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WO1996003436A2 - Gamma-interferon d'origine equine - Google Patents

Gamma-interferon d'origine equine Download PDF

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Publication number
WO1996003436A2
WO1996003436A2 PCT/GB1995/001732 GB9501732W WO9603436A2 WO 1996003436 A2 WO1996003436 A2 WO 1996003436A2 GB 9501732 W GB9501732 W GB 9501732W WO 9603436 A2 WO9603436 A2 WO 9603436A2
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WO
WIPO (PCT)
Prior art keywords
interferon
equine
recombinant
nucleic acid
polynucleotide fragment
Prior art date
Application number
PCT/GB1995/001732
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English (en)
Other versions
WO1996003436A3 (fr
Inventor
David Edward Onions
Lesley Nicholson
Original Assignee
Q-One Biotech Limited
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 Q-One Biotech Limited filed Critical Q-One Biotech Limited
Priority to AU30832/95A priority Critical patent/AU3083295A/en
Publication of WO1996003436A2 publication Critical patent/WO1996003436A2/fr
Publication of WO1996003436A3 publication Critical patent/WO1996003436A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to equine 7-interferon, a polynucleotide fragment encoding equine 7-interferon, a recombinant vector comprising such a polynucleotide fragment, a host cell containing said polynucleotide fragment, or recombinant vector comprising said polynucleotide fragment, an equine ⁇ -interferon polypeptide, antibodies immuno-reactive with said polypeptide and pharmaceutical compositions comprising recombinant equine 7-interferon for use as a prophylactic and/or therapeutic agent and also as an adjuvant in horses.
  • Cyto ines are low molecular weight secreted proteins with immunomodulatory activity. The term applies to interferons, interleukins, lymphokines, monokines, colony stimulating factors and a number of growth factors (Balkwill and Burke 1989) . Interferons are a class of cytokines which exhibit antiviral activity. Three types exist, alph ( ⁇ ) , beta ( ⁇ ) and gamma (7) (Farrar and Schreiber 1993). The type I interferons, a and ⁇ are related and bind to a common cell structure surface receptor, although the multiple forms of ⁇ and the single ⁇ form may have different biological effects.
  • Interferon gamma also known as type II or immune interferon, is distinct from a and ⁇ interferons at a genetic level. Like ⁇ interferon it is encoded by a single gene. IFN7 is produced by natural killer (NK) cells and by T lymphocytes
  • TH1 subset in response to stimulation with antigen or mitogens. It possesses a range of biological effects of immunomodulatory or antiviral nature including enhancement of MHC class I and class II expression, suppression of the TH2 T lymphocyte response, activation of macrophages, enhancement of natural killer cell activity, and modulation of the synthesis and effect of a number of cytokines (Gray 1992).
  • IFN7 has also exhibited adjuvanticity properties in some animal models (reviewed in Health and Playfair 1992) . Although the bovine and ovine IFN7S, which exhibit 93% amino acid identity, are cross-reactive (Radford et al. 1991) , IFN7S tend to be highly species-specific in their biological activity.
  • IFN7 In a particular species generally requires production of recombinant IFN7 of that species. Since the goals of most interferon cloning operations are deduction of the amino acid sequence and production of recombinant IFN, most IFN7 sequences have been derived from mRNA rather than chromosomal DNA. The advent of the polymerase chain reaction has, however, facilitated the cloning of cytokine cDNAs and the nucleotide sequences of the genes or cDNAs encoding a number of mammalian cytokines to been determined.
  • IFN7 interleukin-7
  • the human IFN7 gene encodes a 166 amino acid precursor possessing a 23 amino acid N-terminal signal sequence and two N-linked glycosylation sites. Functional IFN7 is homodimeric with the polypeptides associated in a non- covalent manner (Scahill et al. 1983) . Several forms of human IFN7 exist due to post-translational C-terminal proteolytic cleavage and differential glycosylation (Rinderknecht et al. 1984) . Regions of N-terminal and C- terminal sequence have been determined as being important in maintaining biological activity (Farrar and Schreiber, 1993) .
  • Cytokine and anti-cytokine therapies such as synthesis of inhibitors, soluble cytokine receptors, receptor antagonists or anti-cytokine antibodies, are finding increased clinical application in human medicine (Mire-Sluis, 1993) . Due to the lack of species cross- reactivity of many cytokines and the potential for antibody production against heterologous cytokines (Holmes, 1993), however, the ideal reagents in the horse will be substantially equine-specific.
  • the present invention provides a polynucleotide fragment, such as a DNA fragment, encoding equine 7- interferon.
  • the invention further provides a recombinant equine 7- interferon polypeptide.
  • Polynucleotide fragment refers to a chain of nucleotides such as deoxyribose nucleic acid (DNA) sequences and transcription products thereof, such as RNA, capable of giving rise to an equine 7-interferon protein or physiologically active fragment thereof.
  • DNA deoxyribose nucleic acid
  • RNA transcription products thereof, capable of giving rise to an equine 7-interferon protein or physiologically active fragment thereof. The term excludes the whole naturally ocurring genome.
  • polynucleotide will be in isolated form substantially free of biological material with which the whole genome is normally associated in vivo.
  • this term includes double and single stranded DNA, and RNA sequences derived therefrom.
  • polypeptide refers to a chain or sequence of amino acids displaying a biological activity substantially similar to the biological activity of equine 7-interferon and does not refer to a specific length of the product as such.
  • the polypeptide if required, can be modified in vivo and in vitro. for example by glycosylation, amidation, carboxylation, phosphorylation and/or part translational cleavage; thus inter alia peptides, oligopeptides and proteins are encompassed thereby.
  • a polynucleotide fragment encoding equine 7-interferon can be amplified from equine 7-interferon cDNA, obtained by way of reverse transcription of mRNA, by polymerase chain reaction (PCR) , using primers designed against conserved regions of 7-interferon coding sequences from a single species, or a number of other species.
  • PCR polymerase chain reaction
  • An amplified fragment containing the equine 7-interferon is depicted in Figure 1.
  • the DNA fragment of Figure 1 was shown to encode an open reading frame (ORF) of 166 amino acids.
  • ORF open reading frame
  • a comparison of the amino acid sequence against previously sequenced 7- interferons revealed identities ranging from 45% with murine 7-interferon to 78% with bovine 7-interferon.
  • the two potential N-linked glycosylation sites exist at positions conserved in bovine and pig 7-inteferon precursors at amino acids 39-41 and 106-108 in the equine precursor (data not shown) .
  • equine 7-interferon has a predicted N-terminal signal sequence and possesses no cysteine residues.
  • the present invention includes polynucleotide and polypeptide sequences having at least 80 %, particularly at least 90 % and especially at least 95 % similarity with the sequences of Figure 1.
  • similarity refers to both identical and conservative replacement of nucleotides or amino acids, provided that the functionality of feline 7- interferon is substantially unimpaired.
  • amino acids may be made between amino acids within the following groups:
  • recombinant DNA technology may be used to prepare nucleic acid sequences encoding the various derivatives outlined above.
  • the degeneracy of the genetic code permits substitution of bases in a codon resulting in a different codon which is still capable of coding for the same amino acid, e.g. the codon for the amino acid glutamic acid is both GAT and GAA. Consequently, it is clear that for the expression of a polypeptide with the amino acid sequence shown in Figure 1 or fragment thereof use can be made of a derivative nucleic acid sequence with such an alternative codon composition different from the nucleic acid sequence shown in said Figure 1.
  • fragments derived from the equine 7-interferon polypeptide or from the amino acid sequence depicted in Figure 1 which still display equine 7-interferon properties, or fragments derived from the nucleotide sequence encoding the equine 7-interferon polypeptide or derived from the nucleotide sequence depicted in Figure 1 encoding fragments of said equine 7-interferon polypeptides are also included in the present invention.
  • the equine 7-interferon polynucleotide fragment of the present invention is preferably linked to regulatory control sequences.
  • control sequences may comprise promoters, operators, inducers, ribosome binding sites terminators etc.
  • Suitable control sequences for a given host may be selected by those of ordinary skill in the art.
  • an equine 7-interferon control sequence can be employed in a mammalian host.
  • a polynucleotide fragment according to the present invention can be ligated to various expression controlling sequences, resulting in a so-called recombinant nucleic acid molecule.
  • the present invention also includes an expression vector containing an expressible nucleic acid molecule.
  • Said recombinant nucleic acid molecule can then be used for transformation of a suitable host.
  • hybrid molecules are preferably derived from for example plasmids, or from nucleic acid sequence present in bacteriophages or viruses and are termed vector molecules.
  • a specific bacterial expression vector pHEX has been adapted for 7-interferon production (Reid, submitted for publication) .
  • the pHEX vector has a stretch of 6 histidine residues and a thrombin site downstream of the vector- specified ATG initiation codon.
  • the 6 histidine residues provide crude purification of recombinant proteins using affinity chromatography.
  • the DNA insertion site is positioned 3' to the thrombin site so that should biological activity of the equine 7-interferon produced be compromised by the vector-specified N-terminus, cleavage of extraneous amino-acid sequence from the interferon moiety is possible.
  • the present invention also relates to a transformed cell containing the equine 7-interferon polynucleotide fragment in expressible form.
  • Transformation refers to the introduction of a heterologous polynucleotide fragment into a host cell, irrespective of the method used, for example direct uptake, transfection or transduction.
  • the heterologous polynucleotide fragment may be maintained through autonomous replication or alternatively, may be integrated into the host genome.
  • the recombinant nucleic acid molecules preferably are provided with appropriate control sequences compatible with the designated host which can regulate the expression of the inserted polynucleotide fragment e.g. tetracycline responsive promoter, thymidine kinase promoter and SV-40 promoter.
  • Suitable hosts for the expression of recombinant nucleic acid molecules can be prokaryotic or eukaryotic in origin.
  • the most widely used hosts for expression of recombinant nucleic acid molecules may be selected from bacteria, yeast, insect cells and mammalian cells.
  • yeast and baculovirus systems are preferred.
  • the yeast strain Pichia pastoris exhibits potential for high level expression of recombinant proteins (Clare et al.1991) .
  • the baculovirus system has been used successfully in the production of type 1 interferons (Smith et al. 1983).
  • the cloning and expression of recombinant equine 7-interferon also facilitates in producing reagents for the production of, for example, probes for in situ expression studies, production of anti-equine 7-interferon antibodies (particularly monoclonal antibodies) and evaluation of in vitro and in vivo biological activity of recombinant equine 7-interferon.
  • the antibodies may be employed in diagnostic tests for 7-interferon.
  • the present invention further provides recombinant equine 7-interferon for the manufacture of reagents for use as prophylactic and/or therapeutic agents and also as an adjuvant in horses.
  • the invention provides pharmaceutical compositions comprising the recombinant 7- interferon together with a pharmaceutically acceptable carrier therefor.
  • equine disorders such as disease states attributable to equine 7-interferon deficiencies or over abundance and physiological or immunological abnormalities may benefit from equine 7-interferon therapy and/or prophylaxis.
  • disease states include cancer, endotoxaemia, parasitic and bacterial infections, wound therapy, auto-immune and inflammatory diseases, allergies and viral infections.
  • Equine 7-interferon may also be applied for vaccine adjuvantation.
  • Cytokines such as interferons , ⁇ and 7 and tumour necrosis factor, TNF, are reported as being capable of exhibiting direct antiviral activity in addition to their immunomodulatory properties (Ramsay et al. 1993) .
  • the recombinant equine 7-interferon can prove beneficial as a prophylactic agent to modulate recrudescence of viruses, such as equine herpesvirus (EHV) , under conditions of stress and to reduce the clinical consequence of horizontal transmission of viral agents to in-contact animals.
  • EHV equine herpesvirus
  • Herpes viruses and other viruses with complex genomes play an immunomodulatory role contributing to general or specific immunosuppression in the host.
  • Administration of the equine 7-interferon can also serve to redirect the immune response such that more efficient virus clearance occurs.
  • administration of bovine IFN7 prior to experimental infection of cattle with bovine herpesvirus-1 (BHV-l) has been shown to reduce virus-induced suppression of cell-mediated cyto
  • equine 7-interferon can be used as a tool to study these effects.
  • Non-specific stimulation of the immune response by adjuvant formulations is often beneficial in cases of prophylactic or therapeutic vaccination.
  • a more defined stimulation of the immune response using equine 7-interferon as an adjuvant or co- adjuvant can be more effective.
  • Such a stimulation could take the form of administration of recombinant equine 7-interferon or expression of equine 7-interferon within live or disabled virus vectors.
  • 7-interferon has previously been reported as being associated with adjuvancy in animals (Heath and Playfair, 1992).
  • IFN7 upregulate MHC class II expression enhancing antigen presentation, and suppresses TH2 lymphocyte with resultant inhibition of humoral immunity.
  • Equine 7- interferon may also ameliorate clinical response to live virus vaccination which, providing protective immunity is not compromised, could be a desirable safety feature (Andrew et al. 1991; Ramshaw et al. 1992) .
  • Candidate live virus vectors include poxviruses, adenovirus and herpesvirus, though the latent capacity of the latter coupled with potential immunopathology of cytokines may preclude cytokine insertion in this virus.
  • Administration of equine 7-interferon with live equine herpesvirus vaccines may be used to overcome mechanisms of immune evasion or diversion specified by the virus to provide a longer lasting protective immune response.
  • RNAgents Trademark
  • Total RNA isolation system supplied by Promega Corp.
  • Superscript Trademark
  • oligonucleotides Two oligonucleotides (see below) were designed against interspecies conserved non-coding regions of 7- interferon mRNA.
  • oligonucleotides were then used as primers in a polymerase chain reaction (PCR) experiment to amplify the equine 7-interferon cDNA.
  • the PCR was performed as described by Saiki et al (1987) .
  • Ten ⁇ l of lOOng/ ⁇ l template cDNA from the reverse transcribed mRNA was added to a 40 ⁇ l reaction mixture containing 200 ⁇ M of dATP, dCTP, dGTP, dTTP, 50pmol of both primers (I) and (II) , 1 unit of DNA Polymerase and 5 ⁇ l of lOx reaction buffer.
  • the reaction buffer contained lOOmM Tris-HCl, 500mM potassium chloride, 0.01 per cent (w/v) gelatin and 1.5mM magnesium chloride, ultrapure water, TE (pH8.0).
  • the solution was overlaid with two drops of mineral oil to prevent evaporation.
  • amplification and analysis of the products were carried out. Thirty five cylces of amplification were performed using a Perkin Elmer Cetus thermal cycler. Each cycle consisted of 1 min. at 95°C to denature the DNA, 1 min. at 50°C to anneal the primers to the template and 1 min. at 72°C for primer extension. After the last cycle a further incubation for lOmins. at 72°C was performed to allow extension of any partially completed product. On completion of the amplification, lO ⁇ l of the reaction mixture was electrophoresed through a 1.5 per cent agarose gel. The DNA was visualised by staining with ethidium bromide and exposure to ultraviolet light (320nm) .
  • the pBluescript (Trademark) derived vector, containing the cloned equine 7-interferon DNA was prepared and purified. Double stranded DNA sequencing was carried out on this DNA using the T7 polymerase sequenase (Trademark) kit (supplied by USB Corp.) as per the manufacturers instructions.
  • the DNA sequence obtained is shown in Figure 1.
  • the two underlined regions at the ends of the sequence correspond to the two primers (I) and (II) used to amplify the DNA.
  • the two internal underlined regions represent potential glycosylation sites.
  • ORF open reading frame
  • the pHEX vector was constructed from the vector pGEX- 2T (Smith and Johnson, 1988) .
  • the sequence of the glutathione S-transferase gene was deleted from pGEX-2T and a sequence which contained a consensus to the 5' coding regions of bacterial mRNA and also six histidine residues was inserted 3' to the initiating AUG (see below)

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Abstract

Cette invention concerne un fragment polynucléotidique codant un gamma-interféron d'origine équine, un vecteur de recombinaison comprenant un fragment polynucléotidique de ce type, une cellule hôte contenant ledit fragment polynucléotidique ou ledit vecteur de recombinaison, un polypeptide de gamma-interféron d'origine équine obtenu par recombinaison, et des compositions pharmaceutiques comprenant le gamma-interféron de recombinaison utilisé comme agent prophylactique et/on thérapeutique et également comme adjuvant.
PCT/GB1995/001732 1994-07-21 1995-07-21 Gamma-interferon d'origine equine WO1996003436A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU30832/95A AU3083295A (en) 1994-07-21 1995-07-24 Equine gamma-interferon

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9414753.5 1994-07-21
GB9414753A GB2291645A (en) 1994-07-21 1994-07-21 Equine gamma-interferon

Publications (2)

Publication Number Publication Date
WO1996003436A2 true WO1996003436A2 (fr) 1996-02-08
WO1996003436A3 WO1996003436A3 (fr) 1996-10-03

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WO (1) WO1996003436A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3642096A1 (de) * 1986-12-10 1988-06-16 Boehringer Ingelheim Int Pferde-(gamma)-interferon
NZ239373A (en) * 1990-08-13 1993-10-26 Univ Melbourne Dna sequences encoding ruminant cytokine or cytokine receptors, polypeptides therefrom and vaccines containing an adjuvant containing the polypeptide or mimotope thereof
WO1992005255A1 (fr) * 1990-09-13 1992-04-02 Commonwealth Scientific And Industrial Research Organisation Genes de cytokine d'origine ovine

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GB2291645A (en) 1996-01-31
WO1996003436A3 (fr) 1996-10-03
AU3083295A (en) 1996-02-22
GB9414753D0 (en) 1994-09-07

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