Human immunizing Interféron.

The present invention relates to the field of recombinant DNA technology, to the means and methods utilizing this technology in the discovery of the DNA sequence and the deduced amino acid sequence for human immune interferon, its production and to various products of the production as well as uses thereof.

More particularly the present invention relates to the isolation and identification of DNA sequences encoding the human immune interferon vehicle design and recombinant DNA expression containing said DNA sequences operatively linked to the promoter sequences by performing the expression and thus constructed expression vehicles. In another aspect, the present invention relates to culture systems host, as various cultures of microorganisms and vertebrate cells transformed with such expression vehicles and thus facing the expression of DNA sequences reported above. In yet other aspects, the present invention relates to the means and methods of converting end products of this expression into new entities, as pharmaceutical compositions, useful for prophylactic or therapeutic treatment of humans. In preferred embodiments, the present invention provides particular expression vehicles which are expediently sequenced for that there is production and human immune interferon secretion by the host cell in the mature form. Further, the present invention relates to various methods useful for manufacturing these DNA sequences, these expression vehicles, these culture systems and host of these end products as well as their entities, and their specific embodiments and related.

The present invention is due in part to the discovery of the DNA sequence and the deduced amino acid sequence encoding human immune interferon. In addition the present invention provides a sequential information flanking sequences on the 3 'and 5' of the human immune interferon gene, facilitating its bind in in vitro in the expression vehicles. In particular it provides the segment 5' DNA encoding the polypeptide putative endogenous signal that immediately precedes the amino acid sequence of mature human putative immune interferon. These findings in turn have allowed development to means and methods for the production, via recombinant DNA technology, sufficient amounts of human immune interferon turn to permit the determination of its biochemical properties and its bioactivity.

The publications and other materials thereof used to elucidate finishing method, and in particular cases, to provide additional details regarding its practice, are incorporated herein by reference and, for convenience, they bear a reference number in the text which will follow and are respectively grouped in the appended bibliography.

The human interferons can be classified into three groups based on the antigenicity different and on the biological and biochemical.

The first group comprises a family d * leukocyte interferons (has - interferon, LelF or IFN-α), which normally are produced mainly by constituent cells of human blood by a viral induction. They were produced by the microbial and found to biologically active (1, 2, 3). Their biological properties have suggested their clinical use as therapeutic agents for the treatment of viral infections and malignant conditions (4).

In the second group is the fibroblast interferon (6-interferon, IWF or IFN-b receptor), normally produced by fibroblasts by viral induction, which was also produced by the microbial and which shows a broad spectrum of biological activities (5). Clinical trials also indicate its potential therapeutic value. Interferons leukocyte and fibroblast have similarities very sharp in their biological properties despite the fact that the degree of homology at the amino acid is relatively low. In addition, the two groups of interferons contain 165 to 166 amino acids and proteins are stable to acids.

Human immune interferon (therein - interferon, human gamma interferon or IFN-y), to which the present invention applies, is, in contrast to interferons and 6, leaving at pH 2, produced primarily by inducing lymphocyte mitogen and is also significantly antigenically distinct from. Human immune interferon up recently could not be detected that at very low levels, thereby opening hinder its characterization. Recently, a purifying quite extended, although still partial, human immune interferon has been reported (6). The compound would be produced from cultures of lymphocytes stimulated with a combination of phytohaemagglutine and a phorbol ester, and purified by sequential chromatographic separations. This procedure resulted in a product having a molecular weight of 58,000.

Human immune interferon was produced in very small quantities by translation of mRNA into oocytes, exhibiting interferon activity characteristic of the human immune interferon and giving the hopes that the immune interferon cDNA could be synthesized and cloned (7).

The amount of interferon immune achieved hitherto is certainly insufficient to perform experiments unambiguous on the characterization and biological properties of the purified component. However, studies the in vitro executed with raw preparations, as well as experiments vivo with preparations of murine gamma-interferon, suggest that the primary function of immune interferon can be as an immunoregulatory agent (8, 9). Immune interferon has not only an antiviral activity and antioellulaire in common with all human interferons, but it shows a potentiating effect on these activities with the DNA - and the 0 - interferon (10). Similarly, the antiproliferative effect the in vitro y interferon on the tumor cells is about 10 to 100 indicated times that of the other classes of interferon (8, 11, 12). This result, together with its role immunoregulator (8, 9) pronounced antitumor activity suggests much more pronounced for the ifn-Y-that for 1' INF - a and the ifn-a 6. In fact, experiments vivo with mouse and preparations of ifn-Y-murine show a marked superiority over the interferon induced antiviralement in its antitumor effect against the osteogenic sarcoma (13).

All of these studies, until the present invention, had to be performed with preparations quite coarse, due to the very low availability. However, they suggest certainly biological functions very important immune interferon. Not only immune interferon has antiviral activity associated with powerful, but probably a strong antitumor and immunomodulatory activity, indicating clearly a very promising clinical candidate.

It has been perceived that 1" application of recombinant DNA technology would provide a pathway for more efficient production of greater quantities required human immune interferon. The materials formed that do or do not comprise glycosylation which is considered the characteristic of the native material derived from humans, they would probably have bioactivity that admits their clinical use in the treatment of a variety of conditions or viral diseases, neoplastic and immunosuppressed.

The recombinant DNA technology has reached a certain sophistication in age. The molecular biologists are able to recombine various DNA sequences with relative ease, by creating new entities DNA capable of producing quantities of exogenous protein product copiers in microbes transformed. The means and methods are general in hand for ligation the in vitro a variety of DNA fragments to blunt end or "sticky", producing powerful expression vehicles for use in the transformation of particular organisms, thereby orienting their efficient synthesis of the product desired exogenous. However, on the basis of a single product, the path remains somewhat tortuous and science has not progressed to a point at which predictions can be made regular success. In reality those which the spell positive results without the base experimental, do so with a considerable risk of inoperability.

The plasmid, which is a non-chromosomal loop of dsDNA encountered in bacteria and other microbes, sometimes in multiple copies per cell, remains a base member die the recombinant DNA technology. Is included in the information encoded in the DNA plasmid, that required for reproducing the plasmid in the daughter cells (i.e. an origin of replication or "replicon") and typically one or more phenotypic characteristics of selection as, in the case of bacteria, antibiotic resistance, which allows the clones of the host cell containing the plasmid in question be recognized and preferentially grown in selective media. The utility of plasmids is that they can be selectively cleaved by one or the other restriction endonuclease or "restriction enzymes", each of which recognizes a different site on the DNA plasmid. Subsequently, heterologous genes or fragments of heterologous genes can be inserted into the plasmid by joining end-to-end to the cleavage site or ends reconstructed adjacent to the site of the cleavage. The thus formed are called replicable expression vehicles. The recombinant DNA is carried out outside the cell, but the replicable expression vehicle "a recombinant" resulting, or plasmid, can be introduced into cells by a process known as transformation and obtained large quantities of recombinant carrier obtained by culturing the transformant. Further, when the polymeric gene is inserted in appropriate relation to those portions of the plasmid which govern the transcription and translation of the message DNA encoded, the vehicle resulting expression can be used to produce the polypeptide sequence for which effectively the inserted gene encodes, process called the expression.

Expression is initiated in a region known as the promoter that is recognized by and bound by RNA polymerase. In the phase of transcription of the expression, the DNA is carried out by exposing it as a template for the primed synthesis of messenger RNA from the DNA sequence. Messenger RNA is in turn translated into a polypeptide having the amino acid sequence encoded by the mRNA. Each amino acid is encoded by a triplet of nucleotides or " start codon¹¹ collectively called the "structural gene", it is to say the part which encodes the amino acid sequence of the expressed polypeptide product. The translation is initiated at a "start" signal (ordinarily of sequential, in messenger RNA resulting becomes AUGs). The codons said stop define the translation termination and thus of the production of other amino acid units. The resulting product can be obtained by lysis, if so required, the host cell in microbial systems and recovering the product by a convenient purification from other proteins.

In practice, the use of recombinant DNA technology can express fully the heterologous polypeptides - so-called direct expression - or can express a heterologous polypeptide fused to a portion of the amino acid sequence of a homolog polypeptide. In the latter cases, the bioactive product sought is sometimes rendered bioinactif in the homologous polypeptide fused heterologous /, until it is cleaved extracellular environment. Referring to British Patent no. 2,007 676a and Wetzel, new species in American 68, 664 (1980).

The art cultures of cells or tissues for the study of the genetics and cellular physiology is well established. Is at hand means and methods are provided for maintaining permanent cell lines, prepared successive transfers in series from normal cells isolated. For use in researches, these cell lines are held on a solid support in a liquid medium, or by growth in suspension which contains nutrients support. Scaling to large preparations seems not to place than mechanical problems. As additional knowledge base represents an Microbiology, 2èoe editing, Harper and rowwise, Newspaper, Inc. in Hagerstown, in Maryland (1973), especially pages 1122 and following, as well as at both of which 245, (1981) 66 and following, publications which are incorporated herein by this reference.

The present invention is based on the discovery that recombinant DNA technology can be used to successfully produce human immune interferon, preferably in the form of direct, and in amounts sufficient to initiate and conduct tests on animals and clinical as a prerequisite to approval on the market. The product is suitable for use, in all of its forms, in the prophylactic or therapeutic treatment of humans for viral infections and malignant conditions, immuno3upprimées or immunodeficient. Its possible forms include various forms oligomers which can include glycosylation associated. The product is formed by cell culture systems or microorganisms genetically constructed. Therefore exists thus maintaining the possibility of preparing an isolating human immune interferon as a more effective than has been possible heretofore. A significant factor in the present invention, in embodiments which 3ont especially preferred, is the accomplishment of the direction gene of a microorganism or cell culture for the production of interferon immune human in amounts isolatable, secreted by the host cell in the mature form.

The present invention comprises human immune interferon produced as do the means and the methods for its production. The present invention is further directed towards expression vehicles replicable DNA harboring gene sequences encoding human immune interferon in expressible form. In addition the present invention faces strains of microorganisms or cell cultures transformed with the expression vehicles described above and to microbial cultures or cell strains or transformed cultures, able to produce human immune interferon. In yet other aspects, the present invention is directed towards various methods useful for the preparation of said sequences of immune interferon genes, of mediated DNA expression, strains of microorganisms and cell cultures and towards their specific embodiments. Further, the present invention is directed towards the preparation of fermentation cultures of these microorganisms and cell cultures. Further the present invention is directed to the preparation of human immune interferon, as direct expression product, secreted by the host cell in the mature form. This approach may use the gene encoding the sequence of the human immune interferon recessed wall flanking the DNA to 5' signal encoding the polypeptide. The polypeptide signal is believed to assist the transportation of the molecule to cell wall organisms Kay's host, where it is cleaved during the secretion of the mature human interferon product. This embodiment allows the isolation and purification of the desired mature immune interferon without using procedures involved which are designed to remove contaminants from the intracellular protein of the host or cellular debris.

The need here to the expression "mature human immune interferon" signifies the microbial production or by cell culture of human immune interferon unaccompanied by the signal peptide or peptide presequence which immediately preceding the mRNA translation of human immune interferon. Mature human immune interferon, according to the present invention is thus provided, with, as its first amino acid, methionine (present due to the insertion of the codon start signal TGA in front of the structural gene) or, when methionine is cleaved an intra - or extracellularly, with, as its first amino acid, cysteine normally. Mature human immune interferon can also be produced, in accordance with this, together with a conjugated protein other than conventional signal the polypeptide, the conjugate being specifically cleavable in the intra - or extracellular environment. See in this connection the British Patent no. 2,007 676a. Finally, mature human immune interferon may be produced by direct expression, without the need to disconnect any polypeptide cleavage by unnecessary foreign. This is particularly important in the event that a given host cannot, or at least not efficiently, eliminating a peptide signal in case the expression vehicle for expressing human interferon along with its mature peptide. the signal. Mature human immune interferon thus produced is recovered and purified to a level suitable for use in treating viral conditions, malignant, immunosuppressed or immunocompromised.

Human immune interferon is obtained in accordance with the following:

1. human tissues, for example fabric human spleen or peripheral blood lymphocytes, are cultured with mitogen to stimulate production of immune interferon.

2. Cell pellets are extracted from said cell cultures in the presence of a ribonuclease inhibitor for isolating HRO cytoplasm.

3. A column oligo dT isolates messenger RNA (mRNA) as polyadenylated. This mRNA is fractionated to size using a sucrose density gradient gel electrophoresis and acid urea.

4. The appropriate mRNA (12 to 18 seconds) is converted to the corresponding single-stranded complementary DNA (cDNA) from which is output two-strand cDNA. After stem stretch (tailing off) po Iy-to-DC voltage, it is inserted into a vector such as a plasmid carrying one or more phenotypic markers.

5. The vectors thus prepared are used to transform bacterial cells by providing a "library" of colonies. Used radiolabeled cDNA prepared from mRNA both induced and uninduced, from whose composition described above, to explore separately "libraries" of colonies in duplicate. The cDNA in excess is then removed and the colonies are exposed to X-ray film, so as to identify the ο οη℮ 1 3 cDNA induced.

6. From cDNA clones induced, plasmid DNA is isolated from corresponding and it is sequenced.

7. The DNA sequenced is then custom the in vitro for insertion in a suitable expression vehicle which is used to transform a suitable host cell, which in turn permits the growth in culture and expression of the human immune interferon product desired.

8. Human immune interferon thus produced has 146 amino acids without intellectually in its mature form, starting with cysteine, and it is strongly basic character. Its molecular weight state of monomer was calculated as being of ^ ΐ. 40. Perhaps because of the presence of many basic residues, hydrophobicity, the formation of salt bridges and so on, the molecule is capable of associating with itself in oligosères shapes, for example in the form dimer, trimer, or tetramer. The high molecular weights previously observed with the natural material (6) who cannot enter into consideration on the basis of the amino acid sequence alone, may be due to these forms oligomers as well as the contribution of carbohydrate from posttranslational glycosylation.

9. In some systems of host cells, particularly in case of ligation in a vehicle D.¹ expression so be expressed together with peptide thereof "the signal" of 20 aminoaeides, the mature form of human immune interferon is exported into the cell culture medium, thereby facilitating incommensurableraent methods for recovery and purification.

Cell cultures/microorgani3mes

1. Bacterial strains/promoters

The jobs described herein are performed by employing, inter alia, the microorganism Ξ. Escherichia coli K 12 strain 294 end has, ethi ", HSR STIELL",_{the K} the HSM⁺ , as described in British Patent no. 2055382 has, this strain has been deposited at 1' American Type culture, as ATCC no. 31446. However various other microbial strains are usable, whose known strains of e. coli as the e coli b-, the E.coli 1776 (aîcc N.^I 3153?) and the e coli W. 3110 (F. ", 1", protophic) (A.T.C.C. no. 27325) or other microbial strains which many of them are deposited and (potentially) available from institutions deposits of microorganisms recognized, as 1' American Type culture (from ATCC) - OC. the listing as ATCC catalog. See also German Patent 2644432 of-bone. These other microorganisms comprise for example the bacilli bacteria such as bacillus subtilis and other Enterobacteriaceae among which may be mentioned as examples the Salmonella and Serratia marcesans, using plasmids that can replicate and express the heterologous gene sequences therein.

As examples, the systems promoters to beta lactamase and lactose were advantageously used to initiate and sustain the microbial production of heterologous polypeptides. Details relating to the manufacture and construction of SSO systems promoters have been published by Chang and Ecoli ., 275 in nature, 617 (1978) and willwill itakura coli-and -. Forensic science 198, β (1977) 105, which are incorporated herein by reference. Most recently was developed a system based on the tryptophan, the promoter system called TRPs. Details relating to the manufacture and construction of this system have been published by Goeddet et, Nucleic Large Research in 8, 4057 (1980) and by Kleid and eoll ., request U.S. Patent no. 133" 296, deposited 24 March 1980, which are incorporated herein as a reference. Many other microbial promoters have been discovered and used and details of their nucleotide sequences, enables a worker to functionally link within the plasmid vectors, have been published -, FS. for example Siebenlist et, Glial cell 20, 269 (1980), which is incorporated herein by this reference.

2. Yeast strains/yeast promoters

Their expression system may also be employed YRp7 plasmid (14, 15, 16), which is capable of selecting and replication in both the e. coli and in yeast Saccharornyces cerevisiae strains. For selection in yeast plasmid contains the gene of Trp (14, 15, 16) which complements (permits the growth in the absence of tryptophan) yeast containing mutations in this gene found on chromosome IV (17) yeast. The strain used here is the strain rh218 (18) deposited to 1' American Type culture^: (a.T.C.C. no. 44076) without restriction. However it will be appreciated that any Saccharomyces cerevisiae strain containing a mutation that causes the trpT cell will be an environment effective to 1' expression plasmid containing the expression system. An example of another strain which could be used is the ρ℮ρ 4 - 1 (19). This auxotrophic strain of tryptophan also has a point mutation in the gene of Trp 1.

Being placed on the side 5 'of a non-yeast gene, the DNA sequence flanking 5' (promoter) of a yeast gene (for alcohol dehydrogenase 1) can promote the expression of a foreign gene in yeast when placed in a plasmid used to transform yeast. Side promoter, convenient expression of a gene non-yeast in yeast requires a second sequence of yeast at the end 3' of the non-yeast gene on the plasmid to allow proper termination of transcription and polyadenylation in yeast. The promoter can be suitably used in the present invention as well as other - see infra. In preferred embodiments, the flanking sequence 5 * gene 3 a-yeast phosphoglycerate kinase (20) is placed upstream of the structural gene followed again by 1' DNA containing signals except-terminator polyadényla, for example the gene of Trp (14, 15, 16) or gene of the PGK (20).

Because the flanking sequence 5 'yeast (together with termination DNA yeast * 3) (infrared) may intervene to promote the expression of foreign genes in yeast, it appears likely that flanking sequences 5' of any yeast gene highly expressed could be used for the expression of the relevant gene products. As in some circumstances the yeast expresses due to 65 % its soluble protein has the state of glycolytic enzymes (21) and as this high level appears to result from the production of high levels of mRNA (22) individual, it might be possible to use the flanking sequences 5 'of any other glycolytic genes for of' * expression such applications " such as enolase, . glyceraldehyde-to-3 phosphate dehydrogenase, hexokinase protein, a pyruvate decarboxylase inhibitors, of phosphofructokinase, the glucose-concerns phosphate isooérâse, 3 a-phosphôglycérate mutase is, a pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase and glucokinase. Any flanking sequences 3' of these genes could also be. used for proper termination and polyadenylation of mRNA in such an expression system - εf. above. Certain other genes highly expressed are those for acid phosphatases (23) and those that express high levels of production due to mutations in the regions flanking 5' (mutants that increase the expression) - following ordinarily to the presence.; a transposable element (24) ty1.

All genes mentioned above are believed to be transcribed by RNA polymerase II (24) yeast. It is possible that the promoters for RNA polymerase I and III, which transcribe the genes for tRNAs, RNA 5s and ribosome RNA (24, 25), can also be usable in such expression constructs.

Finally,, many yeast promoters also contain a transcription control, from which they can be closed or opened by varying the growth conditions. Some examples of yeast promoters are the genes that produce the following proteins alcohol dehydrogenase II-τ, isocytochrome c ., acid phosphatase, the degrading enzymes associated with nitrogen metabolism, glyceraldehyde-to-3 phosphate dehydrogenase, and enzymes responsible for maltose and use. (22) galactose. This control region could be very useful in the control of the expression of protein products especially when their production is toxic to the yeast. It could also be possible to set up the control region of a flanking sequence 5' flanking sequence with a 5 * containing a promoter of a gene highly expressed. It would result in a hybrid promoter, this being possible since the control region and the promoter appear constitute DNA sequences physically distinct.

3. Cell culture systems/vectors of cell cultures.^{the R}

The propagation of vertebrate cells in culture (tissue culture) has become a way of " VïïîseæsssssTiLæssaSorters .,, irstemiLsrress. -, editors Kruse and by Patterson, 19t3). The cell line used and the cos-to-7 monkey kidney fibroblasts as a host for the production of interferon immune (25a). However, the experiments detailed herein can be performed in any cell line capable of replication and expressing a compatible vector, for example wi3s, BHK cells, 3t3, CHO-, Vero and of Hela cell lines. Furthermore, what is required of the expression vector is a replication origin and a promoter located faoe of the gene to be expressed, along with ribosome binding sites necessary, splice sites or splice (splice donor) RNA, a polyadenylation site and transcription termination sequences " although these essential elements of sv40 have been exploited here, it will be understood that, although described herein in terms of a preferred embodiment, is not to be taken as limited to SSO sequences. Thus, one can use the replication origin of other viral vectors (e.g. Polyoma, the adeno, VSVs, BPV, etc) die same as cell origins of DNA replication which could operate in, a state non-integrated,

Vector systems^;

T direct expression of innate immune interferon in the e coli

The procedure used to obtain a direct expression of ifn - Y-e eoli state of interferon polypeptide wall (least sequence of signed) is a variant of one employed previously for human growth hormone (26) and for human leukocyte interferon (1), in the since it comprises the combination cDNA and synthetic n-terminal components.

Gum is the deduced from the nucleotide sequence of the pô9 described infrared, and comparison with the known cleavage site between the peptide "signals" and for more mature polypeptide * (2) IFN-α, the ifn Y has a hydrophobic signal peptide of 20 amino acids followed by ifn - 146 amino acids therein (fig. 5) wall. Gum shown in Figure 7, a restriction endonuclease site BstNI is located appropriately to amino acid 4 - IFN γ mature. Two synthetic deoxyoligonucleotides are constructed, which incorporate a translation initiation codon TGA, codons for amino acids 1, 2 and 3 (cysteine-tyrosine-cysteine) and create the EcoRI cohesive end. These deoxyoligonucleotides are connected by ligation to a fragment BstNI-to-PstI 100 base pairs of p69 for constructing a hybrid gene 1115 to synthetic-natural base pairs which encodes 1' - ifn-Y and which is bound by the restriction sites EcoRI and PstI. This gene is inserted into the plasmid pLelF has a Trp 103 between the EcoRI and PstI to give the expression plasmid pIFN γ-Trp - 48. In this plasmid the gene'd ' INF - γ is expressed under the control of the Trp promoter of the e. coextruded 11. (the pLelF has a Trp 103 is a derivative pLelF has 25 wherein the EcoRI site distal to LelF gene has is removed. The procedure used to remove the EcoRI site was described previously (27)).

2. Expression in yeast

For expressing a heterologous gene such that the DnaE for immune interferon in yeast, it is necessary to construct a plasmid vector containing four components. The first component is the portion that allows the transformation of both, 1 'E.•coli and yeast and which must therefore contain a gene that can be selected from each organism. (In this case, it is the gene for ampicillin resistance of the e. coli and gene trp1 yeast). This component requires also an origin of replication which both organisms to be maintained as ADHs plasmid in both organisms. (In this case it is the original E. coli pBR322 originally ars1 of chromosome III of the yeast).

The second component of the plasmid is a flanking sequence 5' of a yeast gene highly expressed for to pro transcription of a structural gene located downstream. In this case, the flanking sequence 5' used is that of the gene of 3 a-phosphoglyeérate kinase inhibitors (PGK promoter) of the yeast. The fragment is constructed to eliminate the ATG of the sequence of the PGK structure 8 as well as LP (base pairs) upstream of this TGA. This sequence is replaced by a sequence containing a restriction site both Xbal and EcoRI for 1 'proper attachment of the flanking sequence 5' to the structural gene.

The third component of the system is a structural gene constructed so as to contain both a start ATG translation and translational stop signals. The isolation and construction of such a gene are described infrared.

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

All of these components being present, 1' immune interferon was produced in yeast.

3. Expression in mammalian cell culture

The strategy for the synthesis of I ' immune interferon in culturing mammalian cells is based on the development of a vector that is capable of autonomous replication and expression of a foreign gene under the control of a heterologous transcriptional unit. The replication of the vector in the tissue culture is accomplished by providing an origin of replication .'d ' DNA (virus derived sv40) and by providing a help function (T antigen) by introducing the vector into a cell line expressing endogènement this

(28, 29) antigen. The last virus promoter sv40 precedes the ' J.

* gene structure of interferon and provides transcription of the

the V; (I-[ '

. ^.

û '

|the vector used to obtain expression of ifn - Y consists of of pBR322 sequences that provide a selectable marker for selection in the e coli (ampicillin resistance) as well as an origin e. ooli replication. These sequences are derived from the plasmid (28) PML and above 1 and enclose the. spanning the restriction sites EcoRl

•and êdmhi. The origin of the sv40 originates from a fragment Pvu-to-HindlII to 342î base pairs spanning this region (30j 31) (both

ends being ' converted EcoRl ends). These sequences, I-•"..

in addition include 'the viral origin of the'. DNA replication, encodlnt the promoter for both the transcription unit. early and late. The orientation of the region of origin of the

, . jjf.■_{the R} Sv40 is such that the promoter for unit, transcription

late is turned near the gene enoodanfc the I ' :•

interferon..

Figure 1 represents the expression of a centrifugation on a sucrose gradient (the sucrose (5 to 25 %) in dimethyl formamide (70 I-)) poly (has)+ peripheral blood lymphocyte RNA (PBLs) armature. Observed two peaks of interferon activity (as indicated by hatched rectangles) with dimensions of 12s and 16s. The positions of the DNA markers ribosomes (centrifuged independently) are marked above the profile of absorbance.

Figure 2 represents the expression of a poly (has) electrophoresis + RNA induced PBLs, on an agarose gel (j of 1.75) - urea (6m) - acid. There is only one peak of activity migrates along with RNA 18s. The positions of RNA markers ribosomes that have electrophoresed in an adjacent passage and which are displayed by dye ethidium bromide are marked above the activity profile.

Figure 3 shows hybridization patterns of 96 colonies with samples (taken) cDNA labeled induced (upper picture) and non-induced (lower picture). 96 individual transformants which are developed in a microtiter plate, the replicas are extended on two membranes. nitrocellulose and then the filters are hybridized with samples of ^ P âbne prepared either from induced mRNA (top) or mRNA isolated from cultures of PBLs not induced (non-induced, bottom). The filters are washed to remove non-hybridized RNA and then exposed to X-ray film for this set of filters is representative of 86 of these games (8300 independent colonies). Clone is g.^{the n} armature " is tagged h12.

Figure 4 is a map of restriction endonucleases 1' cDNA insert of clone 69. The cDNA insert is bounded by Pstl sites (point at both ends) and DC-to-Dc tails trace (simple lines). The number and size of the fragments produced by restriction nuclease cleavage is estimated by electrophoresis through gels has 6 % polyacrylamide. The positions of the sites are confirmees by sequencing nucleic acids (represented in Figure 5). The coding region of the reading frame most widely open is represented by rectangles and the hatched region represents the peptide sequence putatively "signals" to 20 residues, while the region represents the sequence of mature pointilïée IPD (146 aminoacidesj. The end 5 'of the Arno is left while the end 3' is right.

Figure 5 represents the nucleotide sequence of 1' cDNA insert of the plasmid ρό 9 illustrating however the allelic form the most common of the xfn Y. The deduced amino acid sequence of the open reading frame the longest is also presented. The sequence "signal is" putatively is represented by residues labeled ST 320.

Figure 6 is a comparison of the structure of the ifn-Y-mRNA with the leukocyte interferon (α XFN) and fibroblast (IFN-γ), mRNA of clone 69 (labeled immune) contains amounts much larger untranslated sequences.

Figure 7 is a schematic diagram of the construction of plasmid pXFN-Trp - 48 expression therein of the ifn-Y motion. The starting material is 1' insert to 1250 pairs based Pstl p69 DnaE from plasmid.

Figure 8 shows a. diagram of plasmid used for expression of ifn - oellules Y in the monkey.

Figure 9 shows the Southern hybridization of eight different human genomic DNA digested with EeoRI, hybridized with a fragment to 600 base pairs Ddel labeled ^² P from the cDNA insert of the p69 (69 clones), two EcoRI fragments hybridize clearly when sampling in each DNA sample.

Figure 10 shows a Southern hybridization of DNA. human genome digested with six endonucleases - restrictive

except that have experienced different hybridization with a sample

32

marked the P from p69 (clone to 69).

Figure 11 schematically shows the restriction map of 1' insert 3.1 kbp or FBI HindlII vector which has been isolated from the PGK promoter. Is inserting an EcoRI site and Xbal site in the DNA flanking 5' of the gene of the PGK (yeast).

Figure 12 illustrates the sequence flanking 5' plus the initial coding sequence of the gene of the PGK Xbal prior to insertion and the EcoRI sites. (DNA sequence from the end 5' of the structural gene of yeast PGK and flanking DNA).

Figure 13 illustrates schematically the techniques used for inserting a site Xbal in position 8 of the PGK promoter and isolate a fragment (base pairs) 39bp the flanking sequence 5' of the PGK containing this end XbaI and one end Sau3A linker.

Figure 14 illustrates schematically the construction of a fragment to the fragment containing 300 LP 39bp chamber at_O additional sequence (265bp) flanking 5' of the PGK ranging from Pvul to Sau3A linker (see Figure 11), and an EcoRI site adjacent the Xbal.

Figure 15 illustrates schematically the construction of the promoter fragment of the PGK to 1500 LP (the EcoRI/HindlII) which additionally contains fragment constructed in Figure 14, a fragment HindlIIPvul to 1300 to LP from a flanking sequence 5' (see Figure 11).

Figure 16 illustrates the composition of an expression vector for human immune interferon in yeast, the PGK promoter-containing modified, the cDNA of ifn - ^ and the termination region of the PGK gene of yeast as described in more detail below.

Detailed description

A. a source of mRNA of ifn Y (- Y-IFNs mRNA)

Of peripheral blood lymphocytes (little are derived from human donors by leukophorèse. Lymphocytes PBLs are purified by centrifugation on a Fieoll-to-Hypaque gradient into and are then cultured at a concentration of 5 x 13^cells/ml in a solution of RPMI 1640, 1 j-L-glutamine, 25 mm HEPES and 1 if penicillin-streptomycin (Gibco, developing relatively large, ny is). These cells are induced to produce the ïfn γ - under the action of the Staphylococcus Enterotoxin B mitogen (1 μ / ml to gms) and are cultured 24 hours to 48 37° c in 5 % of COGs, is added désacétylthymosine - has -1 (0.1 Μg/ml.) to the cultures PBLs to increase the relative efficiency of the ifn-Y-activity.

B messenger RNA isolation

Total RNA is extracted from cultures of PBLs essentially Berger whiteness, S.L. and ai (33). The cells are pelleted by centrifugation and then resuspended in 10 mm NaCl, 10 mm tris-HCI(pH of 7 * 5), 1, 10 mm and 5 mm of MgClgribonuoléoside vanadyl complex. The cells are lysed by the addition NP-40 (1 $ final concentration) and the cores are brought into pellets by centrifugation. The supernatant contains all of the RNA which is further purified by multiple extractions at phenol and chloroform. TMs aqueous phase is made 0.2 m NaCl and the total RNA is precipitated by addition of two volumes of ethanol. RNA from cultures not induced (unstimulated) is isolated by the same prooédés. Cellulose chromatography is used to purify the oligo dT mRNA of all RNA preparations (34). The yields of 1 to 2 liters of PBLs cultured are typically of 5 to 10 mg of total RNA of 50 to 200 micrograms and poly (has)+ RNA in total.

C size fractionation of the mRNA

Two methodologies are used to fractionate the mRNA preparations. These methods are used independently rather than in unison and each of which gives rise to a significant enrichment of the mRNA of ifn-Y motion.

The centrifugation on sucrose gradient in the presence of formamide denaturing is used to fractionate the mRNA. Sucrose gradient (5 to 25, 50 in formamide<70 %) (32) are centrifuged to 154,000 g for 19 hours at 20° C. (0.5 ml) successive fractions are then removed from the top of the gradient, precipitated with ethanol and an aliquot is injected into oocytes of Xenopus laevis for translation of the AHNm (35).. After 24 hours at room temperature, the incubation medium is tested for antiviral activity by performing a test of inhibition of cytopathic effect using a standard vesicular stomatitis virus (strain in Indiana) or virus of encephalomyocarditis prendre and cells (human amnion) as described by Stewart platform (36), except that the samples are incubated with the cells for 24 hours instead of 4 and brought into the presence of the virus. Observed constantly two peaks of activity in the RNA fractionated on a sucrose gradient (fig. 1). Peak has a sediment with a size calculated from 100 to 400 12s and contains a U/ml of antiviral activity (compared to a standardized IFN-α). The other IEP Asti " quickly in the sedimentation of 16s in size and contains about half of the activity of the udder sedimentation slower. Each of these peaks of activity appears to be from 1 · ΕΝ ≈ γ 1, because no observable activity whem® the ease with fractions are tested on a bovine cell line (MDBK) unprotected by i'i' ifn-Y-human. Both activities of the ifn-a and of the ifn-to-3 being easily detected by the test (5) KBBK.

The fractionation OD ARMA (200 gms therein) is equal " telling by electrophoresis by using gels of agarose-to-rue © acids. The lamella of agarose gel (37, 38) consists of 1.75 % agarose, 0,025 m sodium citrate, pH of 3.8 and 6 M urea. The electrophoresis hard 7 hours to 25 milliamps and 4®C. the gel is then fractionated with a razor blade. The individual wafers are melted to 70° c and extracted twice with phenol and once with chloroform. The fractions are then precipitated with ethanol and subsequently tested for mRNA of ifn-Y-injection in Xenopus laevis oocytes followed by an antiviral assay. A single peak of activity is observed in samples of gel fractionated (fig. 2). This peak migrates simultaneously with RNA 18s and 600 has an activity of a U/ml per microgram of RNA injected. It appears that this activity is also specific 1 'IFN γ-because it does not provide protection MBDE cells.

The discrepancy in size between magpies activity observed on sucrose gradients (12s and 16s) and on of froze urea acids (18s) to be attributable the observation that these methods of fractionation independent are not applied under denaturing conditions total.

D. preparation of a "library" of colonies containing sequences of ifn Y,

3 (ig mRNA gel fractionated cDNA preparations serve two-strand by customary methods (26, 39). The size of the cDNA is fractionated on a gel to 6 % polyacrylamide. Two fractions are électroéluées, one of 800 to 1500 LP (138 nanograms), the other of > 1500 LP (204 nanograms). Shares 35 ng of cDNA of each size are extended with remnants désoxyC using deoxynucleotidyl transferase inhibitors (an HO) and fixed (annealed) with 300 nanograms of pBR322 (41) that has received a similar termination with residues of désoxyC PstI site at (40). Each mixture is then converted into fixed k12 e coli strain 294. About 8000 transformants are obtained with the cDNA to 800 - 1500 BP and 400 transformants are obtained with the cDNA to > 1500 LP.

E sort of a "library" of colonies for the cDNAs induced *

The colonies are inoculated individually in the

E hollow microtiter plates containing lbs (58) + e/ml tetracycline and GP are at rest to -20^I C. after adding DMS0 to 7 %. Grown two copies of the "library" colony nitrocellulose filters and fixing the DNA of each colony at filter technology (42) Grunstein-to-Hogness.

Samples or specimens cDNA labeled ^ P are prepared using mRNA size 18s split over the gel and from cultures of PBLs with or without induction. The oligo dt is₁₂ " iG is used as amoroe LEEs and reaction conditions have been described previously (1). The filters containing 8000 transforming from the fraction cDNA to 600 - 1500 BP and 400 transformants of the fraction cDNA to > 1500 BP are hybridized with 20 x 10 ^ ^ P-induced cDNA bpm. The set of filters obtained by duplication is hybridized with cDNA of OPA 20 x 10 ^ 32?_filtering armature. Hybridization lasts 16 hours these conditions and Al Fritseh (43). The filters are washed abundantly (43) and then exposed to film for X-ray Camera Xb expression 5 with intensifying screens Doe the discharges plus 16 to 48 hours during. Comparing the hybridization pattern of each colony with the two samples. About 40 % colonies have a clear hybridization with both samples while about 50 % colonies do none hybridization with either sample (see Figure 3). 124 colony hybridize significantly-with the induced sample but undetectable or more weakly with the uninduced sample. €@ e colonies are inoculated individually into the recesses or wells of microtiter plates, they are allowed to grow and OH the transfers nitrocellulose filters and the hybrids (45) with the samples induced and the SR ©. induced. 1 'IDs 22's * 5sybsrms tssloaissseoisses * with the induced sample and gives rise to colonies called' "induced" "

F characterization colonies induced

Plasmid DNA is prepared from 5 (46) induced colony is greatly accelerated and serves cDNA inserts characterization. The establishment of the restriction endonuclease in the case of five plasmids induced (ρ 67, ρ β 8, ρ β 9, p71 and p72) suggests that four of them have cards similar restriction nuclease. These four plasmids (ρ 67, p69, p71 and p72) each has four Ddelï site, two sites Hinfl and a single site Bsal in .1 ' inlay cDNA. The fifth plasmid {ρ 68) contains a fragment Ddel common and appears as a short cDNA clone in relation with the other four. The homology suggested by the éta-wrinkling card restriction nuclease is confirmed by hybridization. Prepared a sample DNA labeled 32p (47) from a fragment of the LP differently to 600 plasoide ρ β! 7 and used.

for hybridization (42) towards the other colonies induced, all five colonies with restriction nuclease cards were established form hybrids crossed aveo and OEt sample, as well as 17 other colonies among the 124 selected from the sorting between armature/uninduced. The length of 1' insert ADNo in each of these plasmids cross-hybridization ESCs determined by digestion with PstI and gel electrophoresis. The clone with the longest insert ADNo appears to be the clone 69 aveo insert a length of 1200 - 1400 LP. This DNA is used for all other experiments and his card restriction endonuclease is shown in Figure 4.

It is demonstrated that 1' insert in the ADNo p69 is the ADNo ifn - γ by its expression product, product in three independent expression systems, producing an antiviral activity, as described in more detail below.

G sequence analysis of 1 'cDNA insert of the p69 the full nucleotide sequence of 1' cDNA insert of the plasmid p69 is determined by the method of dideoxynucleotide chain termination (48) aprèis subcloning fragments m13 feed them into the vector, (49) and mp7 chemical process by the Maxam-Gilbert method (52). The open reading frame encodes the longest protein aainoacides 166, shown in Figure 5. The first residue encoded is the first codon puts encountered at the end 5' of the ADNo. The first 20 residues to the amino terminal sequence probably serve "the signal" for secretion of 146 amino acids remaining. This signal sequence possible or putatively has properties in common with other sequences "signal is" characterized as the size ' and hydrophobic character. Further, the four amino acids found in the putative sequence cleavage (Ser Leu glycys) spnt identical to four residues found at the point of cleavage of more interferon leukocyte (LelF B., C., D., F and H, (2)). The amino acid sequence encoded comprising 146 amino acids has a molecular weight of 17,140.

There are two potential positions (50) glycosylation in the second protein sequence encoded amino acids 28 to 30 (of Asn-Gly-Thr) and an amino acid 100 to 102 (-Asn-Tyr-Ser)* the existence of those locations in accordance with glycosylation of ifn - γ human observed (δ, 51). Further the only two cysteine residues (at positions 1 and 3) are stériqueaeat 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 G - mercaptoethanol (51). The sequence of mature aainoacides deduced is generally fully basic with 30 lysine residues, arginine and hystidine in total and only 19 aspartic acid residues and glutamic acid in total.

The structure of ARMA of 1⁹ γ as deduced from the sequence of the ADH OD PKSs © ρ δ 9 is largely different from that of the mRNA of 1 * ifn - (1.2) or with 1 - 1 · ΡΗ (5) receptors. In coma © seen in Figure 6, the coding region of the ïfn-Y is shorter while the untranslated regions 5 'UTR and 3' are much longer than for I ' © XFSba percent the ifm-s-U-.

H direct expression of mature immune interferon in b * Escherichia coli

Referring to Figure 7, 50 μ g of plasmid ρ β 9 are digested with PstI and the 1 * insert to 1250 base pairs is isolated by electrophoresis on a polyacrylamide gel 6 $, about 10 μ g of the insert are éleefcroélues from the gel, 5 P g of the PstI fragment are digested partially with 3 units (the Bethesda Research in predetermined amount) BstNI during 15 minutes at 37®c and the flow medium reaction is purified on a gel 16, 2 polyacrylamide. About 0.5 Pg desired fragment to 1100 base pairs BstHI Pstl is collected. Are synthesized two désoxyoligonueléôti " of, 5 'a-dâattcatgtgttattgtc and 5' a-dTGACAATAACACATG (fig. 7) by applying the method at (53) and phosphotriesfcer their phosphorylation is carried out as follows. 100 pmol of each deoxyoligonucleotide are combined in 30 pL SOaM TrisHCl (pH of 8), ^ MGCs œM of 10, 15 mm β-mercaptoethanol and 240 - Y-IC(γ - ^ Ρ) AFPs (Amersham's, 5000 IC/mmol). Added 12 units 4 Τ polynueléotide kinase and allowed the reaction to progress to C. for 30 min 37°. 1 Μ 1 added ATP 10 Ami and allowed the reaction progress 20 additional minutes. After extraction at 0 OH/CHCl ^ " the olygomères are combined with 0.25 g of the moiety Y-BstNI-to-PstI 1100 base pairs and are precipitated with ethanol. These fragments are ligated to C. for 2 hours in 20° 30 ml of 20 mm TrisHCl (at pH 7.5), 10 mm MgCl₂ , 10 mm dithiothreitol, 0.5 mm ATP and 10 units t4 DNA ligase. The mixture is digested 1 hour with 30 units of EcoRI PstI and 30 units (to remove the polymerization by ligation of cohesive terminal) and undergoes gel electrophoresis 6 % polyacrylamide. The product to 1115 base pairs (110,000 bpm) is reoueili by electroelution.

The plasmid pLelF-Trp(fig. 7) has derived the plasraidepLelF (1) has 25 wherein the EcoRI site distal to the gene LelF has has been removed (27). 3 M-s of pLelF has a Trp 103 are digestion with 20 units EcoRI PstI units 20 and 90 minutes during 37° c and subjected to gel electrophoresis 6 % polyacrylamide. The large fragment (about 3900 base pairs) vector is collected by electroelution. The fragment to 1115 base pair EcoRI PstI DNA of ifn-Y-undergoes a ligation in 0.15 mg of the prepared vector. Transforming the e coli K 12 strain 294 (ATCC designation no. 31446) gives 120 tetracycline resistant colony. The plasmid DNA is prepared from the transformants and 60 EçoRI and Pstl digested with. Three of these plasmids contain the desired fragment to 1115 base pair EcoRI Pstl. The analysis of DNA sequences used to verify that these plasmids have the nucleotide sequence desired to junctions between the Trp promoter, synthetic DNA and cDNA. One of these plasmids pIFN-to-9.ï TRPs 48 was chosen for further study. This plasmid was used to transform the e coli K 12 strain w3110 (A.T.C.C. N.^I 27325).

I gene structure of the coding sequence for 1' - γ IFNs

The structure of the gene encoding the ifn-Y-was analyzed by Southern hybridization. According to this method (5 * 1), is 5 micrograsuaes digesting DNA of human lymphocytes high molecular weight (prepared according to 55) to completion with various restriction endonucleases is treated by electrophoresis on agarose gels to 1 % (56) and dried on ufî nitrocellulose filter (54). Preparing a probe DNA labeled (47) from a fragment of the LP Ddel 600 to 1' cDNA insert

7 of the p69 and hybrid (43) with the spot nitroeéllulose ^ DNA. 10 elements (counts) per minute of the sample are hybridized 16 hours and then washed as described (43). Eight genomic DNA samples from different human donors are digested with the restriction endonuclease EcoRI and hybridized with the retrieval of p6? labeled⁰ E. as shown in Figure 9_O observed two clear signals hybridization with sizes of 8.8 kg base pairs (kbp or) and 2.0 kbp or according to estimate by comparing with the mobilities ADNl digested by HindïII. This could be from two genes of the ïfn-Y or a single gene disrupted by a EcoRI site. As the ABNc p69 contains none of the EcoRI site, a spacer sequence (intron sequence) with an internal EcoRI site would be required to explain a single gene. To distinguish between the two possibilities, a replica of the mimic Southern hybridization with sample 1' against five other digesters to the endonucléqase a single human DNA (fig. 10). Observed two DNA fragments hybridizing two other digestion products to 1' endonuclease, Pvu (6, ? Kbp and kbp or 4.0) and Hindi (2.5 Kbp and kbp or 2.2). However three models of digestion with the endonuclease provide only a single DNA fragment hybridizing; HindïII (9.0 kbp or)_{the R} Bg1ii (11.5 kbp or) and BamHI (kbp or 9.5). Two genes of ifn V should be linked to a distance abnormally close (9.0 kbp or less) to be contained in the same hybridization moiety HindïII. This result suggests that a single gene ifn - V-homologous (unlike many genes of ifn-connected) is present in the DNA of the human genome and that this gene is split by one or more intron containing EcoRI, Pvu and Hindi. This prediction is supported by hybridization of a labeled fragment at (47) prepared accurately from the region 3' untranslated region of the cDNA of the p69 (fragment to 130 BP of Ddel of 860 to 990 BP Figure 5) against the EcoRI digestion product of human genomic DNA. Only the fragment to the EcoRI gives 2.0 kbp or hybridization on this sample, suggesting that this fragment contains the sequences 3' untranslated, while the fragment to 8.8 EcoRl kbp or contains the sequences 5 *. The gene structure of the ifn Y (a gene with at least one intron sequence) is significantly different from that of the ifn-a 3 (multiple genes (2) (56) without intron) or that of IFN - grams (a gene without intron (57)).

J preparation of bacterial extracts

A culture night E. coli w3110 pIFN therein 48 / - Trp-broth Luria Broth + 5 micrograms per ml of tetracycline for innoculer medium (58) containing 0.2 m9 % glucose, 0.5 % of casamino acids and 5 micrograms per ml of tetracycline in dilution of 1:100. Added indole acrylic acid to reach a final concentration of 20 micrograms per ml when a_{the n} and is between 0.1 0.2. Samples of 2 ml are collected or harvested ten centrifugally to ac_5Q =1 and resuspended in 1 ml of saline buffer containing phosphate per ml of 1 mg of bovine serum albumin (PBS-to-BS). The cells are open to ultrasound ôt freed debris by centrifugation. Supernatants are stored, to 4° C to the tests. Interferon activity of the supernatants is determined equal to 250/ml per comparison with U-standards of ifn-by test of inhibition of cytopathic effect (CPE-).

K transformation of yeast strains and appropriate media

Transformed yeast strains (59) coaane hereinbefore described. E coli strain used ja300 (Thrwherein leuBô ethi

- the mR

117 thyAtrpCI willwill hsdra hsdr STRs) (20) to select for the d $P-plasmids containing the functional gene TRPI. Used a yeast strain rh218 having the genotype (-Trp gal2SUC2 improperly CUPI) (18>as host of transformation of the yeast. Rh21ô was deposited without restriction from 1' American Type culture, as ATCC no. 44076. The m9 (minimal medium) comprising 0.25 % of casamino (CAA assay) and 1 © lbs (medium rich) are as described by Miller (58) with the addition of 20 HS / .SL of SAPIs " cilline (delta) after autoclaving and the medium having been cooled. The yeast is grown on the following media T-YEPD containing 1 % de yeast extract, 2 % peptone and 2 %deglucose- 3 % agar DIFCO. ENM + 6.7 g of CAA assay contains yeast nitrogen base (without amino acids) (ENM) (Agar) 10®g of adenine, 10 mg of uracil, 5 grams of CAA, 20 g of glucose and - 30 grams of agar per liter.

L Construetien of the yeast expression vector

1. Is digesting 10 HS of YRsT (14, 13, .16) with EeoRI. The sticky ends or caking DNA thus formed are made blunt with ADH X-polymerase (Klenou fragment). Passed the vector and - 1' insert on agarose gel with d * 1 $(willwill seakera), the shots of the gel, bone subjected to electroelution and extracted twice with equal volumes of chloroform and DS phenol before precipitating with ethanol. The DNA molecules to tersinaispBs blunt and then rejoined fomées ESAW ligated in a final volume of 50 u1 during .12 12 hours^I C AC ligation mixture is then used to transfer allowed " seawater e. colî strain ja300 in ampicillin resistance and prototrophy tryptophan. Isolated plasmids containing the LS TRPT gene in both orientations. pPR or $1 has 1 © of TSP1 gene in the same orientation as YRp7 while pFRW2 has the gene of TRPI in opposite orientation.

20 hS of pFRW2 are linearized with HindlIX and subjected to gel electrophoresis 1 % agarose. The chain molecules are eluted from the gel and an amount of 200 nanograms then undergoes ligation with 500 ng of 1 'Hicdtll 3.1 kb insert of plasmid p31' "' 3) which is a restriction fragment containing the gene of 3 a-yeast phosphoglycerate kinase. ligs except the LS mixture is then used to transform E. coli strain 294 in ampicillin resistance and sensitivity to tetracycline. The plasoide prepared from such a recombinant gene of Trp intact the fragment to 3.1 kbp of HindlII from the DNA insert pb1 HindlII site of the resistance gene to tetracycline. It is plasmid pFRM31. 5 μ g of the pFRM31 are digested with EcoRI completely, extracted twice with phenol and chloroform and then precipitated, to ethanol. The sticky ends of the molecule are filled by DNA polymerase I (Klenow fragment) in a reaction made 250 μΜ in each deoxynucleotide triphosphate. The reaction is made 20 minutes to 14° C., DNA is extracted twice with phenol-chloroform and then precipitated with ethanol. The resuspended DNA is digested completely with based Clal and processed by gel electrophoresis 6 $polyacrylamide. The vector fragment is separated from the gel by elution, phenol-chloroform extracted and precipitated with ethanol.

The six N-terminal amino acids of the enzyme 3 a-phosphoglycerate purified human products are as follows ii

1 - 2 - 3 - 4 - 5 - 6

THE LEU-SER-SER - - - - - - LEU-LYS IN THE HSM

A reading frames translation engendered by the sequence of the restriction fragment 141 LP Sau3A linker - to - Sau3A linker (containing the internal site to hindi; see restriction map of the PGK, fig. 11) produces the following amino acid sequence.

1 _ 2 - 3 - 4 - 5 - 6

THE LEU - - - INVOLVES RCS RCS - LYS-LEU-RCS - - -

After removal of the methionine initiation, see that the amino acid sequence of the PGK-n-terminal homology of 5 to 6 amino acids with the amino acid sequence of human PGK of n-terminal.

The form of sequence thus obtained suggests that the start of the structural gene of yeast PGK is encoded by the DNA restriction fragment in the 141 pb1 Sau3A linker of the LP. Previous work (20) have suggested that the DNA sequences specifying the mRNA of the PGK can be in the area of the fragment HindlII. Another formation of sequence of the fragment 1 - 41 to LP Sàu3A offers more DNA sequence of the promoter of the PGK (fig. 12).

A synthetic oligonucleotide sequence 5 'ATTTGTTGTAAA3' is synthesized by using standard methods (created and Al, Nucleic Large its. 8, 2331 Ο 98 θ)). 100 ng of this primer 3ont marked end 5' using t4 polynueléotide kinase in 20 therein 1 of reaction medium containing 200 yCi FV ^ w) transporters. This solution of labeled primer is used in a repair reaction primers for constituting the first stage of a multi-step process for the EcoRI restriction site in the DNA flanking 5' of the PGK just before the structural gene sequence of the PGK promoter.

It digests 100 HS Pb¹ (20) completely with HaelII then passes on a gel to 6 % polyacrylamide. The upper web éthidum dyed gel (containing the promoter region of the PGK) is isolated by electroelution as described above. That piece of DNA HaelII to 1200 BP is restricted with to hindi and thereafter passed on a polyacrylamide gel to 6 % " The band to 650 BP is isolated by electroelution. 5 Μg DNA is isolated. That piece of DNA HaelIï to HincII to 650 BP is resuspended in 20 GP/HgO, and then mixed with 20 therein 1 of primer solution phosphorylated as described above. This mixture is extracted from a liver phenol-chloroform and then precipitated with ethanol. Dry DNA is resuspended in 50-yl H ^ O and then is heated at boiling water bath for seven minutes. This solution is then rapidly cooled dan3 a bath of dry ice - ethanol (20•10 seconds) and then is transferred into a water-ice bath. With this solution, is added a solution containing 50-yl 10 10x yl derivatives of DNA polymerase I (manufacturer Boehringer Mannheim's) buffer, 10 therein 1 of a solution made of advance 2.5 mm in each deoxynucleoside triphosphate (or dATP, dITP, dGTP and dCTP), 25 w 1 HgO and 5 units of DNA polymerase I, Klenow fragment. This reaction medium

100 μ 1 is incubated at 4 C. for 37° hours. The solution is then extracted once with phenol-chloroform, precipitated with ethanol, dried by lyophilization and restricted exhaustively with 10 Sau3A linker units. This solution is then passed over a gel to 6 % polyacrylamide. The band corresponding to 39 BP of size is cut from the gel and then isolated by electroelution as described above. This strip 39 bp has a blunt end and a cohesive or agglutinating Sau3A linker. This fragment cloned in a vector e3t pFIF TRPs 69 (5) modified. 10 HS of pFIF TRPs 69 are linearized with Xbal, 1x phenol-chloroform extracted, and then precipitated with ethanol. The sticky end Xbal is filled using 1 * Klenow DNA polymerase in 50 μ 1 of a reaction medium containing 250 um in Dane-each nucleoside triphosphate. This DNA is cut BamHI and then passed over a gel to 6 % polyacrylamide. The vector fragment is isolated from the gel by electroelution and resuspended in 20 u1 fl0. Is caused ligation to 20 ng of this vector with 20 nanograms of the fragment to 39 LP prepared herein above 4 hours at room temperature. Fifth of the ligation mixture is used to transform E. coli strain 294 in ampicillin resistance (on plate µg/ml of lb + 20 amps) Y. The plasmids transformants are examined according to a sorting technique (44) fast. A plasmid, pPGK-to-39>is selected for sequence analysis. It digests 20 ng of EO plasmid with Xbal, precipitated with ethanol and then treated with 1000 units of bacterial alkaline phosphase 68° C for 45 min to. DNA is extracted 3x phenol-chloroform, precipitated and then

ethanol. The ends are then labeled dephosphorylated -

32 in a reaction medium containing 200 20 μ 1 PCI (γ^J P) ATP and 10 units in polynucleotide kinase inhibitors. The plasmid is ooupé with its 11 and passed over a 6 $polyacrylamide gel.

The strip insert marked is isolated and sequenced by the method of chemical degradation (52). The DNA sequence at the end 3' of that piece of promoter is such as expected. ' 2. Construction of the promoter fragment of the PGK to 312 Pvul-EcoRl LP

It digests 25 - g of pPGK and 39 (fig. 13) simultaneously with its¹ ! and Xbal' 5 units each) are obtained and processed by electrophoresis gel Jn at 6 ii. The band to 390 LP containing the piece of promoter to 39 BP is isolated by electroelution. The resuspended DNA is restricted Sau3A and then subjected to gel electrophoresis 3 ii polyacrylamide. The strip of the PGK promoter to 39 BP is isolated by electroelution. This DNA contien 39 BP of the end^C. ' on the PGK promoter fragment Sau3A to 3066 basepair.

25 gjnl PBLs are restricted with Pvul KpnI and further processed by gel electrophoresis 6 % polyacrylamide. The CBP are 0.8 to strip. dNA is isolated by electroelution, and then restricted Sau3A and subjected to gel electrophoresis 6 ii polyacrylamide. The band confectioneries 265 BP of the promoter of the PGK (fig.* ' is isolated by electroelution.

Or. then causes this DNA ligation with the promoter fragment to 3^Q lP described above during 2 hours at temperature amnbiante. The mixture undergoes a restriction with XbalPvul and then a gel electrophoresis 6 ii polyacrylamide. the restriction fragment Xba to Pvul to 3¹ 2 bP is isolated by electroelution, then added to year ligation mixture containing 200 nanograms of pBR322 ' 41) (isolated previously without the restriction fragment Pvul to PstX 162 to LP) and 200 nanograms of the cDNA gene Xbal-Pstl LeXF has previously isolated from 20^g of pLelF TRPs has 25. The ligation mixture is used to transform three-factor e coli strain 294 in tetracycline resistance. Transformant colonies (44) undergo minitri and a colony, pPGK above 300 is isolated in a form comprising flanking DNA LP 304 5' fused to the gene in a vector based of pBR322 LeXF. The end 5' gene LelF has has the following sequence:

5' A-CTAGAA7TC AND 3¹ . Thus the melting Xbal site. the PGK promoter fragment in this sequence allows the addition Xbal site of an EcoRI site. The pPGK above 300 thus contains a portion of the promoter of the PGK isolated in a Pvul fragment to the EcoRI.

3. Construction of the PGK promoter fragment EcoRI to the EcoRI to 1500 LP

It digests ΐΟ - g of pb1 with Pvul and EcoRI and passed on a gel to 6 ii polyacrylamide. The band DNA Pvul to the EcoRI to 1.3 kb - DNA flanking 5' of the PGK promoter is isolated by electroelution. It digests 10 Ug of pPGK above 300 aveeEeoRI and Pvul i3ole and the promoter fragment to 312 BP by electroelution after electrophoresis of the digestion mixture on a gel to 6? polyacrylamide. 5 Ug of pFRL4 is cut with EcoRI, precipitated with ethanol and treated with bacterial alkaline phosphatase to C. for 45 min 68°. After three DNA of the with phenol-chloroform, ethanol precipitation, and 20 ml of resuspended in the O ^ ii, is 200 nanograms of ligation of the vector with 100 ng of DNA to 312 LP of the EcoRI-PvuRIpPGK and 300 and 100 ng of DNA EcoRI to Pvul PBL. The ligation mixture is used to transform E. coli strain 294 in ampicillin resistance. One of the transformants obtained is the pPGK above 1500. This plasmid contains the promoter fragment of the PGK to ' 500 BP or in the form of pieces of DNA EcoRI-EcoRI or HindlII-EcoRl.

The digests completely 10 pg of pPGK above 1500 with based Clal and EcoRI followed by electrophoresis of the digestion mixture on a gel to 6 ii polyacrylamide. The fragment to 900 LP promoter is isolated by electroelution pPGK. The digests completely 10 _. g of pIFN Y TRPs 48 with EcoRI and Hindi and subjected to electrophoresis on a polyacrylamide gel to 6 mth. The band to 938 LP containing cDNA of ifn it directly expressible is isolated from the gel by electroelution.

The yeast expression vector is constructed in a reaction medium to three factors connecting together by ligating the promoter fragment of the PGK (on a piece based Clal to the EcoRI), the deleted pFRM and 31 and cDNA of ifn-V compound isolated above. The ligation mixture is inoubé 12 hours to 14° C. the ligation mixture is then used to transform E. coli strain 294 in ampicillin resistance. The transformants are analyzed for the presence of the expression plasmid properly constructed, pPGK IFN-- therein (fig. 16). The plasmids containing the expression system operate to transform yeast spheroplasts strain rh218 in prototropy tryptophan in agar lacking tryptophan. These recombinant yeasts are then tested for the presence of immune interferon.

Prepared yeast extracts as follows! grown ten ml cultures in ENM Agôo CAA assay up to +.¹ "² "^is collection by centrifugation afterwards suspended in 500 UL PBS (20 mm of NaH₂ P0₄ , ph=7.4, 150 mm NaCl solution). Added an equal volume of glass beads (0, 45 - 0, 5 mm) and the mixture is subjected to a vortex during 2 min. Are rotated 30 seconds at 14,000 rpm extracts and by removing the supernatant. Activity is determined interferon of supernatant and found 16,000 a U/ml by comparison to a standard of ifn-, by applying the CPE inhibition test.

In the vector m pSVy69 cell culture

The fragment HindlII-to-Pvu to 342 base pairs encompassing the origin of sv40 is converted to a moiety bonded at EeoRI restriction site. The site HindlII is converted by the adds a synthetic oligomer (5' dAGCTGAATTC) and the site PvuXI is converted by ligation of blunt end in an EcoRI site filled using the polymerase I (Klenow fragment). The resulting EcoRI fragment is inserted into the EcoRI site of the (28) PML and above 1. A plasmid comprising the late promoter (ETL) of sv40 pointing away from the gene amp ** undergoes further modification by removing the EcoRI site nearest the gene amp ** of the PML and above 1 (27).

The fragment is isolated to the HpaI-to-Bgll 1023 basepairs of HBV DNA cloned (60) and converting the Hpal site of hepatitis b virus (HDV) in a EcoRI site with a synthetic oligomer (5' dGCGAATTCGC). This fragment bound EcoRI bg1ii is cloned directly into the sites of the plasmid described above EcoRI the BamHI carrying the origin of the sv40.

In the EcoRI site remaining, is inserted into the gene of ifn -therein on PstI fragment to 1250 base pairs of the Pstl ends p69 after conversion in the EcoRI ends. Isolated clones in which the late promoter of sv40 precedes the manages the ifn-Y-structure. The resulting plasmids are then introduced into tissue culture cells (29) using a technique to the dextran DEAE-(61) modified so that the "transfecting" in the presence of dextran DEAE-hard 8 hours. > •cell media are changed all 2 - 3 days. 200 aicrolitres removed each day for a bioassay of interferon. The yields are typically 50 - 100 U-/ ml for the sample tested three to four days after transfection.

N partially purifying immune interferon derived from cells of monkey

To produce greater amounts of ifn -•derivative monkey cells, the transfection is carried out monecouches of fresh cell cos-to-7 in ten plates 10^W. with a total of 30 in 110 ml 3 pDLIF HS of dextran-AED⁶ ' (200 gms/ml of Y-dextran DEAE-molecular weight 500,000, 0.0 *.^M The tris pH of 7" 5 in DMEM). After 16 hours at 37° C., washed the " plates twice with DMEM. Each plate is added 15 ml 1p DMEM supplemented with 10 %f.b.s., 2 mm glutamine peptide, 50/ml. therein > pénicciline g and 50 mg/ml of streptomycin. Renew the * media the next day with serum-free DMEM. Fresh serum-free media are then added each day.

media collected are kept 4^I C is up to test OS^; * up to link GC. The pooled fractions of échantill™ * 3 and 4 days of transfection show that they contain ** essentially all of the activity.

0.5 G of GPC is added (a controlled pore size on glass, controlled pore glass, Electronucleonics, GPC 350, caliber 120 w '"' meshes, 0,074 to 0,125 mm opening) to 100 ml of cell supernatant and the mixture is stirred 3 hours to 4" C. a centrifuged in a centrifuge brèv * table, and compacted in the URs - * column the beads deposited and washed thoroughly with a mixture of 20 mm NaPO ^, NaCI 1 M., 0.1 % gm - mercaptoétHanoi, at pH 7.2. The activity is then eluted with the same Buff - - " containing éthylèpeglycol (30 $), followed by elution with another said buffer containing ethylene glycol (50 ii). Essentially all of the activity binds to the GPC. Found 75 % the activity eluted in fractions eluted with 30 % of éthylèneglyool. These fractions are combined (pooled) and diluted with 20 mm NaPO ^ "1 the m NaCI, pH of 7" 2 to arrive at a final concentration of 10 9.ï ethylene glycol and are applied to a column of 10 ml-Sepharose chromatography (Pharmacia is) designed. After a serious with the mixture 20 NaPOjj MO, 1 the m NaCI, pH of 7.2, the activity is eluted with a mixture of 20 mm NaPOjj, 1 m in the NaCl, 0.2 m-methyl-D-. an important part of this activity (55 %) he and not bind to the lectin. 45 ii of this activity are eluted with 1' has-methyl-D-.

Pharmaceutical compositions

The compounds of the present invention may be formulated according to known methods for preparing pharmaceutically useful compositions, human immune interferon of these products combined in admixture to a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described in Remington-Peppers pharmaceutique science by E.W. Martin reagent, which is incorporated herein by reference. These compositions contain an effective amount of interferon protein with an appropriate amount of vehicle to prepare pharmaceutically acceptable compositions suitable for effective administration to the hête.

A parenteral administration

Human immune interferon may be administered parenterally to subjects that require antitumor or antiviral and those that reflect conditions of immunosuppression. doses and dosage may be applied by conforming to those currently used in the clinical research other human interferons, e.g. (1 - 10) the X 10⁶ units per day, and for the products of purity greater than 1 %, can be approached presumably up to 50 x 10⁶ units per day. The doses of ifn-Y-may be increased significantly in order to achieve larger effects substantially as a result of the absence.de human proteins other than the ifn V, which proteins when presented in the products of human origin, can induce 5 some undesirable effects.

Examples of suitable dosage form for IFN - - essentially homogeneous coatable parenteral, dissolvable 3 nanograms of ifn - V specific activity, by

The O, example 2 x 10 0/mg in 2 * 5 ml of usp albumin (human) sera; Q-n-5, passing the solution through a bacteriological filter and th aseptically divide the filtered solution in 100 vials ootrfcenant each 6 x 10®units of interferon pure for administration of relatedness to-Epitaxy. The vials are preferably stored in cold (-20 degrees it before use.

13 Biological or biochemical data

A characterization of the antiviral activity

For the various native antibody, diluted samples when necessary to achieve a concentration of 500 - 100/ml to U-with PBS-to-BS. Equal volumes of sample are incubated 2o 12 hours to 2 4° c with serial dilutions of rabbit antihuman leukocyte, fibroblast or immune interferon antisera. The anti-IFN-α and s are from the National Institute prostate. The anti one IFN-- V is prepared using authentic ifn-γ - (5 - 20 purity %) 25 purified from peripheral blood lymphocytes stimulated;

The samples are centrifuged 3 minutes at 12,000 g to 3 min Peppers before testing. For testing the stability at pH 2, the samples are that pH by the addition of HCl iN, incubated 2 hours to 12 4° c and is neutralized by the addition of NaOH 1 n before testing. For testing the sensitivity at 30 sodium dodecylsulfate fSDS), samples are incubated with an equal volume of SDS 2 T- during 2 to 12 hours to 4° c before the test.

B characterization of ifn - 9.ï produced by e. coli and cells of cos-to-7

Antiviral activity (U-/ ml.)

E coli cells/w3110 pIFN γ-Trp-48 the cos-a 7/pSVY69

This table shows the characteristic behavior of ifn-standards, 3 and^Y after various treatments. Interferon activity produced by e coli v/31/pIFN'O' V-Trp-48 and by c0s and 7/VSP 69 is acid-sensitive, at 3ds and is neutralized by the antiserum immune interferon. It is not neutralized by antibodies to IFN-α or 8. These data establish that the interferon produced in these systems is the ifn-Y and that 1'cDNA insert of the plasmid ρ 69 encodes the ifn V.

Hereinafter is described by means of the pattern according to a method for purification of ifn - γ e.g. at start of bacteria.

1. Extracting cells in a buffer medium (about pH 8) sputtering lysis of high conductivity by passage through a high pressure homogenizer, effluent cooling at ice bath.

2. Precipitation of the DNA by the addition of polyethyleneimine with agitation for example to 4° C..

3. Precipitation, under the influence of pH, bacterial proteins once again providing the ifn-Y-in solution.

4. Solids separation by centrifugation at 4° C 5. Concentrating the supernatant (after readjustment of the pH) for example by ultrafiltration.

6. The concentrate dialysis against a buffer of low conductivity.

7. Removing solids by centrifugation leaving i'i' ifn-Y-in solution.

8. Ion exchange chromatography on carboxymethylcellulose, eluting with a gradient of increasing ionic strength.

9. Calcium phosphate gel chromatography by elution with a gradient of increasing ionic strength.

10. Ion-exchange chromatography on carboxyméthylcellul03e with minimal denaturing by elution with a gradient of increasing ionic strength.

H - chromatographic separation gel filtration.

This method provides the product with a purity of greater than 95%.

The immune interferon protein of the invention has been defined using a sequencing determined DNA gene and amino acid derived, see Figure 5. It is understood that for this interferon encompassed herein, natural allelic variations exist from one individual to the other. These variations can be manifested in one or more differences in amino acids in the overall sequence or by deletions, inversions is added or one or more amino acids in this sequence. All these allelic variations are included within the scope of the present invention.

Indeed, the use of recombinant DNA technology offers the opportunity to prepare various derivatives of ifn V human, by performing various modifications of replacements₄ added, deletions or substitutions of single or multiple amino acids. All such modifications which give rise to such derivatized i'i' ifn-Y in humans are included within the scope of the present invention as long as the essential activity, characteristic i'i' ifn Y remains unchanged in its nature.

Having hands the DNA sequences and amino acid i'i' ifn Y (fig. 5), most preferred to reproduce the invention certainly involves the preparation of either the complete gene synthetically (see 26, for example) or that of synthetic deoxyoligonucleotides with which the "library" of human genome or other source of cDNA can be explored to isolate the gene by hybridization assays is normalized. Once obtained the nucleotide sequence which encodes the protein required for the ifn Y, may be adopted the means of expression, isolation and purification process for the production of preparations of ifn - γ very pure consistent with the above description.

Despite the reference to particular and preferred embodiments, it will be understood also that the invention is not limited to the details described above.

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