AT394209B - METHOD FOR PRODUCING NEW MICROORGANISMS FOR PRODUCING HYBRID HUMAN LEUCOCYTE INTERFERON - Google Patents

Description

AT 394 209 B

The present invention relates to a method for producing new microorganisms which are capable of producing polypeptides with the amino acid sequence of hybrid human leukocyte interferons. The method according to the invention is characterized in that a microorganism with a replicable expression vector which contains a DNA sequence coding for hybrid human leukocyte interferon is transformed and cultivated in a manner known per se.

Relatively homogeneous leukocyte interferons have been obtained from leukocytes from normal or leukemic donors. These interferons are a family of proteins that have a strong ability to produce a virus-resistant state in their target cells. Interferon is also able to inhibit cell proliferation and to modulate the immune response. These properties have prompted the clinical use of interferon as a therapeutic agent for the treatment of viral infections and malignant diseases

More recently, recombinant DNA technology has been used to microbially produce various human leukocyte interferons whose amino acid sequences show an order of magnitude of 70% homology with one another (Nature 290.20-26 (1981)). Genes that different leukocyte interferons, e.g. B. LelF A, B, C, D, F, G and H, can encode from that of Η. P. Koeffler and D. W. Golde (Science 200: 1153-1154 (1978)) described cell line KG-1, which is available from the American Type Culture Collection under ATCC no. CRL 8031 was obtained, and can be introduced and expressed for expression using expression plasmids in host bacteria, preferably E. coli K-12 strain 294 (ATCC No. 31445, deposited on October 28, 1978). The combination of DNA sequences which encode parts of naturally occurring interferons and their expression in microorganisms, for example in E. coli K-12, gives new, antiviral polypeptides, so-called hybrid leukocyte interferons, which have an effect differ qualitatively and quantitatively from the known leukocyte interferons. The production of hybrid leukocyte interferons has not yet been described in the literature.

The driving force behind recombinant DNA technology is the plasmid, a non-chromosomal loop of double-stranded DNA in bacteria and other microbes, often in multiple copies per cell. The information encoded in the plasmid DNA contains that required to reproduce the plasmid in daughter cells (ie a " replicon ") and usually one or more selection characteristics, such as in the case of bacteria, resistance to antibioticis, the clones to recognize the host cell containing the plasmid of interest and preferably to grow in selective media. The usefulness of plasmids lies in the fact that one or the other restriction endonuclease or " restriction enzyme " can be cleaved specifically, each recognizing a different location on the plasmid DNA. Thereafter, heterologous genes or gene fragments can be incorporated into the plasmid by connecting the ends at the cleavage site or at reconstructed ends near the cleavage site. DNA recombination occurs outside the cell, but the " recombinant " Plasmid can be introduced into it by a method known as transformation, and large amounts of the heterologous gene-containing recombinant plasmid can be obtained by culturing the transformed cells. Furthermore, if the gene is appropriately used with respect to portions of the plasmid that control the transcription and translation of the encoded DNA message, the resulting expression carrier can actually be used to produce the polypeptide sequence encoded by the gene used, a method , which is called expression.

Expression begins in an area known as a promoter that is recognized and bound by RNA polymerase. In some cases, such as the tryptophan or " trp ,, promoter preferred in the practice of the invention, promoter areas overlap with " operator " areas to form a combined promoter-operator. Operators are DNA sequences that are recognized by so-called repressor proteins that serve to regulate the frequency of the start of a transcription on a special promoter. The polymerase travels along the DNA and transfers the information contained in the coding strand from the 5 'to the 3 end to messenger RNA, which in turn is converted into a polypeptide that has the amino acid sequence that encodes the DNA. Each amino acid is made up of a nucleotide -Triplet or " Codon " encoded into something that is " structural gene " can be designated d. H. the part that encodes the amino acid sequence of the expressed product. After binding to the promoter, the RNA polymerase first transcribes nucleotides, encoding a ribosome binding site, then a translation initiation or " start " signal (usually ATG, which is in the emerging messenger RNA becomes AUG), then the nucleotide codons within the structural gene itself. So-called stop codons are transcribed at the end of the structural gene, whereupon the polymerase can form a further sequence of messenger RNA which, due to the presence of the stop signal, remains untranslated by the ribosomes stay. Ribosomes are bound to the binding site provided on the messenger RNA, usually in bacteria when the mRNA is formed, and generate the encrypted polypeptide themselves, starting at the translation start signal and ending with the aforementioned stop signal. The desired product is formed when the sequences encoding the ribosome binding site are suitably arranged with respect to the AUG initiator codon and when all other codons follow the initiator codon in phase. The product obtained can be obtained by lysing the host cell and recovering the product by suitable purification of other bacterial protein.

Nucleotide sequence studies of genes that encode the various leukocyte interferons allow a -2-

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Recognize the level of generality among various of them in terms of the presence and location of cleavage sites recognized by certain restriction endonucleases. In accordance with the invention, this generality can be used to form new hybrid genes by recombinant DNA which are useful in the microbial generation of hybrid leukocyte interferons which can be expected to have more or less the antiviral and other properties of interferons , encoded by the parental genes, show. In preferred embodiments of the invention, such hybrid leukocyte interferons can develop enhanced activity relative to that encoded by the parental genes.

The parental leukocyte interferon genes that encode the family of leukocyte interferon proteins considered here show individually natural allelomorphic variations. These variations can be detected by one or more amino acid differences in the total protein sequence or by gaps, substitutions, insertions, reversals or additional amino acids in the sequence. For every parental leukocyte interferon, designated LelF A, LelF B ... LelF J etc., allelomorphic variations of this type come under the name or definition and thus fall under the invention.

Further objects, advantages and features of the invention will become apparent from the following detailed description and the figures; of these shows:

Figures la and lb nucleotide sequences of the coding regions of 8 leukocyte interferon (" LeIF ") complementary DNA (" cDNA ") clones. One of these, appropriately labeled LelF E, is an obvious " pseudogen " which does not encode active leukocyte interferon, while another, LelF G, contains less than the full sequence for the corresponding interferon species. The ATG translation start codon and the stop triplet for each LelF is underlined.

Figure 2 Restriction endonuclease maps of eight types of LelF cloned cDNAs (A to H). Plasmids containing the clones were constructed according to the dC: dG tailing method (D.V. Goeddel et al., Nature 287, 411-416 (1980)). Therefore, the cDNA inserts can be excised with Pst I, i.e. H. each end of each insert is a Pst I restriction endonuclease cleavage site. The lines at the end of each cDNA insert represent the flanking homopolymer dC: dG tails. The positions of Pvu II, Eco RI and Bgl Π restriction sites are given. Shading areas in the figure represent the coding sequences of mature LelFs; the hatched areas indicate signal peptide coding sequences; and the free areas show non-coding 3 'and 5 sequences.

Figures 3a and 3b is a comparison of the eight LelF piotein sequences predicted by the nucleotide sequences. The one-letter abbreviations recommended by the IUPAC-IUB Commission from Biochemical Nomenclature are used: A = Alanine; C = cysteine; D = aspartic acid; E = glutamic acid; F = phenylalanine; G = glycine; H = histidine; I = isoleucine; K = lysine; L = leucine; M = methionine; N = asparagine; P = proline; Q = glutamine; R = arginine; S = serine; T = threonine; V = valine; W = tryptophan and Y = tyrosine. The numbers refer to the amino acid position (S refers to signal peptide). The stroke in the 165 amino acid LelF A sequence at position 44 was introduced to align this sequence with the 166 amino acid sequences of the other LelFs. The sequence of LelF E was determined by ignoring the extra nucleotide (position 187 of Figure la) in its coding region. The asterisks denote in-phase stop codons. Amino acids common to all LelFs (except the pseudogene LelF E) are also shown. The underlined residues are amino acids that are also present in human fibroblast interferon.

In Fig. La, the nucleotides +1 to 69 correspond to the S1 to S23 amino acids of Fig. 3a. The codon TGT (nucleotides 70 to 72) of FIG. 1 a corresponds to cysteine (C, amino acid 1) of FIG. 3 a. In Figure 3a, the Pvu II restriction endonuclease cleavage site occurs between the codons for amino acids 92 and 93 in LelF A, B, D, F and G, i. H. between nucleotides 346 and 347 of FIG.

4a and 4b compare the amino acid sequences of mature leukocyte interferons A and D, with a lack of amino acid 44 in LelF A being indicated by dashes. Only those LelF D amino acids that differ from the corresponding amino acids of LelF A are shown: The amino acid sequence of LelF D is otherwise identical to that of LelF A. Figure 4b also shows the relative position of Bgl II and Pvu II restriction endonuclease cleavage sites, used to form preferred hybrid leukocyte genes according to the invention, on the corresponding gene.

5 and 6 illustrate the results of comparative tests of a preferred hybrid leukocyte interferon according to the invention (" LelF-A / D ") for activity against encephalomyocarditis virus (" EMC ") and vesicularstomatitis virus (" VSV ") in mouse cells.

7 and 8 show the results of a comparison test with LelF-A / D and other interferons against EMC virus infections in mice and hamsters, respectively. The data in Fig. 7 come from treatments i. p. 3 h before infection. The doses of LelF-A / D and LeIF-Α are as titrated on Wish cells.

Figures 9a and 9b show the DNA sequences of five LelF proteins including types I and J.

Two microorganisms were used in the described work: E. coli x 1776, as described in US Pat. No. 4,190,495, and E. coli K-12 strain 294 (End A, thf, hsr ", hsmjf +), as in US Pat GB-PS 2 055 382A. Both are with the American Type Culture Collection, ATCC accession number -3-

AT 394 209 B 31537 and 31446, on July 3, 1979 and October 28, 1978, respectively. All work on recombinant DNA was done according to applicable guidelines of the National Institutes of Health.

The invention is described in its most preferred embodiments with reference to E. coli, not only with the strains E. coli x 1776 and E. coli K-12, strain 294 described above, but also with other known E. coli strains , such as E. coli B, or other strains of microbes, many of which are deposited and (possibly) available from recognized depositors for microorganisms, such as the American Type Culture Collection, ATCC. (See the ATCC catalog list and also DE-OS 2 644 432). These other microorganisms include e.g. B. Bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, among which, for example, Salmonella typhimurium and Serratia marcescens can be mentioned, using plasmids that can reduplicate and express heterologous gene sequences. Yeast, such as Saccharomyces cerevisiae, can also advantageously be used as a host organism in the production of the interferon proteins according to the invention by expression of genes coding for them under the control of a yeast promoter.

The LelF hybrids are made according to the invention by using the restriction endonuclease cleavage sites, which are usually in the individual parental genes, and whose ends are used in conjunction with the same sites in carrier expression plasmids (vectors). For example, the large (approx. 3900 bp) fragment of an Xba I-Pst I digest product of the pLelF A trp 25 expression plasmid can be combined with Xba I-Pvu II and Pvu II-Pst I digest fragments of the various LelF element genes to form expression plasmids that make the corresponding hybrid LelF available.

Each LelF expression plasmid was independently constructed with digestive fragments isolated from different LelF plasmids, e.g. B. pLelF A trp 25, pLelF B trp 7, pLelF C Up 35, pLelF D trp 11, pLelF F trp 1, pLelF G, pLelF H and pLelF I etc., the structure of which is described in Nature 287.411 (1980), or from pBR322, the structure of which is described in Gene 2.95 (1977). In certain of these plasmids, " trp " a tryptophan promoter-operator system, most preferred for bacterial expression, as described in published European Patent Application No. 36,776.

Direct expression of a first mature leukocyte interferoa

The procedure followed to express LelF A directly as a mature interferon polypeptide involved the combination of synthetic (N-terminal) and complementary DNAs.

A Sau 3a restriction endonuclease site is conveniently located between codons 1 and 2 of Le-IF A. Two synthetic deoxyoligonucleotides were created which have an ATG translation start codon, which restore the codon for amino acid 1 (cysteine) and which create an Eco Rl cohesive end. These oligomers were attached to a 34 b.p. Sau 3a - Ava II fragment from pL31 stapled The resulting 45 bp product was attached to two further DNA fragments in order to build an 865 base pair synthetic / natural hybrid gene which encodes Le-IF A and is encoded by Eco RI and Pst I restriction sites is bound. This gene was inserted between Eco RI and Pst I sites in pBR322 to give the plasmid pLe-IF Al.

The plasmid pGMl carries the E. coli tryptophan operon with the vacancy ALE1413 (GF Miozzari et al., J. Bacteriology 133.1457-1466 (1978)) and consequently leads to the expression of a fusion protein with the first 6 amino acids of the trp leader and about the last third of the trp-E polypeptide (hereinafter referred to as LE ') and the trp-D polypeptide in its entirety, all under the control of the trp-promoter-operator system. 20 μg of the plasmid were digested with the restriction enzyme Pvu II, which cleaves the plasmid at five sites. The gene fragments were then combined with EcoRI linkers (consisting of a self-complementary oligonucleotide of the sequence pCATGAATTCATG) to form an EcoRI cleavage site for later cloning to form a plasmid with an EcoRI site. The 20 μg of the DNA fragments obtained from pGMl were treated with 10 units of T ^ -DNA ligase in the presence of 200 pico-mol of the 5'-phosphorylated synthetic oligonucleotide pCATGAATTCATG and in 20 μΐ of T ^ -DNA ligase buffer (20 mmol Tris, pH 7.6, 0.5 mmol ATP, 10 mmol MgC ^, 5 mmol dithiothreitol) treated at 4 ° C overnight. The solution was then heated to 70 ° C for 10 minutes to stop binding. The links were broken by EcoRI degradation and the fragments, now with EcoRI ends, were separated by means of 5% polyacrylamide gel electrophoresis (hereinafter " PAGE ") and the three largest fragments of the gel were first stained with ethidium bromide and the fragments were located UV light and cutting out the parts of interest from the gel isolated. Each gel fragment was placed in a dialysis bag with 300 μl 0.1 x TBE and subjected to electrophoresis at 100 V for 1 h in 0.1 x TBE buffer (TBE buffer contains 10.8 g Tris base, 5.5 g boric acid , 0.09 g Na2EDTA in 11 H2O). The aqueous solution was removed from the dialysis bag, extracted with phenol, extracted with chloroform and made 0.2 molar in sodium chloride, and the DNA was recovered in ethanol after ethanol precipitation. The trp promoter-operator-containing gene with EcoRI cohesive ends was identified in the procedure described below, which results in the incorporation of fragments into a tetracycline-sensitive plasmid which becomes tetracycline-resistant after the incorporation of promoter operator. -4-

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The plasmid pBRHL (R.I. Rodriguez et al., Nucleic Acids Research £, 3267-3287 (1979)) expresses ampicillin resistance and contains the gene for tetracycline resistance, but since there is no associated promoter, this resistance is not expressed. The plasmid is therefore sensitive to tetracycline. The plasmid can be made resistant to tetracycline by introducing a promoter-operator system at the EcoRI site.

A pBRHL was digested with EcoRI and the enzyme was removed by phenol extraction and subsequent chloroform extraction and, after ethanol precipitation, recovered in water. The resulting DNA molecule was combined in separate reaction mixtures with the three DNA fragments obtained above and linked with T4 DNA ligase, as described above. The DNA present in the reaction mixture was used to transform an appropriate E. coli K-12 strain 294 (K. Backman et al., Proc. Natl. Acad. Sci. USA 22,4174-4198 (1976)) according to standard techniques (V. Hershfield et al., Proc. Naü. Acad. Sci. USA 21, 3455-3459 (1974)) and the bacteria were applied to LB plates which contained 20 μg / ml ampicillin and 5 μg / ml tetracycline. Several tetracycline-resistant colonies were selected, plasmid DNA isolated and the presence of the desired fragment confirmed by restriction enzyme analysis. The resulting plasmid is called pBRHtrp.

An EcoRI and BamHI degradation product of the viral genome or chromosome set of hepatitis B was obtained by conventional means and cloned to the plasmid pHS32 to EcoRI and BamHI sites from plasmid pGH6 (D.Y. Goeddel et al., Nature 281.544 (1979)). The plasmid pHS32 was cleaved with Xbal, extracted with phenol, extracted with chloroform and precipitated with ethanol. Then it was treated with 1 μE of E. coli polymerase I, Klenow fragment (Boehringer, Mannheim) in 30 ml of polymerase buffer (50 mmol of potassium phosphate, pH 7.4, 7 mmol of MgC ^, 1 mmol of β-mercaptoethanol) with 0 , 1 mmole of dTTP and 0.1 mmole of dCTP treated at 0 ° C for 30 min, then at 37 ° C for 2 h. This treatment results in two of the four nucleotides complementary to the protruding 5-end of the Xbal cleavage site being filled in: 5 'CTAGA-3 ’TCT- 5' CTAGA-3 'T-

Two nucleotides, dC and dT, were introduced and terminated with two protruding 5 'nucleotides. This linear residue of plasmid pHS32 (after phenol and chloroform extraction and recovery in water after ethanol precipitation) was cleaved with EcoRI. The large plasmid fragment was separated from the smaller EcoRI-Xbal fragment by PAGE and isolated after electroelution. This DNA fragment from pHS32 (0.2 μg) was bound under conditions similar to those described above to the EcoRI-Taq I fragment of the tryptophan operon (approx. 0.01 μg) derived from pBRHtrp.

In the method of binding the fragment of pHS32 to Eco RI-Taq I fragment as described above, the protruding Taq I end is bound to the remaining protruding end of Xbal, although it is not fully base-paired according to Watson-Crick : —T CTAGA— —TCTAGA— + - > —AGC TCT— —AGCTCT—

A portion of this binding reaction mixture was transformed into E. coli 294 cells, heat treated and placed on LB plates containing ampicillin. 24 colonies were selected, grown in 3 ml LB medium and plasmid isolated. Six of them were found to have regenerated the Xbal site by E. coli-catalyzed DNA repair and replication: -TCTAGA- -AGCTCT- -TCTAGA- -AGATCT-

It was also found that these plasmids cleaved with both EcoRI and Hpal and gave the expected restriction fragments. A plasmid, designated pTrp 14, was used to express heterologous polypeptides as discussed below.

The plasmid pHGH 107 (D.V. Goeddel et al., Nature 281, 544 (1979)) contains a gene for human growth hormone from 23 amino acid codons, made from synthetic DNA fragments and 163 -5-

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Amino acid codons obtained from complementary DNA produced by reverse transcription of human growth hormone messenger RNA. This gene contains an ATG translation start codon, although the codons of the "before" sequence of human growth hormone are missing. The gene was isolated from 10 µg pHGH 107 after treatment with EcORI and then E. coli polymerase I Klenow fragment and dTTP and dATP as described above. After phenol and chloroform extraction and ethanol precipitation, the plasmid was treated with BamHI.

The human growth hormone (" HGH ") gene-containing fragment was isolated by PAGE, followed by electroelution. The resulting DNA fragment also contains the first 350 nucleotides of the tetracycline-resistant structural gene, but it lacks the tetracycline promoter-operator system, so that when cloning to an expression plasmid, the plasmids containing the insert are localized by restoring the tetracycline resistance can be. Since the EcoRI end of the fragment has been filled in by the Klenow polymerase I procedure, the fragment has a blunt and a cohesive end, which ensures the correct orientation when later inserted into an expression plasmid.

The expression plasmid pTrpl4 was then prepared to obtain the HGH gene-containing fragment prepared above. Thus pTrpl4 was digested with Xbal and the cohesive ends obtained were filled according to the Klenow polymerase I procedure using dATP, dTTP, dGTP and dCTP. After phenol and chloroform extraction and ethanol precipitation, the resulting DNA was treated with BamHI and the large plasmid fragment obtained was isolated by PAGE and electroelution. The fragment derived from pTrpl4 had a blunt and a cohesive end, which allows recombination in a suitable orientation with the fragment containing the HGH gene, previously described.

The HGH gene fragment and the pTrpl4 AXba-BamHI fragment were combined and strung together under conditions similar to those described above. The filled-in Xbal and EcoRI ends connected to each other via the connection over the blunt ends with the regression of both the Xbal and the EcoRI site:

Xbal padded EcoRI padded HGH gene beginning —TCTAG AATTCTATG— —TCTAGAATTCTATG— + - > —AGATC TTAAGATAC— —AGATCTTAAGATAC—

Xbal EcoRI

This structure also regresses the tetracycline-resistant gene. Since the plasmid pHGH 107 expresses tetracycline resistance from a promoter in front of the HGH gene (the Lac promoter), this structure, designated pHGH 207, enables expression of the gene for tetracycline resistance under the control of the tryptophan promoter operator . The binding mixture was transformed into E. coli 294 and colonies were selected on LB plates which contained 5 pg / ml tetracycline.

The plasmid pHGH207 was digested with Eco RI and the trp promoter, a 300 b.p. Containing Eco RI fragment, obtained by PAGE and subsequent electroelution. The 300 b.p. Eco RI fragment contains the E. coli trp promoter, operator and trp leader ribosome binding site, but it lacks an ATG sequence for the introduction of translation. This DNA fragment was cloned into the Eco R1 site of pLe-IF A.

The trp fragment just mentioned is an analogue of the E. coli tryptophan operon, the so-called trp attenuator has been removed in order to controllably increase expression values. Expression plasmids containing the modified trp regulon can then be increased to predetermined values in culture media containing additional tryptophan in sufficient amounts to suppress the promoter-operator system, then freed from tryptophan in order to rid the system of repressor , and cause the expression of the desired product.

In detail, 250 μg of plasmid pL31 were digested with Pst I and the 1000 b.p. insert was isolated by gel electrophoresis on a 6% polyacrylamide gel. About 40 μg insert were electroeluted from the gel and divided into three portions for further digestion: a) a 16 pg sample of this fragment was partially digested with 40 units Bgl II45 'at 37 ° C. and the reaction mixture was purified on a 6% polyacrylamide gel . About 2 pg of the desired 670 b.p. fragment was obtained, b) Another sample (8 pg) of the 1000 b.p.-Pst I insert was treated with Ava II and Bgl II. 1 pg of the stated 150 b.p.-fragment was obtained after gel electrophoresis, c) 16 pg of the 1000 b.p.-piece were treated with Sau 3a and Ava II. After electrophoresis on a 10% polyacrylamide gel, approximately 0.25 pg (approximately 10 pmol) of the 34 b.p. fragment was obtained. The two specified deoxyoligonucleotides, 5'-dAATTCATGTGT (fragment 1) and 5'-dGATCACACATG (fragment 2) were synthesized according to the phosphotriester method (Maxam and Gilbert, Methods Enzymol. 6 £, 499-560 (1980)). Fragment 2 was phosphorylated as follows. 200 pl (approx. 40 pmol) (y ^ P) ATP (Amersham, 5000 Ci / mmol) were thoroughly dried and again in 30 pl 60 mmol. -6-

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Tris-HCl (pH 8), 10 mmol. MgC ^, 15 mmol. ß-mercaptoethanol, containing 100 pmol DNA fragment and 2 units of T4 polynucleotide kinase, suspended. After 15 min at 37 ° C 1 μΐ 10 mmol. AIP added and the reaction allowed to proceed for a further 15 min. The mixture was then heated to 70 ° C for 15 min, combined with 100 pmol of 5'-OH fragment 1 and 10 pmole of the 34 b.p. Sau 3a Ava II fragment. Binding was carried out for 5 h at 4 ° C in 50 μΐ 20 mmol. Tris-HCl (pH 7.5), 10 mmol. MgCl2, 10 mmol. Dithiothreitol, 0.5 mmol. ATP and 10 units of T4 DNA ligase. The mixture was treated electrophoretically on a 6-polyacrylamide gel and the 45 b.p. product obtained by electroelution. 860,000 Cerenkov-cpm were obtained (approx. 30 ng, 1 pmol) combined with 0.5 pg (5 pmol) of the 150 bp Ava II-Bgl II fragment and 1 pg (2 pmol) of the 670 bp-Bgl-II -Pst-I fragments. Binding was carried out at 20 ° C for 16 h using 20 units of T4 DNA ligase. The ligase was inactivated by heating at 65 ° C for 10 minutes. The mixture was then digested with Eco RI and Pst I to remove polymers of the gene. The mixture was purified by 6% polyacrylamide gel electrophoresis. 36,000 cpm (approx. 0.04 pmol, 20 ng) 865 b.p. product were isolated. Half of this (10 ng) was bound to pBR322 (0.3 pg) between Eco RI and Pst I sites. The transformation of E. coli 294 resulted in 70 tetracycline-resistant ampicillin-sensitive transformants. Plasmid DNA isolated from 18 of these transformants was digested with Eco RI and Pst I. 16 of the 18 plasmids had an Eco Rl-Pst I fragment of 865 b.p. Length. 1 pg of one of these, pLe-IF Al, was digested with Eco RI and attached to a 300 b.p. Eco RI fragment (0.1 pg) containing E. coli trp promoter and trp leader ribosome binding site, prepared as described above. Transformants containing the trp promoter were identified with a ^ P-trp probe by the Grunstein-Hogness colony screening method - Grünstem et al. (Proc. Natl. Acad. Sci. (USA) 72, 3961 (1975)). An asymmetrically arranged Xba I site in the trp fragment allowed the determination of recombinants in which the trp promoter was oriented in the direction of the Le-IF-A gene.

Extracts were prepared for the IF assay as follows: 1 ml cultures were grown in L broth with 5 pg / ml tetracycline to an A55Q of about 1.0, then diluted in 25 ml M9 media with 5 pg / ml tetracycline. 10 ml samples were collected by centrifugation when A ^ q reached 1.0 and cell cakes were placed in 1 ml of 15% sucrose, 50 mmol. Tris-HCl (pH 8.0), 50 mmol. EDTA suspended. 1 mg of lysozyme was added and after 5 min at 0 ° C the cells were disrupted by sonication. The samples were centrifuged for 10 min at 15,000 rpm in a Sorvall SM-24 rotor. The interferon activity in the supernatants was determined by comparison with Le-IF standards using the inhibition test of the cy topathic effect (CPE). To determine the number of IF molecules per cell, a Le-IF

Specific activity of 4 x 10 units / mg was used (Rubinstein et al., Proc. Natl. Acad. Sci. (USA) T £, 640 (1979)).

The clone pLe-IF-A-trp 25, in which the trp promoter had been inserted in the desired orientation, gives high activity values (even 2.5 x 10 ^ units / 1). The IF formed by E. coli K12 strain 294 / pLe-IF A-trp 25 behaves like authentic human Le-IF; it is stable to treatment at pH 2 and is neutralized by rabbit anti-human leukocyte antibodies. The interferon has an apparent molecular weight of approximately 20,000.

The isolation of cDNAs for additional steering interferon DNA from the fully characterized Le-IF A cDNA-containing plasmid was excised with Pst I, electrophoretically isolated and according to a published procedure (Taylor et al., Biochem. Biophys. Acta. 442, 324 (1976)) marked with. The radiolabelled DNA was used as a probe for the screening of other E. coli 294 transformants by an in situ colony assay, Grunstein et al., As above. Colonies were isolated that hybridized to the probe in varying amounts. Plasmid DNA from these colonies and the 10 hybridizing colonies mentioned above were isolated by excision with Pst and characterized by three different methods. First, these Pst fragments were characterized by their restriction endonuclease degradation patterns with the Bgl II, Pvu II and Eco RI enzymes. This analysis allowed the classification of at least 8 different types (Le-IF A, Le-IF B, Le-IF C, Le-IF D, Le-IF E, Le-IF F, Le-IF G and Le-IF H ), which comes close to the location of various restriction cuts relative to the currently known pre-sequence and coding sequence of Le-IF A. One of these, Le-IF D, is believed to be identical to that described by Nagata et al., Nature 284, 316 (1980).

Then certain of the DNAs were tested for their ability to selectively remove Le-IF mRNA from poly-A-containing KG-1 cell RNA according to a published hybridization selection test, Cleveland et al., Cell 2Q, 95 (1980) . In this test, Le-IF A, B, C and F were positive. Third, the latter Pst fragments were inserted into an expression plasmid, E. coli 294 was expressed with the plasmid and the fragments were expressed. The expression products, presumably pre-interferons, were all positive in the CPE test for interferon activity, although only marginally active in the case of the Le-IF-F fragment. In addition, all of the described Le-IF types were divided into sequences. -7-

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Second mature leukocyte interferon

The sequence of the isolated fragment with the gene for mature Le-IF-B shows that the first 14 nucleotides of types A and B are identical. We therefore proposed to isolate a fragment from pLe-IF A 25 which carries the trp promoter operator, ribosome binding site and the beginning of the Le-IF (A = B) gene, and this with the rest of the Combine B sequence in an expression plasmid.

Around 950 b.p. To obtain Sau 3a - Pst I fragment from the sequence shown in Fig. 7a, several steps were necessary due to the presence of one or more interfering Sau 3a restriction sites, i. i.e.: 1. The following fragments were isolated: a) 110 b.p. from sow 3a - Eco RI; b) 132 b.p. from Eco RI - Xba; c) over 700 b.p. from XBa - Pst. 2. Fragments (la) and (lb) were bound and cut with Xba and Bgl-II to exclude self-polymerization by Sau 3a and Xba end sites (the relevant Sau 3a site was within a Bgl II site; Bgl II- Cuts leave a sow 3a cohesive end). A 242 b.p. fragment was isolated. 3. The products of (2) and (lc) were combined and cut with Pst I and Bgl II, again to prevent self-polymerization. A fragment of approximately 950 b.p., Sau 3a - Pst I, was isolated. This fragment included the part of the Le-IF-B gene that Le-IF A did not have. 4. An approximately 300 b.p. fragment (Hind III - Sau 3a) with trp promoter operator, ribosome binding site, ATG start signal and cysteine codon from Le-IF A was isolated from pLe-IF A 25. 5. An approximately 3600 b.p. fragment Pst I - Hind III was isolated from pBr 322. This included the replicon and encoded tetracycline but not ampicillin resistance. 6. The fragments obtained in steps 3, 4 and 5 were linked in triplicate and transformed with this plasmid E. coli K-12 strain 294.

Small amounts of transformants were mini-tested, Bimboim et al., Nucleic Acid Research 2,1513 (1979), and plasmid samples were digested with Eco RI. The digested material provided 3 fragments, characteristic of 1) the Eco-RI-Eco RI-trp promoter fragment; 2) the inner Eco RI - Eco RI fragment of pL4; and 3) the fragment from the protein translation start signal to the Eco Rl site of pL4.

In the CPE test, bacterial extracts from clones produced in the above manner typically give test values of approximately 10 × IO ** units of interferon activity per 1 at A ^^ q = 1. A representative clone thus produced is 294 / pLIF B trp 7.

More mature Leukozvten Intserferons

Additional full length gene fragments that have other Le-IF types can be tailored and brought into expression carriers for expression, as in the case of Le-IF A. Complete sequence elucidation according to conventional measures will show whether a restriction site is close enough to the first amino acid codon of the mature interferon type to allow a convenient refuge to the solution, the removal of the pre-sequence by restriction cleavage and replacement of the codons amino-terminally together with the pre-sequence lost amino acid codons by attaching a synthetic DNA fragment as described above. If this does not work, Kleid et al. be applied. Briefly summarized, this includes cleavage of the fragment containing the pre-sequence just before the point at which the codon for the first amino acid of the mature polypeptide begins, by 1. converting the double-stranded DNA into single-stranded DNA in a region around this point; 2. Hybridize the single-stranded region formed in step 1 with a complementary starter sequence of single-stranded DNA, the 5 'end of the starter being opposite the nucleotide adjacent to the intended cleavage site; 3. Restoration of the part of the second strand, eliminated in stage 1, which lies in the 3 'direction of the starter, by reaction with DNA polymerase in the presence of deoxynucleotide triphosphates containing adenine, thymine, guanine and cytosine, and 4. Degradation of the remaining Single strand length of DNA that extends beyond the desired cleavage point. -8th-

AT 394 209 B

A short piece of synthetic DNA that ends at the 3 'end of the coding strand with the translation start signal ATG can then, e.g. B. by connecting to the blunt end to which the tailor-made gene for the mature interferons is bound, and the gene is inserted into an expression plasmid and brought under the control of a promoter and the associated ribosome binding site. Similar to the above, gene fragments coding for Le-IF-C and Le-IF-D were suitably constructed for direct bacterial expression. The expression strategy for these additional leukocyte interferons always included the use of the approximately 300 bp fragment (Hind III - Sau 3a) with the trp promoter operator, ribosome binding site, ATG start signal and cysteine codon from Le-IF A by pLe-IF A25. Hereby, gene fragments from further interferon genes were combined, which encode their respective amino acid sequences beyond the common initial cysteine. Each resulting plasmid was used to transform E. coli K-12 strain 294. Bonds to form the respective genes were as follows:

LelF-C

The following fragments are isolated from pLe IF-C: (a) 35 b.p. from sow 3a to sow 96

(b) 900 b.p. Sau 96 to Pst I (c) An approximately 300 b.p. fragment (Hind III-Sau 3a) is isolated from pLe IF A-25 as in part N (4) above. (d) isolate the approximately 3600 b.p. fragment from part N (5) above,

Construction 1) Link (a) and (c). It is cleaved with Bgl II, Hind III and the product is isolated from about 335 b.p. (Base pairs). 2) Triple linking of 1) + (b) + (d) and transforming E. coli with the plasmid obtained.

A representative clone thus produced is E. coli K-12 strain 294 / pLe IF C trp 35.

Le-IF D

The following are isolated from pLe IF-D:

(a) 35 b.p. from sow 3a to ava II

(b) 150 b.p. from Ava II to Bgl II

(c) approx. 700 b.p. from Bgl II to Pst I

From pLe IF A25: (Φ 300 b.p. from Hind III to Sau 3a

One isolated from PBr 322:

(e) about 3600 b.p. from Hind III to Pst I

Construction 1) Link (a) + (b), split with Bgl II and purify: 185 b.p.-product (1). 2) Link 1) + (d), split with Hind III, Bgl II and purify the approx. 500 b.p. product (2). 3) Connect (2) + (c) + (e) and transform E. coli with the resulting plasmid.

A representative clone thus produced is E. coli K-12 strain 294 / pLeIF D trp 11.

Le-IF F

The fragment containing Le-IF F can be tailored for direct expression by reassembly, made convenient by the complete homology of amino acids 1-13 of Le-IF B and Le-IF F. A trp promoter-containing fragment (a) is also suitable configured ends are obtained from pHGH 207, described above, via Pst I and Xba I treatment with subsequent isolation of the approx. 1050 bp fragment. A second fragment (b) is obtained as the larger of the fragments from the Pst I and Bgl II degradation of the plasmid pHky 10, a derivative of pBR322, which contains a Bgl II site between the tetracycline-resistant promoter and the structural gene. Fragment (a) contains approximately half of the -9- coding for ampicillin resistance

AT 394 209 B

Gens; Fragment (b) contains the rest of that gene and the entire gene for tetracycline resistance, with the exception of the associated promoter. Fragments (a) and (b) are combined via T4 ligase and the product treated with Xba I and Bgl II to eliminate dimerization, resulting in a fragment (c) with the trp promoter operator and genes for tetracycline and leads to ampicillin resistance

A fragment (d) of approximately 580 b.p. is obtained from pLe IF-F via Ava II and Bgl II degradation. It has codons for amino acids 14-166 of Le-IF F.

A fragment (e) (49 b.p.) is obtained by Xba I and Ava Π degradation of pLe-IF B. Fragment (s) encodes amino acids 1-13 of Le-IF F.

Fragments (c), (d) and (e) are triply linked in the presence of T4 ligase. The contiguous ends of the respective fragments are such that the assembled plasmid is formed correctly, the tetracycline resistance gene being brought under the control of the trp promoter operator together with the gene for mature Le-IF F, so that with the desired plasmid-transformed bacteria on tetracycline-containing plates can be selected. A representative clone thus produced is E. coli K-12 strain 294 pLelF F trp 1.

Le-IF H

The complete Le-IF H gene can be configured for expression as mature leukocyte interferon as follows: 1. Plasmid pLe-IF-H is used to isolate the Hae II and Rsa I degradation of the 816 base pair fragment by the signal peptide amino acid 10 extends to the 3 'non-coding region. 2. The fragment is denatured and subjected to repair synthesis with Klenow fragment, Klenow et al., Proc. Natl. Acad. Be. USA 65., 168 (1970), using the synthetic deoxyribooligonucleotide starter 5’-dATG TGT AAT CTG TCT. This general procedure is also described by Goeddel et al., US-SN 190 799 (September 25, 1980). 3. The resulting product is digested with Sau 3a and a 452 base pair (" bp ") fragment that represents amino acids 1-150 is isolated. 4. Sau 3a and Pst I degradation of pLelF H and isolation of the resulting 500 bp fragment yields a gene which codes for amino acids 150 to the end of the coding sequence. 5. Those isolated in steps (3) and (4) Fragments are linked together to form the following fragment: 1 met cys ATG TGT .. 166 asp Stop

GAT TGA____Pst I

Sau 3a which encodes the 166 amino acids of Le-IF H 6. pLelF A trp 25 is digested with Xba I, blunt-ended with DNA polymerase I and the product degraded with Pst I. The large resulting fragment can be linked to the product of step (5) to form an expression plasmid which, after transformation of E. coli K-12 strain 294 or other host bacteria, is able to express mature Le-IF H.

LeIF-I

The gene library of the human genome in phage λ Charon 4A, constructed by Lawn et al., Cell 15, 1157 (1978), was developed according to leukocyte interferon genes by von Lawn et al., Supra, and Maniatis et al., Cell 15,687 ( 1978). A radioactive LelF probe derived from the cDNA, clone LelF A (Goeddel et al., Nature 287, 411 (1980)) was used to test approximately 500,000 plaques. 6 LelF genome clones were obtained. After screening and plaque cleaning again, one of these clones, HLeIF2, was selected for further analysis.

According to the method described above, further probes can advantageously be used to isolate additional LelF clones from the human genome. These in turn can be used to produce further leukocyte interferon proteins according to the invention. 1. The 2000 base pair Eco RI fragment of the genome clone (λ HLeIF2) was cloned again at the Eco Rl site in pBR325. The resulting plasmid LelF I was cleaved with Eco RI and the 2000 base pair fragment isolated. The deoxyoligonucleotide dAATTCTGCAG (an Eco RI > Pst I- -10-

AT 394 209 B

Converter) was linked to the 2000 base pair Eco RI fragment and the resulting product was cleaved with Pst I to a 2000 base pair fragment with Pst I ends. This was digested with Sau 96 and a 1100 base pair fragment isolated that had a Pst I end and a Sau 96 end. 2. The plasmid pLelF C trp 35 was digested with Pst I and Xba I. The large fragment was isolated. 3. The small Xba I-Pst I fragment of pLelF C trp 35 was digested with Xba I and Sau 96. A 40 base pair Xba I Sau 96 fragment was isolated. 4. The fragments isolated in steps 1), 2) and 3) were linked to the expression plasmid pLelF I trp 1.

LelF-J 1. The plasmid pLelF J contains a 3800 base Hind ΙΠ fragment of human genomic DNA which has the LelF J gene sequence. A 760 base pair Dde I-Rsa-I fragment was isolated from this plasmid. 2. The plasmid pLelF B trp 7 was digested with Hind III and Dde I and a 340 bP Hind III - Dde I fragment was isolated. 3. The plasmid pBR322 was digested with Pst I, blunt-ended by incubation with DNA Pol I (Klenow fragment), and then digested with Hind III. The large (approximately 3600 bp) fragment was isolated. 4. The fragments isolated in steps 1), 2) and 3) were linked to the expression plasmid pLelF J trp 1.

The methods and materials used in the following assembly processes were as in the description above. The following Table 1 provides the details for the specific assembly of hybrid LelF plasmids: (Table 1 follows) -11- AT 394 209 B LelF amino acid expression front part rear part resulting hybrid * plasmid expression plasmid (vector) fragment amino acid Fragment amino acids / - 'SQ b / - ~ N < B 9 s < a "B / ~ S h b X" S Ü B < 3 Q P cl 5 "> 6 «< 3 < 3 < 3 Ιέ fei fei fei & i 4 & le 1 & 1 & 1 & 1 & D. & < -. rT 00 & ΛΪΪ

VO VO wo VO vo vo wo vo vo vO | g wo vo 1-N O * ^ N ^ N i-N i-N 1 m CS cö co rö 1 CO wo Os Os VO vo Os Os Os i = N

3 £ NN ^ N WO CS i-N ^ N wo cs NN r- _ & & & & OO ft 1 & & o NN < NN Q NN < Ti m NN ft NN o Uh ^ nn rv W YES CU 1 ft ^ 3 £ £ Cu 1 ft ^ 3% CU 1 fe-a ft & a, 3 W CO ft &'S; J3 & CO ft, 3 · & s ^ NN 3 £ ftO r- ^ § 7 NN NN 'S CQ o' f NN 'S CQ J8 l'T NN 3 £ ft © gl NN NN 3 £ o. © c ^ gl NN NN 3 £ c ° C r— g Ϊ > N-X bß CQ &

Sa O where

^ N CS cs co t i-N o wo Os I Os vo VO Os Os er »1-N i-N 1-N ^ N 1-N 1-N 1-N 1-N wo cs & a < ? & cu

CS X) X WO vo Q <

Oh X - where S oo 2 cs.

£ 5 I NN CS X X ft

ft 00 S oo ° G

S

< Q < CO ÜL fo E3 Ί J3 ä > ^ * - «>

& CU co Q 0) * - »= § & m & IS B ω where > cs üi) 2 α CQ PU ff tßü p, CQ Cu ff where cs & NN 3 < > ft.3 CS c X > S & ft ä cu x wo 00 cs

where cs where cs where .2 cs NN < * NN ^ CO X) X Uh NN 'S Ife / - \ Qh - pa ^ bß > - > pi, (¾ S X 3 X - Jä X ^ cu c o > wo 00 cs Sw 'C0 e £ > 5 X ° wo 00 cs S ··' CS C £ II

in IO

VD IO in full

> n VO

in VO m vo Q <

< Q ffl < O < < -12-

AT 394 209 B

LelF amino acid expression front part rear part total hybrid * plasmid expression plasmid (vector) fragment amino acid fragment amino acid / —v < B / —N Q P, HH CQ l-H / “N o P / —N CQ P PQ S 'Λ \ o a CQ s cq a CQ 3 CQ 3 P 3 P 3 P 3 Sfe 6έ 6 έ 1§ le 1 & 4 & 1 & • äe in VO S VO VO Ό VO s VO vo 8 »-H l-H 1-H 1-H cs PC PC PC PC PC PC Os Os Os Ov Os OV Os m FH cs * + r- 1—1 & & & < V§ & "Q" U, HH O ii p _ pq ~ o SS &-S; ° r% & <? ^ ^ ts & ^ i & 'S SS &-s; , 3. &Amp; SS & 'SB „in 5 S in nh λ h ftg _ in r SR ttS shd ri = 5 · ® R Uo 3 C £ B Ug s B 3.0 3gii £ g > L £ gd / £ g sL £ g £ gst £ g ^ CS CS CN (N CS < N CS Os Os Cf. Cf, Os Os Os tH ^ H 1 “H ^ H» H r · - r * Γ- »H iH iH B & P §« «> & & s «5« 1 = 1 PPPBP £ cq ^ £ PQ -v 3 Uh Xba I-Pvu from pLelF (288 bp) 3 PQ Hind III-from pLel (-630 bp 7 H £ V β 9 Jr W 1-1 _ - clO ^ CS 2 gl Hind III-v. PLelF (-630 bp Hind III-: from pLel (-630 bp Xba I-Pv from pLel (288 bp) Xba I-Pv from pLel (288 bp) X > ja Λ X) α ca GJ ss vo VO ss v8 s pH ^ H iH * -H < P PQ Ü CQ fe o CQ cq CQ CQ PPP Xbal-PVUII 1-92 PvuII-Pstl 92-165 pLelFFA from pLelF F trp 1 from pLelF A trp 25 trp (Pvu II) (288 bp) (-550 bp) -13-

AT 394 209 B T3 E ä ε • 8 .2 C wJä §3 S a c x a ω c < υ

οο Ο C ε < _ ΝΗ Ρ 1-1 ο ^ < £ 5 Κ 3 Λ 3 ~ "PU CQ & έ & £. ΐ & 11 so SO SO Ό so Ό SO VO 1— (1— (I 1 1 1 (Ν m CO m * 0 Cs ΟΝ ΟΝ δ < υ W c £

Table 1 (continued) C οε 60 C3

C ε 3 κΰ C / 5 Ο C ~ Γ- £ & ra δ ftη-ι ο. 3 C < ε § Ο ο.χ > ο m '”Ή ί Ο, ο α, CO α, ΙΟ (Ν & < ft, -, 3 | Ο. ο, C 1 ο > CO τ (Ν (Ν (Ν LO 0 \ ΟΝ Os < —1, -Ν < υ Έ ο > 3 < υ & ο Λ C / 5 C Ο1 * 2? * '£ 5 & aS ω Λ ο C $ υ * 3 > ίΧ

Λ X) X Λ § & & & ft Η ft s ft h— < ί — 1 ft 3 ft 3 ft ft ^ 3g λ ί: ί 1 S 'S' S. · 0 έ 3g CU ^ bO PQ 1 3 £ - 00 ε οο ξ < Ν Λ XX r- 00 S 00 ξ η > 'w' ca £ > X - CO ε co § ö Λ X X _ IO ix Λ Λ Ä ο c ε η ε < ο Ö0 Τ3

58 VO Ό VO \ ο

ΙΟ CN

CQ ft it «ο & feb * ca, 3 -14-

AT 394 209 B

With further reference to Table 1, the first four hybrid LelFs described were made from two Lelf expression plasmids. A Bgl II-S part, as common to LelF A and D cDNAs, was used to construct an expression plasmid pLeDF trp AD (Bgl II), which contains the 63 amino acids of LelF A and amino acids ending in carboxyl groups encoded by LelF D. The same site was used in the construction of an expression plasmid pLelF trp DA (Bgl II), which encodes 64 amino acids of LelF D ending in amino groups and 102 amino acids of LelF A ending in carboxyl groups. The Pvu II part was used in the construction of the two other hybrid interferon expression plasmids: 91 amino acids of A terminating in amino groups with 74 amino acids of D (pLelF trp AD (Pvu II)) terminating in carboxyl groups and 92 in amino groups LelF D amino acids ending with 74 LelF A amino acids ending in carboxyl groups (pLelF trp DA (Pvu II)). In summary: pLelF AD trp fPvuIII:

The large (approx. 3900 bp) fragment of an Xba I and Pst I degradation of pLelF A trp 25 was treated with a 285 bp Xba I-Pvu II fragment of pLelF A trp 25 and an approx. 550 bp Pvu II-Pst I fragment of pLelF D trp 11 linked; pLelF DA tm (Pvu ID:

The large (approx. 3900 bp) fragment from an Xba I and Pst I degradation of pLelF A trp 25 was treated with a 288 bp Xba I-Pvu II fragment from pLelF D trp 11 and an approx. 550 bP Pvu Π- Pst I fragment from pLelF A trp 25 linked; pLelF AD trp (Bgl ID:

The large fragment of a Bgl Π, Pst I degradation of pLelF A trp 25 was linked to an approximately 600 bp Bgl II-Pst I fragment of pLelF D trp 11; and nLelF DA tm IBgl ID:

The large fragment from a Bgl II and Pst I degradation of pLelF D trp 11 was linked to an approximately 700 bp fragment obtained by Pst I cleavage of pLelF A trp 25 and subsequent Bgl Π digestion.

For the 5th specified hybrid: pLelF AB trp (Tvu ID:

The large (approx. 3900 bp) fragment from an Xba I and Pst I degradation of pLelF A trp 25 was combined with a 285 bp Xba I-Pvu II fragment from pLelF A trp 25 and an approx. 750 bp Pvu II- ( partial) -Pst I fragment of pLelF B trp 7 linked Similarly, the other compositions shown in Table 1 are specified. As another example, when building a hybrid containing LelF C and / or LelF H part, one can take advantage of common Bbv I sites that occur, for example, at nucleotide 294 (i.e., GCTGC) of the gene sequences. Similarly, plasmids suitable for microbial expression of other new hybrid leukocyte interferons can be formed by appropriate manipulation of the double-stranded DNA encoding all or part of the amino acid sequences of naturally occurring leukocyte interferons. For example, a first double-stranded DNA fragment is selected which encodes the amino end of a first naturally occurring leukocyte interferon amino acid sequence and, progressing in the 3 'direction, a considerable part of the amino acid sequence. The fragment has a restriction endonuclease cleavage site near the codons for the amino acid " n " of the first leukocyte interferon, n amino acids forming a significant part of the amino acid sequence of the first interferon. Restriction endonuclease cleavage provides a fragment with the amino end of the first interferon and codon for approximately n amino acids. A second fragment with all or part of the codons for the amino acid sequence of a second, different leukocyte interferon is selected, the fragment having a cleavage site for an identical restriction endonuclease near codons for the amino acid of the second interferon, the amino acid number (from Progressing amino end of the second interferon) is about 166-n. Cleavage of the second fragment with the restriction endonuclease provides a product complementary to the " n " end portion of the digest product of the first fragment so that the digest product of the second can be linked to that of the first to regress the restriction endonuclease recognition site and re-forming the codon for the amino acid n of the first interferon, if lost during the first digestion. The restriction endonuclease digestion product of the second fragment preferably proceeds from the end that occurs upon 3'-cleavage by nucleotides encoding the carboxyl end of the second leukocyte interferon.

On the other hand, hybrids can be formed which contain substantial parts of the amino acid sequences of more than two naturally occurring leukocyte interferons. B. the second fragment mentioned above is also selected to be a second restriction endonuclease site after the first one.

AT 394 209 B, the second site being identical to a similarly located site within a fragment encoding the carboxyl end part of a third leukocyte interferon, etc. In the example mentioned, the products of the successive restriction endonuclease inserts can be linked three times to produce a hybrid gene encoding the amino end group part of a first interferon, the middle region of the amino acid sequence of the second and the carboxyl end part of the third (or a further variant of the first, the first and the third interferon being the same).

Preferably, the first fragment mentioned above is derived from an expression plasmid, i.e. H. one in which codons for the amino end portion of the first leukocyte interferon have an ATG or other translation initiation codon as a predecessor and a promoter or promoter-operator system. As a result, the end product of the manipulations described above will be a plasmid capable of expressing the polypeptide encoded by the hybrid gene in bacteria or other microbial organisms transformed with the plasmid. Further measures for designing the hybrid gene for microbial expression are obvious to the person skilled in the art.

In preferred embodiments of the invention, the hybrid genes encode a new leukocyte interferon amino acid sequence of approximately 165 to 166 amino acids, a conjugate of essential amino acid sequences representing two or more different leukocyte interferons, selected from LeDF A, LelF B, LelF C , LelF D, LelF E, LelF F, LelF G and LelF H, as shown in Fig. 3a and 3b. Most preferably, the new leukocyte interferons encoded by the hybrid genes have the named and as in the " all " 3a and 3b indicated arranged amino acids. The expression products of plasmids produced according to the invention can be tested for antiviral activity in a conventional manner, as in the biological activity determinations described below.

Detection of the antiviral activity E. coli K-12 strain 294 were transformed in a conventional manner independently with the plasmids pLelF trp A 25, pLelF trp D, pLelF trp A / D (Bgl II) and pLelF trp D / A (Bgl II). The transformants were grown separately in 5 ml cultures in L broth with 5 mg / ml tetracycline to an A ^ Q value of 1.0, then diluted in 11 M9 medium with 5 μg / ml tetracycline. The cells were harvested when A ^ q reached 1.0 and the cells in 10 ml of 15% sucrose, 50 mmol. Tris-HCl (pH 8.0), 50 mmol. EDTA suspended. 10 mg lysozyme was added and after 5 min at 0 ° C the cells were disrupted by sonication. The samples were centrifuged for 10 minutes at 15,000 rpm in a Sorvall SM-24 rotor. The interferon activity of the supernatants was tested for antiviral activity.

The yields per 1 culture of these interferons, titrated on a human cell strain (Wish), are shown in Table 2, from which it is clear that the LelF-A / D activity is caused to a greater extent than that of other interferons. This difference could be due to the greater predetermined activity of the LelF-A / D or greater yield, expressed in mg protein of this interferon. Since the genetic linkage was identical for all of these interferons, it appears most likely that LelF-A / D has practically greater activity than the other interferons.

Table 2

Yield of leukocyte interferons from shake flask cultures of E. coli

Type of interferon

Activity, yield ^ (units at Wish) +) 8x 107 5x 106 2x 108 1 x 106

A

D AD (Bgl II) DA (Bgl II) +) tested by inhibiting the cytopathic effect on Wish cells with VSV as test substance -16-

AT 394 209 B

The effectiveness of the different interferons in a pure of mammalian cell strains was determined (by the human Wish; by the African green monkey Vero; hamster fibroblast BHK; kidney cells from rabbits, RK-13; mouse L-929; and bovine kidney, MDBK cells ). In order to compare the relative activity of the interferons, their activity on different cells, relative to their activity on Wish cells, was set at 100. The results in Table 3 show that LelF-A / D has a very high activity in VERO and L-929 cells, while LeIF-D / A has a low activity in these cell strains. These results show that the combination of the N-end part of LeIF-Α and the C-end part of LelF-D within one molecule (LelF-A / D) contributes to the particular effectiveness of the hybrid protein, which is expressed in various mammalian species finds. Furthermore, these properties are not a simple summation of the properties of the output interferons. This can be clearly seen in the case of activity on L-929 cells (Table 3), in which case neither a mixture of LeIF-Α and LelF-D nor the other hybrid, LeIF-D / A, shows any significant activity.

Table 3

Titration of different leukocyte interferons in cell strains from different mammalian species

Leukocyte interferons +)

Cell strain A D A / D D / A A + D Leukocyte film in centrifuged blood WISH 100 100 100 100 100 100 VERO 250 75 1,670 20 200 200 BHK 400 200 833 2,000 400 20 RK-13 12 500 6 N.D. N.D. 120 L-929 150 5 3,300 2 10 0.1

Note to Table 3: +) Interferons, tested against VSV infection of the different cell strains. Activities expressed as a percentage of the activity observed in WISH cells.

The activity of LelF-A / D against other viruses was also examined. The data in Fig. 5 show antiviral effects against EMC virus infection of L cells, and the data in Fig. 6 show effects against VSV infection of L cells. It is clear from this data that the greater activity of LelF-A / D is not limited to one virus (VSV) and that the greater activity is apparently a common property towards many viruses. Natural human leukocyte film interferon preparations have no effect on mouse cells (see Table 2). The activity of LelF-A / D against EMC virus infection in CD-1 mice was therefore investigated. The results in Fig. 7 show that LelF-A / D is extremely effective against lethal EMC virus infection and LeIF-Α also has antiviral activity, as expected from the activity in cell strains (Table 2). The data in Fig. 7 come from treatments i. p. 3 h before infection. The doses on LelF-A / D and LeIF-Α are titrated as on WISH.

Lethal EMC virus infection of hamsters is also affected by LelF-A / D and LeIF-Α (Fig. 8), the former being the most effective, and leukocyte film interferon shows only a small and statistically insignificant effect. In the case of Fig. 8, all interferons i. p. Given 3 hours before infection in a dose of 5 × 105 μg / kg, titrated on WISH cells.

These results indicate that the pronounced antiviral effects of LelF-A / D in a number of mammalian species are not limited to cell cultures, but are also observed in lethal viral infections. EMC virus can be regarded as a model system, and a demonstration of the antiviral activity against this system may make it possible to predict the antiviral activity against the embodied virus family, e.g. B. the Picornavirus family, for the foot and mouth disease and polio are representatives. VSV virus can be regarded as a model system, and a demonstration of the antiviral activity against this may make it possible to predict the antiviral activity against the embodied virus family, e.g. B. the rhabdovirus family, of which Rabies is an important representative.

Table 4 summarizes the activities of various LelF hybrids on WISH and MDBK cells and their activity ratios: -17-

AT 394 209 B

Table 4

LelF Hybrid (PvuII)

Units / 1 culture WISH cells

Units / 1 culture of MDBK cells

Activity ratio WISH / MDB AB 2.4 x 108 4x 107 6 AD 1.2 x 108 2 x 107 6 AF 6x 107 1 x 107 6 AG 4x 107 1.5 x 107 2.7 AI 3.2 x 107 1.2 x 107 2.7 BA 1.5 x 107 1 x 107 1.5 BD 6x 107 1.5 x 107 4 BF 1 x 106 3.5 x 105 0.3 BG 2x 107 6x 107 0.3 DA 3x 106 1.2 x 108 0.025 DB 2x 106 5x 107 0.04 DF 2x 105 4x 106 0.05 DG 2x 105 1.5 x 107 0.014 FA 2x 105 6x 107 0.003 FB 2x 106 8x 107 0.025 FD 1 x 107 2x 107 0.5 FG 1 x 106 4x 107 0.025 IA 2.4 x 106 6 x 107 0.04 A * 8x 107 1.2 x 108 0.7 B * 8x 107 4x 108 0.2 C * 2x 107 1.5 x 107 1.3 D * 5x 106 2.5 x 107 0.2 F * 2x 107 2x 108 0.1 I * 1.6 x 107 1.2 x 107 1.3 * For comparison purposes

Parenteral administration

The hybrid leukocyte interferons can be administered parenterally to those in need of anti-tumor or anti-viral treatment and those who show immunosuppressive conditions. Dosage and dose rate may be the same as that currently used in clinical studies of human-derived materials, e.g. B. about (1 to 10) x 10 ^ units per day, and in the case of materials of greater purity than 1%, easily up to, for example, 5 x 107 units per day. Preliminary evidence from the monkey study described above suggests that dosages of bacterially obtained Le-IF could be increased significantly for greater potency, thanks to the practical lack of human proteins other than Le-IF that act as fever-producing materials in leukocyte-derived materials can and thereby adverse effects such. B. discomfort, temperature increase, etc. show.

As an example of a suitable dosage form for essentially homogeneous bacterial Le-IF in parenteral form, to be used here with the necessary changes, 3 mg Le-IF of the specific activity of e.g. B. 2 x 108 U / mg in 25 ml of 5N serum albumin (human) - USP, the solution is passed through a bacteriological filter and the filtered solution is divided aseptically into 100 ampoules, of which this 6 x 10 ^ units of pure interferon , suitable for parenteral administration. The ampoules are preferably stored in the cold (-20 ° C) before use.

The compounds according to the invention can be prepared pharmaceutically by known methods for the preparation.

Claims (11)

AT 394 209 B of useful agents, whereby the polypeptide is mixed with a pharmaceutically acceptable carrier. Suitable carriers and their composition are described in Remington's Pharmaceutical Sciences by E.W. Martin, the disclosure of which is hereby incorporated by reference into the present application. Such agents contain an effective amount of the interferon protein along with an appropriate amount of a carrier for the manufacture of pharmaceutically acceptable agents suitable for effective administration. 1. A method for producing new microorganisms which are capable of producing polypeptides with the amino acid sequence of hybrid human leukocyte interferons, characterized in that a microorganism with a replicable expression vector which contains a DNA coding for hybrid human leukocyte interferon Sequence contains, transformed and cultivated in a manner known per se. 2. The method according to claim 1, characterized in that the transformation is carried out with a plasmid. 3. The method according to claim 1 or 2, characterized in that the transformation is carried out with an expression vector which can express a double-stranded DNA sequence, which consists of a first double-stranded DNA fragment, which is the N-terminal region of human Leukocyte interferon D, and a second double-stranded DNA fragment encoding the C-terminal region of human leukocyte interferon A, both of which are connected via a PvuII interface. 4. The method according to claim 1 or 2, characterized in that the transformation is carried out with an expression vector which can express a double-stranded DNA sequence, which consists of a first double-stranded DNA fragment, which is the N-terminal region of human Leukocyte interferon A, and a second double-stranded DNA fragment encoding the C-terminal region of human leukocyte interferon D, both of which are connected via a PvuII interface. 5. The method according to claim 1 or 2, characterized in that the transformation is carried out with an expression vector which can express a double-stranded DNA sequence, which consists of a first double-stranded DNA fragment, which is the N-terminal region of human Leukocyte interferon D, and a second double-stranded DNA fragment encoding the C-terminal region of human leukocyte interferon A, both of which are connected via a BglII interface. 6. The method according to claim 1 or 2, characterized in that the transformation is carried out with an expression vector which can express a double-stranded DNA sequence, which consists of a first double-stranded DNA fragment, which is the N-terminal region of human Leukocyte interferon A, and a second double-stranded DNA fragment encoding the C-terminal region of human leukocyte interferon D, both of which are connected via a BgUI interface. 7. The method according to claim 1 or 2, characterized in that the transformation is carried out with an expression vector which can express a double-stranded DNA sequence, which consists of a first double-stranded DNA fragment, which is the N-terminal region of human -Leukocyte interferon A, and a second double-stranded DNA fragment encoding the C-terminal region of human leukocyte interferon B, both of which are connected via a PvuII interface. 8. The method according to claim 1 or 2, characterized in that the transformation is carried out with an expression vector which can express a double-stranded DNA sequence, which consists of a first double-stranded DNA fragment, which is the N-terminal region of human -Leukozyten-Interferon A encoded, and from a second double-stranded DNA fragment which encodes the C-terminal -19- AT 394 209 B region of human leukocyte interferon F and which are both connected to each other via a PvuII-interface. 9. The method according to any one of claims 1 to 8, characterized in that a bacterium is used as the starting micro-5 organism. 10. The method according to claim 9, characterized in that a strain of E. coli is used as the bacterium. 11. The method according to claim 10, characterized in that the bacterium E. coli K-12 strain 294 is used. 15 Including 13 sheets of drawings 20 -20-