HRP950157A2 - Polypeptide with human interferon (ifn-gamma) characteristics - Google Patents

Description

Field of invention

The present invention relates to the field of recombinant DNA technology, to the means and methods that use such technology in the detection of DNA sequences and derived amino acid sequences for human immune interferon, as well as to their production and various products of such production, and their use.

In particular, such an invention relates to the isolation and identification of DNA sequences that encode human immune interferon, and to the composition of recombinant DNA expression agents containing such DNA sequences, operatively linked to promoter sequences that influence expression, and to expression carriers that are thus composed. In another aspect, the subject invention relates to host culture systems, such as various microorganisms and vertebrate cell cultures transformed with such expression carriers and thereby directed to the expression of the aforementioned DNA sequences. In another aspect, the present invention relates to means and methods of converting the end product of such expression into novel entities, such as pharmaceutical compositions used for the prophylaxis or treatment of humans. In a preferred embodiment, the present invention provides specific expression carriers that are in the correct sequence, so that human immune interferon is produced and secreted from host cells in a fully developed form. In addition, this invention relates to various methods used to obtain said DNA sequences, expression carriers, host culture systems and their final products and entities, as well as to their special and accompanying forms.

The subject invention derives in part from the discovery of the DNA sequence and the derived amino acid sequence encoding human immune interferon. In addition, the present invention provides sequence information for sequences on the 3'- and 5'-flanking sequences of the human immunointerferon gene, facilitating their in vitro splicing into an expression carrier.

In particular, the 5'-DNA segment that encodes the so-called endogenous signal polypeptide that immediately precedes the sequence of amino acids, the so-called of mature human immunointerferon. These discoveries enabled the development of means and methods for obtaining - through recombinant DNA technology - a sufficient amount of human immunointerferon and determining its biochemical properties and bioactivity.

Publications and other materials used to explain the background of the invention, and particularly cases, to obtain additional details in relation to use, are herein incorporated by reference, numbered and grouped in the attached bibliography.

The origin of the invention

A. Human immune interferon

Human immune interferon can be divided into three groups based on different antigenic activity and biological and biochemical properties.

The first group includes the family of leukocyte interferons (α-interferon, LeIF or IFN-α), which are mainly produced by cells that are an integral part of human blood, after virus induction. They are obtained microbially and have been found to be biologically active (1, 2, 3). Their biological properties have accelerated their use in hospitals as a medicine for the treatment of viral infections, malignant conditions, immune suppression and immune deficiency (4).

In the second group is human stem cell interferon (β-interferon, FIF or IFN-β), which is usually produced by stem cells after viral induction and is also microbially derived. It was found that there is a wide range of biological activators (5). Clinical trials also indicate its potential value in treatment. Interferons of leukocytes and stem connective cells show clear similarities in their biological properties despite the fact that the degree of homology at the amino acid level is relatively low. In addition, both groups of interferons contain from 165 to 166 amino acids and both are acid-resistant proteins.

Human interferon (γ-interferon, IIF or IFN-γ), to which this invention refers, is opposite to α- and β-interferon, pH 2 labile, obtained mainly after mitogenic induction of lymphocytes and also has antigenic activity. Until recently, human interferon could only be found in very small quantities, which certainly prevented its characterization. A rather large but still partial purification of human immunointerferon has recently been reported (6). The compound was produced from lymphocyte cultures stimulated with a combination of phytohemogglutin and phorbol ester, and purified by sequential chromatographic separations. This procedure gave a product with a molecular weight of 58,000.

Human immune interferon was obtained in very small quantities by translation of mRNA in oocytes, which show interferon activity characteristic of human interferon, which gives hope that interferon cDNA could be synthesized and cloned (7).

The amount of immunointerferon that has been obtained so far is certainly not sufficient for carrying out clear experiments for the purpose of characterizing and biological properties of the purified component. However, in vitro studies with crude preparations, as well as in vivo experiments with murine γ-interferon preparations, show that the primary function of immune interteron could be a means of regulating immunity (8, 9). Immune interferon does not only have antiviral and anti-cellular effects that all human interferons possess, but also shows an increased influence on these effects with α- and (β-interferon (10). It has also been reported that the in vitro antiproliferative effect of γ-interferon on tumor cells cells about 10 to 100 times higher than other categories of interferons (8, 11, 12). This result, together with its emphasized role as an immune regulator (8, 9), indicates a more pronounced antitumor potency for IFN-γ than for IFN-α and IFN-β In vivo experiments with mouse and murine IFN-γ preparations show a clear superiority over antiviral-induced interferons in their antitumor effect on osteogenic sarcoma (13).

All these studies, until the present invention, had to be carried out with rather crude preparations, due to their very low availability, but they certainly indicate very important biological functions for immune interferon. Not only does immune interferon have a strong antiviral effect, but probably also a strong immunoregulatory and antitumor effect, which clearly indicates its future use in hospitals.

It was noted that the application of recombinant DNA technology could be the most effective way to obtain the necessary large quantities of human immune interferon. Whether or not the substances thus obtained include the glycosylation that is considered characteristic of a natural substance derived from human cells or not, they are likely to show bioactivity on the basis of which their use in hospitals will be allowed for the treatment of a wide range of viral and malignant conditions or diseases, and diseases due to immunosuppressants or lack of immunity.

B. Recombinant DNA technology

Recombinant DNA technology has been perfected to some extent. Molecular biologists can relatively easily recombine various DNA sequences, creating new DNA units that produce large amounts of exogenous protein products in transformed microbes. General tools and methods are available for in vitro ligation of various blunt-ended or "sticky"-ended DNA fragments, thereby obtaining strong expression carriers that are used to transform certain organisms and thereby direct their efficient synthesis of the desired exogenous product. However, on the basis of an individual product, the path to obtaining a product is difficult and science has not reached a stage where sure success could be achieved. Those who predict successful results without taking into account the experimental base, do so at great risk, that their results will be missing.

Plasmid, a nonchromosomal double-stranded DNA opening found in bacteria and other microbes, often in many copies per cell, remains a basic element of recombinant DNA technology. The information encoded in plasmid DNA requires the plasmid to be amplified in daughter cells (ie, the origin of replication) and usually one or more phenotypic selection characteristics, such as antibiotic resistance in the case of bacteria, which allow clones of host cells containing the plasmid to , recognize and preferentially grow into a selective substance. The advantage of plasmids lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or "restriction enzyme", each of which comes to different positions on the DNA plasmid. Afterwards, heterologous genes or gene fragments can be inserted into the plasmid, by splicing at the cleavage site or at reconstructed ends close to the cleavage site. This is how the so-called replicating means of expression. DNA recombination is carried out outside the cell, but the resulting "recombinant" replicating expression vector, or plasmid, can be introduced into the cell by a process known as transformation and large quantities of recombinant agents are obtained by growing the transformant. In addition, when the gene is inserted correctly with respect to the portions of the plasmid that control the transcription and translation of the encoded DNA order, the resulting expression carrier can be used for the actual production of the polypeptide sequence for which the inserted gene is coded (hereinafter, the process is called expression).

Expression begins at a region called a promoter known to be joined by RNA polymerase. In the transcription phase of expression, the DNA unfolds, exposing itself as a template for the initiation of messenger RNA synthesis from the DNA strand. The messenger DNA is translated into a polypeptide with the amino acid sequence encoded by the mRNA. Each amino acid is encoded by a nucleotide triplet or "codon" which together form a "structural gene", i.e. the part that codes for the amino acid sequence of the expressed polypeptide product. Translation starts at the "start" signal (usually ATG, which becomes AUG in the resulting messenger RNA). So-called stop codons determine the end of translation and therefore the production of further amino acid units. The resulting product can be obtained by dissolving - if necessary, the host cell in a microbial system, and the recovery of the product can be achieved by appropriate purification from other proteins.

In practice, the use of recombinant DNA technology can express exclusively heterologous polypeptides - so-called direct expression - or alternatively, it can express heterologous polypeptides joined to a part of the amino acid sequence of a homologous polypeptide. In further cases, the bioactive product is sometimes bioinactive within the fused homologous/heterologous polypeptide, until cleaved in the extracellular environment. See British Patent Publ. But. 2007676A and Wetzel, American Scientist 68, 664 (1980)

C. Cell culture technology

The methodology of cell or tissue cultures for the study of cell genetics and physiology has been established. There are known ways and methods for maintaining permanent cell lines, prepared by successive serial transfer from isolated normal cells. For use in research, such cell lines are maintained on a solid support in a liquid medium, or by cultivation in a suspension containing nutrients. When preparing larger quantities, there are only mechanical problems. More information on the invention is contained in Microbiology, 2nd edition, Harper and Row, Inc. publishers. Hagerstown, Maryiand (1973), especially p. 1122 et seq. and Scientific American 245,66 (1981).

Summary of the invention

The subject invention is based on the discovery that recombinant DNA technology can be successfully used for the production of human immune interferon, preferably in direct form, and in quantities sufficient to initiate and conduct animal tests and clinical tests as a condition for offering on the market. The product is suitable for use in all its forms, in the prophylaxis and treatment of people suffering from viral infections, malignant diseases and immune suppression or lack of immunity. Its forms include various oligomeric forms that may include accompanying glycosylation. The product is obtained by genetically controlled microorganisms or cell culture systems. In this way, there is a possibility of obtaining and isolating human immune interferon in a more efficient way than it was until now. A significant factor of the subject invention in its most advantageous form is the achievement of genetic guidance of microorganisms or cell cultures, in order to produce human immune interferon in amounts that can be isolated, that is, secreted in a mature form from the host cell.

The present invention includes human immune interferon produced in this way, as well as means and methods for its production. The subject invention is further directed to replicating DNA expression carriers in which gene sequences encoding human immune interferon are found in an expressible form. Furthermore, the present invention is directed to strains of microorganisms or cell cultures transformed with the expression carriers described above and to microbial or cell cultures of such transformed strains or cultures, capable of producing human immune interferon. In a further aspect, the subject invention relates to various procedures, useful for the preparation of immune interferon gene arrays, DNA expression carriers, strains of microorganisms and cell cultures, and their specific forms. Furthermore, this invention relates to the preparation of fermentation cultures of the mentioned microorganisms and cell cultures. Next, this invention deals with the preparation of human immuno-interferon as a direct expression product, secreted in mature form from the host cell. This approach can use the gene encoding the mature human immunointerteron sequence plus the 5' flanking DNA encoding the signal polypeptide. It is believed that the signal polypeptide helps transport the molecule to the cell wall of the host organism where it is cleaved during the process of secretion of the mature human interferon product. This form allows the isolation and purification of mature immunointerferon without procedures designed to eliminate host intracellular protein contaminants or cellular debris.

The term "mature human immunointerferon" refers to the microbial or cell culture production of human immunointerferon without a signal peptide or peptide presequence that directly participates in the translation of human immunointerferon mRNA. In this way, a mature human immune interferon according to the present invention is obtained, which contains methionine as the first amino acid (present through the ATG start signal of codon insertion in front of the structural gene) or, in positions where methionine is cleaved inside and outside the cell, and cysteine is the first amino acid acid. Mature human immune interferon can also be obtained together with a conjugated protein other than a conventional signal polypeptide. The conjugate has the ability to cleave inside or outside the cell. See British patent publication no. 2007676A. Finally, mature human immune interferon can be obtained by direct expression without the need to cleave off the extraneous polypeptide. This is particularly important in cases where the host cannot efficiently remove the signal peptide where the expression carrier is intended to express mature human interferon, together with its signal peptide. The mature human immune interferon thus produced is regenerated and purified to a level suitable for use in the treatment of viral and malignant diseases, immune suppression and immunodeficiency.

Human immune interferon is obtained as follows:

1. Human tissue, for example spleen tissue or peripheral blood lymphocytes, are cultured with mitogens, in order to stimulate the production of immune interferon.

2. Fragments of such cell cultures are extracted in the presence of ribonuclease inhibitors, in order to isolate all cytoplasmic RNA.

3. Oligo-dT column isolates messenger RNA (mRNA) in polyadenylated form. This mRNA was granulometrically fractionated using sucrose density gradient and acid urea gel electrophoresis.

4. The corresponding mRNA (12 to 18 S) is converted into suitable complementary single-stranded DNA (cDNA) from which double-stranded cDNA is obtained. After poly-dC is generated, it is introduced into a vector such as a plasmid with one or more phenotypic markers.

5. Vectors prepared in this way are used to transform bacterial cells creating colonies. Radiolabeled cDNA prepared from induced and uninduced mRNA, derived as described above, are used to screen duplicate colonies separately. Excess cDNA is then removed, and colonies are exposed to X-ray film to identify clones of the induced cDNA.

6. The corresponding DNA plasmid was isolated from the induced cDNA clones and sequenced.

7. The sequenced DNA is then shaped in vitro for introduction into the appropriate expression carrier used to transform the host cell, which in turn is allowed to grow in culture and express the desired human immunointerferon product.

8. Human immune interferon thus produced undoubtedly has 146 amino acids in its mature form, starting with cysteine, and is very basic. Its monomer molecular weight is 17,140. Probably due to the presence of numerous basic residues, hydrophobicity, salt bridge formation, etc., the molecule can self-associate in an oligomeric form, i.e., in a dimeric, trimernomial, or tetrameric form. The high molecular weights observed in natural substances (6), which cannot be attributed to the amino acid sequence alone, are probably caused by such oligomeric forms, as well as by the contribution of carbohydrates from post-translational glycosylation.

9. In certain host cell systems, especially when fused to an expression carrier, to be expressed together with its 20 amino acid signal peptide, the mature form of human immunointerferon is placed in the cell culture medium, greatly aiding regeneration and purification methods. .

Description of the preferred form

Microorganisms/cell cultures

1. Bacterial strains/promoters

The process described here is carried out using, among others, the microorganism E. coli K-12 strain 294 (terminus A, thi-, hsr-, khsm+), as described in British Patent Publication no. 2055382 A. This strain is deposited with the American Type Culture Collection, ATCC Accession no. 31446. However, other microbial strains may be used, including known E: coli strains such as E. coli B, E. coli X 1776 (ATCC No. 31537) and E. coli W 3110 (F-, γ-, protropic) (ATCC No. 27325) or other mycorrhizal strains, many of which are stored and can (eventually) be obtained from recognized microorganism depository institutions, such as the American Type Culture Collection (ATCC)--cf. ATCC catalog. See also German Offenlegungsschrift 2644432. These other microorganisms include, for example, bacilli such as bacillus subtilis and other enterobacteria, of which we cite as examples salmonella typhimurium and serratia marcesans, with the use of plasmids that can replicate and express sequences of heterologous genes in them.

As an example, the beta lactamase and lactose promoter system has been successfully used to initiate and maintain microbial production of heterologous polypeptides. Details regarding the preparation and composition of these promoter systems were published by Chang et. al., Nature 275, 617 (1978) and Itakura et al., Science 198, 1056 (1977), which publications are incorporated herein by reference. Recently, a tryptophan-based system, the so-called trp promoter system, has been developed. Details regarding the preparation and composition of this system are published by Goeddel et al., Nucleic Acids Research 8, 4057 (1980) and Kleid et al., U.S.S.N. 133, 296. Those publications were filed March 24, 1980, and are incorporated herein by reference. Numerous other microbial promoters have been discovered and used, and details of their nucleotide sequences, which allow one skilled in the art to incorporate them into plasmid vectors, have been published, see, e.g., Siebenlist et al., Cell 20, 269 (1980), which is incorporated herein by reference. .

2. Yeast strains/yeast promoters

With this expression system, the plasmid YRp7 (14, 15, 16) can also be used, which enables selection and replication both in E.coli and in the yeast, saccharomyces cerevisiae. For selection in yeast, the plasmid contains the TRP1 gene (14, 15, 16) which complements (enables growth in the absence of tryptophan) yeast containing mutations in that gene found on chromosome IV of yeast (17). The strain used here is strain RH218 (18) deposited in the American Type Culture Collection without restriction (ATCC No. 44076). However, it is understood that any strain of saccharomyces cerevisiae containing a cell-forming mutation must be an efficient environment for the expression of the plasmid containing the expression system. An example of another strain that can be used is pep4-1 (19). This tryptophan auxotrophic strain also has a point mutation in the TRP1 gene.

When placed at the 5' position of a non-yeast gene, the 5'-flanking DNA sequence (promoter) from a yeast gene (for alcohol dehydrogenase 1) can accelerate expression of the foreign gene in yeast when placed in a plasmid used to transform yeast. In addition to the promoter, the correct expression of the non-yeast gene in yeast requires a second yeast sequence that is placed at the 3'-end of the non-yeast gene on the plasmid, to allow for proper termination of transcription and polyadenylation in yeast. This promoter can be used in the subject invention in the same way as others - see infra.

In a preferred embodiment, the 5' flanking sequence of the yeast 3-phosphoglycerate kinase gene (20) is located at a higher position than the structural gene, which is again followed by DNA containing the final polyadenylation signals, for example the TRP1 (14, 15, 16) gene or PGK (20) gen.

Since the yeast 5'-flanking sequence (in conjunction with the yeast 3' end DNA) (infra) can accelerate the expression of a foreign gene in yeast, it is very likely that the 5'-flanking sequences of any highly expressed yeast gene could be used for expression important gene products. Since, under some conditions, yeast expressed up to 65 percent of its soluble protein as glycolytic enzymes (21 ) and since this high level appears to result from the production of high levels of individual mRNAs (22), it would be possible to use the 5'-flanking sequences of any other glycolytic genes for such expression purposes - eg enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Each of the 3'-side sequences of these genes can also be used for the exact termination of mRNA polyadenylation in such an expression system - cf. Supra. Some other highly expressed genes are those for acid phosphatase (23) and those that express high levels of production due to mutations in the 5'-flanking regions (mutants that increase expression) - mainly due to the presence of the TY1 transposable element (24).

All of the above genes are considered to be transcribed by yeast RNA polymerase (II). Promoters for RNA polymerase I and III that transcribe genes for ribosomal RNA, 5S RNA, and tRNa (24, 25) can also be used in such expression constructs.

Finally, many yeast promoters also contain transcriptional control, so they can be turned off or on by varying growth conditions. Some examples of such yeast promoters are genes that produce the following proteins:

alcohol dehydrogenase II, isocytochrome-c, acid phosphatase, degradative enzymes connected with nitrogen metabolism, glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for the utilization of maltose and galactose (22). Such control regions would be very useful in controlling the expression of a protein product - especially if its production is toxic to yeast. It would also be possible to insert the control region of one 5'-flanking sequence with the 5'-flanking sequence containing the promoter of a highly expressed gene. This would lead to a hybrid promoter and would be possible, as the control region and promoter appear to be physically different DNA sequences.

3. Cell culture systems/cell culture vectors

Propagation of vertebrate cells in cultures (tissue culture) has become a routine procedure in recent years (see Tissue Culture, Academic Press, Kruse and Patterson edition, 1973). In this procedure, COS-7 stem connective cells of the monkey kidney were used as a host for the production of immunointerferon (25a). The experiment described herein can be performed in any cell line capable of replicating and expressing a compatible vector, eg W138, BHK, 3T3, VERO and HeLa cell lines. In addition, what is required of an expression vector is a source of replication and a promoter located upstream of the gene being expressed, along with all necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. easily these essential elements of SV40 are used in the process described herein, it is understood that the invention described herein in terms of the preferred form should not be construed as being limited to these sequences. For example, the source of replication of other viral (eg, polyoma, adeno, VSV, BPV, etc.) vectors can also be used as cellular sources of DNA replication, which can function in a non-integrated state.

B. Systems of vectors

1. Direct expression of mature immune interferon in E. coli

The procedure used to obtain the direct expression of IFN-γ in E.coli as a mature interferon polypeptide (minus the signal sequence) was a variant of the one used previously for human growth hormone (26) and human leukocyte interferon (1 ), insofar as it includes combinations of synthetic (N-terminal) and cDNA.

As deduced from the nucleotide sequence of p69, described infra and by comparison with the known cleavage position between the signal peptide and the mature polypeptide for some IFN-α, s (2), IFN-γ has a hydrophobic signal peptide of 20 amino acids, followed by 146 amino acids of mature IFN-γ (Figure 5). As shown in Figure 7, the BstNI restriction endonuclease site is located at amino acid 4 of mature IFN-γ. Two synthetic deoxyoligonucleotides were identified that incorporate the ATG translation start codon, codons for amino acids 1, 2, and 3 (cysteine-tyrosine-cysteine) and create an EcoRI cohesive terminus. These deoxyoligonucleotides were ligated to a 100 bp BstNI-PstI fragment of p69, to obtain a 1115 bp synthetic-natural hybrid gene, which codes for IFN-γ and was ligated via EcoRI and PstI restriction sites. This gene was inserted into the plasmid pLeIF A trp 103 between the EcoRI and PstI positions, to obtain the expression plasmid pIFN-γ trp 48. In this plasmid, the IFN-γ gene is expressed under the control of the E. coli trp promoter. (pLeIF A trp 103 is a derivative of pLelF A 25 in which the EcoRI position distal to the LeIF A gene was removed). The procedure used to remove this EcoRI position has been previously described (27).

2. Expression in yeast

In order to express a heterologous gene such as cDNA for immune interferon in yeast, a plasmid vector containing four components had to be obtained. The first component is the part that enables the transformation of E.coli and yeast and therefore must contain a gene that can be selected from each organism (in this case, it is the ampicillin resistance gene from E.coli and the TRP1 gene from yeast). That component also requires a source of replication from both organisms, to be maintained as plasmid DNA in both organisms (in this case, it is an E.coli source from pBR322 and an ars1 source from yeast chromosome III).

Another plasmid component is a 5'-flanking sequence from a highly expressed yeast gene, to accelerate transcription of a downstream structural gene. In this case, the 5'-flanking sequence used is that of the yeast 3-phosphoglycerate kinase (PGK) gene. The fragment was designed to remove the ATG of the PGK structural sequence as well as 8 bp upstream of that ATG. This sequence was replaced with a sequence containing both XbaI and EcoRI restriction sites for convenient joining of this 5'-flanking sequence to the structural gene.

The third component of the system is a structural gene designed to contain an ATG translational start and translational stop signal. Isolation and cloning of such a gene is described infra.

The fourth component is the yeast DNA sequence containing the 3'-flanking sequence of the yeast gene, which contains the correct signals for transcription termination and polyadenylation.

With all these components, immune interferon was obtained in yeast.

Expression in mammalian cell culture

The strategy for the synthesis of immunointerferon in mammalian cell culture depends on the development of a vector capable of autonomous replication and expression of a foreign gene under the control of a heterologous transcription unit. Replication of this vector in tissue culture is completed with a source of DNA replication (derived from the SV40 virus) and a helper function (T antigen), by introducing the vector into a cell line endogenously expressing that antigen (28, 29). The late promoter of that SV40 virus precedes the interferon structural gene and ensures transcription of the gene.

The vector used to obtain IFN-γ expression consisted of pBR322 arrays that provided a selection marker in E.coli (ampicillin resistance) as well as an E.coli source of DNA replication. These sequences are derived from plasmid pML-1 (28) and surround the region bridging the EcoRI and BamHI restriction sites. The SV40 source was derived from a 342 bp PvuII-HindIII fragment flanking this region (30,31) (both ends converted to EcoRI termini). These sequences, in addition to containing the viral sources of DNA replication, encode the promoter for the early and late transcription units. The orientation of the SV40 source region was such that the promoter for the late transcription unit was positioned proximal to the gene encoding the interferon.

Brief description of the drawing

Figure 1 shows sucrose gradient centrifugation of induced peripheral blood lymphocyte (PBL) poly(A)+RNA. Two points of maximal interferon activity were observed with sizes of 12S and 165. The positions of the ribosomal RNA markers (each of which was centrifuged separately) are marked above the extinction profile.

Figure 2 shows electrophoresis of induced PBL Polx(A)+RNA through acid urea agarose. Only one point of maximum activity was observed, which comigrates with 18S RNA. The positions of ribosomal RMA markers that were subjected to electrophoresis in the adjacent space and visualized by staining with ethidium bromide are indicated above the activity profile.

Figure 3 shows hybridization patterns of 96 colonies with induced and uninduced 32 P-labeled cDNA test substances. 96 individual transformants were grown on a microtiter plate, transferred to two nitrocellulose membranes, and then the filters were hybridized with 32P-CDNA test substances, which were obtained from either induced mRNA (top) or mRNA isolated from non-induced PBL cultures (non-induced, bottom). Filters were washed thoroughly to remove unhybridized RNA and then exposed to X-ray film. This filter set is representative of 86 such sets (8300 independent colonies). An example of an "induced" clone is designated as H 12.

Figure 4 is a restriction endonuclease map of the cDNA entry of clone 69. The cDNA entry was joined by PstI positions (dots at both ends) and oligo dC-dG tails (single lines). The number and size of fragments obtained by nuclease restriction digestion were evaluated by electrophoresis through 6 percent acrylamide gels. The positions of the sides were confirmed by nucleic acid sequencing (shown in Figure 5). The coded area of the largest open frame for reading is a separated and crossed-out area and represents the so-called sequence of 20 remaining signal peptides, while the dotted area represents the mature IIF sequence (146 amino acids). The 5' end of the mRNA is on the left side, while the 3' end is on the right side.

Figure 5 presents the nucleotide sequence of the p69 plasmid entry. The deduced amino acid sequence of the longest open reading frame is also shown. The so-called signal sequence is shown by residues labeled S1 to S20.

Figure 6 is a comparison of IFN-γ mRNA structure with that of leukocyte interferon (IFN-α) and stem cell interferon (IFN-β). Clone 69 mRNA (immunolabeled) contains a significantly higher amount of untranslated sequences.

Figure 7 is a schematic diagram of the composition of the IFN-γ expression plasmid pIFN-β trp 48. The starting material is a 1250 base pair entry of PstI from plasmid p69.

Figure 8 shows a diagram of the plasmid used to express IFN-γ in monkey cells.

Figure 9 shows a Southern hybridization of eight different EcoRI-digested human genomic DNAs hybridized with a 32p-labeled Ddel fragment of 600 base pairs from the p69 tyhe cDNA input. Two EcoRI fragments clearly hybridize with the test substance in each DNA sample.

Figure 10 shows the Southern hybridization of human genomic DNA digested with six different restriction enzymes. endonuclease hybridized with a 32P-labeled p69 test substance.

Figure 11 schematically shows the restriction map of the entry of the 3.1 kbp HindIII vector pB1 from which the PGK promoter was isolated. The entry of the EcoRI position and the XbaI position into the 5'-side DNA of the PGK gene is indicated.

Figure 12 shows the 5'-flanking sequence plus the initial coding sequence for the PGK gene before the insertion of the XbaI and EcoRI positions.

Figure 13 schematically shows the techniques used to introduce an XbaI site at position -8 in the PGK promoter and to isolate a 39bp fragment of the 5'-flanking sequence of PGK containing that XbaI terminus and the Sau3A terminus.

Figure 14 schematically shows the composition of the 300 bp fragment containing the upper 39bp fragment, an additional PGK 5'-flanking sequence (265bp) from PvuI to Sau3A (see Figure 1 1 ) and an EcoRI position adjacent to XbaI.

Figure 15 schematically shows the composition of the 1500 bp promoter fragment (HindIII EcoRI) which contains, in addition to the fragment shown in Figure 14, a 1300 bp HindII to Pvul fragment from the PGK 5'-side sequence (see Figure 1 1).

Figure 16 shows the composition of an expression vector for human immune interferon in yeast, which contains a modified PGK promoter, IFN-v cDNA and the terminal region of the yeast PGK gene as described in detail below.

Detailed description

A. Source of IFN-γ mRNA

Peripheral blood lymphocytes (PBL) were obtained by leukophoresis, and blood was obtained from voluntary donors. PBL are later purified by FicoII-Hapaque gradient centrifugation and then cultured at a concentration of 5x10 6 cells/ml in RPMI 1640, 1 percent L-glutamine, 25 mM HEPES, and 1% penicillin/streptomycin solution (Gibco, Grand Islan, Ny). These cells were induced to produce IFN-γ with the mitogen staphylococcal enterotoxin B (1 µg/ml) and cultured for 24 to 48 hours at 37ºC in 5% CO2. Desacetylthymosin-alpha-1 (0.1 µg/ml) was added to PBL cultures to increase the relative amount of IFN-γ activity.

B. Journal of RNA Isolation

Total RNAs from PBL cultures were extracted essentially as described by Berger, S.L. et al. (33). The cells were pelleted by centrifugation and then resuspended in 10 mM NaCl, 10 mM tris-HCl (pH 7.5), 1.5 mM MgCl2 and 10 mM ribonucleoside vanadyl complex. The cells were dissolved by adding NP-40 (1 percent concentration), and the nuclei were pelleted by centrifugation. The supernatant contained total RNA previously purified by multiple phenol and chloroform extractions. The aqueous phase was made 0.2 M in NaCl, and then the total RNA was precipitated by adding two amounts of ethanol. RNA from non-induced (non-stimulated) cultures was isolated using the same methods. Oligo-dT cellulose chromatography was used to purify mRNA from total RNA preparations (34). Typically, the amounts obtained from 1-2 liters of cultured PBL were 5-10 milligrams of total RNA and 50-200 micrograms of Poly(A)+RNA.

C. Fractionation of mRNA

Two methods were used for fractionation of mRNA preparations. These methods were used independently of each other (rather than together) and each resulted in a significant increase in IFN-γ mRNA.

Sucrose gradient centrifugation in the presence of the denaturing agent formamide was used for mRNA fractionation. At 19 hours and at 20ºC, gradients from 5 percent to 25 percent sucrose in 70 percent formamide (32) were centrifuged at 154,000 x g. Successive fractions (0.5 ml) were then removed from the top of the gradient, ethanol precipitated, and an aliquot was injected into Xenopus laevis. oocytes for mRNA translation (35). After 24 hours at room temperature, the incubation medium is tested for antiviral activity in a standard cytopathic inhibition assay using vesicular stomatitis virus (Indian strain) or encephalomyocarditis virus on WISH cells (human amnion) as described by Stewart (36), except , which samples were inhibited with cells for 24 hours (instead of 4) before working with the virus. Two points of maximum activity were consistently observed in RNA fractionated by a sucrose gradient (Figure 1). A single peak precipitated at a calculated amount of 125 and contained 100-400 units/ml of antiviral activity (compared to an IFN-alpha standard) per microgram of injected RNA. The second maximum activity point deposited as 16S in size contained about half the activity of the slower settling maximum activity point.

Each of these highest activity points is due to IFN-γ, as no activity was observed when the same fractions were tested on bovine cells (MDBK) that were not protected by human IFN-γ. Both activities, i.e. both IFN-alpha activity and IFN-beta activity, would be easily detected with the MDBK test (5).

Fractionation of mRNA (200 µg) was also performed by electrophoresis through acid urea agarose gels. The dense agarose gel (37, 38) was composed of 1.75 percent agarose, 0.025 M citrate, pH 3.8, and 6 M urea. Electrophoresis was carried out for 7 hours at 25 milliamps. and 4ºC. Then the gel was fractionated with a razor blade. Individual lobes were melted at 70ºC and extracted twice with phenol and once with chloroform. Fractions were then precipitated in ethanol and subsequently tested for IFN-γ mRNA by injection into Xenopus laevis oocytes and antivirally. In the gel-fractionated samples, only one point of maximum activity was observed (Figure 2). This point of maximum activity co-localized with 18S and had an activity of 600 units/ml per microgram of injected RNA. This activity seems to be also IFN-γ specific, as it does not protect MDBK cells.

The discrepancy in size between the maximal activities observed on sucrose gradients (12S and 16S) and urea acid gels (18S) can be explained by the observation that these two independent fractionation methods are not performed under total denaturing conditions.

D. Colony preparation containing IFN-γ arrays

3 µg of gel-fractionated mRNA was used in standard double-stranded cDNA preparation procedures (26, 39). cDNA was fractionated on a 6% polyacrylamide gel. Two size fractions were electroeluted, 800 - 1500 bp (138 ng) and less than 1500 bp (204 ng). A 35 ng portion of each cDNA size was extended with a deoxyl C residue using terminal deoxylnucleotidyl transferase (40), and digested with 300 ng of plasmid pBR322 (41) similarly fused with a deoxylG residue at the PstI position (40). Each fused mixture was then transformed into E. coli K12 strain 294. About 8000 transformants were obtained with 800 1500 bp cDNA, and 400 transformants were obtained with less than 1500 'bp cDNA.

E. Separation of colonies for induced cDNAs

Colonies were individually inoculated into microtiter plates containing LB (58) + 5 µg/ml tetracycline, and stored at temp. from - 20°C after adding DMSO up to 7%. Two copies of the colony were grown on nitrocellulose filters, and DNA from each colony was fixed on the filter using the Grunstein-Hogness procedure (42).

32p-labeled cDNA of the test substance was prepared using 18S size gel-fractionated mRNA from induced and non-induced PBL cultures. dT 12-18 was used, and the reaction conditions were described earlier (1). Filters with 8000 cDNA transformants with a size of 600 - 1500 bp and 400 cDNA transformants with a size of less than 1500 bp were hybridized with 20 x 106 cpm induced 32pcDNA. A double set of filters was hybridized with 20 x 106 cpm of uninduced 32pcDNA. In the hybridization, the conditions described by Fritsch et al. were used for 16 hours. (43). Filters were thoroughly washed (43) and exposed to Kodak XR-5 X-ray film with DuPont Lightning-Plus amplification filters for 16 - 48 hours. The hybridization model of each colony with two test substances was compared. About 40% of colonies hybridized with both test substances, while approximately 50% of colonies did not hybridize with either test substance (shown in Figure 3). 124 colonies significantly hybridized with the induced test substance, but weakly or slightly with the non-induced test substance. These colonies were individually inoculated onto microtiter plates, grown and transferred onto nitrocellulose filters, and hybridized with the same two test substances as described above. Plasmid DNA isolated by the rapid procedure (44) from each of the colonies was also fixed on nitrocellulose filters and hybridized (45) with induced and uninduced test substance. DNAs from 22 colonies were hybridized only with the induced test substance, and were called "induced" colonies.

F. Characteristics of induced colonies

Plasmid DNA was prepared from 5 induced colonies (46), and was used to characterize the cDNA entry. Restriction endonuclease display of the five induced plasmids (p67, p68, p69, p71 and p72) indicates that the four plasmids have similar restriction nuclease appearance. Those four plasmids (p67, p69, p71 and p72) had four DdeI positions, 2 HinfI positions and one RsaI position in the cDNA input. The fifth plasmid (p68) contained a normal Ddel fragment and, compared to the other four, represented a short cDNA clone.

The match indicated by the restriction nuclease map was confirmed by hybridization. A 32p-labeled DNA test substance was prepared (47) from a 600 bp Ddel fragment of plasmid p67 and used for hybridization (42) with other induced colonies. All five colonies with expressed restriction nuclease were cross-hybridized with the test substance in question, and the same was the case with 17 other colonies out of 124 selected in the induced/non-induced separation.

The length of the cDNA insert in each of these cross-hybridized plasmids was measured by PstI digestion and gel electrophoresis. The clone with the longest cDNA input was clone 69 with an input length of 1200-1400 bp. This DNA was used in all further experiments, and a representation of its restriction endonuclease can be seen in Figure 4.

In the case of plasmid p69, it was shown that the cDNA entry, by the expression of its product, is IFN-γ cDNA, produced in three independent expression systems, with antiviral activity, as described in more detail below.

G. Array analysis of the cDNA insert in the p69 plasmid

The entire nucleotide sequence of the cDNA entry in the p69 plasmid was determined by the dideoxyl nucleotide chain termination method (48) after subcloning the fragments in the M13 vector mp7 (49), and by the Maxam-Gilbert chemical procedure (52). The longest read form comprises a protein of 166 amino acids, shown in Figure 5. The first coded residue is the first codon found in the 5' end of the cDNA. The first 20 residues at the amino ends probably serve as a signal sequence for the secretion of the remaining 146 amino acids. This so-called signal sequence has features in common with other signal sequences, namely size and hydrophobicity. Furthermore, the four amino acids found at the cleavage site (ser - leu - gly - cis) are identical to the four residues found at the cleavage point of several leukocyte interferons (LeIF B, C, D, F, and H, (2)). The encoded mature amino acid sequence with 146 amino acids has a molecular weight of 17,140.

There are two potential glycosylation sites (50) in the encoded protein sequence, at amino acids 28 to 30 (asn - gly - thr) and amino acids 100 to 102 (asn - tyr - ser). The existence of these positions is consistent with the observed glycosylation of human IFN-γ (6, 51 ). Furthermore, the only two cysteine residues (positions 1 and 3) are spatially too close to form a disulfide bridge that is consistent with the observed stability of IFN-γ in the presence of reducing agents such as beta-mercaptoethanol (51). The derived developed amino acid sequence is mostly quite basic, with a total of 30 residues of lysine, arginine and histidine, and only 19 residues of aspartic and glutamic acid.

The mRNA structure of IFN-γ derived from the DNA sequence of plasmid p69 is significantly different from IFN-α (1, 2 or IFN-(3 (5)) mRNA. As shown in Figure 6, the coding region of IFN-γ is shorter while 5 ' untranslated and 3' untranslated regions much longer than IFN-α or IFN-β.

H. Direct expression of immune interferon in E. coli

Referring to Figure 7, 50 µg of plasmid p69 digested with PstI and with input of 1250 base pairs isolated by gel electrophoresis on a 6% polyacrylamide gel. Approximately 10 µg of this input was electroeluted from the gel. 5 µg of the Pstl fragment is partially digested for 15 minutes with 3 units of BstNl (Bethesda Research Labs) at a temperature of 37ºC, and the reaction mixture is purified on a 6% polyacrylamide gel. Of the desired 1100 base pair BstNl - Pstl fragment, about 0.5 µg is recovered. The two indicated deoxyloligonucleotides, 5' dATTCATGTGTTATTGTC and 5' - dTGACAATAACACATG (Figure 7) were synthesized by the phosphotriester method (53) and phosphorylated as follows:

100 p moles of each deoxyloligonucleotide were combined in 30 µl 60 mM Tris-HCl (pH 8), 10 mM MgCl2, 15 mN beta-mercaptanethanol and 240 µCi (y-32p)ATP (Amersham, 5000 Ci/mmole). 12 units of T4 polynucleotide kinase are added and the reaction takes place for 30 minutes at a temperature of 37°C. 1 µl of 10 mM ATP is added and the reaction is continued for a further 20 minutes. After the o-OH/CHCl3 extraction, oligomers are combined with 0.25 µg fragment of the base pair BstNl-Pstl 1100, and ethanol is precipitated. These fragments are bound at a temperature of 20ºC, for 2 hours in 30 µl of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 0.5 mM ATP and 10 units of T4 DNA ligase. The mixture is digested for 1 hour with 30 units of PstI and 30 units of EcoRl (to eliminate polymerization via cohesive end joining), and electrophoresed on a 6% polyacrylamide gel. 1115 base pair product (110,000 cpm) is recovered by electroelution.

Plasmid pLeIF A trp 103 (Figure 7) is a derivative of plasmid pLeIF A 25 (1) in which the EcoRl position distal to the LeIF A gene (27) has been removed. 3 µg of PLeIF A trp 103 is digested with 20 units of EcoRl and 20 units of Pstl for 90 minutes at a temperature of 37ºC, and is subjected to electrophoresis on a 6 percent polyacrylamide gel. A large (3900 base pair) fragment of the vector is recovered by electroelution. A fragment of base pair 1115 EcoRl-Pstl IFN-γ DNA is joined in 0.15 µg of the prepared vector. Transformation of E. coli K12, strain 294 (ATCC No. 31446) resulted in 120 colonies resistant to tetracycline. Plasmid DNA is prepared from these 60 transformants and digested with EcoRl and PstI. Three of the plasmids contain the desired fragment of the 1115 base pair EcoRl-PstI. DNA sequence analysis confirms that these plasmids contain the desired nucleotide sequence at the junction between the trp promoter, synthetic DNA and cDNA. One of these plasmids, pIFN-γ trp 48, was chosen for further consideration. This plasmid was used to transform E. coli K-12 strain W3110 (ATCC No. 27325).

I. Structure of the gene encoding the IFN-γ sequence

The structure of the gene coding for IFN-γ was analyzed by Southern hybridization. In this procedure (54), 5 micrograms of human lymphocyte DNA (prepared as in 55), of high molecular weight, is digested completely with various restriction endonucleases, subjected to electrophoresis on a 1.0 percent agarose gel (56), and applied to a nitrocellulose filter (54). . A 32p-labeled DNA test substance is prepared (47) from a 600 bp Ddel fragment of the p69 cDNA entry and hybridized (43) to a nitrocellulose-DNA stain. 107 counts per minute of test substance are hybridized for 16 hours and then washed as described (43). Eight genomic DNA samples, from different donors, were digested with restriction endonuclease EcoRl and hybridized with p69 32p-labeled test substance. As shown in Figure 9, two clear hybridization signals were observed with sizes of 8.8 kilobase pairs (kbp) and 2.0 kbp, as assessed by mobility comparison with Hind III digested -DNA. This could be the result of two IFN-γ genes or one gene cleaved by the EcoRl position. Since the p69 cDNA does not contain an EcoRl position, an intervening sequence (intron) with an internal EcoRl position would be required to explain only one gene. To distinguish between these two possibilities, another Southern hybridization was performed with the same test substance on five other endonuclease digests of individual DNA (Figure 10). Two hybridizing DNA fragments were observed with two other endonuclease digests, Pvull (6.7 kbp and 4.0 kbp) and Hincll (2.5 kbp and 2.2 kbp). However, three endonuclease digestion samples yield only one hybridizing DNA fragment: HindIII (9.0 kbp), BglII (11.5 kbp) and BamHI (9.5 kbp). The two IFN-γ genes would have to be linked at an unusually short distance (less than 9.0 kbp) to be contained in the same HindIII hybridizing fragment. This result indicates that only one homologous IFN-v gene is present in human genomic DNA, and that this gene cleaves one or more introns containing EcoRl, Pvull, and Hincll positions. This assumption is supported by hybridization of a 32p-labeled (47) fragment prepared from only the 3' untranslated region of cDNA from p69 (130 bp Ddel fragment from 860 to 990 bp in Figure 5) with an EcoRl digest of human genomic DNA. Only the 2.0 kbp EcoRl fragment hybridized with this test substance, indicating that this fragment contains the 3' untranslated sequence, while the 8.8 kbp EcoRl fragment contains the 5' sequence. The gene structure of IFN-γ (one gene with at least one intron) is significantly different from IFN-α (multiple genes (2) without introns (56)) or IFN-β (one gene without introns (57)).

J. Preparation of bacterial extracts

An overnight culture of E. coli W3110/pIFN-γ trp 48 in Luria nutrient solution + 5 micrograms/ml tetracycline is used to inoculate M9 (58) medium containing 0.2% glucose, 0.5% casamic acids and 5 micrograms/ml tetracycline diluted 1:100. Indole acrylic acid is added to a final concentration of 20 micrograms/ml, when A550 is between 0.1 and 0.1. Ten ml of the sample is obtained by centrifugation at A550 = 1.0 and immediately diluted with 1 ml of physiological phosphate buffer solution containing 1 mg/ml of bovine serum albumin. Cells are opened by sonication and cleaned of debris by centrifugation. The supernatants are stored at a temperature of 4°C until the start of the analysis. The interferon activity in the supernatants was found to be 250 units/ml compared to IFN-α standards as measured by the cytopathic effect inhibition (CPE) assay.

K. Transformation of yeast / strains and medium

Yeasts were transformed as previously described (59). E. coli strain JA300 thr IeuB6 thyA trp C1117 hsdm- hsdR- strR) (20) is used in the selection of plasmids containing a functional TRPI gene. Yeast strain RH218 of genotype trp1 gal2 SUC2 mal CUPI) (18) is used as host for yeast transformation. RH 218 has been deposited without restriction in the American Type Culture Collection, ATTC no. 44076. M9 (minimal medium) with 0.25% casamic acids (CAA) and LB (full medium) was described by Miller (58) with the addition of 20 µg/ml ampicillin (Sigma) after the medium was autoclaved and then cooled. Yeasts were grown in the following media: YEPD containing 1% yeast extract, 2% peptone and 2% glucose +/- 3% Difco agar. YNB + CAA contained 6.7 grams of yeast nitrogen base (without amino acids) (YNB) (Difco), 10 mg of adenine, 10 mg of uracil, 5 grams of CAA, 20 grams of glucose and +/- 30 grams of agar per liter.

L. Composition of yeast expression vectors

1. 10, ug YRp& (14, 15, 16) digested with EcoRl. The resulting sticky DNA ends were blunted using DNA polymerase I (Klenow fragment). The vector and input were loaded onto a 1% (SeaKem) agarose gel, removed from the gel, electroeluted, and extracted 2X with equal amounts of chloroform and phenol before precipitation with ethanol. The obtained DNA molecules with blunt ends were then combined for 12 hours in a volume of 50 µl at a temperature of 12°C. This mixture was then used to transform E. coli strain JA300 for resistance to ampicillin and tryptophan prototrophy. Plasmids containing the TRP1 gene in both orientations were isolated. pFRW1 had the TRP1 gene in the same orientation as YRp7, while pFRW2 had the TRP1 gene in the opposite orientation.

20 µg of pFRW2 was linearized with Hind III and electrophoresed on a 1% agarose gel. Linear molecules were eluted from the gel and 200 ng were mixed with 500 ng of the 3.1 kb HindIII entry of plasmid pB1 (13), which is a restriction fragment with the yeast 3-phosphoglycerate kinase gene. This mixture was used to transform E.coli strain 294 to ampicillin resistance and tetracycline sensitivity. A plasmid made from such a recombinant had an intact TRP1 gene with a 3.1 kbp HindIII fragment from the pB1 DNA entry at the HindIII position of the tetracycline resistance gene. This is the plasmid pFRM3l. 5 µg of pFRM31 was completely digested with EcoRl, extracted twice with phenol and chloroform and then precipitated with ethanol. The cohesive ends of the molecules are filled in by DNA polymerase I (Klenow fragment) in a reaction of 250 µM in each deoxynucleoside triphosphate. The reaction is carried out for 20 minutes at a temperature of 14ºC, during which time the DNA is extracted twice with phenol - chloroform, and then precipitated with ethanol.

The re-diluted DNA is then completely digested with Clal and electrophoresed on a 6% acrylamide gel. The vector fragment is removed from the gel with a solvent, extracted with phenol-chloroform and precipitated with ethanol.

The six N-terminal amino acids of the 3-phosphoglycerate enzyme obtained from a human subject are as follows:

1 - 2 - 3 - 4 - 5 - 6

SER - LEU - SER - HSM - LYS - LEU

One of the translational reads generated from the DNA sequence of the 141 bp Sau3A-to-Sau3A restriction fragment (containing the internal Hincll position; see the PKG restriction map in Figure 1 1 ) produces the following sequence of amino acids:

1 - 2 - 3 - 4 - 5 - 6

MET-SER - LEU - SER - SER - LYS - LEU

After removing the initiator - methionine, it is evident that the N-terminal amino acid sequence of PGK has 5 out of 6 amino acids homologous with the N-terminal amino acid sequence of human PGK.

The sequence obtained in this way indicates that the beginning of the yeast PGK structural gene is coded by DNA in the 141 bp Sau3A restriction fragment of pBl. The preceding procedure indicates that DNA sequences specifying PGK mRNA may exist in this region of the HindIII fragment. Further sequencing of the 141 bp Sau3A fragment yields multiple DNA sequences of the PGK promoter (Figure 12).

A synthetic oligonucleotide with the sequence 5'ATTTGTTGTAAA3' was synthesized by standard methods (Crea et al., Nucleic Acids Res. 8, 2331 (1980)). 100 ng of this basic element was labeled at the 5' end with 10 units of T4 polynucleotide kinase in a 20 µl reaction that also contained 200 µCi of /y32-P/ ATP. This labeled stock solution was used in a primer repair reaction intended to be the first step in the complex process of placing an EcoRl restriction site in the PGK 5'-flanking DNA preceding the PGK sequence of the structural gene.

100 µg of pB1 (20) is completely digested with HaelII and placed on a 6% polyacrylamide gel. The uppermost layer on the ethidium-stained gel (containing the PGK promoter region) is isolated by electroelution as described above. This 1200 bp Haelll piece of DNA is restricted with Hincll and then run on a 6% acrylamide gel. The 650 bp layer is isolated by electroelution. 5 µg of DNA was isolated. This 650 bp Haelll - to - Hincll DNA fragment is re-diluted in 20 µl H2O, then mixed with 20 µl phosphorylated stock solution. This mixture is extracted 1x with phenol - chloroform, then precipitated with ethanol. The dried DNA is diluted in 50 µl of H2O, and heated in a boiling water bath for 7 minutes. This solution is then rapidly cooled in a dry ice-cold ethanol bath (10 - 20 seconds) and transferred to an ice-water bath. 50 µl is added to this solution. solution containing 10 µl of 10X DNA polymerase 1 buffer (Boehringer Mannheim), 10 µl of a previously prepared solution of 2.5 mM in each deoxynucleoside triphosphate (dATP, dTTP, dGTP and dCTP), 25 µl of H2O and 5 units of DNA polymerase I, Klenow fragment. the reaction mixture of 100 µl is incubated for 4 hours at a temperature of 37° C. Then the solution is extracted 1x with phenol - chloroform, precipitated with ethanol, dried by lyophilization and then exhaustively limited with 10 units of Sau3A. Then the solution is placed on a 6% acrylamide gel A layer corresponding to 39 bp in size is excised from the gel and isolated by electroelution as described earlier. This 39 bp layer has one blunt end and one Sau3A sticky end. The fragment is cloned into a modified pFIF trp 69 vector (5). 10 µg p FIF trp 69 is linearized with Xbal, extracted 1x with phenol - chloroform, and precipitated with ethanol. The Xbal sticky end is loaded with DNA polymerase I Klenow fragment in a 50 µl reaction containing 250 µM in each nucleoside triphosphate. This DNA is reduced with BamHl, and placed on a 6 percent acrylamide gel. The vector fragment is isolated from the gel by electroelution and diluted in 20 µl H2O. 20 ng of this vector is ligated to 20 ng of the 39bp fragment prepared for 4 hours at room temperature: one fifth of the ligation mixture is used to transform E. coli strain 294 to ampicillin resistance (on LB + 20 µg/ml amp plates). Plasmids from transformants are screened by rapid screening (44). One plasmid, pPGK-39 is selected for array analysis. 20 µg of this plasmid is digested with Xbal, precipitated with ethanol and then treated for 45 minutes with 1000 units of bacterial alkaline phosphase at a temperature of 68ºC. DNA is extracted 3X with phenol - chloroform, then precipitated with ethanol. The dephosphorylated ends are then labeled in a 20 µl reaction containing 200 µl /y32-p/ ATP and 10 units of T4 polynucleotide kinase. The plasmid is reduced with SalI and loaded on a 6% acrylamide gel.

The labeled input layer is isolated from the gel and sequenced by a chemical degradation method (52). The DNA sequence at the 3' end of this promoter was as expected.

2. Composition of the 312 bp Pvul - to - EcoRl PGK promoter fragment

25 µg of pPGK-39 (Figure 13) is simultaneously digested with SalI and XbaI (5 units of each), then subjected to electrophoresis on a 6 percent gel. The 390 bp layer containing the 39 bp promoter is isolated by electroelution. Diluted DNA is restricted with Sau3A, and subjected to electrophoresis on an 8 percent acrylamide gel. The 39 bp promoter layer is isolated by electroelution. This DNA contained 39 bp of the 5' end of the PGK promoter to the Sau3A - to -XbaI fragment.

25 µg of pB1 is restricted with Pvul and Kpn1 and then electrophoresed on a 6% acrylamide gel. A 0.8 kbp layer of DNA is isolated by electroelution and then restricted with Sau3A. and subjected to electrophoresis on a 6 percent acrylamide gel. The 265 bp layer from the PGK promoter (Figure 11) is isolated by electroelution.

This DNA is then combined for 2 hours with 39 bp of the promoter fragment described above at room temperature. The ligation mixture is restricted with Xbal and Pvul, and subjected to electrophoresis on a 6% acrylamide gel. The 312 bp Xba - to - Pvul restriction fragment is isolated by electroelution. it is then added to a ligation mixture containing 200 ng of pBR322 (41 ) and 200 ng of the Xbal - to - Pstl LeIF A cDNA gene previously isolated from 20 µg of pLeIF trp A 25. This three-factor ligation mixture is used to transform E. coli strain 294 to tetracycline resistance. Transformant colonies were separated (44) and one of them, pPGK-300 was isolated with 304 bp of PGK 5'-flanking DNA fused to the LelF A gene in the pBR322 vector. The 5' end of the LeIF A gene has the following sequence: 5'- CTAGAATTC - 3'. Thus, the fusion of the XbaI position from the PGK promoter fragment in this sequence allows the addition of one EcoRl position to the XbaI position. Thus, pPGK-300 contains part of the PGK promoter isolated in the Pvul - to -EcoR1 fragment.

3. Composition of the 1500 bp EcoRl -to-EcoRl fragment of the PGK promoter

10 µg of pB1 is digested with Pvul and EcoRl and placed on a 6% acrylamide gel. 1.3 kb of Pvul -to- EcoRl DNA layer from PGK 5' - flanking DNA is isolated by electroelution. 10 µg of pPGK-300 is digested with EcoRl and Pvul, and the 312 bp fragment of the promoter is isolated by electroelution after electrophoresis of the digestion mixture on a 6% acrylamide gel. 5 µg of pFRL4 is reduced with EcoRl, precipitated with ethanol and then treated with bacterial alkaline phosphatase for 45 minutes at a temperature of 68°C. After three extractions of DNA with phenol/chloroform, precipitation with ethanol and re-dilution in 20 ml H2O; 200 ng of vector is ligated with 100 ng of 312 bp EcoRl-to-Pvul DNA from pPGK-3oo and 100 ng of EcoRl-to Pvul DNA from pB1. The ligation mixture is used to transform E. coli strain 294 to ampicillin resistance. One of the obtained transformants is pPGK1500. This plasmid contains a 1500 bp PGK promoter fragment in the form of an EcoRl-to-EcoRl or HindIII-to-EcoRl piece of DNA.

10 µg of pPGK-1500 is completely digested with ClaI and EcoRl, then the digested mixture is subjected to electrophoresis on a 6% acrylamide gel. A 900 bp fragment containing the PGK promoter is isolated by electroelution. 10 µg of pIFN-γ trp 48 is completely digested with EcoRl and Hincll, and subjected to electrophoresis on a 6% acrylamide gel. A 938 bp layer containing the direct IFN-γ cDNA was isolated from the gel by electroelution.

The yeast expression vector was composed of three reaction factors by ligating the PKG fragment of the promoter (on a Clal-to-EcoRl piece), nulled pFRM-31 and isolated IFN-γ cDNA. The ligation reaction is incubated for 12 hours at a temperature of 14°C. The ligation mixture is used to transform E. coli strain 294 to ampicillin resistance. Transformants were analyzed for the presence of appropriate expression of the plasmid, pPGK-IFN-γ (Figure 16). Plasmids containing the expression system were used to transform spheroplasts of yeast strain RH218 into tryptophan prototropy in tryptophan-deficient agar. These yeast recombinants were then tested for the presence of immunointerferon.

Yeast extracts were prepared as follows: ten ml of the culture was grown in YNB + CAA until it reached A 660- 1 - 2, collected by centrifugation and diluted in 500 µl PBS buffer (20 mM NaH2PO4, pH - 7.4, 150 mM NaCl). An equal amount of glass beads (0.45 - 0.5 mm) is added and the mixture is then rapidly spun for 2 hours. The extracts are rotated for 30 seconds at a speed of 14,000 rpm, and the supernatant is removed. In the supernatant, an interferon activity of 16,000 units/ml was determined by comparison with the IFN-α standard using the CPE inhibition test.

M. Composition of cell culture vector pSVγ69

342 fragment of the HindIII - Pvull base pair surrounding the SV40 gene was converted to an EcoR1 restriction site fragment. The Hindlll position is converted by adding a synthetic oligomer (5'dAGCTGAATTC), and the Pvull position is converted by blunt-end ligation to an Ecorl position filled in by polymerase I (klenow fragment). The resulting EcoRl fragment is inserted into the EcoRl position of pML-1 (28). The plasmid with the SV40 promoter facing away from the ampR gene is further modified by removing the EcoRl position closest to the ampR gene of pML-1 (27).

A 1023 base pair HpaI - BglII fragment of cloned HBV DNA (60) is isolated, and the HpaI position of hepatitis B virus (HBV) is converted using a synthetic oligomer (5'dGCGAATTCGC) to the EcoRl position. This EcoRl - Bglll linked fragment is cloned directly into the EcoRl - BamHl positions of the SV40-carrying plasmid described above.

In the remaining EcoRl position, the IFN-γ gene is inserted at the 1250 base pair fragment of Pstl after the conversion of the Pstl ends into EcoRl ends. A clone was isolated in which the SV40 promoter precedes the IFN-γ structural gene. The obtained plasmids are then introduced into the tissue culture (29) using the DEAE-dextran method (61) modified in such a way that the transfection in the presence of DEAE-dextran is carried out for 8 hours. The cell medium was changed every 2-3 days. 200 microliters were taken every day for the interferon bioassay. Typical values are 50-100 units/ml in samples examined three or four days after transfection.

N. Partial purification of monkey cell-derived immune interferon

To produce larger amounts of human IFN-γ from monkey cells, fresh layers of COS-7 cells in ten 10 cm plates were transfected with a total of 30 µg pDLIF3 in 110 ml DEAE-Dextran (200 µg/ml DEAE Dextran 500,000 MW, 0.5 M Tris pH 7.5, in DMEM). After 16 hours at a temperature of 37ºC, the plates were washed twice with DMEM. 15 ml of fresh DMEM supplemented with 10% f.b.s., 2 mM glutamine, 50 mg/ml penicillin G and 50 mg/ml streptomycin was then added to each plate. The next day, the medium was replaced with serum-free DMEM. Fresh medium without serum was added every day. The collected medium was kept at 4ºC until assayed or binding to CPG. It was found that after transfection, parts of the 3- and 4-day-old samples possess almost all activities.

0.5 g of CPG (controlled porous glass, Elektronucleonika, CPG 350, sieve mesh size 120/200) is added to 100 m) of cell supernatant and the mixture is stirred for 3 hours at a temperature of 4ºC. After a short centrifugation in a table centrifuge, the precipitated beads are placed in the column and thoroughly washed with 20 mM NaPO4 1 M NaCl 0.1 % beta-mercaptoethanol pH 7.2. The activity is then eluted with the same buffer containing 30% ethylene glycol, followed by elution with a buffer containing 50% ethylene glycol. Basically, all activity was linked to CPG. 75 percent of eluted activity was determined in fragments eluted with 30% ethylene glycol. These fragments were separated and diluted with 20 mM NaPO4 1 M NaCl pH 7.2 to a concentration of 10% ethylene glycol, and directly introduced into a column of 10 ml Con A Sepharose (Pharmacia). After thorough washing with 20 mM NaPO4 1 M NaCl pH 7.2, the activity is eluted with 20 mM NaPO4 1 M NaCl 0.2 M alpha-methyl-Dmannoside. A significant amount of activity (55%) did not bind to that lectin. 45 percent of the activity was eluted from alpha-methyl-D-mannoside.

Pharmaceutical compositions

The compounds of the present invention can be formulated in accordance with known methods for the preparation of pharmaceutically useful compositions. The human immunointerferon product, in this context, is combined in admixture with a pharmaceutically acceptable carrier. Suitable carriers and their compositions are described in Remington's Pharmaceutical Sciences, E.W. Martin. Such a composition will contain an effective amount of interferon protein together with a suitable amount of carrier to provide pharmaceutically acceptable compositions suitable for effective administration.

A. Parenteral administration

Human immune interferon can be given parenterally to people who are being treated for tumors or viruses, and to those who exhibit immune suppression. The dosage is in accordance with the practice used in the clinical testing of other human interferons, for example: about (1 - 10) x 106 units per day, and if it concerns substances with a purity of more than 1%, the dose is 50 x 106 units per day. Doses of IFN-γ can be significantly increased in order to achieve a greater effect, due to the absence of other human proteins than IIN-γ, which proteins in substances derived from human subjects can produce certain unwanted consequences.

Example of appropriate dosage for essentially homogeneous 1FN-γ in parenteral administration: 3 mg of 1FN-γ specific activity, say. 2 x 108 U/mg is dissolved in 25 ml of 5 N albumin serum (human) - USP, the solution is passed through a bacteriological filter, and the filtered solution is aseptically distributed into 100 vials, each of which contains 6 x 106 units of pure interferon, suitable for parenteral administration. It is preferable to keep the bottles in a cold place (temp. of - 20°C) until use.

Bio - testing

A. Characterization of antiviral activity

For antibody neutralization, the samples are, if necessary, diluted to a concentration of 500 - 1000 units/ml with PBS-BSA. Equal amounts of the sample are incubated for 2 - 12 hours at 4 degrees with serial dilutions of rabbit leukocytes, stem binding cells or immune interferon antisera. Anti-IFN-α and β were obtained from the National Institute of Allergy and Infectious Diseases. Anti-IFN-γ was prepared using authentic IFN-γ (5 - 20% purity), purified from stimulated peripheral blood lymphocytes. Before testing, the samples were centrifuged for 3 minutes at 12,000 x g for 3 minutes. For testing pH 2 stability, samples were adjusted to pH 2 by adding 1 N HCI, incubated for 2 - 12 hours at 4 degrees and neutralized by adding 1 N NaOH before testing. To test sensitivity to sodium dodecyl sulfate (SDS), samples were incubated for 2 - 12 hours with an equal amount of 0.2% SDS at a temperature of 4ºC.

Characteristics of IFN-γ produced by E. coli and COS-7 cells

[image]

This table shows the characteristic behavior of IFN-alpha, beta and gamma standards after different procedures. The activity of interferon produced by E.coli W3110/pIFN-γ trp 48 and COS-7/pSVγ69 is acid-sensitive, sensitive to SDS, and neutralized by antiserum of immune interferon. Antibodies do not neutralize it to IFN-alpha or beta. These data confirm that the interferon produced in such systems is IFN-γ and that the plasmid p69 cDNA entry encodes IFN-γ.

The immune interferon protein is defined by a specific DNA gene and deductive sequencing of the --cf amino acid. Figure 5. It is understandable that with interferon from this context, natural variations exist and occur from case to case. These variations can be demonstrated by amino acid differences in the overall sequence or by deletions, substitutions, insertions. inversion or addition of amino acid to said sequence. All such allelomorphic variations are included within the scope of this invention.

Although particular preferred forms have been described, it is to be understood that the present invention is not limited to them.

Claims (31)

1. Human immune interferon, characterized in that it has the sequence of amino acids shown in Figure 5 and their alleles purified from other proteins with which it is usually combined. 2. Derivative of human immune interferon, characterized in that it has the sequence of amino acids shown in Figure 5 and their alleles, whereby the said derivative has the action of human immune interferon. 3. Human immunointerferon derivative according to claim 2, characterized in that the amino acid is methionine at the N-terminus. 4. Human immune interferon according to claim 3, characterized in that it has the amino acid methionine at the N-terminus. 5. Human immune interferon, characterized in that it has the sequence of amino acids shown in Figure 5 or their alleles or derivatives that have the effect of human interferon, and are not accompanied by natural glycolysis. 6. Human immune interferon, characterized in that it has the sequence of amino acids shown in Figure 5 or their alleles or derivatives that have the action of human interferon, obtained by expression in a recombined host cell using the coded DNA for the sequence of amino acids of interferon. 7. Human immune interferon according to claim 6, characterized in that it is obtained by expression of the recombined sequence of coded DNA in a mammalian cell. 8. Human immune interferon according to claim 7, characterized in that the COS-7 cell line is a monkey cell line. 9. DNA isolate, characterized by the fact that it contains the human immune interferon DNA code that has the sequence of amino acids shown in Figure 5 or its alleles or derivatives, which act as human immune interferon. 10. Recombinant cloning vector, characterized by the fact that it contains the human immune interferon DNA code of the sequence of amino acids shown in Figure 5 or its alleles or derivatives that act as human immune interferon. 11. A replicating expression vector, characterized by the fact that it can secrete, in a transformed microorganism or cell culture, human immunointerferon DNA with the sequence of amino acids shown in Figure 5 or its alleles or derivatives that act as human immunointerferon. 12. Recombinant microorganism or cell culture, characterized by the fact that it is capable of secreting immune interferon of the sequence of amino acids shown in Figure 5 or its alleles or derivatives that act as human immune interferon. 13. The microorganism according to claim 12, characterized in that it is an E. coli strain. 14. The microorganism according to claim 12, characterized in that it is a yeast strain. 15. Cell culture according to claim 12, characterized in that it is a mammalian cell line. 16. Cell culture according to claim 15, characterized in that the COS-7 cell line is a monkey cell line. 17. A preparation, characterized in that it contains a therapeutically effective amount of human immunointerferon according to any one of claims 1 to 8 together with a pharmaceutically acceptable carrier. 18. The preparation according to claim 17, characterized in that it is suitable for parenteral administration. 19. The method, characterized in that it comprises the secretion of human immune interferon in a mammalian cell line transformed by the DNA shown in Figure 5 or variant alleles thereof. 20. The method according to claim 19, characterized in that the COS-7 cell line is a monkey cell line. 21. The method according to claim 19 or 20, characterized in that it includes detection of human immunointerferon from the culture. 22. The method, characterized in that it comprises the secretion in the recombined host cell of the DNA code of human immunointerferon, which can be obtained by the method according to claim 21. 23. The method, indicated by the fact that it comprises the secretion in the recombined host cell of the DNA code of human immunointerferon of the sequence of amino acids shown in Figure 5 or its alleles or derivatives that have an effect as human immunointerferon. 24. The method according to claims 22 or 23, characterized in that it additionally comprises the step of detecting said human immunointerferon. 25. The process of obtaining human immune interferon, characterized in that it includes: (a) culturing recombinant host cells that have been transformed with an expression vector containing the human immunointerferon DNA code of the amino acid sequence shown in Figure 5 or its alleles or derivatives having human immunointerferon activity, under conditions that enable the secretion of said DNA sequence to the aforementioned human immune interferon was obtained; and the discovery of the aforementioned human immune interferon. 26. The method according to claim 25, characterized in that it includes preliminary steps (i) preparation of a replicating expression vector capable of secreting the DNA of said human immunointerferon in a host cell; you (ii) transforming the host cell culture to obtain said recombinant host cell. 27. The method according to any of claims 22 to 26, characterized in that the host is an E. coli strain. 28. The method according to any one of claims 22 to 26, characterized in that the host is a yeast strain. 29. The method according to any one of claims 22 to 26, characterized in that the host cell is a mammalian cell line. 30. The method according to claim 29, characterized in that the COS-7 cell line is a monkey cell line. 31. The use of human immunointerferon according to any claim from 1 to 8, or prepared by a process according to any claim from 19 to 30, characterized in that it serves for the preparation of pharmaceutical preparations.