|Publication number||WO1983002460 A1|
|Publication date||21 Jul 1983|
|Filing date||11 Jan 1983|
|Priority date||15 Jan 1982|
|Also published as||CA1282353C, EP0098864A1|
|Publication number||PCT/1983/35, PCT/US/1983/000035, PCT/US/1983/00035, PCT/US/83/000035, PCT/US/83/00035, PCT/US1983/000035, PCT/US1983/00035, PCT/US1983000035, PCT/US198300035, PCT/US83/000035, PCT/US83/00035, PCT/US83000035, PCT/US8300035, WO 1983/002460 A1, WO 1983002460 A1, WO 1983002460A1, WO 8302460 A1, WO 8302460A1, WO-A1-1983002460, WO-A1-8302460, WO1983/002460A1, WO1983002460 A1, WO1983002460A1, WO8302460 A1, WO8302460A1|
|Inventors||Corporation Cetus, Michael A. Innis|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (1), Non-Patent Citations (4), Referenced by (8), Classifications (5), Legal Events (2)|
|External Links: Patentscope, Espacenet|
Description Technical Field
The invention is in the field of biotech nology. - More particularly it relates to a polypeptide having interferon (IFN) activity, DNA that codes for the polypeptide, a recombinant vector that includes the DNA, a host organism transformed with the recombinant vector that produces the polypeptide, pharma ceutical compositions containing the polypeptide, and therapeutic methods employing the polypeptide.
IFNs are proteins with antiviral, immunomodulatory, and antiproliferative activities produced by mammalian cells in response to a variety of inducers (see Stewart, W.E., The Interferon System, Springer-Verlag, New York, 1979). The activity of IFN is largely species specific (Colby, C, and Morgan, M. J., Ann. Rev. Microbiol. 25:333-360 (1971) and thus only human IFN can be used for human clinical studies. Human IFNs are classified into three groups, α, β, and γ, (Nature, 286:110, (1980)). The human IFN-α genes compose a multigene family sharing 85%-95% sequence homology (Goeddel, D. V., et al, Nature 290:20-27 (1981) Nagata, S.,. et al, J. Interferon Research
1:333-336 (1981)). Several of the IFN-α genes have been cloned and expressed in E.coli (Nagata, S., et al , Nature 284: 316-320 (1980 ) ; Goeddel, D. V. , et al , Nature 287 : 411-415 (1980 ) . Yelverton, E . , et al , Nucleic Acids Research, 9 : 731-741 , ( 1981 ) ; Streuli , M . , et al , Proc Nat Acad Sci (USA) , 78: 2848-2852. The resulting polypeptides have beep, purified and tested for biological activities associated with partially purified native human IFNs and found to possess similar activities . Accordingly such polypeptides are potentially useful as antiviral, immunomodulatory, or antiproliferative agents .
A principal object of the present invention is to provide a polypeptide having interferon activity that is produced by an organism transformed with a newly isolated and newly characterized IFN-α gene . This polypeptide is sometimes referred to herein as
IFN-α74. Other objects of the invention are directed to providing the compositions and organisms that are used to produce this polypeptide and to therapeutic compositions and methods that use this polypeptide as an active ingredient.
Disclosure of the Invention
One aspect of the invention is a polypeptide having interferon activity and comprising the amino acid sequence :
CysAspLeuProGln ThrHisSerLeuGly AsnArgArgAlaLeu IleLeuLeuAlaGln MetGlyArglleSer HisPheSerCysLeu LysAspArgHisAsp PheGlyPheProGlu GluGluPheAspGly HisGlnPheGlnLys ThrGlnAlalleSer ValLeuHisGluMet IleGlnGlnThrPhe AsnLeuPheSerThr GluAspSerSerAla AlaTrpGluGlnSer Leu LeuGluLysPhe SerThrGluLeuTyr GlnGlnLeuAsnAsp LeuGluAlaCysVal IleGlnGluValGly ValGluGluThrPro LeuMetAsnValAsp SerlleLeuAlaVal ArgLysTyrPheGln ArglleThrLeuTyr LeuThrGluLysLys TyrSerProCysAla
TrpGluValValArg AlaGluIleMetArg SerLeuSerPheSer ThrAsnLeuGlnLys ArgLeuArgArgLys Asp A second aspect of the invention is a DNA unit or fragment comprising a nucleotide sequence that encodes the above described polypeptide.
A third aspect of the invention is a cloning vehicle or vector that includes the above described DNA.
A fourth aspect of the invention is a host organism that is transformed with the above described cloning vehicle and that produces the above described polypeptide.
A fifth aspect of the invention is a process for producing the above described polypeptide comprising cultivating said transformed host organism and collecting the polypeptide from the resulting culture. Another aspect of the invention is a pharmaceutical composition having interferon activity comprising an effective amount of the above described polypeptide admixed with a pharmaceutically acceptable carrier. Still another aspect of the invention is a method of providing interferon therapy to a human comprising administering a therapeutically effective amount of the above described polypeptide to the human.
Brief Description of the Drawings
Figure 1 is a partial restriction map which shows the two XhoII restriction sites that produce a homologous 260 base pair DNA fragment from the IFN-α1 and IFN-α2 structural genes. Data for this map are from Streuli, M., et al Science, 209:1343-1347 (1980).
Figure 2 depicts the sequencing strategy used to obtain the complete DNA sequence of the IFN-α74 gene coding region. Bacteriophage mp7:α74-1 DNA served as the template for sequences obtained with primers A, H and F and bacteriophage mp7:α74-2 DNA was the template for sequences obtained with primers E and G. The crosshatched area of the gene depicts the region that encodes the 23 amino acid signal polypeptide and the open box depicts the region that encodes the mature polypeptide. The scale, in base pairs, is numbered with 0 representing the ATG start codon of preinterferon. The arrows indicate the direction and extent of sequencing with each primer.
Figure 3 is the nucleotide sequence of the structural gene coding for IFN-α74 including some of the flanking 5'- and 3'- noncoding regions of the gene. The region coding for preinterferon and the mature polypeptide begins with the ATG codon at position 19 and terminates with the TGA codon at position 586.
Figure 4 is a partial restriction map of the coding region of the IFN-α74 gene. The crosshatching represents the region that encodes the 23 amino acid signal peptide and the open box represents the gene coding sequence for the mature polypeptide. The scale, in base pairs, is numbered with 0 representing the ATG start codon of preinterferon. Figure 5 shows the amino acid sequence of the 23 amino acid signal polypeptide and the 166 amino acid mature IFN-α74 coded for by the gene depicted in Figure 3. The 189 amino acid sequence is displayed above the corresponding nucleotide sequence. Amino acid 24, cysteine, is the first amino acid of the mature IFN-α74 protein.
Figure 6 is the DNA sequence of the E. coli trp promoter and the gene of Figure 3 which was inserted between the EcoRI and Hindlll sites of the plasmid pBR322. The amino acid sequence of Figure 5 is written above the corresponding DNA sequence and the location of the restriction sites used in the construction of the expression plasmid are indicated. Figure 7 is a diagram of the expression plasmid, pGC7.
Modes for Carrying Out the Invention
In general terms IFN-α74 was made by identifying and isolating the IFN-α74 gene by screening a library of human genomic DNA with an appropriate IFN-α DNA probe, constructing a vector containing the IFN-α74 gene, transforming microorganisms with the vector, cultivating transformants that express IFN-α74 and collecting IFN-α74 from the culture. A preferred embodiment of this procedure is described below.
DNA Probe Preparation
Total cytoplasmic RNA was extracted from human lymphoblastoid cells, Namalwa, which had been induced for IFN production by pretreatment with 5-bromodeoxyuridine (Tovey, M.G., et al, Nature 267:455-457 (1977)) and Newcastle Disease Virus (NDV). The poly(A) (polyadenylic acid)-containing messenger RNA (mRNA) was isolated from total RNA by chromatography on oligo(dT)-cellulose (type 3 from Collaborative Research; Aviv, H., and Leder, P., Proc Natl Acad Sci (USA) , 69:1408-1412, (1972)) and enriched for IFN mRNA by density gradient centrifugation on 5%-20% sucrose gradients. Fractions containing IFN mRNA were identified by translating the mRNA by microinjecting aliquots of each fraction into Xenopus oocytes and determining the IFN activity of the products of the translations according to a method described by Colman, A., and Morser, J., Cell, 17:517526 (1979).
The Namalwa cell human IFN enriched mRNA was used to construct complementary DNA (cDNA) clones in E. coli by the G/C tailing method using the Pstl site of the cloning vector pBR322 (Bolivar, F., et al, Gene, 2:95-113 (1977)). A population of transformants containing approximately 50,000 individual cDNA clones was grown in one liter of medium overnight and the total plasmid DNA was isolated.
The sequences of two IFN-α clones (IFN-α1 and IFN-α2) have been published (Streuli, M., et al, Science, 209:1343-1347 (1980)). Examination of the DNA sequences of these two clones revealed that the restriction enzyme XhoII would excise a 260 bp fragment from either the IFN-α1 or the IFN—α2 gene (see Figure 1). XhoII was prepared in accordance with the process described by Gingeras, T.R., and Roberts, R.J., J Mol Biol, 118:113-122 (1978). One mg of the purified total plasmid DNA preparation was digested with XhoII and the DNA fragments were separated on a preparative 6% polyacrylamide-gel. DNA from the region of the gel corresponding to 260 bp was recovered by electroelution and recloned by ligation into the BamHI site of the single strand bacteriophage ml3:mp7. Thirty-six clones were picked at random, the single stranded DNA isolated therefrom, and the DNA was sequenced. The DNA sequences of four of these clones were homologous to known IFN-α DNA sequences. Clone mp7:α-260, with a DNA sequence identical to IFN-α1 DNA (Streuli, M. et al, Science, 209:1343-1347 (1980)) was chosen as a highly specific hybridization probe for identifying additional IFN-α DNA sequences. This clone is hereinafter referred to as the "260 probe." Screening of Genomic DNA Library
In order to isolate other IFN-α gene sequences, a 32P-labelled 260 probe was used to screen a library of human genomic DNA by in situ hybridiza tion. The human gene bank, a gift from F. Blattner, University of Wisconsin (unpublished), was generated by partial cleavage of fetal human DNA with EcoRI and cloned into bacteriophage λ Charon 4A. Approximately 200,000 clones were screened, of which about 30 hybridized with the 260 probe. Each individual clone was further characterized by restriction enzyme mapping and comparison with the published restriction maps of 10 chromosomal interferon genes (Nagata, S., et al, J. Interferon Research, 1:333-336 (1981)). One of the clones, hybrid phage λ4Aα74 containing a 24.5 kb insert, was characterized as follows. A DNA preparation of λ4A:α74 was cleaved with Hindlll, Bglll, and EcoRI respectively, the fragments separated on an agarose gel, transferred to a nitrocellulose filter (Southern, E.M., J Mol Biol, 98:503-517 (1977)) and hybridized with 32P-labelled 260 probe. This procedure localized the IFN-α74 gene to a 1.2 kb HindlII restriction fragment which was then isolated and recloned, in both orientations, by ligation of the fragment into Hindlll cleaved ml3:mp7. The two subclones are designated mp7:α74-l and mp7:α74-2. The -1 designation indicates that the single-stranded bacteriophage contains insert DNA complementary to the mRNA (the minus strand) and the -2 designation indicates that the insert DNA is the same sequence as the mRNA (the plus strand). Sequencing of the IFN-α74 Gene
The Sanger dideoxy-technique was used to determine the DNA sequence of the IFN-α74 gene. The strategy employed is diagrammed in Figure 2, the DNA sequence thus obtained is given in Figure 3, and a partial restriction enzyme map of the IFN-α74 gene is illustrated in Figure 4. Unlike many genes from eukaryotic organisms, but analogous to other IFN chromosomal genes which have been characterised, the DNA sequence of this gene demonstrates that it lacks introns. Homology to protein sequence information from these known IFN-α genes made it possible to determine the correct translational reading frame and thus allowed the entire 166 amino acid sequence of IFN-α74 to be predicted from the DNA sequence as well as a precursor segment, or signal polypeptide, of 23 amino acids (Figure 5).
The DNA sequence of the IFN-α74 gene and the amino acid sequence predicted therefrom differ substantially from the other known IFN-α DNA and IFN-α amino acid sequences. Nagata, S., et al, (J Interferon Research, 1:333-336, (1981) describe isolating two IFN-α genes, IFN-α4a and IFNα4b, that differ by five nucleotides which entails 2 amino acid changes in the proteins expressed thereby. The sequence of IFN-αb is given in European Patent Application No. 81300050.2. The IFN-α74 structural gene differs from the IFN-α4b gene by 3 nucleotides which entails 2 amino acid changes in the corresponding proteins: a single nucleotide change creates an amino acid substitution of alanine for threonine at amino acid number 14 of the mature protein; and a double nucleotide change creates an amino acid substitution of alanine for glutamine at amino acid number 19 of the mature protein. Plasmid Preparation and Host Transformation
Assembly of the plasmid for direct expression of the IFN-α74 gene involved replacing the DNA fragment encoding the 23 amino acid signal polypeptide of preinterferon with a 120 bp EcoRI/Sau3A promoter fragment (E.coli trp promoter, operator, and trp leader ribosome binding site preceding an ATG initiation codon) and using the naturally occurring Hindlll site, 142 bp 3'- of the TGA translational stop codon, to insert the gene into a vector derived from the plasmid pBR322. The complete DNA sequence of the promoter and gene fragments inserted between the EcoRI and Hindlll sites of pBR322 is shown in Figure 6 which also shows the exact location of relevant cloning sites. Details of the construction are described below.
The coding region for mature IFN-α74 encompasses a Sau3A site between codons for amino acids 2 and 3 and an Aval site between codons for amino acids 39 and 40. The 111 bp Sau3A to Aval fragment was isolated on a 6% polyacrylamide gel following a Sau3A/AvaI double-digest of the 1.2 kb Hindlll genomic fragment. Similarly, the 528 bp fragment from the Aval site between codons for amino acids 39 and 40 and the Hindlll site 142 nucleotides 3'- of the translational stop codon was isolated on a 5% polyacrylamide gel. These two fragments, together with a 120 bp EcoRI to Sau3A E.coli promoter fragment were ligated together in a four way directed ligation into the EcoRI to Hindlll site of pBR322. The promoter fragment, which contains a synthetic Hindlll restriction site, ATG inititation codon, the initial cysteine codon (TGT) common to all known IFN-αs, and Sau3A "sticky end", had been constructed previously. The ligation mixture was used to transform E.coli MM294 (Backman, K., et al, Proc Natl Acad Sci (USA) 73:41744178 (1974)). The desired correct transformation products, 23 out of 24 screened, were identified by restriction enzyme mapping of colonies which hybridized to a 32P-labelled IFN-α genomic fragment. Figure 7 is a diagram of the final expression plasmid obtained, which is designated pGC7. Other prokaryotic hosts such as bacteria other than E.coli may, of course, be transformed with this or other suitable constructs to replicate the IFN-α74 gene and/or to produce IFN-α74.
IFN-α74 produced in accordance with the invention, is believed to be distinct from the corresponding native protein in several respects. Firstly, because the IFN-α74 gene was expressed by bacterial hosts that utilize N-formyl-methionine and/or methionine to initiate translation, some or all of the bacterially produced IFN-α74 molecules are preceded by an N-formyl-methionine or methionine group. Some of the N-formyl-methionine or methionine groups could be removed by natural in vivo bacterial cleavage mechanisms. This would result in a mixture of molecules, some of which would include an initial N-formylmethionine or methionine and others that would not. All such IFN-α74 molecules, those containing an initial N-formyl-methionine or methionine, those not containing an N-formyl-methionine or methionine and any mixture thereof, are encompassed by the present invention. Secondly, the amino acid residues of the bacterially produced polypeptide are unsubstituted whereas the residues of the native protein may be substituted with sugar groups, ACTH or other moieties. Also, native IFN-α extracts consist of mixtures of various IFN molecules whereas the bacterially produced IFN-α74 is homogeneous; that is, bacterially produced IFN-α74 does not contain functionally related polypeptides. Accordingly, the invention contemplates producing IFN-α74-containing compositions having biological activity that is attributable solely to IFN-α74 and/or said terminal N-formyl-methionine or methionine derivatives thereof.
Cultivation of Transformants
Bacteria transformed with the IFN-α74 gene may be cultivated in an appropriate growth medium, such as a minimum essential medium, that satisfies the nutritional and other requirements needed to permit the bacteria to grow and produce IFN-α74. If the bacteria are such that the protein is contained in their cytoplasm, the IFN-α74 may be extracted from the cells by lysing the cells such as by sonication and/or treatment with a strong anionic solubilizing agent such as sodium dodecyl sulfate. Further purification of the extract may be achieved by affinity chromatography, electrophoresis, or other protein purification techniques.
Biological Testing of IFN-g74 IFN-α74-containing cell sonicates were tested in vitro and found to have the following activities: (1) inhibition of viral replication of vesicular stomatitis virus (VSV) and herpes simplex virus-1 (HSV-1); (2) inhibition of tumor cell growth; (3) inhibition of colony formation by tumor cells in soft agar; (4) activation of natural killer (NK) cells; (5) enhancement of the level of 2',5'-oligo adenylate synthetase (2',5'-A); and (6) enhancement of the double-stranded RNA-dependent protein kinase. The sonicates were active in inhibiting viral infection in both human and other mammalian cells such as hamster, monkey, mouse, and rabbit cells.
The tests show that IFN-α74 exhibits antiviral activity against DNA and RNA viruses, cell growth regulating activity, and an ability to regulate the production of intracellular enzymes and other cell-produced substances. Accordingly, it is expected IFN-α74 may be used to treat viral infections with a potential for interferon therapy such as chronic hepatitis B infection, ocular, local, or systemic herpes virus infections, influenza and other respiratory tract virus infections, rabies and other viral zoonoses, arbovirus infections, and slow virus diseases such as Kuru and sclerosing panencephalitis. It may also be useful for treating viral infections in immunocompromised patients such as herpes zoster and varicella, cytomegalovirus, Epstein-Barr virus infection, herpes simplex infections, rubella, and progressive multifocal leukoencephalopathy. Its cell growth regulating activity makes it potentially useful for treating tumors and cancers such as osteogenic sarcoma, multiple myeloma, Hodgkin's disease, nodular, poorly differentiated lymphoma, acute lymphocytic leukemia, breast carcinoma, melanoma, and nasopharyngeal carcinoma. The fact that IFN-α74 increases protein kinase and 2',5'-oligoadenylate synthetase indicates it may also increase synthesis of other enzymes or cell-produced substances commonly affected by IFNs such as histamine, hyaluronic acid, prostaglandin E, tRNA methylase, and aryl hydrocarbon hydrolase. Similarly, it may be useful to inhibit enzymes commonly inhibited by IFNs such as tyrosine amino transferase, glycerol-3-phosphate dehydrogenase glutamine synthetase, ornithine decarboxylase, S adenosyl-1-methionine decarboxylase, and UDP-N acetylglucosamine-dolichol monophosphate transferase. The ability of the IFN-α74 to stimulate NK cell activity is indicative that it may also possess other activities such as the abilities to induce macrophage activity and antibody production and to effect cell surface alterations such as changes in plasma membrane density or cell surface charge, altered capacity to bind substances such as cholera toxin, concanavalin A and thyroid-stimulating hormone, and change in the exposure of surface gangliosides. Pharmaceutical compositions that contain
IFN-α74 as an active ingredient will normally be formulated with an appropriate solid or liquid carrier depending upon the particular mode of administration being used. For instance, parenteral formulations are usually injectable fluids that use pharmaceutically and physiologically acceptable fluids such as physiological saline, balanced salt solutions, or the like as a vehicle. Oral formulations, on the other hand, may be solid, eg tablet or capsule, or liquid solu- tions or suspensions. IFN-α74 will usually be formulated as a unit dosage form that contains in the range of 104 to 107 international units, more usually 106 to 107 international units, per dose.
IFN-α74 may be administered to humans in various manners such as orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, and subcutaneously. The particular mode of administration and dosage regimen will be selected by the attending physician taking into account the par ticulars of the patient, the disease and the disease state involved. For instance, viral infections are usually treated by daily or twice daily doses over a few days to a few weeks; whereas tumor or cancer treatment involves daily or multidaily doses over months or years. IFN-α74 therapy may be combined with other treatments and may be combined with or used in association with other chemotherapeutic or chemopreventive agents for providing therapy against viral infections, neoplasms, or other conditions against which it is effective. For instance, in the case of herpes virus keratitis treatment, therapy with IFN has been supplemented by thermocautery, debridement and trifluorothymidine therapy. Modifications of the above described modes for carrying out the invention, such as, without limitation, use of alternative vectors, alternative expression control systems in the vector, and alternative host microorganisms and other therapeutic or related uses of IFN-α74, that are obvious to those of ordinary skill in the biotechnology, pharmaceutical, medical and/or related fields are intended to be within the scope of the following claims.
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|Citing Patent||Filing date||Publication date||Applicant||Title|
|WO1984000776A1 *||18 Aug 1983||1 Mar 1984||Cetus Corp||Interferon-alpha 6l|
|WO1985002862A1 *||20 Dec 1984||4 Jul 1985||Monash University||PRODUCTION OF HUMAN INTERFERON-alpha|
|WO1986006744A1 *||14 May 1986||20 Nov 1986||Kabivitrum Ab||A cDNA MOLECULE CODING FOR THE EXPRESSION OF AN INTERFERON alpha TYPE POLYPEPTIDE, A BACTERIAL OR CELLULAR HOST TRANSFORMED WITH SUCH MOLECULE AND A POLYPEPTIDE SHOWING INTERFERON ACTIVITY PREPARED BY SUCH HOST|
|EP0174143A1 *||23 Aug 1985||12 Mar 1986||Genentech, Inc.||Novel, distinct family of human leukocyte interferons, compositions containing them, methods for their production, and DNA and transfected hosts therefor|
|EP0194006A1 *||17 Jan 1986||10 Sep 1986||Imperial Chemical Industries Plc||Analogous interferon polypeptides, process for their preparation and pharmaceutical compositions containing them|
|US5028422 *||13 Apr 1989||2 Jul 1991||Schering Corporation||Treatment of basal cell carcinoma intralesionally with recombinant human alpha interferon|
|US5231176 *||9 Jun 1992||27 Jul 1993||Genentech, Inc.||Distinct family DNA encoding of human leukocyte interferons|
|US7399464||11 Dec 2003||15 Jul 2008||Genodyssee Sa||Polypeptides of the IFNα-7 gene|
|International Classification||A61K38/00, C07K14/56|
|Cooperative Classification||C07K14/56, A61K38/00|
|21 Jul 1983||AK||Designated states|
Designated state(s): AU JP
|21 Jul 1983||AL||Designated countries for regional patents|
Designated state(s): AT BE CH DE FR GB LU NL SE