NZ214261A - Human immune interferon: production by recombinant dna technology - Google Patents

Description

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C o Vvi 0 lef-e Specification has been ante-date^ tx>...„A5...Q..d?Ja&£ »9 %Z.i NEW ZEALAND PATENTS ACT. 1953 No#? Divided out of No. 202.190 15 October 1982 Date: COMPLETE SPECIFICATION PRODUCTION OF HUMAN IMMUNE INTERFERON cWWe, GENENTECH, INC., a corporation of the State of California, U.S.A., of 460 Point San Bruno Boulevard, South San Francisco, California 94080, U.S.A. hereby declare the invention for which^ic/ we pray that a patent may be granted to gjse/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - (followed by page la) 214261 Field of the Invention The present invention relates to the field of recombinwfrtf DNA technology, to means and methods utilizing such technology in the production of human immune interferon and to the various products arrived at during such production and their uses.

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 -la- 214261 expression vehicles containing such DNA sequences operably linked to expression-effecting promoter sequences and to the expression vehicles so constructed. In another aspect, the present invention relates to host culture systems, such as various microorganism and vertebrate cell cultures transformed with such expression vehicles and thus directed in the expression of the DNA sequences referred to above. In preferred embodiments, this invention provides particular expression vehicles that are sequenced properly such that human immune interferon is produced and secreted from the host cell in mature form. In addition, this invention relates to various processes useful for producing said DNA sequences, expression vehicles, host culture systems and end products and entities thereof and to specific and associated embodiments thereof.

This is a divisional of New Zealand Patent Specification No. 202,190 which describes and claims a composition of matter consisting essentially of a polypeptide comprising the amino acid sequence of recombinant human immune interferon or of des-Cys-Tyr-Cys recombinant human immune interferon.

The present invention arises in part from the discovery of the DNA sequence and deduced amino acid sequence encoding human immune interferon. In addition, the present invention provides sequence information on the 3'- and 5'- flanking sequences of the human immune interferon gene, facilitating the in vitro linkage thereof into expression vehicles. In particular, there is provided the 5'-DHA segment encoding the putative endogenous signal polypeptide which immediately precedes the amino acid sequence of the putative mature human immune interferon. These discoveries, in turn, have enabled the development of the means and methods for producing, via recombinant DNA technology, sufficient amounts of human immune interferon-, so as to enable, in turn, the determination of its •emical properties and bioactivity. r r ! The publications and other materials hereof used to illuminate the background of the invention, and in particular cases, to provide additional details respecting its practice are incorporated herein by reference, and for convenience, are numerically referenced by the following text and respectively grouped in the appended bib 1iography.

Background of the Invention A. Hunan Immune Interferon Human interferons can be classified in three groups on the basis of different antigenicity and biological and biochemical properties.

The first group comprises a family of leukocyte interferons (a-interferon, LeIF or IFN-a), which are normally produced mainly by constituent cells of human blood upon viral induction. These have been microbially produced and found to be biologically active (1, 2, 3). Their biological properties have prompted their use in the clinic as therapeutic agents for the treatment of viral infections and malignant conditions In the second group is human fibroblast interferon (B-interferon, FIF or IFN-s), normally produced by fibroblasts upon viral induction, which has likewise been microbially produced and found to exhibit a wide range of biological activities (5). Clinical trials also" indicate its potential therapeutic value. The leukocyte and fibroblast interferons exhibit 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, both groups of interferons contain from 165 to 166 amino acids and are acid ! ' stable proteins.

The hunan immune interferon (iFN-y), otherwise also referred to as human gamma interferon or IIF, (4) ( ( 7 | /?, 7 (h to which this invention is directed, is, fn contrast to the a-and 3 - i n t e r f e ron s, pH 2 labile, is produced mainly upon mitogenic induction of lymphocytes and is also clearly antigenica 11y distinct. Until recently human immune interferon could only be detected in very mi nor levels, which evidently hampered its characterization. Recently, a rather extensive but still partial purification of human immune interferon has been reported (6). The compound was said to be produced from lymphocyte cultures stimulated with a combination of phytohaemaggl uti nin and a phorbol ester and purified by sequential chromatographic separations- This procedure resulted in a product having a oolecular weight of 58,000.

Human immune interferon has been produced in very Tow amounts by translating mRNA in oocytes, showing interferon activity characteristic of human immune interferon and expressing the hope that immune interferon cOfIA could be synthesized and cloned (7).

The amount of immune interferon obtained untiT now is certainly insufficient to carry out unambiguous experiments on the characterization and biological properties of the purified component. However, in vitro studies performed with crude prepara ti ens, as well as i_n vi vo experiments with murine Y~interferon preparations , suggest that the primary function of immune interferon may be as an immunoregulatory agent (8, 9). Immune interferon has not only a'n antiviral and anticellular activity in common to all human interferons, but shows a potentiating effect on these activities with <x- and 6-interferon (10). Also, the in vitro antipro 1iferative effect of f-interferon on tumor cells is reported to be approximately 10- to 100-fold that of the other interferon classes (8, 11, 12). This result, together with its pronounced immunoregu1 a tory role (8, 9), suggests a much more pronounced antitumoral potency for IFN—r than for IFM-a and —frte&t -4- r r "1 i -) IFM-s. Indeed, ijn vivo experinents with mice "and murine IFfl-f preparations show a clear superiority over antivirally induced interferons in its antitumoral effect against osteogenic sa rcona (13).

All of these studies, until the present invention, had to be performed with rather crude preparations, due to the very low a va i 1 ab i 1 i ty . However, they certainly suggest very important biological functions for immune interferon. JJot only has immune interferon a potent associated antiviral activity, but probably also a strong immunoregulatory and antitumoral activity, clearly pointing to a potentially very promising clinical candidate.

It was perceived that the application of recombinant DMA , technology would be a most effective way of providing the requisite larger quantities of human immune interferon.

Whether or not the materials so produced would include glycosylation which is considered characteristic of native, human derived material, they v/ould probably exhibit bioactivity admitting of their use clinically in the treatment of a wide range of viral, neoplastic, and immunosuppressed conditions or diseases.

B. Recombinant DMA Technology Recombinant DMA technology has reached the age of some sophistication. Molecular biologists are able to recombine various DMA sequences with some facility, creating new "DNA entities capable of producing copious amounts of exogenous protein product in transformed microbes. The general means and methods are in hand for the i_n vi tro ligation of various blunt ended or "sticky" ended fragments of DNA, producing potent expression vehicles useful in transforming particular organisms, thus directing their efficient synthesis of desired exogenous product. However, on an individual product basis, / f r the pathway remains somewhat tortuous and the science has not advanced to a stage where regular predictions of success can be made. Indeed, those who portend successful results without the underlying experimental basis, do so with considerable risk of i noperab i 1 i ty .

The plasmid, a no nch romosonjal loop of doub 1 e-s tranded DMA found in bacteria and other microbes, oftentimes in multiple copies per cell, remains a basic element of recombinant DNA technology. Included in the information encoded in the plasmid OMA is that required to reproduce the plasraid in daughter cells (i.e., an origin of replication) and ordinarily, one or more phenotypic selection character!sties such as, in the case of bacteria, resistance to antibiotics, which permit clones of the host cell containing the plasmid of interest to be recognized and preferentially grown in selective media. The utility of plasmids lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or "re.stri cti on enzyme", each of which recognizes a different site on the plasmid DNA. Thereafter heterologous genes or gene fragments may be inserted into the plasmid by endwise joining at the cleavage site or at reconstructed ends adjacent to the cleavage site. Thus formed are so-called replicable expression vehicles* DNA recombination is performed outside the cell, but the resulting "recombinant" replicable expression vehicle, or plasmid, can be introduced into cells by a process known as transformation and large quantities of the recombinant vehicle obtained by growing the transformant. Moreover, where the gene is properly inserted with reference to portions of the plasmid which govern the transcription and translation of the encoded DMA message, the resulting expression vehicle can be used to actually produce the polypeptide sequence for which the inserted gene codes, a process referred to as expression.

Expression is initiated in a region known as the pVoao'te'r which is recognized by and bound by RNA polymerase. In the transcription phase of expression, the DNA unwinds, exposing it as a template for initiated synthesis of messenger RNA from the DMA sequence. The messenger RNA is, in turn, translated into a polypeptide having the amino acid sequence encoded by the mP.NA. Each amino acid is encoded by a nucleotide triplet or "codon" which collectively make up the "structural gene", i.e. that part which encodes the amino acid sequence of the expressed polypeptide product. Translation is initiated at a "start" signal (ordinarily ATG, which in the resulting messenger Rk'A becomes AUG). So-called stop codons define the end of translation and, hence, of production of further amino acid units. The resulting product may be obtained by lysing, if necessary, the host cell, in microbial systems, and recovering the product by appropriate purification from other protei ns.

In practice, the use of recombinant DMA technology can express entirely heterologous polypeptides—so-called direct expression--or alternatively may express a heterologous polypeptide fused to a portion of the amino acid sequence of a homo!ogous polypeptide. In the latter cases, the intended bioactive product is sometimes rendered bioinactive within the fused, homo 1ogous/hetero1ogous polypeptide until it is cleaved in an extracellular environment. See N.Z. Patent Specification No. 188,836 and Wetzel, American Scienti-st 68, 664 (1980).- C. Cell Culture Technology The art of cell or tissue cultures for studying genetics and cell physiology is well established. Means and methods are in hand for maintaining permanent cell lines, prepared by successive serial transfers from isolate normal cells. For use in research, such cell lines are maintained on a solid —«reet- 214261 support In liquid medium, or by growth In suspension containing support nutriments. Scale-up for large preparations seems to pose only mechanical problems. For further background, attention Is directed to Mlcrobtoloov. 2nd Edition, Harper and Row, Publishers, Inc, Hagerstown, Maryland (1973) especially pp. 1122 et seq. and Scientific American 245. 66 et seq. (1981), each of which Is Incorporated herein by this reference.

■Summary of the Invention The present Invention Is based upon the discovery that recombinant DNA technology can be used to successfully produce human Immune Interferon, preferably In direct form, and In amounts sufficient to Initiate and conduct animal and clinical testing as prerequisites to market approval. The product Is suitable for use. In all of Its forms. In the prophylactic or therapeutic treatment of human beings for viral Infections and malignant and Immunosuppressed or fmmunodefIclent conditions. Its forms Include various possible ollgomerlc forms which may include associated glycosylation. The product is produced by genetically engineered transformant microorganisms or transformant cell culture systems. As used herein, the terms "transformant cell" and "transformant microorganism" refer respectively to a cell or a microorganism into which has been introduced DNA, said DNA arising from exogenous DNA recombination, and to the progeny of any such cell or microorganism which retains the DNA so introduced. Thus, the potential now exists to prepare and isolate human immune interferon in a more efficient manner than has been possible. One significant factor of the present 214261 Invention, In Its most preferred embodiments. Is the accomplishment of genetically directing a inIcroorganIsm or cell culture to produce human Immune Interferon In Isolatable amounts, secreted from the host cell In mature form.

Accordingly, In one embodiment the present Invention can be broadly said to consist In a DNA sequence comprising a sequence coding for a polypeptide comprising the amino acid sequence of recombinant human Immune Interferon.

In a further embodiment, the Invention can be said to consist In a DNA sequence comprising a sequence coding for a polypeptide comprising the amino acid sequence of des-Cys-Tyr-Cys recombinant human Immune Interferon.

In a further embodiment, the Invention can be said to consist In a DNA sequence as described above operably linked with a DNA sequence capable of effecting expression of a polypeptide as described above.

In still a further embodiment, the invention can be broadly said to consist In a replicable expression vehicle capable, In a transformant microorganism or cell culture, of expressing a polypeptide as described above.

In still a further embodiment, the Invention can be said to consist In a microorganism or cell culture transformed with a vehicle as described above.

In still a further embodiment, the invention can be said to consist In a culture of transformant cells capable of producing a 9 214261 polypeptide as described above.

This approach may utilize the gene encoding the sequence of the mature human Immune Interferon plus the 5' flanking DNA encoding the signal polypeptide. The signal polypeptide Is believed to aid In the transport of the molecule to the cellular wall of the host organisms where It Is cleaved during the secretion process of the mature human Interferon product. This embodiment enables the Isolation and purification of the Intended mature Immune Interferon without resort to Involved procedures designed to eliminate contaminants of Intracellular host protein or cellular debris.

In still a further embodiment, the Invention can be said to consist In a process for producing an expression vehicle as described above comprising constructing a first DNA sequence coding for said polypeptide and operably linking said first DNA sequence with a second DNA sequence capable of effecting expression of said first DNA sequence.

Reference herein to the expression "mature human Immune interferon" connotes the microbial or cell culture production of human Immune Interferon unaccompanied by the signal peptide or presequence peptide that immediately attends translation of the human Immune Interferon mRNA. A first recombinant human Immune Interferon, according to the present Invention, Is thus provided, having methionine as Its first amino acid (present r r 2 "? A 2 6 by virtue of the ATG start signal codon insertion in front of the structural gene) or, where the methionine is intra- or ex trace11ula rl y cleaved, having its normally first amino acid cysteine. Mature human immune interferon can also be produced, in accordance herewith, together with a conjugated protein other than the conventional signal polypeptide, the conjugate being specifically cleavable in an intra- or extracel1ular environment. See N.Z. Patent Specification Ho. 188,836. Finally, the mature human immune interferon can be produced by direct expression without the necessity of cleaving away any extraneous, superfluous polypeptide. This is particularly important where a given host may not, or not efficiently, remove a signal peptide where the expression vehicle is designed to express the mature human interferon together with its signal peptide. The thus produced nature human immune interferon is recovered and purified to a level fitting it for use in the treatment of viral, malignant, and immunosuppressed or immunodeficient conditions.

Human immune interferon was obtained according to the following: 1. Human tissues, for example human spleen tissue or peripheral blood lymphocytes, were cultured with mitogens to stimulate the production of immune inte rferon. 2. Cell pellets from such cell cultures were extracted in the presence of ribonuclease inhibitor to isolate all cytoplasmic RNA. 3. An oligo-dT column isolated the total messenger RNA (mRNA) in polyadenyl ated form. This niRf.'A was size-fractionated using sucrose density gradient and ( c acid-urea gel e1ectrophoresis. 214 261 The appropriate mRNA (12 to 18 S) was converted to corresponding single stranded complementary DNA (cD'IA) from which was produced double stranded cDMA. After poly-dC tailing, it was inserted into a vector, such as a plasmid bearing one or more phenotypic markers.

The thus prepared vectors were used to transform bacterial cells providing a colony library. Radiolabeled cDMA prepared from both induced and uninduced mRNA, derived as described above, was used to separately probe duplicate colony libraries. The excess cDNA was then removed and the colonies exposed to X-ray film so as to identify the induced cDNA clones.

From the induced cDNA clones the corresponding plasmid DNA was isolated and sequenced.

In a first embodiment, sequenced DNA was then tailored in vitro for insertion into an appropriate expression vehicle which was used to transform an ^ coli host cell which was, in turn, permitted to grow in a culture and to express the desired human immune interferon product.

Human immune interferon thus expressed doubtless has 146 amino acids in its mature form, beginning with cysteine, and is very basic in character. Its monomeric molecular weight has been calculated at 17,140. Perhaps because of the presence of numerous 214261 basic residues, hydrophobicity, salt bridge formation and so forth, the molecule may associate itself in oligomeric forms, e.g., in dimer, trimer or tetramer form. The high molecular weights previously observed with natural material (6) which can not be accounted for on the basis of the amino acid sequence alone may be due to such oligomeric forms as well as to the contribution of carbohydrate from post-trans-lational glycosylation. 9. In certain host cell systems, particularly when ligated into an expression vehicle so as to be expressed together with its signal peptide, the mature form of human immune interferon is exported into the cell culture medium, immeasurably aiding in recovery and purification methods.

. In a second embodiment, human immune interferon is produced which lacks the Cys-Tyr-Cys terminal portion of recombinant human immune interferon above. The des-Cys-Tyr-Cys human immune interferon thus produced consists of 143 amino acids..

Description of Preferred Embodiments A. Microorganisms/Cell Cultures 1. Bacterials Strai n s/Promoters The work described herein was performed employing, inter alia, the microorganism E. coli K-12 strain 294 (end A, thi~, hsr~, j(hsm + ) , as described in N.Z. Patent Specification Nos 194,043 and 201,312. This strain has been deposited with the American Type Culture Collection, ATCC Accession No. 31446. However, various other microbial strains are useful, including known ji. col i strains such as col i B, col i X 1776 (ATCC Ho. 31537 ) and Ji. col i W 3110 (F", x~, prototrophic (ATCC No. 27325 ), or other microbial strains many of/which are deposited and (potentially) available from recognized microorganism depository institutions, such as the ^ r 214261 American Type Culture Collection (ATCC)--cf. the ATCC catalogue listing. — .

I These other microorganisms include, for ex ampie, Bacilli such as Bacillus s u b t i1i s and other enterobacteriaceae amcng which can be mentioned as examples Salmonel1 a tyehi mu rium and S e r r a t i a marcescens, utilizing plasmids that can replicate and express heterologous gene sequences therein.

As examples, the beta lactamase and lactase promoter systems have been advantageously used to initiate and sustain microbial production of heterologous polypeptides. Details relating to the make-up and construction of these promoter systems have been published by Chang e_t aj_., Mature 275, 617 I (1978) and Itakura e_t Sc i ence 198, 1056 (1977), which are hereby incorporated by reference. More recently, a system based upon tryptophan, the so-called trp promoter system, has been developed. Details relating to the make-up and construction of this system have been published by Kleid et al ., m u c 1 e i c Ac ids Resea rch 8^, 4-057 (1980) and n.z.

Patent Specification No- 196,584, which--are = : — hereby incorporated by reference. Numerous other microbial promoters have been discovered and utilized and details concerning their nucleotide sequences, enabling a skilled worker to ligate them functionally within plasmid vectors, have been published -- see, e.g., Siebenlist et jH., Cell 20, 269 (1980), which is incorporated herein by this reference. 2. Yeast Strains/Yeast Promoters The expression system hereof may also employ the plasmid YRp7 (14, 15, 16), which is capable of selection an replication in both _E. col i and the yeast, Saccharomyces cerevi siae. For selection in yeast the plasmid contains the / TRP1 gene (14, 15, 16) which complements (allows for growth in the absence of tryptophan) yeast containing mutations in this o ^ gene found on chromosome IV of yeast (17). The strain used here was the strain RH218 (18) deposited at the American Type Culture Collection without restriction (ATCC Mo. 44076). However, it will be understood that any Saccha romyces cerevisiae strain containing a mutation which makes the cell trp1 should be an effective environment for expression of the plasmid containing the expression system. An example of another strain which could be used is pep4-l (19). This tryptophan autotroph strain also has a point nutation in TRP1 gene.

When placed on the 5' side of a non-yeast gene the 5'-flanking DMA sequence (promoter) from 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. Besides a promoter, proper expression of a non-yeast gene in yeast requires a second yeast sequence placed at the 3"-end of the non-yeast gene on the plasmid so as to allow for proper transcription termination and polyadenylation in yeast. This promoter can be suitably employed in the present invention as well as others -- see infra. In the preferred embodiments, the 5'-flanking sequence of the yeast 3-phosphoglycerate kinase gene (20) is placed *0 upstream from the structural gene followed again by DMA containing termination - polyadenylation signals, for example, the TRP1 (14, 15, 16) gene or the PGK (20) gene.

Because yeast 5'-flanking sequence (in conjunction with 3' yeast termination DNA) (infra) can function to promote expression of foreign genes in yeast, it seems likely that the 5'-flanking sequences of any highly-expressed yeast gene could be used for the expression of important gene products. Since under some circumstances yeast expressed up to 65 percent of / • its soluble protein as glycolytic enzymes (21) and since this high level appears to result from the production of high iy ( r levels of the individual mRNAs (22), it should use the 5 '-flanking sequences of any other glycolytic genes for such expression purposes - e.g., enolase, glyceraldehyde -3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, pho sphofruetokinase , glucose - 6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucofcinase. Any of the 3'-flanking sequences of these genes could also be used for proper termination and mRNA polyadenylation in such an expression system - cf. Supra. Some other highly expressed genes are those for the acid phosphatases (23) and those that express high levels of production due to mutations in the 5'-flanking regions (mutants that increase expression) -usually due to the presence of a TY1 transposable element (24).

All of the genes mentioned above are thought to be transcribed by yeast RNA polymerase II (24). It is possible that the promoters for RNA polymerase I and III which transcribe genes for ribosouial RNA, 5S RMA, and tRHAs (24, 25), may also be useful in such expression constructions.

Finally, many yeast promoters also contain transcri pti onal control so they may be turned off or on by variation in growth conditions. Some examples of such yeast promoters are the genes that produce the following proteins: Alcohol dehydrogenase II, isocytcchrooe-c, acid phosphatase, degradative enzymes associated with nitrogen metabolism, glyceraldehyde -3-phosphate dehydrogenase, and enzymes " responsible for maltose and galactose utilization (22). Such a control region would be very useful in controlling expression of protein product - especially when their production is toxic to yeast. It should also be possible to put the control region of one 5'-flanking sequence with a r ' ■'-flanking sequence containing a promoter from a highly expressed gene. This would result in a hybrid promoter and r r 1 1) /! °J) should be possible since the control region and the promoter"' ^ appear to be physically distinct DNA sequences. 3. Cell Culture Systems/Cell Culture Vectors Propogation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (see Tissue Culture, Academic Press, Kruse and Patterson eds, 1973). Employed herein was the COS-7 line of monkey kidney fibroblasts as the host for the production of immune interferon (25a). However, the experiments detailed here could be performed in any cell line which is capable of the replication and expression of a compatible vector, e.g., WI38, BHK, 3T3, CHO, VERO, and HeLa cell lines. Additionally, what is required of the expression vector is an origin of replication and a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. While these essential elements of SV40 have been exploited herein, it will be understood that the invention, although described herein in terms of a preferred embodiment, should not be construed as limited to these sequences. For example, the origin of replication of other viral (e.g., Polyoma, Adeno, VSV, Bovine Papilloma Virus (BPV), and so forth) vectors could well be used, as well as cellular origins of DNA replication which could function in a nonintegrated state. 8. Vector Systems 1. Direct Expression of Mature Immune Interferon in E. coli The procedure used to obtain direct expression of IFM-y in £. coli as a mature interferon polypeptide (minus signal sequence) was a variant of that employed earlier for human growth hormone (26) and human leukocyte interferon (1), n i n c i. f r 214261 insofar as it involved the combination of synthetic (JJ-termina 1 ) and cDNAs.

As deduced from the nucleotide sequence of p69, described i n f r a , and by comparison with the known cleavage site between signal peptide and mature polypeptide for several IF'J-aS (2), IFH-y has a hydrophobic signal peptide of 20 amino acids followed by 146 amino acids of mature IFN-t (Figure 5).

As shown in Figure 7, a BstNI restriction endonuclease site is conveniently located at amino acid 4 of mature IFN-r. Two synthetic . oligodeoxynucleotides were designed which incorporate an ATG transiational initiation codon, codons for amino acids 1, 2 and 3 (cysteine-tyrosine-cysteine) and create an EcoRI cohesive end. These oligodeoxynucleotides were ligated to a 100 base pair BstNI-PstI fragment of p69 to construct a 1115 base pair synthetic-natural hybrid gene which codes for IFN —r and which is bounded by EcoRI and PstI restriction sites. This gene was inserted into the plasmid pLelF A trp 103 between the EcoRI and PstI sites to give the expression plasmid pi FN-r trp 48. In this plasmid the IFN-y gene is expressed under the control of the E. coli trp promoter. (pLelF A trp 103 is a derivative of pLelF A 25 in which the EcoRI site distal to the LeIF A gene was removed.

"T fc N ?• The procedure used to remove this EcoRI site has been , ^ -4 i described previously (27)). * • i28NOVim WeEI<lV 2. ExpressioninYeast " * To express a heterologous gene such as the cDNA for immune interferon in yeast, it was necessary to construct a plasmid vector containing four components. The first component is the part which allows for transformation of both E. col i and yeast and thus must contain a selectable gene from t each organism. (In this case, this is the gene for ampicillin resistance from E. co1i and the gene TRP1 from yeast.) This C f A j ^ component also requires an origin of replication fron both organisms to be maintained as a plasmid DMA in both organisms. (In this case, this is the col i origin from p B R 3 2 2 and the a rs 1 origin from chromosorne III of yeast.) The second conponent of the plasmid is a 5'-flankihg sequence from a highly expressed yeast gene to promote transcription of a downstream-?1aced structural gene. In this case, the 5'-flanking sequence used is that from the yeast 3-phosphoglycerate kinase (PGK) gene. The fragment was constructed in such a way so as to remove the ATG of the PGK structural sequence as well as 8 bp upstreae fron this ATG.

This sequence was replaced with a sequence containing both an XbaI and EcoRI restriction site for convenient attachment of this 5'-flanking sequence to the structural gene.

The third component of the system is a structural gene constructed in such a manner that it contains both an ATG trans!ational start and trans 1ational stop signals. The isolation and construction of such a gene is described i nfra.

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

With all these components present, immune interferon has been produced in yeast. 3. Expression in Mammalian Cell Culture The strategy for the synthesis of immune interferon in mammalian cell culture relied on the development of a vector capable of both autonomous replication and expression of a foreign gene under the control of a heterologous transcriptional unit. The replication of this vector in tissue culture was accomplished by providing a DMA replication origin (derived from SV40 virus), and providing helper r r 314261 function (T antigen) by the introduction of the vector into a cell line endogenously expressing this antigen (28, 29). The late promoter of SV40 virus preceded the structural gene of interferon and ensured the transcription of the gene.

The vector used to obtain expression of des-Cys-Tyr-Cys recombinant human IFN-y consisted of pBR322 sequences which provided a selectable marker for selection in _E. coli (ampicillin resistance) as well as an _E. col i origin of DNA replication. These sequences were derived from the plasmid pML-1 (28) and encompassed the region spanning the EcoRI and BamHI restriction sites. The SV40 origin is derived from a 342 base pair PvuII-Hindlll fragment encompassing this region (30, 31) (both ends being converted to EcoRI ends). These sequences, in addition to comprising the viral origin of DMA replication, encode the promoter for both the early and late transcriptional unit. The orientation of the SV40 origin region was such that the promoter for the late transcriptional unit was positioned proximal to the gene encoding interferon.

Brief Description of the Drawings Figure 1 depicts a sucrose gradient centrifugation of induced Peripheral Blood Lymphocyte (PBL) Poly(A}+ RNA. Two peaks of interferon activity were observed (as shown by the hatched boxes) with sizes of 12S and 16S. The positions of ribosomal RNA markers (centrifuged independently) are labeled above the absorbance profile.

Figure 2 depicts an electrophoresis of induced PBL Poly(A)+ RNA through an acid-urea-agarose . Only one peak of activity was observed, which comigrated with 18S RNA.. The positions of ribosomal RNA markers which were el ectrophoresed in ?- - r ' ri '' ^ o adjacent lane and visualized by ethidiun bromide staining are labeled above the activity profile.

Figure 3 shows hybridization patterns of 96 colonies with 32 induced and um'nduced P-labelled cDMA probes. 96 individual trarsformants were grown in a microtiter pTate, replica plated on tv/o nitrocellulose membranes, and then the 32 filters were hybridized with P-cDMA probes prepared from either induced mRNA (above) or mRNA isolated from unfnduced PBL cultures (unfnduced, below). The filters were washed to remove non-hybridized RMA and then exposed to X-ray fila.

This set of filters is representative of 86 such sets (8300 independent colonies). An example of an "induced* clone is 1abel1ed as H12 .

Figure 4 is a restriction endonuclease map of the clone 69 cDHA insert. The cDMA insert is bounded by PstI sites (dots at both ends) and olioo dC-dG tails (single lines). The number and size of fragments produced by restriction nuclease cleavage was estimated by electrophoresis through 6 percent acrylamide gels. Positions of sites was confirmed by nucleic acid sequencing (presented in Figure 5). The coding region of the largest open reading frame is boxed and the hatched region represents the putative 20 residue signal peptide sequence, while the stipled region represents the mature IIF sequence (146 amino acids). The 5' end of the mRNA is to the left while the 3' end is to the right.

Figure 5 illustrates the nucleotide sequence of the plasxaid p69 cDNA insert, however, illustrating the most common allelic form of IFN-t- The deduced amino acid sequence of the longest open reading frame is also presented. The putative signal sequence is represented by the residues labelled Si to S20. 4U-£1£4- r r2 14 26 Figure 6 is j comparison of rFM-y mRNA structure with that of leukocyte (IFM-a) and fibroblast (IFH-a) interferons- The clone 69 mRMA (labelled immune) contains s igni f ican tly greater amounts of untranslated sequences- Figure 7 is a schematic diagram of the construction of the IFH-t expression plasmid pIFN-Y trp 48. The starting material is the 1250 base pair Ps ti cD'JA insert from plasmid p69.

Figure 8 shows a diagram of plasmid used for expression of IFN-y in monkey cells.

Figure 9 depicts a Southern hybridization of eight different EcoRI digested human genomic DMAs hybridized with a "^P-labelled 600 base pair Ddel fragment front the cDf/A insert of p69- Two EcoRI fragments clearly hybridize with the probe in each DNA sample.

Figure 10 depicts a Southern hybridization of human genomic DNA digested with six different restriction endonucleases 32 hybridized with the P-labelled probe from p69- Ffgure 11 schematically illustrates the restriction map of the 3.1 kbp H i n d111 insert of vector pBl from which the PGK promoter was isolated. Indicated is the insertion of an EcoRI site and an XbaI site in the 5'-flanking DNA of the PGK gene.

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

Figure 13 schematically illustrates techniques used to insert an XbaI site at position - 8 in the PGK promoter and to - r\ i r\r i. ^ -1 A /f isolate a 39bp fragment of the 5'-fIankfng sequence of "PC YT" containing this Xbal end and a Sau3A end.

Figure 14 sc hetna t i cal 1 y illustrates the construction of a 300 bp fragment containing the above 39bp fragment, additional PCX S'-flan.king sequence ( 2 6 5 b p ) from Pvul to Sau3A (see Fig. II), and a EcoRI site adjacent to Xbal.

Figure 15 schematically illustrates the construction of the 1500 bp PGK promoter fragment (HindiII/EcoRI) which contains, in addition to the fragment constructed fn Fig. 14, a 1300bp Hi n d 111 to Pvu I fragment from PGK 5'-flanking sequence (see Fig. 11).

Figure 16 illustrates the composition of an expression vector for human' immune interferon in yeast, containing the modified PGK promoter, the IFN —r cDHA and the terminator region of the yeast PGK gene as described in more detail herein.

Detailed Description A. Source of IFN-y mRMA Peripheral Blood Lymphocytes (PBLs) were derived from human donors by 1eukophoresis. PBLs were further purified by Ficol1-Hypaque gradient centrifugation and then cultured at a concentration of 5x10® cells/ml in RPfll 1640, 1 percent* L-glutamine, 25 mM HEPES, and 1 percent penicillin-streptomycin solution (Gibco, Grand Island, MY). These cells were induced to produce IFfi-y by the mitogen staphlococcal enterotoxin B (1 ug/rnl) and cultured for 24 to 48 hours at 37°C in 5 percent CO^. Desace ty 1 thymosin-a-1 (0.1 ug/ml) was added to PBL cultures to increase the relative yield of IFN-T activity.. r 2 1 4 2 6 1 B. Messenger RNA Isolation Total RNA from PBL cultures was extracted essentially as reported by Berger, S.L. e_t al_. (33). Cells were pelleted by centrifugation and then re suspended in 10 mM NaCI, 10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl^ and 10 mM ri bonucleoside vanadyl complex. Cells were lysed by the addition of MP-40 (I percent final concentration), and nuclei were pelleted by centrifugation. The supernatant contained the total RNA which was further purified by multiple phenol and chloroform extractions. The aqueous phase was made 0.2 M in NaCT and then total RNA was precipitated by the addition of two volumes of ethanol. RMA from uninduced (nonstimulated) cultures was isolated by the same methods. Oligo-dT cellulose chromatography was utilized to purify mRNA from the total RNA preparations (34). Typical yields from 1-2 liters of cultured PBLs were 5-10 milligrams of total RNA and 5C-200 nicrograras of Poly(A)+ RNA.

C. Size Fractionation of mRNA Tv/o methods were used to fractionate mRMA preparations.

These methods were used independently (rather than in unison) and each resulted in a significant enrichment of IFN-T mRMA.

Sucrose gradient centrifugation in the presence of the denaturant formamide was used to fractionate mRNA. Gradients of 5 percent to 25 percent sucrose in 70 percent forsiaraide (32) were centrifuged at 154, 000 x g for 19 hours at 20<rC.

Successive fractions (0.5 ml) were then removed from the top of the gradient, ethanol precipitated, and an aliquot was injected into Xe noous 1 a e v is oocytes for translation of the mRNA (35). After 24 hrs. at room temperature, the incubation medium was then assayed for antiviral activity in a standard cytopathic effect inhibition assay employing Vesicular Stomatitis Virus (Indiana strain) or Encephalomyocard i ti s ■ i - 7 r r Virus an WISH (human amnion) cells as desert' bed' by 'Stewart^ ^ -v ^ r Stewart' - J ... (36), except that the samples were incubated with the cells for 24 hours (instead of 4) prior to challenge with the virus- Two activity peaks were consistently observed in sucrose gradient fractionated RNA (Figure 1). One peak sedimented with a calculated size of 12S and contained 1CC-4-00 units/ml of antiviral activity (compared with a IFM-C standard) per microgram of RNA injected- The other peak of activity sedimented as 16S in size and contained about half the activity of the slower sedimenting peak. Each of these activity peaks appears to be due to IFN-Y, since no activity was observed when the same fractions were assayed on a bovine cell line (ND8K) which is not protected by human IFN-y. Both IFN-a activity and IFN-a activity would have been easily detected with the MDBK assay (5).

Fractionation of mRNA (200 tig) was also performed by electrophoresis through acid urea agarose gels. The slab agarose gel (37, 38) was composed of 1.75 percent agarose, 0.025 M sodium citrate, pH 3.8 and 6 M urea. Electrophoresis was performed for 7 hours at 25 milliamp and 4°C. The gel was then fractionated with a razor blade. The individual slices were melted at 70°C and extracted twice with phenol and once with chloroform- Fractions were then ethanol precipitated and subsequently assayed for IFU-y mRNA by injection into Xenopus 1 a e v i s oocytes and antiviral assay. Only one peak of activity was observed in gel fractionated sarapTes (Figure 2). Thi's peak comigrated with 18S RNA and had an activity of 600 units/ml per microgram of injected RNA. This activity also appeared to be IFN-y specific, since it did not protect nDBK cells.

The size discrepancy between activity peaks observed on sucro'se gradients (12S and 16S) and acid urea gels (18S) may be explained by the observation that these independent ? 1 fractionation methods are not performed under total denatur^g^i? conditions. 0. Preparation of a Colony Library Containing IFN-y Sequences 3 ug of gel - frac ti ona ted mR.NA was used for the preparation of double stranded cOfJA by standard procedures ( 26 , 39 ). The cDNA was size fractionated on a 6 percent polyacry1 amide gel. Two size fractions were electroeluted, 8G0-1500 bp (138 ng) and >1500 bp (204 ng). 35 ng portions of each size cDNA was extended with deoxyC residues using terminal deoxynucleoti dyl transferase (40) and annealed with 300 ng of the plasmid p8R322 (41) which had been similarly tailed with deoxyG residues at the PstI site (40). Each annealed mixture was then transformed into col i K12 strain 294. Approximately 8000 transformants were obtained with the 800-1500 bp cDMA and 400 tran s f o rman ts were obtained with the >1500 bp cDNA.

E. Screening of Colony Library for Induced cDffAs The colonies were individually inoculated into wells of microtitre plates containing LB (58) + 5 pg/ml tetracycline and stored at -20°C after addition of D.MS0 to 7 percent. Two copies of the colony library were grown up on nitrocellulose filters and the DNA from each colony fixed to the filter by the Grunstein-Hogness procedure (42).

P-labelled cDMA probes were prepared using 18S size gel fractionated mRNA from induced and uninduced PBL cultures. Oligo dT ^ 2-18 was Primer usec^ anc* reaction conditions have been previously described (1). Filters containing 8000 trans f ormants from the 600-1500 bp cDf.'A size cut and 400 tra n sf o rma n ts from the >1500 bp cDfJA size cut were hybridized wi th 20 x 10^ cprn of induced "^P-cDNA. A duplicate set of filters was hybridized with 20 x 10^ cpm of ( c uninduced P-cDNA. Mybri di zation was for 16 hours using conditions described by Fritsch e_t a_l_- (43). Filters were extensively washed (43) and then exposed to Kodak XR-5 X-ray film with OuPont Lightning-Plus intensifying screens for 16-48 hours. Each colony's hybridization pattern with the two probes was compared. Approximately 40 percent of the colonies clearly hybridized with both probes, whiTe approximately 50 percent of the colonies failed to hybridize with either probe (presented in Figure 3). 124 colonies hybridized significantly with the induced probe but undetectably or more weakly v/i th the uninduced probe. These colonies were individually inoculated into wells of microtitre plates, grown and transferred to nitrocellulose filters, and hybridized with the same two probes, as described above. Plasmid DMA isolated from each of these colonies by a rapid method (44) was also bound to nitrocellulose filters and hybridized (45) with the induced and uninduced probes. DNA from 22 colonies hybridized with only the induced probe and were termed "induced" colonies.

F. Character!zation of Induced Colonies Plasmid DNA was prepared from 5 of the induced colonies (46) and used for characterization of the cDMA inserts. Restriction endonuclease mapping of five induced plasmids (p67, p68, p69, p 71 and p 7 2) suggested that four had similar restriction nuclease maps. These four (p67, p69, p71 and p72) each had four Ddel sites, 2 HinfI sites, and a single RsaI site in the cDNA insert. The fifth plasoid (p68) contained a common Ddel fragment and appeared to be a short cOHA clone related to the other four. The homology suggested by restriction nuclease mapping was confirmed by hybridization. 3 2 A P-labelled DNA probe was prepared (47) from a 6C0 bp Dde'I' fragment of the p67 plasmid and used for hybri di zati on (42) to the other induced colonies. All five of the m n&i v 7 restriction nuclease mapped colonies cro ss-hybrijFzedgwi^ this probe, as did 17 other colonies of the 124 chosen in the induced/uninduced screening. The length of cDMA insert in each of these cross-hybridizing plasm ids was determined by PstI digestion and gel electrophoresis. The clone with the longest cD.liA insert appeared to be clone 69 with an insert length of 1200-1400 bp. This DMA was used for all further experiments, and its restriction endonuclease map is shown in Figure 4.

The cOHA insert in p69 was demonstrated to be IFM—r cDNA by its expression products:, produced in three independent expression systems, yielding antiviral activity, as described in more detail infra.

G. Sequence Analysis of cDNA Insert of p69 The complete nucleotide sequence of the plasmid p69 cDNA insert was determined by the dideoxynuc1eotide chain ternination method (48) after subcloning fragments into the M13 vector nip7 (49) and by the Haxam-Gi 1 bert chemical procedure (52). The longest open reading frame encodes a protein of 166 amino acids, presented in Figure 5. The first residue encoded is the first met codon encountered in the 5" end of the cDMA. The first 20 residues at the amino terminus probably serves as a signal sequence for the secretion of the remaining 146 amino acids. This putative signal sequence has features in common with other characterized signal sequences such as size and hydrophobicity. Furthermore, the four amino acids found at the putative cleavage sequence (ser-1eu-gly-cys) are identical with four residues found at the cleavage point of several leukocyte interferons {LeIF B, C, D, F, and H, (2)). The encoded mature amino acid sequence of 146 amino acids (hereinafter referred to as "recombinant human iranune / interferon") has a molecular weight of 17,140.

There are two potential glycosylation positions (50) in ■61CuL —1-3 the encoded prote in sequence, at amino acids 23 to 30 (asn-gly-thr) and amino acids 100 to 102 (asn-tyr-ser) . The existence of these positions is consistent with the observed glycosy1 ation of human IFN-Y (6, 51). In addition, the only two cysteine residues (positions 1 and 3) are sterically too close to form a disulfide bridge, which is consistent with the observed stability of IFH-y in the presence of reducing agents such as a-mercaptoethano 1 (51). The deduced mature amino acid sequence is generally quite basic, with 30 total lysine, arginine, and histidine residues and only 19 total aspartic acid and glutamic acid residues.

The mRNA structure of IFN-y as deduced from DNA sequence of plasmid p69 is distinctively different from IFN-a (1, 2) or IFN-a (5) mRNA. As presented in Figure 6, the coding region of IFM-y is shorter while the 5' untranslated and 3" untranslated regions are much longer than either IFN-a or IFN-B.

H. Expression of Recombinant Human Immune Interferon in E. co1i With reference to Figure 7, 50 ug of plasmid p69 were digested with Ps11 and the 1250 base pair insert isolated by gel electrophoresis on a 6 percent polyacry 1 ami de gel. Approximately 10 ng of this insert was electroeluted from the gel. 5 pg of this PstI fragment was partially digested with 3 units of BstNI (8ethesda Research Labs") for 15 minutes at 37°C and the reaction mixture purified on a 6 percent polyacrylamide gel. Approximately 0.5 yg of the desired 1100 base pair BstNI - PstI fragment was recovered. The two indicated deoxyol igonucleotides, 5 '-dAATTCATGTGTTATTGTC and 51-dTGACAATAACACATG (Figure 7) were synthesized by the phosphotriester method (53) and phosphorylated as follows. 100 pmoles of each deoxyo1igonucleotide were combined in 30 ul v •<! -1 P ' -.» z7, ir3 of '50 mM Tris-HCI (pit 8), 10 mM MgClj, 15 mM 3-me reap toethanol and 240 uCi (y-^P)ATP (Amersham, 5000 Ci/mmole). 12 units of T4 polynucleotide kinase were added and the reaction allowed to proceed at 37°C for 30 minutes. 1 U1 of 10 mM ATP was added and the reaction allowed to proceed an additional 20 minutes. After rf-OH/CHCl^ extraction the oligomers were combined with 0.25 of the BstMI-PstI 1100 base pair fragment and ethanol precipitated. These fragments were ligated at 20°C for 2 hours in 30 iil of 20 mM Tris-HCI ( pH 7.5), 10 in H HgCl2, 10 mM di t hi othrei to! , 0.5 mM ATP and 10 units T4 DNA ligase. The mixture was digested for 1 hour with 30 units of PstI and 30 units of EcoRI (to eliminate polymerization through ligation of cohesive termini) and el ectropho resed on a 6 percent po lya cry 1 amide gel. The 1115 base pair product (110,000 cpm) was recovered by 'el ectroel ution .

The plasmid pLelF A trp 103 (Figure 7) is a derivative of the plasmid pLelF A.25 (1) in which the EcoRI site distal to the LeIF A gene has been removed (27). 3 yg of pLelF A trp 103 was digested with 20 units of EcoRI and 20 units of Psti for 90 minutes at 37°C and el ectrophoresed on a 6 percent polyac ryl amide gel. The large ("3900 base pair) vector fragment was recovered by el ectroel ution. The 1115 base pair Ec oRI -P s ti IFM-y DMA fragment was ligated into 0.15 jig of this prepared vector. Transformation of £. col i K-12 strain 294 (ATCC No. 31446) gave 120 tetracycline resistant colonies. Plasmid DMA was prepared from 60 of these transfo rmants and digested with EcoRI and PstI. Three of these plasmids contained the desired 1115 bass pair EcoRI -PstI fragment. DI.'A sequence analysis verified that these plasmids had the desired nucleotide sequence at the junctions between the trp promoter, synthetic DMA and cDMA. One of these plasmids pIFN-T trp 48 was chosen for additional study. This plasmid was used to 010GL transform the E. coif K-12 strain U3110 (ATCC Mo. 27325 ).

I. Gene Structure of the IFN-r Coding Sequence The structure of the gene coding for IFH-r was analyzed by Southern hy b ri d i za ti on . In this procedure (54), 5 micrograms of high molecular weight human lymphocyte DMA (prepared as in 55) is digested to completion with various restriction endonuc1 eases, el ectropho resed on 1.0 percent agarose gels (56), and blotted to a nitrocellulose filter (54). A 3 2 P-labelled DMA probe was prepared (47) from a SOO bp Ddel fragment of the cDNA insert of p69 and hybridized (43) with the nitrocel1ulose-DNA blot. 10^ counts per minute of the probe were hybridized for 16-hours and then washed as described (43). Eight genomic DNA samples from different human donors were digested with the EcoRI restriction 3? •endonucl ease and hybridized with the p69 P-labelled probe. As presented in Figure 9, two clear hybridization signals are obse rved. wf th sizes of 8.8 fcilobase pairs (kbp) and 2.0 kbp as estimated by comparison of mobilities with H i n d 111 digested xDtJA. This could be the result of two IFN-t genes or a single gene split by an EcoRI site. Since the p69 cDMA contains no EcoRI site, an intervening sequence (intron) with an internal EcoRI site would be necessary to explain a single gene. To distinguish between these possibilities, another Southern hybridization was performed with the same probe against five other endonucl ease-digestions of a single human DMA (Figure 10). Two hybridizing DNA fragments were observed with two other endonuclease digests, P vul I (6.7 kbp and 4.0 kbp) and HincII (2.5 kbp and 2.2 kbp). However, three endonuclease digestion patterns provide only a single hybridizing DMA fragment: H i n d 111 (9.0 kbp), Bgl 11 (11.5 kbp) and BamH I (9.5 kbp). Two IFH-y genes would have to be linked at an unusually close distance (less than 9.C kbp) to be -OlOGL ( rn '1 £ J f c ■ /L contained within the saue H f n d 111 hybridizing fragment. This result suggests that only a single homologous IFM-? gene (unlike the many related IFN-a genes) is present in human genomic DfIA and that this gene is split by one or more introns containing EcoRI, P vu11, and HincII sites. This prediction 32 was supported by hybridization of a P-labeTled (4-7) fragment prepared from just the 3' untranslated region of the cO.'JA from p59 (130 bp Ddel fragment from 350 bp to 990 bp in Figure 5) against an EcoRI digest of human genomic DNA. Only the 2.0 kbp EcoRI fragment hybridized to this probe, suggesting that this fragment contains the 3' untranslated sequences, while the 8.8 kbp EcoRI fragment contains the 5' sequences. The gene structure of IFM-r (one gene with at least one intron) is distinctly different from IFN-a (multiple genes (2) without introns (56)) or IFM-s (one gene with no i n t ro n s (57)).

J. P re pa ra ti on. of bacterial extracts An overnight culture of col i M3110/pIFN-r trp 4-3 in Luria broth + 5 micrograms per ml tetracycline was used to inoculate f-19 (53) medium containing 0.2 percent glucose, 0.5 percent casamino acids, and 5 micrograms per ml tetracycline at a 1:100 dilution. Indole acrylic acid was added to a final concentration of 20 micrograms pern! when A^gg was between 0.1 and 0.2. Ten ml samples were harvested by centrifugation at Aj5q = 1.0 and resuspended immedia-tely in 1 nl phosphate buffered saline containing 1 mg per ml bovine serum albumin (PBS-BSA). Cells were opened by sonication and cleared of debris by cen tri fuga ti on . The supernatants were stored at 4*C until assay. Interferon activity in the supernatants was determined to be 250 units/ml by comparison with IF.'i-a i" / _ standards by the cytopathic effect (CPE) inhibition assay.

K. Transformation of Yeast/Strains and Media Yeast strains were transformed as previously described (59). E. co 1 i strain JA300 (th r 1euB6 th i thyA troC1117 p hsdm hsdR str ) (20) was used to select for plasmids containing functional TRP I gene. Yeast strain RH218 having the genotype (a trp1 gal 2 SUC 2 ma 1 CUPI) (18) was used as yeast transformation host. RH218 has been deposited without restriction in the American Type Culture Collection, ATCC No. 44076. M9 (minimal medium) with 0.25 percent casamino acids (CAA) and LB (rich medium) were as described by Miller (58) with the addition of 20 pg/ml ampicillin (Sigma) after media is autoclaved and cooled. Yeast were grown on the fallowing media: YEPD contained 1 percent yeast extract, 2 percent peptone and 2 percent glucose +3 percent Difco agar. YKB+CAA contained 6.7 grams of yeast nitrogen base (without amino acids) (YNB) (Difco), 10 mg of adenine, 10 nig of uracil, 5 grams CAA, 20 grams glucose and +30 grams agar per liter.

L. Construction of yeast expression vector 1. 10 ng of YRp7 (14, 15, 16) was digested with EcoRI. Resulting sticky DNA ends were made blunt using DMA Polymerase I (Klenow fragment). Vector and insert were run on 1 percent agarose (SeaKem) gel, cut from the gel, electroeluted and extracted 2X with equal volumes of chloroform and phenol before precipitation v/ith ethanol. The resulting blunt end DNA molecules were then ligated together in a final volume of 50 nl for 12 hours at 12*C. This ligation mix was then used to transform col i strain JA300 to ampicillin resistance and tryptophan prototrophy. Plasmids containing the TRPI gene in both orientations were isolated. pFRWl had the TRPI gene in the same orientation as Yftp7 while pFRW2 had the TRPI gene in the opposite orientation. ug of pFRW2 was linearized with Hindi11 and r r <5£ia ij —(j el ectrophoresed on a 1 percent agarose gel. Linear molecules were eluted from the gel and 200 ng were then ligated with 500 ng of the 3.1 kb Hindi II insert of plasmid pBl (13) which is a restriction fragment containing the yeast 3-phosphoglycerate kinase gene. The ligation mix was used to transform E. col i strain 294 to ampicillin resistance and tetracycline sensitivity. Plasmid prepared from one such recombinant had an intact TRPl gene v/ith the 3.1 kbp H i n d 111 fragment fron pBI insert DMA in the H i n d111 site of the tetracycline resistance gene. This plasmid is pFRM31. 5 ug of pFRM31 was completely digested with EcoRI, extracted twice with phenol and chloroform then ethanol precipitated- The cohesive ends of the molecule were filled in using DNA Polymerase I (Klenow fragment) in a reaction which was made 250 pM in each deoxynuc1eoside triphosphate. The reaction was performed for 20 minutes at 14°C at which time the DHA was extracted two times with phenol-chloroform, and then precipitated with ethanol. The resuspended DMA was then completely digested with CI a I and el ectrophoresed on a 5 percent acrylaciide gel. The vector fragment was eluted from the gel, phenol-chloroform extracted and ethanol precipitated.

The six N-terminal amino acids of the 3-phosphoglycerate enzyme purified from humans are as follows: One of the trans 1 ation a 1 reading frames generated from the DMA sequence of the 141 bp Sau3A-to-Sau3A restriction fragment (containing the internal HincII site; see PGK restriction map Figure 11) produces the following amino acid sequence. 1 2 3 4 6 SER - LEU - SER - HSK - LYS - LEU f ^ $ A /f i 2 3 4 6 MET - SER - LEU - SER - SER - LYS - LEU After removal of initiator methionine, it is seen that PGK N-terminal amino acid sequence has 5 of 6 amino acid homology with H-terminal amino acid sequence of human PGK.

This sequencing result suggested that the start of the yeast PGK structural gene is coded for by DNA in the 141bp Sau3A restriction fragment of pBl. Previous work (20) has suggested that the DNA sequences specifying the PGK mRNA may reside in this area of the Hindi II fragment. Further sequencing of the 141 bp Sau3A fragment gives more DMA sequence of the PGK promoter (Figure 12).

A synthetic oligonucleotide with the sequence 'ATTTGTTGTAAA31 was synthesized by standard methods (Crea e t a 1 ., Nucleic Ac i ds Res. 8 , 2331 (198G)). 100 ng of this primer was labeled at the 5* end using 10 units of T4 polynucleotide kinase in a 20 ul reaction also containing 200 3 2 iiCi of [y -P] ATP. This labeled primer solution was used in a primer-repair reaction designed to be the first step in a multi-step process to put an EcoRI restriction site in the PGK 5'-flanking DHA just preceding PGK structure gene sequence. 100 ug of p31 (20) was completely digested with Hael11 then run on a 6 percent polyacrylamide gel. The uppernost band on the ethidum stained gel (containing PGK promoter region) was isolated by e1ectroelution as described above-This 1200 bp Ha e111 piece of DNA was restricted with Hi nc11 then run on a 6 percent acrylamide gel. The 650 bp band was isolated by el ectroelution. 5 ug of DHA was isolated. This 650 bp Haeiri-to-HincII piece of DNA was resuspended in 20 ul H?0, then mixed with the 20 ul of the phosphory1ated primer solution described above. This mixture was IX phenol-chloroform extracted then ethanol precipitated. Dried - DNA was resuspendcjd in 50 U1 of and then heated in a boiling water bath For seven minutes. This solution was then quickly chilled in a dry ice-ethanol bath {10-20 seconds) then transferred to an ice-water bath. To this solution was added 50 ul of a solution containing 10 pi of 10X DMA polymerase I buffer (3oehringer Mannheim), 10 ul of a solution previously made 2.5mfl in each deoxynucl eos i de triphosphate (dAT?, dTTP, dGTP and dCTP), 25 ul of H^O and 5 units of DMA Polymerase I, Klenow fragment. This 100 ul reaction was incubated at 37°C for 4 hours. The solution was then IX phenol-chl o rofortn extracted, ethanol preci pi tated , dried by lyophil ization then exhaustively restricted with 10 units of Sau3A. This solution was then run on a 6 percent acrylaaide gel. The band corresponding to 39 bp in size was cut from the gel then isolated by el ectroel ution described above. This 39 bp band has one blunt end and one Sau3A sticky end. This fragment was cloned into a modified pFIF trp 59 vector (5). 10 u9 of pFIF trp 69 was linearized with Xbal , IX phenol chloroform extracted, then ethanol precipitated. The Xbal sticky end was filled in using DMA Polymerase I Klenow fragment in a 50 ul reaction containing 250 yM in each nucleoside triphosphate. This DHA was cut with BamHI then run on a 6 percent acrylamide gel. The vector fragment was isolated from the gel by electroelution then resuspended in 20 ul 20 ng of this vector was ligated with 20 ng of the 39bp fragment prepared above for 4 hours at room tempe rature." One-fifth of the* ligation mix was used to transform E. col i strain 294 to ampicillin resistance (on LB +20 ug/ml amp plates. Plasmids from the t ran s fo rman ts were examined by a quick screen procedure (44). One plasmid, pPGK-39 was selected for sequence analysis. 20 ug of this plasmid was digested with / t Xbal , ethanol precipitated then treated with 1000 units of bacterial alkaline phosphase at 63*C for 45 nin. The DMA was 3X phenol-chloroform extracted, then ethanol precipitated-The depho sphoryl ated ends were then labeled In a 20 pi reaction containing 200 pCi of Cy -P] ATP and 10 units of T^ polynucleotide kinase. The plasmid was cut with Sa11 and run on a 6 percent acrylamide gel.

The labeled insert band was isolated from the gel and sequenced by the chemical degradation method (52). The DMA sequence at the 3'-end of this promoter piece was as expected. 2. Construction of 312 bp PvuI-to-EcoRI PGK Promoter Fragment pg of pPGK-39 (Fig. 13) was simultaneously digested with Sal I and XbaI (5 units each) then el ectrophoresed on a 6 percent gel. The 390 bp band countaining the 39 bp promoter piece was isolated by electroelution. The resuspended DNA was restricted with Sau3A then electrophoresed on an 8 percent acrylamide gel. The 39 bp PGK promoter band was isolated by electroelution. This Df.'A contained 39 bp of the 5' end of the PGK promoter on a Sau3A-to-Xba I fragment. pg of pBl was restricted with Pvul and Kpnl then • electrophoresed on a 6 percent acrylamide gel. The .8 kbp band of DNA was isolated by electroelution, then restricted with Sau3A and electrophoresed on a 6 percent acrylamide gel. The 265 bp band from the PGK promoter (Fig. 11) was isolated by el ectroel ution.

This DNA was then ligated with* the 39 bp promoter fragment from above for two hours at room teiaperature. The ligation mix was restricted with Xbal and Pvul then electrophoresed on a 6 percent acrylamide gel. The 312 bp Xba-to-Pvul restriction fragment was isolated by electroelution, then added to a ligation mix containing 200 ng of pBR322 (41) (previously isolated missing the 162 bp Pvu I-to-Pstl restriction fragment) and 200 ng of the Of C ( 214261 X ba I - to-P s tl LeIF A cDfJA gene previously isolated from 20 ug of pLelF A 25(1). This t hre e-fac to r-1 i ga ti on mix was used to transform E_. col i strain 294 to tetracycline resistance. Transformant clonies were mi ni sc reened (44) and one of the colonies, pPGK-300 was isolated as having 304 bp of PGK 5'-Flanking DMA fused to the LeIF A gene in a pBR322 based vector. The 5' end of the LeIF A gene has the following N sequence: 51-CTAGAATTC-3' . Thus fusion of the X ba I site from the PGK promoter fragment into this sequence allows for the addition to the Xbal site an EcoRI site. pPGK-300 thus contains part of the PGK promoter isolated in a Pvul-to-EcoRI ) fragment. 3. Construction of a 1500 bp EcoRI-to-EcoRI PGK P romote r Fragment 10 ug of pBl was digested with Pvul and EcoRI and run on a 6 percent acrylamide gel. The 1.3 kb Pvul-to-EcoRI DNA band from the PGK 5-'-flanking DMA was isolated by el ectroel uti on. 10 ug of pPGK-300 was digested with EcoRI and Pvul and the 312 bp promoter fragment was isolated by el ectroel uti on after el ec tropho res i ng the digestion mix on a 6 percent acrylamide gel. 5 ug of pFRL4*was cut with EcoRI, ethanol precipitated then treated with bacterial alkaline phosphatase at 68*C for 45 minutes. After three extractions of DNA with phenol/chloroform, ethanol precipitation, and resuspension in 20 ml of H^O; 200 ng "of the vector was ligated with 100 ng of 312 bp EcoRI-to-Pvul DNA from pPGK-300 and 100 ng of EcoRI-to-P vuI DMA from pBl. The ligation mix was used to transform E_. col i strain 294 to ampicillin resistance. One of the transformants obtained was pPGK-1500. Thi s /pi asmi d contains the 1500 bp PGK promoter fragment as an Stjftfil - to-EcoRI or Hindlll-to-EcoRI piece of DNA. , ' 10 ug of pPGK-1500 was complete1y digested with CI a I 2 4 f 107 " c%"",*/See N.Z. Patent Specification No. 199,722 y and EcoRI then the digestion mix was el ec t ro p ho resed on a 6 percent acrylamide gel. The 900 bp fragment containing the PGK promoter was isolated by e1ectroelution. 10 pg of pIFN-r trp 48 was completely digested with EcoRI and HincII and el ectrop ho resed on a 6 percent acrylamide gel. The 938 bo band containing the directly expressable IFN-y cDNA was isolated from the gel by el ectroel ution.

The yeast expression vector was constructed in a three factor reaction by ligating together the PGK promoter fragment (on a CI al-to-EcoRI piece), the deleted pFRM-31 and the above isolated IFN-r cDNA. The ligation reaction was incubated at 14*C for 12 hours. The ligation mix was then used to transform £. coli strain 294 to ampicillin resistance.

Transfo rmants were analyzed for the presence of the properly constructed expression plasmid, pPGK-IFN-y (Figure 16). Plasmids containing the expression system were used to transform spheroplasts of yeast strain RH218 to tryptophan prototropy in agar missing tryptophan. These recombinant yeast were then assayed for the presence of recombinant human immune interferon.

Yeast extracts were prepared as follows: Ten ml cultures were grown in YNB+CAA until reaching Agg^^-'. collected by centrifugation then resuspended in 500 ul PBS buffer (20 mM NaH2P04, pH=7.4, 150 mM NaCl). An equal volume of glass beads (0.45-0.5 mm) were added and the mixture was then vortexed for 2'. The extracts ware spun 30 seconds at 14,000 rpm and supernatant removed-. Interferon activity in the supernatant was determined to be 16,000 units/ml by comparison with IFN-a standard using the CPE inhibition assay.

M. Construction of cell culture vector pSVr69 The 342 base pair H i n di11-P vu II fragment encompassing the SV40 origin was converted to an EcoRI restriction site bound fragment. The Hind 111 site was converted by the addition of a c C 214261 synthetic oligomer (51dAGCTCAATTC) and the Pvu11 site was converted by blunt-end ligation into an EcoRI site filled in using Polymerase I (Klenow fragment). The resulting EcoRI fragment was inserted into the EcoRI site of pML-1 (28). A plasmid with the SV40 late promoter oriented away from the 0 amp gene was further modified by removing the EcoRI site D nearest the amp gene of pML-1 (27).

The 1023 base pair Hpa I -D gl II fragment of cloned HBV DNA (60) was isolated and the Hpa I site of hepatitis B virus (HBV) converted to an EcoRI site with a synthetic oligomer (51dGCGAATTCGC). This EcoRI-BgllI bounded fragment was directly cloned into the EcoRI-BamHI sites of the plasmid described above carrying the origin of SV40.

Into the remaining EcoRI site was inserted the IFN-t gene on a 1250 base pair Psti fragment of p69 after conversion of the Ps11 ends to EcoRI ends. Clones were isolated in which the SV40 late promoter preceded the structural gene of IFN-y.

The resulting plasmids were then introduced into tissue culture cells (29) using a DEAE-dextran technique (61) modified such that the trans feetion in the presence of DEAE-dextran was carried out for 8 hours. Cell media was changed every 2-3 days. 200 microliters was removed daily for interferon bioassay. Typical yields were 50-100 units/ml on samples assayed three or four days after transfection.

Analysis demonstrates the product of expression to lack the Cys-Tyr-C.ys N-terminal portion of recombinant human immune interferon (compare Fig. 5), supporting the occurrence of signal peptide cleavage at the Cys-Gln junction (amino acids 3 and 4 in Fig. 5) such that the mature polypeptide would in fact consist of 143 amino acids.

N. Partial purification of des-Cys-Tyr-Cys recombinant human immune interferon In order to produce greater quantities of the monkey cell derived Human IFN-y» fresh monolayers of COS-7 cells in ten 10 cm plates were transfected with a total of 30 ug pDLIF3 in 110 mis DEAE--flextran (200 ug/ml DEAE Dextran 500,000 MW, .05 M Tris pH 7.5, (in DMEM).

After 16 hrs. at 37°, the plates were washed twice with DMEM. 15 mis fresh DMEM supplemented with percent f.b.s., 2 mM glutamine, 50 u/ml penicillin G, and 50 mg/.nl streptomycin was then added to each plate. The medi was replaced the following day with serum-free DMEM. Fresh serum-free media was then added every day. The media collected was kept at 4° until either assayed or bound to C?G. The pooled fractions from 3 and 4 day pos t-trans fee ti on samples were found to contain essentially all of the activity 0.5 g of CPG (controlled pore glass, Electronucleonics, CPG 350, mesh size 120/200 ) were added to 100 ml of cell supernatant and the mixture stirred for 3 hrs at 4*C. After short centrifugation in a bench top centrifuge the settled beads were packed into a column and thoroughly washed with 20 mM MaPO^ 1 M NaCl 0.1 percent s-mercaptoethanol pH 7.2. The activity was then eluted with the same buffer containing 30 percent ethyl eneglycol followed by further elution with the above buffer containing 50 percent ethyl eneglycol . Basically all the activity bound to the CPG. 75 percent of the eluted activity was found in the fractions eluted with 30 percent ethy 1 eneglycol . These fractions were pooled and diluted with 20 mM NaP04 1 M NaCl pH 7.2 to a final concentration of 10 percent ethyl eneglycol and directly applied to a 10 nil Con A Sepharose (Pharmacia) column. After a thorough wash with 20 mM NaPO^ 1 M NaCl pH 7.2 the activity was eluted with 20 mM HaPO^ 1 M NaCl 0.2 M a-methyl-D-mannoside. A substantial amount of the activity (55 percent) did not bind to this lectin. 45 percent of the activity efuted with a-methyl-D-mannoside.

Pharmaceutical Compositions The compositions of the parent invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the human imaune interferon product C 1 -■ -1 hereof is combined in admixture with a pharmaceutical^ acceptable carrier vehicle. Suitable vehicles and their f o mm 1 a t i on are described in Remington's P ha raaceuti cal Sciences by E.W. Martin, which is hereby incorporated by reference- Such compositions will contain an effective amount of the interferon protein hereof together with a suitable amount of vehicle in order to prepare p ha rmaceuti cal ly acceptable compositions suitable for effective administration to the patient.

Parenteral Administration The human immune interferon hereof may be parenterally administered to subjects requiring antitumor, or antiviral treatment, and to those exhibiting immunosuppressive .conditions. Dosage and dose rate may parallel that currently in use in clinical investigations of other human interferons, e.g., about (1-10) x 10® units daily, and in the case of materials of purity greater than 1 percent, likely up to, e.g., 50 x 10® units daily. Dosages of IFN-t could be significantly elevated for greater effect owing to the essential absence of human proteins other than IFN--r» which proteins in human derived materials may induce certain untoward effects.

As one example of an appropriate dosage form for essentially homogeneous IFN-t in parenteral form applicable -o herein, 3 mg. IFN-y of specific activity of, say, 2 x 10 U/mg may be dissolved in 25 ml. 5 N serum albumin (human) -USP, the solution passed through a bacteriological filter and the filtered solution aseptically subdivided into 100 vials, each containing 6 x 105 units pure interferon suitable for parenteral administration. The vials are preferably stored in the cold ( - 2 0 ° C ) prior to use. c c Bioassay Data f A. Characterization of antiviral activity For antibody neutralizations, samples were diluted, if necessary, to a concentration of 50C-1C00 units/ml with P8S-BSA. Equal volumes of sample were incubated for 2-12 hrs at 4 degrees with serial dilutions of rabbit antihuman leukocyte, fibroblast, or immune interferon antisera. The anti-lFN-a and 8 were obtained from the National Institute of Allergy and Infectious Dise.ases. The anti-IFM—r was prepared using authentic IFM—r (5-20 percent purity) purified from stimulated peripheral blood lymphocytes. Samples were centrifuged 3 minutes at 12,000 x g for 3 min before assay. To test pH 2 stability, samples were adjusted to pH 2 by addition of 1 M HC1, incubated for 2-12 hrs at 4*C,and neutralized by addition of IN NaOH before assay. To test sodium dodecyl sulfate (SDS) sensitivity, samples were incubated with an equal volume of 0.2 percent SDS for 2-12 hrs at 4°Cbefore assay.

B. Character!* zation of IFN-f Produced by E. col i and COS-7 cells Antiviral Activity (Units/ml) E. coli W3110/ COS-7 ce11/ Treatment IFM-a IFM-B IFM-T pIFN-Ttrp48 extract pSVy69 Supernatant Untre a ted 375 125 250 250 62.5 pH 2 375 125 <5 * <12 " <4 0.1 pe rc en t SDS 375 <4 <8 — Rabbit anti -IFN-e <8 125 250 250 187 Rabbit anti -1 FfJ-a 375 <8 187 250 125 Rabbit anti -IFN-y 375 125 <4 <8 <4 This table shows the characteristic behavior of IFN-a, s t and y standards after various treatments- The interferon activity produced by E. coli W3110/pIFN-r trp 48 and by C0S-7/pSVY69 is acid-sensi t i ve, SOS-sensitive, and neutralized by immune interferon antiserum. It is not neutralized by antibodies to IFN-a or 8. These data confirm that the products produced in these systems are immune interferons and that the cDNA insert of plasmid p69 codes for IFN-y.

Purification One method by which IFN-r can be purified from, e.g., bacteria is described by the following general scheme: 1. Extraction of the cells in high conductivity lysis buffer (at about pH 8) by passage through a homoqenizer at high pressure, cooling the effluent in an ice bath. 2. Precipitation of DNA by polyethylene-imine addition under stirring, for example, at 4*C. 3. pH precipitaton of bacterial proteins, again leaving IFN-r in solution. 4. Separation of the solids by centrifugation at 4*C.

. Concentrat ion of the supernatant (after readjusting the pH) as by ultrafiltration. 6. Dialysis of the concentrate against a low conductivity buffer. 7. Removing solids by centrifugation leaving the IFN-t in solution. 8. Ion exchange chromatography on carboxymethyl cellulose, eluting with a gradient of increasing ionic s tr eng th . 9. Chromatography on calcium phosphate gel by eluting with a gradient of increasing ionic strength.

. Ion exchange chromatography on carboxymethyl cellulose under weak denaturing conditions by eluting with a gradient of increasing ionic strength. / 11. Separation by gel filtration chromatography.

The above process enables yields of material of >95 percent purity by weight. 214261 The immune interferon proteins hereof have been defined by means of determined DNA gene and deductive amino acid sequencing—cf. Figure 5. It will be understood that for these particular interferons, embraced herein, natural allelic variations exist and occur from individual to individual.

These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. All such allelic variations are included within the scope of this invention. Indeed, the potential exists in the use of recombinant DNA technology for the preparation of various human IFN-y derivatives, variously modified by resultant single or multiple amino acid substitutions, deletions, additions or replacements. All such modifications resulting in such derivatives of human IFN-y are included within the ambit of this invention so long as the essential, characteristic human IFN-y activity remains unaffected in kind.

With the DNA and amino acid sequences of IFN-y in hand (see Figure 5), the most preferable course in reproducing the present invention doubtless would involve the preparation of either the complete gene by synthesis means (see 26, for example), or synthetic deoxyoligonucleotides with which the human genomic library or other, cDNA source could be probed in order to isolate the gene by standard hybridization techniques. Once having obtained the nucleotide sequence encoding the requisite IFN-y protein, the means for achieving expression, isolation and purification to afford highly pure preparations of IFN-y could be followed according to the above description.

Notwithstanding that reference has been made to particular / preferred embodiments, it will be further understood that the " .;/* i ^present invention is not to be construed as limited to such, rather to the lawful scope of the appended claims. - dFi - o '" ' '-!•<' ' ■>{■■■ ■ /... , w_... i Murfim,:.* c c 2 3 i bl i o g ra o hy 1. Goeddel e_t a_T_. , Mature 287, 411 (1930). 2. Goeddel et al_., Mature 290, 20 (1981 ). 3. Yelverton e_t aj_. , Nucleic Acids Research 9_, 731 (1981 ). 4. Gutterman £_t > Anna 1 s of Int. Med. 93, 399 (1980).

. Goeddel e_t aj_. , Nucleic Acids Reseach 8, 4057 (1980). 6. Yip e_t a_l_. , Proc. Mat! . Acad. Sci. (USA) 78, 1501 (1981) 7. Taniguchi et al., Proc. Natl. Acad. Sci. (USA) 78, 3469 (1981). - — 8. Bloom, Nature 289, 593 (1980). 9. Sonrienfeld eit: a_l_., Cel 1 ul ar Immunol . 40, 285 (1978).

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. Chambon, Ann. Rev. 8i oc hemi s t ry , 44, 613 (1975 ). 25a. Gluzman, Cell 23, 175 (1981). 26. Goeddel e_t al_. , Nature 281 , 544 (1979). 27. Itakura et al . , Science 198, 1056 (1977 ). t ' " —— 23. Lusky et al_. , Nature 293 , 79 (1981 ) . 29. Gluzman et al., Cold Spring Harbor Symp. Quant. Biol. 44 , 293 Tf9B"U) .

• " '-I-"-''1"■ 7 r~\ (Z * '" " " > . Fiers e_t a]_. , Na tu re 273 , 113 (1978). 31. Reddy et al., Science 200, 494 (1973). 32. Boedtker et al . , Prog, in Nucleic Acids Res. Hoi. Biol. 19 , 253 (T775T. 33. Berger _et_ aj_. , Bi oc hemi s try 18 , 5143 (1979). 34 . A vi v £t a_I_. , Proc. Natl. Acad. Sci. USA 59 , 1408 (1972 ).

. Gu rdon et aj_. , J. Molec. Biol. 80, 539 (1975 ). 36. Stewart, The Interferon System. Springer, New York d. 13-25 (197TT 37. Lehrach e_t aj_. , Bi oc hemi stry 16 , 4743 (1977). 38. Lynch e_t aj_. , V i ro 1 o gy 98 , 251 (1979). 39. Wickens e_t aj_. , J. Biol. Chem. 253, 2483 (1978). 40. Chang ert aj_. , Na tu re 275, 617 (1978). 41. Bol i va r e_t £l_. , Gene 2, 95 (1977 ). 42. Grunstein et al., Proc. Natl. Acad- Sci. U.S.A. 72, 3961 (1975). — 43 . Fri tsc h et: £l_. , Cell 19 , 959 (1980). 44. Birnboim ejt £]_. , Nucleic Acids Res. 7, 1513 (1979 ). 45. Kafatos et a^. , Nucleic Acids Res. 7, 1541 (1979). 46. Clewell e_t a_I_. , Biochemistry 9, 4428 (1970). 47 . Tayl o r ejt £]_. , Biochim. 8iophys. Acta 442 , 324 (1976 ). 48. Smith, Methods Enzymol . 65, 560 (1980). 49. Messing e_t £l_. , Nucleic Acids Res. 9, 309 (1931 ). 50. Winzler, Hormonal Proteins and Peptides (ed. Li) Academic Press, New York, p~! I (1973 J. 51. Nathan et , Nature 292 , 842 (1981 ). 52. Maxam e_t a_l_., Methods in Enzymol. 65, 490 (1980). 53. Crea ejt a_l_. , Proc. Natl. Acad. Sci. (USA) 75, 5765 -(1978). 54. Southern, J. MoT ec. Biol . 98, 503 (1975). 55. Blin e_t aj_-, Nucleic Acids Res. 3, 2303 (1976 ). 55. Lawn et , Science 212 , 1159 (1981 ). 57. Lawn et a]_. , Nucleic Acids Res. 9, 1045 (1981 ). 58. Miller, Experiments in Molecular Genetics , p. 431-3, Cold ' Spring Harbor Lab., Cold Spring Harbor, New York (1972 ). 59. Beggs , Ma tu re 2 75 , 104 (1978). c c 60. Valenzuela et aj_. , An i ma 1 Virus Genetics (ed. Fields, Jaenisch ancT"Fox) p. 57, Academic Press, Hew York (1980) 61. McCuthan et al . , J ■ Nat] . Cancer Inst. 41, 351 (1968).

•SKt) 214261

Claims (33)

WHAT WE CLAIM IS:

1. A DNA sequence comprising a sequence coding for a polypeptide comprising the amino acid sequence of recombinant human immune interferon.

2. A DNA sequence comprising a sequence coding for a polypeptide comprising the amino acid sequence of des-Cys-Tyr-Cys recombinant human immune interferon.

3. A DNA sequence according to claim 1, coding for a polypeptide comprising the amino acid sequence of recombinant human immune interferon containing the amino acid methionine attached to the N-terminus of the ordinarily first amino acid of said interferon.

4. A DNA sequence according to claim 1, coding for a polypeptide comprising the amino acid sequence of recombinant human immune interferon containing a cleavable conjugate or signal peptide attached to the N-terminus of the ordinarily first amino acid of said interferon.

5. A DNA sequence according to claim 1 operably linked with a DNA sequence capable of effecting expression of a polypeptide comprising the amino acid sequence of recombinant human immune interferon.

6. A DNA sequence according to claim 2 operably linked with a DNA sequence capable of effecting expression of a polypeptide comprising the amino acid sequence of des-Cys-Tyr-Cys recombinant human immune interferon.

7. A DNA sequence according to claim 3 operably 214261 linked with a DNA sequence capable of effecting expression of a polypeptide comprising the amino acid sequence of recombinant human immune Interferon containing the amino acid methionine attached to the N-terminus of the ordinarily first amino acid of said Interferon.

8. A DNA sequence according to claim 4 operably linked with a DNA sequence capable of effecting expression of a polypeptide comprising the amino acid sequence of recombinant human Immune Interferon containing a cleavable conjugate or signal peptide attached to the N-termlnus of the ordinarily first amino acid of said Interferon.

9. A replicable expression vehicle capable, In a transformant microorganism or cell culture, of expressing a polypeptide comprising the amino acid sequence of recombinant human Immune Interferon.

10. A replicable expression vehicle capable, In a transformant microorganism or cell culture, of expressing a polypeptide comprising the amino acid sequence of des-Cys-Tyr-Cys recombinant human Immune Interferon.

11. A replicable expression vehicle capable. In a transformant microorganism or cell culture, of expressing a polypeptide comprising the amino acid sequence of recombinant human Immune interferon containing the amino acid methionine attached to the N-terminus of the ordinarily first amino acid of said Interferon.

12. A replicable expression vehicle capable, In a transformant mIcroorganIsm or cell culture, of expressing a polypeptide comprising 50 214261 o @ the amino acid sequence of recombinant human immune interferon containing a cleavable conjugate or signal peptide attached to the N-termlnus of the ordinarily first amino acid of said interferon.

13. A microorganism or cell culture transformed with the vehicle according to any one of claims 9 to 12.

14. A cell culture according to claim 13, obtained by transforming the COS-7 line of monkey kidney fibroblasts.

15. A microorganism according to claim 13, obtained by transforming an £. col I straln.

16. A microorganism according to claim 13, obtained by transforming a yeast strain.

17. A plasmid selected from the group consisting of pIFN-Ytrp 48, pSV ^ 69, and pPGK-IFN- X(each as herein defined).

18. A microorganism or cell culture transformed with any of the plasmids according to claim 17.

19. A culture of transformant cells capable of expressing a polypeptide consisting essentially of the amino acid sequence of recombinant human immune interferon.

20. A culture of transformant cells capable of expressing a polypeptide consisting essentially of the amino 51 214261 acfd sequence of des-Cys-Tyr-Cys recombinant human Immune Interferon.

21. A process which comprises expressing a gene encoding recombinant human Immune Interferon In a microorganism or cell culture.

22. A process which comprises expressing a gene encoding des-Cys-Tyr-Cys recombinant human Immune Interferon In a microorganism or eel I culture.

23. A process for producing a polypeptide comprising the amino acid sequence of recombinant human immune interferon, said process comprising causing a culture of a microorganism or cell culture, transformed with an appropriate replicable expression vehicle, to grow and effect production of said polypeptide, and recovering said polypeptide.

24. A process for producing a polypeptide comprising the amino acid sequence of des-Cys-Tyr-Cys recombinant human immune interferon,said process comprising causing a culture of a microorganism or cell culture, transformed with an appropriate replicable expression vehicle, to grow and effect production of said polypeptide, and recovering said polypeptide.

25. A process for producing a polypeptide comprising the amino acid sequence of recombinant human Immune Interferon containing the amino acid methionine attached to the N-termlnus of the ordinarily first amino acid of said Interferon, said process comprising causing a culture of a microorganism or cell culture, transformed with an appropriate replicable expression vehicle, to grow and effect production of said polypeptide, and recovering said polypeptide. i: 2 II 4 2 f-

26. A process for producing a polypeptide comprising the amino acid sequence of recombinant human immune Interferon containing a cleavable conjugate or signal peptide attached to the N-termlnus of the ordinarily first amino acid of said interferon, said process comprising causing a culture of a m I croorgan I sm or cell culture, transformed with an appropriate replicable expression vehicle, to grow and effect production of said polypeptide, and recovering said polypeptide.

27. A process for producing an expression vehicle according to claim 9 comprising constructing a first DNA sequence coding for said polypeptide (defined In claim 9) and operably linking said first DNA sequence with a second DNA sequence capable of effecting expression of said first DNA sequence.

28. A process for producing an expression vehicle according to claim 10 comprising constructing a first DNA sequence coding for said polypeptide (defined In claim 10) and operably linking said first DNA sequence with a second DNA sequence capable of effecting expression of said first DNA sequence.

29. A process for producing an expression vehicle according to claim 11 comprising constructing a first DNA sequence coding for said polypeptide (defined In claim 11) and operably linking saTd first DNA sequence with a second DNA sequence capable of effecting expression of said first DNA sequence.

30. A process for producing an expression vehicle according to claim 12 comprising constructing a first DNA sequence coding for safd - 53 - 214261 polypeptide (defined in claim 12) and operably linking said first DNA sequence with a second DNA sequence capable of effecting expression of said first DNA sequence.

31. A product of the process according to any one of claims 21 to 30.

32. A culture of transformant cells capable of expressing a polypeptide of amino acid sequence essentially as follows: 1 40 80 10 Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu Ala Glu Asn 20 Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala 30 Asp Asn Gly Thr Leu Phe Leu Gly lie Leu Lys Asn Trp 50 Lys Glu Glu Ser Asp Arg Lys lie Met Gin Ser Gin He 60 Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp 70 Asp Gin Ser He Gin Lys Ser Val Glu Thr He Lys Glu 120 90 Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys 100 Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr 110 Asp Leu Asn Val Gin Arg Lys Ala He His Glu Leu lie 130 Gin Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly 140 Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg 146 Ala Ser Gin. 214261

33. A culture of transformant cells capable of expressing a polypeptide of amino acid sequence essentially as follows: 1 10 Gin Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys 20 Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly 30 Thr Leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu 40 50 Ser Asp Arg Lys lie Met Gin Ser Gin lie Val Ser Phe 50 Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser 70 lie Gin Lys Ser Val Glu Thr lie Lys Glu Asp Met Asn 80 90 Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp 100 Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn 110 Val Gin Arg Lys Ala He His Glu Leu He Gin Val Met 120 130 Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys 140 143 Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser Gin. Bjtta«rTh;.jir Authorised Agents, A. J. PARK & SON