain) which, in association with a 75 kD protein ( ⁇ chain), form the high affinity IL-2 Receptor. Both the ⁇ chain, which is constitutively expressed on T cells, and the ⁇ chain bind I -2 independently with intermediate and low affinity respectively although it is the association of IL-2 with its high affinity receptor which mediates the biological response.
  • a process for identifying nucleotide sequences coding for a polypeptide exhibiting specific ruminant cytokine or cytokine receptor activity or a fragment thereof which process includes providing a vector including a complementary DNA (cDNA) sequence derived from ruminant cells or an extract thereof capable of being expressed in a unicellular organism; a unicellular organism; and a DNA probe for a homologous cytokine; introducing the vector into the said unicellular organism; culturing the organism to express a polypeptide encoded therein; probing the organism with the DNA probe; and isolating a vector containing a cDNA sequence encoding for a polypeptide exhibiting specific ruminant cytokine or cytokine receptor activity.
  • cDNA complementary DNA
  • Cytokines and cytokine receptors of interest include Interleukin-1 alpha (I -l ⁇ ) Interleukin-1 beta (IL-l ⁇ ) Interleukin-1 Receptor (IL-1R) Interleukin-2 (IL-2)
  • Interleukin-2 Receptor Interleukin-3 (IL-3) Interleukin-3 Receptor (IL-3R) Interleukin-4 (IL-4) Interleukin-4 Receptor (IL-4R) Interleukin-5 (IL-5) Interleukin-5 Receptor (IL-5R) Interleukin-6 (IL-6) Interleukin-6 Receptor (IL-6R) Interleukin-7 (I -7)
  • Interleukin-7 Receptor Interleukin-8 (IL-8) Interleukin-8 Receptor (IL-8R) Interleukin-9 (IL-9) Interleukin-9 Receptor (IL-9R) Interleukin-10 (IL-10) Interleukin-10 Receptor (IL-10R) Interferon-gamma (IFN- ⁇ ) Interferon-gamma Receptor (IFN- ⁇ R) Tumor Necrosis Factor-alpha (TNF- ⁇ ) Tumor Necrosis Factor-alpha Receptor (TNF- ⁇ R) Granulocyte Macrophage Colony Stimulating Factor (GMCSF) Granulocyte Macrophage Colony Stimulating Factor (GMCSFR)
  • TGF- ⁇ Transforming Growth Factor-beta
  • TGF- ⁇ R Transforming Growth Factor-beta Receptor
  • IL-l ⁇ , IL-l ⁇ , 11-2, IL-2R, IL-6, IFN- ⁇ and TNF ⁇ are IL-l ⁇ , IL-l ⁇ , 11-2, IL-2R, IL-6, IFN- ⁇ and TNF ⁇ .
  • the cDNA sequences in the vector are derived from ruminant cells.
  • the ruminant cells may be derived from ovine, caprine or bovine animals, preferably ovine animals.
  • Sources of cytokine and cytokine specific receptor mRNA include macrophages, lymphocytes, fibroblasts, endothelial cells and liver cells.
  • the process for identifying nucleotide sequences coding for specific ruminant cytokines may include the preliminary steps of providing a source of ruminant macrophage or extract thereof; and a suitable cloning vector; isolating cytokine or cytokine-specific messenger RNA (mRNA) from the ruminant macrophage; treating the messenger RNA to produce complementary DNA (cDNA) ; and deploying the cDNA sequence into the cloning vector.
  • mRNA messenger RNA
  • the source of ruminant macrophages may be alveolar macrophages.
  • Alveolar macrophages may be isolated from ovine lung tissue. The lungs may be removed and flushed with, for example, a saline solution to remove cells lining the inner surface thereof.
  • the cells so isolated may be stimulated in vitro to enhance cytokine specific messenger RNA production.
  • the cells may be contacted with a lipopolysaccharide.
  • mRNA may be isolated therefrom, for example using an oligo dT column.
  • the isolated mRNA may be probed with a DNA probe for an equivalent homologous cytokine, for example an equivalent human cDNA probe.
  • cDNA probes encoding human IL-l ⁇ and human IL-l ⁇ and human TNF ⁇ were generously provided by Immunex Research and Development Corporation and Genentech Inc. of the United States, respectively.
  • the process for identifying nucleotide sequences coding for specific ruminant cytokines and cytokine receptors may include the preliminary step of providing a source of ruminant lymphocytes or extract thereof; and a suitable cloning vector; isolating cytokine or cytokine receptor-specific messenger RNA (mRNA) from the ruminant lymphocyte; treating the mRNA to produce complementary DNA (cDNA); and deploying the cDNA into the cloning vector.
  • the source of ruminant lymphocytes may be ruminant lymph node cells.
  • the lymph node cells may be extracted from popliteal nodes of, for example, normal sheep.
  • the cells so isolated may be stimulated in vitro to enhance cytokine receptor-specific messenger RNA production.
  • the cells may be stimulated with mitogen Concanavalin A (Con A) and then cultured in the presence of the specific cytokine for the receptor of interest.
  • Con A mitogen Concanavalin A
  • the production of complementary DNA from the messenger RNA may be undertaken in any suitable manner. A number of techniques are known per se in the art for this production.
  • the commercial Amersham cDNA synthesis system may be used.
  • the suitable cloning vector according to the present invention may be selected from plasmid and phage vectors.
  • the bacteriophage virus vector ⁇ gtlO and the like have been found to be suitable.
  • the complementary DNA (cDNA) sequence may be ligated into the phage ⁇ gtlO in any suitable manner.
  • the commercial Arrtersham cDNA cloning kit may be used.
  • a representative cDNA library may be produced and then screened by hybridisation with DNA probes for homologous cytokines.
  • a representative ovine macrophage cDNA library was produced and then screened by hybridisation with the human cDNA probes for IL-l ⁇ , IL-l ⁇ , TNF ⁇ and IL-6.
  • the relative numbers of each is a reflection of the length of stimulation of the original macrophages. A shorter stimulation would be expected to increase the representation of IL-l ⁇ and TNF ⁇ clones. All the IL-l ⁇ , TNF ⁇ and IL-6 clones were further examined in addition to 9 of the IL-l ⁇ isolates. They were found to contain a range of DNA inserts with the largest of each species as follows:
  • IL-6 - approx. 1100 bp TNF ⁇ - approx. 1690 bp Accordingly, in a further aspect of the present invention there is provided a DNA sequence coding for a polypeptide exhibiting ruminant cytokine or cytokine receptor activity, or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof.
  • the polypeptide preferably exhibits ovine cytokine or cytokine receptor activity.
  • the DNA sequences may be modified in any suitable manner.
  • the DNA sequences may be modified to improve expression of the polypeptide encoded thereby.
  • the DNA sequence may be modified to modify the properties of the peptide encoded thereby. For example, pyrogenic or inflammatory characteristics of the peptide may be reduced or eliminated.
  • fusion genes may be formed with gene sequences encoding an antigen.
  • the nucleotide sequence thereof may be illustrated as in Figure 1A.
  • the cDNA was found to be 1781 base pairs in length extending from the 5' untranslated region to a 32 bp poly A tail.
  • This sequence contained an open reading frame of approximately 804 base pairs encoding a protein of approximately 268 amino acids with a predicted molecular weight of 30,953.
  • the amino terminal amino acid of mature ovine Il-l ⁇ is predicted to be the glutamine residue at nucleotide 407 (amino acid 119) giving a mature protein of 150 amino acids with a molecular weight of 17,230.
  • the nucleotide sequence thereof may be illustrated as in Figure 2A.
  • Ovine IL-l ⁇ cDNA as shown in Figure 2 was found to be 1429 bp in length including an 11 bp poly A tail.
  • the 3' region contains a polyadenylation signal and several ATTTA motifs.
  • This sequence contained an open reading frame extending from approximately nucleotide 46 to nucleotide 846 encoding a protein of approximately 266 amino acids with a predicted molecular weight of 30,692.
  • the amino terminal amino acid of mature ovine IL-l ⁇ would be expected to be the alanine residue at nucleotide 385 (amino acid 114) giving a protein of 153 amino acids with a molecular weight of 17 , 708 .
  • the nucleotide sequence thereof may be illustrated as in Figure 3A.
  • Ovine IL-6 cDNA as shown in Figure 3 was found to be 1103 bp in length including a 10 bp poly A tail.
  • the 3* region contains a polyadenylation signal and six ATTTA motifs.
  • This sequence contained an open reading frame of approximately 624 bp encoding a protein of approximately 208 amino acids with a predicted molecular weight of 23,448.
  • the amino terminal amino acid of mature ovine IL-6 would be expected to be the proline residue at nucleotide 131 (amino acid 29) giving a protein of 180 amino acids with a molecular weight of 20,549.
  • TNF ⁇ tumor necrosis factor ⁇
  • bp base pairs
  • the cDNA was found to be 1675 base pairs in length with a long (160 bp) 5* untranslated region and a 32 bp poly A tail.
  • This sequence contained an open reading frame of approximately 702 base pairs encoding a protein of approximately 234 amino acids with a predicted molecular weight of 25,539.
  • the amino terminal amino acid of mature ovine TNF ⁇ is predicted to be the leucine residue at nucleotide 392 (amino acid 78) giving a mature protein of 157 amino acids with a molecular weight of 17,242.
  • IFN- ⁇ ovine interferon
  • the nucleotide sequence thereof may be illustrated as in Figure 5D.
  • This cDNA was found to be 553 bp in length and contains an open reading frame of approximately 498 bp encoding a precursor protein of approximately 166 amino acids with a predicted molecular weight of 17.5 kDa.
  • the nucleotide sequence may be illustrated as in Figure 5A.
  • the cDNA was found to be 2650 bp in length with a 245 bp 5' untranslated region and a very long (1477 bp) 3' untranslated region ending in a 12 bp poly A tail.
  • This sequence contained an open reading frame of approximately 825 base pairs encoding a protein of approximately 275 amino acids with a predicted molecular weight of 30,869.
  • the cytokine receptor DNA sequence so formed is important, since it is the up regulation of the ⁇ chain which occurs in the generation of an immune response to an infection in animal models.
  • Study of the IL-2 receptor ⁇ chain for example may permit design of synthetic drugs which may mimic the effect of cytokines.
  • PCR Polymerase Chain Reaction
  • a DNA sequence coding for a polypeptide exhibiting ovine interleukin-2 (IL-2) activity and having a length of approximately 501 base pairs or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof.
  • the nucleotide sequence thereof may be illustrated as in Figure 5B.
  • This cDNA was found to be 501 base pairs in length with an open reading frame of approximately 465 base pairs encoding a precursor protein of approximately 155 amino acids with a predicted molecular weight of 17,657.
  • the mature protein of 135 residues has a predicted molecular weight of 15,542.
  • DNA sequences discussed above may be utilised in the production of corresponding polypeptides. This may be accomplished in any suitable manner. Standard recombinant techniques may be used including the use of live vectors (e.g. vaccinia) with or without additional protective antigens injected or infected into suitable host species. Alternatively a recombinant expression vector including DNA squences may be injected directly into the tissue of suitable host animals and the peptide directly expressed.
  • live vectors e.g. vaccinia
  • additional protective antigens injected or infected into suitable host species.
  • a recombinant expression vector including DNA squences may be injected directly into the tissue of suitable host animals and the peptide directly expressed.
  • a process for the production of a recombinant polypeptide having ovine cytokine or cytokine receptor activity which process includes the steps of providing a recombinant expression vector including a DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity, or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof, and capable of being replicated, transcribed and translated in a eukaryotic or prokaryotic organism; and an animal host; introducing said vector into said organism, such that the recombinant polypeptide encoded therein is expressed; and optionally isolating said polypeptide from said host.
  • the recombinant expression vector may be injected directly into the tissue of the animal host.
  • the animal host is a mamma1.
  • the DNA sequences may be transferred to a recombinant plasmid expression vector capable of being replicated, transcribed and translated in a unicellular organism.
  • the vectors pGEM3Zf(+) and pGEM5Zf(+) have been found to be suitable.
  • a process for the production of a recombinant polypeptide having ovine cytokine or cytokine receptor activity which process includes the steps of providing a recombinant expression vector including a DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity or a fragment thereof, and capable of being replicated, transcribed and translated in a eukaryotic or prokaryotic organism; and a eukaryotic or prokaryotic organism; introducing said vector into said organism by transformation, transduction or transfection; culturing the resulting organism; expressing the recombinant polypeptide encoded by said DNA sequence; and isolating said polypeptide from the culture.
  • the DNA sequence codes for a polypeptide exhibiting ovine IL-l ⁇ , IL-l ⁇ , IL-2, IL-2R, IL-6, TNF ⁇ or IFN- ⁇ activity.
  • the eukaryotic or prokaryotic organism is preferably a prokaryotic organism, for example a bacterial strain.
  • the bacterial strain may be a strain of E.coli.
  • IB392 strain has been found to be suitable.
  • the successful expression of eukaryotic polypeptides exhibiting cytokine or cytokine receptor activity in bacteria such as E.coli may require modification of the recombinant expression vector and/or the DNA sequences of interest to provide the various regulatory elements necessary for bacterial transcription and translation.
  • the recombinant expression vector may contain a ribosome binding site within its promoter region, and a suitable restriction site for insertion of the DNA sequence distal to the ribosome binding site.
  • the recombinant expression vector used in this aspect of the present invention may be selected from the following, (a) Celltech vector This vector system utilises a dual origin of replication developed by Yarranton, G.T., Wright, E. , Robinson, M.K., and Humphreys, G.O. at Celltech, United Kingdom (Gene 2 ⁇ :293-300).
  • plasmid copy number may be maintained at a low level under the control of a pSClOl origin of replication during growth of the host at 30°C or copy number may be increased dramatically by raising the culture temperature to 42°C which activates a second origin of replication under the control of the ⁇ PR promoter and a thermolabile repressor.
  • This vector contains a tac promoter region including the lac operator site just upstream of a polycloning site.
  • the vector also contains the lacl gene coding for the lac repressor which in the presence of IPTG cannot bind the lac operator. Therefore transcription of inserted genes can be induced by the addition of IPTG to the cultures.
  • the presence of strong transcription terminators downstream of the polylinker ensure that plasmid copy number is not affected by this induction.
  • Both vectors contain ribosome binding sites (RBS) within their promoter regions and DNA sequences of interest may be inserted into these vectors so that the
  • ATG initiation codon of the protein is correctly positioned relative to the RBS.
  • the Celltech vector used to express porcine growth hormone contains the pGH cDNA as a B ⁇ lll/EcoRl insert with the ATG translation initiation codon just downstream of the B ⁇ l II site. This places the ATG 14bp 3" of the vector RBS, a distance empirically determined by Celltech to be optimal for expression of various eukaryotic proteins.
  • the pMMB66EH vector requires the DNA sequence of interest to be inserted into an EcoRl or Smal site so that the ATG initiation codon lies within 10-14 bp of the vector RBS.
  • Cytokines of other species have been found in the prior art to be suitable for expression in bacteria since post translational events such as glycosylation are not required for biological activity.
  • a DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity including a deletion in the untranslated 5' region to remove a signal sequence for transport of the cytokine across the endoplasmic reticulum of a eukaryotic cell. Such features are not required for prokaryotic expression.
  • the DNA sequence according to this aspect of the present invention may further include a second DNA sequence containing an ATG initiation codon introduced in a suitable restriction site within the first DNA sequence.
  • the ATG initiation codon may be placed in front of the sequence coding for the mature protein.
  • the DNA sequence coding for a polypeptide is modified to include an ATG initiation codon correctly positioned relative to a suitable restriction site.
  • the process according to the present invention may include the preliminary steps of providing a first DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity, or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof; a second DNA sequence containing an ATG initiation codon; and inserting the second DNA sequence into a suitable restriction site within the first DNA sequence such that the ATG initiation codon is placed in front of the sequence coding for the mature protein.
  • the 5* portion of the cytokine cDNAs contain an untranslated region plus a signal sequence for transport of the cytokine across the endoplasmic reticulum of the eukaryotic cell. Since these features are not required for prokaryotic expression this region of each cytokine cDNA may be deleted and an ATG initiation codon placed in front of the sequence coding for the mature protein. This ATG should also be correctly positioned relative to a suitable restriction site to facilitate cloning into the two different vectors so that the ATG is at the required distance from the RBS.
  • the process may further include subjecting the first DNA sequence to a digestion step to remove a signal sequence in the untranslated 5' region for transport of the cytokine across the endoplasmic reticulum of a eukaryotic cell.
  • the digestion step may be conducted utilising any suitable restriction enzyme.
  • the process may include the preliminary steps of providing a first DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity; and an oligonucleotide sequence duplicating the nucleotide sequence of the first DNA sequence from the amino terminal amino acid to a restriction site and containing an ATG codon at a suitable restriction site upsteam of the duplicated sequence; and subjecting the first DNA sequence to in vitro mutagenesis to produce an ATG codon and a suitable restriction site therein.
  • the cDNAs for IL-l ⁇ , TNF ⁇ and IL-6 may be modified by inserting DNA fiagments synthesised as complementary oligonucleotides into restriction sites within the cDNA molecules.
  • these unique restriction sites may be generated by in vitro mutagenesis, changing the sequence to produce a restriction site without altering the amino acid sequence.
  • the cDNA already contains an appropriate site.
  • Oligonucleotide derived fragments duplicating the nucleotide sequence from the amino terminal amino acid to the restriction site and containing an ATG codon and Bql II site upstream of these sequences may then be inserted into this restriction site. This regenerates a protein with the correct amino acid sequence but the oligonucleotides are designed to alter the nucleotide sequence to include those codons commonly used in E.coli.
  • the cDNAs for IL-l ⁇ and IL-2 may be modified by oligonucleotide directed in vitro mutagenesis.
  • the DNA upstream of the sequence coding for the mature proteins may be mutated to produce an ATG codon and a B ⁇ l II site.
  • IL-l ⁇ cDNA sequence CATTACAGCTTC CAG AGT AAC Gin Ser Asn Changed to:
  • the polypeptide exhibiting cytokine or cytokine receptor activity may be selected from IL-l ⁇ , IL-l ⁇ , IL-1R, IL-2, IL-2R, IL-3, IL-3R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-7, IL-7R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-10R, IFN- ⁇ , IFN- ⁇ R, TNF- ⁇ , TNF- ⁇ R, GMCSF, GMCSFR, TGF- ⁇ and TGF- ⁇ R, preferably IL-l ⁇ , IL-l ⁇ , IL-2, IL-2R, IL-6, TNF ⁇ or IFN-
  • a veterinary composition including a recombinant polypeptide having ovine cytokine or cytokine receptor activity, or mimotope thereof, or fragment thereof preferably selected from IL-l ⁇ , IL-l ⁇ , IL-1R, IL-2, IL-2R, IL-3, IL-3R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-7, IL-7R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-10R, IFN- ⁇ , IFN- ⁇ R, TNF- ⁇ , TNF- ⁇ R, GMCSF, GMCSFR, TGF- ⁇ and TGF- ⁇ R
  • the veterinary composition may function as a therapeutic agent for example in the treatment of trauma such as burns, wounds or the like, and/or may be utilised as an adjuvant or co-adjuvant in combination with a further active agent, for example a therapeutic agent.
  • the veterinary composition may be utilised in the treatment of ruminant animals, especially sheep, goats and cattle, it may also be utilised in the treatment of other animal species, in particular pigs, cats, dogs and horses.
  • cytokines may be used to induce, enhance or modulate an immune response against an antigen. Accordingly, in a further aspect of the present invention there is provided a vaccine composition including an antigen against a disease of interest; and a non-toxic adjuvant including a recombinant polypeptide having ovine cytokine or cytokine receptor activity, or mimotopes, derivatives or fragments thereof.
  • the cytokine and antigen can be incorporated in any pharmaceutically acceptable vehicle with or without added co-adjuvants or immunostimulatory molecules.
  • the antigen may be derived from any source including viral, fungal, bacterial or parasite antigens, auto-immunity related antigens or tumor-associated antigens.
  • the antigen may also be derived from plants, for example allergens and plant toxins.
  • Other applications may also include the elicitation of an immune response to stimulate or inhibit the stability or interaction of cellular modifiers, including hormones with their corresponding receptors or binding components.
  • the immune response can be used to inhibit/enhance growth, reproduction, differentiation and overall performance.
  • the quality of the immune response can be manipulated to optimize the desired protective response.
  • the recombinant polypeptide may be utilised as a non-toxic adjuvant in combination with a vaccine, for example to enhance vaccination against various diseases, including parasitic diseases.
  • These parasites include, for example, Haemonchus contortus, Trichostrongylus colubriformus and Ostertagia circumcincta (gastrointestinal nematodes), Bacteroides rodosus (foot rot), Lucilia cuprina (blowfly strike), Staph. aureus (mastitis) and C.ovis (cheesy gland).
  • the cytokines may function as adjuvants for existing vaccines or new recombinant vaccines that are being developed.
  • cytokines or cytokine receptors may be more suitable.
  • the route of delivery of the cytokine may vary for both different cytokines and different vaccine preparations.
  • the recombinant polypeptide may also be utilised as a non-toxic adjuvant in combination with a viral vector.
  • the recombinant cytokines may be utilised in receptor studies as discussed above.
  • the recombinant cytokine may be radiolabelled or linked to a detectable ligand or hapten.
  • the ligand or hapten may be selected from biotin, fluorochromes, FITC and the like.
  • cytokine As cytokines act through receptors the effect of a cytokine may be mimicked or blocked utilising an agent that will bind the receptor. Such agents may be generated using both the cytokine and its specific receptor. Accordingly, in a still further aspect of the present invention, there is provided a monoclonal or polyclonal antibody against a recombinant polypeptide exhibiting ruminant, preferably ovine, cytokine or cytokine receptor activity; or a fragment thereof.
  • a receptor specific monoclonal antibody may bind the receptor and either block the activity of a cytokine or alternatively mimmic its effect. Further, immunization of animals with monoclonal or polyclonal antibodies may have both agonistic and antagonistic effects. Thus immunization with the antibody may lead to upregulation of endogenous cytokine production enhancing vaccination.
  • circulating cytokine specific monoclonal or polyclonal antibodies may be utilised to
  • cytokines "mop up" excesses of undesirable cytokines. This may occur in stress situations, for example during transport or animals or during docking or castration of animals.
  • soluble receptors may be utilised to "mop up" circulating excess cytokine.
  • soluble receptors may function to enhance the activity of cytokines.
  • cytokine-receptor complexes may be formed, and used to generate monoclonal or polyclonal antibodies.
  • a veterinary composition including a monoclonal or polyclonal antibody against a recombinant polypeptide exhibiting ruminant, preferaoly ovine, cytokine or cytokine receptor activity, or mimotopes thereof, or fragments thereof.
  • compositions according to the invention may take any form suitable for administration including forms suitable for oral or parental (including implant) use.
  • oral administration the compositions may take the form of, for example solutions, syrups or suspensions e.g. in aqueous buffer, or solid compositions such as tablets or capsules, prepared by conventional means.
  • parental use the compositions may for example take a form suitable for injection, such as a suspension, solution or emulsion in an aqueous or oily vehicle optionally containing formulatory agents such as suspending, stabilising, solubilising and/or dispersing agents.
  • the aqueous or oily vehicle may include any other adjuvant known per se, for example aluminium hydroxide (Al(OH)_).
  • FIGURE 1A is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.
  • FIGURE 1A is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.
  • the 3* polyadenylation signal is overlined and the ATTTA motifs are underlined.
  • Nucleotide sequence and predicted amino acid sequence of ovine Il-l ⁇ modified for bacterial expression The ATG initiation codon added in front of the amino terminal amino acid of the mature protein is underlined.
  • Nucleotide sequence and predicted amino acid sequence of ovine IL-l ⁇ The presumed amino terminus (Ala 114) of mature IL-l ⁇ is marked with an asterisk.
  • the 3' polyadenylation signal is overlined and the ATTTA motifs are underlined.
  • Nucleotide sequence and predicted amino acid sequence of ovine Il-l ⁇ modified for bacterial expression The ATG initiation codon added in front of the amino terminal amino acid of the mature protein and the bases changed for E. Coli codon usage are underlined.
  • FIGURE 3A The Hindlll site created by in vitro mutagenesis is marked.
  • FIGURE 4A Nucleotide sequence and predicted amino acid sequence of ovine II-6 modified for bacterial expression. The ATG initiation codon added in front of the amino terminal amino acid of the mature protein is underlined.
  • FIGURE 4A Nucleotide sequence and predicted amino acid sequence of ovine II-6 modified for bacterial expression. The ATG initiation codon added in front of the amino terminal amino acid of the mature protein is underlined.
  • FIGURE 4A Nucleotide sequence and predicted amino acid sequence of ovine II-6 modified for bacterial expression. The ATG initiation codon added in front of the amino terminal amino acid of the mature protein is underlined.
  • FIGURE 4A Nucleotide sequence and predicted amino acid sequence of ovine II-6 modified for bacterial expression. The ATG initiation codon added in front of the amino terminal amino acid of the mature protein is underlined.
  • FIGURE 4A Nucleotide sequence and predicted amino acid sequence of ovine II-6 modified for bacterial expression. The ATG initiation codon added in front of
  • FIGURE 5A Nucleotide sequence and predicted amino acid sequence of ovine IL-2 receptor cDNA.
  • FIGURE 5B Nucleotide sequence and predicted amino acid sequence of ovine IL-2 receptor cDNA.
  • FIGURE 7 Northern blot analysis of induction of Human IL-l ⁇ , IL-l ⁇ , TNF ⁇ .
  • FIGURE 9 Diagrammatic representation of the Fill expression vector used to express the ovine IL-1 cDNAs. The cDNA inserts used to construct the expression derivatives are shown, with the thick lines representing the coding regions. The full length derivatives contained the entire cDNA with the 3* untranslated regions intact. The truncated derivatives had most of the 3* untranslated regions including the ATTTA sequences deleted.
  • FIGURE 10 Diagrammatic representation of the Fill expression vector used to express the ovine IL-1 cDNAs. The cDNA inserts used to construct the expression derivatives are shown, with the thick lines representing the coding regions. The full length derivatives contained the entire cDNA with the 3* untranslated regions intact. The truncated derivatives had most of the 3* untranslated regions including the ATTTA sequences deleted.
  • FIGURE 10 Diagrammatic representation of the Fill expression vector used to express the ovine IL-1 cDNAs. The cDNA inserts used to construct the expression derivatives are shown, with the thick lines representing the coding
  • FIGURE 11 Northern blot analysis of induction of IL-l ⁇ and
  • IL-l ⁇ mRNA by endotoxin and phorbol ester 20 ⁇ g of total RNA extracted from alveolar macrophages cultured for 5 hours (lanes 1, 3, 5 and 7) or 20 hours (lanes 2, 4, 6 and 8) in the presence of RF10 alone (lanes 1 and 2) or RF10 supplemented with LPS (lanes 2 and 4), PMA (lanes 5 and 6) or PMA plus ionomycin (lanes 7 and 8) was hybridized with probes specific for IL-l ⁇ (A) or IL-l ⁇ (C) . Autoradiographic exposure was for 24 hours. 28S ribosomal RNA is shown to indicate relative RNA loading (B and D) and the position of the 28S and 18S bands relative to the hybridizing band is also indicated.
  • FIGURE 13 Diagrammatic representation of the Fill expression vector used to express the ovine TNF ⁇ cDNA.
  • the cDNA inserts used to construct the expression derivatives are shown, with the thick lines representing the coding regions.
  • the full length derivatives contained the entire cDNA with the 3' untranslated region intact.
  • the truncated derivative had most of the 3' untranslated region including the ATTTA sequences deleted.
  • FIGURE 15 Southern blot analysis of ovine genomic DNA using a cDNA probe specific for ovine TNF ⁇ . Genomic DNA was digested with BamHI (B) , Hindlll (H) and EcoRI (E) . The molecular size markers are from Hindlll digested bacteriophage ⁇ DNA.
  • B BamHI
  • H Hindlll
  • EcoRI EcoRI
  • IL-l ⁇ The arrow marks the induced recombinant IL-l ⁇ band.
  • Il-l ⁇ cultures showing the production of biologically active IL-1.
  • Controls included recombinant human IL-1 and as a negative control, crude fractions from an induced culture carrying ovine IL-6.
  • Protein SDS-PAGE showing induction of proteins in a culture containing an expression vector carrying ovine IL-l ⁇ .
  • the arrow marks the induced recombinant IL-l ⁇ band.
  • Controls included recombinant human IL-1 and as a negative control, crude fractions from an induced culture carrying ovine IL-2.
  • Bovine thymocyte co-stimulation assay of E. coli derived ovine IL-l ⁇ 5 fold dilutions of a crude fraction of ovine IL-l ⁇ with or without 3 ⁇ g/ml of PHA were used to stimulate proliferation of bovine thymocytes. Controls included thymocytes in medium alone and with PHA but no
  • FIGURE 22 Protein SDS-PAGE showing induction of proteins in a culture containing an expression vector carrying ovine
  • the arrow marks the induced recombinant IL-2 band.
  • FIGURE 24 7TD1 assay of supernatants from transfections with ovine IL-6. A lOx concentrate of IL-6 transfection supernatant was assayed in the presence and absence of anti-human IL-6 Ab to block COS derived IL-6 activity. Controls included media alone and recombinant human IL-6 with and without Ab.
  • FIGURE 25
  • FIGURE 27 IL-6 assay of a crude fraction from induced IL-6 cultures showing the production of biologically active IL-6. Controls included Media alone (no activity) and lOOU/ml recombinant human IL-6.
  • FIGURE 27
  • IL-2R from equilibrium binding assays using i25l labelled IL-2.
  • Lungs and trachea were removed intact from one-two year old merino ewes housed at the Department of Veterinary Science, University of Melbourne. Lungs were then flushed via the trachea with 400-800 ml of sterile phosphate buffered saline (PBS) . Cells were recovered from lung washings by centrifugation, and washed twice in RPMI-1640 supplemented with 10% v/v fetal calf serum, 2mM glutamine, and 100 U/ml pencillin and 0.1 mg/ml streptomycin (RF10). Cell populations were cultured in 80
  • Macrophages were recovered for flow cytometric analysis and RNA preparation by trypsinisation.
  • mAbs monoclonal antibodies
  • mAbs reactive with MHC class II molecules included 49.1 (all class II molecules), 42.20 (DR ⁇ specific), 38.27 (DQ ⁇ specific) and 28.1 (DP ⁇ specific).
  • mAbs against bovine r ⁇ lFN IFN2 and IFN9 were the gift of Dr. P. Wood, CSIRO Division of Animal Health, Melbourne, and have also been previously described. Immunofluorescence Staining and FLow Cytometry
  • NOB-1/CTLL assay for determination of IL-1 has been previously described. Briefly, NOB-1 cells were washed three times in RF10, resuspended at 2X10 /ml and 0.1 ml added to "V" bottom microtitre plates containing 0.1 ml of appropriately diluted test SNF. After incubation for 24 hours at 37°C, plates were centrifuged and 50 ⁇ l of SNF transferred to a replicate flat bottom microtitre plate together with 50 ⁇ l contai.ni.ng 5X103 CTLL cells.
  • DNA Probes cDNA probes encoding human IL-l ⁇ and IL-l ⁇ and human TNF ⁇ were generously provided by Immunex Research and Development Corporation and Genentech, Inc. respectively. cDNA probes encoding ⁇ -tubulin or GAPDH were used to indicate relative levels of RNA. 32P-labelled probes were generated by random priming.
  • RNA samples were fractionated by electrophoresis through denaturing formaldehyde-agarose gels and transferred to Hybond-N nylon membranes (Amersham) as previously described. Following overnight transfer RNA was fixed to membranes by UV cross-linking and hybridised to appropriate 32P-labelled probes.
  • ovine probes blots were washed under conditions of high stringency (0.5 X SSC, 60°C) following hybridisation. For cross-species probes low stringency (1 X SSC, 50°C) conditions were employed.
  • Alveolar macrophages were recovered from lung washings and subjected to immunofluorescent staining with a panel of mAbs specific for ovine leukocyte cell surface antigens. Results are presented in Table 1 and represent pooled data from at least three sheep. TABLE 1 PHENOTYPE OF OVINE ALVEOLAR MACROPHAGES Surface Marker Reactivity
  • Macrophages were recovered from lung washings and cultured in RF10 alone or in RF10 supplemented with 10 ⁇ g/ml of LPS.
  • Total RNA was extracted from these macrophages at various time intervals and also from freshly isolated cells. The RNA was electrophoresed, blotted to nylon membranes then hybridised with cDNA probes specific for human IL-l ⁇ , IL-l ⁇ , TNF ⁇ or control GAPDH or ⁇ -tubulin probes. None of the cytokine specific probes hybridised with RNA from freshly isolated macrophages or macrophages cultured in medium alone. In contrast, message encoding all three appeared at various time points following stimulation with LPS ( Figure 6) .
  • TNF ⁇ specific message peaked at 1 hour after initiation of culture and declined thereafter with only a trace being detected at 24 hours but nothing at 48 hours (Figure 6D) .
  • IL-l ⁇ specific message was also detected after 1 hour of stimulation with a similar level observed after 5 hours culture. No mRNA encoding this cytokine was detected at 30 mins stimulation nor at 24 or 48 hours stimulation ( Figure 6A) .
  • mRNA encoding IL-l ⁇ was not detected until after 5 hours of stimulation. A low level of message remained at 24 hours but this had disappeared by 48 hours (Figure 6B) .
  • a dose response curve for the secretion of IL-1 following stimulation with LPS was determined using the NOB-1/CTLL bioassay. Macrophages were cultured in RF10 supplemented with 10 fold dilutions of LPS from 10 mg/ml - 0.1 pg/ml. SNF was sampled at 24 hours and assayed for IL-1. Results show that maximal IL-1 secretion was induced with concentrations of LPS greater than 10 ng/ml ( Figure 8A) . At concentrations below this the level of IL-1 secretion decreased until at 0.1 pg/ml of LPS only background levels were detected.
  • RNA being extracted from macrophages stimulated with the different concentrations of LPS.
  • the RNA was electrophoresed, blotted to a nylon membrane, and hybridised with cDNA probes specific for IL-l ⁇ and IL-l ⁇ .
  • the level of detectable mRNA declined with concentrations of LPS less than 10 pg/ml for both probes, implying a similar dose response for both IL-l ⁇ and IL-l ⁇ .
  • a similar blot to the one just described was used to determine the dose response for induction of TNF ⁇ by LPS.
  • Results presented in Figure 8B show that the level of detectable TNF ⁇ specific message declined over the dose range tested.
  • Production of IL-l ⁇ , IL-l ⁇ and TNF ⁇ by alveolar macrophages freshly isolated or cultured in the presence or absence of LPS was examined.
  • Northern blot analysis we failed to detect mRNA encoding these cytokines in total RNA from freshly isolated macrophages.
  • no specific mRNA was detected in total RNA from macrophages cultured in medium alone.
  • Addition of LPS upregulated production of all three cytokines. With regard to IL-1 this was reflected by increased levels of specific mRNA as well as increa 3d extracellular bioactive protein.
  • IL-l ⁇ mRNA was induced to detectable levels within 1 hour of stimulation, the level peaking at 5 hours with no mRNA remaining at 24 hours. In contrast IL-l ⁇ mRNA was not detected until 5 hours post stimulation with low levels remaining at 24 hours.
  • IL-l ⁇ mRNA was not detected until 5 hours post stimulation with low levels remaining at 24 hours.
  • the frequency of IL-l ⁇ clones was 20 fold greater than that of IL-l ⁇ clones (or TNF ⁇ clones - results not shown) .
  • IL-1 mRNA's peaked at 4-6 hours and IL-l ⁇ message increased as a proportion with time after stimulation.
  • EXAMPLE 2 EXPRESSION OF RECOMBINANT IL-1 MATERIALS AND METHODS Cloning of ovine IL-l ⁇ and IL-l ⁇ cDNAs
  • Ovine alveolar macrophages were isolated as previously described and cultured in RPMI-1640 supplemented with 10% v/v fetal calf serum for 5 or 16 hours with 10 ⁇ g/ml LPS to induce expression of IL-1 specific mRNA.
  • Messenger RNA was isolated via standard acid phenol- guanidine thiocyanate extraction and oligo dT cellulose purification.
  • cDNA was synthesised, cloned into ⁇ gtlO, then packaged in vitro using Amersham cDNA synthesis and ⁇ gtlO cloning kits. Phage were then used to infect E.coli strain NM514. 100,000 independent plaques were transferred onto nylon filters and screened with 32p labelled human IL-l ⁇ and IL-l ⁇ probes (Immunex Research and Development Corporation) . Positive plaques were purified and the EcoRI inserts cloned into pGEM03Zf(+) (Promega Corp.). DNA sequencing of exonuclease III deletion derivatives was performed using the dideoxy chain termination method. Expression of recombinant IL-1
  • the cDNAs for both IL-l ⁇ and IL-l ⁇ were cloned into an expression vector containing the SV40 origin of replication, enhancer, and polyadenylation signal and a human metalothionine promoter hMTII A ( Figure 9).
  • Two constructs for each gene were made, one containing the full length cDNA and a second with a deletion of the 3* untranslated region containing the ATTTA sequences.
  • the EcoRI inserts of both IL-l ⁇ and IL-l ⁇ containing the entire cDNA were cloned into the unique EcoRI site in the vector.
  • the 3' deletion derivative of IL-l ⁇ was constructed from one of the original plaque isolates.
  • This clone was found to contain an EcoRI insert of 1220bp extending from nucleotide 1 to nucleotide 1220. This was thought to have resulted from oligo dT priming at a run of A residues within the IL-l ⁇ mRNA during 1st strand cDNA synthesis.
  • the IL-l ⁇ 3' deletion derivative was cloned as a BamHI/Bglll fragment, covering nucleotides 1 to 985, into the unique BamHI site of the vector.
  • NOB-1/CTLL assay for measuring IL-1 activity was performed as previously described. Briefly, NOB-1 cells were washed three times in RF10, resuspended at 2 x
  • CTL 3 ⁇ l containing 5 x 10 CTL cells.
  • CTL were pulsed at 20 hours with 3 H-thymidine and harvested 4 hours later. All test samples were assayed in duplicate.
  • RNA For preparations of RNA, fresh or cultured macrophages were washed twice in phosphate buffered saline.
  • Genomic DNA was isolated from sheep peripheral blood leukocytes via standard techniques. 10 ⁇ g of DNA was digested with the appropriate enzyme, electrophoresed on a 0.7% agarose gel, transferred to a nylon membrane and hybridised with 32P labellleedd oovviinne cDNA probes.
  • IL-l ⁇ cDNA probe A number of positive plaques were identified and their EcoRI inserts cloned into pGEM-3Zf(+).
  • the cDNA was found to be 1781 base pairs in length extending from the 5' untranslated region to a 32 bp poly A tail. This sequence contained an open reading frame of 804 base pairs encoding a protein of 268 amino acids with a predicted molecular weight of 30,953. Based on the sequence of human IL-l ⁇ , the amino terminal amino acid of mature ovine IL-l ⁇ is predicted to be the glutamine residue at nucleotide 407 (amino acid 119) giving a mature protein of 150 amino acids with a molecular weight of 17,230.
  • the 3* untranslated region contains an AATAAA polyadenylation signal and includes six copies of the ATTTA motif common to other cytokine cDNAs. These ATTTA motifs are thought to affect mRNA stability.
  • Ovine IL-l ⁇ cDNA as shown in Figure 2 was found to be 1429 bp in length including an llbp poly A tail. The 3' region contains a polyadenylation signal and several ATTTA motifs.
  • An open reading frame extending from nucleotide 46 to nucleotide 846 was found to encode a protein of 266 amino acids with a predicted molecular weight of 30,692.
  • the amino terminal amino acid of mature ovine IL-l ⁇ would be expected to be the alanine residue at nucleotide 385 (amino acid 114) giving a protein of 153 amino acids with a molecular weight of 17,708.
  • DNA fragments from pGEM-3Zf(+) derivatives were inserted into a mammalian expression vector as described in Materials and Methods. Previous studies had shown that the AT rich sequences found in the 3' untranslated regions of many cytokine and oncogene cDNAs were involved in RNA stability. These sequences are also present in the ovine genes. To examine the effects of these ATTTA motifs on the levels of IL-1 expression, plasmids with and without the 3' portion of the cDNAs containing these sequences were constructed. Controls were transfections with plasmids in which the gene fragments were inserted in the wrong orientation with respect to the vector promoter.
  • Plasmid DNA was transfected into COS cells and the supernatant harvested 73 hours after transfection. These supernatants were then assayed for IL-1 activity and the results from a single experiment are shown in Table 2. TABLE 2
  • IL-l ⁇ transfection SNF assay The level of IL-1 detected after transfection with control full length or truncated cDNAs for both IL-l ⁇ and IL-l ⁇ was not significantly greater than this.
  • the cDNA encoding ovine IL-l ⁇ was modified in vitro to place a Bql II restriction site and an ATG initiation codon proximal to the amino terminus of the mature polypeptide. This was achieved using in vitro mutagenesis to alter the DNA sequence from nucleotides 399 to 406 ( Figure 1A) to produce the desired sequence.
  • IL-l ⁇ cDNA sequence CATTACAGCTTC CAG AGT AAC Gin Ser Asn
  • the modified sequence was transferred as a
  • Protein induction was monitored by SDS-PAGE (see Figure 17) and crude extracts were then assayed for IL-1 activity.
  • the protein gel shows good induction of a protein band running just below the 21,500 MW marker, the size expected for recombinant ovine IL-l ⁇ .
  • An IL-1 assay of a crude extract from this induced culture showed the presence of biologically active IL-1 ( Figure 18) .
  • Bacterial Expression of Ovine IL-l ⁇ The cDNA encoding ovine IL-l ⁇ was modified in vitro to place a Bglll restriction site and an ATG initiation codon proximal to the amino terminus of the mature polypeptide.
  • the modified sequence was then transferred as a
  • E.coli derived recombinant ovine IL-l ⁇ was assayed in a bovine thymocyte co-stimulation assay. Briefly, a single cell suspension was prepared from the thymus of a 3 month old calf using standard procedures. The thymocytes were then cultured in 200 ⁇ l (in flat bottom microtitre plates) of
  • ovine IL-l ⁇ would be expected to exert biological activity on all bovine cell populations responsive to IL-1.
  • Macrophages from three sheep were recovered from lung washings and cultured independently in RF10 alone or in RF10 supplemented with either LPS, PMA or PMA plus ionomycin at the concentrations described in Materials and Methods.
  • Total RNA was extracted from these macrophages at 5 hours and 20 hours post stimulation. The RNA was electrophoresed, blotted to nylon membranes and then hybridised with ovine IL-l ⁇ or IL-l ⁇ cDNA probes. The results shown in Figure 5 are from one animal, however no differences in expression were detected between the three sheep examined. Without exogenous stimulation a low level of mRNA encoding both IL-l ⁇ and IL-l ⁇ was detected at 4 hours but not 20 hours.
  • TNF ⁇ cDNA was cloned into the Fill expression vector previously described ( Figure 12) . Two constructs were made, one containing the full length cDNA and a second with a deletion of the 3' untranslated region containing the ATTTA sequences. The 1675 bp EcoRI insert containing the entire TNF ⁇ cDNA was cloned into the unique EcoRI site of the vector. The 3' deletion derivative of TNF ⁇ was constructed by cloning a 1050 bp BamHI/Bglll fragment covering nucleotides 139 to 1189 into the unique BamHI site of the vector.
  • CsCl gradient DNA of each derivative, containing fragments in both orientations, were transfected into COS-7 cells by the DEAE dextran technique and the supernatants harvested 72 hours post transfection.
  • IL-6 activity transfected cells were cultured in DM10 and supernatants assayed neat.
  • TNF ⁇ activity DM10 was replaced at 24 hours with the same volume of serum free Opti-mem media (GIBCO Laboratories) . The supernatant recovered 48 hours later was concentrated 15X using Centriprep 10 concentrators (Amicon Corp.) and stored frozen prior to assay.
  • the urine IL-6 dependent hybridoma line 7TD1 was used to assay IL-6 levels in transfection supernatants.
  • 7TD1 cells cultured in DM10 supplemented with 20 U/ml of recombinant human IL-6 (Boehringer Mannheim), were washed three times in DM10 without IL-6 and resuspended at
  • % cytolysis was calculated from the formula 100(a-b)/a where a and b are respectively the mean adsorbances of duplicate wells without or with the test sample.
  • Genomic DNA was isolated from sheep peripheral blood leukocytes via standard techniques. 10 ⁇ g of DNA was digested with the appropriate enzyme, electrophoresed on a 0.7% agarose gel, transferred to a nylon membrane and
  • LPS stimulated ovine alveolar macrophages produced TNF ⁇ specific messenger RNA. Therefore an LPS stimulated ovine alveolar macrophage cDNA library was constructed as described in
  • the amino terminal amino acid of mature ovine TNF ⁇ is predicted to be the leucine residue at nucleotide 392 (amino acid 78) giving a mature protein of 157 amino acids with a molecular weight of 17,242.
  • the 3' untranslated region contains an AATAAA polyadenylation signal and includes nine copies of the ATTTA motif common " to other cytokine cDNAs. These ATTTA motifs are thought to affect mRNA stability.
  • TNF ⁇ cDNA DNA fragments from pGEM-3Zf(+) derivatives were inserted into a mammalian expression vector as described in Materials and Methods. Previous studies have suggested that the AT rich sequences found in the 3* untranslated regions of many cytokine and oncogene cDNAs may be involved in RNA stability. These sequences are also present in ovine TNF ⁇ . To examine the effects of these ATTTA motifs on the levels of TNA ⁇ expression, plasmids with and without the 3' portion of the cDNA containing these sequences were constructed. Controls were transfections with plasmids in which the gene fragments were inserted in the wrong orientation with respect to the vector promoter.
  • the cDNA encoding ovine TNF ⁇ was modified in vitro to place a Bglll restriction site and an ATG initiation codon proximal to the amino terminus of the mature polypeptide. This was achieved by changing the DNA sequence at nucleotide 438 ( Figure 4A) using in vitro mutagenesis to produce a Pmll restriction site whilst still maintaining the correct amino acid sequence.
  • this oligonucleotide introduced a Bglll site and an ATG triplet in front of this sequence. Other bases were altered within this oligonucleotide to provide codons commonly used by E.coli. These changes, depicted in Figure 4B, did not alter the amino acid sequence.
  • Macrophages were recovered from lung washings and cultured in DM10 alone or in DM10 supplemented with either
  • Oligonucleotide primers (based on the bovine and ovine IL-2 cDNA sequences) were used in a PCR reaction to amplify from the pool of cDNA molecules, the cDNA corresponding to ovine IL-2. These oligonucleotide primers (shown below) were designed to bind sequences upstream of the initiation codon (5* primer) and downstream of the termination codon (3' primer) to amplify a fragment of ovine IL-2 covering the entire protein coding region.
  • oligonucleotides are flanked by restriction enzyme recognition sequences (shown in brackets) to facilitate cloning of this 520 bp fragment into a suitable vector such as pGEM3Zf(+) .
  • the sequence of the ovine IL-2 cDNA obtained by PCR is shown in Figure 5B. Mammalian cell expression of ovine IL-2 The ovine IL-2 cDNA fragment generated by PCR was then cloned into the Fill expression vector previously used to express IL-l ⁇ , IL-l ⁇ and TNF ⁇ . Plasmid DNA was transfected into COS-7 cells using DEAE-Dextran and supernatants were harvested after 72 hrs. Supernatants from two separate transfections plus a 15x concentrate of pooled supernatant obtained from these two transfections were assayed for IL-2 activity by their ability to induce proliferation of ovine Con A blasts. Briefly, lymph node cells cultured in 5 ⁇ g/ml of Concanavalin A for 72 hours
  • IL-2 cDNA sequence GTTGCAAACGGT GCA CCT ACT Ala Pro Thr
  • the modified sequence was then transferred as a Bqlll/EcoRl fragment (Figure 5C) into the previously described Celltech vector and then transformed into the E.coli host strain IB392. Cultures were grown in minimal medium at 30°C until they reached an OD600 of 1.00. They were induced at 42°C for 20 mins. and then grown at 38°C for 5 hours to allow protein expression.
  • Protein induction was monitored by SDS-PAGE (see Figure 22) and crude extracts were then assayed for IL-2 activity.
  • the protein gel shows good induction of a protein band running just above the 14,400 MW marker, the size expected for recombinant ovine IL-2.
  • An IL-2 assay of a crude extract from this induced culture showed the presence of biologically active IL-2 ( Figure 23).
  • EXAMPLE 5 CLONING AND EXPRESSION OF RECOMBINANT IL-6 Materials and Methods Cloning of ovine IL-6 cDNA The cDNA library described in the previous examples was screened with a 32P labelled human IL-6 probe. A positive plaque was purified and the EcoRI insert cloned into pGEM-3ZF(+) and M13. DNA sequencing of exonuclease III deletion derivatives was performed using the dideoxy chain termination method.
  • a 3 ' deletion derivative of the ovine IL-6 cDNA was cloned into the Fill expression vector previously described ( Figure 12) .
  • the deletion derivative of IL-6 was constructed by cloning a 686 bp Sacl-Bsml fragment covering nucleotides 18 to 703 into the Smal site of the vector.
  • CsCl gradient DNA of the derivative, containing fragments in both orientations, was transfected into COS-7 cells by the DEAE dextran technique and supernatants harvested 72 hrs later.
  • DM10 media was replaced at 24 hrs with the same volume serum free Opti-mem media.
  • the supernatant recovered 48 hrs later was concentrated 10X using a centriprep 10 concentrator and stored frozen prior to assay.
  • Example 3 was performed as described in Example 3 using the 7TD1 cell line.
  • Recombinant human IL-6 was supplied by Boehringer Mannheim and polyclonal anti-human IL-6 antibodies by British Biotechnology.
  • the ovine IL-6 cDNA isolated from the LPS stimulated alveolar macrophage cDNA library was found to be 1103 bp in length.
  • the sequence contained an open reading frame of approximately 624 bp encoding a protein of approximately 208 amino acids with a predicted molecular weight of 23,448 ( Figure 3A) .
  • the amino terminal amino acid of mature ovine IL-6 would be expected to be the proline residue at nucleotide 131 (amino acid 29) giving a protein of 180 amino acids with a molecular weight of 20,549.
  • Plasmid DNA was transfected into COS cells and the supernatants (Opti. mem) harvested and concentrated 10 x 72hrs later. This SNF was then assayed for IL-6 activity.
  • COS-7 cells produce Simian IL-6 which is also active in the 7TD1 IL-6 assay. To overcome this problem the transfection supernatants were assayed in the presence of 10 ⁇ g/ml of polyclonal anti-human IL-6 Ab. Simian IL-6 is closely related to human IL-6 and is therefore, unlike the ovine IL-6, neutralised by this polyclonal anti-serum.
  • the cDNA encoding ovine IL-6 was modified in vitro to place a Bglll restriction site and an ATG initiation codon proximal to the amino terminus of the mature polypeptide. This was achieved by cloning a short oligonucleotide containing a Bglll site and an ATG into a Smal site (nucleotide 132, Figure 3A) spanning the amino terminus of the mature protein.
  • the modified sequence was then transferred as a Bglll/EcoRl fragment (Figure 3B) into the previously described Celltech vector and then transformed into the E.coli host strain IB392. Cultures were grown in minimal medium at 30°C until they reached an OD600 of 1.00. They were induced at 42°C for 20 mins. and then grown at 38°C for 5 hours to allow protein expression.
  • a protein gel of induced and uninduced cultures shows induction of a band running at a molecular weight corresponding to the expected MW of recombinant ovine IL-6 ( Figure 25) .
  • An IL-6 assay of a crude extract from this induced culture showed the presence of biologically active ovine IL-6 ( Figure 26) .
  • RNA was isolated from ovine alveolar macrophages stimulated with LPS as described in Examples 2, 3 and 5.
  • Reverse transcriptase and oligo dT primer was then used to produce a pool of cNDAs using standard techniques.
  • Oligonucleotide primers (based on the bovine ⁇ -IFN cDNA sequence) were then used in a PCR reaction to amplify from the pool of cDNA molecules, the cDNA corresponding to ovine ⁇ -IFN.
  • oligonucleotide primers (shown below) were designed to bind sequences upstream of the initiation codon (5' primer) and downstream of the termination codon (3' primer) to amplify a fragment of ovine ⁇ -IFN covering the entire coding region.
  • oligonucleotides are flanked by restriction enzyme recognition sequences (shown in brackets) to facilitate cloning of the 553 bp fragment into a suitable vector such as pGEM3Zf(+).
  • the sequence of the ovine ⁇ lFN cDNA obtained by PCR is shown in Figure 5D. Mammalian cell expression of recombinant ovine IFN- ⁇
  • the ovine IFN- ⁇ cDNA fragment generated by PCR was then cloned into the Fill expression vector as previously described. Plasmid DNA was transfected into COS-7 cells using DEAE-dextran and supernatants were harvested after 72hrs. Mock transfection with no cDNA was used as a negative control. Supernatants were assayed for IFN- ⁇ using 2 monoclonal antibodies 9mAb) specific for bovine IFN- ⁇ and an ELISA based assay. Briefly, micro ⁇ titre plates were coated overnight at 4°C with a 1:1000 dilution of IFN- ⁇ mAb IFN-9 in pH 9.2 bicarbonate buffer.
  • RNA was isolated from ovine ConA blasts as previously described (Example 4). Reverse transcriptase and oligo dT primer was then used to produce a pool of cDNAs using standard techniques. Oligonucleotide primers (based on the bovine IL-2R ⁇ cDNA sequence) were then used in a PCR reaction to amplify from the pool of cDNA molecules, a portion of the cDNA corresponding to ovine IL-2R ⁇ chain. Cloning of an ovine IL-2R a chain cDNA
  • a cDNA library from activated ovine T cells produced using methods described in the previous examples was screened with the 32P labelled ovine IL-2R probe described above. A positive plaque was purified and the
  • the IL-2R ⁇ cDNA was cloned as an EcoRI fragment into the Fill expression vector previously described ( Figure 12) .
  • CsCl gradient DNA of this derivative was transfected into CHO cells along with a pSV2 XGRPT selection plasmid.
  • Stable transfectants expressing the ovine IL-2R were screened by association with radiolabelled I 125 -IL-2. Eguilibrium binding assay of CHO transfectants
  • Transfected CHO cells were screened for IL-2R expression by their ability to bind radiolabelled IL-2.
  • the ovine IL-2R ⁇ chain cDNA isolated from the activated T cell cDNA library was found to be 2650 bp in length.
  • the sequence contained and open reading frame of approximately 825 bp encoding a protein of approximately 275 amino acids with a predicted molecular weight of 30,869 ( Figure 5A) .
  • CHO cells expressing the ovine IL-2R were produced and then assayed as described above. The results from these assays were then subjected to Scatchard analysis (Figure 28) to determine a Kd of 27 nM for binding of IL-2 to the transfected ovine IL-2 Receptor ⁇ chain.

Abstract

Procédé d'identification de séquences nucléotidiques codant pour un polypeptide présentant une activité spécifique de cytokine de ruminant ou de récepteur de cytokine ou d'un fragment de celui-ci, procédé qui consiste à fournir un vecteur comprenant une séquence d'ADN complémentaire dérivé de cellules de ruminant ou un extrait de cette séquence pouvant être exprimé dans un organisme unicellulaire; un organisme unicellulaire; et une sonde d'ADN pour une cytokine homologue; à introduire le vecteur dans ledit organisme unicellulaire; à cultiver l'organisme pour exprimer un polypeptide qui y est codé; à sonder l'organisme avec la sonde d'ADN; et à isoler un vecteur contenant une séquence d'ADNc codant pour un polypeptide présentant une activité spécifique de cytokine de ruminant ou de récepteur de cytokine.A method of identifying nucleotide sequences encoding a polypeptide having specific activity of ruminant cytokine or cytokine receptor or a fragment thereof, which method comprises providing a vector comprising a complementary DNA sequence derived from ruminant cells or an extract of this sequence which can be expressed in a unicellular organism; a single-celled organism; and a DNA probe for a homologous cytokine; introducing the vector into said unicellular organism; culturing the organism to express a polypeptide encoded therein; to probe the organism with the DNA probe; and isolating a vector containing a cDNA sequence encoding a polypeptide having specific activity of ruminant cytokine or cytokine receptor.

Description

CYTOKINE PRODUCTION The present invention relates to cytokine production, particularly ovine cytokine production and to pharmaceutical compositions including ovine cytokines. Cytokines are important polypeptides which display significant immuno-regulatory and inflammatory activities in animals. Whilst the ovine immune system has been extensively utilised in lymphocyte recirculation studies and as a large animal model for the study of immune responses to infectious diseases, little is known about either the production of cytokines by ovine leukocytes or the regulation of cellular function by cytokines.
Two important cytokines produced by macrophages are Interleukin-1 (IL-1) and Tumor Necrosis Factor α
(TNFα) . The pleiotropic bioactivities of these two cytokines are in many instances overlapping. The bioactivities ascribed to IL-1 are produced by two molecules (IL-lα and IL-lβ) encoded for by two distinct genes. The amino acid sequence of these two molecules is only 29% homologous, nevertheless, they bind to the same receptor and exert the same biological activity. As a mediator of the inflammatory response IL-1 induces secretion of acute phase proteins, stimulates release of prostaglandin E2 and proteolytic enzymes, and is chemotactic for neutrophils.
As a product of activated macrophages, TNFα also plays an important role in the inflammatory response. Bioactivities that overlap with IL-1 include mitogenicity for non-immune cells such as fibroblasts and endothelial cells and induction of secretion of prostaglandins, proteolytic enzymes and possibly acute phase proteins. TNFα serum levels are associated with onset of septic shock and, in chronic conditions, cachexia. Other important cytokines include Interleukin-6
(IL-6) and the Interleukin-2 (IL-2).
The production of interleukin-2 and expression of its high affinity receptor is essential for T cell proliferation and differentiation in the development of an immune response.
Interleukin-2 (IL-2) is produced by activated T cells. It induces the expression of a 55 kD Receptor protein (α chain) which, in association with a 75 kD protein (β chain), form the high affinity IL-2 Receptor. Both the β chain, which is constitutively expressed on T cells, and the α chain bind I -2 independently with intermediate and low affinity respectively although it is the association of IL-2 with its high affinity receptor which mediates the biological response.
Although the importance of IL-2 and its Receptor in humans and various other species has been demonstrated, its involvement in the ovine immune response to infection and disease is as yet uncharacterised. While studies have been conducted on various cytokines in the human and mouse systems, research has been slowed in ovine animals by the absense of coding sequences and means for producing large quantities of the desired proteins. Accordingly, it would be a significant advance in the art if nucleotide sequence data could be generated on the DNAs coding for, and the amino sequences of, proteins exhibiting ovine cytokine and/or cytokine receptor activity, as well as a method of making substantial and essentially pure quantities of such materials. This is of particular importance due to the potential application of cytokines such as in therapeutic agents (e.g. wound healing) and as adjuvants.
Accordingly, it is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties relating to the prior art.
Accordingly, in a first aspect of the present invention there is provided a process for identifying nucleotide sequences coding for a polypeptide exhibiting specific ruminant cytokine or cytokine receptor activity or a fragment thereof, which process includes providing a vector including a complementary DNA (cDNA) sequence derived from ruminant cells or an extract thereof capable of being expressed in a unicellular organism; a unicellular organism; and a DNA probe for a homologous cytokine; introducing the vector into the said unicellular organism; culturing the organism to express a polypeptide encoded therein; probing the organism with the DNA probe; and isolating a vector containing a cDNA sequence encoding for a polypeptide exhibiting specific ruminant cytokine or cytokine receptor activity.
Utilising the process according to this aspect of the present invention, a range of nucleotide sequences coding for polypeptides may be isolated which exhibit specific ruminant cytokine activity. Cytokines and cytokine receptors of interest include Interleukin-1 alpha (I -lα) Interleukin-1 beta (IL-lβ) Interleukin-1 Receptor (IL-1R) Interleukin-2 (IL-2)
Interleukin-2 Receptor (IL-2R) Interleukin-3 (IL-3) Interleukin-3 Receptor (IL-3R) Interleukin-4 (IL-4) Interleukin-4 Receptor (IL-4R) Interleukin-5 (IL-5) Interleukin-5 Receptor (IL-5R) Interleukin-6 (IL-6) Interleukin-6 Receptor (IL-6R) Interleukin-7 (I -7)
Interleukin-7 Receptor (IL-7R) Interleukin-8 (IL-8) Interleukin-8 Receptor (IL-8R) Interleukin-9 (IL-9) Interleukin-9 Receptor (IL-9R) Interleukin-10 (IL-10) Interleukin-10 Receptor (IL-10R) Interferon-gamma (IFN-γ) Interferon-gamma Receptor (IFN-γR) Tumor Necrosis Factor-alpha (TNF-α) Tumor Necrosis Factor-alpha Receptor (TNF-αR) Granulocyte Macrophage Colony Stimulating Factor (GMCSF) Granulocyte Macrophage Colony Stimulating Factor Receptor (GMCSFR)
Transforming Growth Factor-beta (TGF-β) Transforming Growth Factor-beta Receptor (TGF-βR) Of particular interest are IL-lα, IL-lβ, 11-2, IL-2R, IL-6, IFN-γ and TNFα. The cDNA sequences in the vector are derived from ruminant cells. The ruminant cells may be derived from ovine, caprine or bovine animals, preferably ovine animals. Sources of cytokine and cytokine specific receptor mRNA include macrophages, lymphocytes, fibroblasts, endothelial cells and liver cells. Accordingly, in a further aspect of the present invention the process for identifying nucleotide sequences coding for specific ruminant cytokines may include the preliminary steps of providing a source of ruminant macrophage or extract thereof; and a suitable cloning vector; isolating cytokine or cytokine-specific messenger RNA (mRNA) from the ruminant macrophage; treating the messenger RNA to produce complementary DNA (cDNA) ; and deploying the cDNA sequence into the cloning vector.
The source of ruminant macrophages may be alveolar macrophages. Alveolar macrophages may be isolated from ovine lung tissue. The lungs may be removed and flushed with, for example, a saline solution to remove cells lining the inner surface thereof.
The cells so isolated may be stimulated in vitro to enhance cytokine specific messenger RNA production. For example, the cells may be contacted with a lipopolysaccharide. mRNA may be isolated therefrom, for example using an oligo dT column. The isolated mRNA may be probed with a DNA probe for an equivalent homologous cytokine, for example an equivalent human cDNA probe. cDNA probes encoding human IL-lα and human IL-lβ and human TNFα were generously provided by Immunex Research and Development Corporation and Genentech Inc. of the United States, respectively.
In an alternative aspect of the present invention, the process for identifying nucleotide sequences coding for specific ruminant cytokines and cytokine receptors may include the preliminary step of providing a source of ruminant lymphocytes or extract thereof; and a suitable cloning vector; isolating cytokine or cytokine receptor-specific messenger RNA (mRNA) from the ruminant lymphocyte; treating the mRNA to produce complementary DNA (cDNA); and deploying the cDNA into the cloning vector. The source of ruminant lymphocytes may be ruminant lymph node cells. The lymph node cells may be extracted from popliteal nodes of, for example, normal sheep.
The cells so isolated may be stimulated in vitro to enhance cytokine receptor-specific messenger RNA production. For example, the cells may be stimulated with mitogen Concanavalin A (Con A) and then cultured in the presence of the specific cytokine for the receptor of interest. The production of complementary DNA from the messenger RNA may be undertaken in any suitable manner. A number of techniques are known per se in the art for this production. The commercial Amersham cDNA synthesis system may be used. The suitable cloning vector according to the present invention may be selected from plasmid and phage vectors. The bacteriophage virus vector λgtlO and the like have been found to be suitable. The complementary DNA (cDNA) sequence may be ligated into the phage λgtlO in any suitable manner. The commercial Arrtersham cDNA cloning kit may be used.
Utilising procedures known in the art per se, a representative cDNA library may be produced and then screened by hybridisation with DNA probes for homologous cytokines.
For example a representative ovine macrophage cDNA library was produced and then screened by hybridisation with the human cDNA probes for IL-lα, IL-lβ, TNFα and IL-6.
A number of positive clones were isolated: approximately 100 IL-lβ positives 6 IL-lα 5 TNFα 1 IL-6
The relative numbers of each is a reflection of the length of stimulation of the original macrophages. A shorter stimulation would be expected to increase the representation of IL-lα and TNFα clones. All the IL-lα, TNFα and IL-6 clones were further examined in addition to 9 of the IL-lβ isolates. They were found to contain a range of DNA inserts with the largest of each species as follows:
IL-lα - approx. 1780 bp IL-lβ - approx. 1430 bp
IL-6 - approx. 1100 bp TNFα - approx. 1690 bp Accordingly, in a further aspect of the present invention there is provided a DNA sequence coding for a polypeptide exhibiting ruminant cytokine or cytokine receptor activity, or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof.
The polypeptide preferably exhibits ovine cytokine or cytokine receptor activity. The DNA sequences may be modified in any suitable manner. The DNA sequences may be modified to improve expression of the polypeptide encoded thereby. The DNA sequence may be modified to modify the properties of the peptide encoded thereby. For example, pyrogenic or inflammatory characteristics of the peptide may be reduced or eliminated.
In a preferred aspect, fusion genes may be formed with gene sequences encoding an antigen. In a preferred aspect, there is provided a DNA sequence coding for a polypeptide exhibiting ovine interleukin-lα (IL-lα) activity and having a length of approximately 1781 base pairs (bp), or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof. The nucleotide sequence thereof may be illustrated as in Figure 1A.
The cDNA was found to be 1781 base pairs in length extending from the 5' untranslated region to a 32 bp poly A tail. This sequence contained an open reading frame of approximately 804 base pairs encoding a protein of approximately 268 amino acids with a predicted molecular weight of 30,953. Based on the sequence of human IL-lα, the amino terminal amino acid of mature ovine Il-lα is predicted to be the glutamine residue at nucleotide 407 (amino acid 119) giving a mature protein of 150 amino acids with a molecular weight of 17,230.
In a further preferred aspect, there is provided a DNA sequence coding for a polypeptide exhibiting ovine interleukin-lβ (IL-lβ) activity and having a length of approximately 1439 base pairs (bp), or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof. The nucleotide sequence thereof may be illustrated as in Figure 2A.
Ovine IL-lβ cDNA as shown in Figure 2 was found to be 1429 bp in length including an 11 bp poly A tail. The 3' region contains a polyadenylation signal and several ATTTA motifs. This sequence contained an open reading frame extending from approximately nucleotide 46 to nucleotide 846 encoding a protein of approximately 266 amino acids with a predicted molecular weight of 30,692. By comparison with human IL-lβ, the amino terminal amino acid of mature ovine IL-lβ would be expected to be the alanine residue at nucleotide 385 (amino acid 114) giving a protein of 153 amino acids with a molecular weight of 17 , 708 .
In a further preferred aspect, there is provided a DNA sequence coding for a polypeptide exhibiting ovine interleukin-6 (IL-6) activity and having a length of approximately 1103 base pairs (bp) , or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof. The nucleotide sequence thereof may be illustrated as in Figure 3A.
Ovine IL-6 cDNA as shown in Figure 3 was found to be 1103 bp in length including a 10 bp poly A tail. The 3* region contains a polyadenylation signal and six ATTTA motifs. This sequence contained an open reading frame of approximately 624 bp encoding a protein of approximately 208 amino acids with a predicted molecular weight of 23,448. Based on the sequence of human IL-6, the amino terminal amino acid of mature ovine IL-6 would be expected to be the proline residue at nucleotide 131 (amino acid 29) giving a protein of 180 amino acids with a molecular weight of 20,549. In a further preferred aspect, there is provided a DNA sequence coding for a polypeptide exhibiting ovine tumor necrosis factor α (TNFα) activity or a portion thereof and having a length of approximately 1675 base pairs (bp), or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof. The nucleotide sequence thereof may be illustrated as in Figure 4A.
The cDNA was found to be 1675 base pairs in length with a long (160 bp) 5* untranslated region and a 32 bp poly A tail. This sequence contained an open reading frame of approximately 702 base pairs encoding a protein of approximately 234 amino acids with a predicted molecular weight of 25,539. Based on the sequence of human TNFα, the amino terminal amino acid of mature ovine TNFα is predicted to be the leucine residue at nucleotide 392 (amino acid 78) giving a mature protein of 157 amino acids with a molecular weight of 17,242.
Utilising the PCR techniques and other procedures known in the art per se, it has been possible to clone the coding region of ovine interferon (IFN-γ).
In a still further aspect there is provided a DNA sequence coding for a polypeptide exhibiting ovine interferon-gamma (IFN-γ) activity or a portion thereof and having a length of approximately 553 base pairs (bp) or sequences substantially homologous therewith, derivatives thereof, mutants thereof, or fragments thereof. The nucleotide sequence thereof may be illustrated as in Figure 5D. This cDNA was found to be 553 bp in length and contains an open reading frame of approximately 498 bp encoding a precursor protein of approximately 166 amino acids with a predicted molecular weight of 17.5 kDa.
Utilising similar techniques as described above, it has been possible to clone in sequence the coding region of the IL-2 receptor α chain (55 kD) . Accordingly in an alternative aspect of the present invention there is provided a DNA sequence coding for a polypeptide exhibiting ovine interleukin-2 receptor α activity, and having a length of approximately 2650 bp, or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof. The nucleotide sequence may be illustrated as in Figure 5A.
The cDNA was found to be 2650 bp in length with a 245 bp 5' untranslated region and a very long (1477 bp) 3' untranslated region ending in a 12 bp poly A tail. This sequence contained an open reading frame of approximately 825 base pairs encoding a protein of approximately 275 amino acids with a predicted molecular weight of 30,869. The cytokine receptor DNA sequence so formed is important, since it is the up regulation of the α chain which occurs in the generation of an immune response to an infection in animal models.
Study of the IL-2 receptor α chain for example may permit design of synthetic drugs which may mimic the effect of cytokines.
Utilising the Polymerase Chain Reaction (PCR) technique and other procedures known in the art per se, it has been possible to clone the coding region of ovine interleukin 2. Accordingly, in a further preferred aspect there is provided a DNA sequence coding for a polypeptide exhibiting ovine interleukin-2 (IL-2) activity, and having a length of approximately 501 base pairs or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof. The nucleotide sequence thereof may be illustrated as in Figure 5B.
This cDNA was found to be 501 base pairs in length with an open reading frame of approximately 465 base pairs encoding a precursor protein of approximately 155 amino acids with a predicted molecular weight of 17,657. The mature protein of 135 residues has a predicted molecular weight of 15,542.
The DNA sequences discussed above may be utilised in the production of corresponding polypeptides. This may be accomplished in any suitable manner. Standard recombinant techniques may be used including the use of live vectors (e.g. vaccinia) with or without additional protective antigens injected or infected into suitable host species. Alternatively a recombinant expression vector including DNA squences may be injected directly into the tissue of suitable host animals and the peptide directly expressed.
Accordingly, there is provided a process for the production of a recombinant polypeptide having ovine cytokine or cytokine receptor activity which process includes the steps of providing a recombinant expression vector including a DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity, or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof, and capable of being replicated, transcribed and translated in a eukaryotic or prokaryotic organism; and an animal host; introducing said vector into said organism, such that the recombinant polypeptide encoded therein is expressed; and optionally isolating said polypeptide from said host.
As stated above, the recombinant expression vector may be injected directly into the tissue of the animal host. In the process according to this aspect of the present invention, preferably the animal host is a mamma1.
Alternatively, the DNA sequences may be transferred to a recombinant plasmid expression vector capable of being replicated, transcribed and translated in a unicellular organism. The vectors pGEM3Zf(+) and pGEM5Zf(+) have been found to be suitable.
In a preferred aspect of the present invention, there is provided a process for the production of a recombinant polypeptide having ovine cytokine or cytokine receptor activity which process includes the steps of providing a recombinant expression vector including a DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity or a fragment thereof, and capable of being replicated, transcribed and translated in a eukaryotic or prokaryotic organism; and a eukaryotic or prokaryotic organism; introducing said vector into said organism by transformation, transduction or transfection; culturing the resulting organism; expressing the recombinant polypeptide encoded by said DNA sequence; and isolating said polypeptide from the culture.
Preferably the DNA sequence codes for a polypeptide exhibiting ovine IL-lα, IL-lβ, IL-2, IL-2R, IL-6, TNFα or IFN-γ activity.
In the process according to the present invention the eukaryotic or prokaryotic organism is preferably a prokaryotic organism, for example a bacterial strain. The bacterial strain may be a strain of E.coli. The E.coli
IB392 strain has been found to be suitable.
The successful expression of eukaryotic polypeptides exhibiting cytokine or cytokine receptor activity in bacteria such as E.coli may require modification of the recombinant expression vector and/or the DNA sequences of interest to provide the various regulatory elements necessary for bacterial transcription and translation. For example the recombinant expression vector may contain a ribosome binding site within its promoter region, and a suitable restriction site for insertion of the DNA sequence distal to the ribosome binding site.
The recombinant expression vector used in this aspect of the present invention may be selected from the following, (a) Celltech vector This vector system utilises a dual origin of replication developed by Yarranton, G.T., Wright, E. , Robinson, M.K., and Humphreys, G.O. at Celltech, United Kingdom (Gene 2^:293-300). In this vector, plasmid copy number may be maintained at a low level under the control of a pSClOl origin of replication during growth of the host at 30°C or copy number may be increased dramatically by raising the culture temperature to 42°C which activates a second origin of replication under the control of the λ PR promoter and a thermolabile repressor. The presence of two origins of replication allow only neglible expression of foreign and potentially toxic proteins when cells are grown at 30°C however when cells have reached an appropriate biomass, the switch to 42°C allows high level expression and the formation of large amounts of protein. Transcription of inserted cDNAs is under the control of the E.coli trp promoter. The trp promoter/operator sequence promotes efficient binding of RNA polymerase and thus almost constitutive expression of the gene of interest. The gene for the trp repressor is not present in the vector therefore during high level expression where the plasmid copy number is greatly elevated, tryptophan in the growth media does not influence expression. (b) An autorepressed broad host-range expression vector - PMMB66EH
This vector contains a tac promoter region including the lac operator site just upstream of a polycloning site. The vector also contains the lacl gene coding for the lac repressor which in the presence of IPTG cannot bind the lac operator. Therefore transcription of inserted genes can be induced by the addition of IPTG to the cultures. The presence of strong transcription terminators downstream of the polylinker ensure that plasmid copy number is not affected by this induction.
Both vectors contain ribosome binding sites (RBS) within their promoter regions and DNA sequences of interest may be inserted into these vectors so that the
ATG initiation codon of the protein is correctly positioned relative to the RBS.
The Celltech vector used to express porcine growth hormone contains the pGH cDNA as a Bαlll/EcoRl insert with the ATG translation initiation codon just downstream of the Bαl II site. This places the ATG 14bp 3" of the vector RBS, a distance empirically determined by Celltech to be optimal for expression of various eukaryotic proteins. Similarly, the pMMB66EH vector requires the DNA sequence of interest to be inserted into an EcoRl or Smal site so that the ATG initiation codon lies within 10-14 bp of the vector RBS.
Cytokines of other species have been found in the prior art to be suitable for expression in bacteria since post translational events such as glycosylation are not required for biological activity.
Accordingly in a further aspect of the present invention there is provided a DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity including a deletion in the untranslated 5' region to remove a signal sequence for transport of the cytokine across the endoplasmic reticulum of a eukaryotic cell. Such features are not required for prokaryotic expression.
The DNA sequence according to this aspect of the present invention may further include a second DNA sequence containing an ATG initiation codon introduced in a suitable restriction site within the first DNA sequence.
The ATG initiation codon may be placed in front of the sequence coding for the mature protein.
Accordingly, in a preferred form in the process according to this aspect of the present invention, the DNA sequence coding for a polypeptide is modified to include an ATG initiation codon correctly positioned relative to a suitable restriction site.
Preferably the process according to the present invention may include the preliminary steps of providing a first DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity, or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof; a second DNA sequence containing an ATG initiation codon; and inserting the second DNA sequence into a suitable restriction site within the first DNA sequence such that the ATG initiation codon is placed in front of the sequence coding for the mature protein.
The 5* portion of the cytokine cDNAs contain an untranslated region plus a signal sequence for transport of the cytokine across the endoplasmic reticulum of the eukaryotic cell. Since these features are not required for prokaryotic expression this region of each cytokine cDNA may be deleted and an ATG initiation codon placed in front of the sequence coding for the mature protein. This ATG should also be correctly positioned relative to a suitable restriction site to facilitate cloning into the two different vectors so that the ATG is at the required distance from the RBS.
Accordingly the process may further include subjecting the first DNA sequence to a digestion step to remove a signal sequence in the untranslated 5' region for transport of the cytokine across the endoplasmic reticulum of a eukaryotic cell. The digestion step may be conducted utilising any suitable restriction enzyme.
Each cDNA may be modified in a different way: Accordingly, in an alternative aspect of the present invention the process may include the preliminary steps of providing a first DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity; and an oligonucleotide sequence duplicating the nucleotide sequence of the first DNA sequence from the amino terminal amino acid to a restriction site and containing an ATG codon at a suitable restriction site upsteam of the duplicated sequence; and subjecting the first DNA sequence to in vitro mutagenesis to produce an ATG codon and a suitable restriction site therein.
The cDNAs for IL-lβ, TNFα and IL-6 may be modified by inserting DNA fiagments synthesised as complementary oligonucleotides into restriction sites within the cDNA molecules. In the case of IL-lβ and TNFα these unique restriction sites may be generated by in vitro mutagenesis, changing the sequence to produce a restriction site without altering the amino acid sequence. In the case of IL-6 the cDNA already contains an appropriate site.
Oligonucleotide derived fragments duplicating the nucleotide sequence from the amino terminal amino acid to the restriction site and containing an ATG codon and Bql II site upstream of these sequences may then be inserted into this restriction site. This regenerates a protein with the correct amino acid sequence but the oligonucleotides are designed to alter the nucleotide sequence to include those codons commonly used in E.coli.
The cDNAs for IL-lα and IL-2 may be modified by oligonucleotide directed in vitro mutagenesis. The DNA upstream of the sequence coding for the mature proteins may be mutated to produce an ATG codon and a Bαl II site.
IL-lα cDNA sequence: CATTACAGCTTC CAG AGT AAC Gin Ser Asn Changed to:
CATT AGATCT ATG CAG AGT AAC Bαlll Met Gin Ser Asn site
II-2 cDNA sequence:
GTTGCAAACGGT GCA CCT ACT Ala Pro Thr Changed to:
GTTGC AGATCT ATG GCA CCT ACT Bαlll Met Ala Pro Thr site
Accordingly, in a still further aspect of the present invention, there is provided a recombinant polypeptide exhibiting ruminant, preferably ovine, cytokine or cytokine receptor activity; or fragment thereof in substantially pure form. The polypeptide exhibiting cytokine or cytokine receptor activity may be selected from IL-lα, IL-lβ, IL-1R, IL-2, IL-2R, IL-3, IL-3R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-7, IL-7R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-10R, IFN-γ, IFN-γR, TNF-α, TNF-αR, GMCSF, GMCSFR, TGF-β and TGF-βR, preferably IL-lα, IL-lβ, IL-2, IL-2R, IL-6, TNFα or IFN-γ. The recombinant polypeptide so formed may exhibit surprisingly good in vivo activity relative to the naturally occurring polypeptides, and may be used in the treatment of animals, for example as therapeutic agents and/or as adjuvants. Thus, according to a further aspect of the present invention, there is provided a veterinary composition including a recombinant polypeptide having ovine cytokine or cytokine receptor activity, or mimotope thereof, or fragment thereof preferably selected from IL-lα, IL-lβ, IL-1R, IL-2, IL-2R, IL-3, IL-3R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-7, IL-7R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-10R, IFN-γ, IFN-γR, TNF-α, TNF-αR, GMCSF, GMCSFR, TGF-β and TGF-βR, preferably IL-lα, IL-lβ, IL-2, IL-2R, IL-6 or TNFα, or mixtures thereof; and a carrier or excipient therefor for veterinary use.
The veterinary composition may function as a therapeutic agent for example in the treatment of trauma such as burns, wounds or the like, and/or may be utilised as an adjuvant or co-adjuvant in combination with a further active agent, for example a therapeutic agent.
Whilst the veterinary composition may be utilised in the treatment of ruminant animals, especially sheep, goats and cattle, it may also be utilised in the treatment of other animal species, in particular pigs, cats, dogs and horses.
The cytokines may be used to induce, enhance or modulate an immune response against an antigen. Accordingly, in a further aspect of the present invention there is provided a vaccine composition including an antigen against a disease of interest; and a non-toxic adjuvant including a recombinant polypeptide having ovine cytokine or cytokine receptor activity, or mimotopes, derivatives or fragments thereof.
The cytokine and antigen can be incorporated in any pharmaceutically acceptable vehicle with or without added co-adjuvants or immunostimulatory molecules.
The antigen may be derived from any source including viral, fungal, bacterial or parasite antigens, auto-immunity related antigens or tumor-associated antigens. The antigen may also be derived from plants, for example allergens and plant toxins.
Other applications may also include the elicitation of an immune response to stimulate or inhibit the stability or interaction of cellular modifiers, including hormones with their corresponding receptors or binding components. In this fashion, the immune response can be used to inhibit/enhance growth, reproduction, differentiation and overall performance. Alternatively, the quality of the immune response can be manipulated to optimize the desired protective response.
Utilisation of the recombinant polypeptide is particularly suitable where a site specific immune response is sought, for example in the mucosa, mammary gland, gut, mouth, skin or the like.
The recombinant polypeptide may be utilised as a non-toxic adjuvant in combination with a vaccine, for example to enhance vaccination against various diseases, including parasitic diseases.
These parasites include, for example, Haemonchus contortus, Trichostrongylus colubriformus and Ostertagia circumcincta (gastrointestinal nematodes), Bacteroides rodosus (foot rot), Lucilia cuprina (blowfly strike), Staph. aureus (mastitis) and C.ovis (cheesy gland). The cytokines may function as adjuvants for existing vaccines or new recombinant vaccines that are being developed.
For different diseases, different cytokines or cytokine receptors, or combinations thereof may be more suitable. Similarly, the route of delivery of the cytokine may vary for both different cytokines and different vaccine preparations.
The recombinant polypeptide may also be utilised as a non-toxic adjuvant in combination with a viral vector.
The recombinant cytokines may be utilised in receptor studies as discussed above. In this form the recombinant cytokine may be radiolabelled or linked to a detectable ligand or hapten. The ligand or hapten may be selected from biotin, fluorochromes, FITC and the like.
As cytokines act through receptors the effect of a cytokine may be mimicked or blocked utilising an agent that will bind the receptor. Such agents may be generated using both the cytokine and its specific receptor. Accordingly, in a still further aspect of the present invention, there is provided a monoclonal or polyclonal antibody against a recombinant polypeptide exhibiting ruminant, preferably ovine, cytokine or cytokine receptor activity; or a fragment thereof.
A receptor specific monoclonal antibody (MAb) , for example, may bind the receptor and either block the activity of a cytokine or alternatively mimmic its effect. Further, immunization of animals with monoclonal or polyclonal antibodies may have both agonistic and antagonistic effects. Thus immunization with the antibody may lead to upregulation of endogenous cytokine production enhancing vaccination.
In addition, circulating cytokine specific monoclonal or polyclonal antibodies may be utilised to
"mop up" excesses of undesirable cytokines. This may occur in stress situations, for example during transport or animals or during docking or castration of animals.
Further, where cytokines are in excess and exert a harmful effect in vivo, for example with IL-1 and TNFα, soluble receptors may be utilised to "mop up" circulating excess cytokine.
Alternatively, soluble receptors may function to enhance the activity of cytokines. For example, cytokine-receptor complexes may be formed, and used to generate monoclonal or polyclonal antibodies.
Accordingly, in a further aspect of the present invention there is provided a veterinary composition including a monoclonal or polyclonal antibody against a recombinant polypeptide exhibiting ruminant, preferaoly ovine, cytokine or cytokine receptor activity, or mimotopes thereof, or fragments thereof.
The compositions according to the invention may take any form suitable for administration including forms suitable for oral or parental (including implant) use. For oral administration the compositions may take the form of, for example solutions, syrups or suspensions e.g. in aqueous buffer, or solid compositions such as tablets or capsules, prepared by conventional means. For parental use, the compositions may for example take a form suitable for injection, such as a suspension, solution or emulsion in an aqueous or oily vehicle optionally containing formulatory agents such as suspending, stabilising, solubilising and/or dispersing agents. The aqueous or oily vehicle may include any other adjuvant known per se, for example aluminium hydroxide (Al(OH)_).
The present invention will now be more fully described with reference to the following examples. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above. In the Figures: FIGURE 1A
Nucleotide sequence and predicted amino acid sequence of ovine IL-lα. The presumed amino terminus
(Gin 119) of mature IL-lα is marked with an asterisk.
The 3* polyadenylation signal is overlined and the ATTTA motifs are underlined.
Figure IB
Nucleotide sequence and predicted amino acid sequence of ovine Il-lα modified for bacterial expression. The ATG initiation codon added in front of the amino terminal amino acid of the mature protein is underlined.
FIGURE 2A
Nucleotide sequence and predicted amino acid sequence of ovine IL-lβ. The presumed amino terminus (Ala 114) of mature IL-lβ is marked with an asterisk. The 3' polyadenylation signal is overlined and the ATTTA motifs are underlined.
Figure 2B
Nucleotide sequence and predicted amino acid sequence of ovine Il-lβ modified for bacterial expression. The ATG initiation codon added in front of the amino terminal amino acid of the mature protein and the bases changed for E. Coli codon usage are underlined.
The Hindlll site created by in vitro mutagenesis is marked. FIGURE 3A
Nucleotide sequence and predicted amino acid sequence of ovine IL-6. The presumed amino terminus (Pro 29) is marked with an asterisk. The 3* polyadenylation signal is overlined and the ATTTA motifs are underlined. Figure 3B
Nucleotide sequence and predicted amino acid sequence of ovine II-6 modified for bacterial expression. The ATG initiation codon added in front of the amino terminal amino acid of the mature protein is underlined. FIGURE 4A
Nucleotide sequence and predicted amino acid sequence of ovine TNFα. The presumed amino terminus (Leu 78) of mature TNFα is marked with an asterisk. The 3' polyadenylation signal is overlined and the ATTTA motifs are underlined. Figure 4B
Nucleotide sequence and predicted amino acid sequence of ovine TNFα modified for bacterial expression. The ATG initiation codon added in front of the amino terminal amino acid of the mature protein and the bases changed for E. coli codon usage are underlined. The Pml I site created by in vitro mutagenesis is marked. FIGURE 5A Nucleotide sequence and predicted amino acid sequence of ovine IL-2 receptor cDNA. FIGURE 5B
Nucleotide sequence and predicted amino acid sequence of ovine IL-2 cDNA obtained by PCR. Figure 5C
Nucleotide sequence and predicted amino acid sequence of ovine II-2 modified for bacterial expression. The ATG initiation codon added in front of the amino terminal amino acid of the mature protein is underlined. Figure 5D
Nucleotide sequence and predicted amino acid sequence of ovine IFNγcDNA obtained by PCR. FIGURE 6
Northern blot analysis of induction of Human IL-lα, IL-lβ , TNFα. FIGURE 7
NOB-1/CTLL IL-1 bioassay illustrating the kinetics of IL-1 secretion. FIGURE 8
Dose response curve for the secretion of IL-l(A) and TNFα(B) following stimulation with LPS using the NOB-1/CTLL bioassay. (C) Indicates relative RNA levels. FIGURE 9 Diagrammatic representation of the Fill expression vector used to express the ovine IL-1 cDNAs. The cDNA inserts used to construct the expression derivatives are shown, with the thick lines representing the coding regions. The full length derivatives contained the entire cDNA with the 3* untranslated regions intact. The truncated derivatives had most of the 3* untranslated regions including the ATTTA sequences deleted. FIGURE 10
Southern blot analysis of ovine genomic DNA with cDNA probes specific for IL-lα and IL-lβ. Genomic DNA was digested with Ba Hl (B), Hindlll (H) and EcoRl (E) . the molecular size markers are from Hindlll digested bacteriophage λ DNA. FIGURE 11 Northern blot analysis of induction of IL-lα and
IL-lβ mRNA by endotoxin and phorbol ester. 20 μg of total RNA extracted from alveolar macrophages cultured for 5 hours (lanes 1, 3, 5 and 7) or 20 hours (lanes 2, 4, 6 and 8) in the presence of RF10 alone (lanes 1 and 2) or RF10 supplemented with LPS (lanes 2 and 4), PMA (lanes 5 and 6) or PMA plus ionomycin (lanes 7 and 8) was hybridized with probes specific for IL-lα (A) or IL-lβ (C) . Autoradiographic exposure was for 24 hours. 28S ribosomal RNA is shown to indicate relative RNA loading (B and D) and the position of the 28S and 18S bands relative to the hybridizing band is also indicated. FIGURE 12
Diagrammatic representation of the Fill expression vector used to express the ovine TNFα cDNA. The cDNA inserts used to construct the expression derivatives are shown, with the thick lines representing the coding regions. The full length derivatives contained the entire cDNA with the 3' untranslated region intact. The truncated derivative had most of the 3' untranslated region including the ATTTA sequences deleted. FIGURE 13
Assay of COS-7 transfection supernatants for TNFα and IL-6 activity. Supernatants of COS-7 cells transfected with an expression vector containing the 3* truncated (B-E) or full length ( - ) TNFα cDNA were assayed for TNAα and IL-6 activity. Control supernatant (■-■) was from COS-7 cells transfected with a vector containing the truncated cDNA in the wrong orientation with respect to the vector promoter. FIGURE 14
Southern blot analysis of ovine genomic DNA using a cDNA probe specific for ovine TNFα. Genomic DNA was digested with BamHI (B) , Hindlll (H) and EcoRI (E) . The molecular size markers are from Hindlll digested bacteriophage λDNA. FIGURE 15
Northern blot analysis of induction of TNFα mRNA by LPS, PMA and ionomycin. 10 μg of total RNA extracted from alveolar macrophages cultured for 1 hour, 5 hours or 24 hours in the presence of medium (DM10) alone or DM10 supplemented with LPS, PMA, ionomycin, or PMA plus ionomycin was 1*. bridized with a probe specific for ovine TNFα. Autoradi;. raphic exposure was for 24 hours. 18S ribosomal RNA is shown to indicate relative RNA loading and the position of the 28S and 18S bands relative to the hybridizing band is also indicated. FIGURE 16
Assay of COS-7 transfection supernatants for IL-2 activity. Supernatants from two separate transfections (trans 1 and trans 2) with an expression vector containing the ovine IL-2 cDNA were assayed for IL-2 activity. Pooled supernatant from these two transfections, concentrated 15x using an Amicon centriprep 10 concentrator, was also assayed (15x (1+2)). Control levels of proliferation induced by medium alone (med) or
100 U/ml recombinant human IL-2 are also shown.
Supernatants from normal COS-7 cells did not induce proliferation of the CON A blasts.
FIGURE 17
Protein SDS-PAGE showing induction of proteins in a culture containing an expression vector carrying ovine
IL-lα. The arrow marks the induced recombinant IL-lα band.
FIGURE 18
IL-1 assay of a crude fraction from induced
Il-lα cultures showing the production of biologically active IL-1. Controls included recombinant human IL-1 and as a negative control, crude fractions from an induced culture carrying ovine IL-6.
FIGURE 19
Protein SDS-PAGE showing induction of proteins in a culture containing an expression vector carrying ovine IL-lβ. The arrow marks the induced recombinant IL-lβ band.
FIGURE 20
IL-1 assay of a crude fraction from induced Il-lβ cultures showing the production of biologically active
IL-1. Controls included recombinant human IL-1 and as a negative control, crude fractions from an induced culture carrying ovine IL-2.
FIGURE 21
Bovine thymocyte co-stimulation assay of E. coli derived ovine IL-lβ. 5 fold dilutions of a crude fraction of ovine IL-lβ with or without 3 μg/ml of PHA were used to stimulate proliferation of bovine thymocytes. Controls included thymocytes in medium alone and with PHA but no
IL-lβ.
FIGURE 22 Protein SDS-PAGE showing induction of proteins in a culture containing an expression vector carrying ovine
IL-2. The arrow marks the induced recombinant IL-2 band.
FIGURE 23
IL-2 assay of a crude fraction from induced IL-2 cultures showing the production of biologically active IL-2. Controls included Media alone (no activity) and lOOU/ml recombinant human IL-2. FIGURE 24 7TD1 assay of supernatants from transfections with ovine IL-6. A lOx concentrate of IL-6 transfection supernatant was assayed in the presence and absence of anti-human IL-6 Ab to block COS derived IL-6 activity. Controls included media alone and recombinant human IL-6 with and without Ab. FIGURE 25
Protein SDS-PAGE showing induction of proteins in a culture containing an expression vector carrying ovine IL-6. The arrow marks the induced recombinant IL-6 band. FIGURE 26
IL-6 assay of a crude fraction from induced IL-6 cultures showing the production of biologically active IL-6. Controls included Media alone (no activity) and lOOU/ml recombinant human IL-6. FIGURE 27
Assay of IFNγ COS-7 transfection supernatants using an ELISA based IFNγ assay. Controls included 2ng.ml recombinant bovine IFN and supernatant from a mock transfection. FIGURE 28
Scatchard analysis of CHO cells expressing ovine
IL-2R from equilibrium binding assays using i25l labelled IL-2.
EXAMPLE 1 MATERIALS AND METHODS
Isolation and Culture of Alveolar Macrophages
Lungs and trachea were removed intact from one-two year old merino ewes housed at the Department of Veterinary Science, University of Melbourne. Lungs were then flushed via the trachea with 400-800 ml of sterile phosphate buffered saline (PBS) . Cells were recovered from lung washings by centrifugation, and washed twice in RPMI-1640 supplemented with 10% v/v fetal calf serum, 2mM glutamine, and 100 U/ml pencillin and 0.1 mg/ml streptomycin (RF10). Cell populations were cultured in 80
2 cm tissue culture flasks in RF10 at 37°C with 5% C02 in air. After 1 hour cell monolayers were gently flushed with two changes of RF10 to remove any non-adherent cells. Where indicated macrophages were then stimulated with bacterial lipopolysaccharide at 10 μg/ml.
Macrophages were recovered for flow cytometric analysis and RNA preparation by trypsinisation.
Monoclonal Antibodies The monoclonal antibodies (mAbs) used in this study were directed against the ovine cell surface antigens CD4, CD8, T19, CD5, CD45, CD45R, MHC class I and II and ILA-24 and have been previously described. mAbs reactive with MHC class II molecules included 49.1 (all class II molecules), 42.20 (DRα specific), 38.27 (DQα specific) and 28.1 (DPα specific). mAbs against bovine rγlFN (IFN2 and IFN9) were the gift of Dr. P. Wood, CSIRO Division of Animal Health, Melbourne, and have also been previously described. Immunofluorescence Staining and FLow Cytometry
Single color immunofluorescence staining of macrophages was performed as described previously. Briefly, 1-2X10 cells were incubated with mAb as undiluted supernatant fluid (SNF) in microtitre trays. After washing, cells were reacted with a 1:30 dilution of FITC-conjugated F(ab)2 fragments of sheep anti-mouse immunoglobulin (Silenus, Melbourne). All reactions were performed at 4°C. All samples were fixed with 1% formaldehyde, 2% glucose in PBS and analysed on a fluorescent activated cell analyser (FACScan, Becton Dickinson, California) . Bioassavs
The NOB-1/CTLL assay for determination of IL-1 has been previously described. Briefly, NOB-1 cells were washed three times in RF10, resuspended at 2X10 /ml and 0.1 ml added to "V" bottom microtitre plates containing 0.1 ml of appropriately diluted test SNF. After incubation for 24 hours at 37°C, plates were centrifuged and 50 μl of SNF transferred to a replicate flat bottom microtitre plate together with 50 μl contai.ni.ng 5X103 CTLL cells.
For determination of proliferation CTLL were pulsed at 20
3 hours with H-thymidine (Amersham) and harvested two hours later. The actinomycin D treated WEHI-164 TNFα assay was performed as previously described.
DNA Probes cDNA probes encoding human IL-lα and IL-lβ and human TNFα were generously provided by Immunex Research and Development Corporation and Genentech, Inc. respectively. cDNA probes encoding β-tubulin or GAPDH were used to indicate relative levels of RNA. 32P-labelled probes were generated by random priming.
RNA Isolation and Northern Blot Analysis
For preparation of RNA, fresh or cultured macrophages were washed two times in PBS. Total RNA was then prepared from the cell pellet by acid phenol-guanidine thiocyanate extraction. RNA samples were fractionated by electrophoresis through denaturing formaldehyde-agarose gels and transferred to Hybond-N nylon membranes (Amersham) as previously described. Following overnight transfer RNA was fixed to membranes by UV cross-linking and hybridised to appropriate 32P-labelled probes. When using ovine probes blots were washed under conditions of high stringency (0.5 X SSC, 60°C) following hybridisation. For cross-species probes low stringency (1 X SSC, 50°C) conditions were employed.
THE CELL SURFACE PHENOTYPE OF OVINE ALVEOLAR MACROPHAGES
Alveolar macrophages were recovered from lung washings and subjected to immunofluorescent staining with a panel of mAbs specific for ovine leukocyte cell surface antigens. Results are presented in Table 1 and represent pooled data from at least three sheep. TABLE 1 PHENOTYPE OF OVINE ALVEOLAR MACROPHAGES Surface Marker Reactivity
CD4 CD5
CD8 T19
CD45 +++
CD45R OLAC 1 +++
OLAC 11 DR
>90% DP +++ heterogenous
DQ ILA-24 ++
Esterase +++
Macrophages stained negatively for the T cell surface antigens CD4, CD8, CD5 and T19. They also stained negatively for expression of ovine CD45R. All macrophages expressed CD45, or leukocyte common antigen, and all macrophages were MHC class I positive. Greater than 90% of macrophages stained positively, but in a' heterogenous fashion, with mAbs specific for the MHC class II molecules DRα, DPα and DQα. A similar pattern of staining was observed with mAb 49.1 which reacts with all ovine class II molecules. All alveolar macrophages were esterase positive.
This staining pattern was typical of that observed for tissue macrophages in other species. In the following experiments we examined the ability of bovine rγlFN to modulate this pattern of expression. CYTOKINE PRODUCTION BY LPS STIMULATED ALVEOLAR MACROPHAGES Kinetics The production of the cytokines IL-lα, IL-lβ, and TNFα was characterised by resting and LPS stimulated alveolar macrophages. At the molecular level, cross species homology permitted us to use the comparable human cDNA probes to detect specific mRNA. With regard to biological activity only the production of IL-1 was readily characterised due to the failure of ovine TNFα to function in cross species bioassays.
Macrophages were recovered from lung washings and cultured in RF10 alone or in RF10 supplemented with 10 μg/ml of LPS. Total RNA was extracted from these macrophages at various time intervals and also from freshly isolated cells. The RNA was electrophoresed, blotted to nylon membranes then hybridised with cDNA probes specific for human IL-lα, IL-lβ, TNFα or control GAPDH or β-tubulin probes. None of the cytokine specific probes hybridised with RNA from freshly isolated macrophages or macrophages cultured in medium alone. In contrast, message encoding all three appeared at various time points following stimulation with LPS (Figure 6) . The level of TNFα specific message peaked at 1 hour after initiation of culture and declined thereafter with only a trace being detected at 24 hours but nothing at 48 hours (Figure 6D) . IL-lα specific message was also detected after 1 hour of stimulation with a similar level observed after 5 hours culture. No mRNA encoding this cytokine was detected at 30 mins stimulation nor at 24 or 48 hours stimulation (Figure 6A) . In contrast to IL-lα message, mRNA encoding IL-lβ was not detected until after 5 hours of stimulation. A low level of message remained at 24 hours but this had disappeared by 48 hours (Figure 6B) .
Using the NOB-1/CTLL IL-1 bioassay we next determined the kinetics of IL-1 secretion into the surrounding medium. Macrophages were cultured in RF10 alone or in RF10 supplemented with 10 μg/ml LPS. SNF was sampled from these cultures after 1, 5, 24 and 48 hours. Results show that in the absence of exogenous stimulation small amounts of IL-1 were secreted into the SNF (Figure 7A) . No. IL-1 was detected after 1 hour culture. 5 units/ml was observed after 5 hours and the level peaked at approximately 80 units/ml after 24 hours. To ensure that secretion in the absence of stimulation did not affect determination of kinetics following specific stimulation we studied secretion of IL-1 by freshly isolated macrophages as well as macrophages that had been pre-cultured for 24 hours in RF10. Kinetics varied markedly for these two macrophage populations (Figures 7B and 7C) . In the presence of LPS the kinetics of IL-1 secretion by freshly isolated macrophages was similar to that observed for unstimulated macrophages, although the actual level of IL-1 secreted was increased by approximately 150 fold. A comparitively low level of IL-1 was detected after 5 hours of culture (320 units/ml) and this increased to approximately 10,000 units/ml after 24 hours (figure 2B) . In contrast, macrophages pre-cultured for 24 hours had secreted high levels of IL-1 within 5 hours of addition of LPS and this level increased only slightly over the next 19 hours. Dose Response
A dose response curve for the secretion of IL-1 following stimulation with LPS was determined using the NOB-1/CTLL bioassay. Macrophages were cultured in RF10 supplemented with 10 fold dilutions of LPS from 10 mg/ml - 0.1 pg/ml. SNF was sampled at 24 hours and assayed for IL-1. Results show that maximal IL-1 secretion was induced with concentrations of LPS greater than 10 ng/ml (Figure 8A) . At concentrations below this the level of IL-1 secretion decreased until at 0.1 pg/ml of LPS only background levels were detected. As this bioassay does not differentiate between IL-lα and IL-lβ a similar experiment was performed with RNA being extracted from macrophages stimulated with the different concentrations of LPS. The RNA was electrophoresed, blotted to a nylon membrane, and hybridised with cDNA probes specific for IL-lα and IL-lβ. The level of detectable mRNA declined with concentrations of LPS less than 10 pg/ml for both probes, implying a similar dose response for both IL-lα and IL-lβ. A similar blot to the one just described was used to determine the dose response for induction of TNFα by LPS. Results presented in Figure 8B show that the level of detectable TNFα specific message declined over the dose range tested. Production of IL-lα, IL-lβ and TNFα by alveolar macrophages freshly isolated or cultured in the presence or absence of LPS was examined. Using Northern blot analysis we failed to detect mRNA encoding these cytokines in total RNA from freshly isolated macrophages. Similarly, no specific mRNA was detected in total RNA from macrophages cultured in medium alone. Addition of LPS upregulated production of all three cytokines. With regard to IL-1 this was reflected by increased levels of specific mRNA as well as increa 3d extracellular bioactive protein. IL-lα mRNA was induced to detectable levels within 1 hour of stimulation, the level peaking at 5 hours with no mRNA remaining at 24 hours. In contrast IL-lβ mRNA was not detected until 5 hours post stimulation with low levels remaining at 24 hours. We have confirmed the dominance of IL-lβ message with time after stimulation during cDNA cloning of ovine IL-lα and IL-lβ. In a library prepared from pooled RNA extracted from macrophages stimulated with LPS for 5 and 16 hours the frequency of IL-lβ clones was 20 fold greater than that of IL-lα clones (or TNFα clones - results not shown) . In these macrophage populations both IL-1 mRNA's peaked at 4-6 hours and IL-lβ message increased as a proportion with time after stimulation.
The results presented herein demonstrate that functional capacity of ovine alveolar macrophages, and other ovine macrophage populations, is subject to the same regulatory mechanisms demonstrated for macrophage populations in other species. Exposure of macrophages to LPS led to a rapid upregulation in the synthesis of the cytokines IL-lα, IL-lβ and TNFα.
EXAMPLE 2 EXPRESSION OF RECOMBINANT IL-1 MATERIALS AND METHODS Cloning of ovine IL-lα and IL-lβ cDNAs Ovine alveolar macrophages were isolated as previously described and cultured in RPMI-1640 supplemented with 10% v/v fetal calf serum for 5 or 16 hours with 10 μg/ml LPS to induce expression of IL-1 specific mRNA. Messenger RNA was isolated via standard acid phenol- guanidine thiocyanate extraction and oligo dT cellulose purification. cDNA was synthesised, cloned into λgtlO, then packaged in vitro using Amersham cDNA synthesis and λgtlO cloning kits. Phage were then used to infect E.coli strain NM514. 100,000 independent plaques were transferred onto nylon filters and screened with 32p labelled human IL-lα and IL-lβ probes (Immunex Research and Development Corporation) . Positive plaques were purified and the EcoRI inserts cloned into pGEM03Zf(+) (Promega Corp.). DNA sequencing of exonuclease III deletion derivatives was performed using the dideoxy chain termination method. Expression of recombinant IL-1
The cDNAs for both IL-lα and IL-lβ were cloned into an expression vector containing the SV40 origin of replication, enhancer, and polyadenylation signal and a human metalothionine promoter hMTIIA (Figure 9). Two constructs for each gene were made, one containing the full length cDNA and a second with a deletion of the 3* untranslated region containing the ATTTA sequences. The EcoRI inserts of both IL-lα and IL-lβ containing the entire cDNA were cloned into the unique EcoRI site in the vector. The 3' deletion derivative of IL-lα was constructed from one of the original plaque isolates. This clone was found to contain an EcoRI insert of 1220bp extending from nucleotide 1 to nucleotide 1220. This was thought to have resulted from oligo dT priming at a run of A residues within the IL-lα mRNA during 1st strand cDNA synthesis. The IL-lβ 3' deletion derivative was cloned as a BamHI/Bglll fragment, covering nucleotides 1 to 985, into the unique BamHI site of the vector.
CsCl gradient DNA of each derivative, containing fragments in both orientations, were transfected into COS-7 cells by the DEAE dextran technique and the supernatants harvested 72 hours post transfection. These supernatants were then assayed for IL-1 activity. IL-1 bioassay
The NOB-1/CTLL assay for measuring IL-1 activity was performed as previously described. Briefly, NOB-1 cells were washed three times in RF10, resuspended at 2 x
10 /ml and 0.1 ml added to U bottom microtitre plates containing 0.1 ml of appropriately diluted transfection supernatant. After incubation for 24 hours at 37°C plates were centrifuged at 1000 rpm and 50 μl transferred to a replicate flat bottom microtitre plate together with 50
3 μl containing 5 x 10 CTL cells. For determination of proliferation, CTL were pulsed at 20 hours with 3 H-thymidine and harvested 4 hours later. All test samples were assayed in duplicate.
Northern blot analysis
For preparations of RNA, fresh or cultured macrophages were washed twice in phosphate buffered saline.
Total RNA was then prepared from the cell pellet by acid phenol-guanidine thiocyanate extraction. Samples were electrophoresed in denaturing formaldehyde-agarose gels and transferred to nylon membranes as previously described. Filters were hybridised to 32P labelled ovine IL-1 cDNA probes and washed under conditions of high stringency (0.5xSSC, 60°C).
Genomic DNA analysis
Genomic DNA was isolated from sheep peripheral blood leukocytes via standard techniques. 10 μg of DNA was digested with the appropriate enzyme, electrophoresed on a 0.7% agarose gel, transferred to a nylon membrane and hybridised with 32P labellleedd oovviinne cDNA probes. RESULTS
Ovine IL-lα cDNA sequence
A previous study had shown that LPS stimulated ovine alveolar macrophages produced IL-1 specific messenger
RNA. Therefore an LPS stimulated ovine alveolar macrophage cDNA library was constructed as described in the Materials and Methods and screened with a 32P labelled human
IL-lα cDNA probe. A number of positive plaques were identified and their EcoRI inserts cloned into pGEM-3Zf(+).
Using exonuclease III deletion derivative the nucleotide sequence of ovine IL-lα was determined and is shown in
Figure 1. The cDNA was found to be 1781 base pairs in length extending from the 5' untranslated region to a 32 bp poly A tail. This sequence contained an open reading frame of 804 base pairs encoding a protein of 268 amino acids with a predicted molecular weight of 30,953. Based on the sequence of human IL-lα, the amino terminal amino acid of mature ovine IL-lα is predicted to be the glutamine residue at nucleotide 407 (amino acid 119) giving a mature protein of 150 amino acids with a molecular weight of 17,230.
The 3* untranslated region contains an AATAAA polyadenylation signal and includes six copies of the ATTTA motif common to other cytokine cDNAs. These ATTTA motifs are thought to affect mRNA stability. Ovine IL-lβ cDNA sequence
Screening the ovine alveolar macrophage cDNA library with a 32P labelled human IL-lβ cDNA probe resulted in the isolation of a large number of positive plaques. Two full length isolates were subcloned into pGEM-3Zf(+) and their sequences determined. Ovine IL-lβ cDNA as shown in Figure 2 was found to be 1429 bp in length including an llbp poly A tail. The 3' region contains a polyadenylation signal and several ATTTA motifs. An open reading frame extending from nucleotide 46 to nucleotide 846 was found to encode a protein of 266 amino acids with a predicted molecular weight of 30,692. By comparison with human IL-lβ (6), the amino terminal amino acid of mature ovine IL-lβ would be expected to be the alanine residue at nucleotide 385 (amino acid 114) giving a protein of 153 amino acids with a molecular weight of 17,708. Mammalian Expression of recombinant ovine IL-lα and IL-lβ
To test that the cloned cDNAs actually encode proteins with IL-1 activity, DNA fragments from pGEM-3Zf(+) derivatives were inserted into a mammalian expression vector as described in Materials and Methods. Previous studies had shown that the AT rich sequences found in the 3' untranslated regions of many cytokine and oncogene cDNAs were involved in RNA stability. These sequences are also present in the ovine genes. To examine the effects of these ATTTA motifs on the levels of IL-1 expression, plasmids with and without the 3' portion of the cDNAs containing these sequences were constructed. Controls were transfections with plasmids in which the gene fragments were inserted in the wrong orientation with respect to the vector promoter.
Plasmid DNA was transfected into COS cells and the supernatant harvested 73 hours after transfection. These supernatants were then assayed for IL-1 activity and the results from a single experiment are shown in Table 2. TABLE 2
* the background level of IL-2 produced by culture of NOB-1 cells in medium alone was 2669 ± 399 for the IL-lβ transfection supernatant assay and 463 ± 179 for the
IL-lα transfection SNF assay. The level of IL-1 detected after transfection with control full length or truncated cDNAs for both IL-lα and IL-lβ was not significantly greater than this.
Similar results were observed in at least three separate transfections (data not shown) .
From these results it can be seen that the cloned cDNAs did produce biologically active IL-1 and that the level of expression was higher in derivatives lacking the
3' portion of the genes.
Bacterial Expression of Ovine IL-lα
The cDNA encoding ovine IL-lα was modified in vitro to place a Bql II restriction site and an ATG initiation codon proximal to the amino terminus of the mature polypeptide. This was achieved using in vitro mutagenesis to alter the DNA sequence from nucleotides 399 to 406 (Figure 1A) to produce the desired sequence.
IL-lα cDNA sequence: CATTACAGCTTC CAG AGT AAC Gin Ser Asn
Changed to:
CATT AGATCT ATG CAG AGT AAC Bglll Met Gin Ser Asn site
The modified sequence was transferred as a
Bglll/EcoRI fragment (Figure IB) into the previously described Celltech vector and then transformed into the E.coli host strain IB392. Cultures were grown in minimal medium at 30°C until they reached an OD600 of 1.00. They were induced at 42°C for 20 mins. and then grown at 38°C for 5 hours to allow protein expression.
Protein induction was monitored by SDS-PAGE (see Figure 17) and crude extracts were then assayed for IL-1 activity. The protein gel shows good induction of a protein band running just below the 21,500 MW marker, the size expected for recombinant ovine IL-lα. An IL-1 assay of a crude extract from this induced culture showed the presence of biologically active IL-1 (Figure 18) . Bacterial Expression of Ovine IL-lβ The cDNA encoding ovine IL-lβ was modified in vitro to place a Bglll restriction site and an ATG initiation codon proximal to the amino terminus of the mature polypeptide. This was achieved by changing the DNA sequence at nucleotides 411 and 414 (Figure 2A) using in vitro mutagenesis to produce a Hindlll rsetriction site whilst still maintaining the correct amino acid sequence. A double stranded oligonucleotide constructed in vitro was then cloned into this site to duplicate the IL-lβ amino acid sequence from nucleotides 385 to 414. In addition, this oligonucleotide introduced a Bglll site and an ATG triplet in front of this sequence. Other bases were altered within this oligonucleotide to provide codons commonly used by E.coli. These changes, depicted in Figure 2B, did not alter the amino acid sequence.
The modified sequence was then transferred as a
Bqlll/EcoRI fragment into the previously described
Celltech vector and then transformed into the E.coli host strain IB392. Cultures were grown in minimal medium at 30°C until they reached an OD600 of 1.00. They were induced at 42°C for 20 mins, and then grown at 38°C for 5 hours to allow protein expression.
Protein induction was monitored by SDS-PAGE (see
Figure 19) and crude extracts were they assayed for IL-1 activity. The protein gel shows good induction of a protein band running just below the 21,500 MW marker, the size expected for recombinant ovine IL-lβ. An IL-1 assay of a crude extract from this induced culture showed the presence of biologically active IL-1 (Figure 20). Demonstration of the biological activity of ovine IL-lβ on bovine cells
To demonstrate the biological activity of ovine cytokines across species (i.e. in the bovine) E.coli derived recombinant ovine IL-lβ was assayed in a bovine thymocyte co-stimulation assay. Briefly, a single cell suspension was prepared from the thymus of a 3 month old calf using standard procedures. The thymocytes were then cultured in 200 μl (in flat bottom microtitre plates) of
RF10 supplemented with 3 μg/ml of phytoaemaggulutinin and the indicated dilutions of the ovine IL-lβ. Controls were thymocytes cultured in medium alone, PHA alone (3 μg/ml) and IL-lβ alone (indicated concentrations). Proliferation
3 was measured at 48 hrs after pulsing with H-thymidine for the final 4 hrs. Results presented in Figure 21 clearly demonstrate that the ovine IL-lβ with PHA was able to co-stimulate the proliferation of bovine thymocytes.
Thus ovine IL-lβ would be expected to exert biological activity on all bovine cell populations responsive to IL-1. Southern blot analysis of ovine IL-1 genomic DNA
Southern blots of ovine genomic DNA using IL-lα and IL-lβ cDNA probes (Figure 10) revealed restriction fragment sizes unique for each gene. IL-lα showed hybridizing bands at 4.3kb following Hindlll digestion, 3kb following EcoRI digestion and 8kb plus one larger than lOkb following BamHI digestion. Probing with IL-lβ resulted in a number of bands for Hindlll (8.5kb, 7kb, 6kb) , 2 bands for EcoRI (6kb and 3kb) and a single large band ( lOkb) for BamHI. These results suggest that IL-lα and IL-lβ probably exist as single copy genes in the ovine genome. IL-1 mRNA expression
Using Northern blot analysis and the cDNA probes described above we next examined induction of transcription of IL-lα and IL-lβ in ovine alveolar macrophages.
Macrophages from three sheep were recovered from lung washings and cultured independently in RF10 alone or in RF10 supplemented with either LPS, PMA or PMA plus ionomycin at the concentrations described in Materials and Methods. Total RNA was extracted from these macrophages at 5 hours and 20 hours post stimulation. The RNA was electrophoresed, blotted to nylon membranes and then hybridised with ovine IL-lα or IL-lβ cDNA probes. The results shown in Figure 5 are from one animal, however no differences in expression were detected between the three sheep examined. Without exogenous stimulation a low level of mRNA encoding both IL-lα and IL-lβ was detected at 4 hours but not 20 hours. For IL-lα addition of LPS, PMA and PMA plus ionomycin increased the level of mRNA detected at 4 hours. In addition, stimulation with both LPS and PMA plus ionomycin (but not PMA alone) led to the detection of mRNA at 20 hours post stimulation. For IL-lβ a different pattern emerged. Stimulation with LPS led to high levels of mRNA at both 5 hours and 20 hours. In contrast, neither PMA nor PMA plus ionomycin induced levels of IL-lβ mRNA above background at 4 hours. Both however induced high levels of IL-lβ mRNA by 20 hours post stimulation. These results serve to demonstrate differential regulation of transcription of IL-lα and IL-lβ.
EXAMPLE 3 EXPRESSION OF RECOMBINANT TNFα MATERIALS AND METHODS
Cloning of Ovine TNFα cDNA
The cDNA library previously described was screened
32 with a P labelled human TNFα probe. Positive plaques were purified and the EcoRI inserts from a number of isolates cloned into pGEM-3Zf(+) (Promega Corp.). DNA sequencing of exonuclease III deletion derivatives was performed using the dideoxy chain termination method.
Mammalian Expression of Recombinant TNFα
TNFα cDNA was cloned into the Fill expression vector previously described (Figure 12) . Two constructs were made, one containing the full length cDNA and a second with a deletion of the 3' untranslated region containing the ATTTA sequences. The 1675 bp EcoRI insert containing the entire TNFα cDNA was cloned into the unique EcoRI site of the vector. The 3' deletion derivative of TNFα was constructed by cloning a 1050 bp BamHI/Bglll fragment covering nucleotides 139 to 1189 into the unique BamHI site of the vector.
CsCl gradient DNA of each derivative, containing fragments in both orientations, were transfected into COS-7 cells by the DEAE dextran technique and the supernatants harvested 72 hours post transfection. For determination of IL-6 activity transfected cells were cultured in DM10 and supernatants assayed neat. For determination of the TNFα activity DM10 was replaced at 24 hours with the same volume of serum free Opti-mem media (GIBCO Laboratories) . The supernatant recovered 48 hours later was concentrated 15X using Centriprep 10 concentrators (Amicon Corp.) and stored frozen prior to assay. IL-6 Bioassay
The urine IL-6 dependent hybridoma line 7TD1 was used to assay IL-6 levels in transfection supernatants. 7TD1 cells, cultured in DM10 supplemented with 20 U/ml of recombinant human IL-6 (Boehringer Mannheim), were washed three times in DM10 without IL-6 and resuspended at
5 1x10 cells/ml. 100 μl of the cell suspension was then added to flat bottom microtitre wells containing 100 μl of appropriately diluted test supernatant. 7TD1 metabolic activity was measured after 72 hours incubation at 37°C.
100 μl was removed from each test well and 10 μl of a
10 mg/ml solution of MTT (Sigma Chemical Co.) in saline added. Following a further 4 hours at 37°C 100 μl of a solution consisting of 20% w/v SDS in 50% v/v dimethyl formamide was added to dissolve crystals and the optical density determined 24 hours later using a Titertek Multiscan plate reader (filters at 540 nm and 690 nm) . Activity induced by exogenous IL-6 was determined by subtracting from the test well value the level observed with 7TD1 cells cultured in DM10 alone. All test samples were assayed in duplicate and standard errors, which are not shown, were always less than 10%. TNFα Bioassay
The standard TNFα bioassay using actinomycin D treated WEHI-164 cells was used to measure TNFα activity in transfection supernatants. Briefly, WEHI-164 cells cultured in DM10 were washed three times in DM10 and
5 resuspended at 8x10 cells/ml in DM10 supplemented with
2 μg/ml of actinomycin D (Sigma Chemical Co.). 50 μl of this suspension was then added to 50 μl of appropriately diluted test supernatant in flat bottom microtitre plates. After incubation for 24 hours at 37°C cell death was determined using MTT as described above.
All test samples were assayed in duplicate with standard errors, which are not shown, always less than 10%. The % cytolysis was calculated from the formula 100(a-b)/a where a and b are respectively the mean adsorbances of duplicate wells without or with the test sample.
Northern Blot Analysis For preparations of RNA, fresh or cultured macrophages were washed twice in phosphate buffered saline. Total RNA was then prepared from the cell pellet by acid phenol-guanidine thiocyanate extraction. Samples were electrophoresed in denaturing formaldehyde-agarose gels and transferred to nylon membranes as previously described. Filters were hybridised to a 32P labelled ovine TNFα cDNA probe and washed under conditions of high stringency (0.5xSSC, 60°C). Genomic DNA Analysis
Genomic DNA was isolated from sheep peripheral blood leukocytes via standard techniques. 10 μg of DNA was digested with the appropriate enzyme, electrophoresed on a 0.7% agarose gel, transferred to a nylon membrane and
32 hybridised with a P labeaillleedd oovvine TNFα cDNA probe.
RESULTS
Ovine TNFα cDNA sequence
A previous study had shown that LPS stimulated ovine alveolar macrophages produced TNFα specific messenger RNA. Therefore an LPS stimulated ovine alveolar macrophage cDNA library was constructed as described in
32 the Materials and Methods and screened with a P labelled human TNFα cDNA probe. A number of positive plaques were identified and their EcoRI inserts cloned into pGEM-3Zf(+). Using exonuclease III deletion derivatives the nucleotide sequences of two of these ovine TNFα cDNA isolates were determined and found to be identical (Figure 4). The cDNA was found to be 1675 base pairs in length with a long (160 bp) 5' untranslated region and a 32 bp poly A tail. This sequence contained an open reading frame of 702 base pairs encoding a protein of 234 amino acids with a predicted molecular weight of 25,539. Based on the sequence of human TNFα, the amino terminal amino acid of mature ovine TNFα is predicted to be the leucine residue at nucleotide 392 (amino acid 78) giving a mature protein of 157 amino acids with a molecular weight of 17,242.
The 3' untranslated region contains an AATAAA polyadenylation signal and includes nine copies of the ATTTA motif common" to other cytokine cDNAs. These ATTTA motifs are thought to affect mRNA stability. Ma malian Expression of Recombinant Ovine TNFα
To test that the cloned TNFα cDNA actually encoded a protein with TNF activity, DNA fragments from pGEM-3Zf(+) derivatives were inserted into a mammalian expression vector as described in Materials and Methods. Previous studies have suggested that the AT rich sequences found in the 3* untranslated regions of many cytokine and oncogene cDNAs may be involved in RNA stability. These sequences are also present in ovine TNFα. To examine the effects of these ATTTA motifs on the levels of TNAα expression, plasmids with and without the 3' portion of the cDNA containing these sequences were constructed. Controls were transfections with plasmids in which the gene fragments were inserted in the wrong orientation with respect to the vector promoter.
Plasmid DNA was transfected into COS cells and the supernatants harvested 72 hours after transfection. These supernatants were then assayed for both TNFα and IL-6 activity. Synthesis and secretion of IL-6 is induced in many cells by TNFα and it was thought that measurement of COS cell derived IL-6 induced by the ovine TNFα might prove more sensitive than the direct assay of TNFα activity. Results of experiments using each assay are shown in Figure 13. Similar results were observed in at least two separate transfections (data not shown) .
From these results it can be seen that the cloned cDNAs did produce biologically active TNFα and that the level of expression was higher in the derivative lacking the 3' portion of the gene.
Production of a Modified TNFα cDNA for Bacterial Expression
The cDNA encoding ovine TNFα was modified in vitro to place a Bglll restriction site and an ATG initiation codon proximal to the amino terminus of the mature polypeptide. This was achieved by changing the DNA sequence at nucleotide 438 (Figure 4A) using in vitro mutagenesis to produce a Pmll restriction site whilst still maintaining the correct amino acid sequence. A double stranded oligonucleotide constructed in vitro ws then cloned into this site to duplicate the TNFα amino acid sequence from nucleotides 391 to 439. In addition, this oligonucleotide introduced a Bglll site and an ATG triplet in front of this sequence. Other bases were altered within this oligonucleotide to provide codons commonly used by E.coli. These changes, depicted in Figure 4B, did not alter the amino acid sequence. Southern Blot Analysis of Ovine Genomic DNA
Southern blots of ovine genomic DNA using an ovine TNFα cDNA probe (Figure 14) revealed hybridising bands of 2.2kb and l.δkb following Hindlll digestion, 4kb following EcoRI digestion and 5.8kb following BamHI digestion. A second large band in the BamHI digested DNA greater than 20kb may reflect a large BamHI fragment carrying a portion of the TNFα gene or may result from incomplete BamHI digestion. These results suggest that TNFα exists as a single copy gene in the ovine genome. TNFα mRNA Expression
Using Northern blot analysis and the cDNA probe described above we next examined induction of transcription of TNFα in ovine alveolar macrophages.
Macrophages were recovered from lung washings and cultured in DM10 alone or in DM10 supplemented with either
LPS, PMA, ionomycin or PMA plus ionomycin at the concentrations described in Materials and Methods. Total
RNA was extracted from these macrophages at 4 hour, 5 hours and 24 hours post stimulation. The RNA was electrophoresed, blotted to nylon membranes and then hybridised with the ovine TNFα cDNA probe. The results shown in Figure 15 are from one animal, however no differences in expression were detected in other sheep examined. Without exogenous stimulation a low level of mRNA encoding TNFα was detected at 1 hour but not 5 hours or 24 hours post stimulation. Addition of LPS, PMA or PMA plus ionomycin altered this pattern of expression. For LPS, increased mRNA encoding TNFα was detected at 1 hour and 5 hours post stimulation but not at 24 hours. PMA alone led to an increase in TNFα mRNA detected at 5 hours while the combination of PMA and ionomycin increased the level :
TNFα mRNA detected at all time points tested. Ionomyc.n alone did not increase the level of TNFα mRNA detected at any of the time points examined. EXAMPLE 4 CLONING AND EXPRESSION OF OVINE IL-2 MATERIALS AND RESULTS Isolation of an ovine IL-2 cDNA using PCR Lymph node cells from a normal sheep were cultured in vitro with Con A for 72 hrs and then cultured in the presence of IL-2 for a further 72 hrs. Messenger RNA isolated from these cells was then used to produce a pool of complementary DNAs using standard techniques involving Reverse Transcriptase and oligo dT primer. Oligonucleotide primers (based on the bovine and ovine IL-2 cDNA sequences) were used in a PCR reaction to amplify from the pool of cDNA molecules, the cDNA corresponding to ovine IL-2. These oligonucleotide primers (shown below) were designed to bind sequences upstream of the initiation codon (5* primer) and downstream of the termination codon (3' primer) to amplify a fragment of ovine IL-2 covering the entire protein coding region.
5' primer (derived from the bovine sequence) -
5* - 3' (GGTTGGATCC)TCAACTCCTGCCACAATG
3' primer (derived from the ovine sequence) - 3' - 5'
TAACTGTGTGCTTCTCAT(GAATTCGGTA)
These oligonucleotides are flanked by restriction enzyme recognition sequences (shown in brackets) to facilitate cloning of this 520 bp fragment into a suitable vector such as pGEM3Zf(+) .
The sequence of the ovine IL-2 cDNA obtained by PCR is shown in Figure 5B. Mammalian cell expression of ovine IL-2 The ovine IL-2 cDNA fragment generated by PCR was then cloned into the Fill expression vector previously used to express IL-lα, IL-lβ and TNFα. Plasmid DNA was transfected into COS-7 cells using DEAE-Dextran and supernatants were harvested after 72 hrs. Supernatants from two separate transfections plus a 15x concentrate of pooled supernatant obtained from these two transfections were assayed for IL-2 activity by their ability to induce proliferation of ovine Con A blasts. Briefly, lymph node cells cultured in 5 μg/ml of Concanavalin A for 72 hours
(Con A blasts) were cultured with 200 μl of an appropriate dilution of test sample. IL-2 dependant
3 proliferation was measured by pulsing with H-thymidine for 4 hrs at 68 hrs. These results (Figure 16) demonstrate that COS cells transfected with an expression vector carrying the ovine IL-2 cDNA fragment produce biologically active ovine IL-2. Bacterial Expression of Ovine IL-2 The cDNA encoding ovine IL-2 was modified in vitro to place a Bql II restriction site and an ATG initiation codon proximal to the amino terminus of the mature polypeptide. This was achieved using in vitro mutagenesis to alter the DNA sequence from nucleotides 89 to 95 (Figure 5B) to produce the desired sequence.
IL-2 cDNA sequence: GTTGCAAACGGT GCA CCT ACT Ala Pro Thr
Changed to:
GTTGC AGATCT ATG GCA CCT ACT
Bglll Met Ala Pro Thr site
The modified sequence was then transferred as a Bqlll/EcoRl fragment (Figure 5C) into the previously described Celltech vector and then transformed into the E.coli host strain IB392. Cultures were grown in minimal medium at 30°C until they reached an OD600 of 1.00. They were induced at 42°C for 20 mins. and then grown at 38°C for 5 hours to allow protein expression.
Protein induction was monitored by SDS-PAGE (see Figure 22) and crude extracts were then assayed for IL-2 activity. The protein gel shows good induction of a protein band running just above the 14,400 MW marker, the size expected for recombinant ovine IL-2. An IL-2 assay of a crude extract from this induced culture showed the presence of biologically active IL-2 (Figure 23).
EXAMPLE 5 CLONING AND EXPRESSION OF RECOMBINANT IL-6 Materials and Methods Cloning of ovine IL-6 cDNA The cDNA library described in the previous examples was screened with a 32P labelled human IL-6 probe. A positive plaque was purified and the EcoRI insert cloned into pGEM-3ZF(+) and M13. DNA sequencing of exonuclease III deletion derivatives was performed using the dideoxy chain termination method.
Mammalian Expression of Recombinant IL-6
A 3 ' deletion derivative of the ovine IL-6 cDNA was cloned into the Fill expression vector previously described (Figure 12) . The deletion derivative of IL-6 was constructed by cloning a 686 bp Sacl-Bsml fragment covering nucleotides 18 to 703 into the Smal site of the vector.
CsCl gradient DNA of the derivative, containing fragments in both orientations, was transfected into COS-7 cells by the DEAE dextran technique and supernatants harvested 72 hrs later. DM10 media was replaced at 24 hrs with the same volume serum free Opti-mem media. The supernatant recovered 48 hrs later was concentrated 10X using a centriprep 10 concentrator and stored frozen prior to assay.
IL-6 Bioassay
Was performed as described in Example 3 using the 7TD1 cell line. Recombinant human IL-6 was supplied by Boehringer Mannheim and polyclonal anti-human IL-6 antibodies by British Biotechnology.
RESULTS Ovine IL-6 cDNA Sequence
The ovine IL-6 cDNA isolated from the LPS stimulated alveolar macrophage cDNA library was found to be 1103 bp in length. The sequence contained an open reading frame of approximately 624 bp encoding a protein of approximately 208 amino acids with a predicted molecular weight of 23,448 (Figure 3A) . Based on the sequence of human IL-6, the amino terminal amino acid of mature ovine IL-6 would be expected to be the proline residue at nucleotide 131 (amino acid 29) giving a protein of 180 amino acids with a molecular weight of 20,549. Expression of Recombinant Ovine IL-6 To ensure that the cloned ovine IL-6 cDNA actually encoded a protein with IL-6 activity a 3' deletion derived was cloned into the Fill mammalian expression vector as described above. The deletion derivative lacked the non-coding ATTTA motifs shown previously to be involved in mRNA instability.
Plasmid DNA was transfected into COS cells and the supernatants (Opti. mem) harvested and concentrated 10 x 72hrs later. This SNF was then assayed for IL-6 activity. COS-7 cells produce Simian IL-6 which is also active in the 7TD1 IL-6 assay. To overcome this problem the transfection supernatants were assayed in the presence of 10 μg/ml of polyclonal anti-human IL-6 Ab. Simian IL-6 is closely related to human IL-6 and is therefore, unlike the ovine IL-6, neutralised by this polyclonal anti-serum. Results presented in Figure 24 demonstrate that even in the presence of anti-human IL-6 Ab the transfection supernatant was able to stimulate the proliferation of 7TD1 cells thus demonstrating the presence of recombinant ovine IL-6. Bacterial Expression of Ovine IL-6
The cDNA encoding ovine IL-6 was modified in vitro to place a Bglll restriction site and an ATG initiation codon proximal to the amino terminus of the mature polypeptide. This was achieved by cloning a short oligonucleotide containing a Bglll site and an ATG into a Smal site (nucleotide 132, Figure 3A) spanning the amino terminus of the mature protein.
The modified sequence was then transferred as a Bglll/EcoRl fragment (Figure 3B) into the previously described Celltech vector and then transformed into the E.coli host strain IB392. Cultures were grown in minimal medium at 30°C until they reached an OD600 of 1.00. They were induced at 42°C for 20 mins. and then grown at 38°C for 5 hours to allow protein expression.
A protein gel of induced and uninduced cultures shows induction of a band running at a molecular weight corresponding to the expected MW of recombinant ovine IL-6 (Figure 25) . An IL-6 assay of a crude extract from this induced culture showed the presence of biologically active ovine IL-6 (Figure 26) .
EXAMPLE 6
CLONING AND EXPRESSION OF RECOMBINANT OVINE IFN-γ
Methods and Results Messenger RNA was isolated from ovine alveolar macrophages stimulated with LPS as described in Examples 2, 3 and 5. Reverse transcriptase and oligo dT primer was then used to produce a pool of cNDAs using standard techniques. Oligonucleotide primers (based on the bovine γ-IFN cDNA sequence) were then used in a PCR reaction to amplify from the pool of cDNA molecules, the cDNA corresponding to ovine γ-IFN. These oligonucleotide primers (shown below) were designed to bind sequences upstream of the initiation codon (5' primer) and downstream of the termination codon (3' primer) to amplify a fragment of ovine γ-IFN covering the entire coding region.
5' primer 5' - 3' (GCTAGGATCC)TTTCAACTACTCCGGCC
3' primer
3' - 5'
(TACCGAATTC)ATATTGCAGGCAGGAGAA
These oligonucleotides are flanked by restriction enzyme recognition sequences (shown in brackets) to facilitate cloning of the 553 bp fragment into a suitable vector such as pGEM3Zf(+). The sequence of the ovine γlFN cDNA obtained by PCR is shown in Figure 5D. Mammalian cell expression of recombinant ovine IFN-γ
The ovine IFN-γ cDNA fragment generated by PCR was then cloned into the Fill expression vector as previously described. Plasmid DNA was transfected into COS-7 cells using DEAE-dextran and supernatants were harvested after 72hrs. Mock transfection with no cDNA was used as a negative control. Supernatants were assayed for IFN-γ using 2 monoclonal antibodies 9mAb) specific for bovine IFN-γ and an ELISA based assay. Briefly, micro¬ titre plates were coated overnight at 4°C with a 1:1000 dilution of IFN-γ mAb IFN-9 in pH 9.2 bicarbonate buffer.
Plates were then washed 3 x with PBS plus 0.05% Tween 20 (wash buffer) . Vacant binding sites were then blocked for lhr at 37°C with PBS plus 0.5% Tween 20. After 3 further washes 40 μl of test sample at the appropriate dilution was added to each well and plates incubated at 37°C for lhr. Wells were then washed 3x and 50μl of peroxidase conjugated mAb IFN-2 added to each well. After lhr at room temperature all plates were washed and binding of the second mAb detected using the TNB (Aldrich Chemicals) substrate.
Results shown in Figure 27 demonstrated that in comparison to the supernatant from mock transfections, supernatant from test transfections contained recombinant ovine IFN-γ.
EXAMPLE 7 CLONING AND EXPRESSION OF THE OVINE IL-2 RECEPTOR α CHAIN Materials and Methods
Production of an ovine IL-2Rα probe using PCR
Messenger RNA was isolated from ovine ConA blasts as previously described (Example 4). Reverse transcriptase and oligo dT primer was then used to produce a pool of cDNAs using standard techniques. Oligonucleotide primers (based on the bovine IL-2Rα cDNA sequence) were then used in a PCR reaction to amplify from the pool of cDNA molecules, a portion of the cDNA corresponding to ovine IL-2R α chain. Cloning of an ovine IL-2R a chain cDNA
A cDNA library from activated ovine T cells produced using methods described in the previous examples was screened with the 32P labelled ovine IL-2R probe described above. A positive plaque was purified and the
EcoRI insert cloned into pGEM-3ZF(+) . DNA sequencing of exonuclease III deletion derivatives was performed using the dideoxy chain termination method.
Production of a stable CHO cell line expressing the ovine IL-2 Receptor α chain
The IL-2Rα cDNA was cloned as an EcoRI fragment into the Fill expression vector previously described (Figure 12) . CsCl gradient DNA of this derivative was transfected into CHO cells along with a pSV2 XGRPT selection plasmid. Stable transfectants expressing the ovine IL-2R were screened by association with radiolabelled I125-IL-2. Eguilibrium binding assay of CHO transfectants
Transfected CHO cells were screened for IL-2R expression by their ability to bind radiolabelled IL-2.
The clone bearing the highest number of receptors was examined in equilibrium binding assays for receptor binding affinity. Cells were harvested with 5 mM EDTA in
PBS. The cells were washed three times in culture media by
7 5 mm centrifugations at 1000 RPM and suspended at 10 cells/ml in α MEM binding media (α MEM containing 5%
FCS, 1% BSA, P/S, G and lOmM Hepes pH 7.0). 100 μls of cells was incubated with doubling dilutions of I 125-IL-2. (final volume 200 μls) in siliconised eppendorf tubes for 20min at 37°C. The cells were then layered over 100 μls of binding oil in 400 μl mini microfuge tubes and spun for 30 seconds at maximal speed in an eppendorf centrifuge. The tips containing the cell pellets were cut off and the tips and columns placed into separate counting tubes for determination of bound and free I 125-IL-2. between the range of 5pM and 20 nM was tested in triplicate. Non specific binding was determined by incubating cells with 124I-IL-2 in the presence of an excess (>500 fold) of unlabelled HurlL-2 (Cetus corp) . This was subtracted from total bound CPM values to give specifically bound CPM.
RESULTS Ovine IL-2R α chain cDNA Sequence The ovine IL-2R α chain cDNA isolated from the activated T cell cDNA library was found to be 2650 bp in length. The sequence contained and open reading frame of approximately 825 bp encoding a protein of approximately 275 amino acids with a predicted molecular weight of 30,869 (Figure 5A) .
Eguilibrium binding assay of CHO transfectants
CHO cells expressing the ovine IL-2R were produced and then assayed as described above. The results from these assays were then subjected to Scatchard analysis (Figure 28) to determine a Kd of 27 nM for binding of IL-2 to the transfected ovine IL-2 Receptor α chain.
Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims

Claims
1. A process for identifying nucleotide sequences coding for a polypeptide exhibiting specific ruminant cytokine or cytokine receptor activity or a fragment thereof, which process includes providing a vector including a complementary DNA (cDNA) sequence derived from ruminant cells or an extract thereof capable of being expressed in a unicellular organism; a unicellular organism; and a DNA probe for a homologous cytokine; introducing the vector into the said unicellular organism; culturing the organism to express a polypeptide encoded therein; probing the organism with the DNA probe; and isolating a vector containing a cDNA sequence encoding for a polypeptide exhibiting specific ruminant cytokine or cytokine receptor activity.
2. A process according to claim 1, further including the preliminary steps of providing a source of ruminant macrophage or extract thereof; and a suitable cloning vector; isolating cytokine or cytokine-specific messenger RNA (mRNA) from the ruminant macrophage; treating the messenger RNA to produce complementary DNA (cDNA); and deploying the cDNA sequence into the cloning vector.
3. A process according to claim 2, wherein the ruminant macrophages are alveolar macrophages stimulated in vitro to enhance cytokine specific messenger RNA production.
4. A process according to claim 1, further including the preliminary steps of providing a source of ruminant lymphocytes or extract thereof; and a suitable cloning vector; isolating cytokine or cytokine receptor-specific messenger RNA (mRNA) from the ruminant lymphocyte; treating the mRNA to produce complementary DNA (cDNA); and deploying the cDNA into the cloning vector.
5. A process according to claim 4, wherein the ruminant lymphocytes are ruminant lymph node cells stimulated in vitro to enhance cytokine receptor specific messenger RNA production.
6. A process according to claim 1, wherein the DNA probe is a human cDNA probe specific for a cytokine selected from IL-lα, IL-lβ, TNFα, IL-6 and IFN-γ.
7. A DNA sequence coding for a polypeptide exhibiting ruminant cytokine or cytokine receptor activity, or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof. 8. A DNA sequence coding for a polypeptide exhibiting ovine interleukin-lα (IL-lα) activity having a length of approximately 1781 base pairs (bp) or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof. 9. A DNA sequence according to claim 8, wherein the sequence contains an open reading frame of approximately 804 base pairs (bp) encoding a protein of approximately 268 amino acids.
10. A DNA sequence coding for a polypeptide exhibiting ovine interleukin-lβ (IL-lβ) activity and having a length of approximately 1439 base pairs (bp) or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof.
11. A DNA sequence according to claim 10, wherein the sequence contains an open reading frame of approximately
801 base pairs (bp) encoding a protein of approximately 266 amino acids.
12. A DNA sequence coding for a polypeptide exhibiting ovine interleukin-6 (IL-6) activity and having a length of approximately 1103 base pairs (bp) or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof.
13. A DNA sequence according to claim 12, wherein the sequence contains an open reading frame of approximately
624 base pairs encoding a protein of approximately 208 amino acids.
14. A DNA sequence coding for a polypeptide exhibiting ovine tumor necrosis factor α (TNFα) activity and having a length of approximately 1675 base pairs (bp) or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof.
15. A DNA sequence according to claim 14, containing an open reading frame of approximately 702 base pairs encoding a protein of approxiately 234 amino acids.
16. A DNA sequence coding for a polypeptide exhibiting ovine interleukin-2 receptor α activity, and having a length of approximately 2650 bp or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof.
17. A DNA sequence according to claim 16, wherein the sequence contained an open reading frame of approximately 825 base pairs encoding a protein of approximately 275 amino acids.
18. A DNA sequence coding for a polypeptide exhibiting ovine interferon-gamma (IFN-γ) activity or a portion thereof and having a length of approximately 553 base pairs (bp) or squences substantially homologous therewith, derivatives thereof, mutants thereof, or fragments thereof.
19. A DNA sequence according to claim 18 wherein the DNA sequence contains an open reading frame of approximately 498 bp encoding a precursor protein of approximately 166 amino acids.
20. A DNA sequence coding for a polypeptide exhibiting ovine interleukin-2 (IL-2) activity having a length of approximately 501 base pairs or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof.
21. A DNA sequence according to claim 20, wherein the DNA sequence contains an open reading frame of approximately 465 base pairs, encoding a precursor protein of approximately 155 amino acids.
20. A process for the production of a recombinant polypeptide having ovine cytokine or cytokine receptor activity which process includes the steps of providing a recombinant expression vector including a
DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity or sequences substntially homologous therewith, derivatives or mutants thereof, or fragments thereof, and capable of being replicated, transcribed and translated in a eukaryotic or prokaryotic organism; and an animal host; introducing said vector into said organism, such that the recombinant polypeptide encoded therein is expressed; and optionally isolating said polypeptide from said host.
23. A process according to claim 22 wherein the recombinant expression vector is injected directly into the tissue of the animal host.
24. A process for the production of a recombinant polypeptide having ovine cytokine or cytokine receptor activity which process includes the steps of providing a recombinant expression vector including a
DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity, or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof, and capable of being replicated, transcribed and translated in a eukaryotic or prokaryotic organism; and a eukaryotic or prokaryotic organism; introducing said vector into said organism by transformation, transduction or transfection; culturing the resulting organism; expressing the recombinant polypeptide encoded by said DNA sequence; and isolating said polypeptide from the culture.
25. A process according to claim 24 wherein the DNA sequence codes for a polypeptide exhibiting ovine IL-lα, IL-lβ, IL-2, IL-2R, IL-6, TNFα or IFN-γ activity.
26. A process according to claim 24, wherein the recombinant expression vector contains a ribosome binding site within its promoter region, and a suitable restriction site for insertion of the DNA sequence distal to the ribosome biding site.
27. A process according to claim 24, wherein the DNA sequence coding for a polypeptide is modified to include an ATG initiation codon correctly positioned relative to a suitable restriction site.
28. A process according to claim 27, including the preliminary steps of providing a first DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof; a second DNA sequence containing an ATG initiation codon; and inserting the second DNA sequence into a suitable restriction site within the first DNA sequence such that the ATG initiation codon is placed in front of the sequence coding for the mature protein.
29. A process according to claim 28, further including subjecting the first DNA sequence to a digestion step to remove a signal sequence in the untranslated 5' region for transport of the cytokine across the endoplasmic reticulum of a eukaryotic cell.
30. A process according to claim 24, further including the preliminary steps of providing a first DNA sequence coding for a polypeptide having ruminant cytokine or cytokine receptor activity,or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof; and an oligonucleotide sequence duplicating the nucleotide sequence of the first DNA sequence from the amino terminal amino acid to a restriction site and containing an ATG codon at a suitable restriction site upsteam of the duplicated sequence; and subjecting the first DNA sequence to in vitro mutagenesis to produce an ATG codon and a suitable restriction site therein. 31. A process according to claim 29, wherein the oligonucleotide sequence contains an ATG codon and a Bglll restriction site.
32. A recombinant polypeptide exhibiting ruminant cytokine or cytokine receptor activity, or sequences substantially homologous therewith, derivatives or mutants thereof, or fragments thereof, in substantially pure form.
33. A recombinant polypeptide according to claim 32, selected from IL-lα, IL-lβ, IL-2, IL-2R, IL-6, TNF-α and IFN-γ. 34. A veterinary composition including a recombinant polypeptide having ovine cytokine or cytokine receptor activity or mimotope thereof; or fragment thereof; and a carrier or excipient therefor for veterinary use.
35. A monoclonal or polyclonal antibody against a recombinant polypeptide exhibiting ruminant cytokine or cytokine receptor activity or mimotope thereof; or a fragment thereof. 36. A vaccine composition including an antigen against a disease of interest; and a non-toxic adjuvant including a recombinant polypeptide having ovine cytokine or cytokine receptor activity, or mimotopes, derivates or fragments thereof. 37. A process according to claim 1, substantially as hereinbefore described with reference to any one of the examples.
38 A process according to claim 22, substantially as hereinbefore described with reference to any one of the examples.
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WO1988009382A1 (en) * 1987-05-28 1988-12-01 Immunex Corporation BOVINE INTERLEUKIN-1alpha

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Title
CYTOKINE vol. 4, no. 6 , November 1992 pages 418 - 428 C.E. FISKERSTRAND ET AL 'Clonig, expression and characterizationof ovine interleukins 1alpha and beta' *
JOURNAL OF CELLULAR BIOCHEMISTRY vol. 15F , April 1991 page 123 A. ANDREWS ET AL 'Molecular cloning and functional characterization of the ovine cytokines IL-1alpha, IL-1beta and TNFalpha' *
NUCLEIC ACIDS RESEARCH vol. 16, no. 18 , September 1988 , ARLINGTON, VIRGINIA US page 9053 S.R. LEONG ET AL 'The nucleotide sequence for the cDNA of bovine interleukin-1 alpha' *
See also references of WO9203574A1 *

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