BG61060B2 - Human immune inteferon - Google Patents

Abstract

The invention relates to the preparation of a new pure polypeptide by the recombinant DNA technique with the participation of any of the known expression vectors and host cultures. The polypeptide, human immune (gamma) interferon (ifn), was isolated and characterized by its DNA and amino acid sequences, physical properties and biological activity. 5 claims

Description

The present invention relates to the field of recombinant DNA technology, to the means and methods used by this technique for the disclosure of the DNA sequence and the putative amino acid sequence for human immune interferon and its preparation, as well as various products for its preparation and use.

More particularly, the present invention relates to the isolation and identification of DNA sequences encoding human immune interferon and to structures of recombinant DNA expression agents containing such DNA sequences operatively linked to expression promoter sequences, as well as to expression constructs constructed therein. way. In another aspect, the present invention relates to host culture systems, such as different cell cultures, similar expression agents have been transformed and thus targeted for expression of the DNA sequences mentioned above. In another aspect, this invention relates to the means and methods of converting end products of similar expression into novel ones, such as pharmaceutical compositions useful for the prophylaxis or therapy of humans. In preferred embodiments, the present invention provides special expression agents that are co-ordinated as human immune interferon is produced and secreted by the host cells in mature form. In addition, the present invention relates to various methods applicable to the production of said DNA sequences, host culture systems and end products and to specific and related embodiments thereof.

The present invention derives in part from the discovery of the DNA sequence and putative amino acid sequences encoding human immune interferon. In addition, the present invention provides sequential information on the 3 and 5 flanking sequences for the human immunointerferon gene facilitating their in vitro binding to expression agents. In particular, there is provided 5 DNA segments encoding the putative endogenous signal polypeptide that immediately precedes the amino acid sequence of the putative mature human immune interferon. These findings, in turn, allow the development of the means and methods of obtaining, through DNA technology, a sufficient amount of human immune interferon to make it possible, on the other hand, to determine its biochemical properties and bioactivity. Publications and other materials used to illuminate the level of the invention and, in special cases, to provide additional details for their practical use, are included in the accompanying references.

BACKGROUND OF THE INVENTION

A. Human immune interferon

Human interferon is classified into three groups based on differences in its antigenicity and biochemical properties.

The first group comprises a family of leukocyte interferons (? -IFN, LelF or IFN -?), Which are normally produced mainly by constitutive human blood cells upon viral induction. They are also produced by a microbiological pathway and are biologically active (1,2,3). Their biological properties suggest their use in the clinic as therapeutic agents for the treatment of viral infections and malignancies (4).

In the second group is human fibroblast interferon (βinterferon, FIF or IFN-β), normally produced by fibroblasts in viral induction, also produced by a microbiological pathway and found to exhibit a wide range of biological activities (5). studies and their potential therapeutic relevance have been identified. Leukocyte and fibroblast interferons show very clear similarities in their biological properties, irrespective of the fact that the degree of homology at the amino acid level is relatively low. In addition, the two interferon groups contain from 165 to 166 amino acids and are acidic proteins.

1. Human immune interferon (γ - interferon, ΙΙΝ or IFN-γ) to which this interferon is assigned, in contrast to α and β-interferons, is pH 2 labile, produced mainly by mitogenic induction of lymphocytes and is clearly antigenic distinguishable. Insofar as human immune interferon can now be detected in very small amounts, its characterization is very difficult. However, the very extensive still partial purification of human immune interferon (6) has recently been reported. The compound is produced from lymphocyte cultures stimulated by the phinochemistry of phytohaemagglutinin and phorbol ether purified by sequential chromatographic separation. This procedure results in a product with a molecular weight of 58,000.

2. Human immune interferon is produced in very small amounts by translating mRNA into oocytes, exhibiting a characteristic of interferon activity similar to that of human immunointerferon, and thus expressing the hope that immunointerferon cDNA can be synthesized (and) cloned (7) .

3.The amount of immunointerferon obtained so far is indeed insufficient to conduct unambiguous experiments to characterize and establish the biological properties of the purified components. Nevertheless, in vitro experiments with the crude product as well as in vivo experiments with murine γ - interferon preparations suggest that the primary function of the immunointerferon may be as of one immunoregulatory agent ¢ 8, 9}. only antiviral and anticellular activity, which is common to all human interferons, but also shows a potentiating effect on these activities with α- and β-interferon (10). In addition, the in vitro antiproliferative effect of γ - interferon on tumor cells has been reported to be 10 to 100-fold greater than that of other interferon classes (8, 11, 12) .This result, together, is its immunoregulatory role (8, 9) suggests much more pronounced antitumor potential for IFN γ than for IFN-α IFN-β. Indeed, in vivo experiments in mice and murine IFN - γ show a clear superiority over antiviral induced interferons in their antitumor effect against osteogenic sarcoma Q3).

All these studies, up to the present invention, were to be carried out with too harsh preparations. Nevertheless, they did suggest very important biological functions for immunointerferon.

The latter has not only potentially associated antiviral activity but is also likely to have strong immunoregulatory and antitumor activity, which clearly point to potentially promising clinical agents.

It is clear that claiming recombinant DNA technology would be the most effective way to obtain sufficient amounts of human immune interferon. Whether the materials thus produced would include glycosylations that are considered to be characteristic of the natural human-derived material, they are likely to exhibit biological activity that suggests their clinical application in the treatment of a wide range of viral, neoplastic and immunosuppressive conditions or diseases.

B. Recombinant DNA technology

Recombinant DNA technology has reached some maturity as it ages. Molecular biologists are able to recombine different DNA sequences with ease, creating new DNA structures capable of producing copies of the amount of exogenous protein product in transformed microorganisms. Basic tools and methods are needed for in vitro binding of different DNA fragments to blind ends or adhesive ends, thus producing potential expression products applicable to

I

I transform specific organisms, thereby directing them to the synthesis of the desired exogenous product. Nevertheless, based on the individual product, the path of exchange remains volatile and science is not always able to predict the successful outcome. Indeed, one who predicts successful results without being based on experimental data does so with relative risk.

Plasmid, a non-chromosomal strand of double stranded DNA found in bacteria and other microorganisms, often in multiple copies per cell, remains a core element of recombinant DNA technology. The information encoded in the plasmid DNA is that which is necessary for the reproduction of the plasmid in daughter cells (ie, a source of replication) and usually one or more phenotypes: breeding characteristics such as, in the case of a bacterium, antibiotic resistance that allows clones to host cells containing the plasmid of interest to you are cultured preference for a selective medium. The applicability of the plasmids is based on the fact that they can be specifically degraded by one or the other. tion endonucleases or restriction enzymes, each of which recognizes different sites of the plasmid DNA. The heterologous genes or gene fragments can then be inserted into the plasmid by joining the ends at the degradation sites or at the reconstructed ends at the same sites. Thus, the so-called replication expression agents are formed. DNA recombination takes place outside the cell, but the resulting recombinant replication-capable expression agents or plasmids can be introduced into the cells by a method known as transformation, and thus a large number of expression agents can be obtained by culturing the transformants. Moreover, when a gene is inserted into portions of a plasmid that regulates the transcription and translation of encoded DNA (shDNA), the resulting expression agent can be used to produce a polypeptide sequence that encodes the inserted gene for a method directed to this expression.

Expression is initiated in an area known as a promoter that is recognized by and binds to RNA polymerase. In the transcriptional phase of expression, DNA is desirculated, thereby serving as a template for the initiation of synthesis of information RNA from the DNA sequence. The information RNA is, in turn, translated into a polypeptide having an amino acid sequence encoded by the mRNA. Each amino acid is encoded by a nucleotide triplet or codon that generally yields the structural gene, i. that portion that encodes the amino acid sequence of the expressed polypeptide product. The translation is initiated at a single start signal (usually ATG, which in the resulting RNA becomes AUG). The so-called stop codons determine the termination of the translation end, therefore, the production of other amino acid units. The resulting product can be obtained by lysis, if necessary, of a host cell, in microbial systems, and recovery of the product by appropriate purification from other proteins.

In practice, the use of recombinant DNA technology can express fully heterologous polypeptides - the so-called direct expression - or vice versa can express heterologous polypeptides fused to a portion of the amino acid sequence of a homologous polypeptide. In the literal case, the expected bioactive product is sometimes of varying activity within the fused homologous / heterologous polypeptide as it degrades in the extracellular environment. See British Pat.Publ. No. 2007676A and Wetzel, American Scientist 68, 664 (1980).

C. Cell culture technology

The field of cell or tissue cultures for the study of genetics and cell physiology is well developed. The means and methods of this technique serve to maintain permanent cell lines obtained by successful serial transfers from isolated normal cells. For the purposes of research, such cell lines are maintained on solid surfaces in liquid media or by culturing in suspensions containing solid nutrients. Increasing production seems to be a mechanical problem only. For further overview of the field, attention should be focused on Microbiology, 2nd Eddition, Harper and Row, Publishers, Inc, Md. (1973), esp. pp. 1122 et seq. and Scientifc American 245, 66 et seq. (1981), each of which is included in the accompanying references.

TOTAL INVENTION

The present invention is based on the finding that recombinant DNA technology can be used to successfully obtain human immune interferon, preferably in direct form, and in amounts _? sufficient to initiate and conduct animal and human testing, which is a prerequisite for market approval. The product is suitable for use in all its forms, for the prevention and treatment of humans in viral infections and in malignant and immunosuppressive or immunodeficient conditions. Its forms include various possible oligomeric forms, which may include associated glycosylations. ProductTT obtained from genetically engineered microorganisms or cell culture systems. As used herein, the term cellular transformers refers to a cell into which DNA is introduced, this DNA being obtained by exogenous DNA recombination, and as a precursor to any such cell that stores the DNA so introduced. In this new, more effective than known manner, human immune interferon is produced and isolated. One significant factor in the present invention, in its preferred embodiment, is the production of human immune interferon by the production of genetically targeted microorganisms or cell cultures in isolable amounts, thereby secreted by the host cell in mature form.

The present invention encompasses human immune interferon thus obtained and the means and methods for its preparation. The present invention is further directed to replication DNA expression agents that harbor gene sequences that encode human immune interferon in expression form. Further, the present invention is directed to strains of microorganisms or cell cultures of such transformed strains or cultures capable of producing human immune interferon. In other aspects, the present invention is directed to various methods that are applicable to the preparation of said gene sequences for human immune interferon, DNA expression agents, strains of microorganisms and cell cultures, and to specific embodiments thereof. Still further, this is her

the invention is directed to the preparation of fermentation cultures of said microorganisms and cell cultures. In addition, the present invention is directed to the preparation of human immune interferon as a direct expression product secreted by the host cell in mature form. This approach may utilize the gene encoding the sequence of mature human immune interferon plus the 5 'flanking DNA encoding the signaling polypeptide. The signaling polypeptide is thought to assist the transport of the molecule to the cell wall of the host organisms, where it degrades during the secretion process of the mature human interferon product. This embodiment allows isolation and purification of the expected mature immune interferon without resorting to procedures designed to eliminate contaminants from intracellular host proteins or cellular debris.

Referring here to the expression of mature human immune interferon means its production by the microbial or cellular culture pathway, without being accompanied by a signal peptide or a sequencing peptide that results in immediate translation of human immunointerferon mRNA. The first recombinant human immune interferon of the present invention is that having methionine as the first amino acid (represented by an ATG start signal codon insertion against the structural gene) or, where methionium? is intra- or extracellularly degraded and normally as the first amino acid has cysteine. Murine human immunointerferon can also be prepared, in conjunction with conjugated protein other than the conventional signal polypeptide, the conjugate in turn is specifically degraded in intra- or extracellular media. See British

Pat. publication No. 2007676A. Finally, mature human immunointerferon can be produced by direct expression without the need for degradation of any side, excess peptide.

The mature human immune interferon thus obtained is recovered and purified to the extent necessary for use in the treatment of viral, malignant as well as immunosuppressive or immunodeficient conditions.

Human immune interferon is prepared as follows:

1. Human tissues, such as human spleen or peripheral blood lymphocytes, are cultured with mitogens to stimulate the production of immune interferon.

2. Cell centrifuges from such cell cultures are extracted in the presence of ribonuclease inhibitors to isolate all cytoplasmic RNA.

3. Oligo-dT column isolates total amount of RNA in polyadenylation form. This mRNA is size fractionated using a sucrose density gradient and uric acid gel electrophoresis.

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

5. The vectors thus obtained are used to transform bacterial cells forming a colonial library. Radiolabeled cDNA generated from induced and non-induced mRNA, obtained as described above, is used to separately probe duplicate colonial libraries. The excess cDNA is then taken up

I removed and the colonies were exposed to a UV film to identify the induced cDNA clones.

6. Corresponding cDNAs are isolated from the induced cDNA clones and matched.

7. In the first embodiment, the matched DNA is attached for in vitro insertion into a suitable expression agent, which is used to transform an E. coli host cell, which in turn can be cultured and can express the desired human immunointerferon product.

8. The human immune interferon thus expressed undoubtedly possesses 146 amino acids in their mature form, beginning with cysteine, and is highly basic in character. Its monomeric molecular weight is estimated to be 17,140. Probably due to the presence of many basic residues, hydrophobicity, salt bridge formation, etc., the molecule may be associated in an oligomeric form such as e.g. dimeric, three-dimensional or tetrameric form. The high molecular weights observed previously for natural products (6), which cannot be measured solely on the basis of the amino acid sequence, may be due to such oligomeric forms as well as to the assistance of carbohydrate by post-translational glycosylation.

9. In host systems, especially when coupled to the expression system so that they are expressed together with their signal peptide, the mature form of human immune interferon is produced in the cell culture medium without dependence on the methods of recovery and purification.

PREFERRED EMBODIMENTS

A. Microorganisms / Cell cultures

1. Bacterial Strains / Promoters

The development described herein was accomplished by, inter alia, the micro-organism E. coli strain 294 (end A, thi -, _k hsm +), as described in British Pat. Publication No. 2055382 A. This strain is deposited in the American Type Culture Collection, ATCC Accession No. 31446. Notwithstanding this fact, many other bacterial strains, including known E. coli strains such as E. coli B, E. coli X 1776, are applicable ( ATCC No. 31537) and E. coli W 3110 (F-, λ-, protrophic) (ATCC Ν 27325), or other microbial strains, many of which are deposited and (most likely) available from deposit institutes, such as ATCC - under the catalog list. See also German Offenlegungschrift No 2644432. These other microorganisms include, for example, Bacillus such as e.g. Bacillus subtilis and other enterobacteriaceae, among which may be, for example, Salmonella typhimurium and Serratia marcesans, using plasmids that can replicate and express the heterologous gene sequences of the invention.

By way of example, beta lactamase and lactose promoter systems have been used predominantly to initiate and maintain the microbial production of heterologous polypeptides. Details relating to the creation and construction of these promoter systems have been published by Chang et al., Nature 275, 617 (1978) and Itakura et al., Science 198, 1056 (1977), which are incorporated by reference. Recently, a system based on tryptophan, designated as the trp promoter system, has been developed.

Details of its creation and construction have been published by Goeddel et al., Nucleic Acids Research 8, 4057 (1980) and Kleid et al., U.S. Pat. Sir. Nos. 133, 296, filed March 24, 1980, which are incorporated by reference. Many other bacterial promoters have been discovered and details regarding their nucleotide sequence allowing those of skill in the art to include them functionally in plasmid vectors have been published - see e.g. Siebenlist et al., Cell 20, 269 (1980), which are included in the literature.

2. Yeast strains / Yeast promoters

The expression system of the invention may also use the plasmid YRp7 (14, 15, 16), which can be selected and replicated in both E. coli and yeast, Saccharomyces cerevisiae. For selection in yeast, the plasmid contains the TRP1 gene (14, 15, 16), which supplements (permits development in the absence of tryptophan) yeast containing mutations in this gene found in the IV yeast chromosome (17). The strain used here is RH218 deposited in ATCC without restriction No. 44076. However, it is apparent that any strain having a mutation that makes the trp 1 cell may be an effective expression medium for a plasmid containing an expression system. An example of another strain that may be used is rep4-1 (19). This tryptophan auxotrophic strain also has a point mutation in the TRP1 gene.

Placed at the 5'-end of a non-yeast gene, the 5'-flanking sequence (promoter) of a yeast gene (for alcohol dehydrogenase 1) can develop expression for a foreign gene in yeast when placed in a plasmid used to transform yeast. In addition to the promoter, expression of the non-yeast gene in yeast requires a second yeast sequence inserted at the 3'-end of the non-yeast gene from the prusside to allow actual transnational / ribic termination and polyadenylation in yeast. This promoter can be used appropriately in the present invention and in others, see below. In preferred embodiments, the 5 ' flanking sequence of the yeast 3-phosphoglycerate kinase gene (2 (5) is inserted upstream of the structural gene, followed by DNA containing terminator-polyadenylation signals, e.g., TRPI Q4, 15, 10 gene from PGK (2 (5) gene).

Due to the fact that the 5 'flanking sequence (linked to 3'-yeast termination DNA) (below) may function to develop expression of foreign genes in yeast, as if the 5'-flanking sequence of any strong expressed yeast gene can be used for the expression of important gene products. While in some circumstances the yeast expresses up to 65 percent of its soluble protein as glycolytic enzymes (21), and while this high level appears to be due to the production of high levels of individual mRNAs (72), it would be possible to use 5 ' the flanking sequence of any glycolytic genes for similar expression purposes - such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase. Each of the 3 'flanking sequences of these genes can also be used for termination and mRNA polyadenylation in a similar expression system - see. above. Some other highly expressed genes are those for acidic phosphatases (20 and those that express high levels of production due to mutations in the 5'-flanking regions (mutants that increase expression) - usually due to the presence of a ΊΎ1 transportable element (24).

All of the above-mentioned genes are still transcribed by yeast RNA polymerase II (24). It is possible that RNA polymerase I and III promoters that transcribe ribosomal RNA, 5S RNA and tRNA genes (24,25) may also be useful in similar expression structures.

Finally, many yeast promoters also contain transcriptional control so that they can be switched on or off by variations in cultivation conditions. Some examples of such yeast promoters are the genes that produce the following proteins: Alcohol dehydrogenase II, isocytochrome c, acid phosphatase, degrading enzymes related to nitrogen metabolism, glyceraldehyde-3-phosphate dehydrogenase, and enzyme galactosinase, and enzymatic actinase, and enzyme activity. ). Such control areas of a protein product can be very useful in controlling the expression of protein products - in particular when their production is toxic to yeast. It would also be possible to insert a control region of a 5'-flanking sequence with a 5'-flanking sequence containing a promoter of a highly expressive gene. This would lead to a hybrid promoter and would be possible as long as the control region and the promoter have apparently physically distinct DNA sequences.

3. Cell culture systems / Cell culture vectors

The propagation of vertebrate cells into tissue (tissue cultures) has become a routine procedure in recent years (see Tissue

Culture, Academic Press, Kruse and Patterson eds, 1973). It is used here

COS-7 monkey kidney fibroblast line like

I host for the production of immune interferon (25a). However, the experiments described in detail herein may be carried out in any cell line capable of replication and expression of a competing vector, e.g. W138, BHK, 3T3, CHO, VERO, and HeLa cell lines. In addition, what is required for the expression vector is a source of replication and a promoter located in front of the expression gene, along the required ribosomal binding sites, RNA split sites, polyadenylation sites, and transcriptional terminator sequences. Insofar as these essential elements of the SV40 are used herein, it is clear that the invention, although described herein in a preferred embodiment, is not limited to these sequences. For example, they can be used as a source of replication and other viral (Polioma, Adeno, VSV, BPV, etc.) vectors, as well as cellular sources of DNA replication that function in a non-integrated state.

B. Vector systems

1. Direct expression of mature immune interferon in E. coli The procedure used to obtain direct expression of IFN - γ 1 E. coli as a mature interferon polypeptide (minus the signal sequence ^ is a variant of that used previously for human growth hormone (26) and human leukocyte interferon (T) as far as the combination of synthetic (N-terminal) and cDNA is involved.

As might be expected from the nucleotide sequence of p69 described below and from comparison with known degradation sites between the signal peptide and the mature polypeptide

For several IFNs (7), IFN-γ possesses a hydrophobic signal peptide of 20 amino acids, followed by 146 amino acids for mature IFN-γ (Fig. 5). As shown in FIG. 7, the BstNI restriction endonuclease site is conveniently located at the 4th amino acid of the mature CMM. Two synthetic oligonucleotides have been constructed that incorporate the ATG translator initiation codon, amino acid codons 1, 2 and 3 (cysteine-cysteine-cysteine-cysteine-cysteine) EcoRI cohesive end. These deoxyoligonucleotides are linked to 100 pairs of base PstNI - PstI fragment of p69 to construct a synthetic-natural hybrid gene of 1,115 base pairs that encodes IFN-γ and which is linked via EcoRI and PstI restriction sites. This gene is inserted into plasmid pLelF A trp 103 between EcoRI and PstI sites to produce the expression plasmid pIFN-γ trp 48. In this plasmid, IFN-γ is expressed under the control of the E. coli trp promoter. (pLelF A trp 103 was derived from pLelF A 25 _g in which the EcoRI site distal to the LelF A gene was removed. The procedure used to remove this EcoRI site was described previously (27).

2. Expression in yeast

For expression of a heterologous gene such as cDNA for immune interferon in yeast, it is necessary to construct a plasmid vector containing four components. The first component is the part that allows the transformation of both E. coli and yeast and therefore should contain a selective gene from each organism. (In this case, it is the ampicillin resistance gene of E. coli and the yeast TRP 1 gene.) This component requires that the source of replication from both organisms be maintained as plasmid DNA in both organisms. (In this case, this is the E. coli source from pBR322 and the ars I source from chromosome III from yeast.)

The second component of the plasmid is the 5 'flanking sequence of a highly expressed yeast gene to develop 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 ATG from the PGK structural sequence as 8 bp upwards from that ATG. The sequence is shifted from a sequence containing two Xbal and EcoRI restriction sites to conveniently attach this 5'flange sequence to the structural gene.

The third component of the system is a structural gene designed to contain both ATG translation start and stop signals. The isolation and construction of such a gene is described below.

The fourth component is a yeast DNA sequence comprising a 3'-flanking sequence of a yeast gene that contains signals for transcriptional termination and polyadenylation.

Yeast immune interferon was obtained with all the components thus presented.

3. Expression in mammalian cell culture

The synthesis of immune interferon in mammalian cell cultures is based on the creation of a vector capable of both autonomous replication and expression of a foreign gene under the control of a heterologous transcription unit. Replication of this vector in tissue culture is accompanied by the provision of a DNA replication source (derived from the SV40 virus) and the provision of ancillary function (T antigen) by introducing the vector into a cell line endogenously expressing this antigen (28, 20. The late promoter of The SV40 virus precedes the structural gene of interferon and provides for gene transcription.

The vector used to obtain IFN-γ expression consists of pBR322 sequences that provide a selective marker for E. coli (ampicillin resistant) as well as an E. coli replication source. These sequences are derived from plasmid pML-1 (28) and span the region extending between EcoRI and BamH1 restriction sites. The SV40 source was derived from the PvuII-HindIII fragment with 342 base pairs spanning this region (30, 31) (both ends converted to EcoRI ends). These sequences, in addition to encompassing the viral source of DNA replication, encode a promoter for both the early and the last transcriptional unit. The orientation of the SV40 source region is such that the promoter of the last transcription unit is positioned proximal to the interferon encoding gene.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1-sucrose gradient of peripheral blood lymphocyte-induced centrifugation (PBL) Poly (A) + RNA. Two peaks of interferon activity (as shown in the shaded boxes) of 12S and 16S sizes are observed. The positions of the ribosomal DNA markers (centrifuged independently) are marked above the absorption profile.

FIG. 2 shows PBL induced poly (A) + RNA electrophoresis on uric acid agarose. Only one activity peak migrating with 18S RNA was observed. The positions of ribosomal markers, which are subjected to electrophoresis in the neighborhood and visualized by ethidium bromide staining, are noted above the activity profile.

FIG. 3 shows hybridization patterns of 96 colonies with induced and uninduced ³² P-labeled cDNA probes. 96 individual transformants were grown in a microtiter plate, re-plated on two nitrocellulose membranes, and then the filters were hybridized with P ³² -sDNK probes prepared from either induced mRNA (above) or mRNA isolated from uninduced PBL cultures (uninduced , below), the filters are washed to remove non-hybridized RNA and then exposed to a UV film. This filter network is representative of 86 such networks (8300 independent colonies). An exemplary induced clone is designated H12.

FIG. 4 is a restriction endonuclease map for clone 69 cDNA insertion. cDNA inserts linked via PstI sites (dots at both ends) and oligo dC-dG tail (single line). The number and size of the fragments produced by restriction endonuclease digestion were evaluated by electrophoresis on 6 percent acrylamide gels. The positions of the sites were confirmed by nucleic acid matching (presented in Fig. 5). The coding region of the largest open reading frame is framed and the fold / cleavage portion represents the putative signal peptide sequence consisting of 20 amino acids, while the dotted portion represents the mature IFN sequence (146 amino acids). The 5'-end of the mRNA is to the left, while the 3'-end is to the right.

FIG. 5 illustrates the nucleotide sequence of the plasmid p69 cDNA insert. The putative amino acid sequence for the longest open reading frame is also provided. The probable signal sequence is represented by the residue designated S1 to S20.

FIG. 6 is a comparison of the IFN-γ mRNA structure with that of leukocyte (IFN-α) and fibroblast (IFN-β) interferons. The 69 mRNA clone contains a significantly larger amount of untranslated sequences.

FIG. 7 is a chemical diagram of the structure of the IFN-γ expression plasmid pIFN-γ trp 48. The starting material is 1250 base pairs PstI cDNA insert from plasmid p69.

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

FIG. 9 presents Southern hybridization of eight different EcoRI-degraded human genomic DNAs hybridized with ^{32 32-} labeled 600 base pairs Ddel fragment from p69 insert cDNA. Two EcoRI fragments were clearly hybridized to the probe in each DNA sample.

FIG. 10 is a Southern hybridization of human genomic DNA digested with different restriction endonucleases hybridized with the P ³² -labeled probe from p69.

FIG. 11 schematically illustrates the restriction map of a 3.1 kbp Hindlli insert from the vector ρΒΙ, οτ which is the PGK promoter isolated. The insertion of one EcoRI site and one Xbal site into the 5'-flanking DNA of the PGK gene is indicated.

FIG. 12 illustrates the 5'-flanking sequence for the PGK gene prior to insertion of Xa1 and EcoRI sites.

FIG. 13 schematically illustrates the methods used to insert the Xbal site at position 8 in the PGK promoter and to isolate a 39 bp fragment from the 5'-flanking sequence of the PGK containing that Xbal end and the Sau3A end.

FIG. 14 schematically illustrates the structure of a 300 bp fragment containing the above 39 bp fragment, an additional PGK 5'-flanking sequence (265 bp) from Pvul to Sau3A (see Fig. 11) and an EcoRI site adjacent to Xbal.

FIG. 15 schematically illustrates the structure of a 1500 bpPGK promoter fragment (HindIII / EcoRI) _? which comprises, in addition to the fragment constructed in FIG. 14, 1300 bp HindIII to Pvul fragment of the PGK 5'-flanking sequence (see Fig. 11).

FIG. 16 illustrates the structure of an expression vector for human immune interferon in yeast containing a modified PGK promoter, an IFN-γ promoter, an IFN-γ cDNA, and a terminator region of the yeast PGK gene as described in detail herein.

DETAILED DESCRIPTION

A. Source of IFN-γ mRNA

Human peripheral blood (PBL) lymphocytes obtained by leucophoresis are purified by Ficoll-Hypaque gradient centrifugation and then cultured at a concentration of 5 x 10 ⁶ cells / ml in RPMI 1640, 1 percent L-glutamine, 25 mM HEPES, and 1 percent penicillin-streptomycin solution (Gibco, Grand Island, NY). Te3H cells were induced to produce IFN-γ by mitogenic staphylococcal enterotoxin B (1 pg / ml) and cultured for 24 hours to 48 hours at 37 ° With a 5 percent CO,. Desacetylthymosin-a-1 (0.1 pg / ml) was added to PBL cultures to increase the relative yield of IFN-γ activity.

C. Isolation of RNA

The total amount of RNA from PBL cultures was extracted mainly by the method reported by Berger, SL et al. (33). Cells were precipitated by centrifugation and then resuspended in 10 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl ₂ and 10 mM ribonucleoside vanadyl complex. Cells were lysed by addition of NP-40 (1 percent final concentration) and nuclei were precipitated by centrifugation. The supernatant contains total RNA, which is further purified by multiple phenolic and chloroform extractions. The aqueous phase was adjusted to 0.2 M in NaCl and then the total amount of RNA was precipitated by the addition of two volumes of ethanol. The RNA of the induced (unstimulated) cultures was isolated by the same methods. Oligo-dT cellulose chromatography has been used to purify mRNA from total RNA production (34). It is usually obtained from 1-2 liters of cultured PBL 5-10 milligrams of total RNA and 50-200 micrograms of Poly (A) + RNA.

C. TRNA size fractionation

Two methods were used to fractionate the mRNA production. These methods were used independently (preferably in unison) and each resulted in significant enrichment with IFN-γ mRNA.

Sucrose gradient centrifugation was used to fractionate the mRNA in the presence of denatured pharmamide at 19 C. at 200 C. Successful fractions were removed from the top of the gradient, precipitated with ethanol, and injected into the Xenopus laevis oocytes for translation. (35). After 24 hours at room temperature, the incubation medium was tested for anti-viral activity in a standard cytopathic sample with Vesicular Stomatitis Virus (Indian strain) or Encephalomyocarditis Virus on WISH (human amniotic) cells as described by

Stweart Q> 5), except that the samples were incubated for 24 hours (instead of 4) before being challenged with the virus. Substantially two peaks of activity in sucrose-graded fractionated RNA were observed (Fig. 1). One peak is precipitated size calculated at 12S and containing 100-400 units / ml of antiviral activity (compared to IFN-α standard) per microgram of injected RNA. The other activity peak is precipitated as 16S in size and contains about half of the activity of the slower precipitated peak. Each of these activity peaks is apparently due to IFN-γ until activity is observed when the same fractions are tested on calf cell nnHuji (MDBK), which is not protected by human IFN-γ. Both IFN-α activity and IFN-β activity would be easier to detect with MDBK samples (5).

The fractionation of gpRNA (200 pg) was also accomplished by electrophoresis on uric acid agarose gels. The agarose gel plate (37, 38J is composed of 1.75 percent agarose, 0.025M sodium citrate, pH 3.8 and 6M urea. Electrophoresis is carried out for 7 hours at 25 milliamps and 4 ° C. The gel is then fractionated with The individual sections were then ground at 70 ° C and extracted twice with phenol and once chloroform, the fractions were then precipitated with ethanol, followed by IFN-γ mRNA injection by injection into Xenopus laevis oocytes and assay. Only one activity peak was observed in the gel fractionated samples (Fig. 2) the peak migrates with 18S RNA and has an activity of 600 units / ml per microgram of injected RNA, which is also IFN-γ specific insofar as it does not protect MDBK cells.

The discrepancy in the magnitude of the activity peaks observed in sucrose gradients (12S and 16S) and uric acid gels (18S) can be explained by the observation that these two independent fractionation methods were not carried out under totally denaturing conditions.

D. Preparation of a colony library containing IFN-γ sequences

For the production of double stranded cDNA by standard methods 06, 393, 3 pg of gel-fractionated mRNA is used. The cDNA was size fractionated on a 6 percent polyacrylamide gel. Two fractions were electroeluted, 800-1500 bp (138 ng) and 22 1500 bp (204 ng). 35 ng portions of each cDNA was lengthened with deoxynucleotidyl transferase 06) and renatured with 300 ng of plasmid pBR322 (4f), which was similarly lengthened with deoxyC residues at the PstI site (40). Each renatured mixture was then transformed into E coli K 12 strain 294. Approximately 8000 transformants with 800-1,500 bp cDNA and 400 transformants with greater than 1500 bp cDNA were obtained.

E. Colonial library screening for induced cDNAs

The colonies were inoculated individually into wells of microtiter plates containing LB (58) + 5 pg / ml tetracycline and kept at -20 ° C after addition of DMSO up to 7 percent. Two copies of the colony library were cultured on nitrocellulose filters and DNA from each colony was fixed to the filter using the Grunstein-Hogness method (42).

Probes P ³² cDNA were prepared using 18S gel fractionated mRNA from the induced and non-induced PBL cultures. The primer is <Ρζ ₂ _ ₁₈ and the reaction conditions are as described previously (T). filters containing 800 transformants from 60027

1500 bp cDNA was excised and 400 transformants from the cut> 1500 bp cDNA was hybridized with 20 χ 10 ⁶ cpm of induced ³² P -sDNK. Another network of filters was hybridized with 20x 10 ⁶ cpm of induced ³² P - cDNA. The hybridization was carried out for 16 hours under the conditions described by Fritsch et al. 03}. the filters are extensively washed with 00 and then exposed to a UV-violet Kodak XR-5 film is a DuPont Lightning-Plus reinforcement screen for 16-48 hours. Each colony-hybridization sample is compared with the two probes. Approximately 40 percent of the colonies hybridize clearly with the two probes, while approximately 50 percent of the colonies fail to hybridize (presented in Fig. 3). The 124 colonies hybridize significantly with the induced probe, but without detecting this or weakly with the non-induced probe. These colonies were inoculated individually into the wells of microtiter plates, grown and transferred to nitrocellulose filters, hybridizing with the same two probes as described above. Plasmid DNA isolated from each of these colonies by rapid method 04) also binds to nitrocellulose filters and hybridizes 051 with induced and non-induced probes. Does 22 colonies DNA hybridize with the induced probe only _? which is why they are called induced colonies.

F. Characterization of induced colonies

Plasmid DNA 00 was prepared from five of the induced colonies, which was used to characterize the cDNA inserts. Endonuclease mapping of five induced plasmids (p67, p68, p69, p71, and p72) suggests that four have similar restriction maps. Each of these four (p67, p69p p71, and p72) has four Ddel sites, 2HinfI sites, and one Rsal site in insert cDNA. The fifth plasmid (p68) contains a common Ddel fragment and is a short cDNA clone bound to the other four. The homology postulated after restriction endonuclease mapping is confirmed by hybridization. P ³² -labelled DNA probe is prepared from 600 bp dDel fragment from plasmid p67 and used for hybridization 02) to the other induced colonies. All five of the restriction endonuclease mapped colonies cross-hybridized with this probe, as did the 17 other colonies of the 124 selected on induced / non-induced screening. The length of the insert in each of these cross-hybridized plasmids was determined by PstI digestion and gel electrophoresis. The clone that has the longest cDNA insert is clone 69 with an insertion length of 1200-1400 bp. This DNA was used for further experiments, and its restriction map is shown in FIG. 4.

the insert cDNA in p69 represents the IFN-γ cDNA shown by its expression products, obtained in three independent expression systems, thereby achieving antiviral activity as described in detail below.

G. Sequential analysis of insert cDNA in p69

The complete nucleotide sequence of the plasmid p69 cDNA insert was determined by the method for termination of the dideoxynucleotide chains 0-0 after subcloning of fragments into M13 sr7 00 by the Mahat-Gilbert chemical procedure (52). The longest open reading frame encodes a 166 amino acid protein shown in FIG. 5. The first encoded residue is the first meth codon counted at the 5'-end of the cDNA. The first 20 residues at the amino terminus probably serve as a signal sequence for secretion of the remaining 146 amino acids. This putative signal sequence has the most common properties of other signal sequences, such as size and hydrophobicity. Moreover, the four amino acids found at the putative sequence (ser-leu-cys) are identical to the four residues found at the point of degradation for several leukocyte interferons (LelF B, C, D, F, and H (2)). The encoded mature amino acid sequence of 146 amino acids (referred to herein as recombinant human immune interferon) has a molecular weight of 17 140.

There are two potential glycosylation positions (50) in the encoded protein sequence, for 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 (6,5G). In addition, only two cysteine residues (positions 1 and 3) are stereo too close to form a disulfide bridge, which is in agreement with the observed stability of IFN-γ in the presence of reducing agents such as β-mercaptoethanol (5Ϊ). The putative mature amino acid sequence is usually too basic, with a total of 30 lysine, arginine and histidine residues and only a total of 19 aspartic and glutamic acid residues.

The mRNA structure of IFN-γ as suggested by the DNA sequence of plasmid p69 is clearly distinguishable from IFN-β (5) and IFNα (1,2) mRNA. As shown in FIG. 6, the coding region of IFN-γ is shorter, while the 5'-untranslated and 3'-untranslated regions are slightly longer than both IFN-α and IFN-β.

N. Expression of recombinant human immune interferon in

E. coli

In accordance with FIG. 7, 50 pg of plasmid p69 was digested with PstI and an insert of 1250 base pairs was isolated by gel electrophoresis on a 6% percent polyacrylamide gel. Approximately 10 pg of this insert is electrodepositioned from the gel. 5 pg of this PstI fragment was partially digested with 3 units of BstNI (Bethesda Research Labs) for 15 minutes at 37 ° C and the reaction mixture was purified on a 6 percent polyacrylamide gel. Approximately 0.5 pg of desired; 1,100 base pairs of the BstNI-PstI fragment were recovered. The two labeled deoxyoligonucleotides, 5'-dAATTCATGTGTTATTGTC and 5'dTGACAATAACACATG (Fig. 7), were synthesized by the phosphotriester method (53) and phosphorylated as follows: 100 pmoles of each deoxyoligonucleotide 30 HCl combined 60 HCl ), 10 mM MgCl ₂ , 15 mM β-mercaptoethanol and 240 pCi (γ-P ³² ) ATF (Amersham, 5000 Ci / mmole). 12 units of T4 polynucleotide kinase were added and the reaction was allowed to stand at 37 ° C for 30 minutes. 1 pl 10 mM ATf was added and the reaction was left for an additional 20 minutes. After ¢-OH- / CHCl / extraction, the oligomers were combined with 0.25 µg BstNI-PstI 1100 base pair fragment and ethanol precipitated. These fragments were ligated at 20 ° C for 2 hours in 30 pl 20 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 10 mM dithiothreitol, 0.5 mM ATf g 10 units of T4 DNA ligase. The mixture was decomposed in 1 hour with 30 units of PstI and 30 units of EcoRI (to eliminate polymerization by bonding the cohesive ends) and subjected to electrophoresis on a 6 percent polyacrylamide gel.

Plasmid pLelF A trp 103 (Fig. 7) is derived from plasmid pLelF A 25 (T), in which the EcoRI site distal to the LelF A gene is removed (27). 3 pg of pLelF A trp 103 was digested with 20 units of EoRI and 20 units of PstI for 90 minutes at 37 ° C and subjected to electrophoresis on a 6 percent polyacrylamide gel. The large (-3900 base pair) vector fragment is recovered by electroelution. The 1115 base pairs of the EcoRI-PstI IFN-γ DNA fragment were included in 0.15 pg of the vector thus prepared. Transformation of E. coli K-12 strain 294 (ATCC N 31446) yielded 120 tetracycline resistant colonies. Plasmid DNA was obtained from 60 of these transformants and digested with EcoRI and PstI. Three of these plasmids contain the desired 1,115 base pairs of the EcoRI-PstI fragment. DNA sequence analysis confirms that these plasmids have the desired nucleotide sequence at the point of attachment between the trp promoter, synthetic DNA and cDNA. One of these plasmid pIFN-γ trp 48 was selected for further study. This plasmid was used to transform E. coli K-12 strain W3110 (ATCC No. 27325).

I. Gene structure of the IFN- γ coding sequence

The structure of the gene encoding IFN-γ by Southern hybridization. In this procedure (50, 5 micrograms of high molecular weight lymphocyte DNA (obtained as in 55) is subjected to complete degradation with various restriction endonuclease followed by electrophoresis on 10% agarose gels (56) and transferred to a nitrocellulose filter (54 ). P ³² -labelled DNA probe is prepared from 600 bp Ddel fragment of the cDNA insert of p69 and hybridized 00 with a nitrocellulose-DNA filter. Quantity amounting to 10 ⁷ per minute of the probe were hybridized for 16 hours and then washed as described in 00. Eight genomic DNA probes from pa personal human donors were digested with EcoRI are restriction endonuclease and hybridized with the p69 ³² P-labeled probe. As shown in Fig. 9, two clear hybridization signals are observed with sizes of 8.8 kilobase pairs (kbp) and 2 kbp as determined by degraded with HindIII λDHK This could be the result of two IFN-γ genes or the degradation of a separate gene from one EcoRI site.

I

Since p69 does not contain an EcoR1 site, it would be necessary to include one sequence (intron) with an internal EcoR1 site to amplify a single gene. To differentiate between these two possibilities, another Southern hybridization was performed with the same sample against five other single human DNA endonucleases (Fig. 10). Two hybridizing fragments with two other endonuclease cleavages, PvuII (6.7 kbp and 4.0 bp) and HincII (2.5 kbp and 2.2 kbp), were observed. However, three samples of endonuclease digestion provide only one hybridization fragment: HindIII (9.0 kbp), Bglll (11.5 kbp) and BamHI (9.5 kbp). Two IFN-γ genes would need to be linked at an unusually close distance (less than 9.0 kbp) to be contained in the same hybridization fragment. This result suggests that only one homologous IFN-γ gene (other than many related IFN-cc genes) is present in human genomic DNA and is cleaved by one or more introns containing EcoR1, PvuII, and HincII sites. This assumption is confirmed by the hybridization of the Z ^'32 labeled 07) fragment derived from a properly 3-untranslated region of p69 cDNA (130 bp Ddel fragment of 850 bp in Fig. 5) against an EcoR1 digested fragment of human genomic DNA. Only the 2.0 kbp EcoR1 fragment hybridizes with this probe, suggesting that this fragment contains 3'-untranslated sequences, whereas the 8l8 kbp EcoRl fragment contains 5 'sequences. The gene structure of IFN-γ (one gene with at least one intron) is clearly different from IFN-α (multiple genes (2) without introns (56) or IFN-β (one gene without introns C57)) ·

J. Preparation of bacterial extracts

The overnight culture of E. coli W3110 / pIFN-γ trp 48 in Luria broth + 5 micrograms per ml 3 of tetracycline was used to inoculate M9 (58) medium containing 0.2 percent glucose, 0.5 percent casamic acid and 5 micrograms per ml tetracycine at a dilution of 1: 100. Indole acrylic acid is added to a final concentration of 20 micrograms per ml when D ₅₀ is between 0.1 and 0.2. Ten ml of samples were centrifuged at D ₅₀ = 1.0 and collected and resuspended immediately in 1 ml of phosphate buffered solution containing 1 mg per ml of bovine serum albumin (PBS-BSA). The cells were opened by sonication (sonication) and cleared of debris by centrifugation. The supernatants were stored at 4 ° C until tested. Interferon activity in supernatants was determined to be 250 units / ml by comparing IFN-α cytopathic inhibition (CPE) standards.

K. Transformation of yeasts / strains into a nutrient medium

Yeast strains are transformed as described previously (59). E. coli strain JA300 (thr leuB6 thi thyA trpC1117 hsdm-hsdR- ° str ^R ) (20) was used to select a plasmid containing a functional TRPI gene. The yeast strain RH218, which has the genotype (a trpl gal2 SUC2 mal CUPI) (18), is used as a yeast host for transformation. RH218 is deposited in American Type Culture Collection, ATCC N0 44076. Mediums M9 (minimum culture medium) with 0.25 percent casamic acids (CAA) and LB (culture medium) are as described by Miller (58) with addition of 20 pg / ml ampicillin (Sigma) after autoclaving and cooling. Yeast is cultured on the following culture medium: YEPD containing 1 percent yeast extract, 2 percent peptone and 2 percent glucose ± 3 percent Difco agar. YNB + CAA containing 6.7 grams of yeast nitrogen base (no amino acids)

I (YNB) (Difco), 10 mg adenine, 10 mg uracil, 5 grams CAA, 20 grams glucose and ± 30 grams agar per liter.

L. Construction of a yeast expression vector

1. 10 pg YRp (14, 15, 16) was digested with EcoRI. The resulting adhesive DNA ends are made blind using DNA polymerase I (Klenow fragment). The vector and the insert were passed through a 1 percent agarose (SeaKem) gel, cut off from the gel, electroeluted, and extracted twice with equal volumes of chloroform and phenol prior to precipitation with ethanol. The resulting blank end-stranded DNA molecules were then bound together in a final volume of 50 μΐ for 12 hours at 12 ° C. This mixture was then used to transform E. coli JA300 ampicillin-resistant and trygophan prototrophic strain. Plasmids containing the TRPI gene in both orientations were isolated. pFRW1 has the TRPI gene in the same orientation as YRp7, while pFRW2 has the TRPI gene in the opposite orientation.

pg pFRW2 was linearized with HindIII and electrophoresed on a 1% agarose gel. The linear molecules were eluted from the gel and 200 ng were then coupled to 500 mg of the 3lb HindIII insert of plasmid pB1 (13), which is a restriction fragment containing the yeast 3-phosphoglycerate kinase gene. The mixture was then used to transform E. coli strain 294 into ampicillin-resistant and tetracycline-sensitive ones. The plasmid derived from one such recombinantTu harbors an intact TRP1 gene with a 3.1 kbp HindIII fragment of pB1 insertion DNA in HindIII with the tetracycline resistant gene. This plasmid is FRM31. 5 pg of pFRM31 was completely digested with EcoRI, extracted twice with phenol and chloroform and precipitated with ethanol. The cohesive ends of the molecule are filled using DNA polymerase I (Klenow fragment) in a reaction that is brought to 250 μ до for each deoxynucleoside triphosphate. The reaction was carried out for 20 minutes at 14 ° C, at which time the DNA was extracted twice with phenol-chloroform and then precipitated with ethanol. The resuspended DNA was then completely digested with Clal and electrophoresed on a 6 percent alkylamide gel. The vector fragment was eluted from the gel, extracted with phenol-chloroform and precipitated with ethanol. The six N-terminal amino acids of the 3phosphoglycerate kinase enzyme purified by humans are as follows:

- 2- 3- 4- 5- 6SER-LEU-SER-HSM-LYS-LEUOne of the translation open reading frames generated by the DNA sequence of the 141 bp Sau3A-to-Sau3A restriction fragment (containing an internal Hine II site, see PGK restriction site) map of Fig. 11) produces the following amino acid sequence:

1-2-3-4-5-6

MET-SER-LEU-SER-SER-LYS-LEUSAfter removal of the initiating methionine, it is seen that the PGK N-terminal amino acid sequence has 5 of 6 amino acid homology with the N-terminal amino acid sequence of human PGK.

This sequential result suggests that the start of the yeast PGK structural gene is encoded by DNA in the 141 bp Sau3A restriction fragment of pB1. Previous developments (2 (J) suggest that DNA sequences that specify PGK, mRNA may be located in the HindIII fragment. Further alignment of the 141 bp Sau3A fragment yields more DNA sequences from the PGK promoter.

A synthetic oligonucleotide is a sequence

5 'ATTTGTTGTAAA 3' was synthesized by standard methods (Ccsa et al., Nucleic acids Res. 8, 2331 (1980)). 100 ng of this primer was scored at the 5 'end using 10 units of T4 olinucleotide kinase in a 20 μΐ reaction mixture also containing 200 pCi (/ ^2- P) ATf. This labeled primer solution is used for a primer recovery reaction designed to be the first step in a multi-step process of inserting an EcoRI restriction site into PGK 5'-flanking DNA preceding the PGK structural gene sequence.

100 pg pB1 Q6) was completely digested with NaHCO3, then passed through a 6 percent polyacrylamide gel. The upper band was isolated in ethidium-colored gel (containing the PGK promoter region) by electrophoresis as described above. This 1200 pb Our DNA strand is restriction is HincII, then passed through a 6 percent acrylamide gel. The 650 bp band was isolated by electroelution. 5 pg of DNA was isolated. This 650 bp Hae-to-HincII portion of the DNA was resuspended in 20 µL of H ₂ O and then mixed with 20 µl of the phosphorylated primer solution as described above. This mixture was extracted once with phenol-chloroform and then precipitated with ethanol. The dried DNA was resuspended in 50 pl H ₂ O and heated in boiling water for seven minutes. The solution was then rapidly cooled in an ice-ethanol bath (10-20 seconds) and then transferred to an 9 ice water bath. To this solution were added 50 µl of a solution containing 10 µl of 10 x DNA polymerase I buffer (Boeringer Mannheim), 10 µl of the solution previously brought to 2.5 mM in each deoxynucleoside triphosphate (dATP, dGTP and dCTP), 25 µl H ₂ O and 5 units of DNA polymerase I, Klenow fragment. This 100 µl reaction was incubated at 37 ° C for 4 hours. The solution was then extracted with 1 x phenol-chloroform, precipitated with ethanol, dried by lyophilization, and then completely extracted with 10 units of Sau3A. This solution was then passed through a 6 percent acrylamide gel. The band corresponding to 39 bp in size was cut from the gel, then isolated by electroelution as described above. This 39 bp strip has one blind end and one Sau3A sticky end. This fragment was cloned into a modified pFIF trp 69 vector (5). 10 pg pFIF trp 69 was linearized with Xbal, 1 x extracted with phenol-chloroform. The xbal adhesive end was filled using DNA polymerase I Klenow fragment in a 50 μΐ reaction containing 250 μΜ in each nucleoside triphosphate. This DNA was excised into BamHI, then passed through a 6 percent acrylamide gel. The vector fragment was isolated from the gel by electroelution, then resuspended in 20 μΐ H ₂ O. 20 ng of this vector was coupled with 20 ng of 39 bp fragment obtained as above for 4 hours at room temperature. One-fifth of the ligation mixture was used to transform E. coli strain 294 into ampicillin-resistant (on LB + 20 pg / ml amp plates). Transformant plasmids were tested by rapid screening (44). One plasmid pPGK-39 was selected for sequence analysis. 20 pg of this plasmid was digested with Xbal, precipitated with ethanol, and then treated with 1000 units of bacterial alkaline phosphatase at 68 ° C for 45 minutes. The DNA was extracted three times with phenol-chloroform and then precipitated with ethanol.

The dephosphorylated ends were then labeled in a 20 μΐ reaction containing 200 pCi [γ ³² - P] ATf and 10 units of T4 polynucleotide kinase. The plasmid was excised with SalI and passed through a 6 percent acrylamide gel.

The labeled insert is isolated from the gel and co-ordinated by the chemical degradation method. The DNA sequence at the 3 'end of this promoter portion is as expected.

2. Construction of the 312 bp Pvu-to-EcoRI PGK promoter fragment pg pPGK-39 (Fig. 13) were digested simultaneously with Sall and Xbal (five units each) and then electrophoresed on a 6 percent gel. The 390 bp band containing the 39 bp promoter portion was isolated by electroelution. The resuspended DNA was digested with restriction endonuclease Sau3A and then electrophoresed on a 6 percent acrylamide gel. The 39 bp promoter band was isolated by electroelution. This DNA contains 39 bp from the 5 'end of the PGK promoter of the Sau3A-to-XbaI fragment.

pg of pB1 was subjected to restriction with Pvul and ΚρηΙ, and then subjected to electrophoresis on a 6 percent acrylamide gel. The 0.8 kbp band of DNA was isolated by electroelution, then restrained with Sau3A and electrophoresed on a 6 percent acrylamide gel. The 265 bp band from the PGK promoter (Fig. 11) was isolated by electroelution.

This promoter is then ligated with the 39 bp promoter fragment above for two hours at room temperature. The ligation mixture was subjected to restriction with Xbal and Pvul, then electrophoresed on a 6 percent acrylamide gel. The restriction fragment 12bp Xba-to-PvuI was isolated by electroelution and then added to the ligation mixture containing 200 ng pBR322 01} (previously isolated, lacking 162 bp Pvul-to-Pstl restriction fragment) and 200 ng Xbal-to-PstI LelF A cDNA gene previously isolated from 20 pg pLelF trp A 25.This three-factor ligation mixture was used to transform E.stam 294 into tetracycline-resistant. Transform clones were subjected to mini-screening (44) and one of the 3PGK-300 colonies was isolated as having 304 bp PGK 5'-flanking DNA fused to the LelF A gene in the pBR322 main vector. The 5 'end of the LelF A gene has the following sequence: 5' CTAGAATTC - 3 '. This fusion of the Xbal site from the PGK promoter fragment in the given sequence allows one EcoRI site to be added to the Xbal site. pPGK-300 thus contains a portion of the PGK promoter isolated in the Pvul -to-EcoRI fragment.

p

3. Construction of 1500 bp EcoRI-to-EcoRI PGK promoter fragment pg pB1 was digested with Pvul and EcoRI and passed through 6 percent acrylamide gel. The 1.3 kb Pvulto-EcoRI lane from the 5'-flanking DNA was isolated by electroelution. 10 pg pPGK-30 was digested with EcoRI and Pvul and the 312 bp promoter fragment was isolated by electroelution after electrophoresis of the digested mixture in 6 percent acrylamide gel. 5 pg of pFRL4 was cut with EcoRI, precipitated with ethanol and then treated with bacterial alkaline phosphatase at 68 ° C for 45 minutes. After three extractions of phenol / chloroform DNA, ethanol precipitation and resuspension in 20 ml of H ₂ O, 200 ng of the vector was ligated with 100 ng of 312 EcoRI-to-Pvul DNA from pPGK-30O and 100 ng of EcoRI-to-Pvul DNA from pBl. The ligation mixture was used to transform E. coli strain 294 into ampicillin resistant. One of the transformants obtained is pPGK-1500. The plasmid contains a 1500 bp PGK promoter fragment, such as an EcoRI-to-EcoRI or HindIII-EcoRI portion of DNA.

pg of PGK-1500 was completely digested with Clal and EcoRI, after which the digested mixture was electrophoresed on a 6 percent 40 acrylamide gel. The 900 bpq fragment containing the PGK promoter was isolated by electroelution. 10 pg pIFN- γ trp 48 was completely digested with EcoRI and HincII and electrophoresed on a 6 percent acrylamide gel.

The yeast expression vector was constructed in a three-focal reaction by binding into one of the promoter fragment PCCK (on the Clal-EcoRI portion), the deletion pFRM-31 and the IFN-γ DNA isolated above. The coupling reaction was incubated at 14 ° C for 12 hours. This mixture was then used to transform E. coli strain 294 into ampicillin resistant. Transformants were analyzed for the expression of the expression plasmid actually generated, namely for pPGK-INF-γ (Fig. 16). Plasmids containing the expression system are used to transform spheroblasts of the yeast strain RH218 into tryptophan prototrophic in agar, in which this compound is absent. These recombinant yeasts are then tested for the presence of recombinant human immune interferon.

Yeast extracts were obtained as follows: Ten milliliters of cultures were cultured in ΥΝΒ + CAA to reach A _m = 1 - 2, collected by centrifugation, then resuspended in 500 μΐ PBS buffer (20 mM NaH ₂ PO ₄ , pH = 7.4, 150 µM NaCl). An equal amount of glass beads were added and the mixture was then stirred vigorously for 2 '. The extracts were rotated for 30 seconds at 14,000 rpm and the supernatants were removed. The interferon activity in the supernatants was 16,000 units / ml compared to the IFN-α standard using the CPE inhibitory sample.

M. Construction of the cell culture vector pSVy69

A 342 bp HindIII-PvuII fragment spanning the SV40 source is bound to a single fragment at the EcoRI restriction site.

The HindIII site is transformed by the addition of a synthetic oligomer (5 'dAGCTGAATTC) and the PvuII site is transformed by binding the blind end to an EcoR1 site filled with polymerase I (Klenow fragment). The resulting fragment was inserted into the EcoR1 site of pML-1 (28). Plasmid with SV40 late promoter targeted further by amp * gene _? is further modified by deletion of the EcoR1 site closest to the amp * gene of pML-1 (27).

The 1023 bp Hpal-Bglll fragment from the cloned HBV DNA (60) was isolated and the Hpal site from hepatitis B virus (HBV) converted to an EcoR1 site with a synthetic oligomer (5 'dGCGAATTCGC). This EcoR1-Bglll binding fragment was cloned directly into EcoR1-BamHI sites by the plasmid described above carrying the SV40 source.

The remaining EcoR1 site inserts the IFN - γ gene into 1250 base pairs of the PstI fragment of p69 after converting the PstI ends into EcoR1 ends. The clones in which the SV40 late promoter precedes the IFN-y structural gene were isolated = The resulting plasmids were then introduced into tissue culture cells (20 using DEAE-dextran technique (10, modified so that transfection in the presence of of DEAE-dextran is carried out for 8 hours. The cellular (.target is changed every 2-3 days. 200 microliters per day are removed for bioassays. Typical yield is 50-100 units / ml in samples taken three to four days after transfection .

The expression product does not contain the CYS-TYR-CYS N-terminal portion of recombinant human immune interferon (compare with Figure 5) _? which supports the emergence of signal peptide degradation at the CYS-GLN junction (amino acids 3 and 4 in Fig. 5) such that the mature polypeptide would actually consist of 143 amino acids.

N. Partial purification of des-CYS-TYR-CYS recombinant human interferons

In order to obtain larger amounts of des-CYS-LYSCYS recombinant human interferon, fresh monolayers of COS-7 cells in ten 10 cm plates were transfected with a total of 30 pg pDLIF3 in 10 ml DEAE - Dexstran (200 pg / ml DEAE Dexstran 500,000 MW, 0.05 Tris pH 7.5, in DMEM). After 16 hours at 37 ° the Dishwashers were washed twice with DMEM. Then, 15 ml of fresh DMEM replaced with 10 percent f was added to each plate. b. s., 2 mM glutamine, 50 μ / ml penicillin D and 50 mg / ml streptomycin. The medium is replaced with DMEM serum free the next day. Serum free medium is then added daily. The collected medium was stored at 4 ° C until tested or coupled to CPG. The fractions obtained from 3 and 4-day post-transfection samples were found to contain substantially all of the activity.

O. 5 g CPG (Controlled Porous Glass, Electronucleotics, CPG 350, mesh size 120/200) were added to 100 ml of cell supernatant and the mixture was stirred for 3 hours at 4 ° C. After brief centrifugation, place the glass beads in a column and wash thoroughly with 20 mM NaPO ₄ IM NaCl 0.1 percent β mercaptoethanol pH 7.2. The activity was then eluted with the same buffer containing 30 percent ethylene glycol, followed by further elution with the above buffer containing 50 percent ethylene glycol. Essentially, all activity is associated with CPG. 75 percent of the eluted activity is recovered in the fraction eluted with 30 percent ethylene glycol. These fractions were poured into 20 µM NaPO ₄ IM NaCl pH 7.2 to a final concentration of 10 percent ethylene glycol and applied directly to a 10 ml Con Sepharose (Pharmacia) column. After washing with 20 mM NaPO ₄ IM NaCl pH 7.2, the activity was eluted with 20 mM NaPO ₄ IM NaCl 0.2M α-methyl-D-mannose.

A significant amount of activity (55 percent) did not bind to this lectin. 45 percent of the activity was eluted with α-methyl-Dmanoside.

PHARMACEUTICAL COMPOSITIONS

The compounds of the present invention may be incorporated according to known methods into pharmaceutical compositions wherein the human immunointerferon product is combined with a pharmaceutically acceptable carrier. Suitable vehicles and their forms are described in Remington's Pharmaceutical Sciences by E.W. Martin, who is included in the reference. Such compositions will comprise an effective amount of interferon protein, together with an appropriate amount of vehicle for the preparation of pharmaceutical compositions suitable for administration to the host.

A. Parenteral administration

Human immune interferon can be administered parenthetically in subjects with antitumor or antiviral therapy or in those exhibiting an immunosuppressed condition. Doses and administration may be those used in clinical trials of other human interferons, i.e., about (1 10) χ 10 ⁶ units per day, and in the case of materials with a purity greater than 1 percent , almost to e.g. 50 χ 10 ⁶ . Doses of IFN-γ can be significantly increased for greater effect in the absence of substantially human proteins other than IFN-γ, which proteins in human-derived materials can cause undesirable effects.

As an example of a suitable dose for substantially homogeneous parenteral IFN-γ, 3 mg IFN-γ with a specific activity of, for example, 2x10 ⁸ U / mg can be dissolved in 25 ml of 5N albumin (human) - USP, the solution being was filtered through a bacteriological filter and the filtered solution was aseptically distributed into 100 tubes, each containing 6 x 10 ⁶ units of pure interferon suitable for parenteral administration. The tubes are preferably stored cold (-20 ° C) before use.

DATA ON BIOLOGICAL RESEARCH

A. Characterization of antiviral activity

To neutralize the antibodies, the samples were diluted, if necessary, to a concentration of 500-1000 units / ml with PBS-BSA. Equal volumes of the sample were incubated for 2-12 hours at 4 ° C with serial dilutions of rat anti-human leukocyte, fibroblast or immune interferon antiserum. Anti-IFN-oc and β were obtained from the National Institute of Allergy and Infectious Diseases. Anti-IFN-γ was obtained using authentic IFN-γ (5- 20 percent purity purified from stimulated peripheral blood lymphocytes. Samples were centrifuged 3 minutes at 12,000 x g 3 minutes before testing. adjusted to pH 2 by the addition of 1N HCl, incubated for 2 to 12 hours at 4 ° C and neutralized by the addition of 1 N NaOH prior to assay. SDS for 2 to 12 hours at 4 ° C before testing.

C. Characterization of

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

Antivirus activity (Units! T1)

E.coli W3110 / pIFN- COS-7 cells / pSVy69 Treats- IFN IFN -β IFN -γ ytrp48 cynep- no extract nantant Net- 375 125 250 250 62.5 wounds pH 2 375 125 <6 <12 <4 0.1% SDS 375 - <4 <8 - Plush <8 125 250 250 187 anti- IFN- α Plush 375 <8 187 250 125 anti- IFN-β Plush 375 125 <4 <8 <4 anti-IFN-γ

This table shows the characteristic behavior of IFN - α, β and γ standards under different treatments. Interferon activity derived from E. coli W 3110 / pIFN-γ trp 48 COS-7 / pSV γ69, is acid-sensitive, SDS-sensitive and neutralized by immuno-interferon antiserum. It is not neutralized by antibodies to IFN-α or β. These data confirm that the products obtained from these systems are immunointerferons and that the plasmid p69 cDNA insert encodes IFN-γ.

The immunolesterin protein is herein defined by a specific DNA gene and deductively consistent with amino acids, cf. 5. It is clear that for this particular interferon there are natural allelic differences that occur from individual to individual. These differences can be demonstrated by amino acid (s) differences throughout the sequence or by deletions, substitutions, insertions, inversions or additions of amino acid (s) in the same sequence. All such allelic differences are within the scope of the present invention.

Although reference is made to the particularly preferred embodiments, it should be understood that the present invention has been created, not limited to, but to the legitimate scope of the appended claims.

Claims (5)

A DNA molecule comprising a recombinant DNA molecule or cDNA molecule encoding a polypeptide having the following amino acid sequence: 1 10 CYS TYR CYS OLN ASP PRO TYR VAL LYS GLU ALA GLU ASN LEU LYS LYS TYR PHE ASN ALA GLY HIS SER ASP VAL ALA ASP ASN GLY THR LEU OHE LEU GLY ILE LEU LYS ASN TRP LYS GLU GLU SER ASP ARG LYS ILE MET 50 60 GLN SER GLN ILE VAL SER PHE TYR PHE LYS LEU PHE LYS ASN PHE LYS ASP ASP GLN SER ILE GLN LYS SER VAL GLU THR ILE LYS GLU ASP MET ASN VAL LYS PHE PHE ASN SER ASN LYS LYS LYS ARG ASP ASP PHE GLU 100 LIS LEU THR ASN TYR SER VAL THR ASP LEU ASP VAL 110 120 GLN ARG LYS ALA ILE HIS GLU LEU ILE GLN VAL MET 130 ALA GLU LEU SER PRO ALA ALA LYS THR GLY LYS 140 ARG LYS ARG SER GLN MET LEU PHE ARG GLY ARG 146 ARG ALA SER GLN. 2. A DNA molecule that includes a recombinant DNA molecule or cDNA molecule encoding a polypeptide having the following amino acid sequence: 1 10 GLN ASP PRO TYR VAL LYS GLU ALA GLU ASN LEU LYS LYS TYR PHE ASN ALA ALY HIS SER ASP VAL ALA ASP ASN GLY THR LEU PHE LEU GLY ILE LEU LYS ASN TRP LYS GLU GLU SER ASP ARG LYS ILE MET GLN SER GLN 50 60 ILE VAL SER PHE TYR PHE LYS LEU PHE LYS ASN PHE LYS ASP ASP GLN SER ILE GLN LYS SER VAL GLU THR ILE LYS GLU ASP MET ASN VAL LYS PHE PHE ASN SER ASN LYS LYS LYS ARG ASP ASP PHE GLU LYS LEU THR 100 ASN TYR SER VAL THR ASP LEU ASN VAL GLN ARG LYS 110 120 ALA ILE HIS GLU LEU ILE GLN VAL MET ALA GLU LEU 130 SER PRO ALA ALA LYS THR GLY LYS ARG LYS ARG SER 140 143 GLN MET LEU PHE ARG GLI ARG ARG ALA SER GLN. A DNA molecule according to claim 1 or 2 operatively linked to a DNA sequence capable of expressing DNA encoding a given polypeptide. A molecule consisting essentially of a DNA molecule encoding the amino acid sequence of recombinant human immune interferon. 5. A molecule consisting essentially of a DNA molecule encoding the amino acid sequence of des-CYS-TYR-CYS recombinant human immune interferon.