KR910009901B1 - Hybrid human leukocyte interferons - Google Patents

Abstract

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Description

Hybrid human leukocyte interferon

Figure 1 depicts the nucleotide sequence of the coding region of eight leukocyte interferon ("LeIF") complementary DNA ("cDNA") clones.

2 is a restriction endonuclease map of eight forms of LeIF cloned cDNA (A-H).

3 is a comparison of the eight LeIF protein sequences expected from the nucleotide sequence.

4 is a comparison of the amino acid sequences of mature leukocyte interferon A and D.

5 and 6 show the activity of the preferred hybrid leukocyte interferon (“LeIF-A / D”) of the present invention against brain myocarditis virus (“EMC”) and vesicular stomatitis virus (“VSV”) in mouse cells, respectively. The results of comparative tests are shown.

7 and 8 show the results of comparative tests using LeIF-A / D and other interferons for EMC virus infection in mice and hamsters, respectively.

Figure 9 shows the DNA sequences of the five LeIF proteins, including the I and J forms.

The present invention relates to a method for microbiologically preparing hybrid leukocyte interferon useful for the treatment of viral diseases and neoplastic diseases, by means of recombinant DNA techniques, and to means and end products in such methods.

Relatively homogeneous leukocyte interferon is obtained from leukocytes of normal or leukemia patients. These interferons are a class of proteins characterized by their strong ability to confer virus-resistance in their target cells. In addition, interferon can inhibit cell proliferation and modulate immune responses. These characteristics suggest that interferon is clinically used as a therapeutic agent in the treatment of viral infections and malignancies.

More recently, recombinant DNA techniques have been used to microbiologically prepare a number of different leukocyte interferons, each amino acid sequence being about 70% homologous, as described in David V. Goeddel et. al., Nature 290, 20-26 (1981). In particular, genes encoding amino acid sequences of various leukocyte interferons designated LeIF A, B, C, D, F, G and H are described in David V. Goeddel and Sidney Pestka, respectively; Koeffler, H.P. And the cell line KG-1 described in Golde, D.W., Science 200, 1153-1154 (1978). Cell line KG-1 has been deposited with the American type culture collection under ATCC Accession No. CPL 8031. Using these genes properly deployed in plasmidic vehicles for bacterial expression, E. coli deposited as host bacteria, preferably E. coli, deposited in the American Type Culture Collection on October 28, 1978. K-12 strain 294 can be transformed.

Agonists in recombinant DNA technology are plasmids, which are non-chromosomal loops of double-stranded DNA found in bacteria and other microorganisms, sometimes in multiple copies per cell. Information encoded in plasmid DNA generally includes one or more optional properties (e.g. resistance to antibiotics that enable cloning of host cells containing important plasmids that are recognized and selectively grown in the selection medium for bacteria) and autologous cells. Necessary for replicating the plasmid, i.e., the "replicon". The utility of the plasmids is that the plasmids can be specifically delivered by one or more restriction endonuclease or “restriction enzymes”, each of which recognizes different sites on the plasmid DNA. Therefore, a heterologous gene or gene fragment can be inserted into the plasmid by terminal linkage at the cleavage site or at the reconstructed end site adjacent to the cleavage site. DNA recombination is performed extracellularly, but the resulting “recombinant” plasmid can be introduced into the cell through a process known as transformation, and the transformant is grown to obtain a recombinant plasmid containing a large amount of heterologous genes. Moreover, when the gene is properly inserted in relation to the site of the plasmid that regulates the transcription and translation of the encoded DNA message, the resulting expression vehicle can be used to produce polypeptide sequences encoded by the actually inserted gene. This process is called expression.

Expression begins at the promoter region which is recognized and bound by RNA polymerase. As in the case of tryptophan or “trp” promoters, which are preferred for the practice of the present invention, in some cases the promoter regions are overlapped by the operator regions to form the associated promoter-operator region. An operator is a DNA sequence recognized by a so-called repressor protein that regulates the frequency of transcription initiation at a particular promoter. Moving along the polymerase DNA, the information contained in the encoded backbone is transcribed from its 5 'end to the 3' end into messenger RNA, which is then translated into a polypeptide having an amino acid sequence encoded by DNA. Each amino acid is encoded by a nucleotide triplet or “codon” in a site that may be referred to as the “structural gene”, ie, the site that encodes the amino acid sequence of the expressed product. RNA polymerase, after binding to a promoter, first encodes a ribosome binding site, and then a translational initiation or "start" signal (usually ATG, resulting in AUG in the messenger RNA produced), and then its structural genes. Nucleotide codons are transcribed. Stop codons are transcribed at the ends of structural genes, and the polymerase can then form additional sequences of messenger RNA that remain untranslated by the ribosomes due to the presence of stop signals. In bacteria, once the mRNA is formed, ribosomes bind to the binding sites on the messenger RNA, which produce encoded polypeptides that begin at the translational start signal and end at the stop signal mentioned above. The desired product is produced when the sequence encoding the ribosomal binding site is properly located relative to the AUG start codon and all remaining codons are present after the start codon in turn. The resulting product can be obtained by dissolving the host cell and then appropriately purifying and recovering it from other bacterial proteins.

Studies of the nucleotide sequences of genes encoding various leukocyte interferons have found some of them have some commonalities with respect to the presence and location of cleavage sites recognized by certain restriction endonucleases. According to the present invention, using this commonality, novel hybrid genes useful for the microbiological production of hybrid leukocyte interferon, which can be expected to exhibit some of the antiviral and other properties of interferon encoded by the parent gene by DNA recombination, are described. You can take advantage of it. In a preferred embodiment of the invention, such hybrid leukocyte interferon may exhibit enhanced activity than that encoded by the parent gene.

The parental leukocyte interferon genes encoding the class of leukocyte interferon proteins of interest in the present invention each exhibit a natural allele difference. These differences can be explained by difference (s) of amino acids in the overall protein sequence or by deletion, substitution, insertion, inversion or addition of amino acid (s) in the sequence. Each parental leukocyte interferon in the present invention, ie labeled LeIF A, LeIF B... In the case of LeIF J et al, such allelic differences are included in the present invention as they are within the scope of the label or term defining them.

These and other objects and advantages of the present invention will become more apparent from the following detailed description and the accompanying drawings:

Figure 1 depicts the nucleotide sequence of the coding region of eight leukocyte interferon complementary DNA clones. Of these, one sequence correspondingly labeled LeIF E is an apparent “pseudogene” that fails to encode active leukocyte interferon, and the other labeled LeIF G is less than the complete sequence for the corresponding interferon species. It contains. The ATG detoxification initiation codon and termination triplets for each LeIF are underlined.

2 shows restriction endonuclease maps of eight forms of LeIF cloned cDNAs (A to H). Plasmids containing clones are constructed by the dC: dG tailing method (Goeddel, D. V. et al., Nature 287, 411-416 (1980)). Therefore, cDNA inserts can be cleaved using Pst I, ie each end of each insert is a pst I restriction endonuclease cleavage site. Lines at each end of each cDNA insert are flanking monopolymeric dC: dG ends. Pvu II, Eco RI and Bgl II restriction sites are shown. Dark areas in the figures show the coding sequence of mature LeIF; Hatched portions represent signal peptide coding sequences; Blank portions indicate 3 'and 5' unencoded sequences.

3 shows a comparison of the eight LeIF protein sequences expected from the nucleotide sequence. Use the one letter abbreviation recommended by the IUPAC-IUB Commission on Biochemical Nomenclature: A, Alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine, G, glycine; H, histidine; I, isoloincin; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, gulutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; And Y, tyrosine. Numbers indicate amino acid positions, where S represents signal peptide. A dash (-) at position 44 of the 165 amino acid sequence of LeIF A was introduced to fit the LeIF A sequence to the 166 amino acid sequence of the other LeIF. The LeIF E sequence was determined ignoring extra nucleotides (position 187 in Figure 1) in its coding region. Asterisks indicate stop codons in the same phase. Amino acids common to all LeIF (except pseudo gene LeIF E) are also shown. Underlined residues are amino acids that are also present in human fibroblast interferon.

Nucleotide +1 to 69 in FIG. 1 corresponds to S 1 to S 23 amino acids in FIG. Codon TGTs (nucleotides 70-72) of FIG. 1 correspond to cysteine (C, amino acid 1) of FIG. In FIG. 3, the Pvu II restriction endonuclease cleavage site is between the codons for amino acids 92 and 93 at LeIF A, B, D, F and G, ie between nucleotides 346 and 347 of FIG.

4 shows a comparison of the amino acid sequences of mature leukocyte interferon A and D, where the deletion of amino acid 44 in LeIF A is shown in dashes. For LeIF D amino acids only those different from the corresponding amino acids of LeIF A are shown: The amino acid sequence of LeIF D is identical to LeIF A in other parts. Figure 4 also shows the relative positions on the corresponding genes of the Bgl II and pvu II restriction endonuclease cleavage sites used to prepare the preferred hybrid leukocyte genes of the present invention.

5 and 6 show the activity of the preferred hybrid leukocyte interferon (“LeIF-A / D”) of the invention against brain myocarditis virus (“EMC”) and vesicular stomatitis virus (“VSV”) in mouse cells, respectively. It shows the results obtained by comparing the test.

7 and 8 show the results of comparative tests using LeIF-A / D and other interferons for EMC virus infection in mice and hamsters, respectively. The data in FIG. 7 is the result of intraperitoneal administration 3 hours prior to infection. The dose of LeIF-A / D and LeIF-A is the result of titer on WISH cells.

Figure 9 depicts the DNA sequences of five LeIF proteins, including the I and J forms.

[Description of the Desirable Sun]

[Microorganisms Used]

Two microorganisms are used in the described study: E. coli X1776 as described in US Pat. No. 41,90495, and E. coli K-12 strain 294 (end as described in British Patent No. 2053538A). A, thi-, hsr-, hsm _K +). Each of these has been deposited as ATCC Accession Nos. 31537 and 31446 to the American Type Culture Collection on July 3, 1979 and October 28, 1978, respectively. All recombinant DNA studies are conducted according to the guidelines of the National Institute of Health.

In the most preferred aspect of the present invention, the present invention relates to E. coli, including strains E. coli X1776 and E. coli K-12 strain 294 as defined above, as well as other known E. coli strains such as E. coli B, Or in relation to many other microbial strains (potentially available) that have been deposited with an accredited microbial depository, such as the American Type Culture Collection (ATCC), and (potentially) (see also the ATCC catalog and JP-A-2644432). These other microorganisms include Bacillus, such as Bacillus subtilis, and Salmonella typhimurium, for example, using plasmids capable of replicating and expressing heterologous gene sequences therein. ) And other enterobacteriaceae to which Serratia marcesans may be mentioned. FIG's yeast such as three Levy jiae (Saccharomyces cerevisiae), I for the production of interferon proteins of the invention by the under the control of a yeast promoter Sikkim expressing the gene encoding it can be advantageously used as a host organism.

According to the present invention, LeIF hybrids are prepared by advantageously taking the restriction endonuclease cleavage sites which are common in the individual parent genes and their respective ends, together with the same sites in the carrier expression plasmid (vector). For example, large (˜3900 bp) fragments of xba I to Pst I digests of pLeIF A trp 25 expressing plasmids can be linked to xba I to Pvu II and Pvu II to Pst I digest fragments of various LeIF parent genes to obtain corresponding Expression plasmids can be provided that can be operated to yield a hybrid LeIF.

Each LeIF expression plasmid is independently a variety of LeIF plasmids such as pLeIF A trp 25, pLeIF B trp 7, pLeIF C trp 35, pLeIF D trp 11, pLeIF F trp 1, pLeIF G, pLeIF H and pLeIF I. (The methods for their construction are described in Nature 287, 411 (1980)), either by using digest fragments separated from pBR 322 (the construction of Gene 2, 95 (1977)). Construct using the digest fragments isolated from. In some of these plasmids, “trp” represents the tryptophan promoter-operator system that is most preferred for bacterial expression (published European Patent Application No. 36776).

[Direct Expression of First Mature Leukocyte Interferon]

Methods of directly expressing LeIF A as a mature interferon polypeptide include a combination of synthetic (N-terminal) DNA and complementary DNA.

The Sau-3a restriction endonuclease site is conveniently located between codons 1 and 2 of LeIF A. Two synthetic deoxyoligonucleotides are designed to import ATG translational initiation codons, repairing the codons for amino acid 1 (cysteine) and generating EcoRI adhesion ends. These oligomers are linked to the 34 bp Sau 3a-Ava II fragment of pL 31. The resulting 45 bp product is linked to two additional DNA fragments to construct a 865 bp synthetic-natural hybrid gene that encodes LeIF A and is bound by EcoRI and Pst I restriction sites. This gene is inserted between the EcoRI site and the Pst I site of pBR322 to give plasmid pLeIF A1.

Plasmid pGM 1 carries E. coli tryptophan operon containing the defect ΔLE1413 (GFMiozzari et al., J. Bacteriology 133, 1457-1466 (1978)), under the control of the trp promoter-operator system It expresses a fusion protein consisting of the entire trp D polypeptide as well as the first six amino acids of the leader and the approximately last third amino acid of the trp E polypeptide (highly referred to as LE '). 20 μg of plasmid is digested with restriction enzyme Pvu II, which cleaves the plasmid at five sites. The gene fragments are then combined with an EcoRI linker (consisting of the sequence's own complementary oligonucleotide pCATGAATTCATG) which provides an EcoRI cleavage site for cloning into a plasmid containing the EcoRI site. 20 μg of DNA fragment obtained from pGM 1 was transferred to 20 μl T ₄ DNA ligase buffer (20 mM Tris, pH 7.6, 0.5 mM ATP, 10 mM MgCl ₂ , 5 mM in the presence of 200 picomolar 5′-phosphorylated synthetic oligonucleotide pCATGAATTCATG). Dithio traceol), at 4 ° C. overnight with 10 units of T ₄ DNA ligase. The solution is then heated at 70 ° C. for 10 minutes to stop the linking reaction. The linker was cleaved by EcoRI digestion, whereby fragments with EcoRI ends were separated using 5% polyacrylamide gel electrophoresis (highlighted as “PAGE”) and the largest three fragments were first stained with ethidium bromide The location of the fragments is then confirmed by ultraviolet light and the desired portion is cut from the gel, thereby separating from the gel. Each gel fragment is placed in a dialysis network with 300 microliters of 0.1 × TBE (0.2 × TBE buffer (TBE buffer contains 10.8 gm of Tris base, 5.5 gm of boric acid, 0.09 gm of Na ₂ EDTA per liter of H ₂ O). ) Electrophoresis at 100V for 1 hour.

The aqueous solution is recovered from the dialysis network, extracted with phenol, extracted with chloroform to make 0.2 M sodium chloride, and then precipitated with ethanol to recover DNA in water. A trp promoter-operator containing gene having an EcoRI adhesion terminus was identified by the method described below, and then inserted into the tetracycline sensitive plasmid, the fragment became tetracycline resistant as the promoter-operator was inserted.

Plasmid pBRH 1 [R.I. Rodriguez, et al., Nucleic Acids Research 6, 3267-3287 (1979), express an ampicillin resistance site and contain genes for tetracycline resistance, but when it is not linked to a promoter, Can not express. This plasmid is thus tetracycline sensitive. Plasmids can be made tetracycline resistant by introducing a promoter-operator system into the EcoRI site.

The pBR H1 was digested with EcoRI, followed by phenol extraction followed by chloroform extraction to remove the enzyme, precipitated with ethanol and recovered in water. The resulting DNA molecules are combined with each of the three DNA fragments obtained above in a separate reaction mixture and linked using T ₄ DNA ligase as described above. Using DNA present in the reaction mixture, V. Hershfield et al., Proc. Natl. Acad. Sci. USA 71, 3455-3459 (1974), which are appropriately defined by the standard methods described in E. coli K-12 strain 294 (K. Backman et al., Proc. Natl. Acad. Sci. USA 73, 4174-4198 (1976), and bacteria are plated on LB plate medium containing 20 μg / ml ampicillin and 5 μg / ml tetracycline. Several tetracycline-resistant colonies are selected, plasmid DNA is isolated and the presence of the desired fragment confirmed by restriction enzyme assay. The resulting plasmid is represented by pBRH trp.

EcoRI and BamH I degradation products of the hepatitis B virus genome were obtained by conventional methods and then added to the EcoRI and BamH I sites of plasmid pGH 6 (DVGoeddel et al., Nature 281, 544 (1979)). Cloning to form plasmid pHS 32. Plasmid pHS 32 was cut with Xba I, phenol extraction and chloroform extraction followed by precipitation with ethanol. This was then added to 1 μl E. coli polymerase I cloneno fragment (Behringer) in 30 μl polymerase buffer (50 mM potassium phosphate pH 7.4, ₇ mM MgCl ₂ , 1 mM β-mercaptoethanol) containing 0.1 mM dTTP and 0.1 mM dCTP. Mannheim) for 30 minutes at 0 ° C and then 2 hours at 37 ° C. This treatment fills two of the four nucleotides complementary to the 5 'protruding end of the Xba I cleavage site.

By importing two nucleotides dC and dT, a terminal with two 5 ′ overhang nucleotides is formed. The linear residue of this plasmid pHS 32 (extracted with phenol and chloroform, precipitated with ethanol and recovered in water) was then cleaved with EcoRI. Large plasmid fragments are separated from smaller EcoRI-Xba I fragments by PAGE, eluted and then separated. This DNA fragment (0.2 μg) from pHS 32 is linked to an EcoRI-Taq I fragment (˜0.01 μg) of tryptophan operon derived from pBRH trp under similar conditions as described above.

As described above, in the process of linking fragments from pHS 32 to EcoRI-Taq I fragments, the Taq I overhanging end of the Xba I, although it does not completely form Watson-Crick base pairs, Connected to the remaining protruding end:

A portion of this ligation mixture is used to transform E. coli 294 cells and then heat treated to plate onto LB plate medium containing ampicillin. Twenty-four colonies are selected, grown in 3 ml of LB medium and the plasmids isolated. Six of these have been found to have Xba I sites regenerated through E. coli catalyzed DNA repair and replication:

These plasmids were also found to provide the restriction fragments expected when cleaved with both EcoRI and Hpa I. One of these plasmids pTrp 14 is used for expression of heterologous polypeptides, as described below.

Plasmid pHGH 107 [D.V. Goedded et al, Nature, 281, 544, (1979) describe a human body consisting of 23 amino acid codons generated from synthetic DNA fragments and 163 amino acid codons obtained from complementary DNA produced through reverse transcription of human growth hormone messenger DNA. Contains genes for growth hormone. This gene contains no codons for the "pre" sequence of human growth hormone, but contains an ATG translational start codon. Genes are isolated after treatment with EcoRI followed by E. coli polymerase I Klenow fragment and dTTP and dATP from 10 μg of pHGH 107 as described above. After phenol and chloroform extraction and ethanol precipitation, the plasmid is treated with BamHI.

Fragments containing human growth hormone (“HGH”) genes are isolated by PAGE followed by electroelution. The resulting DNA fragment also contains the first 350 nucleotides of the tetracycline resistance structural gene, but since there is no tetracycline promoter-operator system, when subsequently cloned into the expression plasmid, the plasmid containing the insert recovers tetracycline resistance by You can set the location. Since the EcoRI terminus of the fragment is filled by the Klenow Polymerase I method, the fragment has one smooth end and one adhesive end so that accurate orientation is possible when later inserted into the expression plasmid.

The expression plasmid pTrp 14 is then prepared to receive the HGH gene-containing fragments prepared above. That is, pTrp 14 is decomposed to Xbal and the resulting adhesive ends are filled by the Klenow Polymerase I method using dATP, dTTP, dGTP and dCTP. After phenol and chloroform extraction and ethanol precipitation, the resulting DNA is treated with BamHI, PAGE and electroelute to separate the resulting large plasmid fragment. Since the pTrp 14-derived fragment has one smooth end and one adhesion end, it can be recombined with the HGH gene-containing fragment described above in an appropriate orientation.

The HGH gene fragment and the pTrp 14 ΔXba-BamHI fragment are combined and linked under conditions similar to those described above. Filled XbaI terminus and EcoRI terminus are linked together by smooth end ligation to regenerate both XbaI and EcoRI sites: Filled EcoRI terminus HGH gene initiation

This construction also regenerates the tetracycline resistance gene. Since plasmid pHGH 107 expresses tetracycline resistant sites from a promoter located on top of the HGH gene, this construct plasmid pHGH 207 allows expression of genes for tetracycline resistant sites under the control of tryptophan promoter-operators. . E. coli 294 was transformed with this ligation mixture and colonies were selected on LB plate medium containing 5 μg / ml tetracycline.

Plasmid pHGH 207 was digested with EcoRI to recover the 300 bp EcoPI fragment containing the trp promoter by PAGE followed by electroelute. The 300 bp EcoRI fragment contains the E. coli trp promoter, operator and trp leader ribosomal binding site, but no ATG sequence for the translation start site. This DNA fragment is cloned into the EcoRI site of pLeIF A.

The trp fragment mentioned above is an E. coli tryptophan operon analog that can control the expression level due to a lack of trp attenuator. Expression plasmids containing the modified trp regulon can be grown to a predetermined degree in a nutrient medium containing additional tryptophan in an amount sufficient to inhibit the promoter-operator system, followed by removal of the tryptophan Release the inhibition and cause expression of the desired product.

More particularly, 250 μg of plasmid pL 31 is digested with Pst I and gel electrophoresed on a 6 percent polyacrylamide gel to separate the 1000 bp insert. About 40 μg of the insert is electroeluted from the gel and divided into three aliquots for further degradation:

a) 16 μg sample of this fragment is partially digested with 40 units of Bgl II at 37 ° C. for 45 seconds and the reaction mixture is purified on a 6 percent polyacrylamide gel. Recover about 2 μg of the desired 670 bp fragment.

b) Limit another sample (8 μg) of 1000 bp Pst 1 insert to Ava II and Bgl II. After gel electrophoresis, 1 μg of the desired 150bp fragment is recovered.

c) Treat 16 μg of 1000bp with Sau 3a and Ava II.

After electrophoresis on a 10 percent polyacrylamide gel, about 0.25 μg (˜10 pmole) of the 34 bp fragment is recovered. Two desired deoxyoligonucleotides, namely 5'-dAATTCATGTGT (fragment 1) and 5'-dGATCACACATG (fragment 2), were used in the phosphoester ester method [see; Maxam and Gilbert, Methods Enzymol. 65, 499-560 (1980). Fragment 2 is phosphorylated as follows. 200 μl (˜40 pmole) of (r ³² P) ATP (Amersham, 5000 Ci / mmole) was dried and then 30 μl of 60 mM Tris-HCl (pH) containing 100 pmole of DNA fragment and 2 units of T4 polynucleotide kinase. 8), resuspend in 10 mM MgCl ₂ , 15 mM β mercaptoethanol. After 15 minutes at 37 ° C., 1 μl of 10 mM ATP is added to allow the reaction to proceed further for 15 minutes. The mixture is then heated at 70 ° C. for 15 minutes and then combined with 100 pmole of 5′-OH fragment 1 and 10 pmole of 34 bp Sau 3a-Ava II fragment. 50 μl of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 10 mM dithiothreitol, 0.5 mM ATP and 10 units of T4 DNA ligase were ligated at 4 ° C. for 5 hours. The mixture is electrophoresed on a 6 percent polyacrylamide gel and electroeluted to recover the 45 bp product. 860,000 Serenkov Cpm was recovered (˜30 ng, 1 pmole) and combined with 0.5 μg (5 pmole) of 150 bp Ava II-Bgl II fragment and 1 μg (2 pmole) of 670 bp Bgl II-Pst I fragment. Using 20 units of T4 DNA ligase, the ligation reaction is carried out at 20 ° C. for 16 hours. Ligase was inactivated by heating to 65 ° C. for 10 minutes.

The mixture is then digested with EcoRI and Pst I to remove the polymer of the gene. The mixture is purified by 6% polyacrylamide gel electrophoresis. Separate 3865 cpm (~ 0.04 pmole, 20 ng) of 865 bp product. Half of this (10 ng) is linked between the EcoRI site of pBR 322 (0.3 μg) and the Pst 1 site. E. coli 294 is transformed to yield 70 tetracycline resistant and ampicillin sensitive transformants. Plasmid DNA isolated from 18 of these transformants is digested with EcoRI and Pst I. 16 of 18 plasmids have an EcoRI-Pst I fragment of 865 bp in length. One of them, i.e., 1 µg of pLeIF A 1, was digested with EcoRI and then to 300 bp EcoRI fragment (0.1 µg) containing the E. coli trp promoter and trp leader ribosomal binding site, prepared as described above. Connect it. Transformants containing the trp promoter were run using ³² P-trp probes, as described by Grunstein et al., Proc. Natl. Acad. Sci. (USA) 72, 3961 (1975), by the Grunstein-Hogness colony screening procedure. The asymmetrically located Xba I site in the trp fragment allows the determination of the preparation in which the trp promoter is oriented in the direction of the LeIF A gene.

Extracts are prepared as follows for IF analysis. 1 ml of culture is grown in L broth containing 5 μg / ml tetracycline until A ₅₅₀ is about 1.0 and then diluted in 25 ml of M9 medium containing 5 μg / ml tetracycline. Once A ₅₅₀ reaches 1.0, centrifuge to collect 10 ml of the sample and suspend the cell pellet in 1 ml of 15 percent sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA. 1 mg of lysozyme is added and after 5 minutes at 0 ° C., the cells are sonicated and destroyed. Samples are centrifuged for 10 minutes at 15,000 rpm on a Sorvall SM-24 rotor. Interferon activity in the supernatants is determined by comparison with LeIF standards by cytopathic effect (CPE) inhibition test. LeIF with 4 × 10 ⁸ units / mg inactivity is used to determine the number of IF molecules per cell. See Rubinstein et al., Proc. Natl. Acad. Sci. (USA) 76, 640 (1979)].

The inserted clone pLe IF A trp 25, oriented by the trp promoter as desired, exhibits high activity (as high as 2.5 × 10 ⁸ units per liter). IF produced by E. coli K-12 strain 294 / pLe IF A trp 25 acts similar to true human LeIF; Stable to treatment at pH 2 and neutralized by rabbit anti-human leukocyte antibody. This interferon has an apparent molecular weight of about 20,000.

[Isolation of cDNA for Additional Leukocyte Interferon]

DNA from a fully characterized LeIF A cDNA-containing plasmid was digested with Pst I, electrophoretically isolated, and then ³² P was used to determine the performance of Taylor et al., Biochem. Biophys. Acta. 442, 324 (1976). Additional E. coli 294 transformants are selected by in situ colony screening methods (see Grunstein et al., Supra) using the resulting radiolabeled DNA as a probe. Colonies are separated and hybridized to the probe in various amounts. Plasmid DNA from these colonies and the ten hybridized colonies mentioned above are cleaved with Pst to separate and characterized in three different ways. First, these Pst fragments are characterized by their restriction endonuclease digestion using the enzymes Bgl II, Pvu II and EcoRI. This analysis classifies at least eight different forms (LeIF A, LeIF B, LeIF C, LeIF D, LeIF E, LeIF F, LeIF G, and LeIF H), which are known to be the presequences and coding sequences of LeIF A. It is possible to infer the position of various restriction cuts with respect to. One of these, LeIF D, is thought to be the same as reported in Nagata et al., Nature 284, 316 (1980).

Second, some of the DNAs are tested by hybridization screening tests described in the following literature for the ability to selectively remove LeIF mRNA from poly-A containing KG-1 cell RNA. et al., Cell 20, 95 (1980). LeIF A, B, C and F were positive by this test method. Third, the last Pst fragment is inserted into the expression plasmid, and E. coli 294 is transformed with the plasmid to express the fragment. Expression products thought to be precursor interferon were all positive for interferon activity by the CPE test, although it showed the lowest activity for the LeIF F fragment. All forms of LeIF were sequenced in addition to those described above.

[Second Mature Leukocyte Interferon]

The sequence of the isolated fragments consisting of genes for mature LeIF B showed that the first 14 nucleotides of the A and B forms were identical. Thus, fragments can be isolated from pLEif A 25, which contains the trp-promoter-operator, ribosomal binding site, and the start of the LeIF (A = B) gene, and then bind it to the rest of sequence B in the expression plasmid. .

In order to obtain a Sau 3a to Pst I fragment of about 950 bp from the sequence shown in Figure 7a, several steps are required due to the presence of one or more intervening Sau 3a restriction sites. In other words:

1. Isolate the following fragments: a) 110 bp from Sau 3a to EcoRI; b) 132 bp from EcoRI to Xba; c) at least 700 bp from Xba to Pst.

2. Link fragments (1a) and (1b) and cleave with Xba and Bgl II to prevent self-polymerization reaction through Sau 3a and Xba ends (the corresponding Sau 3a site is in the Bgl II site; Bgl II Cut to leave Sau 3a adhesive ends). 242bp fragment is isolated.

3. The product of (2) and the fragment of (1c) are linked and cleaved with Pst I and Bgl II to prevent self-polymerization again. Approximately 950 bp fragments from Sau 3a to Pst I in FIG. 7a are isolated. This fragment contains a portion of the LeIF B gene that is not common with LeIF A.

4. A 300 bp fragment (Hind III to Sau 3a) consisting of the trp promoter-operator of the LeIF A, the ribosomal binding site, the ATG start signal and the cysteine codon is isolated from pLeIF A25.

5. Isolate approximately 3600 bp fragments from Pst I to Hind III from pBR 322. This fragment contains a replicon and encodes a tetracycline resistant site but not an ampicillin resistant site.

6. Triple-link the fragments obtained in steps 3, 4 and 5 and transform E. coli K-12 strain 294 with the resulting plasmid.

Transformants are miniscreened by the method described in Birnboim et al., Nucleic Acid Research Z, 1513 (1979), and then plasmid samples are digested with EcoRI. The digestion results in three fragments having the following characteristics: 1) EcoRI-EcoRI trp promoter fragment; 2) internal fragment from EcoRI to EcoRI of pL4; And 3) fragments from the protein translational start signal of pL4 to EcoRI.

In the CPE test, bacterial extracts from clones prepared in this manner are typically tested at about 10 × 10 ⁶ units of interferon activity per liter at A ₅₅₀ = 1. One representative clone prepared in this manner is 294 / pLIF B trp 7.

[Additional Mature Leukocyte Interferon]

Additional full-length gene fragments of different LeIF forms are made and placed into expression vehicles for expression as in the case of LeIF A. Complete sequencing in a conventional manner, using the removal of the presequence by restriction cleavage, and replacement of the codon for the N-terminal amino acid lost in the presequence removal by ligation of the synthetic DNA fragment, as described above. It is possible to determine whether the restriction site is close enough to the first amino acid codon in the form of a mature interferon to allow a convenient way to try. Otherwise, Claude et al. (See above) may be used. In short, this method consists of precisely cutting the fragment containing the presequence just before the start of the codon for the first amino acid of the mature polypeptide, which is as follows:

1. converting the double stranded DNA to single stranded DNA in a region around the position; 2. Hybridize the single stranded DNA complementary primer to the single stranded region generated in step (a), wherein the 5 'end of the primer is placed in the opposite direction to the nucleotide adjacent to the desired cleavage site; 3. The part of the second strand removed in step 1, placed in the 3 'direction from the primer, is recovered by reacting with DNA polymerase in the presence of oxynucleotide triphosphate containing adenine, thymine, guanine and cytosine ; 4. Decompose the remaining part of the Japanese strand of DNA that protrudes in addition to the desired cleavage site.

The short length synthetic DNA terminated with the translational start signal ATG at the 3 ′ end of the encoding disruption is then linked to a gene prepared for mature interferon, for example by blunt end ligation, and the gene is inserted into an expression plasmid to provide a promoter and Under control of the ribosomal binding site associated with the. In a manner similar to that used above, the gene fragments encoding LeIF C and LeIF D are appropriately placed for direct bacterial expression. These additional leukocyte interferon expression methods in each case comprise a fragment of about 300 bp (Hind III to Sau 3a) consisting of the trp promoter-operator of LeIF A from pLeIF A25, a ribosomal binding site, an ATG start signal and a cysteine codon How to use is included. Here, the gene fragments from additional interferon genes encoding their respective amino acid sequences other than the starting cysteines common to all are joined. Each of the resulting plasmids is used to transform E. coli K-2 strain 294. The linkages that form each gene are as follows:

[LeIF C]

The following fragments are separated from pLeIF C:

(a) 35 bp fragment from Sau 3a to Sau 96.

(b) a fragment of at least 900 bp from Sau 96 to Pst I.

(c) As in Part N (4) above, about 300 bp fragment (Hind III-Sau 3a) is isolated from pLeIF A-25.

(d) Isolate about 3600 bp fragment of Part N (5) above.

How to create:

(1) The fragments (a) and (c) are linked. Cleavage with Bgl II and Hind III separates about 335 bp of product.

(2) Triple linking of (1), (b) and (d), and transformed E. coli with the resulting plasmid.

An exemplary clone prepared in this manner is E. coli K-12 strain 294 / pLeIF C trp 35.

[LeIF D]

Separate the following fragments from pLeIF D:

(a) 35 bp fragment from Sau 3a to Ava II.

(b) 150 bp fragments from Ava II to Bgl II.

(c) about 700 bp fragment from Bgl II to Pst I.

The following fragments are isolated from pLeIF A25:

(d) 300 bp fragment from Hind III to Sau 3a.

The following fragments are separated from pBR 322:

(e) about 3600 bp fragment from Hind III to Pst I.

How to create:

(1) After linking (a) and (b), digest with Bgl II to purify 185 bp of product (1).

(2) Link (1) and (d), then cleave with Hind III and Bgl II to purify product (2) of about 500 bp.

(3) (2) and (c) and (e) are linked, and then the E. coli is transformed with the resulting plasmid.

An exemplary clone prepared in this way is E. coli K-12 strain 294 / pLeIF D trp 11.

[LeIF F]

Fragments containing LeIF F can be prepared for direct expression via a preparation conveniently prepared by the complete homology of amino acids 1 to 13 of LeIF B and LeIF F. A trp promoter-containing fragment (a) having an appropriately arranged end is obtained by digesting Pst I and Xba I from pHGH 207 described above and then separating about 1050 bp of fragment. The second fragment (b) is obtained as a larger fragment produced by Pst I and Bgl II digestion of plasmid pHKY 10 (a derivative of pBR322 containing a Bgl II site between the tetracycline resistance promoter and the structural gene). Fragment (a) contains about half of the gene encoding ampicillin resistance; Fragment (b) contains the entire gene for tetracycline resistance, except for the rest of the gene encoding ampicillin resistance, and the associated promoter. Fragments (a) and (b) were combined using T4 ligase and the product was treated with Xba I and Bgl II to eliminate dimerization, consisting of trp promoter-operator and genes for tetracycline and ampicillin resistance Prepare fragment (c).

A fragment (d) of about 580 bp is obtained by digesting pLeIF F with Ava II and Bgl II. It contains codons for amino acids 14-166 of LeIF F.

Fragment (e) (49 bp) is obtained by digesting pLeIF B with Xba I and Ava II. Fragment (e) encodes amino acids 1 to 13 of LeIF F.

Fragments (c), (d) and (e) are triple linked in the presence of T4 ligase. The adhesive ends of the fragments allow the hybrid plasmid to be correctly circularized, with tetracycline resistance genes under the control of the trp promoter-operator along with the gene for mature LeIF F so that the bacteria transformed with the desired plasmid Allow selection on cyclin-containing plates. An exemplary clone prepared in this manner is E. coli K-12 strain 294 / pLeIF F trp 1.

[LeIF H]

The complete LeIF H gene can be placed to be expressed as mature leukocyte interferon as follows:

1. The plasmid pLeIF H is digested with Hae II and Rsa I to separate 816bp fragments from signal peptide amino acids 10 to 3 ′ unencoded region.

2. Denature the fragments and then recover synthetically with the Klenow fragment using synthetic deoxyribooligonucleotide nucleotide primer 5′-dATG TGT AAT CTG TCT. Klenow et al., Proc. Natl. Acad. Sci. USA 65, 168 (1970). This general method is also described in US Patent Application No. 190799 to Goeddel et al., Filed September 25, 1980.

3. The resulting product is cut with Sau 3a to separate 452bp fragments representing amino acids 1-150.

4. PLeIF H is digested with Sau 3a and Pst I and the resulting 500 bp fragment is isolated to obtain a gene encoding 150 amino acids up to the end of the coding sequence.

5. The fragments separated in steps (3) and (4) are joined to produce the following fragments encoding 166 amino acids of LeIF H:

6. PLeIF A trp 25 is digested with Xbal, blunt terminated with DNA polymerase I, and the product is digested with Pst I. The resulting large fragments can be isolated and linked to the product of step (5) to transform E. coli K-12 strain 294 or other host bacteria to produce an expression plasmid capable of expressing mature LeIF H.

[LeIF I]

Phage λ Charon 4A recombinant stores of human genomes constructed according to the methods described in Lawn et al., Cell 15, 1157 (1978) are described in Lawn et al., Supra. The leukocyte interferon gene is screened according to the methods described in the references and Maniatis et al., Cell 15, 687 (1978). Approximately 500,000 plaques are screened using a radioactive LeIF probe derived from cDNA clone LeIF A (Goeddel et al., Nature 287, 411 (1980)). This screening yields six LeIF genome clones. After rescreening and plaque purification, one of these clones, λ HLeIF 2, is selected for further analysis.

Using the methods described above, other probes may also be advantageously used to isolate additional LeIF clones from human genomes. These can also be used to generate additional leukocyte interferon proteins according to the invention.

1. Subcloning the 2000 bp EcoRI fragment of the genome clone (λ HLeIF 2) at the EcoRI site of pBR 325. The resulting plasmid LeIF I was digested with EcoRI to separate the 2000 bp fragment. The deoxy oligonucleotide dAATTCTGCAG (EcoRI> PstI convertor) was linked to a 2000 bp EcoRI fragment and then the resulting product was cut with Pst I to yield a 2000 bp fragment containing the Pst I terminus. This fragment was cut into Sau 96 to separate 1100 bp fragment with one Pst I terminus and one Sau 96 terminus.

2. Decompose plasmid pLeIF C trp 35 into Pst I and Xba I. Separate large fragments.

3. The small Xba I-Pst I fragments generated from pLeIF C trp 35 are digested with Xba I and Sau 96. Isolate 40 bp of Xba I-Sau 96 fragment.

4. Link the fragments separated in steps 1), 2) and 3) to obtain the expression plasmid pLeIF I trp 1.

[LeIF J]

1. Plasmid pLeIF J contains a 3.8 kb Hind III fragment of human genome DNA comprising the LeIF J gene sequence. A 760 bp Dde I-Rsa I fragment is isolated from this plasmid.

2. Cleavage the plasmid PLeIF B trp 7 with Hind III and Dde I and then isolate the 340 bp Hind III-Dde I fragment.

3. Cut plasmid pBR 322 into Pst I, incubate with DNA polymerase I (Clenow fragment) to blunt ended, and digest with Hind III. Separate large (~ 3600bp) fragments.

4. Link the fragments separated in fragments 1), 2) and 3) to obtain the expression plasmid pLeIF J trp 1. The methods and materials used in the construction below are the same as in the above description. Table 1 below provides a detailed description of the specific construction of the hybrid LeIF plasmids of the present invention:

TABLE 1

With regard to Table 1, the first four hybrid LeIFs described were generated from two LeIF expression plasmids. The Bal II site common to LeIF A and D cDNA was used to construct the expression plasmid pLeIF trp AD (Bgl II) encoding 63 amino terminal amino acids of LeIF A and 102 carboxy terminal amino acids of LeIF D. The same site is used to construct the expression plasmid pLeIF trp DA (Bgl II) encoding the 64 amino terminal amino acids of LeIF D and 102 carboxy terminal amino acids of LeIF A. The Pvu II site is used to construct two different hybrid interferon expression plasmids: the expression plasmids pLeIF trp AD (Pvu II) encoding Le 91 A and 74 carboxy terminal amino acids of LeIF D, and LeIF D. Expression plasmid pLeIF trp DA (Pvu II), encoding 92 amino terminal amino acids of and 74 carboxy terminal amino acids of LeIF A. In short, it looks like this:

pLeIF AD trp (Pvu II): Large (˜3900 bp) fragments of the Xba I and Pst I digests of pLeIF A trp25 were selected from the 288 bp Xba I-Pvu II fragment of pLeIF A trp11 and approximately 550 bp Pvu II-Pst I fragment of pLeIF D trp11. Connect with

pLeIF DA trp (Pvu II): Large (˜3900 bp) fragments of the Xba I and Pst I digests of pLeIF A trp25, 285 bp Xba I-Pvu II fragments of pLeIF D trp25 and about 550 bp Pvu II-Pst I fragments of pLeIF A trp25 Connect with

pLeIF AD trp (Bgl II): Links large fragments of Bgl II and Pst I digests of pLeIF A trp25 with ˜600 bp Bgl II-Pst I fragments of pLeIF D trp11.

pLeIF DA trp (Bgl II): A large fragment of the Bgl II and Pst I digests of pLeIF D trp11 is linked with the approximately 700 bp fragment obtained by transferring pLeIF A trp25 to Pst I and then partially digesting with Bgl II.

In 15 hybrids: pLeIF AB trp (Pvu II): Large (˜3900 bp) fragments of Xba I and Pst I digests of pLeIF A trp25 were replaced with 285 bp Xba I-Pvu II fragments of pLeIF A trp25 and approximately 750 bp Pvu of pLeIF B trp7. Linkage with II (partial) -Pst I fragment.

In a similar manner, the following constructs shown in Table 1 are also defined. As a further example, in constructing a hybrid containing the LeIF C and / or LeIF H moieties, one may advantageously take the conventional BbV I site present at approximately nucleotide 294 (ie, GCTGC) of the gene sequence.

In a similar manner, plasmids suitable for microbiological expression of other novel hybrid leukocyte interferons can be prepared by appropriately manipulating double stranded DNA encoding some or all of the naturally occurring amino acid sequence of leukocyte interferon. That is, the first double-stranded DNA fragment is selected to encode the first naturally occurring leukocyte interferon amino acid sequence and the amino terminus of a substantial portion of its amino acid leading in the 3 'direction therefrom.

The fragment contains a restriction endonuclease cleavage site located adjacent to the codon for amino acid “n” of the first leukocyte interferon (which constitutes a substantial portion of the amino acid sequence of the first interferon of n amino acids). Cleavage with a restriction endonuclease yields a fragment consisting of the amino terminus of the first interferon and a codon for approximately n amino acids. The second fragment, consisting of some or all of the codons for the amino acid sequence of the second different leukocyte interferon, for the amino acid of the second interferon whose fragment is about 166-n amino acid number (starting from the amino terminus of the second interferon) It is selected to contain a cleavage site for the same restriction endonuclease located adjacent to the codon. Cleavage of the second fragment with a restriction endonuclease yields a product complementary to the “n” terminal portion of the degradation product of the first fragment, linking the degradation product of the second fragment to the degradation product of the first fragment. The clease recognition site can be regenerated and the codons for amino acid n of the first interferon lost in the initial degradation can be reconstructed. The restriction endonuclease degradation product of the second fragment preferably proceeds from the end produced by cleavage in the 3 'direction to the nucleotide encoding the carboxy terminus of the second leukocyte interferon.

In addition, hybrids containing a substantial portion of two or more amino acid sequences of naturally occurring leukocyte interferon, for example, may further contain a second restriction endonuclease site, as described above, with the second fragment further downward from the first fragment. (The second site is identical to the site similarly located in the fragment encoding the carboxy terminal site, such as the three serpentine leukocyte interferon). In the example mentioned, the products of consecutive restriction endonuclease acts are triple-linked so that the amino terminus of the first interferon, the intermediate amino acid sequence of the second interferon and the carboxy terminus of the third interferon (or the first and third If the first interferon is the same, a hybrid gene encoding the first interferon may be generated).

Preferably, the first fragment mentioned above is derived from an expression plasmid, ie an expression plasmid preceded by an ATG or other detoxification codon and a promoter or promoter-operator system for the codon for the amino terminal region of the first leukocyte interferon. As a result, the end product of the engineering operation described above is a plasmid capable of expressing a polypeptide encoded for a hybrid gene in bacteria or other microbial organisms transformed with the plasmid. Other methods of placing hybrid genes for microbial expression will be apparent to those skilled in the art.

In a preferred embodiment of the invention, the hybrid gene is at least two selected from the group consisting of LeIF A, LeIF B, LeIF C, LeIF D, LeIF E, LeIF F, LeIF G and LeIF H as shown in FIG. The new leukocyte interferon amino acid sequence is encoded, which is about 165 to 166 amino acids that constitute a conjugate of substantial amino acid sequences derived from different leukocyte interferon. Most preferably, the new leukocyte interferon encoded by the hybrid gene consists of amino acids that are specified and located as indicated in sequence “A11” in FIG. 3. Expression products of the plasmids prepared according to the invention can be tested for antiviral activity in a conventional manner, as in the biological activity assays described below.

[Measurement of Antiviral Activity]

E. coli K-12 strain 294 is conveniently transformed with independent plasmids pLeIF trp A25, pLeIF trp D, pLeIF trp A / D (Bgl II) and pLeIF trp D / A (Bgl II). Transformants are grown separately in L broth containing 5 mg / ml tetracycline in 5 ml cultures until A ₅₅₀ is about 1.0, then diluted with 1 L of M9 medium containing 5 μg / ml tetracycline. Once A ₅₅₀ reaches 1.0 the cells are harvested and the cell pellet suspended in a 10 ml solution of 15% sucrose, 50 mM Tris-HCl, pH 8.0, 50 mM EDTA. 10 mg of lysozyme is added and after 5 minutes at 0 ° C., the cells are sonicated and destroyed. Samples are centrifuged for 10 minutes at 15,000 rpm on a Sorvall SM-24 rotor. Interferon activity in the supernatant is tested for antiviral activity.

Activity yields per liter of these interferon cultures determined by titer on human cell lines (WISH) are shown in Table 2, which clearly shows that LeIF-A / D activity is produced in much greater amounts than other interferons. This difference may be due to greater intrinsic activity of LeIF-A / D or higher yields expressed in mg protein of this interferon. Since the genetic link-up is the same for all of these interferons, it seems most likely that LeIF-A / D has radically greater activity than other interferons.

TABLE 2

Measure the efficacy of various interferons in a range of mammalian cell lines (human, WISH; African green monkey, VERO; hamster fibroblasts, BHK; rabbit kidney cells, RK-13; mouse, L-929; and bovine) Kidney, MDBK cells). To compare the relative activity of interferon, their activity on various cells is calculated relative to their activity on WISH cells as 100. The results in Table 3 show that LeIF-A / D has very high activity in VERO and L-292 cells, while LeIF-D / A has low activity in these cell lines. These results show that the combination of the N-terminal portion of LeIF-A and the C-terminal portion of LeIF-D in one molecule (LeIF-A / D) confers hybrid proteins with specific activities that are apparent in many mammalian species. Suggest. In addition, these specifics are not simply the sum of the properties of the parent interferon. This is clearly seen in the case of activity on L-929 cells (Table 3), as well as the mixture of LeIF-A and LeIF-D as well as other hybrids, LeIF-D / A, which do not show significant activity.

TABLE 3

LeIF-A / D activity against other viruses is also tested. The data in FIG. 5 shows the antiviral effect on EMC virus infection of L-cells and the data in FIG. 6 shows the effect on VSV infection of L-cells. From these data it can be seen that the greater activity of LeIF-A / D is not limited to one virus (VSV) and its greater activity is a general property for many viruses. Normal human buffer-coat interferon products have no effect on mouse cells (see Table 2). Therefore, the activity of LeIF-A / D against EMC virus infection in CD-1 mice is measured. The results shown in FIG. 7 show that LeIF-A / D is highly effective against lethal EMC virus infection, and as expected from activity in cell lines (Table 2), LeIF-A also has antiviral activity. Indicates. The data in FIG. 7 is the result of treatment intraperitoneally 3 hours before infection. Doses of LeIF-A / D and LeIF-A are as determined by titer on WISH.

Fatal viral infections of hamsters are also affected by LeIF-A / D and LeIF-A (Figure 8), with LeIF-A / D being the most effective, and stratified interferon showing only minor effects that are not statistically significant. In FIG. 8 all interferons are administered intraperitoneally at a dose of 5 × 10 ⁵ μg / kg titered on WISH cells 3 hours prior to infection.

These results indicate that significant antiviral effects of LeIF-A / D within the mammalian species are not limited to cell cultures but are also observed in lethal viral infections.

EMC viruses are thought to be a model system in explaining antiviral effects that can be expected to be antiviral effects, for example, to the representative viral family of picornaviruses that cause foot and mouth disease and polio. Can be. VSV viruses can be thought of as a model system in explaining the antiviral effects that can be expected, for example, against a representative viral class of the rabies virus class that causes rabies.

Table 4 tabulates the activity and ratio of activity of the various LeIF hybrids of the present invention on WISH and MDBK cells:

TABLE 4

Parenteral administration

The hybrid leukocyte interferon of the present invention can be administered parenterally to patients in need of antitumor or antiviral therapy and to patients with immunosuppressive responses. Doses and dose ratios are similar to those currently used in clinical studies of human derived substances, for example, about (1-10) × 10 ⁶ units per day, and 5 × per day for substances with purity greater than 1%. Up to 10 ⁷ units. The preliminary findings in the monkey study described above indicate that the dose of LeIF obtained bacterially is not the same as that of human proteins other than LeIF (in leukocyte-inducing substances, this protein may act as a pyrogen that has adverse effects such as anxiety, temperature rise, etc.). This implies that the necessary absence can significantly increase the effect.

As an example of a suitable dosage form for the nearly homogeneous bacterial LeIF, the parenteral dosage form applicable mutatis mutandis in the present invention, 25 mg of 5N serum albumin (human body)-with 3 mg of LeIF having an inactivation of 2 × 10 ⁸ μ / mg Dissolve in Usp, pass the solution through a bacteriological filter and distribute the filtered solution aseptically into 100 vials so that each vial contains 6 × 10 ⁶ units of pure interferon suitable for parenteral administration. The vial is preferably stored at low temperature (˜20 ° C.) before use.

The compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions wherein the polypeptides of the present invention are combined in the form of a mixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulations are described in the literature, which is incorporated herein by reference (E.W. Martin, Remington's Pharmaceutical Sciences). Such compositions contain an appropriate amount of an interferon protein of the present invention, together with an appropriate amount of vehicle, to prepare a pharmaceutically acceptable composition suitable for effective administration to a host.

Claims (10)

Consists of approximately 165 to 166 amino acids, the amino acid sequence of which, in amino acid type and number, consists of distinct sub-sequences corresponding to sub-sequences of naturally occurring different leukocyte interferons, the overall composition In a double stranded DNA containing a strand encoding a polypeptide different from the amino acid sequence of a naturally occurring leukocyte interferon. The double-stranded DNA of claim 1, wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1 to 92 of LeIF-D, and the carboxy terminal portion thereof essentially consists of amino acids 92 to 165 of LeIF-A. . The double-stranded DNA of claim 1, wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-91 of LeIF-A, and the carboxy terminal portion thereof essentially consists of amino acids 93-166 of LeIF-D. . The double-stranded DNA of claim 1 wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-63 of LeIF-D, and the carboxy terminal portion thereof essentially consists of amino acids 63-165 of LeIF-A. . The double-stranded DNA of claim 1 wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-62 of LeIF-A, and the carboxy terminal portion thereof essentially consists of amino acids 64-166 of LeIF-D. . The double-stranded DNA of claim 1 wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-91 of LeIF-A, and the carboxy terminal portion thereof essentially consists of amino acids 93-166 of LeIF-B. . The double-stranded DNA of claim 1 wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-91 of LeIF-A, and the carboxy terminal portion thereof essentially consists of amino acids 93-166 of LeIF-F. . In a transformant microorganism, a replicable plasmid expression vehicle capable of expressing a polypeptide encoded by the DNA sequence according to any one of claims 1 to 7. A plasmid selected from the group consisting of pLeIF AD trp (Bgl II), pLeIF AD trp (Pvu II), pLeIF DA trp (Bgl II) and pLeIF DA trp (Pvu II). a plasmid selected from the group consisting of pLeIF AB (Pvu II) and pLeIF AF (Pvu II).

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