NO177863B - Method for Preparation of Recombinant Human IFN Type I, IFN1, IFN-Pseudo-2, -3, and -4, and Expression Vectors - 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

The present invention relates to a method for the production of recombinant DNA molecules that code for new human interferons of type I IFNiol (Figure 1), which contain

168 to 174 amino acids, for interferon-pseudo-u>2 (Figure 12),

for interferon-pseudo-o)3 (Figure 13) or for interferon-pseudo-o)4 (Figure 14); an expression plasmid for IFN-ul, which

is a derivative of plasmid PBR322; and a method for the production of new type I human interferons, wherein the finished interferons have a divergence of 30 to 50% in relation to the human α-interferons, and of their N-glycosylated derivatives.

Interferon is a term that has been coined to describe

a selection of proteins that are endogenous to human cells and are characterized by having partly overlapping and partly divergent biological activities. These proteins modify the body's specific immune reaction, and it is assumed that they contribute to effective protection against viral diseases. The interferons were e.g. divided into three classes, namely a-,

/3- and t-interferon.

Of jS- and 7-interferon only one subtype is known in the human organism (eg S. Ohno et al., Proe. Nati.

Acad. Pollock. 78, 5305-5309 (1981); Gray et al., Nature 295, 503-508 (1982); Taya et al., EMBO Journal 1(8, 953-958 (1982)). In contrast, different subtypes of α-interferon have been described (eg Phil. Trans. R. Soc. Lond. 299, 7-28 (1982)). The finished α-interferons differ among themselves at a maximum of 23% divergence and are approx. 166 amino acids long.

Furthermore, a report of an α-interferon is noteworthy,

which has a surprisingly high molecular weight (26,000 determined by SDS polyacrylamide/gel electrophoresis) (Goren, P. et al., Virol-

and ogy 130, 273-280 (1983)). This interferon is called IFN-a 26K. It has been found that this has the highest speci-

acquired antiviral and anti-cellular activity.

The known interferons appear to work in various diseases, but show little or no effect in many other diseases (eg, Powledge, Bio/Technology, March 1984, 215-228 "Interferon On Trial"). Interferons also cause side effects. For example, investigations into the anti-cancer effect of recombinant α-interferon found that doses of approx. 50 million units, which according to the results of phase I studies were considered safe, produced acute states of confusion, joint pain leading to inability to work, severe fatigue, lack of appetite, loss of orientation, epileptic seizures and liver toxicity. In 1982, the French government banned research with IFN-a after cancer patients who had been treated with it had fatal heart attacks. At least two cardiac deaths have also been reported in recently carried out investigations in America. In addition, it has become clear that at least some of the side effects, such as fever and malaise, are directly related to the interferon molecule itself and cannot be attributed to contamination.

Due to the great hopes that the interferon had raised, and stimulated by the desire to find new interferon-like molecules with reduced side effects, the task of the present invention was to find and prepare such new compounds.

The present invention provides new interferons of type I, which may contain a Leader peptide, and their N-glycosylated derivatives (here called omega-interferon or IFN-u)), which contain 168-174, preferably 172 amino acids, and which have a divergence of 30 to 50%, preferably 40-48% in relation to the hitherto known subtypes of α-interferon, and a divergence of approx. 70% compared to β-interferon, and on the one hand show similar effects to the α-interferon, but on the other hand do not have the many therapeutic disadvantages of known compounds.

The object of the invention is thus the production of new interferons in completely pure form, their non-glycosylated and glycosylated forms, further expression plasmids containing the corresponding gene sequences.

According to the invention, the initially mentioned recombinant DNA molecules are produced by hybridizing cDNA from a B-lymphocyte cell line with, under stringent conditions that give a homology greater than 85%, the IFN-ul insert or a degenerate variant thereof, in The Pstl seat i

a) the plasmid E76E9 with Dep. No. DSM 3003

b) the plasmid P9A2 with Dep. No. DSM 3004

and inserted into an expression vector pRWHIO (Fig. 6), pRWH11

or pRWH12 (Fig. 6) with subsequent transformation of E. coli.

The expression plasmid for IFN-11 is characterized by the fact that the expression plasmid for IFN-11 is a pBR322 plasmid, which instead of the plasmid-specific, shorter EcoRI-BamHI fragment, contains the DNA sequence with the formula:

The method for producing the new human interferons of type I is characterized by the fact that a suitable host organism is transformed with an expression plasmid according to claim 10 with the genetic information that a recombinant DNA molecule according to claims 1 to 6 contains, is expressed, and the thus produced interferon peptide is isolated from this host organism.

A preferred object of the invention is the production of co-interferons with the following formula, which have the corresponding gene sequences:

In position 111, the sequence GGG (which codes for Gly) can be replaced with GAG (which codes for Glu). The preferred molecules also contain derivatives which are N-glycosylated at amino acid position 78.

For example, the two aforementioned o>-interferons may contain the leader peptide of formula

Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly. Comment on the drawings:

Fig. 1. Restriction map of clone E76E9

Fig. 2. Restriction map of clone P9A2

Fig. 3. DNA sequence of clone P9A2

Fig. 4. DNA sequence of clone E76E9

Fig. 5. Genomic Southern Blot analysis in use

of DNA from clone P9A2 as a probe

Fig. 6. Construction of expression clone pRHW12

Fig. 7. Amino acid and nucleotide differences between type I interferons

Fig. 8a. nucleotide composition of the IFN-αA gene

Fig. 8b. nucleotide composition for the IFN-col gene

Fig. 8c. nucleotide composition of the IFN-αD gene

Fig. 9. schematic presentation of synthesis of interferon- subtype-specific probes Fig. 10. detection of interferon-subtype-specific mRNAs

Fig. 11. DNA sequence for the IFN-col gene

Fig. 12. DNA sequence for the IFN-pseudo-w2 gene

Fig. 13. DNA sequence for the IFN-pseudo-co3 gene

Fig. 14. DNA sequence for the IFN-pseudo-u)4 gene

Fig. 15. corrected arrangement of IFN co-gene sequences

Fig. 16. homology of signal sequences

Fig. 17. homology of "mature" protein sequences

Fig. 18. homology of 4 DNA sequences to each other.

According to the invention, the new cj-interferons and the DNA sequences that code for them are obtained as follows: A human B-lymphoma cell line, e.g. Namalwa cells (see G. Klein et al., Int. j. Cancer 10, 44 (1972)), can be activated by treatment with a virus, e.g. Sendai virus to consent you produce α- and β-interferon. If the mRNA produced is isolated from stimulated Namalwa cells, these can serve as a template for cDNA synthesis. In order to increase the yield of interferon-specific sequences during the cloning process, the mRNA preparation cleaves correspondingly according to different lengths of the individual mRNA molecules through a sugar density gradient. Advantageously, the mRNA is collected in the area around 12S (approx. 800 to 1000 bases length of mRNA). In this area, they deposit specific mRNAs for α- and β-interferons. the mRNAs from this gradient region

is concentrated by precipitation and dissolution in water.

The preparation of a cDNA library essentially follows methods known from the literature (see, for example, E. Dworkin-Rastl, M.B. Dworking and P. Swetley, Journal of Interferon Research 2/4, 575-585 (1982)) . The mRNAs are primed by addition of oligo-dT. Then, by adding the four deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) and the enzyme reverse transcriptase in a corresponding buffered solution, cDNA is synthesized for 1 hour at 45°C. By chloroform extraction and chromatography through a gel column, e.g. through Sephadex G50, purified The cDNA/mRNA hybrid. The RNA is hydrolyzed by alkali treatment (0.3 mol NaOH at 50°C for 1 hour), and the cDNA is precipitated after neutralization with acidic sodium acetate solution with ethanol. After addition of the four deoxynucleoside triphosphates and E. coli DNA polymerase I in the corresponding solution, the double-stranded synthesis is carried out, during which the cDNA simultaneously functions as a template and as a primer by hairpin structure formation at its 3'-end (6 hours at 15°C) (see A. Eftratiadis et al., Cell 7, 279 (1976)). After phenol extraction, Sephadex G50 chromatography and ethanol precipitation, the DNA is treated in a suitable solution with the single-chain-specific endonuclease Sl. This breaks down the hairpin structure, as well as cDNA that has not been converted to a double chain. After k loroform extraction and ethanol precipitation, double-stranded DNA (dsDNA) is separated by size on a sugar gradient. For the following cloning steps, preferably only dsDNA with a size of 600 bp and more is used to increase the probability of obtaining only clones containing the complete coding sequence for the new interferons. dsDNA with a length of more than 600 bp is concentrated from the gradient by precipitation with ethanol and dissolution in water.

In order to multiply the dsDNA molecules, these must first be brought into a corresponding carrier and then introduced into the bacterium E. coli. The carrier used is preferably plasmid pBR322 (F. Bolivar et al., Gene 2, 95 (1977)). This plasmid essentially consists of a replicon and two selection markers. These give the host resistance to the antibiotics Ampicillin and Tetracycline (AP<1>, Tcr). The gene for the /3-lactamase (Ap<r>) contains the recognition sequence for the restriction endonuclease Pstl. pBR322 can be cut with Pstl. The protruding 3' ends are extended with the enzyme terminal deoxynucleotid transferase (TdT) when dGTP is present in a corresponding buffered solution. At the same time, the dsDNA is likewise extended with the enzyme TdT using dCTP on the 3' ends. The homopolymer ends of the plasmid and dsDNA are complementary and hybridize for plasmid DNA and dsDNA are mixed in corresponding concentration ratios and under suitable salt, buffer and temperature conditions (T. Nelson et al., Methods in Enzymology 68, 41-50

(1980)).

E. coli strain HB 101 (Genotype F-, hsdS20 (r-B, m-B) recA13, ara-14, proA2, lacYl, galK2, rpsL20 (Smr), xyl-5, mt-1-1, supE44, lambda-) is prepared for uptake of the recombinant carrier dsDNA molecules by washing with a CaCl2 solution. Competent E. coli HB 101 is mixed with the DNA, and after incubation at 0°C, the plasmid DNA thus obtained is transformed by heat shock at 42°C for 2 minutes (M. Dagert et al., Gene 6, 23-28 (1979)). The transformed bacteria are then plated on tetracycline-containing agar plates (10 µg/ml). On this agar, only E. coli HB 101 that has taken up a carrier or recombinant carrier molecule (Tcr) can grow. Recombinant carrier dsDNA molecules impart the Ap<s>Tc<r> genotype to the host, as the information for the /3-lactamase is destroyed by the introduction of the dsDNA into the /9-lactamase gene. The clones are transferred onto agar plates containing 50 µg/ml Ampicillin. Only approx. 3% grew up, meaning that 97% of the clones contained an inserted dsDNA molecule. From 0.5 µg of dsDNA, more than 30,000 clones were obtained. 28,600 cones thereof were transferred individually into the dishes of microtiter plates containing nutrient medium, 10 µg/ml tetracycline and glycerol. After the clones were grown therein, the plates are kept at -70°C (cDNA library).

When screening the cDNA library for new interferon gene-containing clones, the clones are transferred after thawing onto a nitrocellulose filter. This filter is on nutrient agar containing tetracycline. After the growth of bacterial colonies, the DNA of the bacteria is fixed on the filter.

The insert from the clone pER33 (E. Rasti et al., Gene 21, 237-248 (1983 - see also EP-A-0,115,613)), which contains the gene for IFN-α2-arg, serves as a probe. By nick- translation, this piece of DNA is labeled radioactively, during which DNA polymerase, dATP, dGTP, dTTP and α-32P-dCTP are used. addition of radioactive probe. The filter is then similarly washed under non-stringent conditions. Through the low hybridization stringency and washing, not only interferon-α2-arg-containing clones are captured, but also other interferon-containing clones, whose sequences may deviate considerably from what is known so far interferon-a. After drying, the filter is exposed to an X-ray film. A blackening clearly above the background level indicates the presence of a clone with interferon-specific sequences.

As the radioactivity signals are of different quality, the positive clones, or the clones suspected of reacting positively, are grown on a small scale. The plasmid DNA molecules are isolated, cleaved with restriction endonuclease Pstl and resolved electrophoretically on an agarose gel according to size (Birnboim et al., Nucl. Acid. Res. 7, 1513 (1979)). The DNA in the agarose gel is transferred according to the method of Southern (E.M. Southern, J. Mol. Biol. 98, 503-517 (1975)) onto a nitro-cellulose filter. This filter's DNA is hybridized with the radioactive, IFN gene-containing, denatured probe. Plasmid 1F7 serves as a control (deposited at DSM under DSM number 23 62), which contains the gene for interferon-α2-arg. The autoradiogram clearly shows that clone E76E9 and clone P9A2 contain a sequence which, under non-stringent conditions, hybridizes with the gene for interferon-α2-arg. To characterize the dsDNA input of the clone E76E9 and E9A2 more closely, the plasmids of this clone are produced on a larger scale. The DNA is cleaved with various restriction endonucleases, thus e.g. with Alul, Sau3A, Bglll, Hinfl, Pstl and Haelll. The resulting fragments are separated in an agarose gel. When compared with corresponding size markers, thus e.g. the fragments formed by cleavage of pBR322 with restriction endonuclease Hinfl and Haelll, respectively, the sizes of the fragments are determined. By mapping according to Smith and Birnstiel (H.O. Smith et al., Nucl. Acid. Res. 3, 2387-2398 (1967)), the order of these fragments is determined. From the thus drawn up restriction enzyme maps (fig. 1 and 2), one surprisingly finds that the inserts of clone E76E9 and P9A2 are hitherto unknown interferon genes, namely those for co-interferons.

This information about the co-interferon is used to cleave the cDNA insert with appropriate restriction endonucleases. The fragments formed are ligated into the dsDNA form (replicative form) by the bacteriophage M13mp9 (J. Messing et al., Gene 19, 269-276

(1982)), and sequenced with the Sanger dideoxy method

(F. Sanger et al., Proe. Nati. Acad. Sei. 74, 5463-5467

(1977)). The single-stranded DNA from recombinant phages is isolated. After binding of a synthetic oligomer, two chain syntheses are performed in four separate batches using large fragments of DNA polymerase I from E. coli (Klenow fragment). One of the four dideoxynucleoside triphosphates (ddATP, ddGTP, ddCTP and ddTTP) is added to each of the four partial reactions. This leads to statistically distributed chain breaks in the places where the complementary base of the individual dideoxynucleoside triphosphate is located in the template DNA. For this, radioactively labeled dATP is used. After completed synthesis reactions, the products are denatured, and the single-stranded DNA fragments are separated by size in a denaturing polyacrylamide gel

(F. Sanger et al., FEBS Letters 87, 107-111 (1978)). An X-ray film is then exposed on the gel. The DNA sequence of the recombinant M13 phages can be read from the autoradiogram. The sequences of the inserts of the various recombinant phages are processed using suitable computer programs (R. Staden, Nucl. Acid, Res. 10, 4731-4751 (1982)).

Fig. 1 and 2 show the strategy for the sequencing. Fig. 3 shows the DNA sequence of the clone's P9A2 insert, Fig. 4 for clone E76E9. The non-coding DNA strand is shown in the 5'-> 3' direction together with the amino acid sequence deduced from it.

The isolated cDNA from clone E76E9 for co-(Glu-) interferon is 858 base pairs long and has a 3' untranslated region. This region coding for finished co-(Glu-) interferon runs from nucleotide 9 to nucleotide 524. The isolated cDNA from clone P9A2 for co-(Glu-) interferon is 877 base pairs long, below which the sequence coding for finished co-interferon extends from nucleotide 8 to nucleotide 523. The 3' untranslated region in the case of P9A2 extends to the poly-A section.

The DNA sequences that code for finished co-interferon are completely present in clone E76E9 and P9A2. They begin at the N-terminal end with the amino acids cysteine-aspartic acid leucine. Surprisingly, the two finished co-interferons are 172 amino acids long, which clearly differs from the length of the previously known interferons, e.g. 166 (respectively 165) amino acids in α-interferons. Surprisingly, the two co-interferons have a potential N-glycosylation site at amino acid position 78 (asparagine-methionine-threonine).

A comparison of DNA from clone E76E9 and P9A2 shows a difference. The triplet in clone E76E9 that codes for amino acid 111 is GAG and codes for glutamic acid, while this triplet in clone P9A2 is GGG and codes for glycine. The two co-interferon proteins therefore differ in one amino acid and are designated co-(Glu-) interferon (E76E9) and co-(Gly-) interferon (P9A2).

The comparison of the two co-interferons with the previously known human α-interferon subtypes gives the following picture:

E. coli HB 101 with the plasmid E76E9 and E. coli 101 with the plasmid P9A2 have been deposited with the Deutschen Sammlung fur Mikroorganismen (DSM Gottingen) under the number DSM 3003 and DSM 3004 respectively on 3 July 1984.

To substantiate that the newly found clones produce

an interferon-like activity, is cultivated e.g. the clone E76E9

and the lysate of the bacteria after growth is tested in a plaque reaction test. As expected, the bacteria produced interferon-like activity (see example 3).

Furthermore, as support for the fact that the two newly discovered interferons were members of a new interferon family, total DNA was isolated from Namalwa cells and cleaved with different restriction endonucleases.

In this way, the number of genes that code through the cDNAs of the clones P9A2 and E76E9 can be determined. For this purpose, the obtained DNA fragments are cleaved on agarose gel according to the method of Southern (E.M. Southern et al., J. Mol. Biol. 98, 503 - 517

(1975)), are placed on nitrocellulose filters and hybridized under relatively strict conditions with radioactively labeled specific DNA from clone P9A2. The result obtained after hybridization with DNAs from the plasmid P9A2 and pER33 is shown in fig. 5a: Below, the individual tracks are characterized by letters showing the different cleaved DNA probes (E = EcoRI, H=HindIII, B=BamHI, S=SphI, P=PstI, C=ClaI). The left half of the filter was hybridized with the interferon-<X gene probe ("A") and the right half with the cDNA insert from clone P9A2

("0"). The band set that either hybridizes with the α-interferon gene probe or with the new interferon gene probe is different. With the two different probes, no cross-hybridization could be observed when considering the corresponding traces.

In fig. 5b, the cDNA from clone P9A2 and the fragment used for the hybridization are depicted. The cutoff sites of some restriction enzymes are shown (P=PstI, S=Sau3A, A=AluI). The probe comprises only two of the three possible Pstl fragments. The hybridization pattern shows only a hybridizing fragment with approx. 1300 base pairs (bp), belonging to the homologous gene.

The smaller 120 bp fragment had run out of the gel. The band belonging to the 5' part of the gene cannot be detected, as the probe does not contain this region. At least 6 different bands can be detected in the PstI trace. This means that another gene related to the new sequences must be present in the human genome. Should one or more Pstl cut-off sites be present in these genes, it can be expected that at least three further genes could be isolated.

These genes could preferably be isolated by means of hybridization from a human gene library present in a plasmid carrier, phage carrier or cosmid carrier (see example 4e).

It should be mentioned here that the co-interferon produced according to the invention is not only about two ready-made interferons which are described in detail, but also about any modification of these polypeptides which does not affect the IFN-co-activity. These modifications include e.g. shortening of the molecule at the N- or C-terminal end, replacement of amino acids with other residues, whereby the activity does not change significantly, chemical or biochemical bonds of the molecule to other molecules that are inert or active. In the case of the latter modifications, it can e.g. involve hybrid molecules of one or more co-interferons and/or known α- or β-interferons.

In order to be able to compare the differences of the amino acid and nucleotide sequences of the new interferons, especially the co(Glu) and co (Gly) interferon in comparison with the published amino acid nucleotide sequences for the α-interferon and for the /3-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., Proe. Nat., Acad. Pollock. 77, 4003-4006 (1980); K. Tokodoro et al., EMBO J. 3, 669-670 (1984)) the corresponding sequences are arranged in pairs and the differences are counted at the individual positions.

Of the results shown in fig. 7 shows that the DNA sequences of the clones P9A2 and E76E9 are related to the sequences of the type I interferon (e.g. α- and β-interferon). On the other hand, it is shown that the differences in amino acid sequences between the individual interferons of the new sequences are greater than 41.6% and less than 47.0%. The differences between the new sequences and those for the individual interferons and for /3-interferons are of the order of magnitude approx. 70%. Based on the results in example 4, in which the existence of a whole set of related genes is documented, and taking into account the proposed nomenclature for the interferons (J. Vilceck et al., J. Gen. Virol. 65, 669-670 ( 1984)) the cDNA inserts of clones P9A2 and E76E9 are believed to encode a new class of type I interferon designated as interferon-co.

Furthermore, it is substantiated that co-interferon gene expression takes place analogously to that of an interferon type I gene. In addition, the transcription for the individual members of the multigene family a- and co-interferons is examined on the basis of the S1 mapping method (A.J. Berk et al., Cell 12, 721 (1977)) (see example 7), and the it is shown that the expression co-l-mRNA is virus inducible. Since the transcripts of such a gene family only differ by a few bases of approx. 1000, the hybridization alone is not a sufficiently delicate distinguishing feature to separate the different IFN mRNAs.

For this, the mRNA sequences of 9 α-interferons, of interferon-co-1 and of β-interferon are prepared. The bases are indicated here with capital letters that are specific to the top sequence. Such specific locations can easily be found by a trivial computer program. A hybridization probe starting from a specific point can only hybridize perfectly with the mRNA of a specific subtype. All other mRNAs cannot hybridize to the specific point of the subtype. When the hybridization probe is radioactively labeled at its specific 5' end, only the radioactive labels are protected from cleavage by a single-strand specific nuclease (preferably S1 nuclease) that has hybridized with the interferon subtype mRNA for which the probe was designed.

This principle is not limited to interferon, but can, on the contrary, be applied to any type of known sequence having the specific points described in fig. 8.

The above-mentioned specific points for subtypes are in most cases no restriction points, so that the cutting of cDNAs with restriction endonucleases is not suitable for producing subtype-specific hybridization probes. The probes used in this example are therefore prepared by extension of a 5'-end radioactively labeled oligonucleotide which is complementary to the mRNA for interferon-col above the specific point.

From fig. 10 shows that, as expected, col mRNA is inducible in Namalwa and NC37 cells.

The object of the present invention is therefore not only the production of gene sequences that code specifically for the specified co-interferons, but also modifications that can be easily and routinely obtained by mutation, degradation, trans-position or addition. Any sequence encoding the described human co-interferons (ie having the spectrum of biological activity described herein) and degenerate in comparison to that shown can be similarly prepared, and one skilled in the art is able to degenerate -re the DNA sequences of the coding regions. Likewise, any sequence that codes for a polypeptide with the activity spectrum of IFN-co, and that hybridizes with the shown sequences (or parts thereof) under stringent conditions (e.g., conditions that select for more than 85%, preferably more than 90% homology), are produced.

The hybridization is carried out in 6 x SSC/5 x Denhardf's solution/0.1% SDS at 65°C. The degree of stringency is determined in each washing step. Thus, the conditions 0.2 x SSC/0.01%, SDS/65°G are suitable for a selection on DNA sequences with approx. 85% or more homology and for a selection on DNA sequence with more than 90% or more homology the conditions 0.1 x SSC/0.01% SDS/65°C are suitable.

When screening a cosmid library under these conditions (0.2 x SSC), some cosmids are obtained that hybridize with an IFN-col probe. The sequence analysis of restriction enzyme fragments isolated from these gives the authentic IFN-11 gene (see Fig. 11) as well as three further related genes called IFN-pseudo-co2, IFN-pseudo-o)3 and IFN-pseudo-o) 4 (see fig. 12-14) .

The DNA comparisons show an around 85% homology of the pseudo-gene to the IFN-col gene (interferon-col gene).

Furthermore, the IFN-tol gene shows that upon transcription the mRNA contains the information for a functional interferon protein, i.e. it codes for a 23 amino acid long signal peptide with the formula

Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly

which is followed by the finished 172 amino acid long IFN-col.

Interferon-10 genes can be introduced under conditions into any organism that lead to high yields. Suitable hosts and carriers are well known to those skilled in the art, and as an example reference is made to EP-A-0,093,619.

In particular, prokaryotes are preferred for the expression. As an example, E. coli K 12, strain 294 (ATCC No. 31 446) is suitable. Other strains that are suitable are E. coli X1776 (ATCC No. 31,537). Similar to the aforementioned strains, E. coli W3110 (F", Lambda", Prototroph, ATCC no. 27325), bacilli such as Bacillus subtilis and other Enterobacteriaceae such as Salmonella typhimurium or Serratia marcenses and various Pseudo-monads can also be used.

In general, plasmid carriers containing replicon and control sequences derived from species compatible with the host cells can be used in conjunction with these hosts. The carrier normally contains, next to a point of replication, recognition sequences that make it possible to select the transformed cells genotypically. For example, E. coli is normally transformed with pBR322, a plasmid derived from E. coli species (Bolivar, et al., Gene 2, 95 (1977)). pBR322 contains genes for Ampicillin and Tetracycline resistance and thereby provides easy means to identify transformed cells. The pBR322 plasmid or other plasmids must therefore themselves contain promoters or must be sufficiently modified in such a way that they contain promoters that can be used by the microbial organism for expression of its own proteins. The promoters most often used for the production of recombinant DNA contain beta-lactamase (Penicillinase) and lactose promoter systems (Chang et al., Nature, 275, 615 (1978); Itakura et al., Science 198, 1056 (1977) ; Goeddel et al., Nature 281, 544 (1979)) and Tryptophan (trp) promoter systems (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980); EP-A-0,036,776). Although the ones mentioned are the most common promoters, other microbial promoters have also been developed and used. The gene sequence for IFN-10 can e.g. is used under the control of the Leftward promoter of the bacteriophage Lambda (PL). This promoter is a promoter known to be particularly strong and controllable. The control is made possible with the Lambda repressor for which the neighboring restriction cutoff sites are known.

A temperature-sensitive allele for this repressor gene can be inserted into a vector, which contains a complete IFN-w sequence. If the temperature is increased to 42°C, the repressor is inactivated, and the promoter is expressed to its maximum concentration. The sum of mRNA produced under these conditions should be sufficient to obtain a cell which, among its new synthetic ribonucleic acids, contains approx. 10% originating from the PL promoter. In this way, it is possible to establish a clone bank, in which a functional IFM co-sequence is placed in the vicinity of a ribosome binding point at varying distances from the Lambda-PL promoter. These clones can then be examined, and the one with the highest yield selected.

Expression and translation of the IFN co-sequence can also take place under the control of other regulatory systems that apply to "homologous" organisms in their non-transformed form. Thus contains e.g. chromosomal DNA from a lactose-dependent E. coli a lactose or lac operon, which enables lactose breakdown by secretion of the enzyme Ø-galactosidase.

The lac control elements can be obtained from the bacterial phage lambda-plac<5> which can infect E. coli. The lac operon of the phage can, by transduction, originate from the same bacterial species. The regulatory system which finds use in the method according to the invention can originate from plasmid DNA which is unique to the organism. The lac promoter-operator system can be induced by

IPTG.

Other promoter-operator systems or parts thereof may equally well be used: e.g. arabinose operator, Colicine Ex operator, galactose operator, alkaline phosphatase operator, trp operator, xylose-A operator, tac promoter and others.

In addition to prokaryotes, eukaryotic micro-organisms such as yeast cultures can also be used. Saccharomyces cerevisiae is the most widely used of the eukaryotic microorganisms, although several other species are generally available. For expression in Saccharomyces, e.g. the plasmid YRp7 (Stinchcomb et al. Nature 282, 39 (1979); Kingsman et al., Gene 7, 141

(1979); Tschumper et al., Gene 10, 157 (1980)) and the plasmid YEpl3 (Bwach et al., Gene 8, 121-133 (1979)) normally. The plasmid YRp7 contains the TRP1 gene, which is a selection marker for a yeast mutant unable to grow in tryptophan-containing medium; e.g. ATCC No. 44076.

The presence of TRP1 Schadens as a characteristic of the yeast host genome then constitutes an effective aid to detect the transformation, as one cultivates without tryptophan. The situation is similar with the plasmid YEpl3, which contains the yeast gene LEU 2, which can be used for complementing a LEU 2-minus mutant. Suitable promoter sequences for yeast carriers include the 5'-flanking region of the gene of ADH I (Ammerer G., Methods of Enzymology 101, 192-201 (1983)), 3-phosphoglycerate kinase (Hitzeman et al., J. Biol . Chem. 255,2073 (1980)) or other glycolytic enzymes (Kawaski and Fraenkel, BBRC 1008, 1107-1112 (1982)) such as enolase, glyceraldehyde-3-phosphate, dehydrogenase, hexokinase, pyruvate, decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglucose isomerase and -glucokinase. When concentrating suitable expression plasmids, the termination sequence in connection with these genes can likewise be used in the expression vector at the 3' end of the sequence to be expressed to produce polyadenylation and termination of the mRNA.

Other promoters that also have the advantage of transcription controlled by growth conditions are the promoter regions of the gene for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase degrading enzymes associated with nitrogen metabolism, the above-mentioned glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for the reactions of maltose and galactose. Promoters that are regulated by the yeast feeding type locus, e.g. promoters for the genes BARI, MFal, STE2, STE3, STE5 can be used in temperature-regulating systems through the use of temperature-dependent reed mutations.

(Rhine PH.D. in Thesis, University of Oregon, Eugene, Oregon

(1979), Herskowitz and Oshima, The Molecular Biology of the yeast Saccharomyces, Part I, 181-209 (1981), Cold Spring Harbor Laboratory). These mutations affect the expression of yeast resting feeding type cassettes and thereby indirectly feeding type dependent promoters. In general, however, any plasmid carrier containing a yeast-tolerable promoter, origin of replication and termination sequences is suitable. In addition to microorganisms, cultures of multicellular organisms are also suitable host organisms. In principle, any of these cultures can be used, both by vertebrate or invertebrate animal cultures. However, the greatest interest is in vertebrate cells, so that the propagation of vertebrate cells in culture (tissue culture) has become a routine method in recent years (Tissue, Culture, Academic Press, Kruse and Patterson, publishers (1973)). Examples of such useful host cell lines are VERO and HeLa cells, golden hamster ovary (CHO) cells, and W138, BHK, COS-7, and MDCK cell lines. Expression carriers for these cells normally contain (if necessary) a replication site, a promoter located in front of the gene to be expressed, together with a necessary ribosome binding site, RNA splicing site, polyadenylation site and transcriptional termination sequences.

When using mammalian cells, viral material is often used for control functions on the expression carriers. For example, the normally used promoters originate from polyoma Adenovirus 2, and particularly often from Simian Virus 40 (SV 40). The early and late end promoters from SV 40 are also useful, as both are easily obtained from the virus as a fragment, which also contains the viral replication site of SV 40. (Fiers et al., Nature 273, 113 (1978)). Smaller or larger fragments of SV 40 can also be used provided that they contain the approximately 250 bp long sequence that runs from the HindIII cleavage site to the Bgl 1 cleavage site in the viral replication site. In addition, it is also possible and often advisable to use promoter or control sequences which are normally linked together with the desired gene sequences, provided that these control sequences are compatible with the host cell systems.

A replication site can either be produced by corresponding carrier construction to incorporate an exogenous site, e.g. of SV 40 or from other virus sources (e.g. polyoma, adeno, VSV, PVB, etc.) or may be provided by chromosome replication mechanisms from the host cell. If the carrier is integrated into the host cell chromosome, the latter method is usually sufficient.

The gene can nevertheless preferably be expressed in an expression plasmid pER103 (E. Rastl-Dworkin et al., Gene 21, 237-248

(1983) and EP-A-0,115,613 - deposited with DSM under the number DSM 2773 on December 20, 1983), as this carrier contains all regulatory elements leading to a high level of expression of the cloned gene. According to the invention, therefore, in the plasmid pBR322, the plasmid's own shorter EcoRI/BamHI fragment was replaced with the DNA sequence with the formula To achieve the goal of the invention, one proceeds as e.g. shown in fig. 6: I. Production of the individual DNA fragments required here:

Fragment a)

For the production of fragment a) a plasmid containing a gene for IFN-omega is cleaved, e.g. the plasmid P9A2 with the restriction endonuclease Avall. After chromatography and purification of the cDNA insert obtained, this is further cleaved with the restriction endonuclease Ncol and Alul twice, and then isolated by means of chromatography and electroelution. This fragment contains the largest part of the corresponding omega-interferon gene. Thus, e.g. The omega(Gly) interferon gene of clone P9A2 has the following structure:

Fragment b)

To prepare fragment b), the plasmid P9A2 is cleaved with the restriction endonuclease Avall. After chromatography and purification of the obtained cDNA insert, this is cleaved further with the restriction endonuclease Sau3A and the desired 189bp long fragment is isolated by means of chromatography and electroelution. This is the following structure:

Fragment c)

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 cleaved twice with the restriction enzymes EcoRI and PvuII. The 389bp long fragment obtained after agarose gel fractionation and purification, which contains the Trp promoter, the ribosomal binding point and the start codon, is then cleaved with Sau3A. The desired 108bp long fragment is obtained by agarose gel electrophoresis, electroelution and Elutip column purification. This has the following structure:

Ligation of the fragments b) and c)

Fragments b) and c) are ligated with T4 ligase and cut off after destruction of the enzyme with HindIII. This ligated fragment has the following structure:

Alternatively, this DNA fragment which is necessary for the production of the plasmid pRHWI0 can also be produced using two synthetically produced oligonucleotides:

The oligonucleotide of formula

remains dephosphorylated at its 5' end.

The oligonucleotide of formula

kinase is processed using T4~polynucleotide kinase and ATP at the 5' end.

By hybridizing the two oligonucleotides, the following short DNA fragment is obtained:

Thereby, the 5' overhang that is typical for HindIII is formed on one end and the 5* overhang that is typical for Sau3A on the other end.

The fragment b) is dephosphorylated using calf intestinal phosphatase. Fragment b) and the fragment described above are brought together and connected to each other by means of T^-ligase.

As the ligase requires at least one 5'-phosphate-containing end, can

only the synthetic DNA piece with fragment b), or 2 synthetic fragments at their Sau3A ends are joined together. As the two resulting fragments have different lengths, they can be separated by selective isopropanol precipitation. The thus purified fragment is phosphorylated with the help of T4~polynucleotide kinase and ATP.

II. Preparation of the expression plasmids

a) Preparation of the plasmid pRHW 10

The expression plasmid pER103 (E. Rastl-Dworkin et al., Gene 21, 237-384 (1983) and EP-A-0,115,613 - submitted to DSM under DSM number 2773) is linearized with HindIII and then treated with calf intestinal phosphatase . After isolation and purification of the DNA thus obtained, this is dephosphorylated and then ligated with ligated fragments b) and c) (after cleavage with HindIII). E. coli HB 101 is then transformed with the ligation mixture obtained and grown on LB agar plus 50 µg/ml Ampicillin. The plasmid with the desired structure is given the designation pRHWI0 (see fig. 6), which after its replication served as an intermediate for the production of further plasmids.

b) Preparation of the plasmid pRHWI2

To the plasmid pRHWIO which had been cut with BamHI, one puts

Klenow fragment of DNA polymerase I and the four deoxynucleoside triphosphates. The plasmid obtained linearized after incubation and with blunt ends is purified and then cut off with NcoI. The larger fragment, which is obtained by means of agarose gel electrophoresis, electroelution and elutip purification, is ligated with the fragment a). The ligation mixture is then transformed with E. coli HB101 and grown on LB agar plus 50 µg/ml Ampicillin. The plasmid with the desired structure is designated pRHW12 (see Fig. 6), which expresses the desired omega/Gly Joint interferon.

For example, 1 1 of the thus obtained bacterial culture (optical density: 0.6 at 600 nm) contains 1 x 10<6> international units of interferon.

c) Preparation of the plasmid pRHWI1

To the plasmid pRHWIO cut with BamHI sets

man the Klenow fragment of DNA polymerase I and the four deoxynucleoside triphosphates. The plasmid obtained linearized after incubation with straight ends is purified and then cut off with NcoI.

The larger fragment, which is obtained by means of agarose gel electrophoresis, electroelution and elutip purification, is ligated with fragment a) which is analogously prepared from plasmid E79E9, in which only the GGG codon coding for the 111th amino acid (Gly) is replaced with The GAG codon (Glu). E. coli HB 101 is then transformed with the ligation mixture obtained and grown on LB agar. The plasmid which has the desired structure is named pRHWI1 (see analogously to Fig. 6) which expresses the desired omega (Glu) interferon.

Transformation of the cells with the carriers can be achieved through several methods. For example, it can be done with calcium, during which either the cells are washed in magnesium and the DNA is added to the cells suspended in calcium, or the cells are exposed to a coprecipitate of DNA and calcium phosphate. Upon subsequent gene expression, the cells are transferred to media that selects for the transformed cells.

After complete transformation of the host, expression of the gene and fermentation or cell cultivation under conditions under which IFN-omega is expressed, the product can normally be extracted by known chromatographic separation methods to thus obtain a material containing IFN-omega with or without leader and tail sequences. IFN-omega can be expressed with a leader sequence at the N-terminus (Pre-IFN-omega), which can be removed from some host cells. If not, cleavage of the leader polypeptide (if present) is necessary to obtain complete IFN-omega. Alternatively, the IFN-omega clone can be modified so that the finished protein is produced directly in the microorganism instead of pre-IFN-omega. In this case, the precursor sequence of the yeast maturation pheromone MF-alpha-1 can be used to achieve a correct "maturation" of the fused proteins and excretion of the products in the growth medium or in the periplasmic space. The DNA sequence for functional or finished IFN-omega can be joined with MF-alpha-1 at the putative cathepsin-like cut-off point (after Lys-Arg) in position 256 from the start codon ATG (Kurjan, _ Herskowitz, Cell 30, 933 - 943 (1982)).

On the basis of their biological spectrum of action, the new interferons are applicable for any type of treatment for which the known interferons are also suitable. This includes e.g. herpes, rhinovirus, opportunistic AIDS infections, various types of cancer and the like. The new interferons can be used alone or in combination with other known interferons or biologically active products, e.g. with IFN-α, IL-2, other immune modulators and the like.

or antiviral treatment is necessary, and in cases where immunosuppressive properties are shown. Dosage and dosage rate may be similar to what currently applies to clinical investigations of IFN-α materials, e.g. approx.

(1-10) x IO<6> units daily, and in preparations that are less than 1%

clean, up to e.g. 5 x 10<7> units daily.

For example, 3 mg of IFN-omega can be dissolved in 25 ml of 5% human serum albumin for an appropriate dosage form for an essentially homogeneous, bacterially produced IFN-omega for parenteral use. This solution is then passed through a bacteriological filter, and the filtered solution is distributed aseptically into 100 bottles, each of which contains 6 x 106 units of pure IFN-omega suitable for parenteral use. The glasses are stored.

preferably cold (-20°C) before use. The substance can be formulated in a known manner into pharmaceutically usable agents, during which the produced polypeptide is mixed with a pharmaceutically acceptable carrier substance. Useful carriers and their formulation are described by E.W. Martin at Remington's Pharmaceutical Sciences. IFNto- is mixed with a measured amount of the carrier substance to provide suitable pharmaceutical agents suitable for effective use in the recipient (patient). Preferably, a parenteral application is made.

The following examples describe the invention in detail.

EXAMPLE 1

Selection of IFN sequence-specific clones

a) Preparation of the cDNA library

mRNA from Sendai virus-stimulated cells is used as

starting material for the preparation of a cDNA library according to methods known from the literature (E. Dworkin-Rastl et al., Journal of Interferon Research Vol 2/4, 575 - 585 (1982)). The 30,000 clones formed are transferred individually into the dishes of microtiter plates.

The following medium is used for growth and freezing of colonies:

10 g Tryptone

5 g Yeast extract

5 g NaCl 0.51 g Na citrate x 2 H2O

7.5 g K 2 HPO 4 x 2 H 2 O

1.8 g of KH 2 PO 4

0.09 g MgSO 4 x 7 H 2 O

0.9 g of (NH 4 ) 2 SO 4

44 g Glycerol

0.01 g Tetracycline x HC1

ad 1 1 H2O

The microtiter plates are incubated with the individual clones overnight at 37°C and then stored at -70°C.

b) Hybridization probe

The recombinant plasmid pER33 (E. Dworkin-Rastl et al.,

Gene 21, 237-248 (1983)) serves as starting material for the hybridization probe. This plasmid contains the coding region for the finished interferon IFN-0(2arg plus 190 bases of the 3'-untranslated region. 20 µg of pER33 is incubated with 30 units of HindIII restriction endonuclease in 200 µl of reaction solution (10 rtiM Tris-HCl , pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol (DTT), 50 mM NaCl) for 1 hour at 37° C. The reaction is terminated by adding 1/25 volume of 0.5 M ethylenedinitrotetraacetic acid (EDTA) and heating to 70° C for 10 min. After addition of 1/4 volume 5x buffer (80% glycerol, 40 mM tris-acetic acid pH = 7.8, 50 mM EDTA, 0.05% sodium dodecyl sulfate (SDS), 0.1% bromophenyl blue) the formed fragments are separated electrophoretically according to size in a 1% agarose gel (gel and migration buffer

(TBE): 10.8 g/l trishydroxymethylaminomethane (TrisBase), 5.5 g/l boric acid, 0.9 3 g/l EDTA). After incubation of the gel in a 0.5 ug/ml ethidium bromide solution, the DNA bands are visualized in UV light, and the gel area containing the IFN gene-containing piece of DNA (approx. 800 bp long) is excised. By applying a voltage, the DNA is electroeluted in 1/10 x TBE buffer. The DNA solution is extracted once with phenol and four times with ether, and the DNA is precipitated by adding 1/10 volume of 3M sodium acetate (NaAc)

pH = 5.8 and 2.5 volumes of EtOH from the aqueous solution at -20°C. After centrifugation, the DNA is washed once with 70% ethanol

and dried for 5 minutes in a vacuum. The DNA is taken up in 50 µl of water (approx. 50ug/µl). This DNA is radioactively labeled by nick translation (modified according to T. Maniatis et al., Molecular Cloning, ed. CSH). 50 µl incubation solution contains: 50 mM Tris pH = 7.8, 5 mM MgCl2, 10 mM mercaptoethanol, 100 ng insert DNA of pER33, 16 pg DNasel, 25 uMol dATP, 25 uMol dGTP, 25 uMol dTTP, 20 uCicX 32P-dCTP O3000 Ci/mMol) as well as 3 units of DNA polymerase I (E. coli). Incubation took place at 14°C for 45 minutes. The reaction is terminated by the addition of 1 volume of 50 mM EDTA;

2% SDS, 10 mM Tris pH = 7.6 solution and heating to 70°C

for 10 minutes. The DNA is separated by chromatography through Sephadex G-100 in TE buffer (10 mM Tris pH = 8.0, 1 mM EDTA) from non-incorporated radioactivity. The radioactively labeled probe has a specific activity of approx. 4 x 10' cpm/ng.

c) Selection of the clones with IFN gene content

The bacterial culture that was frozen into microtiter plates

thawed (a). One piece of nitrocellulose filter of similar size (Schleicher and Schiill, BA 85, 0.45 µm pore size) is placed on LB agar (LB agar: 10 g/l Tryptone, 5 g/l yeast extract, 5 g/l NaCl, 15 g/l Bacto-agar, 20 mg/l Tetracycline-HCl. With a stamp adapted to the microtitre plates, the individual clones are transferred to the nitrocellulose filter. The bacteria grow overnight at 37°C to colonies with a diameter of about 5 mm. To destroy the bacteria and to denature the DNA, the nitrocellulose filters are placed 1 order on a stack with the Whatman 3MM filter permeated with the following solutions: 1) 8 minutes in 0.5 M NaOH, 2)

2 minutes 1 M Tris pH = 7.4, 3) 2 minutes 1 M Tris pH = 7.4

and 4) 4 min 1.5 M NaCl, 0.5 M Tris pH = 7.4. The filters

air-dried and then kept for 2 hours at 80°C. The pretreatment of the filter took place for 4 hours at 65°C in the hybridization solution consisting of 6 x SSC (1 x SSC corresponds to 0.15 M NaCl; 0.015 M trisodium citrate; pH = 7.0), 5 x Denhardt<1>'s solution (1 x Denhardf's solution corresponds to 0.02% PVP (polyvinylpyrrolidone); 0.02% Ficoll (MG: 40,000 D); 0.02% BSM (Bovine serum albumin)

and 0.1% SDS (sodium dodecyl sulfate). About. 1 x 10 cpm per filter of the probe produced in b) is denatured by boiling and added to the hybridization solution. Hybridization takes place for 16 hours at 65°C. The washing of the filter takes place at 65°C 4 x 1 hour with 3 x SSC/0.1% SDS. The filters are air-dried, covered with Såran Wrap and exposed on Kodak's X-OmatS film.

EXAMPLE 2

Southern Transfer for confirmation of IFN gene-containing recombinant plasmids

From the colonies that react positively or are suspected

to react positively, 5 ml cultures are grown in L medium (10 g/l Tryptone, 5 g/l yeast extract, 5 g/l NaCl, 20 mg/l tetracycline x HCl) overnight at 37°C. The plasmid DNA is treated according to Birnboim and Doly (Nucl. Acid Res. 7, 1513, (1979)) and isolated. The cells from 1.5 ml suspension are centrifuged (Eppendorf centrifuge) and resuspended at 0°C in 100 μl lysozyme solution consisting of 50 mM glucose, 10 mM EDTA, 25 mM Tris-

HCl pH = 8.0 and 4 mg/ml lysozyme. After 5 minutes of incubation at room temperature, 2 volumes of ice-cold 0.2 M NaOH are added,

1% SDS solution and incubated for a further 5 minutes. Then 150 ul of ice-cold sodium acetate solution pH = 4.8 is added and incubated for 5 minutes. The precipitated cell components are centrifuged. The DNA solution is extracted with 1 part by volume of phenol/CHCl^ (1:1)

and the DNA is precipitated by the addition of 2 volumes of ethanol. After no joints, the pellet is washed once with 70% ethanol and dried for 5 minutes in a vacuum. The DNA is dissolved in 50 µl (TE) buffer. 10 µl of this is cleaved in 50 µl reaction solution (10 mM Tris-HCl pH = 7.5, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT) with 10 units of Pstl restriction endonuclease for 1 hour at 37°C. After adding 1/25 parts by volume of 0.5 M EDTA and 1/4 parts by volume

5 x buffer (see example 1b)) is heated for 10 minutes at 70°C,

and the DNA is then electrophoretically separated in a 1% agarose gel

(TBE buffer). The DNA in the agarose gel is transferred onto a nitro-cellulose filter according to the method of Southern (E.M. Southern,

J. Mol. Biol. 98, 503-517 (1975)). The DNA in the gel is denatured by incubating the gel for one hour with a 1.5 M NaCl/0.5 M NaOH solution. It is then neutralized for 1 hour with a 1 M Tris

x HC1 pH = 8/1.5 M NaCl solution. The DNA is transferred with 10 x SSC (1.5 M NaCl, 0.15 M sodium citrate, pH = 7.0) onto the nitrocellulose filter. After the end of the transfer (approx. 16 hours), the filter is rinsed briefly in 6 x SSC buffer, then air-dried and then heated for 2 hours at 80°C. The filter is pretreated for 4 hours with a 6 x SSC/5 x Denhardfs solution/0.1% SDS (see example 1c) at 65°o C. Approx. 2 x 10 6 cpm of the hybridization probe (see example 1b) is denatured by heating to 100°C and then introduced into the hybridization solution. The duration of the hybridization is 16 hours at 65°C. The filter is then washed 4 x 1 hour at 65°C with a 3 x SSC/0.1% SDS solution. After air drying, the filter is covered with Såran Wrap and exposed on Kodak X-OmatS film.

EXAMPLE 3

Detection of interferon activity in clone E76E9

A 100 ml culture of clone E76E9 is grown in L medium (10 g/l Tryptone, 5 g/l yeast extract, 5 g/l NaCl, 5 g/l glucose, 20 mg tetracycline x HC1 per 1) at 37°C up to optical density Ag00 = 0.8. The bacteria are centrifuged from 10 minutes at 7000 rpm, washed once with 50 mM Tris x HC1 pH = 8.0, 30 mM NaCl solution and suspended in 1.5 ml of washing solution. After incubation with 1 mg/ml lysozyme at 0°C for half an hour, the bacterial suspension is frozen and thawed five times. The cell remains are centrifuged at 40,000 rpm for one hour. The supernatant is sterile filtered and examined for interferon activity. As a test, the plaque reduction test with ©6 V3 cells and Vesicular stomatitis virus is used (G.R. Adolf et al., Arch. Virol.

72, 169-178 (1982)). Surprisingly, the clone produces up to 9,000 IU of interferon per liters of starting culture.

EXAMPLE 4

Genomic Southern Blot for determination of the number of genes that

is assigned to the new sequences

a) Isolation of the DNA from Namalwa cells

400 ml of a Namalwa cell culture is centrifuged at 1000

rpm in a JA21 centrifuge to pellet the cells. The obtained pellets are carefully washed, resuspended in NP40 buffer (NP40 buffer: 140 mM NaCl, 1.5 mM MgCl2, 10 mM Tris/Cl pH = 7.4) and pelleted again at 1000 rpm. The obtained clumps are resuspended in 20 ml of NP40 buffer and mixed with 1 ml of a 10% NP40 solution to destroy the cell walls.

After standing for 5 minutes in an ice bath, the intact cell nuclei are pelleted by centrifugation at 1000 rpm, and the supernatant is discarded. The cell nuclei are resuspended in 10 ml of a solution consisting of 40 mM Tris/HCl pH = 8.0, 10 mM EDTA and 200 mM NaCl, resuspended and then mixed with 20 ml of 20% SDS to remove the proteins. The resulting viscous solution is extracted twice with the same amount of phenol (saturated with 10 mM Tris/HCl pH = 8.0) and twice with chloroform. The DNA<*> is precipitated by the addition of ethanol, separated by centrifugation, then the obtained DNA clump is washed once with 70% ethanol, dried for 5 minutes in vacuum and dissolved in 6 ml of TE buffer (TE buffer: 10 mM Tris/Cl pH = 8.0, 1 mM EDTA). The concentration of DNA is 0.8 mg/ml.

b) Restriction endonuclease cleavage of the DNA from Namalwa cells

The restriction endonuclease cleavage is always performed according to the conditions specified by the manufacturer.

1 µg of DNA is digested for 1 hour with 2 units of the appropriate restriction endonuclease in a volume of 10 µl at 37°C for 2 hours or longer. EcoRI, HindIII, BamHI, SphI, PstI and ClaI are used as restriction endonucleases. 20 µg of DNA is used for each cleavage. To check the completeness of the cleavage, always separate at the start of the reaction 10 µl (aliquots) and mix with 0.4 µg of lambda phage DNA. After 2 hours of incubation, these aliquots are checked by means of agarose gel electrophoresis and the completeness of cleavage is assessed by means of a pattern of colored lambda phage DNA fragments.

After carrying out these checks, the reactions are stopped by adding EDTA up to a final concentration of 20 mM and heating the solution at 70°C for 10 minutes. The DNA is separated by adding 0.3 M NaAc pH = 5.6 and 2.5 parts by volume of ethanol. After 30 minutes of incubation at -70°C, the DNA is pelleted in an Eppendorf centrifuge, washed once with 70% ethanol and dried. The DNA obtained is taken up in 30 µl TE buffer.

c) Gel electrophoresis and Southern transfer

The cleaved DNA probes are fractionated according to size i

a 0.8% agarose gel in TBE buffer (10.8 g/l Tris base, 5.5 g/l boric acid, 0.93 g/l EDTA). For this, 15 ul of the DNA probe is mixed with 4 ul of loading buffer (0.02% SDS, 5 x TBE buffer, 50 mM EDTA, 50% glycerol, 0.1% bromophenol blue), briefly heated to 70°C and placed in the gels in the present troughs. Lambda DNA, which is cut off with EcoRI and HindIII, is placed in a corresponding trough and serves to mark the size of the DNA. The gel electrophoresis is carried out for 24 hours at approx. 1 V/cm. Then, according to Southern's method, the DNA is placed on a nitrocellulose filter (Schleicher and Schuell, BA85), and 10 x SSC (1 x SSC: 150 mM trisodium citrate, 15 mM NaCl, pH = 7.0) is used as a transfer buffer. After drying the filter at room temperature, it is heated for 2 hours at 80°C to bind the DNA to it.

d) Hybridization probe

20 µg of the plasmid P9A2 is cut off with Avall, under which

an approx. 1100 bp long fragment is released, which contains the entire cDNA insert. This piece of DNA is recut with Sau3A

and Alul and the largest piece of DNA after agarose gel electrophoresis (see Fig. 5b) is isolated by electroelution and elutip column chromatography. The DNA thus obtained (1.5 µg) is dissolved in 15 µl of water.

20 µg of the plasmid pER33 is cleaved with HindIII, during which this expression plasmid for IFN-ofé-Arg (E. Rastl-Dworkin et al., Gene 21, 237-248 (1983)) is cleaved twice. The smaller DNA fragment contains the gene for interferon-0C2-Ai;g and

is isolated in the same way as for the plasmid P9A2.

Both DNAs are nick-translated according to the method proposed by P.W.J. Rigby et al. (J. Mol. Biol. 113, 237 -

251 (1977)). The Nick translation is performed with 0.2 µg DNA in a solution of 50 µl consisting of 1 x Nick buffer (1 x Nick buffer: 50 mM Tris/Cl pH = 7.2, 10 mM MgSO 4 , 0.1 mM DTT , 50 ug/ml BSA), each 100 JiMOl dATP, dGTP and dTTP, 150 uCi d-32P-dCTP

3,000 Ci/mMol) and 5 units of DNA polymerase I

( 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 by means of G50 column chromatography in TE buffer. The remaining specific radioactivity is approx. 100 x 10 cpm/ug DNA:

e) Hybridization and autoradiography

The cellulose filter is cut into two halves. Each half

contains an identical set of tracks with Namalwa DNA treated with the restriction enzyme discussed in Example 4a. The filter is prehybridized in a solution containing 6 x SSC, 5 x Denhardt's (1 x Denhardt's: 0.02% bovine serum albumin (BSA), 0.02% polyvinylpyrrolidone (PVP), 0.02% Ficoll 400), 0, 5% SDS; 0.1 mg/ml denatured calf thymus DNA and 10 mM EDTA for 2 hours at 65°C. The hybridization is carried out in a solution containing 6 x SSC, 5 x Denhardfs, 10 mM EDTA; 0.5% SDS

and approx. 10 x 106 cpm nick-translated DNA for 16 hours at 65°C. One half of the filter is hybridized with interferon-2-Arg DNA and the other half with interferon DNA that was isolated from the plasmid P9A2. After hybridization, both filters are washed at room temperature 4 times with a solution consisting of 2 x SSC and 0.1% SDS and then twice at 65°C for 45 minutes with a solution consisting of 0.2 x SSC and 0.01% SDS . The filters are then dried and a Kodak X-OmatS film is exposed.

EXAMPLE 5

Preparation of the expression plasmids pRHW 12 and pRHW 11

Preliminary remark:

The preparation of the expression plasmids is shown _L fig.

6 (not true to scale), further all restriction enzyme-

cleavages according to the enzyme manufacturer's instructions.

<a>) Preparation<g> of the plasmid pRHW 10

100 µg of the plasmid P9A2 is cleaved with 100 units of the restric ion endonuclease Avall. After cleavage, the enzyme is inactivated by heating to 70°C, the fragments obtained are fractionated on a 1.4% agarose gel with TBE buffer (TBE buffer: 10.8

g/l Tris-Base, 5.5g/l boric acid, 0.93 g/l EDTA) according to size. The bands containing the entire cDNA insert electro-

eluted and purified on an elutip column. Of the 20 µl obtained, 6 µl are further cleaved with the restric ion endonuclease Sau3A (20 units in a total of 100 µl solution). The fragments are separated using 2% agarose gel in TBE buffer. After staining with ethidium bromide (EtBr), the 189 bp DNA piece is electroeluted and purified as described above (= fragment b in fig-6).

To connect the interferon gene to a promoter, a ribosomal binding site and a start codon, the corresponding piece of DNA is isolated from the expression plasmid pER33 (E. Rastl-Dworkin

et al., Gene 21, 237-248 (1983)). For this purpose, 50 µg of pER33 is cleaved with the restriction enzymes Ecol and PvuII twice and the fragments obtained are fractionated according to the size of

1.4% agarose gel in TBE buffer. 389 bp long DNA pieces containing the Trp promoter, the ribosomal binding site and the start codon are electroeluted and purified over an elutip column. The thus obtained fragment is cleaved with Sau3A, and the desired 108 bp long fragment is obtained by means of agarose gel electrophoresis, electroelution and elutip column purification in a yield of approx. 100 ng (= fragment c in Fig. 6).

20 ng of fragment b is ligated with 20 ng of fragment c i

a volume of 40 µl using 10 units of T4 ligase in a solution containing 50 mM Tris/Cl pH = 7.5, 10 mM

MgCl2, 1 mM DTT and 1 mM ATP at 14°C for 18 h. The enzyme is then destroyed by heating to 70°C, and the DNA obtained is cut with HindIII in a number volume of 50ul.

10 µg of expression plasmid pER103 (E. Rastl-Dworkin

et al., Gene 21, 237-248 (1983)) is linearized with HindIII in a total volume of 100 µl. After 2 hours at 37°C, a volume of 2 x phosphatase buffer (20 mM Tris/Cl pH = 9.2, 0.2 mM

EDTA) together with one unit of calf intestinal phosphatase (CIP). After

After 30 minutes at 45°C, a further unit of CIP is added

and incubate again for 30 minutes. The DNA thus obtained is purified by 2 times phenol extraction, once by chloroform extraction and by precipitation using the addition of 0.3 M sodium acetate (pH = 5.5) and 2.5 volumes of ethanol. It is then dephosphorylated to prevent the deligation of the carrier in the next ligation step.

100 ng of the linear pER103 and the ligated fragments

b and c (after the HindiII cleavage) a solution of 100 µl containing ligase buffer is introduced and ligated at 14°C for 18 hours with T^-DNA ligase.

200 µl of competent E. coli HB 101 (E. Dworkin et al., Dev. Biol. 76, 435-448 (1980)) is mixed with 20 µl of ligation mixture and incubated for 45 minutes on ice. The DNA uptake is then terminated with a 2-minute heat shock at 42°C. The cell suspension is incubated for a further 10 minutes on ice and finally transferred to LB-agar containing 50 mg/l ampicillin. The plasmids present in the 24 colonies obtained are isolated according to the method of Birnboim and Doly (see Nucl. Acid Res. 7, 1513 - 1523 (1979)). After cleavage with the various restriction enzymes, a plasmid has the desired structure. This was named pRHW 10 (see fig. 6).

b) Preparation of the plasmid pRHW 12

About. 10 µg of the plasmid pRHW 10 is cleaved with Barn HI. The Klenow fragment of DNA polymerase I and the four deoxynucleoside triphosphates are then added and incubated for 20 minutes at room temperature. The straight-ended plasmid that has been linearized is purified by phenol extraction and precipitation and then cut with the restriction endonuclease NcoI in a volume of 100 µl. The largest fragment is isolated using agarose gel electrophoresis, electroelution and electrotip purification. The fragment a) (see fig. 6) is obtained by cleavage of 4 µg of the AvaII fragment, which contains the P9A2 cDNA insert (see above) with NcoI and AlUl, whereby approx. 2 (ag of fragment a).

In the last ligation step, fragment a) and the added pRHW 10, which has been cleaved twice with BamHI/NcoI, are ligated in a volume of 10 ul, during which 10 ng of each DNA is used. The ligation of a filled-in BamHI cleavage site on a DNA capped with AluI again yields a BamHI cleavage site.

The obtained ligation mixture is transformed as described above with competent E. coli HB 101. From the 40 colonies obtained, one is selected, and this is designated pRHW 12.

The plasmid is isolated and the EcoRI/BamHI insert is sequenced according to the method of Sanger (F. Sanger et al., Proe. Nat. Acad. Sei. 74, 5463-5467 (1979)). This has the expected sequence.

c) Preparation of the plasmid pRHW 11

This takes place analogously to example 5b. 1 µg of plasmid

pRHW 10 is digested with BamHI. The overhanging (sticky) ends of the obtained DNA are made blunt with the help of the Klemow fragment of DNA polymerase I and the four deoxynucleoside triphosphates, and then the linearized DNA is cut with Ncol. The largest fragment is isolated by agarose gel electrophoresis, electroelution or elutip column chromatography.

The NcoI-AluI fragment is isolated from clone E76E9 analogously to example 5b. 10 ng of the vector part is then ligated with 10 ng of the cDNA part in a volume of 10 ul under suitable conditions and using 1 unit of T^-ligase. After transformation of the obtained DNA mixture into E. coli HB 101 and selection of the obtained 45 colonies on ampicillin-containing LB plates, a clone is selected which is designated pRHW 11. After cultivation of the corresponding clone, plasmid DNA is isolated. This structure is supported by the presence of several specific restriction endonuclease cleavage sites (Alul, EcoRI, HindIII, Ncol, Pstl).

d) Expression of interferon activity with E. coli HB 101

which contains the plasmid pRHW 12

100 ml of the bacterial culture is incubated until an optical density of 0.6 at 600 nm in M9 minimal medium, which contains all amino acids except tryptophan (20 ug/ml per amino acid),

1 µg/ml thiamine, 0.2% glucose and 20 µg/ml indole-(3)-acrylic acid (IAA), the inducer of the tryptophan operon. The bacteria are then clumped by centrifugation (10 minutes at 7000 rpm), washed once with 50 mM Tris/Cl pH = 8, 30 mM NaCl and finally resuspended in 1.5 ml of the same buffer. After 30 minutes of incubation with 1 mg/ml lysozyme on ice, the bacteria are frozen and thawed five times. The cell residue is removed by centrifugation for 1 hour at 40,000 rpm. The supernatant is sterile filtered and tested for interferon activity in the plaque reduction assay using human A549 cells and Encophalo myocarditis virus.

Result: 1 1 of the prepared bacterial culture contains

1 x 10<6> international units interferon (A. Bil-liau, Antiviral Res. 4, 75-98 (1984)).

EXAMPLE 6

COMPILATION OF AMINO ACID AND NUCLEOTIDE SEQUENCE DIFFERENCES FROM TYPI INTERFERONS

a) Comparison of the amino acid sequences

The pairwise comparison of the amino acid sequences gives

one by aligning the first cysteine residue of the finished (/-interferon with the first cysteine residue to the amino acid sequences encoded through the cDNA inserts in the plasmids P9A2

and E76E9. Both sequences are shown in fig. 7 as in IFN-omega, as no differences could be found between the specific sequences for P9A2 or E76E9 clones at the measured values. The only correction made was the introduction of a blank space

at position 45 of interferon-c(k), which was counted as the wrong site. When the sequence of the omega interferon is a partner, the comparison was carried out taking into account the other 166 amino acids. This value is shown in Fig. 7 together with the additional 6 amino acids which is encoded through the clones P9A2 and E76E9. The percentage differences are obtained by dividing the measured differences by the number 1.66. An additional amino acid thus makes the percentage 0.6. This gives 3.6% for the six additional amino acids of IFN- omega, which is already present in the percentage figure.

The comparisons with the Æ-interferon are performed by listing the third amino acid of the finished /i-interferon with the first amino acid of the finished oC-interferon or the first amino acid encoded by plasmids P9A2 and E76E9. The longest comparative structure of a <X-interferon with ^-interferon thus extends over 162 amino acids, which gives two additional amino acids for the <<>*-interferon and the $-interferon. These are counted as fault locations and are shown separately in fig. 7, but is included in the percentage rate figure. The listing of the co-interferon with the amino acid sequences of the clones P9A2 or E76E9 is carried out in the same way. However, this gives a total of 10 additional amino acids.

b) Comparison of nucleotide sequences

The listing of the sequences to be compared takes place

analogous to example 6a. The first nucleotide from the DNA of the finished α-interferon is the first nucleotide of the triplet that codes for cysteine in the finished α-interferon. The first nucleotide from the DNA of plasmids P9A2 or E76E9

is likewise the first nucleotide of the codon for cysteine-1. The first nucleotide from the DNA of the /3-interferon is the first nucleotide of the third triplet. The comparison takes place over a total of 498 nucleotides when the individual DNAs of the a-interferons are compared with the DNA of the /3-interferon, and over 516 nucleotides when the DNA sequences of the individual a-interferons or /3-interferons are compared with such for the plasmids P9A2 or E76E9. The absolute number for fault locations is indicated in the left part of the table in fig. 7 and then in brackets the corresponding percentages.

VIRUS-INDUCIBLE EXPRESSION OF OMEGA-1- mRNA AND NC3 7- CELLS

a) Synthesis of an omega-interferon specific hybridization probe

10 pMOl of the oligonucleotide d(TGCAGGGCTGCTAA) are mixed

with 12 pMol gamma-32-P-ATP (specific activity: > 5,000 Ci/mMol) and 10 units of polynucleotide kinase in a total volume of 10 ul

(70 mM Tris/Cl pH = 7.6, 10 mM MgCl2, 50 mM DTT) and allowed to stand

1 hour at 37°C. The reaction is then interrupted by heating for 10 minutes at 70°C. The radioactively labeled oligonucleotide obtained is hybridized with 5 pMol M13pRHW 12 ssDNA (see Fig. 9) in a total volume of 35 ul (100 mM NaCl) with a 1-hour standing time at 50°C.

After cooling to room temperature, nick-trans-lase ion buffer, the four deoxynucleoside triphosphates and 10 units of Klenow polymerase are added to a total volume of 50 µl (50 mM Tris/Cl pH = 7.2, 1<0> mM <MgCl>2, 5 µg/ml BSA; 1 mM per nucleotide). The polymerization is carried out with 1 hour's rest at room temperature, then the reaction is stopped by heating for 5 minutes at 70°C.

The conversion results in a partially double-stranded circular DNA. This is then cut into a total volume of 500 ul

with 25 units Avall, under which the buffer described by the manufacturer is used. Below, the double-chained areas are cut to uniform sizes. The reaction is then interrupted by heating for 5 minutes at 70°C.

b) Production of RNA from cells infected with virus

100 x 10<6> cells (0.5 x 10<6>/ml) are treated for 48-72 hours with

100 jiMole dexamethasone (16α-methyl-9α-fluoro-A'-hydrocortisone) - the controls contain no dexamethasone. For interferon induction, the expression cells are resuspended in serum-free medium at a concentration of 5 x 10<6>/ml and infected with 2<10> units/ml of Sendai virus. Aliquots of the cell culture supernatants are tested for IFN activity by plaque reduction measurement (see example 5b). The cells are harvested 6 hours after virus infection by centrifugation (1000 g, 10 minutes), washed in 50 ml NP40 buffer (Example 4a), resuspended in 9.5 ml ice-cold NP4 0 buffer and lysed by adding 0, 5 ml 10% NP40 for 5 minutes on ice. After separation of the nuclei by centrifugation (1000 x g, 10 minutes), the supernatant is adjusted with 50 mM Tris/Cl, 0.5% Sarcosine and 5 mM EDTA to pH = 8 and stored at -20°C. To isolate all RNA from the supernatant, extract once with phenol, once with phenol/chloroform/isoamyl alcohol and once with chloroform/isoamyl alcohol. The aqueous phase is placed on a 4 ml 5.7 molar CsCl pad and centrifuged in a SW40 rotor (35 rpm, 20 hours) to free the extract of DNA and residual proteins. The RNA pellet obtained is resuspended in 2 ml TE pH=8.0, and precipitated with ethanol. The precipitated RNA is then dissolved in water at a concentration of 5 mg/ml.

c) Detection of interferon-omega mRNA

0.2 µl of the hybridization sample prepared according to example

7b, is precipitated together with 20 - 50 µg of the RNA prepared according to 7c by the addition of ethanol. In the control experiment, transfer RNA (tRNA) or RNA originating from E. coli transformed with the plasmid pRHW 12 is added instead of the cellular RNA

(Example 5). The pellets obtained are washed salt-free with 70% ethanol, dried and dissolved in 25 µl 80% formamide (100 mM PIPES

pH = 6.8, 400 mM NaCl, .10 mM EDTA). The probe is then heated for 5 minutes at 100°C to denature the hybridization probes, immediately set to 52°C and incubated for 24 hours at this temperature. After the hybridization, the probes are placed on ice and 475 µl of S1 reaction mixture (4 mM Zn(Ac) 2 , 30 mM NaAc, 250 mM NaCl, 5% glycerol, 20 µg of calf thymus DNA, 100 units of S1 nuclease) is added. After 1 hour of cleavage at 37°C, the reaction is interrupted by ethanol precipitation.

The pellets are dissolved in 6 µl formamide buffer and split

substantially similar to the probes from the DNA sequencing reactions on a 6% acrylamide gel containing 8 M urea, (F. Sanger et al., Proe. Nat. Acad. Sci. 74, 5463-5467 (1979)).

For autoradiography, the dried gel is exposed to a DuPont Cronex x-ray film using Kodak Lanex Regular intensifiers at -70°C.

Text to fig. 10

Lanes A to C are controls.

Lane A: 20 µg tRNA

Lane B: 10 µg RNA from pER 33 (E. coli expression strain

for interferon-d2-Arg)

Lane C: 1 ng RNA from pRHW 12 (E. coli expression strain

for interferon-omega 1)

Lane D: 50 µg RNA from untreated Namalwa cells

Lane E: 50 µg RNA from Namalwa cells infected with virus Lane F: 50 µg RNA from Namalwa cells pretreated with dexamethasone and infected with virus

Lane G: 20 µg RNA from untreated NC 37 cells

Lane H: 20 µg RNA from NC 37 cells infected with virus

Lane I: 20 µg RNA from NC 37 cells pretreated with dexamethasone and infected with virus

Track M: size marking (pBR 322 coated with Hinfl)

Tracks B and C show that the expected signal can only be detected when an omega 1-specific RNA is found among the RNA molecules. Furthermore, it is shown that even a large excess of the fake RNA does not produce any background signal (see lane B). Furthermore, the tRNA used as a hybridization partner also does not give any signal (see lane A).

Lanes G to I show induction of the omega 1-specific RNA in NC 37 cells infected with virus. The pre-treatment with dexamethasone also enhances this effect.

Tracks D to F show basically the same result

which is achieved with Namalwa cells. However, the induction with omega1-specific RNA is not as strong as in the NC 37 cells. This result is parallel to the interferon concentrations

which was measured in the individual supernatant.

These relationships to interferon-omega1 gene expression

is also as expected for an interferon type I gene.

EXAMPLE 8

ISOLATION OF THE GENE ENCODING FOR IFN-OMEGA1 AND ITS RELATED GENES:

a) Cosmid sorting

A human cosmid bank (human DNA (from males) cloned in

Cosmid pcos2 EMBL (A. Ponstka, H.R. Rockwitz, A.M. Frischauf, B. Hohn, H. Lehrach Proe. Nati. Acad. Sel. 81, 4129 - 4133

(1984)) with a complexity of 2 x 10^) was examined with respect to IFN-omega and related genes. E. coli DH1 (r„-, m„+, rec.A; gyrA96, sup.E) was used as host. Mg cells (similar to "plating bacteria") were first produced. E. coli DH1 grows overnight in L-medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) supplemented with 0.2% maltose. The bacteria are centrifuged away and taken up in a 10 mM MgSO^ solution to an ODgQQ =2. 5 ml of this cell suspension is incubated with 12.5 x 10 5 colony forming units of prepackaged cosmids for 20 minutes at 37°C. Then 10 vol LB is added and the suspension is kept for 1 hour for expression of the kanamycin resistance mediated by the cosmid at 37°C. The bacteria are then centrifuged away, resuspended in 5 ml LB and streaked in 200 ul aliquots on the nitrocellulose filter (BA85, 132 mm diameter), which lies on LB agar (1.5% agar in L medium) plus 30 ug/ml kanamycin . Per filter grows approx. 10,000 to 20,000 colonies up. The colonies are replica-plated onto additional nitro-cellulose filters which are stored at 4°C.

A batch of colony filters is processed as described in the example

lc), i.e. the dsDNA is denatured, and the single-stranded DNA

fixed on nitrocellulose. The filters are pre-washed for 4 hours at 65°C in a 50 mM Tris/HCl, pH = 8.0, IM NaCl, 1 mM EDTA; 0.1% SDS solution. The filters are then incubated in a 5 x Denhardfs (see example 1c), 6 x SSC, 0.1% SDS solution for 2 hours at 65°C and hybridized with approx. 50 x 10<6> cpm nick-translated, denatured IFN-omega 1 DNA (Hind III-BamHI insert of clone pRHW12,

see fig. 6) 24 hours at 65°C in the same solution. After completed hybridization, the filters are first washed 3 x 10 minutes at room temperature in a 2 x SSC, 0.01% SDS solution and then 3 x 45 minutes at 65°C in a 0.2 x SSC, 0.01% SDS solution . The filters are dried and exposed on Kodak X-OmatS film using an intensifier foil at -70°C. Positively reacting colonies are located on the replica filters, scraped off and resuspended in L-medium + kanamycin (30 µg/ml). From this suspension, a few µl are plated on LB agar + 30 µg/ml kanamycin. The resulting colonies are replica-plated onto nitrocellulose filters. These

32

the filters are hybridized as described above with P-labeled IFN-omega1 DNA. From a hybridizing colony the cosmid was isolated by the method described by Birnboim & Doly (Nucl. Acids Res. 7, 1513 (1979)). With this cosmid DNA preparation, E. coli DH1 was transformed and the transformants were selected on LB agar + 30 ug/ml kanamycin. The transformants

32

were again tested with P-radioactively labeled IFN-omega1-DNA after positively reacting clones. One clone was selected from the original isolate and its cosmid was prepared on a larger scale (Clewell, D.B. and Helinski, D.R., Biochemistry 9, 4428 (1970)). Three of the isolated cosmids are named cos9, cos 10 and cosB.

b) Subcloning of hybridized fragments in PUC8

1 µg of the cosmids cos9, cos10 and cosB were cut with

Hindllll. The fragments are separated electrophoretically on 1% agarose gels in TBE buffer and transferred according to Southern to the nitro-cellulose filter (see example 4c). Both filters are hybridized with nick-translated ul-DNA as described in example 4d, washed and exposed. About. 20 strands of each cosmid are cut with HindIII, and the resulting fragments are separated gel-electrophoretically. The fragments that are hybridized with tol DNA in the initial experiment are electroeluted and purified over elutip columns. These fragments are ligated with HindIII-linearized, dephos-phorylated pUC8 (Messing, J., Vieira, J., Gene 19, 269-276

(1982)). E. coli JM101 (supE, thi, (lac-proAB), (F', traD36, pro AB, lac q Z Ml 5) (e.g. Fa. P.L. Biochemicals) is transformed with ligase reaction solution. The bacteria are plated on LB- agar containing 50 µg/ml ampicillin, 250 µg/ml 5-bromo-4-chloro-3-indolyl-O-D-galactopyranoside (BCIG), and 250 µg/ml isopropyl-Ø-D-thiogalacto-pyranoside (IPTG) .A blue father- ging of the reared colonies shows that an insert is missing in pUC8. Small-scale plasmid DNAs were isolated from some white clones, cut with HindIII and separated on 1% agarose gel. The DNA fragments were transferred onto nitrocellulose filters and hybridized as above with <32>P-omega1 DNA. From cos9 and cos 10, a subclone was chosen and designated pRHW22 and pRH57 respectively. From cosB, two DNA fragments that hybridized well with the omega1 probe were subcloned and designated pRH51 and pRH52, respectively.

c) Sequence analysis

The DNA inserted into pUC8 is separated by cutting with

HindIII and subsequent gel electrophoresis from the support portion. This DNA, approx. 10 µg, ligated using T4~DNA ligase in 50 µl reaction solution, the volume is brought to 250 µl with nick translation buffer (Example 4d) and then broken under ice-cooling using ultrasound (MSE 100 Watt Ultrasonic Disinte -grator, max output at 20 kHz, 5 x 30 seconds). Then, the ends of the breaks are repaired by adding ^100 volume of 0.5 mM dATP; dGTP, dCTP and dTTP as well as 10 units of Klenow fragment of DNA polymerase I for 2 hours at 14°C. The resulting DNA fragments with straight ends are separated by size

a 1% agarose gel. Fragments with sizes between 500 and

1000 bp was isolated and subcloned into the Smal-cut, defos-forylated phage carrier M13 mp8. The recombinant single-stranded DNA phages are isolated and sequenced according to the method developed by Sanger (Sanger, F. et al., Proe. Nati. Acad. Sei 74, 5463-5467 (1976)). The composition of the individual sequences in relation to the total sequence takes place with the help of Computer (Staden,

R., Nucl. Acids Res. 10, 4731-4751 (1982)).

d) Sequence of the subclone pRH57 (IFN-omega1)

The sequence is reproduced in fig. 11. This 1933 bp long

fragment contains the gene for interferon-omega1. The area that codes for protein comprises nucleotides 576 to 1163. The sequence is completely identical to that from the cDNA clone P9A2. The nucleotide section 576 to 574 codes for a 23 amino acid long signal peptide. The TATA box is located at a distance that is characteristic of the interferon type I gene in front of the start codon ATG (positions 476 - 482). The gene has some signal sequences for the polyadenylation during transcription (ATTAAA at positions 1497 - 1502, respectively 1764 - 1796; AATAAA at positions 1729 - 1734, respectively 1798 - 1803), the first of which was used in the clone P9A2.

e) Sequence of the subclone pRHW22 (IFN-pseudo-omega2)

In fig. 2, the 2132 bp long sequence of the HindIII fragment from cosmid cos9 hybridizing with the omega1 DNA probe is shown. An open reading frame is formed from nucleotide 905 to nucleotide 1366. The amino acid sequence that can be derived from it is reproduced. The first 23 amino acids are similar to those of the signal peptide of a typical type I interferon. The subsequent 31 amino acids show similarity to omega 1 interferon up to amino acid 65, during which tyrosine is notable as the first amino acid in the finished protein.

After amino acid position 66 follows the sequence of a potential N-glycosylation site (Asn-Phe-Ser). At this location, the amino acid sequence differs from a type I interferon. However, it can be demonstrated that by suitable insertion and resulting displacement of the protein reading frame, similarity to IFN-omega1 can be obtained (Example 9). From the point of view of the type I interferon, the isolated gene is therefore a pseudogene: IFN-pseudo-omega2.

f) Sequence of the subclone pRH51 (IFN-pseudo-omega3)

One approx. 3,500 bp long and hybridized with an omega1 probe

HindIII fragment originating from cosmid B was partially sequenced (Fig. 13). There is an open reading frame from nucleotide position 92 to 94. The first 23 amino acids show the features of a signal peptide. The subsequent sequence begins with tryptophan and shows similarity to IFN-omega1 until amino acid 42. Thereafter, the derived sequence differs from IFN-omega1

and ends after amino acid 98. The sequence can be changed by insertions so that it then has great homology with IFN-omega1 (example 9). The gene is designated IFN-pseudo-omega3.

g) Sequence of the insert from pRH52 (IFN-pseudo-omega4)

The 3659 bp sequence of the HindIII fragment which

is isolated from cosmid B and hybridizes with the omega1 DNA,

is shown in fig. 14. An open reading frame, whose translation product partially shows homology with IFN-omega1, is located between nucleotide position 2951 and 3250. After the 23 amino acid long signal peptide, the further amino acid sequence begins with phenylalanine. The homology with IFN-1 is already interrupted after the 16th amino acid, continues at the 22nd amino acid and ends at the 41st amino acid. A possible translation will be possible up to amino acid 77. Analogous to examples 8f and 8g, a good homology with IFN-omega1 can also be obtained here by introducing insertions (example 9). The pseudogene isolated here is called IFN-pseudo-omega4.

EXAMPLE 9

EVALUATION OF THE GENE FOR 4 MEMBERS OF THE IFN-OMEGA FAMILY

In fig. 15, the genes for IFN-omega1 to IFN-pseudo-omega4 are listed together with the amino acid translation. To achieve better agreement, gaps are introduced in the individual genes shown by dots. No bases are omitted. The base numbering includes the holes. The amino acid translation of IFN-omega1 is retained (eg, position 352 - 355: "C.AC" codes for His). In the case of pseudogenes, the translation into an amino acid only takes place where it is unambiguously possible. Because of this arrangement, one immediately sees that the four isolated genes are related to each other. Thus, you get e.g. the potential N-glycosylation site (nucleotide positions 301 to 309) in all four genes.

Likewise, a triplet which is a stop codon is also found in IFN-pseudo-omega4 at the nucleotide positions

611 - 614, and as with IFN-omega1 ends a finished protein

with a length of 172 amino acids. However, it exists in

this arrangement in IFN-pseudo-omega2 (nucleotide positions 497 to 499) and in IFN-pseudo-omega4 (nucleotide positions 512 - 514) previous stop codons.

The degree of kinship between the genes and the amino acid translations can be calculated from the arrangement shown in fig. 15. Fig. 16 depicts the DNA homology between the members of the IFN-omega family. Below that, those positions are not counted in pairwise comparisons where one of the two parties or both have a hole. The comparison gives a homology of around 85% between the IFN-omega1 DNA and the sequences of the pseudogenes. IFN-pseudo-omega2-DNA is homologous with the DNAs from IFN-pseudo-omega3 and IFN-pseudo-omega4 respectively in approximately 82%. Fig. 17 shows the comparison results for signal sequences and fig. 18 the comparisons of "finished" proteins. The latter are between 72 and 88%. This homology is nevertheless significantly greater than between IFN-omega 1 and IFN-oCenet and IFN-0> (Example 6). The fact that the individual members of the IFN-omega family are further apart than for the IFN-a family,

can be explained by the fact that three of the four isolated IFN-omega genes are pseudogenes and are obviously no longer exposed to the same selection pressure as functional genes.

EXAMPLE 10

FERMENTATION

Tribe:

A single colony of the strain HB101/pRHW12 on LB-agar (with 25 mg/ml ampicillin) is inoculated into tryptone-soya medium (OXDID CM129) with 25 mg/ml ampicillin and shaken at 250 rpm. minute (R/min) and 37°C until an optical density (546 nm)

of approx. 5 obtained (logarithmic growth phase). Then 10% by weight of sterile glycerol is added, the culture is filled in 1.5 ml portions into sterile ampoules and frozen at -70°C:

Preculture:

The culture medium contains 15 g/l Na2HP04 x 12H20; 0.5 g/l NaCl; 1.0 g/l NH4Cl; 3.0 g/l KH2PO4; 0.25 g/l MgSO 4 x 7H 2 O; 0.011 g/l CaCl2; 5 g/l casein hydrolyzate (merck 2238); 6.6 g/l glucose monohydrate; 0.1 g/l ampicillin; 20 mg/l cysteine and

1 mg/l thiamine hydrochloride. 4 x 200 ml of this medium in each 1000 ml Erlenmeyer flask are each inoculated with 1 ml of a stock culture of HB101/pRHW14 and shaken for 16 - 18 hours at 37°C and 250 rpm. minute.

Main culture:

The medium for the fermentation contains 10 g/l.,.(NH4 ) 2HP04 ; 4.6 g/l K2HPO4 x 3H2O; 0.5 g/l NaCl; 0.25 g/l MgSO 4 x 7H 2 O; 0.011 g/l CaCl2; 11 g/l glucose monohydrate; 21 g/l casein hydrolyzate (merck 2238); 20 mg/l cysteine, 1 mg/l thiamine hydrochloride and 20 mg/l 3-/?-indole acrylic acid. 8 liters of sterile medium in a fermenter with a total volume of 14 liters (height: radius = 3:1) are inoculated with 800 ml of the pre-culture. Fermentation takes place at 28°C; 1000 rpm. (Effigas-Turbine), an air flow rate of 1vvm (volume/volume/minute) and an initial pH

of 6.9. During fermentation, the pH value drops and is then kept constant at 6.0 with 3N NaOH. If an optical density (546 nm) of 18 to 20 is reached (normally after 8.5 to 9.5 hours of fermentation), cool under otherwise identical conditions to 20°C and then adjust the pH to 2.2 with 6N H2S04 (without air recirculation) and stir for 1 hour at 10°C and 800 rpm. (without air blow-through). The biological mass that has been inactivated in this way is then centrifuged in a CEPA laboratory centrifuge type GLE at 30,000 rpm. and frozen and stored at -20°C.

EXAMPLE 11

PURIFICATION OF INTERFERON- OMEGA- Gly

a) Partial purification

All steps are performed at 4°C.

140 g of biomass (E. coli HB101 transformed with the expression clone pRHW12) is resuspended in 1150 ml of 1% acetic acid (pre-cooled to 4°C) and stirred for 30 minutes. The suspension is adjusted to pH 10.0 with 5 M NaOH and stirred further for 2 hours. After the setting with 5 M HC1 at pH = 7.5, stirring is continued for 15 minutes and then centrifuged (4°C, 1 hour at 10,000 rpm, J21 centrifuge (Beckman), JAlO rotor).

The clear supernatant is placed on a 150 ml CPG (controlled pore glass) column (CPG 10-350, mesh size 120/200) at 50 ml/h, washed with 1000 ml 25 mM Tris/1 M NaCl, pH 7.5 and eluted with 25 mM Tris/1 M KCNS/50% ethylene glycol, pH = 7.5 (50 ml/hour).

The protein peak containing interferon activity was collected, dialyzed against 0.1 M Na phosphate, pH = 6.0 and 10% polyethylene glycol 40,000 overnight, and the precipitate formed was centrifuged from (4°C, 1 hour, 10,000 rpm, J21 centrifuge, JA20 rotor) (see Table 1).

b) Further purification

The dialyzed and concentrated CPG eluate is diluted with

buffer A (0.1 M Na phosphate pH = 6.25/25% 1,2-propylene glycol)

1:5 and passed through a "superloop" injector (Pharmacia) at 0.5 ml/min onto the buffer A equilibrated MonoS 5/5 (Pharmacia, cation exchange) column. The elution takes place with 20 ml of a linear gradient of 0 to 1 M NaCl in buffer A, likewise at a flow rate of 0.5 ml/min. The flow-through and 1 ml fractions were pooled and examined using a CPE reduction assay for interferon activity.

The active factions were gathered.

U:units relative to interferon-d2 (see example 2 in EP-A-0.115.613: E. Coli HB101 transformed with the expression clone pER 33) as standard

DL: throughput

EXAMPLE 12

CHARACTERIZATION OF HuIFN- OMEGAI

A. Antiviral activity on human cells

B. Antiviral activity on monkey cells

C. Antiproliferative activity on human Burkitt's lymphocytes (cell line Daudi) D. Antiproliferative activity on human cervical carcinoma cells

(cell line HeLa)

E. Acid stability

F. Serological characterization.

A. Antiviral activity on human cells

Cell line: Human lung carcinoma cell line (A549)

(ATCC CCL 185)

Virus: Mouse encephalomyocarditis virus (EMCV) Test system: Inhibition of cytopathic effect (all potency determinations were performed in quadruplicate)

A partially purified preparation from HuIFN-omegal with a protein content of 9.4 mg/ml was titrated through suitable dilution in the aforementioned test system. The preparation showed an antiviral effect with a specific activity of 8300 IU/mg in relation to the reference standard Go-23-901-527.

B. Antiviral activity on monkey cells

Cell line: GL-V3 monkey kidney cells (Christofinis G.J., J.

With. Microbiol. 3, 251 - 258, 1970)

Virus: Vesicular stomatitis virus (VSV)

Test system: Plaque reduction test (all titrations four times)

The preparation described in example 12A showed a specific antiviral activity of 580 U/mg in said test system.

C. Antiproliferative activity on human Burkitt's lymphoma cells

(cell line Daudi)

The human Burkitt's lymphoma cell line Daudi was cultured

in the presence of different concentrations of HuIFN-omegal. After 2, 4 and 6 days of incubation at 37°C, the cell density was determined, untreated cultures serving as controls. All cultures

was employed three times in parallel. From the subsequent figure it appears that IFN-omega shows a pronounced inhibition of cell proliferation at a concentration of 100 antiviral units (IU, see example 12A) per ml; at a concentration of 10 IU/ml, a partial, transient inhibition was observed (the following symbols are used in the following figure: 0 control, ° 1 IU/ml, □ 10 IU/ml, ▼ 1 00 IÉ/ml):

D. Antiproliferative activity on human cervical carcinoma cells (cell line HeLa)

The human cervical carcinoma cell line HeLa was cultured

in the presence of the following proteins or protein mixtures:

HuIFN-omegal (see Example 12A) 100 IU/ml

HuIFN-gamma (see Example 12A) 100 IU/ml

Human tumor necrosis factor (HuIFN), >_ 98% pure, manufactured by Genentech Inc., San Francisco, USA (see Pennica D.

et al., Nature 312, 724-729; 1984) 100 ng/ml

All binary combinations of the mentioned protein concentrations as above 2 cultures were started in each of 3 cm plastic tissue culture dishes (50,000 cells/dish) and incubated for 6 days at 37°C, and then the cell density was determined. The treatment of the cells with HuIFN-omega or HuIFN-gamma had only a small influence on cell growth, while HuIFN-gamma showed a clear cytostatic effect. Combinations of IFN-gamma with IFN-omega1 showed synergistic cytostatic/cytotoxic action.

The following figure shows the result of the experiment:

The following symbols are used in the figure above: C untreated controls, T HuTNF, 0 HuIFN-omega, G HuIFN-gamma.

E. Acid stability

To investigate the acid stability of HuIFN-omegal, a dilution of the preparation described in example 12A is set up in cell culture medium (E/min.I 1640 with 10% fetal calf serum) using HCl at pH 2, incubated for 24 hours at 4° C and then neutralized with NaOH. This probe was titrated in the antiviral sample (Example 12A) and it showed 75% of the biological activity of a control incubated at neutral pH. IFN-omega1 must therefore be described as acid-stable.

F. Seroloqical characterization

To determine the serological properties of HuIFN-col

in comparison with HuIFN-α2, probes (diluted to 100 IU/ml) were pre-incubated with the same volume of solutions of monoclonal antibodies or polyclonal antisera for 90 minutes at 37°C,

and then the antiviral activity of the probes was determined. The following table shows that HuIFN-omega can only be neutralized with an antiserum against leukocyte interferon in a relatively high concentration, but not with antibodies directed against HuIFN-a2 or HuIFN-/3. HuIFN-col is therefore neither serologically related to HuIFN-a2 nor HuIFN-Ø:

Symbols: - not examined; 0 no neutralization; + partial-neutralization; +++ complete neutralization.

Claims (18)

1. Process for the production of recombinant DNA molecules encoding new human interferons of type I IFNtol-(figure 1), containing 168 to 174 amino acids, for interferon-pseudo-co2 (figure 12), for interferon-pseudo- o>3 (figure 13) or for interferon-pseudo-a>4 (figure 14), characterized in that cDNA from a B-lymphocyte cell line is hybridized with, under stringent conditions that give a homology greater than 85%, IFN -tol insert or a degenerate variant thereof, in the Pstl site in a) plasmid E76E9 with Dep. No. DSM 3003 b) plasmid P9A2 with Dep. No. DSM 3004 and inserted into an expression vector pRWHIO (Fig. 6), pRWH11 or pRWH12 (Fig. 6) with subsequent transformation of E. coli. 2. Method according to claim 1, characterized in that the stringent conditions can only demonstrate a homology of more than 90%. 3. Method according to claims 1-2, characterized in that the recombinant DNA molecule IFN-col contains the sequence: 4. Method according to claims 1 and 2, characterized in that the recombinant DNA molecule IFN-col according to claim 3 contains the nucleotide A instead of the nucleotide G in position 332. 5. Method according to claims 1 to 4, characterized in that the recombinant DNA molecules additionally contain a DNA sequence that codes for a leader peptide with the sequence: 6. Method according to claim 1, characterized in that the recombinant DNA molecule is an interferon-col gene with the formula: 7. Method according to claim 1, characterized in that the recombinant DNA molecule is an interferon pseudo-to2 gene with the formula: 8. Method according to claim 1, characterized in that the recombinant DNA molecule is an interferon-pseudo-co3 gene with the formula: 9. Method according to claim 1, characterized in that the recombinant DNA molecule is an interferon pseudo-w4 gene with the formula: 10. Expression plasmid for IFN-tol, which is a derivative of the plasmid PBR322, characterized in that the expression plasmid for IFN-Q1 is a pBR322 plasmid, which instead of the plasmid-specific, shorter EcoRI-BamHI fragment, contains the DNA sequence with the formula: 11. Process for the production of new type I human interferons, wherein the finished interferons have a divergence of 30 to 50% in relation to the human α-interferons, and of their N-glycosylated derivatives, characterized in that a suitable host organism is transformed with an expression plasmid according to claim 10 with the genetic information that a recombinant DNA molecule according to claims 1 to 6 contains is expressed, and the interferon peptide thus produced is isolated from this host organism. 12. Method according to claims 1-9, characterized in that the finished interferons also have a divergence of approximately 70% in relation to 0-interferon. 13. Method according to claims 11 and 12, characterized in that the finished interferons have a divergence of 40 to 48% in relation to the human α-interferons. 14. Method according to claims 11 to 13, characterized in that the interferons contain a leader peptide of the formula Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly. 15. Method according to claims 11 to 14, characterized in that the finished interferons contain 172 amino acids. * 16. Method according to claim 15, characterized in that the finished interferon contains the amino acid sequence and their N-glycosylated derivatives at amino acid position 78. 17. Method according to claim 15, characterized in that the finished interferon according to claim 16 in position 111 contains the amino acid Glu instead of Gly, and its N-glycosylated derivatives in amino acid position 78. 18. Process for producing the Met derivative according to claims 16 and 17, characterized in that E. coli HB 101 transformed with the plasmid pHRW11 or pRHW12 is cultivated, and the Met derivative formed in situ is isolated.