BG60939B2 - Human immune interferon - Google Patents

Abstract

The interferon is produced by recombinant DNA technology using any of an assortment of expression vectors and host cultures. Recombinant human immune gamma-interferon (ifn) has been isolated and characterized by its DNA and amino acid sequences, physical properties and biological activity. Des-сys-тyr-сys species were also obtained. 13 claims, 16 figures

Description

FIELD OF THE INVENTION

The present invention relates to the field of recombinant DNA technology, to the means and methods it uses to detect the DNA sequence and predicted amino acid sequences of human immune interferon, as well as to its preparation and to various products in its preparation and use.

More particularly, the present invention relates to the isolation and identification of DNA sequences encoding human immune interferon and to the construction of recombinant DNA expression agents containing such DNA sequences operatively linked to expression promoter sequences and to expression constructs thus constructed. In another aspect, the present invention relates to a host culture

system, such as various microorganisms, vertebrate cell cultures, previously transformed with similar expression agents and thus directed to the expression of the DNA sequences mentioned above. According to another aspect, the present invention relates to means and methods for converting the end products of an expression to new ones by nature, e.g. pharmaceutical compositions useful for the prophylaxis or treatment of humans. In preferred embodiments, the present invention provides partially expression agents that are substantially co-ordinated such that human immune interferon is obtained in mature form by host cells. In addition, the invention is

refers to various methods applicable to the preparation of said DNA sequences, to cell culture host systems and their end products, as they are, and to specific and related embodiments thereof.

The present invention arises in part from the discovery of the DNA sequence and predicted amino acid sequences encoding human immune interferon. In addition. the present invention provides sequential information on the 3'- and 5'-flanking sequences of the human immune interferon gene, facilitating in vitro binding within expression systems. In particular, a 5-DNA segment encoding the putative endogenous signal polypeptide is provided that precedes the amino acid sequence of the putative mature human immune interferon. These findings in turn make it possible to refine the means and methods of obtaining, through recombinant DNA technology, a sufficient amount of human immune interferon to make it possible, on the other hand, to determine its biochemical properties and biological activity.

Publications * and other materials illustrating the prior art and, in special cases, illuminating further details of the practice, are included in the literature and conveniently numbered in the text, respectively grouped in the accompanying bibliography.

BACKGROUND OF THE INVENTION

A. Human immune interferon

Human interferon can be classified into three groups based on differences in antigenicity and biological and biochemical properties.

The first group consists of a family of leukocyte interferons (a interferon, LelF or IFN - a), which are normally produced mainly by constitutive human blood cells upon viral induction. They are obtained by a microbiological route and have been found to be biologically active ζ 1,2,3 Y Their biological properties suggest their use in the clinic as therapeutic agents for the treatment of viral infections and malignancies (T).

In the second group is human fibroblast interferon (β interferon, FIF or IFN-β), normally produced by viral-induced fibroblasts, which are also obtained by microbiological pathway and have been found to exhibit a wide range of biological activities®. Clinical studies also show their potential therapeutic relevance. Leukocyte and fibroblast interferons show very clear similarities in their biological properties, despite the fact that the degree of homology at the amino acid level is

relatively low. In addition, the two interferon groups include 165 to 166 amino acids and are acid-stable proteins.

The human immune interferon (γ-interferon, IIF or IFN -γ) to which this interferon is addressed is, in contrast to X and interferons, pH 2 labile, produced mainly by mitogenic induction of lymphocytes and clearly antigenically distinct . As far as human interferon can currently be detected

only in very small quantities does this impede its characterization. Too extensive but still partial purification of human immune interferon® has recently been reported. It is indicated that the compound is produced from lymphocyte cultures stimulated with a combination of phytohaemagglutinin and phorbol ester and purified by sequential chromatographic separation. This method results in a product with a molecular weight of 58, 000.

By translating the mRNA into oocytes in very small amounts, human immune interferon was obtained, showing a characteristic of interferon activity inherent in human interferon and thus expressing the hope that immune inteferon cDNA can be synthesized and cloned (7).

The amount of immune interferon obtained so far is indeed unsatisfactory in order to conduct unambiguous experiments on the characteristics and biological properties of the purified compound. However, in vitro studies with crude preparation as well as in vivo experiments with murine γ-interferon preparations suggest that the primary function of immune interferon is its function of an immunoregulatory agent (8,9). Not only does immune interferon have antiviral and anti-cellular activity common to all human interferons, but it also shows potential effects on these activities with α- and β-interferon (10). In addition, the in vitro antiproliferative effect of interferon ^ on tumor cells has been reported to be 10 to 100 times greater than that of the other interferon classes (8, 11, 12). This result, along with his expressed

immunoregulatory function (8,9) suggests stronger antitumor potential for IFN-γ than for IFN-α and IFN-β. In fact, in vivo experiments with mice and murine IFN-γ preparations show a clear superiority over antiviral-induced interferons in their antitumor effect against osteogenic sarcoma (13).

All these studies of the present invention have been carried out with a too crude preparation. But they do suggest very important biological functions for immune interferon. The latter not only has potential associated antivirus activity, but probably does

it also has strong immunoregulatory and antitumor activity, clearly targeting potentially promising clinical use.

It is understood that, in this case, recombinant DNA technology would be the most effective way to obtain the required greater amount of human immune interferon. Whether the materials thus produced will include glycosylations that are considered inherent in the human-derived material or not, they would exhibit bioactivity allowing 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 adulthood.

Molecular biologists are able to recombine different DNA sequences with ease, creating new DNA agents capable of producing copies of different exogenous products in transformed germs. The main tools and methods at hand are in vitro binding of different blind or sticky DNA fragments,

producing potential expression agents that are used in partially transformed organisms, thereby directing their synthesis to the desired exogenous product. However, with respect to the individual product, the exchange remains volatile and science has made no progress with regard to the stage of prediction and obtaining a permanent result. One who predicts successful results without emphasizing experimental data does so with relative risk.

Plasmid, a non-chromosomal double-stranded DNA strand found in bacteria and other germs, capable of repeated self-reproduction in the cell, remains an essential element of

recombinant DNA technology. Included in the information encoded in plasmid DNA * is what is required for the reproduction of the plasmid in daughter cells (ie origin of replication) and usually one or more phenotypically selected characteristics such as, in the case of bacteria, antibiotic resistance , which allows clones of the host cell containing the desired plasmid * to be recognized and preferentially cultured in the selected medium. The applicability of plasmid lines is based on the ability to be specifically degraded by one or the other endonuclease enzyme or restriction enzyme, each of which recognizes a different site from the plasmid DNA. It is therefore also possible to insert heterologous genes or gene fragments into the plasmid by joining them at the degradation site or at the reconstructed ends adjacent to the degradation site. Thus, the so-called replicative expression agents are formed. DNA recombination takes place outside the cell, but the resulting recombinant replicative expression agent or plasmid can be introduced into cells in a manner known in the art, called transformation, to obtain large amounts of the recombinant agent by

cultivation of transformants. Moreover, when the gene is inserted according to the part of the plasmid that regulates the transcription and translation of the encoded DNA message, the resulting expression means can be used to update the production of the polypeptide sequence that the inserted gene encodes, and the whole process is called the expression process. .

Expression is initiated in an area known as a promoter that is recognized by and binds to RNA polymerase. In the transcription phase of expression, DNA is desirculated, becoming an template for initiating synthesis of transport (m) RNA from the DNA sequence.

The transport RNA is in turn translated into a polypeptide having an amino acid sequence encoded by the nucleotide ¹ triplet or a code which generally gives the structural gene, i. that portion that encodes the amino acid sequence of the expressed polypeptide product. The translation is initiated at the start signal (usually ATG, which in the resulting transport RNA passes into AUG).

The so-called stop codons determine the end of translation and, therefore, the production of subsequent amino acid units.

The final product can be obtained by leasing, if necessary, the host cell into a microbial system and recovering the product with appropriate purification from other proteins.

In practice, C using recombinant DNA technology can express entirely heterologous polypeptides - so-called direct expression - or vice versa heterologous polypeptides that are fused to a portion of the amino acid sequence of a homologous polypeptide. In the latter cases, the expected bioactive product may undergo biologically inactive form within the fusion homologous / heterologous polypeptide until degraded in an extracellular environment. See Eng. patent public No. 2007676 and Wetzel, American Scientist 68,664 (1980).

C. Cell culture technology

Cell or tissue culture technology for studying the genetics and physiology of cells is well-developed. The means and methods of this technology serve to maintain permanent cell lines obtained through successful serial transfers from isolated normal cells. For use in the assay, such cell lines are maintained on a solid surface in liquid medium or by culturing in a suspension containing solid nutrients. Making them on a large scale seems to only pose mechanical problems. For more information on the level of knowledge in the field, see Microbiology, 2nd Eddition, Harper and Row, Publishers, Inc., Hagerstown, Md. (1973), in particular p.1122 and Scientific American 245,66 (1981), each of which is included in the literature.

TOTAL INVENTION

The present invention is based on the discovery that recombinant DNA technology can be used to obtain human immunointerferon, preferably in direct form, and in amounts sufficient to initiate and test animals and humans under clinical conditions as a prerequisite for the approval of market. The product is suitable for use in all its forms in the prevention and treatment of people with viral infection and malignant and immunosuppressive or immunodeficient conditions. These forms include various possible oligomeric forms, which may include associated glycosylation. The product is obtained through genetically engineered micro-organisms or transformed cell culture systems. As used herein, the term cellular transformants refers to cells into which DNA has been introduced, obtained after exogenous DNA recombination, and to the progeny of any such cell that stores the DNA so introduced. Thus, there is currently a potential to produce and isolate human immunointerferon in a more efficient manner. A particularly significant factor of the present invention, in its most preferred embodiment _? is to achieve the genetic targeting of microorganisms or cell cultures to produce human immune interferon in

quantities that allow them to be isolated after secretion by the host cell into mature form.

The present invention encompasses the immune interferon thus obtained, as well as the means and methods for preparing it. The present invention is further directed to replicating DNA expression agents containing gene sequences that encode human immunointerferon in a form suitable for expression. Furthermore, the present invention is directed to strains of microorganisms or cell cultures transformed with the expression agents described above _? and to microbial or cell cultures of such transformed strains or cultures capable of producing human immune interferon. In yet another aspect, the present invention is directed to various methods applicable to the preparation of the gene sequence of said immunointerferon, DNA expression agents, strains of microorganisms and cell cultures and their specific embodiment. Still further, this invention is directed to the preparation of fermentation cultures from the given microorganisms and cell cultures. In addition, this invention is directed to the preparation of human immunointerferon as a direct expression product secreted by the host cell in mature form. Does this approach involve the use of the gene _? encoding the sequence of the mature human immunointerferon plus the 5 'flanking DNA encoding the signaling polypeptide.

The human immunointerferon thus expressed undoubtedly possesses 146 amino acids in their mature form, beginning with cysteine, and is basic in character. The molecular weight of its monomeric form is calculated to be 17,140. Probably due to the presence of many basic residues, hydrophobicity, formation of bridges, etc., the molecule may be associated in oligomeric forms such as e.g. in dimeric, three-dimensional or tetrameric form. The previously observed high molecular weights for natural materials (b), which cannot be calculated solely on the basis of the amino acid sequence, may be due to such oligomeric forms as uka and the assistance of carbohydrate by posttranslational glycosylation.

In actual cellular host systems, especially when bound in an expression agent so that it is expressed together with its signal peptide, the mature form of the human

1] Immunointerferon is transported in the cell culture medium, aided by recovery and purification methods.

DESCRIPTION OF THE PREFERRED METHODS OF IMPLEMENTATION

A. Microorganisms / Cell cultures

1. Bacterial strains / Promoters

The actions described herein are performed with the participation of the E. coli K-12 microorganism strain 294 (and A, thi-, hsr-, hsm +), as described in U.S. Patent No. 2055382 A. This strain is deposited in the American Type Collection ATCC cultures under No. 31446. However, many other microbial strains are also useful, including known strains of E. coli B, E. coli X 1776 (ATCC No. 31537) and E. coli W 3110 (F-, λ-, protrophic) (ATCC No. 27355) or other microbial strains, most of which are deposited and (potentially) available from authorized microbial deposit institutions, such as ATCC. See also German Offenlegungsschrift

2644432. These other microorganisms include, for example, bacteria such as Bacillus subtilis and other enterobacteria, among which can be noted as examples of Salmonella typhimurium nSerratia marcesans, which can replicate and express heterologous gene sequences.

By way of example, the beta lactamase and lactose promoter systems are predominantly used to initiate and maintain the microbial production of heterologous polypeptides. Details regarding the design 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 included in the literature. Recently, the so-called. trp tryptophan based promoter system. Details relating to the construction of such a system are published by Goeddel et al., Nucleic Acid Research 8.4057 (1980) and Kleid et al., USSer.No 133 296, filed March 24, 1980, which are incorporated by reference. literary reference. Many other microbial promoters have been discovered and used, and details regarding their nucleotide sequences allowing those of skill in the art to bind them functionally in plasmid vectors have been published — see e.g. Siebenlist et al., Cell 20, 269 (1980), which is included in the accompanying reference.

2. Yeast strains / Yeast promoters

The expression system used in the invention may be plasmid YRpQ4,15,16), which is capable of selection and replication in both E. coli and yeast Saccharomyces cerevisiae. For selection in yeast, the plasmid contains the gene TRP1Q4,15,10, whose complement (allowing development in the absence of tryptophan) containing mutations in this gene was found on chromosome IV (17). The strain used here is RH218QS) deposited in ATCC without restriction (ATCC No. 44076). But it should be understood that any strain of Saccharomyces cerevisiae containing a mutation that makes the trpl cell should be an effective medium for

expression of the plasmid containing the expression system. An example of another strain that may be used is pep4-lQ9). This tryptophan auxotrophic strain also has a point mutation on the TRP1 gene.

When a 5'-flanking DNA sequence (promoter) of a yeast gene (for alcohol dehydrogenase 1) is placed on the 5'-end of a gene of non-yeast origin, it can promote the expression of a foreign gene in yeast if inserted into a plasmid applicable to yeast transformation. In addition to the promoter, the true expression of a non-yeast gene in yeast requires a second yeast sequence inserted at the 3'-end of the non-yeast gene into a plasmid to allow actual transcriptional termination and polyadenylation in yeast. This promoter can be suitably used in the present invention, as well as others, see above. In preferred embodiments, the 5'-flanking sequence of the yeast 3phosphoglycerate kinase gene (20) is upstream of the structural gene, followed by DNA containing terminator-polyadenylation signals, e.g., TRP1 £ 14,15,16 gene or PGK (2Q ) gene.

Because the 5'-flanking sequence (linked to 3'-yeast termination DNA) (above) can functionally contribute to

expression of foreign genes in yeast, as if those of the 5'-flanking sequences of any highly expressed yeast gene can be used for the expression of important gene products. While in some circumstances yeasts express up to 65 percent of their soluble protein as glycolytic enzymes (21), and while this high level results from the production of high levels of individual mRNA (22), the use of 5'-flanking sequences would be possible of any other glycolytic genes for such

expression targets - such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosophosphosomer isomerase. Any of the 3'-flanking sequences of these genes can also be used for termination and mRNA polyadenylation in such expression systems - see. above. Other high expression genes are those for acidic phosphatases (23) and those that express high levels of productivity due to mutations in the 5'-flanking regions (mutants that increase expression) - usually due to the presence of a TY1 transposing element (24).

All the genes marked above are those that can be transcribed by yeast RNA polymerase 11 (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 such expression structures.

Finally, many yeast promoters also contain transcriptional control * so that they can be excluded or incorporated by variants of developmental conditions. Some examples of such yeast promoters are the genes that produce the following proteins: Alcohol dehydrogenase II, isocytochrome c, acid phosphatase, degrading enzymes associated with nitrogen metabolism, glyceraldehyde-3-phosphate dehydrogenase, and utilization of enzyme maltase and response using ). Such control regions would be very useful in controlling the expression of the protein product - in particular when their production is toxic to yeast. It would also be possible to place the control region of a 5′-flanking sequence with a 5 * flanking sequence containing a promoter of a highly expressed gene. This would lead to a hybrid promoter and would be possible as long as the control region and the promoter are physically distinct DNA sequences.

3. Cell culture systems / Cell culture vectors

Vertebrate cell reproduction (tissue culture) has been a routine procedure in recent years (see Tissue Culture, Academic Press, Kruse and Patterson eds, 1973). The COS-7 line from monkey kidney fibroblasts was used as a host for immunointerferon production (25). However, the experiments described in detail herein may be conducted in any cell line capable of replication and expression of a competing vector, such as WI138, BHK, 3T3, CHO, VERO, and HeLa cell lines. In addition, what is required for an expression vector is a replication source and promoter located in front of the expression gene along any ribosomal binding site, RNA cleavage site, polyadenylation site, and transcription termination sequences. Although these essential elements of the SV40 are used here, it would be understood that the invention, although described within preferred embodiments, should not be limited in its design to those sequences. For example, the source of the invention may be used. replication of other viral vectors (such as Polyoma, Adeno, VSV, BPV, and the like) as well as cellular replication sources that could function in a non-integrated state.

B. Vector systems

1.Direct expression of mature immunointerferon in E. coli

The procedure used to obtain IFN-γ direct expression

in E. coli as a mature interferon polypeptide (minus signal sequence) (a variant of one that uses earlier human growth hormone Q.6) and human leukocyte interferon (T), insofar as it involves the combination of synthetic (N-terminal) and cDNA .

As suggested by the nucleotide sequence of p69 described above and by comparing known degradation sites between the signal peptide and the mature polypeptide for several IFN-α (2), IFN-γ possesses a hydrophobic signal peptide of 20 amino acids, followed by of 146 amino acids of mature IFN-γ (Fig. 5). As shown in Fig. 7, the BStNI restriction endonuclease site is conventionally located at amino acid 4 of mature IFN-J '. Two synthetic oligodeoxynucleotides have been constructed that include an ATG translation initiation codon, codons for amino acids 1, 2, and 3 (cysteine-tyrosine-cysteine), and an EcoRI casseous end is created. These deoxyoligonucleotides are linked to 100 base pairs of the BstNI-PstI fragment of p69 to construct a synthetic-natural hybrid gene with 1,115 base pairs that encodes IFN-? R and which is linked through 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, the IFN-γ gene is expressed under the control of the E. coli trp promoter. pLelF A trp 103 was obtained from pLelF A 25 in which the EcoRI site, distal to the LelF A gene, was removed. The procedure used to remove this site is described previously (27).

2. Expression in yeast

To express a heterologous gene such as cDNA for immunointerferon in yeast, it is necessary to construct a plasmid vector containing four components of both E. coli and yeast and thus should contain a selective gene from each organism. (In this case, this is the ampicillin resistance gene from E. coli and the yeast TRP1 gene). This component also requires a source of replication from both organisms (In this case, it is the source of E. coli from pBR322 and the arsl source from chromosome III of yeast).

The second component of the plasmid is the 5'-flanking sequence of a highly expressed gene to aid in the transcription of the downstream structural gene. In this case, the 5'-flanking sequence used is that which is from the yeast 3-phosphoglycerate kinase (PGK) gene, the fragment is designed to remove the ATG from the PGK structural sequence as 8 bp down from that ATG. This sequence is replaced by the sequence containing both restriction sites XbdI and EcoRI for convenience attached to the 5'-flanking sequence of the structural gene.

The third component of the system is a structural gene constructed in the same way as to contain both an ATG translation start and a translational stop signal. Isolation of structures from such a gene is described above.

The fourth component is a yeast sequence comprising a 3'flange sequence of a yeast gene that contains a substantive signal for transcriptional termination and polyadenylation.

Immunointerferon was obtained in the presence of all these components in yeast.

H. Expression in mammalian cell cultures

The methodology for the synthesis of immune interferon in mammalian cell cultures relies on the creation of a vector capable of both autonomous replication and expression of an external gene under the control of a heterologous transcription unit. Replication of this vector in tissue culture is accomplished by providing a DNA replication source (derived from the SV40 virus), and providing ancillary function (T antigen).

by introducing the vector into a cell line endogenously expressing this antigen (28,29). The late SV virus poromotor precedes the interferon structural gene and guarantees the transcription of the gene.

The vector used to obtain IFN-γ expression consists of pBR322 sequences that provide a selective marker for selection in E. coli (ampicillin resistant) as well as E. coli DNA replication source. These sequences are derived from plasmid pML-1 (28) and span the region between the restriction sites E. coli and BamHI. The SV40 source was obtained from 342 base pairs of the PvuII-HindIII fragment covering this region (30.3 D) (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 late transcription units. The orientation of the region for the SV40 source is that of the promoter for the late transcription unit proximal to the interferon encoding gene.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the sucrose gradient of lymphocyte-induced peripheral blood poly (A) + RNA. Two peaks of interferon activity (as shown in the shaded boxes) of 12S and 16S sizes were observed. The sites of ribosomal RNA markers (centrifuged immediately) are marked above in an absorption profile.

Figure 2 shows PBL poly (A) + RNA induced electrophoresis by acid-urea-agarose. Only one peak of activity was observed for 18S RNA. The position of ribosomal RNA markers, which were electrophoresed in the adjacent line and visualized by ethidium bromide staining, were noted above the activity profile.

FIG. 3 shows the hybridization patterns of 96 colonies with induced and uninduced 32p-labeled cDNA probes. In microtiter plates were cultured 96 individual transformant, transferred to nitrocellulose membranes, and then the filtrates were hybridized with P ³² cDNA probes prepared from either mRNA induction (as _above)? or mRNA isolated from non-induction PBL cultures (non-induced, below). the filters are washed to remove non-hybridized RNA and then exposed to a UV-sensitive film. This filter complex is representative of 86 such complexes (8300 independent colonies). One example of an induced clone is referred to as H12.

Fig. 4 is a map of restriction endonucleases of the insertion clone 69 cDNA. cDNA insert is linked via PstI sites (points at both ends) and oligo dC-dG tails (single lines). The number and size of fragments _? obtained by restriction endonuclease digestion was evaluated by electrophoresis on 6 percent acrylamide gels. The positions of the sites were confirmed by nucleic acid matching (presented in Figure 5). The coding region of the largest open reading frame is enclosed by ¹ , and the shaded portion represents the putative signal peptide sequence of 20

the residue, while the dotted region represents the mature IIF sequence (146 amino acids). The 5 'end of the mRNA is to the left and the 3' end is to the right.

FIG. 5 illustrates the nucleotide sequence of the insertion plasmid p69 cDNA. The putative amino acid sequence of the longest open reading frame is also presented. The putative signal sequence is represented by the residues indicated by 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 (referred to as immune) contains a significantly larger amount of untranslated sequences.

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

FIG. 8 shows a plasmid diagram useful for expression of IFN-γ in monkey cells.

Fig. 9 shows Southern hybridization of EcoRI degraded human genomic DNA hybridized with a 32p-labeled Ddel fragment with 600 pairs of insertion p69 DNA bases. Two EcoRI fragments were clearly hybridized to the probe of each DNA sample.

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

FIG. 11 schematically illustrates the restriction map of a 3.1 kbp HindIII insert from the vector pB1 from which the PCK promoter was isolated. The insertion of one EcoRI site and one Xaa1site into the 5'-flanking DNA of the PGK gene is indicated.

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

FIG. 13 schematically illustrates the techniques used to insert the Xbal site at position 8 in the PGK promoter and to isolate a 39 bp fragment from PbKl containing this Xbal end and Sau3A end.

FIG. 14 schematically illustrates the construction of a 300 bp fragment containing the above 39 bp fragment, an additional PGK 5'flange 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 bp PGK promoter (HindIII / EcoRI), which further comprises, in addition to the fragment constructed in Fig. 14, a 1300 bp HindIII to the PGK 5'flange sequence (see Fig. 11).

FIG. 16 shows the composition of a yeast immunointerferon expression vector comprising a modified PGK promoter, IFN-γ cDNA, and the terminator region of the yeast PGK gene, as described in more detail herein.

DETAILED DESCRIPTION

A. Source of IFN-γ mPNK

Human peripheral blood lymphocytes (PBLs) were obtained by leukophoresis. PBLs were purified by Ficaoll-Hypaque gradient centrifugation and then cultured at a concentration of 5 × 10 ⁶ cells / ml in RPMI1640, 1 percent L-glutamine, 25 mM HEPES and 1 percentage of penicillin-streptomycin pa3TBop (Gibco, Grand Island, NY). These cells were induced to produce IFN-γ by mitogenic staphylococcal enterotoxin B (1 mg / ml) and cultured for 24 to 48 hours at 37 ° C. Desacetylthymosin-α-1 (0.1 mg / ml) was added to PBL cultures to enhance the resulting IFN-h activity.

C. Isolation of transport RNA

The total amount of RNA from PBL cultures was extracted predominantly as reported by Berger, SL et. al. (33). Cells were precipitated by centrifugation and then resuspended in 10 mM NaCl, 10 mM TrisCH1 (pH 7.5) 1.5 mM MgCl ₂ and 10 mM ribonucleoside vanadyl complex. Cells were lysed by the addition of NP-40 (1 percent final concentration), and nuclei were precipitated by centrifugation. The supernatant contains the total amount of RNA, which is further purified by repeated phenolic and chloroform extraction. The aqueous phase was adjusted to 0.2 M in NaCl, after which the total amount of RNA was precipitated by adding two volumes of ethanol. The RNA from the uninduced (unstimulated) cultures was isolated by the same methods. Oligo-d T cellulose chromatography (34) was used to purify the mRNA from the total quantitative RNA precipitate. Typically, 5-10 milligrams of total RNA and 50-200 micrograms of poly (A) + RNA are obtained from 1-2 liters of cultured PBL.

C. mRNA size fractionation

Two methods are used to fractionate tRNA preparations. They are used independently (better than simultaneously) and each of them results in significant enrichment of IFN-γ mRNA.

Sucrose gradient centrifugation in the presence of denatured formamide is used to fractionate the mRNA. From 5 to 25 percent sucrose in 70 percent formamide are centrifuged (32) at 154,000 x g for 19 hours at 20 ° C. The successive fractions (0.5 ml) after

these are removed, precipitated with ethanol, and an aliquot injected into oocytes of Xenopus laevis for translation of mRNA (35). After 24 hours at room temperature, the incubation medium is tested for antiviral activity in a standard cytopathic inhibition effect sample using vesicular stomatitis virus (Indiana strain) or encephalomyocardial virus on Y / 18H (human amnion) cells as described by Stewart ( 36), except that the samples were incubated with cells for 24 hours (instead of 4) before challenge with the virus. . Two constant peaks of activity in the fractionated RNA were observed (Fig. 1). One peak is sedimented at a calculated size of 12S and contains 100-400 units / ml of antiviral activity (compared to IFN-α standard) per microgram of injected RNA. Another activity peak, sedimented as

16S in size contains about half the activity at a slower rate

the sedimented peak. Each of these activity peaks is apparently due to IFN-γ, whereas when the same fraction is tested on calf cell line (MDBK), no activity is observed as they are not protected by human IFN-γ. Both IFN-α activity and IFNβ activity would be readily detectable by MDBK (5).

Fractionation of mRNA (200 mg) was also performed by electrophoresis on uric acid agarose gels. The agarose gel plate (37.30 consists of 1.75 percent agarose, 0.025 M sodium citrate, pH 3.8 and 6M uric acid. The electrophoresis was carried out for 7 hours at 25 milliamps and 4 ° C. The gels were then cut with a razor blade, the individual sections were softened at 70 ° C and extracted twice with phenol and once with chloroform, then the fractions were precipitated with ethanol and sequentially. were tested for IFNγ mRNA by injection into oocytes n Xenopus laevis and antiviral activity Only one peak of activity was observed in the fractionated gel samples (Fig. 2) This peak is due to migration along with 18S RNA and has an activity of 600 units / m2 for micrograms of injected RNA. is probably IFN-γ

specific as it does not protect MDBK cells.

The discrepancy in size between the activity peaks observed on sucrose gradients (12S and 16S) and uric acid gels (188) can be explained by the observation that these independent fractionation methods are impracticable under conditions of complete denaturation.

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

For the production of double stranded DNA by standard procedure, 3 pg gel fractionated mRNA (26.3 ^) · cDNA was fractionated on 6 percent polyacrylamide gel. Two of the fractions were electroeluted, 800-1500 bp (138 ng) and> 1500 bp (204 ng). 35 ng portions of each cDNA were expanded with deoxyC residues using terminal deoxynucleotidyl transferase (4 (Y) and renatured with 300 ng of plasmid pBR3220f, which was similarly extended with deoxyC residues at PstI site 00). Each renatured mixture was then transformed into E. coli K12 strain 294. Approximately 8000 transformants were obtained with 800 - 1500 bp cDNA and 400 transformants with> 1500 bp cDNA.

F. Screening of the colony library for induced cDNAs

The colonies were inoculated individually into wells of microtiter plates containing LB (58) + 5 mg / ml tetracycline and left at -20 ° C after addition of DMSO up to 7 percent. Two copies of the colonies were cultured on nitrocellulose filters and DNA from each colony was fixed on the filter by the Grunstein-Hognes method (45 ·

P ^32- labeled cDNA probes were prepared using 18S gel fractionated mRNA from induced and non-induced PBL cultures. Oligo dT12-18 is the primer used, the reaction conditions described previously (1). Filters containing 8000 transformants in size of the cDNA 600-1500 bp and transformants with size> 1500 bp were hybridized with 20 x 10 ⁶ cpm of induced ³² P -sDNK. A second set of filters hybridized with 20 x 10 ⁶ 230. cf. of the induced P ³² -cDNA. Hybridization was performed for 16 hours under the conditions described by Fritch et al. (43). the filters are vigorously flushed (43) and then exposed to ultraviolet irradiation on a Kodak XR-5 reinforced Du-Pont LightningPlus film for 16 to 48 hours. 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). 124 colonies hybridize significantly with the induction probe, but without being detectable or less with the non-inductive probe. These

colonies were inoculated individually into the wells of microtiter plates, cultured and hybridized with the same two probes as described above. Plasmid DNA isolated from each of these colonies by Method No. 44) also binds to nitrocellulose filters and hybridizes (45) with induction and non-induction probes. The 22 colonies' DNAs hybridize with an induction probe only and are called induced colonies.

F. Characterization of induced colonies

Plasmid DNA was prepared from five of the induced colonies (46), which was used to characterize cDNA inserts.

The restriction maps of five induced plasmids (p67, p68, p69, p71, and p72) suggest that four have similar restriction maps. These four are p67, p69, p71, and p72, each with four Ddel sites, 2 Hinf1 sites, and one Rsal site in cDNA. The fifth plasmid (p68) contains a common Ddel fragment and is apparently a short cDNA clone bound to the other four. The homology suggested by the restriction maps was also confirmed by hybridization. P ³² - labeled DNA probe is prepared (47) of 600 pb Ddel fragment of the plasmid p67, and used for hybridization 02) to the other induced colonies. All five of the restriction endonucleases mapped colonies hybridized with this probe as did the 17 other colonies of induction / non-induction ^η screening. The length of the insert cDNA in each of these cross-hybridized plasmids was determined by PstI and wire electrophoresis. The clone with the longest cDNA insert is apparently a clone 69 with an insert length of 1200 - 1400 bp. This DNA was used for all further experiments and its restriction map is shown in Figure 4.

By producing expression products in three independent expression systems demonstrating antiviral activity, the insert cDNA in p69 was shown to be IFN-γ cDNA.

c-Sequential analysis of insert cDNA in p69

The complete nucleotide sequence of the p69 plasmid insert was determined by the dideoxynucleotide chain termination method (48) by subcloning the fragments in vector M13 tp709) and the Maxam-Gilbert chemical procedure (52). The longest open reading frame encodes a protein of 166 amino acids shown in FIG. 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 probable signal sequence has features common to the other signal sequences, such as size and hydrophobicity. Moreover, the four amino acids found at the probable degradation site (ser-leu-cys) are identical to the four residues found at the degradation points of several leukocyte interferons (LelF B, C, D, F and H (2)).

The encoded mature amino acid sequence of 146 amino acids

acids (hereinafter referred to as recombinant human immunointerferon) has a molecular weight of 17,140.

There are two potential glycosylation points (50) in the encoded protein sequence, for amino acids 28 to 30 (asn-gly-thr) and amino acids 100-102 (asn-tyr-ser). The existence of these positions is consistent with the observed glycosylation of human IFN-γ (6.5f). In addition, only two cysteine residues are close enough to form disulfide bridges, which is consistent with the observed stability of IFN-γ in the presence of reducing agents such as mercaptoethanol (3 f).

The putative mature amino acid sequence is quite basic, with a total of 30 lysine, arginine and histidine residues and only a total of 19 aspartic and glutamine

acid residues.

The mRNA structure of IFN-γ * as suggested by the DNA sequence of plasmid p69β is distinctly different from IFN-α (1, 2) or IFN-β (5) of mRNA. As shown in FIG. 6, is the coding region of IFN-γ shorter _? while the 5 'untranslated and 3' untranslated regions are slightly longer than both IFN-α and I

IFN-β.

N. Expression of recombinant human immune interferon in E. coli

As shown in FIG. 7, 50 μg of plasmid p69 were digested with PstI and an insert of 1250 base pairs was isolated by

gel electrophoresis in 6 percent polyacrylamide gel. Approximately 10 pg of this insert is electroeluted from bodies. Then, 5 µg of this PstI fragment was digested into 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 the desired BstNI-PstI fragment consisting of 1100 base pairs was recovered. The 7 denoted deoxyoligonucleotides, 5 'dAATTCATGTGTTATTGTC and 5' - dTGACAATAACACATG were synthesized by the phosphotriester method (53) and phosphorylated as follows: 100 pmol of each deoxyoligonucleotide 30 HCl combined 60 HCl , 10MM MgCl ₂ , 15 mM β-mercaptoethanol and 240 μ Ci (γ-32P) ATF (Amersham, 5000 Ci / mmol). 12 units of T4 polynucleotide kinase were added and the reaction was left at 37 ° C for 30 minutes. 1 µl of 10 mM ATf was added and the reaction was allowed to continue for 20 minutes. After--OH / CHClНС 3 extraction, the oligomers were combined with the BstNI-PstI fragment having 1100 base pairs and precipitated with ethanol. These fragments were ligated at 20 ° C for 2 hours in 30 pl 20mM Tris-HCl (pH 7.5),

ΜM MgCl ₂ , 10 mM dithiothreitol, 0.5 mM ATf, and 10 units of T4 DNA ligase. The mixture was digested for 1 hour with 30 units of PstI and 30 units of EcoR1 (to remove polymerization by ligation of the cohesive end) and electrophoresed on a 6 percent polyacrylamide gel. The product of 1,115 base pairs (110,000 ct) is recovered by electroelution.

Plasmid pLelF A trp 103 (Fig. 7) is derived from plasmid pLelF A 25 (T), in which the EcoR1 site distal to the LelF A gene is removed (27). 3pg of pLelF A trp 103 was digested with 20 units of EcoR1 and 20 units of PstI for 90 min at 37 ° C and subjected to electrophoresis on a 6 percent polyacrylamide gel. The large vector fragment (~ 3900 base pairs) is recovered by electroelution. the 1115 base pair fragment, EcoRI-PstI IFN-λ DNA, was 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 EcoPI and Pst1. Three of these plasmids contain the degraded EcoRI - Pstl 1115 base pair fragment. The DNA sequence analysis confirms that these plasmids possess the desired nucleotide sequence and the binding between the trp promoter, synthetic DNA and cDNA. One of these plasmids pIFN-λ trp 48 was selected for further study. This plasmid was used to transform E. coli K-12 strain W3110 (ATCC N 27325).

I. Gene structure of the IFN -γ coding sequence

The structure of the gene encoding IFN-γ was analyzed by Southern hybridization. In this procedure (54), 5 micrograms of high molecular weight human lymphocyte DNA (obtained as in 55) is digested with various restriction endonucleases, electrophoresed in 1.0 percent agarose gels (56) and dried on nitrocellulose filters (54). p ³² -labeled DNA probe was prepared (47) from the 600 bp Ddel fragment of the cDNA insert of p69 and hybridized (43) with the nitrocellulose DNA. An amount of 10 ⁷ per minute from the probe was hybridized for 16 hours and then washed as described in (43). Eight genomic DNA samples from different donors (human) were digested with EcoRI restriction endonuclease and hybridized with the p69 ³² p-labeled probe. As shown in FIG. 9, two clear hybridization signals of 8.8 kilobase pairs (kBp) and 2.0 kbp were observed as can be found when comparing motions with HindIII degraded CDNA. This may be the result of the cleavage of two IFN-γ genes or only one gene at the EcoRI site. While p69 does not contain an EcoRI site, an intermediate structure with an intermediate EcoRI site called an intron would be required to explain the single gene. To differentiate these two options, another Southern hybridization is conducted

with the same probe against five other single human DNA endonucleases (Fig. 10). Two hybridization DNA fragments were observed with two other restriction endonucleases - PvuII (6.7 kbp and 4.0 kbp) and Hindi (2.5 kbp and 2.2 kbp). However, three endonuclease cleavage samples provide only one hybridization DNA fragment: HindIII (0.9 kbp), BdllI (11.5 kbp) and BamHI (9.5 kbp). Two IFN-γ genes should be attached to one unusual site within the same hybridization fragment. This result suggests that only a single homologous IFN-γ gene (unlike many IFN-α related genes) is present in human genomic DNA and that this gene is part of one

or more introns containing EcoRI, PvuII, and Hindi sites. This assumption is confirmed by hybridization of p ³² -labeled (47) fragment derived from the 3 'untranslated region of the cDNA from p69 (130 bp Ddel fragment from 860 bp to 990 bp in FIG. 5) against EcoRI digested human genomic DNA. Only the 2.0 kbp EcoRI fragment hybridizes to this probe, suggesting that this fragment contains 3 'untranslated sequences, while the 8.8 kbp EcoRI fragment contains 5' sequences. The gene structure of IFN-γ (one gene with at least one intron) is clearly distinguishable from IFN-α (multiple genes (2) without introns (56) or IFN-β (one gene without introns (57)).

J. Preparation of bacterial extracts

Overnight culture of E. coli W 3110 / pIFN trp 48 in broth

Luria + 5 micrograms per ml of tetracycline is used to inoculate M9 medium (58) containing 0.2 percent glucose, 0.5 percent casamic acid and 5 micrograms per ml tetracycline at a dilution of 1: 100. Indole acrylic acid is added to a final concentration of 20 micrograms per ml when the A550 is between 0.1 and 0.2. Samples of 10 ml were centrifuged at A550 = 1.0, the precipitate was collected and immediately resuspended in 1 ml of phosphate buffer solution containing 1 mg3a ml of bovine serum albumin (PBSBSA). The cells were sonicated and sonicated. are purified from the cell walls by centrifugation. The supernatants were left at 4 ° C until the test. Interferon activity in supernatants was determined to be 250 units / ml by comparison with IFN-α standards by the Cytopathic Inhibition Effect (CFE) assay.

K. Yeast / strain and medium transformation

Yeast strains were transformed as described previously G9 · For selection of plasmids containing a functional TRPI gene, the E. coli JA300 strain (thr leuB6 thi thyA trpC1117 hsdm-hsdR- (20) was used. The yeast strain RH218 possessed (a trp gal2 SUC2 mal CUPl) (18), is used as the host organism to be transformed.RH218 is deposited in ATCC N 44076. M9 (minimum culture medium) with 0.25 percent casamic acids (CAA) and bB ( rich media) were used as described by Miller (58) with the addition of 20 pg / ml ampicillin (Sigma) after autoclaving and cooling. on the following medium: YEPD containing 1% yeast extract, 2% peptone and 2% glucose ± 3% Difco agar YNB + CAA contain 6.7 grams of yeast nitrogen base (without amino acids) (YNB) (Difco), 10 mg adenine, 10 mg uracil, 5 grams of CAA, 20 grams of glucose and ± 30 grams of agar per liter.

L. Construction of a yeast expression vector

1. 10 pg YRp7 04,15,16) were digested with EcoRI. The resulting adhesive DNA ends are made blind using DNA polymerase I (Klenow fragment). The vector and 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 before precipitation with ethanol. The resulting blind end DNA molecules were ligated together in a final volume of 50 μΐ for 12 hours at 12 ° C. This mixture was then used to transform E. coli strain JA300 into an ampicillin-resistant and tryptophan prototrophic organism. Plasmids containing the TRPI gene from both orientations were isolated. pFRW1 has the TRPI gene in the same orientation as

YRp7 while pFRW2 possesses the TRPI gene in reverse orientation.

pg of pFRW2 is linearized with HindIII and subjected to

electrophoresis in 1 agarose gel. The linear molecules were eluted from the gel, after which 200 ng were ligated with 500 ng of 3.1 kb Hindlll insert from plasmid pB1 03), which is a restriction fragment containing yeast

3-phosphoglycerate kinase gene. The ligation mixture was used to transform E. coli strain 294 into an ampicillin-resistant and tetracycline-sensitive organism. The plasmid prepared from such a recombinant possesses an intact TRP1 gene with a HindIII fragment of 3.1 kbp size from pB1 insertion DNA at the HindIII site of the tetracycline-resistant gene. The plasmid is pFRM31. 5 pg of pFRM31 was completely digested with EcoRI, extracted twice with phenol and chloroform and then precipitated with ethanol. The cohesive ends of the molecule are filled with the participation of DNA Polymerase I (Klenow fragment) in a reaction that is carried out in 250 μΜ of each deoxynucleoside triphosphate. The reaction is carried out for 20 min at 14 ° C, during which time the DNA is extracted twice with phenol -chloroform, then precipitated with ethanol. The resuspended DNA was then completely digested with Clal and electrophoresed on a 6 percent acrylamide 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 3-phosphoglycerate kinase enzyme, purified by humans, are as follows:

- 2 - 3-4-5-6

SER-LEU-SER-HSM-LYS-LEU One of the translational reading frames generated by the DNA sequence of a 141 bp Sau3A-to-Sau3A restriction fragment (containing an intermediate HincII site; see PGK restriction map in Fig. 11) produces the following amino acid sequence.

1-2-3-4-5-6

MET-SER-LEU-SER-SER-LYS-LEU After 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.

The result of the alignment suggests the onset of the yeast PGK structural gene is encoded by DNA in the 141 bp Sau3A restriction fragment of pB1. Earlier developments (20) have suggested that the DNA sequences specifying PGK mRNA are likely located in this region of the HindIII fragment. Further alignment of the 141 bp Sau3A fragment yields more DNA sequence than the PGK promoter (Fig. 12).

A synthetic oligonucleotide of the sequence 5 'ATTTGTTGTAAA 3' was synthesized by standard procedures (Schea et al., Nucleic Acids Res. 8.2331 (1980)). 100 ng of this primer were recorded at

5 'end using 10 units of T4 polynucleotide kinase in 20 μΐ reaction also containing 200 pCi of [γ 32 -P] ATf. This labeled primer solution is used for a recovery reaction created as a first step in a multistage process for inserting an EcoRI restriction site into PGK 5'-flanking DNA preceding the PGK sequence of the structural gene.

100 pg of pB1 (2f was completely digested with NaHCO3 then passed through a 6 percent polyacrylamide gel. The top band of ethidium stained gel (containing PGK) was isolated by electroelution as described above. This 1200 bp Our DNA strand was then digested with Hindi ₉ and then passed through a 6% acrylamide gel, the 650 bp band was isolated by electroelution, 5 pg of DNA was isolated, and this 650 bp HaeIII-to-HincII portion of the DNA was resuspended in 20 pl H2O and then mixed with the 20 pl phosphorylated primer solution described above. extracted 1 x with phenol-chloroform, then precipitated with ethanol. The dried DNA was resuspended in 50 µl of H2O and then heated in a boiling water bath for seven minutes. This solution was then rapidly cooled in a dry ice-ethanol bath ( 10n - 20 seconds), then transferred to an ice water bath. To this solution were added 50 µl of 10 x DNA polymerase I buffer (Boeringer Mannheim), 10 µl of pre-prepared 2.5 mM solution for each deoxynucleoside triphosphate (dATP, dTTP, dGTP and dCTP), 25 pl H2O and 5 units of DNA polymerase I, Klenow fragment. This 100 pl reaction was incubated at 37 ° C for 4 o'clock. The solution was then extracted once with phenol-chloroform, precipitated with ethanol, dried by lyophilization, and then subjected to complete depletion of the enzyme 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 off from the gel and isolated by elution as described above. This 39 bp strip has a blind end. The fragment was cloned into a modified pFIF trp 69 vector (5). 10 gg of pFIF trp 69 was linearized with Xbal, extracted once with phenol-chloroform and then precipitated with ethanol. The sticky Xbal end was filled with the participation of DNA polymerase I Klenow fragment in a 50 μΐ reaction containing 250 μΜ of each nucleoside triphosphate. This DNA was cut with VasH 2 and then passed through a 6 percent polyacrylamide gel. The vector fragment was isolated from the gel by electroelution, then resuspended in 20 μΐ H2O. 20 ng of this vector were ligated with 20 ng of the 39 bp fragment prepared as above for 4 hours at room temperature. One-quarter of the mixture was used to transform E. coli strain 294 into ampicillin-resistant (on LB + 20 μβ / ml amp). Transformant plasmids were examined by rapid screening procedure (0 l). One plasmid, pPGK-39, was selected for sequence analysis. 20 μg of this plasmid were digested with Xbal, precipitated with ethanol, and then treated with 1000 units of bacterial alkaline phosphatase at 68 ° C for 45 min. The DNA was extracted three times with phenol-chloroform and se

precipitated with ethanol.

The dephosphorylated ends were then labeled with a 20 μl reaction containing 200 μl of {γ32 - P] ATf and 10 units of T4 polynucleotide kinase. The plasmid was cut with Sall and passed through a 6 percent acrylamide gel.

The labeled insertion strip is isolated from the gel and coordinated by the chemical degradation method (52) · The DNA sequence at 3 '- the end of this promoter portion is as expected.

2. Construction of 312 pb Pvul-to-EcoRI PGK promoter fragment μg pPGK-39 (Fig. 13) was digested simultaneously with Sall and Xbal (5 units each) and then subjected to electrophoresis in 6 percent gel. consisting of 390 pb and containing a 39 bp promoter moiety was isolated by electroelution. The resuspended DNA was subjected to restriction with Sau 3 A and then electrophoresed on an 8 percent acrylamide gel. The 39 pb PGK promoter band was isolated by electroelution. This DNA contains 39 bp from the 3 'end of the PGK promoter on the Sau3A - to-Xbal fragment.

gg pB1 is subjected to restriction with Pvul and Krp1, then subjected to electrophoresis in 6 percent acrylamide gel. The 0.8 kbp band of DNA is isolated by electroelution, then restriction with Sau3A and electrophoresis at 6 percent acrylamide gel. The 265 pb band from the PGK promoter (Fig. 11) was isolated by

electrical elution.

This DNA was then bound (ligated) to the 39 pc promoter fragment of the above structure for two hours at room temperature. The ligator mixture was restricted with Xbal and Pvul, then electrophoresed on a 6 percent acrylamide gel. The Xba-to-Pvul restriction fragment of size 312 bp was isolated by electroelution, then added to the ligation mixture containing 200 ng pBR322 (41) (isolated earlier, omitting 162 pb Pvul-to-PstI restriction fragment) and 200 ng from Xbal-to-PstI LelF A cDNA gene isolated earlier than 20 gg pLelF trp A 25 .This three-component ligator mixture is used to transform E. coli strain 294 into tetracycline resistant. Transforming clones are mini-screened (44) and one of the colonies, pPGK-300 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 '. Thus, the fusion of the XbdI site from the PGK promoter fragment in this sequence allows one EcoRI site to be added to the Xbal site. Thus, pPGK -300 contains a portion of the PGK promoter isolated in the Pvul to - EcoRI fragment.

3. Construction of 1500 pb EcoRI-to-EcoRI PGK promoter fragment.

pg pB1 was digested with Pvul and EcoRI was passed through a 6 percent acrylamide gel. 1.3 kb Pvul - to- EcoRI DNA Strip

from the PGK 5 'flanking DNA was isolated by electroelution. 10 pg pPGK-300 was digested with EcoRI and Pvul and the 312 pb 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 treated with bacterial alkaline phosphatase at 68 ° C for 45 minutes. After three extractions of phenol / chloroform DNA, precipitation with ethanol and resuspended in 20 ml of H2O, 200 ng the vector was ligated with 100 ng of 312 pb EcoRI - to - Pvul DNA from pPGK - 300 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. This plasmid contains a 1500 bp PGK promoter fragment such as the EcoRI - to EcoRI or Hindlli - to - EcoRI portion of DNA.

gg of pPGK-1500 was completely digested with Clal and EcoRI, after which the digested mixture was electrophoresed on a 6 percent acrylamide gel. The 900 pb fragment containing the PGK promoter was isolated by electroelution. 10 gg of pIFN-γ trp 48 was completely degraded with EcoRI and Hindi and electrophoresed on a 6 percent acrylamide gel. A 900-pb band containing directly expressed IFN-γ cDNA was isolated from the gel by electroelution.

The yeast expression vector was constructed in three factor reactions by ligation together of the PGK promoter fragment (in the Clal to EcoRI moiety), deletion. pFRM-31 and IFN-γ DNA isolated above. The reaction mixture was incubated at 14 ° C for 12 hours, then used to transform E. coli strain 294 into ampicillin resistant. Transformants were assayed for the presence of a truly engineered expression plasmid, pPGK - IFN - γ (Fig. 16).

Plasmids containing expression systems have been used to transform the spheroplasts of the yeast strain RH218 to tryptophan prototrophic in non-tryptophan agar. These recombinant yeasts are then tested for the presence of recombinant human immune interferon.

Yeast extracts were prepared as follows: Ten ml cultures were grown in YNB + CAA to reach A 660 ~ 1 - 2, collected by centrifugation, then resuspended in 500 μΐ PBS buffer (20 mM NaH2PO4, pH = 7.4 , 150 mM NaCl). I add

an equal volume of glass beads (0.45 - 0.5 mm) was added and the mixture was then swirled for 2 '. The extract was rotated for 30 seconds at 14000 rpm and the supernatant removed: interferon activity in the supernatant was determined to be 16,000 units / ml by comparison with the IFN-α standard using the CPE inhibition assay.

M. Construction of the cell culture vector pSVy69

The 342-pair HindIII-PvuII base fragment enclosing the SV40 source was converted into an EcoRI restriction-bound fragment. The Hindlll site is converted by the addition of a synthetic oligomer (5 'dAGCTGAATTCC) and the PvuII site is converted by blind linking to

EcoRI site filled using polymerase I (Klenow fragment).

The resulting EcoRI fragment was inserted into the EcoRI site of pML-1 ($ 2). A plasmid with an SV40 late promoter oriented away from the amp R gene was further modified by deletion of the EcoRI site near the amp R gene of pML-28 (28).

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

In the remaining EcoRI site, the IFN-γ gene is inserted into 1250 base pairs of the PstI fragment of p69 after conversion of the PstI ends into EcoRI ends. The clones in which the SV40 late promoter precedes the IFN-γ structural gene were isolated. The resulting plasmids are then introduced into tissue culture cells (29) using the DEAEdextran technique (61) modified so that transfection in the presence of DEAE-dextran proceeds for 8 hours. The cell medium was changed every 2-3 days. 200 microliters are collected daily for interferon bioassay. The typical yield of 50-100 units / ml per sample is established three or four days after transfection.

The transfection product lacks the CYS-TYR-CYS N-terminal portion of recombinant human immune interferon (cf. Fig. 5), which supports the emergence of degradation of the signal peptide at CYS-GLY binding (amino acids 3 and 4 in Fig. 5) , so that the mature polypeptide actually consists of 143 amino acids.

Ν. Partial purification of des-CYS - TYR - CYS recombinant human immune interferon

To obtain more des - CYS - TYR - CYS recombinant human immune interferon, a fresh monolayer of COS-7 cells in ten 10 cm plates was transfected with a total of 30 pg pDLIF3 in 110 mis DEAE-Dextran (200 pg / ml DEAE Dextran 500,000 MW, 0.05M Tris pH 7.5, in DMEM) After 16 hours at 37 ° C. the plates were washed twice with DMEM. To each plate was added 15 mis fresh DMEM, replaced with 10 percent f.b.s., 2 mM glutamine, 50 μ / μΐ penicillin G and 50 Mg / μΐ streptomycin. The medium was replaced the following day with

serum free of DMEM. The serum free medium is then added daily. The collected medium was stored at 4 ° until examined or coupled with CPG. The fractions of day 3 and day 4 of the posttransfection samples were found to contain substantially all of the activity.

0.5 g CPG was added to 100 μΐ cell supernatant and the mixture was stirred at 4 ° C for 3 hours. After brief centrifugation, the precipitated pellets were placed in a column and gently washed with 20 μΜ NaPO4 IM NaCL 0.1 percent β-mercaptoethanol at pH 7.2. The activity was then eluted by addition of the same buffer containing 30 percent

ethylene glycol, followed by further elution with the above buffer containing 50 percent ethylene glycol. All activity was associated with CPC. It was found that 75 percent of all activity was in fractions eluting with 30 percent ethylene glycol. These fractions were poured and diluted with 20 mM NaPO4 1 M NaCl pH 7.2 to a final concentration of 10 percent ethylene glycol and directly applied to a 10 ml Con A Sepharosa (Pharmacia) column. After thorough washing with 20 mM NaPO4 IM NaCl pH 7.2, the activity was eluted with 20 mM NaPO4 IM NaCl 0.2M a - 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-methylD-mannose.

PHARMACEUTICAL COMPOSITIONS

The compounds of the present invention may be incorporated by known methods into pharmaceutical compositions wherein the human immunointerferon product is combined with pharmaceutically suitable carriers. Suitable carriers and their forms are described in Remington's Pharmaceutical Sciences by E.W. Martin, as this source is included in the accompanying references. Such compositions would contain an effective amount of interferon protein together with an appropriate amount of agents necessary for the preparation of pharmaceutical compositions suitable for effective administration to the host.

A. Parenteral administration

Human immune interferon can be administered parenterally in subjects in need of antitumor or antiviral treatment and in those with signs of immunosuppression. Doses and routes of administration may be those used recently

in clinical studies of other human interferons, such as about (1-10) χ 10 ⁶ units per day, and in the case of materials with a purity higher than 1 percent, e.g. 50 χ 10 ⁶ units per day. Doses of IFN-γ can be significantly increased for greater effect due to the essentially lack of human proteins, as an example of a suitable dose for substantially homogeneous parenteral IFN-γ, 3 mg IFN-γ with specific activity. of, for example, 2 χ 10 ⁶ U / mg can be dissolved in 25 ml of 5N serum albumin (human) - USP, the solution passed through a bacteriological filter and the filtrate distributed into 100 tubes, each containing 6 χ 10 ⁶ units of pure interferon suitable for parenteral administration. The tubes should preferably be stored cold (- 20 ° C) before use.

DATA FROM BIOLOGICAL TESTS

A. Characterization of antiviral activity

To neutralize the antibodies, samples were diluted, if necessary, to a concentration of 500-1000 units / ml with PBS-BSA. Equal volumes of samples were incubated for 2-12 hours at 4 degrees with serial dilutions of rat anti-human leukocytes, fibroblasts, or immunointerferon antiserum. Anti-IFN-α 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 for 3 minutes at 12,000 x g 3 minutes before testing. For stability testing at pH 2, samples were brought to pH 2 by the addition of IN HCl, incubated for 2 - 12 hours at 4 ° and neutralized by the addition of IN NaOH before testing. To investigate the susceptibility of dodecyl sulfate (SDS), samples were incubated with an equal volume of 0.2 percent SDS for 2-12 hours before the test.

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

Anti-virus activity (units / ml) E. coli W3110 / COS -7 cellular pIFN-γ trp48 pSVy69 Treatment IFN's IFN-β IFN-γ extract the supernatant net- 375 125 250 250 62.5 tyrants pH 2 375 125 <6 <12 <4 0.1 375 - <4 <8 - percentage SDS rats <8 125 250 250 187 anti IFN's rats 375 <8 187 250 125 anti IFN-β rats 375 125 <4 <8 <4 anti IFN-γ

This table shows the characteristic behavior of IFN-α, β and y standards after various treatments. The interferon activity obtained from E. coli W 3110 / pIFN-γ trp 48 and via COS-7 / pSVy69 is acid-sensitive, SDS-sensitive and neutralized by immunointerferon antiserum. It is not neutralized by IFN-α or β antibodies. These data confirm that the products produced in these systems are immunointerferons and that the plasmid p69 insert cDNA encodes

The immunointerferon protein of the invention was determined by a deterministic DNA gene and deductive amino acid matching - FIG. 5. It is clear that for this particular interferon covered herein, natural allelic variants exist and occur from individual to individual. These variants may be demonstrated by amino acid differences in all sequences or by deletions, substitutions, insertions, inversions or additions of one or more amino acids in a given sequence. All such allelic variants are within the scope of this 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 (13)

Patent Claims 1. A transformed microorganism or cell culture transformed with a replicating expression agent capable of expressing into a given microorganism or a given cell culture a polypeptide comprising the amino acid sequence of des-CYSTYR-CYS recombinant human immune interestferon. The cell culture of claim 1, which is a transformed COS line of monkey kidney fibroblasts. A transformed microorganism or cell culture transformed with a replicating expression agent capable of expressing in a given microorganism or cell line a polypeptide comprising the amino acid sequence of human immune interferon. A microorganism according to claim 3 or 1, representing a transformed strain of E. coli. A microorganism according to claim 3 or 1, representing a transformed yeast strain. A transfused microorganism or cell culture according to claims 3 or 1, wherein said polypeptide has a signal peptide adjacent to the N-terminal portion of said interferon. A transformed microorganism or cell culture according to claims 3 or 1, wherein the polypeptide is free of the signal peptide. 8. A replicating expression agent capable of expressing a polypeptide comprising the amino acid sequence of des-CYS-TYR-CYS recombinant human immune interferon in the transformed microorganism or cell culture. A replicating expression agent capable of expressing into a transformed microorganism or cell culture a polypeptide comprising the amino acid sequence of recombinant human immune interferon. The expression agent of claim 8 or 9, wherein said polypeptide has a signal peptide adjacent to the N-terminal portion of said interferon. The expression agent of claim 8 or 9, wherein said polypeptide is free of the signal peptide. 12. A cell culture transformed with a recombinant expression vector capable of expressing a polypeptide with the following amino acid sequence: GLN 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 PHE LEU GLIILE LEU LYS ASN TRP LYS GLU GLU 40 50 SER ASP ARG LYS ILE MET 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 GLYS LU ASP MET ASN 80 90 VAL LYS PHE PHE ASN SER ASN LYS LYS LYS ARG ASP ASP 100 PHE GLU LYS LEU THR ASM TYR SER VAL THR ASP LEU ASN 110 VAL GLN ARG LYS ALA ILE HIS GLU LEU ILE GLN VAL MET 120 130 ALA GLU LEU SER PRO ALA ALA LYS THR GLY LYS ARG LYS 140 ARG SER GLN MET LEU PHE ARG GLU ARG ARG ALA SER 143 GLN. © 13.Culture of transformed cells capable of expressing a polypeptide comprising the amino acid sequence: 1 10 CYS TYR CYS GLN ASP PRO TYR VAL LYS GLU ALA GLU ASN 20 LEU LYS LYS TYR PHE ASN ALA GLY HIS SER ASP VAL ALA _ ASP ASN GLY THR LEU PHE LEU GLY ILE LEU LYS ASN TRP 40 50 LYS GLU GLU SER ASP ARG LYS ILE MET 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 100 ARG ASP ASP PHE GLU LYS LEU THR ASN TYR SER VAL THR 110 ASP LEU ASN VAL GLN ARG LYS ALA ILE HIS GLU LEU ILE 130 GLN VAL MET ALA GLU LEU SER PRO ALA ALA LYS THR GLY 140 LYS ARG LYS ARG SER GLN MET LEU PHE ARG GLY ARG ARG 146 ALA SER GLN.