IE58942B1 - New genetic sequences, the interferon peptides of type 1 encoded by them and the microorganisms producing these interferons - Google Patents

Abstract

The type I interferon peptides encoded by the genetic DNA sequences are called omega-interferons and are produced using appropriate expression vehicles and organisms.

Description

- 1 - 58942 The present invention relates to new Typea interferons as well as recombinant DMA methodsof producing these peptides and products requiredin these methods, for example, genetic sequences,recombinant DNA molecules, expression vehiclesand organisms.

Interferon is a word coined to describe avariety of proteins endogenous to human cells character-ised by partly overlapping and partly divergingbiological activities. These proteins modify thebody’s immune response and are believed to contributesubstantial protection against viruses. For example,interferons have been classified into three broadspecies, a-, 8, and Ϊ -Interferon.

Only one subspecies of 6- and V -interferonis known in humans (see, for example, S. Ohno etal., Proc. Natl. Acad. Sci. 78, 5305-5309 (1981); Gray et al., Nature 295, 503-508 (1982); Taya,et al., EMBO Journal 1/8, 953-958 ,(1982)). Onthe other hand, several subtypes of a-interferonhave been described (See, for example, Phil. Trans. R. Soc. Lond. 299, 7-28 (1982)). The mature c- interferons reveal a maximum divergence of 23¾ among each other and are about 155 amino acids long. Of note is also a report of an a-interferon having an unusually high molecular weight (25,000 2 ΪΟ 15 20 25 30 35 by SDS polyacrylamide gel electrophoresis , describedby Goren, P. et al», Virology 130 , 273-280 (1983) ).

This interferon is called IFN-α 26K. It has beenfound to have the highest known specific anti-viraland anti-cellular activities.

The interferons known to date appear to beeffective against various diseases but demonstratelittle or no efficacy in many others (seef forexample, Powledge, Bio/Technology, March 1984e215-228,, "’Interferon On Trial"). Interferons havealso been plagued by side effects. For example,in trials of the anti-cancer properties of recombinantK-interferon trials, doses, of around 50 millionunits, which had been believed to be safe on thebasis of Phase I trials have been associated withacute confusional states,, disabling arthragia,profound fatigue and anorexia, disorientation,seizures and hepatic toxicity. In 1982, the Frenchgovernment stopped trials with α-interferon aftercancer patients receiving it suffered fatal heartattacks.. At least two cardiac deaths have alsobeen reported in recent American trials. It hasbecome increasingly clear that at least some ofthe side effects, like fever and malaise, appearto be inherent in the interferon molecule itselfand not in impurities present therein.

Due to the great hopes elicited by the interferons,,and spurred by the wish to discover yet new interferon-like molecules with decreased side effects, thepresent inventors set out to search for and producesuch new substances, The present invention therefore relates tocertain new interferons of Type I which may containa leader peptide, and their N-glycosylated derivatives,(called herein omega-interferon, or IFN-omega)which contain 168 to 174, preferably 172, amino acids,and which reveal a divergence of 30 to 503, preferably 3 40-48%, compared to the hitherto known subtypesof c-interferon, and a divergence of about 70%compared to 8-interferon, while showing similareffectiveness to α-interferons, and which do notreveal many of the known therapeutic disadvantagesof these molecules.

The invention therefore provides new interferons,in fully purified fornP the unglycosylated andglycosylated forms thereof? genetic sequences codingtherefor? as well as recombinant molecules containingthe sequences. The invention also provides expressionvehicles such as plasmids containing genetic sequencesas indicated? as well as various hosts? such asmicroorganisms or tissue culture hosts? capableof producing the novel interferons by fermentationor tissue culture methods. ' In a preferred embodiment, the inventionprovides omega-interferons as well as the correspondinggenetic sequences, having the following formulae: 4 Cys Asp Leu Pro 5 Gin Asn His Gly Leu 10 Leu Ser Arg Asn Thr 15 Leu TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG Val Leu Leu His 20 Gin Met Arg Arg He 25 Ser Pro Phe Leu Cys 30 Leu 5 GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC Lys Asp Arg Arg 35 Asp Phe Arg Phe Pro 40 Gin Glu Met Val Lys 45 Gly AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 10 Ser Gin Leu Gin 50 Lys Ala His Val Met 55 Ser Val Leu His Glu 60 Met AGC CAG TTG CAG AAG GCC CAT GTC ATG TGT GTC CTC CAT GAG ATG Leu Gin Gin He 65 Phe Ser Leu Phe His 70 Thr Glu Arg Ser Ser 75 Ala CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 15 Ala Trp Asn Met 80 Thr Leu Leu Asp Gin 85 Leu His Thr Gly Leu 90 His GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT Gin Gin Leu Gin 95 His Leu Glu Thr Cys 100 Leu Leu Gin Val Val 105 Gly 20 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA Glu Gly Glu Se r 110 Ala Gly Ala He Ser 115 Ser Pro Ala Leu Thr 120 Leu GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 25 Arg Arg Tyr Phe 125 Gin Gly He Arg Val 130 Tyr Leu Lys Glu Lys 135 Lys AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA Tvr Se r Asp Cys 140 Ala Trp Glu Val Val 145 Arg Met Glu He Met 150 Lys • TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 30 V Ser Leu Phe Leu 155 Ser Thr Asn Met Gin 160 Glu Arg Leu Arg Ser 165 Lys TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 170 Asp Arg Asp Leu Gly Ser Ser35 GAT AGA GAC CTG GGC TCA TCT 5 In position 111(. the sequence GGG (codingfor gly) can be replaced with GAG (coding for glu).The preferred molecules also include derivative-sthereof which are N-glycosylated at amino acidposition 78.

The two omega-interferons mentioned abovemay. for examplef contain the leader peptide ofthe formula Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu ValMet Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly.

DESCRIPTION OF THE FIGURES Figure 1: Restriction map for clone E75E69. Figure 2: Restriction map for clone P9A2, Figure 3: DNA sequence of the clone P9A2. Figure 4: DNA sequence of the clone E76E9. Figure 5: Genomic Southern Blot Analysis using the DNA of the clone P9A2 as probe Figure δ: Construction of the expression clone pRHWl2 Figure 7: Amino acid and nucleotide differencesbetween Type I inte^f-er-ons of Figure 8a: List the IFN-αΑ gene of singular nucleotide positions of Figure 8b: Listthe IFN-omega 1 gene of t singular nucleotide positions of Figure 8cs List the IFN-aD gene of singular nucleotide positions Figure 9; Schematic representation of thesynthesis of interferon-subtype of specific samples Figure 10: Detection of interferon-subtype _of specific mRNA’s 6 Figure 11: DNA sequence of the IFN-omegal gene Figure 12: DNA sequence of the IFN-pseudo- omega2 geneFigure 13: DNA sequence of the IFN-pseudo- omega3 geneFigure 14: DNA sequence of the IFN-pseudo- omega4 gene Figure 15: corrected list of the IFN-omega gene sequences Figure 16: Homologies of the signal sequences Figure 17: Homologies of the ’’mature" protein sequences Figure 18: Homolog ies of the 4 DNA sequences relative to one another ^.-According to the invention? the new omegainterferons and the DNA sequences coding for themare obtained as follows: A human B-cell lymphoid line? for example? Namalwa cells (see G. Klein et al.P Int. J, Cancer10, 44 (1972)) can be stimulated for the simultaneousproduction of a- and 6-interferons through treatmentwith a virus? for example? Sendai Virus. In theprocess? the mRNA formed is isolated from the stimulatedNamalwa cells? and this can then be used as a templatefor cDNA synthesis. In. order to increase the yieldof interferon-specific sequences during the cloningprocess? the. mRNA preparation is separated in asugar density gradient into regions with differentlengths of individual mRNA molecules.

Preferably? the mRNA in the region around12S (about 800-l?000 bases length of mRNA) iscollected» The mRNA which is specific fore-interferons and 6-interferons will settle inthis region. The mRNA from this gradient 7 region is concentrated through precipitation anddissolution in water.

The preparation of a CDNA library essentiallyinvolves the use of methods known in the literature(see, for example, E. Dworkin-Rastl, M.B. Dworkinand P. Swetly., Journal of Interferon Research2/4 f 575-585 (1982)). The mRNA is primed throughthe addition of oligo-dT. After that,, by addingthe four deoxynucleosidetriphosphates (dATP, dGTP,dCTP, dTTP) and the enzyme reverse transcriptase,,in an appropriately buffered solution,- cDNA issynthesized at 45°C for 1 hour. Through chloroformextraction and chromatography via a gel column, /m for example, via SephadexHsSO, the cDNA/mRNA hybridis purified. The RNA is hydrolysed by means ofalkali treatment (0.3 M NaOH at 50°C for 1 hour),and the cDNA is precipitated with ethanol afterneutralising with acid sodium acetate solution.

Double strand synthesis is performed after additionof the four deoxynucleosidetriphosphatesand E.coli DNA-polymerase I in an appropriatelybuffered solution, whereby the cDNA is used bothas a template and as a primer through hairpin structureformation at its 3’ end (6 hours at 15°C) (seealso A. Eftratiadis et al„, Cell /, 279 (1976)).Following-phenol extraction, Sephadex G50 chromatographyand ethanol precipitation, the DNA is subjected,in a suitable solution, to a treatment with endonucleaseSi, which is specific to single strands. The hairpinstructure as well as any cDNA that was not convertedinfo double stranded is degraded» After chloroformextraction and precipitation with ethanol, thedouble-stranded DNA (dsDNA) is separated in termsof size on a sugar density gradient. In the subsequentsteps of cloning, it is preferred to use only dsDNAof size 600 bp and more in order to increase theprobability of obtaining only clones which containthe complete coding sequence for the new interferons. 8 dsDNA with a length of more than 600 bp is concentratedout of the gradient through precipitation withethanol and dissolution in water.

In order to increase the number of dsDNAmolecules that have been obtainedr they are firstplaced in an appropriate vector, and then introducedinto the bacterium E. coli. The vector used ispreferably the plasmid pBR322 (F. Bolivar et al.PGene 2, 95 (1977)). This plasmid essentially consistsof a replicon and two selection markers. Theyprovide the host with resistance against the antibioticsampicillin and tetracyclin (Apr? Tcr). The genefor 6-lactamase (Apr) contains the recognitionsequence for the restriction endonuclease Pstl.pBR322 can thus be cut with Pstl. The overlapping35-ends are extended with terminal deoxynucleotidetransferase (TdT) along with a premixed quantityof dGTP in an appropriately buffered solution.

At the same time,, dsDNA is likewise extended withthe enzyme TdT£, using dCTP at the 35-ends. Thehomopolymer ends of the plasmid and dsDNA are comple-mentary and will hybridize if plasmid DMA and dsDNAare mixed in the appropriate concentration ratioand under suitable salt, buffer, and temperatureconditions (T. Nelson et al.t, Methods in Enzymology68, 41-50 (1980)). E. coli P HB101 strain (genotype F-, hsdS20(r - B, m - B) recAl3r ara-14r proA2, lacYl? galK2,rpsL20 (Smr) , xyl-5^ mtl-1, supE44t. lambda-) isprepared for transformation of the recombinantvector-dsDMA molecules by means of washing witha CaClj solution. Competent E. coli HB101 aremixed with the DMA and? after incubation at 0°Cfthe plasmid DMA thus obtained is transformed bymeans of heat shock at 42°C for 2 minutes (M. Dagertet si,. Gene 6f 23-28 (1979)). The transformed 9 10 15 20 25 30 bacteria are then spread on tetracyclin-containingagar plates (10 ug per ml)- Only E. coli HB101which have received a vector or recombinant carriermolecule (Tcr) can grow on this agar. Recombinantvector-dsDNA molecules give the host the genotypeAps,pcr because the introduction of the dsDNA intothe 8-lactamase gene destroys the information forβ-lactamase. Clones are then transferred to agarplates, containing 50 pg/ml ampicillin. Only about3% grow, meaning that 97% of the clones containthe insertion of a dsDNA molecule. Starting with0.5 pg dsDNA, more than 30,000 clones were thusobtained; 28,600 clones thereof were individuallytransferred into the cups of microtiter plateswhich contained nutrient medium, 10 ug/ml tetracyclinand glycerin. After the clones had grown, theplates were kept at -70°C for storage (cDNA library).

In order to search the cDNA library for thenew interferon-gene-containing clones, the clonesare transferred after thawing to nitrocellulosefilters. These filters -rest on tetracyclin-containingnutrient agar. The bacterial colonies are allowedto grow and then the DNA of the bacteria is fixedon the filter.

As a probe, one can advantageously use the insert of the clone p£R33 (E. Rastl et al., Gene ____ 21, 237-248 (1983) - see also European Patent applicationWo„ 0.115.613) which contains the gene for IFN-c2-Arg. By means of nick translation, this portionof DNA is marked radioactively, using DNA-polymeraseI, dATP, dGTP, dTTP and a-32P-dCTP. The nitrocellulosefilters are first pretreated under relaxed hybridisatipnconditions, without the addition of the radioactivesample, and after that they are hybridised forabout 16 hours with the addition of the radioactivesample. The filters are then likewise washed underrelaxed conditions. Due to the low stringency 35 10 10 15 20 25 30 of hybridisation and washing, not only are clonesobtained that contain interferon a2-Arg, but alsoother clones containing interferons whose sequencesmay differ considerably from that of the a-interferonknown hitherto. After drying,, the filters areexposed on an x-ray film. A blackening effect,which is substantially above the level of background,shows the presence of clones with interferon-specificsequences.

Because the radioactivity signals are ofdiffering quality, the positive clones or the clonesthat react in a manner leading to a suspicion ofpositive results are then cultured on a small scale.The plasmid DNA molecules are isolated, digestedwith the restriction endonuclease Pstl, and separatedelectrophoretically on an agarose gel accordingto size (Birnboim et al., Nucl. Acid. Res. 7, 1513(1979)). The DNA in the agarose gel is transferredto a nitrocellulose filter according to the methodof Southern (E. M. Southern, J,. Mol. Biol- 98, 503-517 (1975)). The DNA in this filter is hybridisedwith the radio-activa^JCFN-gene-containing, denaturedsample. As positive control is used the plasmid1F7 (deposited at the DSM under the DSM no. 2362)which contains the gene for interferon a2- Arg.

The autoradiogram clearly showed that two clones, E76E9 ana P9A2, contained a sequence that hybridisedwith the gene of interferon a2-Arg under nonsfcringent,relaxed conditions. In order to be able more closely — to describe the dsDNA inserts of the clones E76E9and P9A2, the plasmids of these clones were preparedon a larger scale. The DNA is digested with variousrestriction endonucleases, for example, with Alul,Sau3A, Bglll, Hinfl, Pstl, and Haelll. The resultingfragments are separated in an agarose gel. Throughcomparison with corresponding size markers, forexample the fragments which result from digestionof pBR322 with the restriction endonuclease Hinfl 35 11 or Haelll, one can determine the sizes of the fragments.

By means of mapping according to Smith and Birnsteil (Η. O, Smith et al., Wucl. .

Acid. Res, 2387-2398 (1957)), one can determine-the sequence of these fragments. From the restriction v enzyme maps thus determined (Figures 1 and 2),the surprising finding was made that the insertsof the clones E76E9 and P9A2 involve a hithertounknown interferon gene» that is» the omega-interferongene.

This information on omega-interferons canbe used in order to digest the cDNA insert withsuitable restriction endonucleases. The fragmentsare ligated into the dsDWA form (replicative form)of the bacteriophage M13 mp9 (J. Messing et al., Gene 19» 269-276 (1982)) and are sequenced withthe help of Sanger's dideoxy method (F. Sangeret al., Proc. Natl. Acad. Sci. USA 74» 5463-5467(1977)). The single-strand DWA of recombinantphages is isolated. After the binding of a syntheticoligomer, second-strand syntheses are performedin four separate preparations, using the largefragment of DNA-polymerase I from Ξ. coli (Klenowfragment). For each of the four partial reactions,one of the four didexoynucleosidetriphosphates(ddATP, ddGTP, ddCTP, ddTTP) are added. This leadsto statistically distributed chain breaks at thoseplaces where the base that is complementary withrespect to the particular dideoxynucleosidetriphosphatehappens to be in the template-DWA. Radioactivelymarked dATP is also used. After termination ofthe synthesis reactions, the products are denaturedand the single-strand DWA fragments are separatedaccording to size in a denaturing polyacrylamidegel (F. Sanger et al., FEBS Letters 87» 107-111 12 (1978)). The gel is then exposed to x-ray film.

From the autoradiogram one can read off the DMAsequence of the recombinant Hl3 phage» The sequences g of the inserts of the various recombinant phages are processed by means of suitable computer programs(R. Staden, Nucl. Acid» Res- 10, 4731-4751 (1982)).

Figures 1 and 2 reveal the strategy of sequencing.Figure 3 shows the DNA sequence of the insert of jq the clone P9A2; Figure 4 shows that of the clone Ε76Ε9» The noncoding DNA strand is shown in the5s—-3’ direction, together with the amino acidsequence derived therefrom.

The isolated cDNA of the clone E76E9 for 15 omega(Glu)-interferon is 858 base pairs long and has a 38 nontranslated region- The region whichcodes for mature omega(Glu)-interferon extendsfrom nucleotide 9 to nucleotide 524. The isolatedcDNA of the clone P9A2 for omega(Gly)-interferon 20 is 877 base pairs long, whereby the sequence that codes for mature omega-interferon extends fromnucleotide 8 to nucleotide 523. The 39 nontranslatedregion in the case of P9A2 extends to the poly-Asegment. 25 The DNA sequences that code for mature omega- interferon are completely contained in the clonesE76E9 and Ρ9Ά2. The start at the N-terminal endwith the amino acids cysteine-aspartic acid-leucine.

Quite surprisingly, the two shown mature omega- 30 interferons are 172 amino acids long; this clearly deviates from the hitherto known length of otherknown interferons, that is, 1S6 (or 1S5) aminoacids for α-interferons- Quite surprisingly,the two omega-interferons have a potential l^-glycosylation 35 site at amino acid position 78 (asparagine-methionine- threonine) . A comparison of the DNA of the clones E7SE9and P9A2 yields one difference. The triplet in 13 clone E76E9 which codes for amino acid 111 is GAGand codes for glutamic acid. This triplet in cloneP9A2 is GGG., and codes for glycine. The two omega-interferon proteins therefore differ from eachother by one amino acid and are referred to hereinas omega(Glu)-interferon (76E9) and omega(Gly)-interferon (P9A2). A comparison of the two omega-interferonswith the hitherto known human a-interferonsubtypes gives the following picture: omega alpha Length of protein in amino acids 172 166* Potential N-glycosylation siteat position 78 * Interferon alpha A has only 165 amino acids. ** Interferon alpha H has a potential N-glycosylationsite at position 75 (D. Goeddel et al., Mature290, 20-26 (1981)). E. coli KB 101 with the plasmid Ξ76Ε9 andE. coli 101.with the plasmid P9A2 are depositedat the German Collection for Microorganisms (DSMGSttigen) under the numbers DSM 3003 and 3004,respectively, on 3rd July 1984.

To prove that the newly discovered clonesproduce an activity resembling interferon, the -clone Ξ76Ε9 is cultivated, for example, and thelysate of the bacterium is grown and then testedin a plague reduction test. As expected, the bacteriumproduced interferon-like activity (see Example3) . 14 Moreover, to prove that the two newly discoveredinterferons are members of a new interferon family,all the DNA was isolated from Namalwa cells and-digested with various restriction endonucleases.

In this way it is possible to access thenumber of genes which are coded by the cDNA’s ofthe clones Ρ9Ά2 and E76E9. For this purpose, theDNA fragments obtained are separated on agarosegel using the method of Southern (E.M. Southernet al., J. Mol. Biol. 98, 503-517 (1975)), placedon nitrocellulose filters and hybridised underrelatively stringent conditions with radioactivelylabelled specific DNA of the clone P9A2.

The results obtained under hybridisationwith DNA5s from the plasmid P9A2 and ρΕΡ33 areshown in Figure 5a: The individual traces are marked with lettersto indicate the various DNA samples digested (E=EcoRI,H=HindIII, B=BamHI, S=SphI, P=PstI, C=ClaI). Theleft-hand half of the filter was hybridised withthe interferon-c probe p’A") and the right-handhalf was hybridised with the cDNA insert of theclone P9A2 (’’O") . The set of bands which hybridiseeither with the c-interferon probe or with thenew interferon probe, are different. No cross-hybridisation could be detected with the two differentprobes by assessing the corresponding traces.

Figure 5b illustrates the cDNA of the cloneP9A2 and the fragment used for hybridisation.

The points of intersection of some restrictionensymes are shown (P=PstI, S=Sau3A, A=AluI). Theprobe includes only two of the three possiblePstl fragments. The hybridisation pattern showsonly one hybridising fragment with approximately1300 base pairs (bp), which belongs to the homologousgene. The shorter fragment, 120 bp long, had runout of the gel. The band belonging to the 5’ part 15 of the gene cannot be discovered since the probe does not contain this region. At least 6 differentbands can be discovered in the Pstl trace. Thismeans that some other genes which are related tothe new sequences must be present in the humangenome. If one or more Pstl points of intersectionare present in these genes, one can expect to beable to isolate at least three more additionalgenes.

These genes may preferably be isolated byhybridisation from a human gene library containedin a plasmid vector, phage vector or cosmid vector(see Example 4e).

At this point it should be mentioned thatthe omega-interferons according to the inventiondo not only encompass the two mature interferonswhich are specifically described but also any modificationof these polypeptides which leave the IFN-omegaactivity unaffected. These modifications compriseshortening of the molecules at the N- or C-terminithereof, exchanging amino acid residues for otherresidues without substantially affecting activity,or chemically or biochemically attaching the moleculesto other molecules, which may be inert or otherwise.

Among the latter can be mentioned hybrid moleculesmade from one or more omega interferons and/orknown «- or 8-interferons.

In order to be able to compare the differencesbetween the amino acid and nucleotide sequencesof the new interferons, particularly of the omega(Gly)and omega(Glu) interferon, with the amino acidand nucleotide sequences which have already beenpublished for α-interferons and 8-interferon (C-Weissmann et al,, Phil. Trans. R. Soc. London 299, 7=28 (1982); A. Ullrich et al., J. Molec. Biol. 156, 467-486 (1982);, T.Taniguchi et al., Proc.

Nat. Acad. Sci. 77, 4003-4006 (1980); K. Tokodoro 16 et al., EMBO J» 669-670 (1984)), the corresponding sequences are arranged in pairs and the differencesat individual positions are counted.

The results shown in Figure 7 demonstratethat the DNA sequences of the clones P9A2 and E76E9are related to the sequences of the Type I interferons(e.g. a and 8-interferons). It is also shown thatthe differences in the amino acid sequences betweenthe individual α-interferons and the new sequencesare greater than 41.6% and less than 47.0%. Thedifferences between the new sequences and thoseof the individual α-interferons and that of 8-interferonare in the order of about 70%. Taking into accountthe results of Example 4 in which the existenceof a whole set of related genes is demonstrated,and also taking into account the proposed nomenclaturefor interferons (J. Vilceck et al., J. Gen. Virol. 65, 669-670 (1984)) it is assumed that the cDNAinserts of the clones P9A2 and E76E9 code for anew class of Type I interferon which will be referredto as interferon-omega.

It is also shown—that the omega-interferongene expression occurs analogously to that of aType I interferon gene. Transcription of the individualmembers of the multi-gene families of the a- andomega-interferons based on the SI mapping method(A. J. Berk et al.. Cell 12, 721 (1977)) is investigated(see Example 7) and it is found that the expressionomega-X-mRNA is virus-inducible. Since the transcripts "of a gene family of this kind differ by only afew bases out of approximately 1000, hybridisationalone is not a sufficiently sensitive distinguishingfeature in order to distinguish between the variousIFN mRNA3s.

To do this, the mRNA sequences of 9 G-interferons,interferon-omega-1 and 6-interferon are produced.Capital letters are used to designate those bases 17 which are specific to the top sequence. Such specificsites can easily be found using a simple computerprogramme, ft hybridisation probe which startsfrom a specific site can only hybridise perfectly-with the mRNft of a specific subtype. All othermRNA’s are unable to hybridise at the specificsite of the subtype. If the hybridisation probeis radioactively labelled at its specific end*·only those radioactive labels which are protectedfrom digestion with a single strand-specific nuclease(preferably Si nuclease) are those which have hybridisedwith the interferon subtype mRNft for which theprobe was designed - This principle is not restricted to interferonbut may be applied to any group of known sequenceswhich have the specific sites described in Figure 8.

The above-mentioned specific sites of thesubtypes are not restriction sites*, in most cases,which means that the cutting of the cDNft’s withrestriction endonucleases is not capable of producingsubtype-specific hybridisation probes. The probesused in this example were therefore produced byextending an oligonucleotide radioactively labelledat the 5’ end which is complementary to the mRNftof interferon-omegal above the specific site.

Figure 10 shows that? as expected*, omega- 1-mRNft can be induced in Namalwa and NC37 cells.

The invention therefore comprises not onlythe genetic sequences specifically coding for theomega interferons mentioned*· but also modificationsobtained readily and routinely by mutation*· deletion?transposition or addition, ftny sequence whichcodes for human omega-interferons as described(i.e. having a spectrum of biological activitiesas shown herein) and which is degenerate to thoseactually shown is also included. Those skilled 18 in the art understand how to prepare degenerate DMA sequences of the coding regions- Also? any sequence coding for a polypeptide having the spectrumof activities shown herein for IFN-omegaP and whichhybridises with the sequences (or portions thereof)shown herein under stringent hybridisation conditions(i,e„P selecting for better than about 85%, preferablybetter than about 90% homology) is also covered.

The hybridisations are carried out in 6 x SSC/5 xDenhardt’s solution/0.1% SDS at 65°C. The degreeof stringency is determined in the washing step.

Thus? for a selection to DMA sequences with about85% homology or more, the conditions 0.2 x SSC/0.01%?SDS/65°C are suitable and for selection to DMAsequences with about 90% homology or more, theconditions 0.1 x SSC/0.01% SDS/65°C are suitable.

When looking through a cosmid library underthese conditions (0.2 x SSC) one will find a numberof cosmids which hybridise with an IFK-omegal probe-Sequence analysis of restriction enzyme fragmentsisolated therefrom will give the authentic IFN-omegal gene (see Figure 11) and three other relatedgenes which have been referred to as IFN-pseudo-omega2, XFM~pseudo-omega3 and IFN-pseudo-omega4(see Figures 12-14) . The invention also relatesto these"~and to the peptides which they code.

This is recited in claims 43 to 52.

The DMA comparisons give an approximately85% homology of the pseudo genes with the IFM-omega1gene (interferon-omega-one gene).

Moreover? the IFN-omegal gene shows thatin transcription the mR’NA contains the informationfor a functional interferon protein? i.e. a signalpeptide 23 amino acids long? of the formula 19 10 15 20 25 30 Met Ala Leu Leu Phe Pro Leu LeuAla Ala Leu Val Met Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly is codedwhich is followed by the mature IFN-omegalwhich is 172 amino acids long.

Interferon-omega genes may be introducedinto any organism under conditions which resultin high yields. Suitable hosts and vectors arewell known to anyone skilled in the art; by wayof example, reference is made to EP~A-0.093.619.

In particular, prokaryotes are preferredfor expression. For example, Ξ. coli K12 strain294 (ATCC No. 31446) is particularly useful. Othermicrobial strains which may be used include E.coli X1776 (ATCC No. 31.537). The aforementionedstrains, as well as E. coli W3110 (F , lambda ,prototrophic, ATCC No. 27325), bacilli such asBacillus subtilis, and other enterobacteriasuch as Salmonella typhimurium or Serratia marcesens,and various pseudomonad species may be used.

In general, plasmid vectors containing repliconand control sequences which are derived from speciescompatible with the host cell are used in connectionwith these hosts. The vector ordinarily carriesa replication site, as well as marking sequenceswhich are capable of providing phenotypic selectionin transformed cells- For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. coli species (Bolivar, et al~, Gene __ 2, 95 (1977)). pBR322 contains genes for ampicillinand tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR322plasmid or other plasmids must also contain, orbe modified to contain, promoters which can beused by the microbial organism for expression ofits own proteins. Those promoters most commonly 35 20 used in recombinant DNA construction include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al.? Nature 275 , 615 (1978); Itakura et al.f Science 198. 1056 (1977); Goeddel et al. , Nature 281F 544 (1979)) and tryptophan (trp) promoter system (Goeddel et al.r Nucleic Acids Res» fh 4057 (1980); ΕΡ-Ά-0.036„776).

While these are the most commonly used, other microbial promoters have been discovered and utilized. For example, the genetic sequence for IFN-omega can be placed under the control of the leftward promoter of bacteriophage Lambda (P,-) . This promoter isJL« one of the strongest known promoters which can be controlled. Control is exerted by the lambdarepressor, and adjacent restriction sites are known. A temperature sensitive allele of this genecan be placed on the vector that contains the IFN-omega complete sequence. When the temperatureis raised to 42°C„ the repressor is inactivated,and the promoter will be expressed at its maximumlevel. The amount of mRNA produced under theseconditions should be sufficient to result in acell which contains about 10% of its newly synthesisedRNA originated from the P„ promoter. In this way, 1j it is possible to establish a bank of clones in which a functional IFN-omega sequence is placed adjacent to a ribosome binding sequence, and at varying distances from the lambda P^ promoter.

These clones can then be screened and the one givingthe highest yield selected.

The expression and translation of an IFN-omega sequence can also be placed under control ;of other regulons which may be homologous" tothe organism in its untransformed state. For example The lac control elements may be obtainedfrom bacteriophage lambda plac5, which is infectivefor E. coli. The phage's lac operon can be derivedby transduction from the same bacterial species.

Regulons suitable for use in the process of theinvention can be derived from plasmid DNA nativeto the organism- The lac promoter-operator systemcan be induced by IPTG.

Other promoter-operator systems or portionsthereof can be employed as well: for example, thearabinose-operator, Colicine E.-operator, galactose-operator , alkaline phosphatase-operator, trp-operator,xylose Ά-operator, tac-promoter and the like canbe used.

In addition to prokaryotes, eukaryotic microbes,such as yeast cultures may also be used. Saccharomycescerevisiae is the most commonly used among eukaryoticmicroorganisms, although a number of other speciesare commonly available. For expression in Saccharomyces,the plasmid YRp7, for example (Stinchcomb, et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschumper, et al., Gene 10, 157 (1980))and plasmid ΥΕρ13 (Bwach et al.. Gene 121-133(1979)) are commonly used. The plasmid YRp7 containsthe TRPl gene which provides a selection markerfor a mutant strain of yeast lacking the abilityto grow in tryptophan, for example ATCC No. 44076.

The presence of the TRPl lesion as a characteristicof the yeast host cell genome then provides aneffective environment for detecting transformationby growth in the absence of tryptophan. Similarly,the plasmid YEpl3 contains the yeast LEU2 genewhich can be used to complement a LEU2 minus mutant strain.

Suitable promoting sequences in yeast vectorsinclude the 5’-flanking region of the genes of ADH I (Ammerer, G., Methods of Enzymology 101, 192-201 (1983)),3-phosphoglycerate kinase (Hitzeman, et al., J. 22 Biol. Chenu 255, 2073 (1980)) or other glycolyticenzymes (Kawasaki and Fraenkel, BBRC 108, 1107-1112 (1982)) „ such as enolasef glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate,decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, phosphoglucose isomerase, and glucokinase.

In concentrating suitable expression plasmids,the termination sequences associated with thesegenes are also ligated into the expression vectorat the 39 end of the sequences to be expressed,to provide polyadenylation of the mRNA and termination.

Other promoters, which have the additionaladvantage of transcription controlled by growthconditions are the promoter regions of the genes for alcoholdehydrogenase-2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism,the aforementioned glyceraldehyde-3-phosphate dehydro-genase, and enzymes responsible for maltose andgalactose utilisation. Promoters which are regulatedby the yeast mating type locus, such as the promotersof the genes BARI, MF cl, STE2, STE3, STE5 can beused for temperature regulated systems by usingtemperature dependent siv mutations (Rhine, Ph.D.

Thesis, University of Oregon, Eugene, Oregon (1979),Herskowitz and Oshima, The Molecular Biology ofthe Yeast Saccharomvces, part I, 181-209 (1981), Cold Spring Harbor Laboratory). These mutationsdirectly influence the expressions of the silentmating type cassettes of yeast, and therefore indirectlythe mating type dependent promoters. Generally,however, any plasmid vector containing a yeast-compatible promoter, originating replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate 23 or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture)- has become a routine procedure in recent years a (Tissue Culture, Academic Press, Kruse and Patterson, Editors (1973)}. 'Examples'of such'userui"host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, 3HK, COS-7 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites.» polyadenylationsite, and transcriptional terminator sequences.

For use in mammalian cells, the control functionson the—expression vectors are often provided byviral material. For example, commonly used promotersare derived from polyoma, Adenovirus 2, and mostfrequently Simian Virus 40 (SV40). The early andlate end promoters of SV40 are particularly usefulbecause both are obtained easily from the virusas a fragment which also contains the SV40 viralorigin of replication (Piers et al., Nature 273, 1123 (1978)). Smaller or larger SV40 fragmentsmay also be used, provided .there is included theapproximately 250 bp sequence extending from theHind III site toward the Bgl I site location inthe viral origin of replication. Further, it isalso possible, and often desirable, to utilisepromoter or control -sequences normally associatedwith the desired gene sequence, provided such controlsequences are compatible with the host cell systems- An origin of replication may be provided ·/ either by construction of the vector to includean exogenous origin, such as may be derived from. SV40 or other viral (e-g., Polyoma, Adeno, VSV, BPV, etc.) source, or may be provided by the host 24 cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome,the latter is often sufficient.

The genes mayf however, preferably be expressedin an expression plasmid pERlO3 (E. Rastl-Dworkinet al., Gene 21, 237-248 (1983) and EP-A-0.115-613 - deposited at the DSM under the number DSM2773 on 20th December 1983), since this vectorcontains all the regulons which result in a highexpression rate of the cloned genes. Accordingto the invention, therefore, the shorter EcoRI/BamHIfragment belonging to the plasmid pBR322 was replacedby the DMA sequence of formula EcoRI Sau3A gaattcacgctGATCGCTAAAACATTGTGCAAAAAGAGGGTTGACTTTGCCTTCGCGA 5 9 •mRNA-Start Met Ψ ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAaty"ATG" 116RBS Hindlll Cys Asp TGT GAT C -IFN-omega-Gen-> Sau3A In order to arrive at the objective of theinvention, the following procedure is used, forexample, according to Figure 6: I. Preparation of the individual DMA fragmentsrequired; Fragment a In order to produce fragment a) a plasmidwhich contains a gene for iFN-omega, e.g. the plasmidΡ9Ά2, is digested with the restriction endonucleaseAvail. · After chromatography and purification ofthe resulting cDNA insert, the latter is twiceredigested with the restriction endonucleases Ncol 25 and Alul and then isolated by chromatography andelectroelution. This fragment contains the majorityof the corresponding omega-interferon gene. Thus,for example, the omega(Gly) interferon gene of the clone P9A2 has the following structure: 5 10 15 His Gly Leu Leu Ser Arg Asn Thr Leu c|cAT GGC CTA CTT AGC AGG AAC ACC TTG 28 - Wool 1 20 25 30 Val Leu Leu His Gin Met Arg Arg lie Ser Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 73 35 40 45 Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu Met Val Lys Gly AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 50 55 60 Ser Gin Leu Gin Lys Ala His Val Met Ser Val Leu His Glu Met AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 65 70 75 Leu Gin Gin lie Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 208 80 85 90 Ala Trp Asn Met Thr Leu Leu Asp Gin Leu His Thr Gly Leu His GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253 95 100 105 Gin Gin Leu Gin His Leu Glu Thr Cys Leu Leu Gin Val val Gly CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 110 115 120 Glu Gly Glu Ser Ala Gly Ala lie Ser Ser Pro Ala Leu Thr Leu GAA GGA GAA TC? GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343 125 130 135 Arg Arg Tyr Phe Gin Gly lie Arg Val Tyr Leu Lys Glu Lys Lys AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388 26 Ty r Ser Asp Cys 140 Ala Trp Glu Val Val 145 Arg Met Glu lie Met 150 Lys TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 Ser Leu Phe Leu 155S£ IT TH x Asn Met Gin 160 Glu Arg Leu Arg Ser 165 Lys TCC TTG TTC TTA TCA AC A AAC ATG CAA GAA AGA CTG AGA AGT AAA 478 170 Asp Arg Asp Leu Gly Ser Ser GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 530 10 TAAC ACTTGC ACATGTGACTCTGGTCAATTCAAAAGAC TCTTATTTCGGCTTTAATCAC 589AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 648AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 707TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 766 15 TTTACATTGTATTAAGATAACAAAACATGTTCAGb£ 802 Alul 20 Fragment b: In order to produce fragment b) the plasmid P9A2 is digested with the restriction endonuclease Avail. After chromatography and purification of the resulting cDNA insert, the latter is redigested 2 25 with the restriction endonuclease Sau3A and the desired fragment 189bp long is isolated by chromatography and electroelution. It has the following structure: 5 10 15 30 Asp Leu Pro Gin Asn His Gly Leu Leu Ser Arg Asn Thr LeulGAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 42 Sau3A| Neo I 20 25 30 35 Val Leu Leu His Gin Met Arg Arg lie Ser Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 87 27 Lvs Asp Arg Ar 35 Asp Phe Arg Phe AAG GAC AGA AGA GAC TTC AGG TTC Ser Gin Leu Gin 50 Lvs Ala His Val ACG CAG TTG CAG AAG GGC CAT GTC Leu CTG Gin CAG Gin caI? lie ate Sau3A 40 45 Pro Gin Glu Met Val Lys Gly CCC CAG GAG ATG GTA aaa’ GGG 132 55 60 Met Ser Val Leu His Glu Met ATG TCT GTC CTC CAT GAG ATG 177 189 Fragment c In order to prepare fragment c) the plasmid pER33 (see E« Rastl-Dworkin et aL- Gene 21, 237-248 (1983) and EP-A-0.115.613) is digested twice with the restriction enzymes EcoRI and PvuII. The fragment 389bp long which is obtained after agarose gel fractionation and purification and which contains the Trp promotor p the_ ribosomal binding site and the starting codon, is subsequently digested with Sau3A. The desired fragment 108bp long is obtained by agarose gel electrophoresis, electroelution<§) and Elutip column purification. It has the followingstructure: EcoRI lSau3A qaattcacgctlGATCGCTAAAACATTGTGCAAAAAGAGGGTTGACTTTGCCTTCGCGA 59 5mRWA-Start Met ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG RBS Hindlll Cys Asp TGT gat cSau3A 116 123 28 Ligation of fragments b and c: The fragments b and c are ligated with T4 ligase and? after destruction of the enzyme? cut with Hindlll. This ligated fragment has the following structure: Hindlll Sau3A Neo I ajAGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 50 1 ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 AGATCACACA TCTTTA^qct tSau3A Hindlll Alternatively this DNA fragment necessaryfor the production of the plasmid pRHWlO may alsobe produced by using two synthetically producedoligonucleotides: — The oligonucleotide of formula 5·-AGCTTAAAGATGTGT-3· remains dephosphorylated at its 5s end, The oligonucleotide of formula 5"~GATCACACATCTTTA~3’ is kinased by means of the T^ polynucleotide kinaseand ATP at the 5’ end,. 29 When the two oligonucleotides are hybridised,the following short DMA fragment is obtained: 5 e-AGCTTAAAGATGTGT3 ’ - ATTTCTACACACTAGp 3' 59 This produces at one end the 5’ overlap typicalof Hindi!! and at the other end the 5’ overlaptypical of Sau3A.

Fragment b) is dephosphorylated using calves9intestine phosphatase. Fragment b) and the fragmentdescribed above are combined and joined togetherby means of ligase.

Since the ligase requires at least one endcontaining 59-phosphate, only the synthetic pieceof DMA can be joined to fragment b) or 2 syntheticfragments may be connected at their Sau3A ends.

Since the two resulting fragments are of differentlengths, they may be separated by selective isopropanolprecipitation. The fragment thus purified isphosphorylated using T^ polynucleotide kinase and ATP. II. II. Preparation of the expression plasmids а) Preparation of plasmid pRHWlOs The expression plasmid pERlO3 (E. Rastl-Dworkinet al-? Gene 21, 237-284 (1983) and EP~A-0.115.613,filed at the DSM under DSM Number 2773) is linearisedwith Hindi!! and then treated with calves’ intestinephosphatase. After isolation and purificationof the DMA thus obtained, it is dephosphorylatedand then ligated with ligated fragments b and c(after they have been digested with Hindi!!).

Then E.coli HB 101 is transformed with the resultingligation mixture and cultivated on LB agar plus50 pg/ml of ampicillin. The plasmid having thedesired structure was designated pRHW 10 (see Figure б) , and after replication was used as an intermediate 30 for preparing other plasmids. b) Preparation of the plasmid pRHWl2: The Klenow fragment of the DNA polymeraseX and the 4 deoxynucleotide triphosphates are addedto the plasmid pRHWlO cut with BamHI. The linearisedstraight-ended plasmid obtained after incubationis purified and then cut with NcOI. The largerfragmentr which is obtained using agarose gel electro-phoresis P electroelution and Elutih£^>urificationt-is ligated with fragment a. Then the ligationmixture is transformed with E. coli HB 101 andcultivated on LB agar plus 50 pg/ml of ampicillin.

The plasmid having the structure was designatedpRHWl2 (see Figure 6) which expresses the desiredomega(Gly) interferon.

For example, 1 litre of the incubated bacterial culture thus obtained (optical density: 0-6 at6 600 nm) contains 1 x 10 International Units ofinterferon. c) Preparation of the plasmid pRHWll: The Klenow fragment of DNA polymerase I andthe 4 deoxynucleotide triphosphates are added tothe plasmid pRHWlO cut with BamHI- The linearisedstraight-ended plasmid obtained after incubationis purified and then cut with NcOI. The largerfragmentt, which is obtained using agarose gel electro-phoresis,, electroelution and Elutippurification,,is ligated with fragment a obtained analogouslyfrom the plasmid E76E9,, in which only the GGG codoncoding for the amino acid (Gly) is replacedby the GAG codon (Glu). Subsequently the resultingligation mixture is transformed with E. coli KB101and cultivated on LB agar. The plasmid havingthe desired structure was designated pRHWll (seeanalogously Figure 5) which expresses the desired 31 omega(Glu)-interferon.

Transformation of cells with vectors canbe effected by a number of procedures. For example,it can be carried out by means of calcium? whicheither involves washing cells in magnesium andadding DMA to cells suspended in calcium or exposingcells to a coprecipitate of DNA and calcium phosphate.Following gene expression, the cells are platedon media which select for transformants.

After appropriate transformation of the host,expression of the gene therein and fermentationor cell culture under conditions where IFN-omegais expressed, the product can normally be extractedby means of well known chromatographic separationprocedures to yield a material comprising IFN-omegawith or without leading and trailing sequences.

The IFN-omega may be expressed with a leading sequenceat the N-terminus thereof (to yield pre-IFN-omega) ,which may be removed in some of the host cells.

If not removed, it may be necessary to cleave theleading polypeptide (if^ny is present) to yieldthe mature IFN-omega. Alternatively, the IFN-omegaclone can be modified in such a way that the matureprotein will be directly produced in the microorganisminstead of pre-IFN-omega. In this respect, theprecursor sequence of the yeast mating pheromone —MF-alpfaa-l can be used for precise maturation ofthe fused protein, and for secretion of the productsinto the growth medium or periplasmic space. TheDNA sequence corresponding to functional or matureIFN-omega can be connected to MF-alpha-1 at thepresumed cathepsin-1ike cleavage site (after lys- ,arg) at position 256 from the initiation codonATG (Kurjan, Herskowits, Cell 30, 933-943 (1982)).

On the basis of their biological actions, the new interferons according to the invention are suitable for the treatment of any conditions 32 for which the known interferons have been used.

These include conditions such as herpes? rhinovirus, . opportunistic AIDS infactions, certain cancers,______ and the like- The new interferons can be usedby themselves or in combination with other knowninterferons or other biologically active products,such as IFN-alpha, IL-2, other immune modulators,and the like. IFN-omega may be parenterally administeredto subjects requiring antitumour or antiviral treatment,and to those exhibiting immunosuppressive conditions.Dosage and dose rate may parallel those currentlyin use in clinical investigations of IFN-a materials, e.g. about (1-10) x 10θ units daily, and in the case of materials of purity greater7 than 1%, up to e.g. 5 x 10 units daily.

As one example of an appropriate dosage formfor essentially homogenous bacterial IFN-omegain parenteral form, 3 mg IFN-omega may be dissolvedin 25 ml of 5 N human serum albumin, the solutionis passed through a bacteriological filter andthe filtered solution aseptically subdivided into100 vials, each containing 6 x 10^ units pure IFN-omega suitable for parenteral administration.

The vials are preferably stored in the cold (~20°C)prior to use.

The compounds of the present invention canbe formulated according to known methods to preparepharmaceutically useful compositions, whereby thepolypeptide hereof is combined in admixture witha pharmaceutically acceptable carrier vehicle.

Suitable vehicles and their formulation are describedin Remington’s Pharmaceutical Sciences by E. W.

Martin, to which reference is expressly made. IFN-omega is mixed together with a suitable amountof vehicle in order to prepare pharmaceuticallyacceptable compositions suitable for effective 33 administration to the receiver (patient). The preferred mode of administration is parenteral.

The following examples, which are not exhaustivef will illustrate the invention in greater detail. r 34 Example 1 Finding IFM-sequence-specific clones a) Preparation of a cDMA Library mRMA from Sendai-virus-stimulated cells was used as initial material for the establishment of a cDMA library according to methods known in the literature (E. Dworkin-Rastl et al., Journal of Interferon Research Vol. 2/4 g 575-585 (1982)).

The 30F000 clones obtained were individually transferredinto the wells of microtiter plates. The followingmedium was used for growing and freezing the colonies: 10 g Trypton5 g Yeast Extract5 g MaCl 0.51 g Na-Citrate x 2 H2O----7.5 g K2HPO4 x 2 H2O 1.8 g KH2PO4 0.09 g MgSO4 x 7 HjO 0.9 g (NH4)2SO4 ad 44 g glycerine 0.01 g Tetracycline x HCl 1 1 h2o The microtiter plates with the individualclones were incubated overnight at 37°CJ and werethen stored at -70°C. b) Hybridisation Test As the starting material for the hybridisationtest was used the recombinant plasmid pER33 (E.Dworkin-Rastl et. ai,P Gene 21, 237-248 (1983)).

This plasmid contains the coding region for themature interferon IFN-a2 arg plus 190 basesof the 35 nontranslated region. 20 pg pER33 wereincubated with 30 units of Hind III restrictionendonuclease in 200 pi reaction solution (10 mMTris-HClj. pH-7.5 P 10 mM MgCl2<, 1 mM Dithiotre itol(DTT) t- 50 mM MaCl) for 1 hour at 37°C. The reaction 35 was terminated by the addition of 1/25 vol 0.5 M ethylenedinitrilotetraacetic acid (EDTA) and heating to a temperature of 70°C for 10 minutes,» After the addition of 1/4 vol 5 x buffer (80% glycerine? 40 mM Tris acetate? pH 7.8? 50 mM EDTA? 0.05% Sodium dodecylsulphate (SDS), 0.1% bromophenolblue)? the developing fragments were separatedelectrophoretically according to size in a 1% agarosegel. [Gel and electrophoresis buffer (TBE): 10.8 g/1trishydroxymethylaminomethane (Tris-Base)? 5.5 g/1boric acid? 0.93 g/1 EDTA]. After the incubationof the gel in a 0.5 pg/ml ethidium bromide solution?the DNA strips were made visible in UV-light andthe gel area? which contained the IFN-gene-containingDNA piece (about 800 bp long)? was cut. By applyinga voltage? the DNA was electroeluted into 1/10 x TBEbuffer. The DNA solution was extracted once withphenol and four times with ether? and the DNA wasprecipitated by adding 1/10 vol 3 M sodium acetate(NaAc) pH 5.8 and 2.5 vol EtOH from the aqueoussolution at -20°C. After centrifuging? the DNAwas washed with 70% ethanol and was dried in avacuum for 5 minutes» The DNA was placed in 50 ulof water (about 50 pg/ul). The DNA was markedradioactively by means of nick translation (modifiedaccording—-to T. Maniatis at al.? Molecular Cloning? Ed. CSH). Furthermore? 50 pi incubation solutioncontained the followings 50 mM Tris pH 7.8. 5 mM MgC]^? 1 θ mM mercapfcoethanol, 100 ng DNA insert from pER33? Io pg DNase!? 25 pMdATP? 25 pM dGTP? 25 pM dTTP? 20 pCi a ~32P-dCTP(^/3,,000 Ci/mMol)? as well as 3 units of DNA polymeraseI (E.coli)- Incubation was performed at 14°C for45 minutes. The reaction was terminated throughthe addition of 1 vol 50 mM EDTA? 2% SDS? 10 raMTris pH=7.o solution and heating to 70°C for 10minutes. The DNA was separated by means of chromatography 36 using Sephadex ϋ-100 into TE buffer (10 mM Tris pH=8.0? 1 mM EDTA) from radioactivity that was not internal.

The radioactively marked sample had a specificradioactivity of about 4 x 10^ cpm/pg. c) Screening the Clones for IFN-gene-containing inserts The bacterial cultures, which had been frozeninto the microtiter plates,- were thawed (a) . Apiece of nitrocellulose filter of correspondingsize (Schleicher and Schullr BA 85f 0.45 urn poresize) was placed on LB-agar (LB-agarj 10 g/1 Trypton? 5 g/1 yeast extract t, 5 g/1 NaCl^ 15 g/1 Bacto Agar , 20 mg/1 Tetracycline-HCl). By means of a plungeradapted to the microtiter plates, the individualclones were transferred to the nitrocellulose filter.

The bacteria grew overnight at 37°C to form colonieswith a diameter of about 5 mm. To destroy thebacteria and to denature the DNAt, the nitrocellulosefilters were, one after the other,» placed on astack of Whatman 3mm Filter which had been soakedwith the following solutions: (1) 8 minutes at0.5 M NaOHr (2) 2 minutes at 1 M Tris pH=7.4P (3) 2 minutes at 1 M Tris pH-7.4 and (4) 4 minutes at 1.5 M NaCl,, 0.5 M Tris pH=7.4. The filters were dried in air and were then kept at 80°C for 2 hours. The filters were pretreated for 4 hoursat 65°C in the hybridisation solution? consistingof 6 x SSC (1 x SSC corresponds to 0.15 M MaCl? 0.015 M trisodium citrate? pH = 7.0), 5 x Denhardt’ssolution (1 x Denhardt’s solution corresponds to0.02% PVP (polyvinylpyrrolidone)? 0.02% ficoll (MG: 40,000D)? 0.02% BSM (Bovine Serum Albumin) and 0.1% SDS (sodium dodecylsulphate)- About 1 x 10θ cpmper filter of the sample made in (b) were denaturedby boiling and added to the hybridisation solution.Hybridisation was performed at 65°C for a periodof 16 hours. The filters were washed four times 37 for 1 hour at o5°C with 3 x SSC/0.1S SDS. The filters were dried in air, were covered with SaranWra and exposed on Kodak X-OmatS® film.

Example 2 Southern Transfer to Confirm IFN-gene-containing Recombinant Plasmids Of the positively reacting colonies or thosecolonies that were suspected of reacting positively,. 5 ml cultures were produced in L-broth (10 g/1 Trypton, 5 g/1 Yeast Extract, 5 g/1 NaCl, 20 mg/1 Tetracycline x HC1) at 37°C overnight» The plasmid- DNA was isolated in a modified manner according to Birnboim and Doly (Nucl. Acid. Res. 7, 1513 (1979)). The cells in 1.5 ml suspension were centrifuged (Eppendorf Centrifuge) and resuspended at 0°C in 100 pi lysozyme solution consisting of 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl pH=8.0 and 4 mg/ml oflysozyme. After 5 minutes of incubation at roomtemperature,. 2 vol of ice-cold 0.2 M NaOH, 1% SDSsolution were added, anctincubation continued foranother 5 minutes. Then 150 ul of ice-cold sodiumacetate solution pH=4.8 was added and incubatedfor 5 minutes. The precipitated cell componentswere centrifuged. The DNA solution was extractedwith 1 vol phenol/CHCl^ (1:1), and the DNA wasprecipitated by the addition of 2 vol ethanol.

After centrifuging, the pellet was washed once with 70S ethanol, and dried in a vacuum for 5 minutes.

The DNA was dissolved in 50 pi (TE)-buffer. Ofthat amount, 10 pi were digested in 50 ul reactionsolution (10 mM Tris-HCl pH=7.5, 10 mM MgC^, 50NaCl, 1 mM DTT) with 10 units Pstl-restricfcionendonuclease for 1 hour at 37°C- After the additionof 1/25 vol 0.5 M EDTA as well as 1/4 vol 5 x buffer(see Example lb)), it was heated for 10 minutesand the DNA was then separated electrophoretreally 38 in a 1% agarose gel (TBE-buffer), The DNA in theagarose gel was transferred to a nitrocellulosefilter according to the method of Southern (E. - M. Southern? J- Mol. Biol. 98? 503-517 (1975)) .

The DNA in the gel was denatured by means of 1hour incubation of the gel with a 1.5 M NaCl/0.5 MNaOH solution. This was followed by neutralisationfor 1 hour with a 1 M Tris x HC1 pH=8/1.5 M MaClsolution. The DNA was transferred to the nitrocellulosefilter with 10 x SSC (1.5 M NaCl? 0.15 M sodiumcitratepH=7.0). After completion of transfer(about 16 hours), the filter was briefly rinsedin 6 x SSC buffer and then dried in air; it wasfinally baked at 80°C for 2 hours. The filterwas pretreated for 4 hours with a 6 x SSC/5 x Denhardt’ssolution / 0.1% SDS (see Example 1c) at 65°C. About2 x 10θ cpm of the hybridisation sample (see Examplelb) were denatured by means of heating to a temperatureof 100°C and were then added to the hybridisationsolution. The duration of hybridisation was 16hours at 65°C. Then the filter was washed 4x1hours at 55°C with a—3- x—SSC/0.1% SDS solution.

After air-drying? the filter was covered with SaranWrap® and was exposed on Kodak X-OmatS® film.

Example 3 Detection of Interferon Activity in the Clone Ε76Ξ9 A 100-ml culture of clone E76E9 was culturedin L-broth (10 g/1 Trypton? 5 g/1 yeast extract? -—'δ g/1 NaCl? 5 g/1 glucose? 20 mg per 1 tetracyclinex HC1) at 37°C up to an optical density of Α^θθ=0.8.

The bacteria were centrifuged for 10 minutes at 7,,000 rpm? they were washed once with a 50 mM Tris x HC1 pH=8.0? 30 mM NaCl solution and then were resuspended in 1.5 ml washing solution. After incubation with 1 mg/ml of lysozyme at 0°C for 39 half an hour, the bacterial suspension was frozenand thawed five times. The cells were pelletizedby means of centrifugation at 40,,000 rpm for 1 .hour. The supernatant was sterile filtered and-was tested for interferon activity. The test used was the Plaque Reduction Test with V3 cells andVesicular Stomatitis Virus (G- R. Adolf et al., Arch. Virol. 72, 169-178 (1982)). Surprisingly,the clone produced up to 9,000 IU of interferonper litre of initial culture.

Example 4 Genomic Southern Blot for determining the number of genes associated with the new sequences a) Isolating the DMA from Namalwa cells 400 ml of a^Namalwa cell culture are centrifuged at 1000 rpm in a JA21 centrifuge in order to pelletisethe cells. The resulting pellets are carefullywashed by resuspending them in NP40 buffer (NP40buffer: 140 mM NaCl, 1.5 mM MgClj, 10 mM Tris/Cl pH=7.4) and pelletising them again at 1000 rpm.

The pellets obtained are again suspended in 20 ml solution in order to destroy the cell walls. Afterstanding for 5 minutes in an ice bath the intactcell nuclei are pelletised by centrifuging at 1000 rpmand the supernatant is discarded, The cell nucleiare resuspended in a 10 ml of a solution consistingof 50 mM Tris/Cl pH=8»0, 10 mM SDTA and 200 mMNaClf. and then 1 ml of 20% SDS are added to eliminatethe proteins. The resulting viscous solution isextracted twice with the same quantity of phenol(saturated with 10 mM Tris/Cl pH=8.0) and twicewith chloroform. The DNA is precipitated by adding ethanol,separated off by centrifuging, then the resulting DNA pellet iswashed once with 70% ethanol, dried for 5 minutes in vacuo anddissolved in 6 ml of TE buffer (TE buffer: 10 mM 40 Tris/Cl pH=8.0, 1 mM EDTA). The concentration of the DNA is 0.8 mg/ml. b) Restriction endonuclease digestion of the DNA from Namalwa cells The restriction endonuclease digestion wascarried out in accordance with the conditions specifiedby the manufacturer (New England Biolabs). 1 pg of DNA is digested with 2 units of the suitablerestriction endonuclease in a volume of 10 pi at37°C for 2 hours or longer. EcoRI? Hindlll, BamHI, SphI„ Pstl and Clal are used as restriction endonucleases.20 ug of DNA are used for each digestion. In orderto monitor that the digestion is complete10 pi(aliquot parts) are separated off at the startof the reaction and mixed with 0.4 ug of lambdaphage^-DNA. After 2 hours incubation, these aliquotportions are monitored by agarose gel electrophoresisand the completeness of digestion is assessed withthe aid of a sample of the coloured lambda phaseDNA fragments.

After these checks have been carried outthe reactions are stopped by adding EDTA to a finalconcentration of 20 mM and heating the solutionto 70°C for 10 minutes. The DNA is precipitatedby the addition of 0.3.M NaAc, pH=5.6 and 2.5 volsof ethanol. After 30 minutes incubation at ~70°Cthe DNA is pelletised in an Eppendorf centrifuge,washed once with 70% ethanol and dried. The resultingDNA is taken up in 30 pi of TE buffer. c) Gel electrophoresis and Southern Transfer The digested DNA samples are fractionatedaccording to their size in a 0.8% agar gel in TBEbuffer (10.8 g/1 Tris base, 5.5 g/1 boric acid, 0.93 g/1 EDTA). For this purpose, 15 pi of theDNA sample are mixed with 4 pi of charging buffer 41 (0,02% SDS? 5 x TBE buffer? 50 mM EDTA? 50% glycerine?0.1% bromophenol blue), heated briefly to 70°Cand introduced into the troughs provided in thegel, Lambda-DNA which has been cut with EcoRIand Hindlll is introduced into a correspondingtrough and serves as a marker for the size of theDNA. Gel electrophoresis is carried out for 24hours at about 1 V/cm. Then the DNA is transferredto a nitrocellulose filter using the method ofSouthern (Schleicher and'Schuell, BA85)? usingas the transfer buffer 10 x SSC (1 x SSC: 150 mMtrisodium citrate? 15 mM NaCl? pH=7.0). Afterthe filter has been dried at ambient temperatureit is heated for 2 hours to 80°C in order to bindthe DNA to it. d) Hybridisation probe 20 pg of the plasmid P9A2 are cut with Avail?thereby liberating a fragment approximately 1100 ’fepf"' ··long which contains the entire cDNA insert. ThisDNA fragment is again cut with Sau3A and Alul andthe largest DNA fragment is isolated after agarosegel electrophoresis (see Figure 5b) by electroelution and Elutip^-column chromatography. The resulting DNA (1.5 pg) is dissolved in 15 pi of water. 20 pg of the plasmid pER33 are cut with Hindlll?and this expression plasmid for IFN-c2-Arg (E.

Rastl.-Dworkin et al. Gene 21 ? 237-248 (1983))is cut twice. The smaller DNA fragment containsthe gene for interferon-c,2~Arg and is isolatedin the same way as in the case of the plasmid P9A2.

Both DNA’s are nick-translated using themethod proposed by P.w.J. Rigby et al. (J. Mol.

Biol. 113? 237-251 (1977)). The nick translationis carried out with 0.2 ug of DNA in a solutionof 50 pi? consisting of 1 x nick buffer (1 x nickbuffer: 50 mM Tris/Cl pn=7.2? 10 mM MgSO^? 0.1 mM 42 DTTP 50 pg/ml BSA), 100 umol each of dATP, dGTP and dTTPf 150 uCi α-^^P-dCTP (Amersham, 3000 Ci/mMol) and 5 units of DNA polymerase I (Boehringer-Mannheim, nick translation quality). After 2 hours at 14°C the reaction is stopped by adding the same amount of an EDTA solution (40 mMol) and the unreacted radioactive material is separated off by means of Sephadex G50 column chromatography in TE buffer. Theremaining specific radioactivity is approximately100 x 10^ cpm/pg DNA. e) Hybridisation and autoradiography The nitrocellulose filter is cut into two halves» Each half contains an identical set oftraces with Namalwa DNA which have been treatedwith the restriction enzymes mentioned in Example4a. The filters are pre-hybridised in a solutioncontaining 6 x SSC, 5 x Denhardt’s (1 x Denhardt’s:0.02% bovine serum albumin (BSA), 0.02% polyvinyl-pyrrolidone (PVP), 0.02% Ficoll 400), 0.5% SDS, 0.1 mg/ml of denatured calf thymus DNA and 10 mMEDTA for 2 hours at 65°C. Hybridisation is carriedout in a solution containing 6 x SSC, 5 x Denhardt’s?10 mM EDTA, 0.5% SDS and approximately 10 x 10θ cpmof nick translated DNA for 16 hours at 65°C. Onehalf of the filter is hybridised with interferon-a2-Arg-DNA and the other half with interferon-DNAwhich has been isolated from the plasmid P9A2.

After hybridisation, both filters are washed atambient temperature, four times with a solutionconsisting of 2 x SSC and 0.1% SDS and twice at65°C for 45 minutes with a solution consisting of 0.2 x SSC and 0.01% SDS- The filters are then 6 dried and exposed to a Kodak X-Oraat S film. 43 Example 5 Preparation of the expression plasmids pRHW 12 and PRHW 11 - " Preliminary comment: The preparation of the expression plasmidsis illustrated in Figure 5 (not to scale) , andalso all the restriction enzyme digestions arecarried out in accordance with the instructionsof the enzyme manufacturers. a) Preparation of the plasmid pRHW 10 100 ug of the plasmid Ρ9Ά2 are digested with100 units of the restriction endonuclease Avail(New England Biolabs). After digestion, the enzymeis deactivated by heating to 70°C and the fragmentsobtained are fractionated on a 1.4% agarose gelwith TBE buffer (TBE buffer: 10.8 g/1 Tris base, 5.5 g/1 boric acid, 0.93 g/1 EDTA) according totheir size. The band which contains the entirecDNA insert is electroeluted and purified usingan Elutip-'column (Schleicher & Schuell) . Of the20 ug obtained, δ ug are further digested withthe restriction endonuclease Sau3a (20 units ina total of 100 ul of solution). The fragmentsare separated using 2% agarose gel in TBE buffer.

After staining with ethidium bromide (EtBr) the DNA fragment 189 bp long is electroeluted and purified as described above (= fragment b in Figure S).

In order to link the interferon gene witha promoter,- a ribosomal binding site and a startingcodon, the corresponding DNA fragment is isolatedfrom the expression plasmid pER 33 (E. Rastl-Dworkinet al„, Gene 21, 237-248 (1983)). For this purpose50 ug of pER 33 are digested twice with the restrictionenzymes EcoRI and PvuII and the resulting fragmentsare fractionated according to their size on a 1.4%agarose gel in TBE buffer. The DNA fragment which 44 10 15 20 25 30 is 389 bp long and contains the Trp promotor? the ribosomal binding site and the starting codon? is electroeluted and purified using an Elutip^column.

The fragment thus obtained is then digested withSau3A and the desired fragment 108 bp is obtainedby agarose gel electrophoresis? electroelutionand elutip column purification in a yield of approx-imately 100 ng (= fragment c in Figure 6). 20 ng of fragment b are ligated with 20 ngof fragment c in a volume of 40" pi using 10 unitsof T4 ligase in a solution containing 50 mM Tris/ClpH=7»5? 10 mM MgCl2, 1 mM DTT and 1 mM ATP? at14°C for 18 hours» The enzyme is then destroyedby heating to 70°C and the resulting DNA is cutwith Hindlll in a total volume of 50 ul. 10 pg of the expression plasmid pER 103 (E.

Rastl-Dworkin et al.r Gene 21 ? 237-248 (1983)) is linearised with Hindlll in a total volume of 100 ul. After 2 hours at 37°C 1 volume of 2 x phosphatase buffer (20 mM Tris/Cl pH=9.2? 0.2 mM EDTA) together with one unit of calves intestine phosphatase (CIP) are added. After 30 minutes at 45°Ct- a further unit of CIP is added and the incubation is continued for 30 minutes. The DNA thus obtained is purified by extracting twice with phenol? once with chloroform and precipitating by the addition of 0»3 M sodium acetate (pH=5»5) and 2.5 Vol ethanol- It is then dephosphorylated in order to prevent religation of the vector during the next ligation step. 100 ng of the linearised pER 103 and theligated fragment b and c (after Hindlll digestion)are added to a solution of 100 μΐ which containsligase buffer and ligated with 18 hours at 14°C. 200 pi of competent S. coli HB 101 (E. Dworkinet al.’, Dev. Biol. 75 ? 435-448 (1980)) are mixedwith 20 ul of the ligation mixture and incubatedfor 45 minutes on ice. Then absorption of DNA 35 45 is completed by a heat shock lasting 2 minutesat 40°C. The cell suspension is incubated fora further 10 minutes on ice and finally applied,to LB agar (10 g/1 trypton, 5 g/1 yeast extract, -5 g/1 NaCl, 1.5% agar), containing 50 mg/1 ampicillin.The plasmids contained in the 24 colonies obtainedare isolated using the method of Birnboim and Doly(see Nucl. Acid» Res. 7, 1513-1523 (1979)). Afterdigestion with various restriction enzymes oneplasmid has the desired structure. This is designatedpRHW 10 (see Figure 6). b) Preparation of the plasmid pRHW 12 About 10 pg of the plasmid pRHW 10 are cutwith BamHI. Then the Klenow fragment of DNApolymerase I and "the 4 deoxynucleoside triphosphatesare added and incubated for 20 minutes at ambienttemperature. The linearised and straight-endedplasmid obtained is purified by phenol extractionand precipitation and then cut with the restrictionendonuclease Ncol in a volume of 100 pi. The largerfragment is obtained by agarose -021 electrophoresis,electroelution and elutip purification. The fragmenta) (see Figure 6) is obtained by digestion of 4 ugof Avail fragment which contains the PSA2-CDMAinsert (see above) with Ncol and Alul, therebyobtaining about 2 ug of fragment a).

In the final ligation step, fragment a) andthe pRHW 10 added thereto, which has twice beendigested with BamHI/NcoI, is ligated in a volumeof 10 ul, using 10 ng of each DNA. Ligation ofa filled BamHI cutting site to a DNA cut by Alulagain produces a BamHI cutting site.

The resulting ligation mixture is transformedwith competent E. coli HB 101 as described above-Of the 40 colonies obtained, one is selected? thisis designated pRHW 12. 46 The plasmid is isolated and the EcoRI/BamHIinsert is sequenced using the method of Sanger(F. Sanger et al·, Proc. Nat. Acad. Sci f 5463-5467 (1979)) - This has the expected sequence. c) Preparation of the plasmid pRHW 11 This is carried out analogously to Example5b„ 1 pg of the plasmid pRHW 10 is digested with BamHI. The sticky ends of the resulting DNA areblunted using the Klenow fragment of the DNA polymeraseI and the 4 deoxynucleoside triphosphates and thenthe linearised DNA is cut with Ncol. The largerfragment is obtained by agarose gel electrophoresis,electroelution and SLutip^olumn chromatography.

The Ncol-Alul fragment is isolated from theclone E76E9 analogously to Example 5b. Then 10 ngof the vector part is ligated with 10 ng of thecDNA part in a volume of 10 ul under suitable condTtionsusing 1 unit of T4 ligase. After transformationof the resulting DNA mixture in E. coli HB 101and selection of the 45 resulting colonies on LBplates containing ampicillin, a clone is selected,·which is designated-pRHW 11. After' the correspondingclone has been cultivated, the plasmid DNA is isolated.Its structure is proved by the presence of severalspecific restriction endonuclease cutting sites(Alul, EcoRI, Hinam, Ncol , Pstl) . d) Expression of the interferon activity by E. coli HB 101 corrtaininq the plasmid pRHW 12 100 ml of the bacterial culture are incubatedup to an optical density of 0.6 at 600 nm in M9minimal medium which contains all the amino acidswith the exception of tryptophan (20 pg/ml peramino acid), I ug/ml of thiaminer 0.2% glucoseand 20 pg/ml of indol-(3)-acrylic acid (IAA) , theinductor of the tryphtophan operon. Then the 47 bacteria are pelletised by centrifuging (10 minutesat 7000 rpm) , washed once with 50 m'M Tris/Cl pH=8f30 mM NaCl and finally suspended in 1-5 ml of thesame buffer» After 30 minutes incubation with1 mg/ml of lysozyme on ice the bacteria are frozenand thawed five times. The cell remains are eliminatedby centrifuging for 1 hour at 40,,000 rpm- Thesupernatant is filtered sterile and tested forinterferon activity in a plaque reduction assayusing human A549 cells and encophalo-myocarditisvirus- Result: 1 litre of the bacterial culture producedcontains 1 x 10^ international units of interferon(A, Billiaur Antiviral Res. 4, 75-98 (1984))» Example 6 Summary of the differences between the amino acid and nucleotide sequences of Type I interferons a) Comparison of the amino acid sequences A comparison of amino acid sequences by pairs is obtained by aligning the first cysteine residueof mature a-interferon with the first cysteine residueof the amino acid sequences which are coded bythe cDNA inserts of the plasmids P9A2 and Ξ76Ε9.

The two sequences are shown in Figure 7 as IFN~omega, since no differences could be detected betweenthe specific sequences of the P9A2 or E76E9 clonesin the values obtained- The only correction madewas the insertion of a gap at position 45 of the interferon-cAt. which was counted as an error. _____ If the sequence of the omega-interferon is a partner,,the comparison is carried out taking into accountthe usual 166 amino acids. This value is shownin Figure 7 together with the additional 6 aminoacids coded by the clones P9A2 and Ξ76Ε9» Thepercentage differences are obtained by dividingthe differences by the number 1,66, An additional 48 amino acid thus represents a percentage of 0.6.

This gives 3.8% for the 6 additional amino acidsof IFN-omega which are already contained in the-percentage.

The comparisons with 8-interferon are carriedout by aligning the 3rd amino acid of the mature8-interferon with the first amino acid of the matureα-interferon or the first amino acid which is codedby the plasmids P9A2 and E7SE9. The longest comparisonstructure of an G-interferon with 8-interferonis thus over 162 amino acidse which gives 2 additionalamino acids each for the G-interferon and 8-interferon.These are counted as errors and are shown separatelyin Figure 7 but they are included in the percentage» The listing of 8-interferon with the amino acidsequences of the clones P9A2 or Ε78Ξ9 is carriedout in the same way. However, this gives a totalof 10 additional amino acids. b) Comparison of the nucleotide sequences The sequences which are to be compared arelisted analogously to Example 6b. The first nucleotideof the DNA of the mature G-interferon is the firstnucleotide of the triplet of mature G-interferoncoding for cysteine» The first nucleotide fromthe DNA of the plasmids P9A2 or E76E9 is also the ---first nucleotide of the codon for cysteine-1. Thefirst nucleotide from the DNA of 8-interferon isthe first nucleotide of the third triplet. Thecomparison is made over a total of 498 nucleotidesif the individual DNA's of the G-interferons arecompared with the DNA of e-interferon? and over518 nucleotides if the DNA sequences of the individualG-interferons or of the 8-interferon are comparedwith those of the plasmids P9A2 and Ε76Ξ9. Theabsolute number of gaps is given in the left-handpart of the Table in Figure 7 and then the corresponding 49 percentages are given in brackets.

Example 7 Virus-inducible expression of omega-l-mRNA and NC37 cells a) Synthesis of an omega-interferon specific hybridisation probe 10 pMol of the oligonucleotide d(TGCAGGGCTGCTAA) 32 are mixed with 12 pMol of gamma- P-ATP (specific activity: > 5000 Ci/mMol) and 10 units of polynucleotidekinase in a total volume of 10 pi (70 mM Tris/ClpH = 7.6» 10 mM MgC^» 50 mM DTT) and left to standfor one hour at 37°C. The reaction is then stoppedby heating to 70°C for 10 minutes. The resulting «η radioactively labelled oligonucleotide is hybridisedwith 5 pMol of Ml3pRHW 12 ssDNA (see Figure 9)in a total volume of 35 ml (100 mM NaCl) by standingfor one hour at 50°C.

After cooling to ambient temperature, nicktranslation buffer» the 4 deoxynucleoside triphosphatesand 10 units of Klenow polymerase are added togive a total volume of 50 pi (50 mM Tris/Cl pH= 7.2» 10 mM MgClj? 50 ug/ml BSA» 1 mM per nucleotide) .Polymerisation is carried out by standing for onehour at ambient temperature and then the reactionis stopped by heating to 70°C for 5 minutes.

In the reaction» a partially double-strandedcircular DMA is obtained. This is then cut ina total volume of 500 μΐ with 25 units of Avail»using the buffer described by the manufacturer.

The double stranded regions are cut to uniformsixes. The reaction is then stopped by heatingto 70°C for 5 minutes. b) Preparation of RNA from virus-infected cells 100 x 10° cells (0.5 x 10°/ml) are treatedwith 100 pMol of dexamethasone for 48 to 72 hours 50 - the control contains no dexamethasone. To induceinterferon? the expression cells are suspended in serum-free medium in a concentration of 5 x ΙΟθ/ml 10 . . and infected with 2 units/ml of Sendai virus.Aliquot parts of the cell culture supernatantsare tested for IFN activity in a plaque reductionassay (Example 5b). The cells are harvested 6hours after the virus infection by centrifuging(1000 g? 10 minutes)? washed in 50 ml of NP40^buffer(Example 4a)? resuspended in 9.5 ml of ice coldNP40®buf fer^nd lysed by the addition of 0.5 mlof 10% NP40 ror 5 minutes on ice. After the nucleihave been removed by centrifuging (1000 x gf 10minutes) the supernatant is adjusted to pE=8 with50 mM Tris/Cl? 0.5% Sarcosine^and 5 mM EDTA andthen stored at -20°C. In order to isolate thetotal RNA from the supernatant? it is extractedonce with phenol? once with phenol/chloroform/isoamylalcohol and once with chloroform/isoamyl alcohol.

The aqueous phase is transferred to a 4 ml 5-7molar CsCl pad and centrifuged in an SW40 rotor(35 krpm? 20 hours) in order to free the extractfrom DNA and remaining proteins. The resultingRNA pellet is resuspended in 2 ml of TE? pH=8.0?and precipitated with ethanol. The RNA precipitatedis then dissolved in water at a concentration of5 mg/ml. c) Detection of interferon-omega mRNA 0.2 pi of the hybridisation probe prepared in Example 7b are precipitated together with 20-50 pgof the RNA prepared according to Example 7c bythe addition of ethanol. In the control experiment?instead of the cellular RNA? transfer RNA (tRNA)or RNA originating from E. coli transformed withthe plasmid pRHW 12 (Example 5) is added. Theresulting pellets are washed free from salt with 51 70% ethanol? dried and dissolved in 25 pi of 80%formamide (100 mM PIPES pH=6.8? 400 mM NaCl? 10 mMEDTA)„ Then the samples are heated to 100°C for5 minutes in order to denature the hybridisationsample? adjusted directly to 52°C and incubatedfor 24 hours at this temperature. After hybridisation?the samples are placed on ice and 475 pi of SIreaction mixture (4 mM ZnfAc^? 30 mM NaAc? 250 mMNaCl? 5% glycerine? 20 ug ss calf thymus DNA? 100 units SI nuclease) are added. After digestionat 37°C for 1 hour the reaction is stopped by ethanolprecipitation.

The pellets are dissolved in 8 pi formamidebuffer and separated essentially like samples fromDMA sequencing reactions on a 6% acrylamide gelcontaining 8 M urea (F. Sanger et al.? Proc. Nat.

Acad....Sc i- 74? 5463-5467 (1979)).

For autoradiography? the dried gel is exposedto a DuPont Cronex^-ray film using the Kodak LanexRegular intensivator at -70°C.

Legend relating to Figure 10 Traces A to C represent the controls.

Trace As 20 ug tRNA Trace Bs 10 ug RNA from pER 33 (E. coli - expressionstrain for interferon-c2-Arg) Trace Cs 1 ng RNA from pRHW 12 (E. coli expressionstrain for interferon-omega 1) Trace Ds 50 pg RNA from untreated Namalwa cells Trace E: 50. pg RNA from virus-infected Namalwa cells Trace F s 50 pg RNA from Namalwa cells pretreated with dexamethasone and infected withvirus Trace G: 20 pg of RNA from untreated NC 37 cells.Trace Hs 20 pg RNA from virus-infected NC 37 cellsTrace Is 20 pg RNA from NC 37 cells pretreated 52 with dexamethasone and infected withvirus Trace M: Size marking (pBR 322 cut with. Hinfl); Traces B and C show that the expected signal canonly he detected when an omegal-specific RNA isamong the RNA molecules. They also show that evena large excess of the wrong RNA does not causea background signal (see trace B). Furthermore,the tRNA used as a hybridisation partner does notproduce a signal either (see trace A).

Traces G to I show the induction of the omegal-specific RNA in virus-infected NC 37 cells. Thepretreatment with dexamethasone reinforces thiseffect.

Traces D to F show fundamentally the sameresult as is obtained with Namalwa cells. However,the induction with omegal-specific RNA is not asgreat as in the NC 37 cells. This result is parallelto the interferon titres which were measured inthe associated cell supernatant.

This behaviour of interferon-omegal geneexpression is thus as would be expected from aninterferon Type I gene.

Example &-- Isolation of the gene coding for IFN-omegal or genes related thereto; a) Cosmid screening A human cosmid bank (human DNA (male) cloned in the cosmid vector pcos2 EM'BL (A. Ponstka, H.-R.

Rockwitz, Ά--Μ- Frischaufr B. Hohn, H. Lehrach Proc. Natl. Acad. Sci. 81, 4129-4133 (1984)) withβ a complexity of 2 x 10 ) was searched for IFN~omegaor related genes. E. coli DHl (rK"r rec.A; gyrA96, sup.E) was used as the host. First ofall, Mg+' cells ("plating bacteria") were prepared. 53 Ε. coli DHl grows overnight in L broth (10 g/1 tryptonF 5 g/1 yeast extract, 5 g/1 NaCl) supplementedwith 0.2¾ maltose. The bacteria are removed by-centrifuging and taken up in a 10 mm MgSO^ solutionto give an optical densityggg = 2. 5 ml of this cell suspension are incubated with 12.5 x 10θ colonyforming units of packed cosmids for 20 minutesat 37°C. Then 10 vol of LB are added and the suspensionis kept at 37°C for one hour for the purpose ofexpression of the kanamycin resistance impartedby the cosmid. The bacteria are then removed bycentrifugingj. resuspended in 5 ml of LB and spreadover nitrocellulose filter in 200 ul aliquots (BA85fSchleicher and Schflll, 132 mm diameter) lying onLB agar (1.5S agar in L broth) plus 30 pg/ml kanamycin.About 10,,000 to 20,,000 colonies grow on each filter.

The colonies are replica-plated on further nitrocellulosefilters which are kept at 4°C. A set of the colony filters is processed as described in Example lc) , i.e. the bacteria are denatured, and the single strand DNA is fixed to the nitrocellulose. The filters are washed for 4 hours at 65°C in a 50 mM Tris/HClP pH=8.0r 1 M NaCl, 1 mM EDTA? 0.1S SDS solution. The filters are then incubated at 65°C for 2 hours in a 5 x Dennardt's (see Example le), δ x SSCP 0.1% SDS solution andβ hybridised with about 50 x 10 cpm of nick-translateddenatured IFN-omegal DNA (Hindlil-BamEI insertof the clone PRHW12,, see Figure 6) for 24 hours at 65°C in the same solution. After hybridisation,,______ the filters are washed first 3 x 10 minutes atambient temperature in a 2 x SSCt, 0.01% SDS solutionand then 3 x 45 minutes at S5°C in a 0.2 x SSC^ 0.01% SDS solution. The filters are dried andexposed to Kodak X-Omat S film using an intensifierfilm at -70°C. Positively reacting colonies arelocalised on the replica filters,, scratched off 54 and resuspended in L broth + kanamycin (30 yg/ml) .

Of this suspension, a few ul are spread out on LB agar + 30 pg/ml kanamycin. The resulting colonies are replica-plated on nitrocellulose filters. 32 These filters are hybridised with P-marked IFN- omegal-DNA as described above. From each hybridising colony, the cosmid is isolated using the method described by Birnboim & Doly (Nucl. Acids Res. 7„ 1513 (1979)). With this cosmid DNA preparation, E. coli DHl was transformed and the transformants were selected on LB agar + 30 yg/ml kanamycin. 32 The transformants were again tested with P-radio-actively labelled IFN-omegal DNA for positivelyreacting clones. One clone in each case startingfrom the original material isolated is selectedand the cosmid thereof is produced on a largerscale (Clewellf D.B„ and Helinski, D.R., BiochemistryS), 4428 (1970)) „ Three of the isolated cosmidscarry the names cos9, coslO and cosB. b) Sub-cloning of hybridising fragments in PUC8 1 yg of Cosmids cos9, coslO and cosB werecut with Hindlll under the conditions recommendedby the manufacturer (New England Biolabs). Thefragments are separated on 1% agarose gels in TBEbuffer by electrophoresis and transferred to nitro-cellulose filters according to Southern (Example4c). The two filters are hybridised with nick-translated omegal DNA as described in Example 4d,and washed and exposed. About 20 yg of each cosmidare cut with Hindlll and the fragments formed areseparated by gel electrophoresis. The fragmentshybridising with omegal-ΏΝΑ in the preliminarytests are electroeluted and purified via Elutip-'columns (Schleicher & SchOll). These fragmentsare ligated with Hindlll-linearised dephosphorylatedpUC8 (Messing, J., Vieira, J., Gene 19,, 269-276 55 (1982)} and B. coli JM101 (supE,, thi, (Xae-proAB) i? IF", traD36, pro AB, lac q Z M15) (e-g. F-L. Biochemicals) is transformed with the ligase reaction solution.

The bacteria are spread on LB agar containing 50 pg/ralof ampicillin, 250 pg/ml of 5‘-br©mO"-4-chloro-3-indolyl-S-D-galactopycanoside (BCTG# Sigma) and250 ug/rnl of £sopropyl-8-~D-thiogalacto~pyranoside(IPTG# Sigma). Blue colouration of the coloniesformed indicates the absence of an insert in ©uC8. 10 The plasmid DMA’s were isolated on a small scale from some white clones? then cut with Hindlll and separated on 1% agarose gels. The DNA fragments were transferred to nitrocellulose filters and32 hybridised with F-omegal-DMA as above. Startingfrom cos9 and coslO, a subclone was selected ineach case and designated pRHW22 or pRH57. FromcosBj? two ONA fragments which hybridise well withthe omegal probe were subcloned and designated 20 PRH51 or pRBS2. c) Sequence analysis——___ The DNA inserted in pUC8 is separated from the vector part by cutting with Hindi!! and subsequent 9g gel electrophoresis. This DNA# about 10 pg, is ligated in 50 ul of reaction solution using T4DMA ligase# the volume is adjusted co 350 ul withnick translation buffer (Example 4d) and then decomposed-using ultrasound# whilst cooling with ice (MSB 30 _''"’·~1Ό0 watt ultrasonic disintegrator, maximum output at 20 kHz, five times 30 seconds). Then the endsof the fragments are repaired by adding 1/100 volof 0.5 mM d&TP, dGTP, dCTP and dTTF and 10 unitsof Klenow fragment of the DMA polymerase I for 35 2 hours at 14°C- The resulting DMA fragments having straight ends are separated according to their size on a 1¾ agarose gel. Fragments of sizes between 500 and 1000 bp were isolated and subcloned in 56 the dephosphorylated phage vector M13 mp8 cut withSmal. The single strand DNA of recombinant phagesis isolated and sequenced using the method developedby Sanger (Sanger, F. et al., Proc. Natl. Acad.

Sci 74, 5463-5467 (1976)). The individual sequencesare put together to form the total sequence bymeans of computers (Staden, R. , Nucl. Acids Res. 10, 4731-4751 (1982)). d) Sequence of the subclone PRH57 (IFN-omegal) The sequence is shown in Figure 11. This fragment which is 1933 bp long contains the gene for interferon-omegal. The region coding for protein comprisesthe nucleotides 576 to 1163. The sequence is totallyidentical to that of the cDNA clone P9A2. Thenucleotide portion 576 to 674 codes for a signalpeptide 23 amino acids long. The TATA box is ata spacing characteristic of interferon Type I genesin front of the starting codon ATG (positions 476-482). The gene has a number of signal sequencesfor polyadenylation during transcription (ATTAAAat positions 1497-1502, or 1764-t796; AATAAA atpositions 1729-1734 or 1798-1803), the first ofwhich was used in the clone Ρ9Ά2. e) Sequence of the subclone PRHW22 (IFN-oseudo- omega2) Figure 12 shows the sequence, 2132 bp long,of the Hindi!! fragment from the cosmid cos9 whichhybridises with the omegal-DNA sample. An openreading frame is produced from nucleotide 905 tonucleotide 1366. The amino acid sequence derivedtherefrom is shown. The first 23 amino acids aresimilar to those of the signal peptide of a typicalType I interferon. The following 131 amino acidsshow a similarity to omegal interferon, up to aminoacid 65, whilst tyrosine is notable as the first 57 amino acid of the mature protein.

Following amino acid position 66 is the sequenceof a potential N-glyeosylation site (Asn-Phe—Ser).

From this point onwards the amino acid sequenceis different from that of a Type I interferon.

However, it can be demonstrated that similarityto the IFW-omegal can be established by suitableinsertions and the resulting displacement of theprotein reading frame (see Example 9). Thus^ fromthe standpoint of Type I interferons, the isolatedgene is a pseudogene: IFN-pseudo-omega2. f) Sequence of the subclone PRH51 (IFN-pseudo~omega3) The Hindlll fragment originating from cosmidB and about 3500 bp long which hybridises withthe omegal probe is partially sequenced (Figure 13). -An open reading frame is obtained from nucleotideposition 92 to 394. The first 23 amino acids displaythe features of a signal peptide. The subsequentsequence starts with tryptophan and shows similarityto the IFN"omegal up to amino acid 42. Thereafter,,the derived sequence is different from IFN-omegaland ends after amino acid 78. The sequence canbe altered by insertions so that it is then greatlyhomologous to IFN-omegal (Example 9). The geneis designated IFN~pseudo-omega3. g) Sequence of the insert of pRH52 (IFN-pseudo- omeqa4) The sequence of the Hindlll fragment whichis 3659 bp long,, is isolated from cosmid B and whichhybridises with omegal~DNAt, is shown in Figure 14. An openreading frame the translation product of whichis partially homologous to IFN-omegal is locatedbetween nucleotide positions 2951 and 3250- Aftera signal peptide 23 amino acids long,, the furtheramino acid sequence begins with phenylalanine. 58 Homology to IFN-l Is Interrupted after only the16 th amino acid, continues at the 22nd amino acidand ends at the 41st amino acid. Translation wouldbe possible up to amino acid 77. Analogously toExample 8f) and 8g), good homology can be established-with IFW-omegal 'by- the introduction of·"insertions(Example 9)- The pseudo gene Isolated here isdesignated IFW-pseudo-omega4.

Example 9 Evaluation of the genes for 4 members of the IFN- omega family Figure 15 lists the genes for IFN-omegalto IFN-pseudo-omega4 one below the other togetherwith the amino acid translation. To establishbetter concordance, gaps are inserted In the individualgenes which are indicated by dots- Wo bases areomitted. The numbering of the bases includes thegaps- The amino acid translation of IFW-omegalis retained (e-g- at positions 352-355: "C.AC"codes for His). In the case of the pseudo genes,translation into an amino acid is given only wherethis is clearly possible. This list immediatelyshows that the 4 isolated genes are related toone another- Thus, for example, the potentialW-glycosylafcion point (nucleotide positions 301to 309) is obtained in all 4 genes.

Similarly, apart from the case of IFN-pseudo- omega4, at nucleotide positions 611 to 614, there _____ is a triplet which represents a stop codon andwhich, in the case of IFW-omegal, terminates amature protein with a length of 172 amino acids.Certainly, in this arrangement, there are prematurestop codons in IFW-pseudo-omega2 (nucleotide positions497 to'499) and In IFN-pseudo-omega4 (nucleotidepositions 512 to 514) - 59 The degree of relationship between the genes or theamino acid translations can be calculated from thearrangement shown in Figure 15. Figure 16 .shows the DNAhomologies between the members of the IFN-omega family.

In the comparison in pairs, those positions where one ofthe two partners or both partners have a gap are notincluded in the counting. The comparison gives ahomology of about 85% between IF'N-omegal—DMA and thesequences of the pseudo genes. IFN~pseudo-omega2-DNA isabout 82% homologous to the DNA’s of IFN-pseudo-omega3and IFN-pseudo~omega4„ Figure 17 shows the results ofcomparisons of the signal sequences and Figure 18 showsthe results of the comparisons of the ’’mature" proteins.The latter vary between 72 and 88%. However, thishomology is substantially greater than that between IFN-omega 1 and the IFN-a’s and IFN-0 (Example 6). The factthat the individual members of the IFN-omega family aremore remote from one another than the members of theIFN-g family can be explained by the fact that three ofthe four isolated IFN-omega-genes are pseudo genes andare clearly not subject to^the same selection pressureas functional genes.

Sxample_lO Fermentation Care of the strain: A single colony of the strain H8X01/pRHW12 on LB-agar (with 25 mg/ml of ampicillin) is super-inoculatedwith 25 mg/ml of ampicillin in trypton-soya broth (OXDIDCMX29) and shaken at 250 rpm at 37‘C until an optical—density (546 nm) of about 5 is achieved (logarithmicgrowth phase). Then 10 wt.-% of sterile glycerol areadded, the culture is transferred into sterile ampoulesin 1.5 ml batches and frozen at -70 °C. 60 - Primary culture; The culture medium contains 15 g/1 Na2HPO4 x 12H2O;0.5 g/1 NaCl; 1.0 g/1 NH4C1; 3.0 g/1 KH2PO4;. 0.25 g/1MgSO4 X 7H2O; 0.011 g/1 CaCl2; 5 g/1 caseine hydrolysate(Merck 2238); 6.6 g/1 glucose-monohydrate; 0.1 g/1ampicillin; 20 mg/1 cysteine and 1 ml/1 thiamine-hydrochloride. 4 x 200 ml of this medium, each in a1000 ml Erlenmeyer flask, are inoculated with 1 ml of astock culture of HB101/pRHW14 and shaken for 16 to 18hours at 37°C and at 250 rpm.

Main culture: The medium for fermentation contains 10 g/1(NH4)2HPO4; 4.6 g/1 X 30,0; 0.5 g/1 NaCl; 0.25 g/1 MgSO4 x 7H2O; 0.011 g/1 CaCl2; 11 g/1 glucose- monohvdrate; 21 g/1 casein-hydrolysate (Merck 2238) ; 20 mg/1 cysteine, 1 mg/1 thiamine-hydrochloride and20 mg/1 3-0-indolacrylic acid. 8 litres of sterilemedium in a fermenter with.a total volume of 14 litres(height: radius = 3:1) are inoculated with 800 ml of theprimary culture.

Fermentation is carried out at 28°C, 1000 rpm.(Effigas turbine), at an aeration rate of 1 wm(volume/volume/minute) and at a starting pH of 6.9.During fermentation the pH level falls and is thenmaintained constant at 6.0 using 3N NaOH. Once anoptical density (546 nra) of 18 to 20 is reached (usuallyafter 8.5 to 9.5 hours fermentation), the preparation iscooled to 20°C under otherwise identical conditions andthen adjusted to a pH of 2.2 using 6N H2SO4 (withoutaeration) and stirred for 1 hour at 20°C and at 800 rpm(without aeration). The biomass activated in this wayis then centrifuged at 30,000 rpm in a CEPA laboratorycentrifuge Type GLE and frozen and stored at -20°C. 61 Example 11 Purification of interferon-omega-Gly a) Partial purification All the steps are carried out at 4 °C. 140 g of biomass (Ξ. coli HB1O1 transformed withthe expression clone pRHW12) are resuspended in 1150 mlof 1% acetic acid (pre-cooled to 4°C) and stirred for 30minutes- The suspension is adjusted to pH 10.0 using5 M NaOH and stirred for a further 2 hours. After thepH has been adjusted to 7.5 using 5 M HCl, the mixtureis stirred for a further 15 minutes and then centrifuged(4 °C, 1 hour at 10,000 rpm, J21 centrifuge (Heckman),JA10 rotor).

The clear supernatant solution is poured onto a150 ml CPG(controlled pore glass) column (CPG 10-350,mesh size 120/200) at a rate of 50 ml/h, washed with1000 ml of 25 mM tris/ΙΜ NaCl, pH 7.5, and eluted with25 mM tris/ΙΜ KCNS/50% ethyleneglycol, pH = 7.5(50 ml/h).

The protein peak"coxrcaxning the interferon activityis collected, dialysed against 0.1 M Na-phosphate,pH = 6.0 and 10% polyethyleneglycol 40,000 overnight andthe precipitate formed is removed by centrifuging (4°C, 1 hour, 10,000 rpm, J21 centrifuge, JA20 rotor) (seeTable 1). b) Further purification The dialysed and concentrated CPG eluate is dilutedwith buffer A (0.1 M Na-phosphate pH = 6.25/25% 1,2-propyleneglvcol) 1:5 and applied, by means of at9Superloop9’ injection apparatus (Pharmacia) at a rate of0.5 ml/min, to the MonoS® 5/5 column (Pharmacia, cationexchanger) equilibrated with buffer A. Elution iscarried out with 20 ml of .a linear gradient from 0 to1 M NaCl in buffer A, again at a flow rate of 0.5 ml/min. The through flow and the 1 ail fractions werecollected and tested for interferon activity by means ofa CPS reduction test. The active fractions werecollected.

Table 1 Volume (ml) Biological Test Protein (mg/ml) total (mg) U/mg Ϊ ield g. 0 U/ml+ U total crude 1150 15000 17,3x10® 3,6 4140 4180 100 DL 2200 < 600 < 1,3x10° 0,74 1628 <600 < 5 Eluate 124,3 170000 21,0x10° 16,8 2088 10000 121 after 41 dialysis 300000 12,3χ10δ 12,6 516,6 23800 71 * CPS reduction test; A549 cells, SMC virusU: Units based on interferon-c2 (see Example 2 ofΕΡ-Ά-0.115.613: E. coli HB101 transformed with theexpression clone pSR 33) as standard DL: throughflow Example 12 Characterisation of HuIFN·-omegal A. Antiviral activity on human cells B. Antiviral activity on monkey cells C„ Antiproliferative activity on human Brukittsslymphoma cells (Daudi cell line) 63 D. Antiproliferative activity on human cervicalcarcinoma cells (HeLa cell line) E. , Acid stability F. Serological characterisation A, Antiviral activity on human cells Cell line: Human lung carcinoma cell line A549 (ATCC CCL 185) Virus: Murine encephalomyocarditis virus (EMCV) Test system: Inhibition of cytopathic effect (all titrations were carried out four times) A partially purified preparation of HuIFN-omegalwith a protein content of 9.4 mg/ml was titrated in asuitable dilution in the above-mentioned test system. .zzzrtur.' The preparation showed an antiviral activity with aspecific activity of 8300 Σϋ/rag based on the referencestandard Go-23-901-527. B. Antiviral activity on monkey cells Cell line: G1-V3 monkey kidney cells (Christofinis -----G.J., J. Med. Microbiol. 3, 251-258, 1970) Virus: Vesicular Stomatitis Virus (VSV) Test system: Plaque-Reduction Test (all titrations werecarried out four times) The preparation described in Example 12A showed aspecific antiviral activity of 580 U/mg in the testsystem mentioned. 64 10 15 C. Antiproliferative activity on human Burkitt’slymphoma cells (Daudi cell line) The human Burkitt’s lymphoma cell line Daudi wasgrown in the presence of various concentrations ofHuIFN-omegal. After two, four and six days incubationat 37°C the call density was determined; untreatedcultures' were used as controls. All the cultures werecarried out three times in parallel. The Figure whichfollows shows that IFN-omega exhibits a markedinhibition of cell proliferation at a concentration of100 antiviral units (IU, see Example 12A) per ml; at aconcentration of 10 lU/ml a partial transient inhibitionwas observed (the following symbols are used in theFigure which follows; O control, ο i lU/ml, □ 10 lU/ml,v 100 XU/ml): 20 25 30 65 D, Antiproliferative activity on human cervical -carcinoma cells (cell line HeLa) The human cervical carcinoma cell line HeLa wasgrown in the presence of the following proteins orprotein mixtures: HuIFN-omegal (see Example 12A) 100 IU/ml HuIFN-gamma (see Example 12A) 100 IU/ml Human tumour necrosis factor (HuTNF) >98% pure,made by Genentech Inc., San Francisco, USA (seePennica D. et al., Nature 312, 724-729; 1984) 100 ng/ml All the binary combinations of the said proteins Concentrations as above 2 Cultures were set up in 3 cm plastics tissueculture dishes (50,000 cells per dish) and incubated forsix days at 37°C; then the cell density was determined.Treatment of the cells with HuIFN-omegal or HuTNH-gammahad only a slight effect on cell growth, whereas HuIFN-gamma showed a significant cytostatic effect.

Combinations of IFN-gamma with IFN-omegal showedsynergistic cytostatic/cytotoxic effect.

The following Figure gives the results of theexperiment: 66 10 a 1 i i ΓΠ is s 1 Γ~Τ Mill 1 . 1 - 1 t 1 · . . ; · : 1 1 < 1 1 ; ♦ · t 1 . .7.2 J 2 •dC Ml-==: :·: : |: :: f., :..: ·. -. · ::: · 1:::’j .- ·.:: | ‘::;{.: j : i: (.{4 · 27=:7=127=. i-M • ’·’· . ' · 77: i. i i i 1 :·> ψ::;Ι7Ίΐ!=· 1·: -1 d i 1 ,!:i:: U.f:d: : :·::1: M = Γ -: 1. - :.-2)2:2 ::7 :!Π:ί;Ι;μί7!τ·:Π!κΜ:τί:! ίΚΐ.ΓίϊΗί=1;Η^ϋ\. ί' 1 = :: ί":: 1 . · / : ===1==:1) ' ::51=:=:1 : ::: (-: =/p: j 2 : . 1 i|< = 7:7 ·-·«= 2 : ·. 2 =! ?=ϊτ ΐ:2ί: - -L )22:2p.2 : j :: =:. h:--2.:: ·:: (-=71::/1:. 2: |-l:.; |-f 2 i-i»i::; "-ΞΓ^ΖτϊΤΞγΞ: . Μ: i: /1/ ζ7-ΓΊΓ. . . 2:1 ‘. ί*2:·! · · j - :; ϊ ·. : w ^: ···.·:" J ·iU··! J : *-· · I '·::j · -1 ’· 1 ' : J * ; · - : :Ϊ· - . iitv; 10 ".ν',: τ )=7: Μί-=··|:=·:Ι·===Μ==~:;? 12 ^2-22 = 7221.212=27= Ο:/ ):22-/ }:i:7;2-7./7 r2-|’=T:|* =i’.· L =/) - i 2 i .12:-.-7-7- : Γr2 "=.: till: £7-:=-1=2=12 I is.=272==221 2 22Ξ7 Ml·/: =//': = ) "-.ΛΙ -:=/=/:L·~Γ-Πί 2 L=")="i) 7/227=7:- r=== :rr:· : i".:|·. • I·.:. .. 1: :l . ι "7Γ : ····--(-.:::::1: -Hi 7 r-iJUFr-· 15 ίίΕίΟΜ 1=-. 20 25 M) = • 1.· 4. iii. ./27: 1.=7 il :u: ·„-:1::: 2:122 -·:-- i£L I .: ·: •I - : H Τ'*, r, 4.1. r nr; - ‘1 . ; : J - i :::i : 1 T Γ"ΤΤΓΊ-ΐ 1 . I .- I HHilU.4:?! ?.· 1-: i ---! ·-Ϊ = ! - I t;l - · : I-.' I. .1. -::--1.

:: I 1± "SH 16 w ::·3 · :: .’:v: j · ·.( L 30 A ou 100 " ’ X CELLS / DISH The following symbolsC = untreated controlG = HuIFN-gamma. are used in theT = HuTNF, O = Figures HuIFN-omegal, S„ Acid stability 30 In order to investigate the acid stability of HuIFN-omegal a dilution of the preparation mentioned inExample 12Λ in cell culture medium (U/min.I 1640 with10% foetal calves’ serum) was adjusted to pH 2 usingHCl, incubated at 4°C for 24 hours and then neutralised 35 with NaOH. This sample was titrated in the antiviral test (see Example 12A); it. showed 75% of the biological activity of a control incubated at neutral pH. IFN- - 67 - omega1 can thus he regarded as acid-stable. F. Serological characterisation In order to determine the serological properties of HuIFN-omegal in comparison with HuIFN~c2, samples(diluted to 100 lU/ml) were pre-incubated with equalvolumes of solutions of monoclonal antibodies orpolyclonal antisera for 90 minutes at 37°C; theantiviral activity of the samples was then determined.The following Table shows that HuIFN-omegal can only beneutralised by an antiserum against leukocycteinterferon in relatively high concentrations but cannotbe neutralised by antibodies directed against HuIFN*-a2or HuIFN-beta. HuIFN-omegal is therefore notserologically related either to HuIFN-ck2 or toHuIFNbeta: 68 Antiserum monoclonal antibodies Dilution Mg/xnl Neutralisation of HuIFN-a2 HuIFN-omegal EBI-ln 1 + - 10 100 - 1000 - 0 EBI~3n 1 +++ 10 +++ - 100 +++ - 1000 -++-+ 0 L3B72) 100 4-+4- 0 1000 4* 4-4~ 0 Sheep anti- 1 eukocy te-1FN3 5 1:50,000 +++ - 1: 5,000 0 1: 500 +++ 1: 50 - +++ Rabbit anti- HuIFN-a2 1: 1,000 +++ - 1: 100 +++ 0 1: 10 - 0 Sheep anti- HuIFN-/?° 1: 50 ** 0 Ί) = see SP-Α Ό.. 119.476 25 = A. Berthold et al. in Arzneimitelforschung 35.364-369 (1985) 3) = Research Reference Reagent Catalog No.G-026502-558 = Research Reference Reagent Catalog No.G-028-501-568 Research Resources Branch, National Institute ofAllergy and Infectious Diseases, Bethesda,Maryland, USA. - 69 - Symbols: - not tested, 0 no neutralisation, + partialneutralisation, -r+t complete neutralisation

Claims (5)

70

1. Recombinant DNA molecule containing a coding sequence for new human interferon proteins of type I,containing 168-174 amino acids, for interferon-pseudo-omega2 5 interferon-pseudo~omega3 or interferon-pseudo-omega4 wherein the coding sequence is hybridised under stringent conditions which enable a homologygreater than 85% to be recognised, with the inserts inthe Pst-1 cutting site a) of the plasmid Ξ76Ξ9 in Ξ.coli HB 101, filed at the DSM under the number DSM 3003or b) of the plasmid P9A2 in E. coli HB 101, filed atthe DSM under the number DSM 3004»

2. DNA molecule as claimed in claim 1, wherein the stringent conditions only enable a homologyof more than 90% to be detected.

3. DNA molecule as claimed in claims 1 and 2, wherein the coding sequence is present as an insert in the Pst-1 cutting site a) of the plasmidE76E9 in Ξ. coli HB 101, filed at the DSM under thenumber DSM 3003 or b) of the plasmid P9A2 in E. coli HB101, filed at the DSM under the number DSM 3004, or adegenerate variation of this insert.

4. DNA molecule as claimed in claims 1 to 3, wherein the vehicle is a plasmid which is replicatable in procaryotes or eucaryotes. 5,. DNA molecule according to claim 4, wherein it is an expression vehicle which is replicatablein microorganisms or in mammalian cells. 6. DNA molecule as claimed in claim 5, containing thesequence 71 TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 45 GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 90 AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG ATG GTA AAA GGG 135 AGC CAG TTG CAG AAG GCC CAT GTC nib TCT GTC CTC CAT GAG ATG 180 CTG CAG CAG ATC TTC AGC CTC TTC ACA GAG CGC TCC TCT GCT 225 GCC TGG AAC ATG ACC CTC CTA GAC CTC CAC ACT GGA Qfprp CAT 270 CAG CAA CTG GAA CAC CTG ACC TGC TTG CTG CAG GTA GTG GGA 315 GAA GAA GAA TCT GCT GGG GCA ATT * AGC CCT GCA CTG ACC TTG 360 AGG AGG TAG iprpQ CAG GGA ATC CGT GTC TAG CTG AAA GAG AAG AAA 405 TAG AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 450 TCC TTG TTC TTA TCA ACA AAC ATG GAA AGA CTG AGA AGT AAA 495 GAT AGA GAC CTG GGC TCA TCT 516 7. DNA molecule as claimed in claim 5, wherein the DNA molecule as claimed in claim 6 contains the nucleotide A instead of the nucleotide G in position332 . 8» DNA molecule as claimed in claim 5, wherein it is a) the plasmid Ξ76Ξ9 in E. coli HB 101 filed at the DSM under the number DSM 3003 or b) theplasmid P9A2 in E. coli HB 101, filed at the DSM underthe number DSM 3004. 9 - DNA molecule as claimed in claims 6 and 7, wherein it additionally contains the DNA sequence of formula ATG GCC CTC CTG TTC CCT CTA CTGGCA GCC CTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG GGCcoding for the leader peptide. 72 10. Inrerferon-omegal gene of formula GATCTGGTAAACCTGAA 17GCAAATATAGAAACCTATAGGGCCTGACTTCCTACATAAAGTAAGGAGGGTAAAAATGG 7 δAGGCTAGAATAAGGGTTAAAATTTTGCTTCTAGAACAGAGAAAATGATTTTTTTCATAT 135 5 ATATATGAATATATATTATATATACACATATATACATATATTCACTATAGTGTGTATAC 194 ATAAATATATAATATATATATTGTTAGTGTAGTGTGTGTCTGATTATTTACATGCATAT 253AGTATATACACTTATGACTTTAGTACCCAGACGTTTTTCATTTGATTAAGCATTCATTT 312GTATTGACACAGCTGAAGTTTACTGGAGTTTAGCTGAAGTCTAATGCAAAATTAATAG A 371TTGTTGTCATCCTCTTAAGGTC ATAGGG AGAACACACAAATGAAAACAGTAAAAGAAAC 430 10 TGAAAGTACAGAGAAATGTTCAGAAAATGAAAACCATGTGTTTCCTATTAAAAGCCATG 489CATACAAGCAATGTCTTCAGAAAACCTAGGGTCCAAGGTTAAGCCATATCCCAGCTCAG 548TAAAGCCAGGAGCATCCTCATTTCCCA ATG GCC CTC CTG TTC CCT CTA CTG 599 GCA TGT GCC CTA GTG ATG ACC AGC TAT GGC AGC CTA CCT. GTT GGA TCT CTG GGC 644 689 GAT CTG CCT CAG AAC CAT CTT AGC AGG AAC ACC TTG 15 GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 734 AAG GAC AGA AGA GAC TTC AGG TTC ccc CAG GAG ATG GTA AAA GGG 779 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 824 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 869 GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 914 2C CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 959 GAA GGA GAA TCT GCT GGG GCA ATT__A£C AGC CCT GCA CTG ACC TTG 1004 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 1045 TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 1094 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 1139 25 GAT AGA GAC CTG GGC TCA TCT TGA AATGATTCTCATTGATTAATTTGCCAT 1190 ATAACACTTGCACATGTGACTCTGGTC AATTC AAAAGACTCTTATTTCGGCTTTAATCA 1249C AGAAT7G ACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTT 1308AAAAAG ACTTAGGTTCAGGGGCATC AGTCCCTAAG ATGTTATTTATTTTTACTCATTTA 1367TTTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGC 14 26 30 CTTTACATTGTATTAAGATAACAAAACATGTTCAGCTTTCCATTTGGTTAAATATTGTA 1485 TTTTGTTATTTATTAAATTATTTTCAAAC AAAACTTCTTGAAGTTATTTATTCGAAAAC 1544 CA AAATCCAAACACTAGTTTTCTGAACCAAATCAAGGAATGGACGGTAATATACACTTA 1603 CCTATTCATTCATTCCATTTACATAATATGTATAAAGTGAGTATCAAAGTGGCATATTT 1662TGGAATTGATGTCAAGCAATGCAGCTGTACTCATTGCATGACTGTATCAAAATATCTCA 1721TGTAACCAATAAATATATACACTTACTATGTATCCCACAAAAATTAAAAAGTTATTTTA 1780 35 AAAAAG AAATACAGGTGAATAAACACAGTTTCTTTCCGTGTTGAAGAGCTTTCATTCTT 1839 ACAGGAAAAGAAACAGTAAAGATGTACCAATTTCGCTTATATGAAACACTACAAAGATA 1898AGTAAAAGAAAATGATGTTCTCATACTAGAAGCTT 1933 I 73 11. Interferon-pseudo-omega2-gene of formula AAGCTTGAGCCCCCAGGGAAGCATAACCACATGAACCTGAATGAATATATTCTAGAAGGAGGGAAGCACCAGAGAAGTTCTTTCACTAATAACCATCAACGTCTTCTGTGAATCAAATATCAAACAAAGATAGTCCTAAAAAGTTTAATTTCCAGAGATAGGTAATTTCCTAACTGAATACAGAAACCCATAGGGCCCAGGGATCCTGATTTCCTATGCAAAATGGAGGGTAAAACTGGAGGCTAGGATCTGGGCTAAAAGTATATACTTCTAACAGTAGCACAAAGATGTTTCTCATCTGATTGATCAATATTCATTTGGATTGATATATCTTAAGTTTACTGGGAATATTGAACATCCATTGCAAAAATCAAGAGTGTAGAGTGATGACCTCCTTTTAGGTCATATAGAACAAGGTTTTTCAACCCCCATCCATGGACCGGGGTACTGGTCCTGGCCTGGTAGGAACAGGGCCGCACAGCAGGAGGCAAGCAGGCCAACCAACAAGCATTAACGCCTGAGCTCTGCCTCCTGTCAGATCAGCAGTGGCATTGGA'^i'CTCKAA-AGAiGCAGGAACCCTATTGTGAAGTGCAGATGCGAAGGATCTAGGTTGTGGTCTCCTAATGAGAATCTAATGCCTCTGAAAGCATTCCCTCCCTGACCCCATTTTTCGTGGAAAAATTATCTTCCACCAAACTGGTGGCCAAAAGGTTGTGGATGCTGATATAGAAGACATGTAAATGAAAACAATAAATGGAATTAAAAATTTAGAGAAATGCTCAGAAAAATGAAAACTATTTGTGCTCCATTAAAGCCATGCATAGATAGAATGTCTTCATAGAACCTAGGATCCAAGGTTCTATGAAGACCTCAGCTCAACCAGGCCAAAAGCATCCTGATTTCTCA ATG GCC CTC CTC TTC CCT CTA CTGGCA GCC CTA GAG GTG TGC AGC TGT GGC TCT TCT GGA TCT CTA GGATAT AAC CTG CCT CAG AAC CAT GGC CTG CTA GGC AGG AAC ACC TTGGTG CTT TTG GGC CAA. ATG AGG AGA ATC TCT CGC TTC TTG TGT CTAAAG GAC AGA AGT GAC TTC AGA TTC CCC CAG GAG AAG GTG GAA GTCAGC CAG TTG CAG AAG GCC CAG GCT ATG TCT TTC CTC TAT GAT GTGTTA CAG CAG GTC TTC AAC TTC TCA CAC AAA GCG CTC CTC TGC TGCATG GAA CAT GAC CTT CCT GGA CCA ACT CCA CAC TTT ACG TCA TCAGCA GCT GGA ACA CCT GGA GAC CTG CTT GGT GCA GGA GAT GGG AGAAGG AGA AGC TGG GGG CAG TGG GTG ATT GAG GGC TCT ACA CTG GCCTTG AGG AGG TAT TTC CAG GAA TCC ATC TCT ACC TGA AAGAGAAGAAATAAAGATTGTGCCTGGGAAGTTGTCAGAGTGGAAATCATGAGATCCTTTTCATCCACAAGTTTGCAAGAAAGATTGAGAAGTAAGGATGAAGACCTGGGCTCATCATGAAATGATTCTCATTGACTAATCTGCCATATCACACTTGTACATGTGACTTTGGATATTCAAAAAGCTCATTTCTGTTTCATCAGAAATTATTGAATTAGTTTTAGCAAATACTTTATTAATAGCATAAAGCAAGTTTATGTCAAAAACATTCAGCTCCTGGGGCATCCGTAACTCAGAGAT.AACTGCCCTGATGCTGTTTATTTATCTTCCTTCTTTTTTTTCATGCCTTGTATTTATGATATTTATATATTTTATATTTTCATCTTCACATCGTATTAAAATTTATAAAACATTCACTTTTTCA - 74 - TATTAAGTTTGCATTTTGTTTTATTAAATTCATATCAAAGAAAACTCTGTAAATGTTTC 1852TATTCTAAAAACAATGTCTACTTTCTCTTTTTGTAAACC AAATTGAAAATATGGTAAAA 1911TGTATTAACTCATTCATTTCATTCCTATTATATGTATAAATTGAGTAAATGGCAAACTG 1970TGGGGTTTTCTTAAAGAAATACAGGTGAATAAAGCAAAC ACAGTTTCTCTCAGTCTAAG 2029AGGGAAAGAGACGTAAAAACAGGACAAATATTTATATTATTTCAATTATGTTAAATGCT 2088AC AAAGAGAAGTAAAGAAAAGTGATGTTCTCACATCAGAAGCTT 2132 12. Interferon-pseudo-omeg&3-gene of formula CCATG 5 CATAGCAGGAATGCCTTCAGAGAACCTGAAGTCCAAGGTTCATCCAGACCCCAGCTCAG 64CTAGGCCAGCAGCACCCTCGTTTCCCA ATG GTC CTC CTG CTT CCT CTA CTC 115 GTG GCC CTG CCG CTT TGC CAC TCT GGC CCT GTT GGA TCT CTG AGC 160 TGG GAC CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 205 GCA CTT CTG GGC CAA ATG TGC AGA ATC TCC ACT TTC TTG TGT CTC 250 AAG GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA 295 GTC AGT TGC AGA AGG CCC CGG CCC TGT CTG TCC TCC ATG AGA TGC 340 TTC AGC AGA TCT TCA GCC TCT TCC CCA CAG AGT GCT CCT CTG er©' 383 CCT GGA ACA TGA CCCTCCTGGACCAACTCCACACTGGACTTCATCTGCAGCTGGA 440 ATGCCTGGATGCTTGCTTAGGGCAGACAAAAAGAGAGGAAGAATCTGTGGGGGTGATTG 499 GGGCCCTACACTGGCCTTGAGGACGTACTTTCAGGGAATGCATGGGAATCCAGGGAATC 558TACCTGGAGGAGAAGAAATACAGTGACTGTGCTTGGGAGGTTGTCAGAGTGGAATCGTG 617AAATCCTTCTCTTCATCATCAACAAACTTGCAAGAAGGACTGAGAAGTAAGGATGAAGA 67 5CCTGGGTTCATCCTGAAATTATTCTCATTGATTAATCTGCCATATCACACTTGCACATG 735TGTCTTTGGTCATOCAATAGGTTCTTATTTCTGCAG 772 13. Xnterferon-pseudo-omega4 gene of formula AAGCTTTGGGCATGACCTAGTAGGTGACTCTT 3 2AGTTGGAGTGGTCAGITGTAAGGCTCTTGCTCAGTCACGTGGCTCCCCTGTATTTCCCC 91 ACGTTTGCAGCCCTGCTCCTTCTCAATGCTATGAAAGTGTGGGCTCCTCTCCCACTGGA 150 GTGCTGACTGTAGCTTGTATCTTGGCACTCCCAGGCTGTACATAACAGCTCTGAGGTGA 209 TCTC AGGGTTAATGTTTTCTCCCCGACTTGG AGGCCATTGAAGGAAGGGACCTTAGTAG 268 TAGTTGTAACTGAGGGTCTTTTGCTTGTCTCCTGGGGGCTCCACCCCAG AG ATGC AGGT 327 75 GAGCAATCACTCAGTGCATTCAGCTTGGGATGGGGGTGTCTGTGCTGTGGGCCCAAGAC 386AGGGGTTCCCTGCCTGGTGATGTGAGGGGTGGGTGGTTGACCAATGGCAGACAGACTGG 445CCTCCTCTCTTGGGTTGACAGCAGCTTGTTGGAGGTATGCATAAGGCACTTGGGGTCTT 504GCTCCTTCATTAGTTCCAAGGTAGCTGGGGTAGTACCACTGCAGAGGCAGTGTTAGACA 563 5 GGCTTTCTGTTACCCCTGGGGTCTCCACCTCCTAGAAATGTGAAGTCATGTTAATGGGA 622GTGTTTAGCCAGAGGAGTGGGGTGGCYGCATGCTGGTGTAAGTATGGGACTTCACTTCT 681TGGGAAACAGGGAGGTGGAATCTTACCAGCAGGAGACTGTTCTCCTCACGATGTGTAAC 740CTGCAGTGTGCTGGAAGTTTAGGTGACTGTGGCATGATGTTAGCTTGTAAACAAAGAGC 799TTC AGACTCTTTGTCTCTTCCCCAGACCCAAGGCAGCAAGGATAAAAGCTGCTGCTGTG 858 10 GCAGTGGCAGAGGTAGGATGGTTGTGGGAGCCTCTCCCCAGGGAAACTCTAGTCAACTA 917CCAGTGGATATGCTCAGCCATGGGTAGGGTGACTGTTCTGCAGTCATGGGC AGGGGGCC 976TGCTCCTGAAGAATAGGGACAGGGATTCTCAGGGAAGAGGGGCTGGACTCCTCTTCATA 1035TAGTGGCGA YGATGTGCTGGAGGTGCCAGTGTAGTGAATAGGCCTTTGTTCCTTCCCCA 1094GCC AG AGGGCTGCTGGGGCTGTCCCACTGCAACGGTCATGGTGGATGGGTTATGGGTTG 1153 15 ACTGTGGG ATTTCTTTCTTGGAGA AATGCTGGACTGCCTG ATTG AGGAGACGAGGCAAG 1212 ^GG AAG AATGCCTGTGCTGGAGTCTCTGGTCAGGTGGCTCTGCCACAGAGG AGAGGTGAC 1271 CACC AGGAACTGCGTGGAGAACAGTGTAGCCACTCTTCTGTGAGGCAGTTGCTCTGTTT 1330TGGGGATCTGGAGCAGCCGCTATTCCTTACAGATTCCCAGAGCCTGGAGACAGCAAGGG 1389CAAGAGCTGTGAGAAAGCAAAGATAGCAACCCACCCTTCTCACTGGGAGCTCTGTTCCA 1448 20 GGGAGATGCAGAGCTGCCATTGCTCAATAGCCCCAGCTGGTAGCTGCAGACCCAGGCCT 1507GGCAGACCCACCCAGTGAGCAGATAGGGGATTAGGGACCCACATAACACACAGTCTGGC 1566CACTTTTCCATAGGGCTGCTGAATATGCTGGGGGTCCAATCCAGACCATAGTCACCTCA 1625CATTTTTCAGTACCTGAAGATATCAACAGTGAAGGCTATGAAACAGTGAAGATGGGGAC 1684CTGCCCCTGCCTCTGGACCTCTGTTCCAGAGAGGTACAACCTGTTGCCTCCGACATACA 1743 2 5 TGCAGGAGGTGGCTGGAGACCCGGTGGATATCCCTCCCACTGAGGAGAAGCAGCATCAG 1802 GG AATC AGGTGAAG AAAC AGTCTGGCCACTTTTTGGTAG AGCAGCTGTGCTTGCTGGGG 1861GTCTGCTACCACCCCCAGCAAAAAGAATGGCATTTGCAAGAATGGCTAAGGCTGCTAAA 1920CAGCAACAATGGCAACCTACCATTCCCTTTGGAGCGCCATCCCAGGGATATTCGAAACT 1979GCTGTCCACTAGAAAACAGTGGTGGAGGTGACTGGAGACCCCAGTGGAGAGTTTCACCT 2038 30 GGTGAAAAGAAACAGGATTTGGGATCGACATGAATAACCAATCTGACTGCTTCCCCGTA 209 7GAGCTGCTGGACTGTGCYGGGTGGCTGCTCCAGTCCCTAGCTGCCTTGGACTCCCGAGA 2156ACCCA AAGGCTCC AATAGCT AAG ATTGTG AAAG AGCAAAG ATGGCAGCCCACCCCCTGC 2215CACAGGGAGCTCCATGTCAGGGAGGTATGAGGCTGCTACCAGTGTCTGGCTGGATTCCC 2274AAGTCG AGTGGGTCTTACCCTG AG AC AGGCCATGGAAGGTGGGCCTGTCATTGTC ACTG 2333 35 CCCAGCGCCCTGGATGAAACCCCTTTCCTAGGGGTATGTATAGGGGTCTAGCGTCCTGC 2392 TTGGCTGGAGTTATAGCTTCTTTTGTGGGGAGGCCTGGGTATCTAAGGCTCCAGGGTAC 2451 76 CCATGCATGCGAGAGCGGCTGCTCTGCTGAACCCTACGTAGCCCTGCATGTCAGACTAA 2510ATGCCCTGGTAGAGTGGGTTCACTAGGAGATCTCCTGACCTGAGGATTGCAAAGATCTG 2569TGGGAGAAGCGTGGGTCCCCAGGGCTGCTCACTTACTCACCACTTCCCTGGGCAGGAGA 2628GGCTCCCCTGGCTCTGTGTCATCCTGGGGGGGCAGTTGTCCTGCCTTACTTTGCTTTAT 2687TCTCCATGGGTCAAGTTGTTTTCTTGAGTCTCAATGTGTGCACCTGGTTTTTTCAGTTG 274 6AAGGTGCTGTATTTACTTGCCCCTTCC ATTTCTCTCCATG AGAGTGGC AC AC ACTAGC A 2805GGTTCCAGTCGGCCATCTTGCAACCCCTGAAAACTATTTGTTTCCAGCTATAAGCCATT 2864G AGAG A ACCTGGAGTGGCATAAAAAG AATGCCTCGGGGTTC ATCCCG ACCCC AGCTCAG 2923 CTAGGCCAGCAGCACCCTCGTTTCCCA ATG GTC CTA CTG CTT GTT CTA CTG 2974 GTG GCC CTG CTG CTT TGC CAA TGT GGC CCT GTT GGA TCT CTG GGC 3019 TTT GAC CTG CCT CAG AAC CGT GGC CTA CTT AGC AGG AAC ACC TTG 3064 GCA TTC TGG GCC AAA TGC AGA ATC TCC ACT TTC TTG TGT CTC AAG 3109 GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA GTC 3154 ATT TGC AGA AGG CCC AGG CTG TGT CTG TCC TCC ATG AGA TGC TTC 3199 AGC AGA TCT TCA GCC TCT TCC CCA CAG AGC GCT CCT CTG CTG CCT 3244 GGA AC A TGA CCCTCCTGGACCAGCTCCACACTGGATTTCATCAGCAGCTCGAATAG 3300CCTGGAGTCTTGCTTAGGGCAGGCAACAGGAGAGGAAGAATCTGTGGGGGTGATTGGGA 3359CCCTACACTGGCCTTGAGGAGGTACTTCCAGGGAATCCATGGGAATCC AGAG AATCTAC 3418CTGAAAGAGAAGAAATACAGTGACTGTGCTTAGGAGGTTGTCAGAATGGAATCATGAAA 3477TCCTTCTCTTCATCAACAGACTTGC AAGGACTGAGAAGTAAGGATG AAGACCTGGGGTC 3536TGCTTTACTCTTTCTTATTTTCTTCCTCTTCCTTACTATGTGTTTATTTCTTCTTTTTC 3595TAGTTCCTTAACTTGTAAGTAGTTCACTTGGTTTGAGGTCTTTCTTCTTTTTTAATATA 3654AGCTT . 3659 14. Transformed host organism which contains thegenetic information as claimed in claims 1 to 10. 15. Host organism as claimed in claim 14, wherein the genetic sequence is contained in a vehiclewhich is replicatahle in the host organism. 16. Host organism as claimed in claim 14, wherein it is a mammalian cell line or Ξ. coli. 17. Host organism as claimed in claim 14, wherein it is E. coli with the DSM number 3003 or Ξ.coli with the DSM number 3004. 18. Host organism as claimed in claim 17, wherein the vehicle additionally contains the repliconand control sequences required for expression. 77 19„ Host organism as claimed in claim 18, wherein the vehicle contains the replicon and control sequences of the plasmid pER103 with DSM no- 2773required for expression. 20. Host organism as claimed in claim 20, transformedwith an expression plasmid as claimed in claims 21, 22or 24. 21. Expression plasmid for interferon-omega which is aderivative of plasmid pBR 322, wherein instead of the shorter EcoRI/BamHI fragment inherent inthe plasmid, the plasmid pBR322 contains the DNAsequence of formula EcoRI Sau3A gaattcacgc tGATCGCTAAAACATTGTGCAAAAAGAGGGTTGACTTTGCCTTCGCGA 5 9 .mRNA-Start Met ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG 116 R3S HindiII Cys Asp TGT GAT C - interferon-omega gene- Sau3A 22. Expression plasmid pRHW12 as claimed in claim 21,wherein the interferon-omega-gene contains the DNA sequence of formula 78 TGT GAT CTG CCT CAG AAC CAT GGC Ncol CTA CTT AGC AGG AAC ACC & JL 28 GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 73 AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG. ATG GTA AAM GGG 118 CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC GCT 208 GCC TGG AAC ATG ACC GTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 GAA GGA GAA TCT CCT GAG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343 A/’ AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG A ft ft 388 TAG AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAX ATC ATG AAA 433 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 478 GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 530 1A TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 589AGAATTGACTGAATTAGTTCTGCAAATACTrTCTCGGTATATTAAGCCAGTATATGTTA 648AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTT ATTTTTACTCATTTAT 707TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 7 S 6TTTACATTGTATTAAGATAACAAAACATGTTCAGct 802 AluX 23- Expression plasmid pRHWXl as daisied in claim 21,wherein the interferon-omega-gene contains the DNA sequence of formula TGT GAT CTG CCT <** « /*Vs AAC CAT Neo I GGC • CTA i·· A «« AGC AGG a&c ul^ U> wb & \Z3 28 X CTG CAC CAA AGG AGA ATC TCC CCT A A Vew TTG TGT CTC £ 3 AAG GAC AGA AGA GAC AGC CCC CAG GAG ATG GTA AAA GGG 118 AGC ,1*9 !A CAG AAG GCC CAT GTC ATG a & GTC CTC CAT GAG ATG 163 CTG CAG CAG ATC •WY* Jo A V» AGC CTC B®*STC CAC GAG CGC TCC TCT GOT 208 GCC AAC ATG ACC CTC CTA CAA CTC CAC ACT GGA CTT CAT 253 CAG CAA CTG CAA CAC CTG ,4*·* ft X"’ ft AVV TGC TTG CAG GTA isA Vjs GGA 298 55 79 GAA GGA GAA TCT GCT GAG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388 TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 478 GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 530 TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 589AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCC AGTATATGTTA 648AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 707TTATTCTTACATTTTATCATATTTATACTATTTATATTCTT ATATAACAAATGTTTGCC 766TTTACATTGTATTAAGATAACAAAACATGTTCAGct . 802 Alul 24. Plasmid PRHW10 which is a derivative of plasmidpBP 322, wherein the DNA fragment of formula HindiII aAGCTTAAAG Sau3A Ncol ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 50 ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 AGATCACACA TCTTTAaqct t Sau3A HindIXI is inserted in the plasmid pSP103, DSM no. 2773,,in the Hindlll site. 25. Ξ. coli HB 101 transformed with a plasmid asclaimed in claim 21, 22, 23 or 24. 26. New human type I interferon proteins, wherein they are coded by a DNA molecule as claimed inone of claims 1 to 10. 27. New human interferons as claimed in claim 26, wherein the mature interferons have a 80 divergence of 30 to 50% compared with human α-interferons. 28. New human interferons of type I as claimed in claim 26, wherein a) the mature interferons have a divergence of 30 to 50%compared with human c-interferons and a divergence ofapproximately 70% compared with β-interferon and b) they are 168 to 174 amino acids long, and theN-glycosylated derivatives thereof. 29. Interferons as claimed in claim 28, wherein they have a divergence of from 40 to 48%compared with human α-interferons, and theN-glycosylated derivatives thereof. 30. Interferons as claimed in claims 26 to 29, wheeein they contain a leader peptide. 31. Interferons as claimed in claims 26 to 29, wherein they contain 172 amino acids. 32. Interferon as claimed in claim 31, whereinit contains the amino acid sequence Cys 5 10 Leu Ser Arg Asn Thr 15 Leu Asp Leu Pro Gin Asn His Gly Leu 20 25 30 Val Leu Leu His Gin Met Arg Arg lie Ser .Pro Phe Leu Cys Leu 35 40 45 Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu Met Val Lys Gly 81 50 '55 60 Ser Gin Leu Gin Lvs Ala His Val Met Ser Val Leu His Glu Met Leu Gin Gin 65 Phe Ser Leu Phe His 70 Glu Arg Ser Ser 75 Ala Ala Trp Asn Met 80 Thr Leu Leu Asp Gin 85 Leu His Thr Gly Leu 90 His Gin Gin Leu Gin 95 His Leu Glu Thr Cys 100 Leu Leu Gin Val Val 105 Gly Glu Gly Glu Ser 110 Ala Gly Ala lie Ser 115 Ser Pro Ala Leu Thr 120 Leu Arg Arg Tyr & β λ *— 125 Gin Gly X x θ Arg Val i· 130 Tyr Leu Lys Glu Lys 135 Lys Tyr Ser Asp Cys 140 Ala Trp Glu Val Val 145 Arg Met Glu Xie Met 150 Lys Ser Leu Phe Leu 155 Ser Thr Asn Met Gin 160 Glu Arg Leu Ser 165 Lys Asp Arg Asp Leu 170 Gly Ser Ser and the derivatives thereof N-glycolysed in amino acidposition 78. 33. Interferon as claimed in claim 31, wherein the interferon as claimed in claim 32 contains inposition 111 the amino acid Glu instead of Gly, and thederivatives thereof N-glycolysed in amino acid position78. 34. Interferons as claimed in claims 30 to 33, wherein they contain the leader peptide of formula Met Ala Leu Leu Phe Pro Leu LeuAla Ala Leu Val Met Thr Ser Tyr Ser Pro Val Glv Ser Leu Uly . 35. Process for preparing the interferon peptides asclaimed in claims 26 to 34, wherein a suitable host organism is transformed with geneticinformation coding for the interferon proteins as 82 claimed in claims 26 to 34, this information is expressed in order to produce thecorresponding interferon in the host organism and the interferon peptide thus obtained is isolated fromthis host organism. 36. Process as claimed in claim 35, wherein the information is contained in each recombinantDMA molecule as claimed in claims 1 to 10. 37. Process as claimed in claim 36, wherein after transformation the host organism is definedaccording to one of the claims 15 to 20. 38. Process as claimed in claim 35, wherein - the interferon is defined according to claims 32 to 34- 39. Process as claimed in claim 35, wherein E. coli HB101 is used as the host and a plasmid asclaimed in claim 21, 22 or 23 is used as the plasmid. 40. Omega-interferon which can be prepared by theprocess as claimed in claim 35 or 39. 41. Mixture suitable for antitumour or antiviraltreatment, containing in addition to one or more inertcarriers and/or diluents an effective quantity ofinterferon peptide as claimed in one of claims 26 to 34. 42. Use of omega-interferon as claimed in claims 30 to34 for preparing a mixture suitable for antitumour orantiviral treatment. 43. Process for preparing the plasmid pPHWlO, wherein the DMA fragment of formula Hindlll Sau3A Mcol aAGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 50 ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 83 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC AGATCACACA TCTTTAaqct tSau3A HindJII is inserted into the plasmid pER103, DSM no. 27733 at the Hindlll site by ligase reaction*, and the resulting plasmid is transformed for replication inE. coli HB 101 and cultivated» 44. Process for preparing the expression plasmidpRHWl2, wherein the fragment of formula C CAT GGC Neo I CTA CTT AGC AGG AAC ACC TTG GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA TAG AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATATAACACTTGCAGATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCACAGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT TTATTCTTACATTT3?ATCATATTTATACTATTTATATTCTTATATAACAA,ATGTTTGCC TTTACATTGTATTAAGATAACAAAACATGTTCAGct—Alul obtained by digesting the Avail fragment of clone P9A2(DSM no. 3004) with Wool and Alul is inserted by ligasereaction into the larger fragment of plasmid pRHWIO,which contains m the Hindlll site of the plasmidpER103, DSM no. 2773, the DNA fragment of formula 84 Hindlll Sau3A Ncol aAGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA ACACGTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC AGATCACACA TCTTTAaqct tSau3A Hindlll this larger fragment of the plasmid pRKWIO having beenobtained by restriction endonuclease digestion withBamHI, filling the cutting sites with the aid of theKlenow fragment of DNA polymerase I and the 4deoxynucleoside triphosphates and subsequent cuttingwith Ncolt. and the resulting plasmid is transformed and cultivatedin Ξ. coli HB 101 for replication. 45. Process for preparing the expression plasmidpHWRli, wherein the fragment of formula c CATJGGC CTA CTT AGC AGG AAC ACC TTGiS!col GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC AAG GAC AGA AGA GAG TTC AGG TTG CCC CAG GAG ATG GTA AAA GGG AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA GAA GGA GAA TCT GCT GAG GCA ATT AGC AGC CCT GCA CTG ACC AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG TAG AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT GAT AGA GAC CTG GGG TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATATAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCACAGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 85 AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 707TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 766TTTACATTGTATTAAGATAACAAAACATGTTCAGct 802 Alul 5 obtained by digesting the Avail fragment of clone Ξ75Ε9(DSM no. 3003) with Ncol and Alul is inserted by ligasereaction into the larger fragment of plasmid pRHWio,which contains in the Hindi!! site of the plasmid pER103 10 (DSM no. 2773, see Fig. 6) the DNA fragment of formula HindiII Sau3A Ncol aAGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 50 15 ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 20 AGATCACACA TCTTTAagct tSau3A Hindlll 25 this larger fragment of the plasmid pRHWIO having been obtained by restriction endonuclease digestion withBarnHI, filling the cutting sites with the aid of theKlenow fragment of DNA polymerase I and the 4deoxynucleoside triphosphates and subsequent cutting 30 with Ncol, and the resulting plasmid is transformed andcultivated in 3. coli HB 101 for replication. 46. Synergistic mixture according to claim 41,wherein it additionally contains γ~ interferon. 47. Synergistic mixture according to claim 41, wherein it additionally contains human tumour necrosis factor. - 86 - Dated this 31st day of July, 1985. BY:- TOMKINS & CO.,Applicants' Agents, (Signed)

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