BG60889B2 - Human immune interferon - Google Patents

Abstract

The invention relates to the production of new, recombinant human interferon and des-syn-syn-recombinant human interferon by recombinant DNA techniques using any of the known expression vectors and host cultures. Human immune (gamma) interferon (ifn) is isolated characterized by DNA and amino acid sequences, physical motions and biological activity.

Description

FIELD OF THE INVENTION

This invention relates to the field of recombinant DNA technology, to the means and methods used by this technology to detect the DNA sequence and to predict the amino acid sequence for human immune interferon and its production, and to the various products of this production and their use. .

More particularly, the invention relates to the isolation and identification of DNA sequences encoding human immune interferon and to the construction of recombinant expression agents containing such DNA sequences operatively linked to expression-promoting promoter sequences and to the expression constructs thus constructed. In another aspect, the present invention relates to host culture systems, such as e.g. various vertebrate microorganisms and cell cultures transformed with such expression agents and thus directed to the expression of the DNA sequences indicated above. In other respects, the invention relates to means and methods for converting end products of similar expression to novel ones, such as pharmaceutical compositions useful for the prophylaxis or treatment of humans. In preferred embodiments, this invention provides special

expression agents that are coordinated with a view to producing and secreting human immune interferon in its mature form from host cells. In addition, the present invention relates to various processes applicable to the preparation of said DNA sequences, expression agents, host culture systems and end products, and to specific and associated embodiments.

The present invention arises in part from the discovery of the DNA sequence and the predicted amino acid sequence encoding human immune interferon. In addition, the present invention provides information on the 3'- and 5'-flanking sequences of the human immune interferon gene, facilitating their in vitro binding in expression agents. In particular, a 5'-DNA segment encoding the probable endogenous signal polypeptide is provided immediately preceding the amino acid sequence of the probable human immune interferon. These findings, in turn, allow the development of the means and methods of obtaining, through DNA technology, a sufficient amount of human immune interferon, so that, in turn, its biochemical properties and biological activity can be determined. Publications and other materials, used herein to illuminate the level of the invention and, in special cases, to provide further details pertinent to the practice, are included in the literature and are conveniently numbered in the following below and grouped in the attached bibliography.

BACKGROUND OF THE INVENTION

A. Human immune interferon

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

The first group comprises a family of leukocyte interferons (ainterferon, LelF or IFN-a), which are usually produced mainly by constitutive blood cells upon viral induction. They have been produced microbiologically and have been shown to have biological activity (1,2,3). Their biological properties are the reason for their use in the clinic as therapeutic agents for the treatment of viral infections and malignancies (4).

The second group includes human fibroblast interferon (β-interferon, FIF or IFN-β), usually produced by viral induction fibroblasts, which are also produced by a microbiological pathway and for which a wide range of biological activities have been identified (5). Clinical trials have also shown their potential therapeutic value. Leukocyte and fibroblast interferons show very clear similarities in their biological properties due to the fact that the degree of homology at the amino acid level is relatively low. In addition, the two interferon groups contain 165 to 166 amino acids and are acid-stable proteins.

The human immune interferon (γ-interferon, IIF or IFN-γ) to which the present invention is directed is, contrary to α- and β-interferons, pH 2 labile, and is mainly produced by mitogenic induction of lymphocytes and is also clearly antigenic distinguishable. Until recently, human immune interferon could only be detected at very low levels, which apparently impeded its characterization. Too extensive but still partial purification of human immune interferon has recently been reported (6). The compound is produced from lymphocyte cultures stimulated with a combination of phytohaemagglutinin and phorbol ester and purified by sequential chromatographic separation. This procedure results in a product having a molecular weight of 58,000.

By translating mRNA into oocytes, very small amounts of human immune interferon are produced, exhibiting a characteristic of interferon activity similar to human immune interferon, which gives hope that immunointerferon cDNA can be synthesized and cloned (7).

The amount of immune interferon obtained so far is indeed insufficient to conduct independent experiments to characterize and determine the biological properties of the purified component. However, in vitro studies with the crude preparation as well as in vivo experiments with murine γ-interferon preparations suggest that the primary function of the immune interferon can be as much as an immunoregulatory agent (8, 9). Immunointerferon not only has antiviral and anti-cellular activity like all other human interferons, but also shows a potentiating effect on these activities with α- and β-interferon (10). In addition, the in vitro antiproliferative effect of γ-interferon on tumor cells is approximately 10- to 100-fold greater than that reported for other interferon classes (8, 11, 12). This result, together with its pronounced immunoregulatory role (8, 9), suggests slightly higher antitumor potential for IFN-? Than for IFN-? And IFN- ?. In fact, in vivo experiments with mouse and murine IFN-γ preparations

show a clear superiority over antiviral-induced interferons in their antitumor effect against osteogenic sarcoma (13).

All of these studies up to the present invention have been carried out with too crude preparations. Nevertheless, they actually suggest very important biological functions for immune interferon. Immune interferon has not only potential associated antiviral activity, but probably also strong immunoregulatory and antitumor activity, clearly targeting potentially promising clinical applications.

It is claimed that the application of recombinant DNA technology is the most effective way of providing the greater amount of human immune interferon required. Whether or not the materials thus produced include glycosylations that are considered to be characteristic of natural human-derived material, they are likely to exhibit biological activity suggesting their clinical application for the treatment of a wide variety of viral, neoplastic and immunosuppressive conditions or diseases .

B. Recombinant DNA technology

Recombinant DNA technology has reached adulthood. Molecular biologists are able to recombine different DNA sequences with ease, creating new DNA agents capable of producing copies of various endogenous products in transformed microorganisms. The main agents and methods used are in vitro binding of various blind or sticky DNA fragments producing potentially expressing agents that are used in partially transformed organisms, thereby directing them to synthesize the desired exogenous product. But for the individual product, the exchange remains volatile and science is unable to predict a permanent result. The one who predicts successful results without providing experimental data does so with relative risk.

Plasmid, a non-chromosomal double strand DNA strand isolated from bacteria and other microorganisms capable of multiple reproduction, remains an essential element of recombinant DNA technology. Included in the information encoded in the plasmid DNA is what is required to reproduce the plasmid in daughter cells (ie, the source of replication) and usually one or more phenotypically selected characteristics, such as in the case of bacteria, antibiotic resistance, allows clones of the host cell containing the desired plasmid to be recognized and preferentially cultured in the selected medium. The applicability of plasmids lies in the ability to be specifically degraded by one or the other endonuclease enzyme or restriction enzyme, each of which recognizes a different site in plasmid DNA. It is therefore possible to insert heterologous genes or gene fragments into the plasmid by

joining at the degradation site or at the reconstructed edges adjacent to the degradation site. Thus, so-called replicative expression agents are formed. DNA recombination takes place outside the cell, but the resulting recombinant replicative expression agent or plasmid can be introduced into the cell in a manner known in the art, called transformation, and thus obtain large quantities of the recombinant agent by culturing the transformants. Moreover, when the gene is inserted according to the part of the plasmid that regulates transcription and translation, and the encoded DNA message, the resulting expression means can be used to update the production of the polypeptide sequence that the inserted gene encodes, and the whole process is called expression.

Expression is initiated in an area called a promoter that is recognized and bound by RNA polymerase. In the transcriptional phase of expression, DNA is desirculated, becoming an template for initiating synthesis of transport (III) RNA from the DNA sequence. The transport RNA is in turn translated into a polypeptide having an amino acid sequence encoded by the nucleotide triplet or code that generally gives the structural 'gene, i. that portion that encodes the amino acid sequence of the expressed polypeptide product. The translation is initiated at the start signal (usually ATG, which in the resulting transport RNA passes into AUG). The so-called stop codons determine the end of translation and, therefore, the production of subsequent amino acid units. The final product can be obtained by lysis, if necessary, of the host cell from the microbial system and recovery of the product by appropriate purification from others

proteins.

In practice, using heterologous DNA technology, heterologous peptides - the so-called direct expression - can be expressed, or vice versa, heterologous polypeptides that are fused to part of the amino acid sequence of a homologous polypeptide can be expressed. In the latter cases, the expected bioactive product may undergo biologically inactive form within the fusion homologous / heterologous polypeptide until degraded in an extracellular environment. See Eng. patent publication 2007676A and Wetzel, American Scientist 68, 664 (1980).

C. Cell culture technology

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

TOTAL INVENTION

The present invention is based on the discovery that recombinant DNA technology can be used to produce human immunointerferon, preferably in direct form and in quantities sufficient to initiate and conduct animal and human testing under clinical conditions as a prerequisite for market approval. The product is suitable for use in all its forms in the prevention and treatment of people with viral infection and malignant and immunosuppressive or immunodeficient conditions. These are its forms.

include the various possible oligomeric variants containing associated glycosylation .; The product is obtained by genetically engineered microorganisms or by transformed cell culture systems. As used herein, the term cellular transformants refers to cells into which DNA has been introduced, obtained after exogenous DNA recombination, and to the progeny of any of those cells that stores the DNA so introduced. Thus, there is currently a potential to produce and isolate human immunointerferon in a more efficient manner. A particularly important factor of the present invention in its preferred embodiment is the genetic targeting of microorganisms for the production of human immune interferon in amounts allowing them to be isolated after secretion by the host cell into mature form.

The present invention encompasses the immune interferon thus obtained as well as the means and methods for preparing it. It is further directed to DNA replicating host expression agents

gene sequences that encode human immunointerferon in an expression form. The present invention is further directed to strains of microorganisms or cell cultures transformed with the expression agents described above and to microbial or cell cultures of such transformed strains or cultures capable of producing human immune interferon. In another aspect, the invention is directed to various methods applicable to obtain the gene sequence of said immune interferon, DNA expression agents, strains of microorganisms and cell cultures and their specific embodiment. Still further, this invention is directed to the preparation of fermentation cultures from the given microorganisms and cell cultures. In addition, this invention is directed to the preparation of human immune interferon as a direct expression product secreted by the host cell in mature form. This approach involves the use of the gene encoding the sequence of mature human immune interferon plus the 5 'flanking DNA encoding the signaling polypeptide. The signaling polypeptide is thought to assist the transport of the molecule to the cell wall of the host organism, where it degrades during the secretion process of the mature human immunointerferon product.

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This option allows isolation and purification of the expected mature immune interferon without resorting to procedures for eliminating contaminants of the intracellular host protein or cellular debris.

The term mature product, as used herein, refers to human immune interferon as a product of a microbial cell or cell culture, with no signal peptide or pre-sequencing peptide that results directly in translation of human immune interferon mRNA. The first recombinant immune interferon provided according to the present invention is one having methionine as the first amino acid (represented by the ATG start signal codon insertion against the structural gene) or, where methionine is intra- or extracellularly degraded and possesses, as usual, cysteine. Mature human immune interferon can also be produced, in accordance with the present invention, in conjunction with a conjugated protein other than the conventional signal peptide, the conjugate can be specifically degraded in intra- or extracellular media. See Eng. patent description Ν 2007676A. Finally, mature human immune interferon can be produced by direct expression without the need for further degradation of any foreign, redundant polypeptide. This is particularly important when a host may ineffectively remove the signal peptide or not remove it at all when the expression agent is designed to express mature human immune interferon together with its signal peptide. The mature human immune interferon thus produced is recovered and purified to the level necessary for use in the treatment of viral, malignant and immunosuppressive or immunodeficient conditions.

Human immune interferon is obtained in the following sequence:

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

2. The cell granules of such cell cultures are extracted in the presence of a ribonuclease inhibitor to isolate all cytoplasmic RNA.

3. The oligodT column isolates the total information (mRNA) in polyadenylation form. This mRNA was fractionated by size using sucrose density gradient and uric acid electrophoresis.

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

5. The vectors thus created are used to transform bacterial cells providing a colonial library. Radiolabeled cDNA prepared from induced and non-induced mRNA prepared as described above is used to separately probe the double colony libraries. The excess cDNA is then removed and the colonies are exposed to a radioactive film to identify the induced cDNA clones.

6. The corresponding plasmid DNA is isolated and matched by the cDNA-induced clones.

7. The first matched DNA is then extended in vitro for insertion into a suitable expression agent, which is used to transform E. coli host cells, which in turn are able to grow and express the desired human immune interferon product.

8. The immune interferon thus expressed undoubtedly possesses 146 amino acids in its mature form, beginning with cysteine and is highly basic in character. Its monomeric molecular weight is estimated at 17, 140. Perhaps due to the presence of many basic residues, hydrophobicity, the formation of bridges from salts, etc., the molecule can be associated in oligomeric forms, such as e.g. in the dimer, trimmer or tetramer. The previously observed high molecular weights for the naturally occurring material (6), which cannot be ascertained solely by amino acid counting, can be due to such oligomeric forms so as to promote the carbohydrate from post-translational glycosylation.

9. In true host cell systems, especially when bound in an expression agent so that it is expressed together with its signal peptide, the mature form of human immune interferon is brought to the cell culture medium, which promotes recovery and purification.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Microorganisms / Cell cultures

1. Bacterial Strains / Promoters

The actions described herein were performed with, inter alia, the microorganism E. coli K-12 strain 294 (and A, thi-, hsr-, _k hsm +), as described in Eng. patent publication N 2055382 A. This strain is deposited in ATCC under N 31446. However, many other microbial strains may be used, including E. coli B, E. coli X 1766 (ATCC N 31537) or other microbial strains, most of which are deposited and (potentially) made available by depository institutions, such as. ATCC, see also German Offenlegungssrift 2644432. These other microorganisms include, for example, bacteria such as Bacillus subtilis and other enterobacteriaceae, among which Salmonella typhimurium and Serratia marcerans, which can replicate and exaggerate sequences, may be observed.

For example, the ft-lactamase and lactose promoter systems are predominantly used to initiate and maintain the microbial production of heterologous polypeptides. Details relating to the design of these promoter systems are published by Chang et al., Nature 275 617 (1978) and Itacura et al., Science 198 1056 (1977), which are included in the literature. Recently, a so-called trp-based tryptophan promoter system was created. Details regarding the construction of such a system have been published by Goeddel et al., Nucleic Acid Research 8.4057 (1980) and Kleid et al., U. S. Ser. No. 133,296, filed March 24, 1980, incorporated by reference. Many other bacterial promoters have been discovered and used, and details regarding their nucleotide sequences allowing those of skill in the art to bind them functionally in plasmid vectors have been published, see e.g. Siebenlist et al., Cell 20, 269 (1980), incorporated herein by reference.

2. Yeast strains / Yeast promoters

The expression system used in the invention may be the plasmid YRp (14, 15, 16), which is capable of selection and replication in both E. coli and the yeast Saccharomices cerevisiae. For selection in yeast, the plasmid contains the TRP1 gene (14, 15, 16), whose complement developmental mutations in tryptophan and containing mutations in this gene have been found in chromosome IV (17). The strain used here is RH218 (18) deposited in the ATCC without restriction below N 44076. However, it should be known that any strain of Saccharomyces cerevisiae containing a mutation that makes the trpl cell should be an effective expression medium for the plasmid containing the expression system. An example of another strain that could be used is rep 4-1 (19). This tryptophan auxotrophic strain also has a point mutation on the TRP1 gene.

When a 5'-flanking DNA sequence (promoter) of a yeast gene (for alcohol dehydrogenase) is placed at the 5'-end of a gene other than yeast origin, it can promote expression of a foreign gene in yeast if placed in a plasmid applicable to yeast transformation. In addition to the promoter, the true expression of a non-yeast gene in yeast requires a second yeast sequence placed at the 3'-end of the non-yeast gene in a plasmid to allow actual transcriptional termination and polyadenylation in yeast. This promoter, as well as others, can be used appropriately in the present invention - see above. In preferred embodiments, the 5'-flanking sequence of the yeast 3- phosphoglycerate kinase gene (20) is upstream of the structural gene, followed by DNA

containing a terminator - polyadenylation signals, for example the TRP1 (14, 15, 16) gene or the PGK (20) gene.

Because the 5'-flanking sequence (linked to 3'-yeast termination DNA) (above) can functionally contribute to the expression of foreign genes in yeast, it seems that those of the 5'-flanking sequences of any high an expression yeast gene, can be used to express important gene products. Whereas in some circumstances yeasts express up to 65 percent of their soluble protein as glycolytic enzymes (21), and as far as this high level results from the production of high levels of individual mRNA (22), the use of 5'-flanking sequences of any other glycolytic genes for similar expression purposes such as e.g. enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructo kinase, glucose-6-phosphate isomerase, phosphoglucose isomerase and glucokinase. Any of the 3'-flanking sequences of these genes can also be used for termination and mRNA polyadenylation in such expression systems - see above. Other high expression genes are the genes for acid phosphatases (23) and genes that express high levels of productivity due to mutations in the 5'-flanking regions (expression-enhancing mutants), usually due to the presence of a ΊΎ1 transposing element (24). All the above mentioned genes are those that can be transcribed by yeast RNA polymerase II (24). It is possible that RNA polymerase I and III promoters that transcribe ribosomal RNA, 5S RNA and tRNA genes (24, 25) may also be applicable to similar expression structures.

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Finally, many yeast promoters also contain transcriptional control so that they can be excluded or switched on depending on the variants of the culture conditions. Examples of such yeast promoters are the genes that produce the following proteins: alcohol dehydrogenase II, isocytochrome c, acid phosphatase, degrading enzymes associated with nitrogen metabolism, glyceraldehyde-3-phosphate dehydrogenase and enzymatic enzyme, and enzymatic enzyme utilization ). Such control regions would be useful in controlling the expression of

protein product - especially when their production is toxic to yeast. It would also be possible to place the control region of a 5'-flanking sequence with a 5'-flanking sequence containing a promoter of a highly expressed gene. This would lead to a hybrid promoter and would be possible as long as the control region and the promoter are physically distinct DNA sequences.

3. Cell culture systems / Cell culture vectors

The propagation of vertebrata cells in culture (tissue culture) has been a routine procedure in recent years (see Tissue Culture, Academic Press, Kruse and Patterson eds, 1973). The COS-7 line from monkey kidney fibroblasts was used here as a host for the production of immune interferon (25). However, the exterminates described herein can be conducted in any cell line capable of replicating and expressing a competing vector such as, for example. WI138, BHK, 3T3, CHO, VERO and HeLa cell lines. In addition, the requirement for an expression vector is the source of replication and the promoter localized to the expression gene along any required ribosomal binding site, RNA cleavage site, polyadenylation site and transcription termination sequences. Although these basic elements of the SV40 are used here, it can be understood that, although described within the preferred embodiments, the invention should not be construed as being limited within this scope. For example, the source of replication of other viral vectors (such as Polioma, Adeno, VSV, BPV, and the like) may be used, as well as sources of replication from the cell that could function in a non-integrated state.

B. Vector systems

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

The procedure used to obtain direct expression of

IFN-γ in E. coli as a mature interferon polypeptide (no signal sequence) is a variant of one that uses earlier human growth hormone (26) and human leukocyte interferon (1) insofar as it involves the combination of synthetic (N-terminal) ) and cDNA.

As suggested by the nucleotide sequence of p69 described above, and by comparing known degradation sites between the signal peptide and the mature polypeptide for several IFN-? (2), IFN-? Has a hydrophobic signal peptide of 20 amino acids, followed by of 146 amino acids of mature IFN-γ (Fig. 5). As shown in FIG. 7,

The BStNI restriction endonuclease site is conventionally located at amino acid 4 of mature IFN-γ. Two synthetic oligo deoxynucleotides have been constructed that include an ATG translation initiation codon, amino acid codons 1, 2, and 3 (cysteine tyrosine cysteine) and an EcoRI cohesive end is created. These deoxyoligonucleotides are coupled to 100 base pairs of the BstNI-PstI fragment of p69 to construct a synthetic, 1115 base pair synthetic hybrid gene that encodes IFN-γ and which is linked via EcoRI and PstI restriction sites. This gene is inserted into plasmid pLelF A trp 103 between EcoRI and PstI sites to produce the expression plasmid pIFN-γ trp 48. In this plasmid, the IFN -γ gene is

expressed under the control of E. coli trp promoter. (pLelF A trp 103 was obtained from pLelF A 25 in which the EcoRI site distal to the LelF A gene was removed. The procedure used to remove this site was described previously (27).

2. Expression in yeast

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

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

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

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

Immune interferon is obtained in the presence of all these components in yeast.

3. Expression in mammalian cell cultures

The methodology for the synthesis of immune interferon in mammalian cell cultures relies on the creation of a vector capable of both autonomous replication and expression of an external gene under the control of a heterologous transcription unit. Replication of this vector in tissue culture is accomplished by providing a DNA replication source (derived from the SV40 virus) and providing an auxiliary function (T antigen) by introducing the vector into a cell line endogenously expressing this antigen (28, 29). The late promoter of the SV virus precedes the structural gene of interferon and guarantees the transcription of the gene.

The vector used to produce IFN-γ expression consists of pBR322 sequences that provide a selective marker for selection in E. coli (ampicillin resistant) as well as E. coli DNA replication source. These sequences are derived from plasmid pML-1 (28) and span the region between the restriction sites E. coli and BamHI. The SV40 source was obtained from 342 base pairs of the PvuII-Hindlli fragment covering this region (30,31) (both ends converted to EcoRI ends). These sequences, in addition to encompassing the viral source of DNA replication, encode a promoter for both the early and late transcription units. Area orientation for SV40 ί

the source is as it is at the promoter for the late transcription unit - proximal to the interferon encoding gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sucrose gradient of centrifugation of lymphocytes induced by peripheral blood Poly (A) + RNA. Two peaks of interferon activity (as indicated by shaded boxes) of 12S and 16S sizes were observed. The sites of ribosomal RNA markers (centrifuged immediately) are marked above the absorption profile.

FIG. 2 shows PBL induced poly (A) + RNA electrophoresis on uric acid agarose. There is only one activity peak observed for

18S RNA. The position of ribosomal RNA markers, which are subjected to electrophoresis in the adjacent line and visualized with ethidium bromide, are noted above the activity profile.

FIG. 3 shows the hybridization patterns of 96 colonies with induced and uninduced P ³² - labeled cDNA probes. 96 individual transformants were cultured in a microtiter plate, transferred to a nitrocellulose membrane, and then the filtrates were hybridized to P ³² cDNA probes obtained either from induction mRNA (as above) or from mRNA isolated from non-induction PBL cultures ( below), the filters are washed to remove non-hybridized RNA and then exposed to a UV-sensitive film. This filter complex is representative of 86 such complexes (8300 independent colonies). An exemplary induced clone is designated H12.

FIG. 4 is a restriction endonuclease map of the insertion clone 69 cDNA. CDNA insert is linked via PstI sites (dots at both ends) and oligo dC-dG tails (single lines). The number and size of the fragments obtained by restriction endonuclease digestion was evaluated by electrophoresis on 6 percent acrylamide gels. The positions of the sites were confirmed by nucleic acid matching (presented in Fig. 5). The coding region of the largest open reading frame is enclosed, and the shaded portion represents the putative signal peptide sequence of 20 residues, while the dotted region represents the mature IIF sequence (146 amino acids). The 5'-end of the mRNA is to the left and the 3'-end is to the right.

FIG. 5 illustrates the nucleotide sequence of the insertion plasmid p69 cDNA. The putative amino acid sequence of the longest open reading frame is also provided. The putative signal sequence is represented by the residues indicated from S1 to S20.

FIG. 6 presents a comparison of the IFN-γ mRNA structure with that of leukocyte (IFN-?) And fibroblast (IFN-?) Interferons. The 69 mRNA clone (referred to as immune) contains a significantly larger amount of untranslated sequences.

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

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

FIG. 9 shows a Southern hybridization of digested with EcoRI human genomic DNA hybridized with a P ³² - labeled Ddel fragment with 600 base pairs of the DNA insert of plasmid p69. Two EcoRI fragments were clearly hybridized to the probe from each DNA sample.

FIG. 10 shows Southern hybridization of human genomic DNA,

digested with six different restriction endonucleases hybridized with the P ³² - labeled probe from p69.

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

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

FIG. 13 schematically shows the methods used to insert the Xbal site at position-8 in the PGK promoter and isolate the 39 bp fragment from the PGK containing that Xbal end and the Sau3A end.

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

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

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

DETAILED DESCRIPTION

A. IFN source in the tRNA

Human peripheral blood leukocytes (PBLs) were obtained by leukophoresis. PBLs were purified by Ficoll-Hypaque gradient centrifugation, then cultured at a concentration of 5 x 10 ⁶ cells / ml in RPMI1640, 1 percent L-glutamine, 25 mM HEPES, and 1 percent penicillin-streptomycin solution (Gibco, Grand Island, NY). These cells were induced to produce IFN-γ by mitogenic staphylococcal enterotoxin B (1 mg / ml) and cultured for 24 to 48 hours at 37 'C. Desacetylthymosin-α-1 (1 mg / ml) was added to PBL cultures. to increase IFN- γ obtained

activity.

B. Transport RNA Isolation

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

C. TRNA size fractionation

Two methods are used to fractionate the mRNA preparations. They are applied independently (rather than simultaneously) and each results in a significant increase in the amount of IFN-γ mRNA.

Sucrose gradient centrifugation in the presence of denatured formamide is used to fractionate the mRNA. 5 to 25 percent sucrose in 70 percent formamide were centrifuged (32) at 154,000 xg for 19 hours at 20 ° C. The successive fractions (0.5 ml) were then removed, precipitated with ethanol, and an aliquot injected into oocytes of Xertopus laevis for translation of tRNAs (35). After 24 hours at room temperature, the incubation medium was tested for antiviral activity in a standard sample for the effect of cytopathic inhibition using vesicular stomatitis virus (Indiana strain) or WISH (human amnion) cell encephalomyocardial virus, as described by Stewart (36 ), except that the samples were incubated with cells for 24 hours (instead of 4) before challenge with the virus. Two constant peaks of activity in the fractionated RNA were observed (Fig. 1). One peak is sedimented at a calculated size of 12S and contains 100-400 units / ml of antiviral activity (compared to IFN-α standard) per microgram of injected RNA. Another activity peak, sedimented as 16S in size, contains half of the activity of the slower sedimented peak. Each of these activity peaks is apparently due to IFN-γ, whereas when the same fraction is tested on calf cell line (MDBK), no activity is observed since they are not protected by human IFN-γ. Both IFN-α activity and IFN-β could be readily detected by MDBK (5).

The mRNA fractionation (200 mg) was also performed by electrophoresis on uric acid agarose gels. The agarose gel plate (37, 38) consists of 1. 75 percent agarose, 0. 025 M sodium citrate, pH 3. 8, and 6M uric acid. The electrophoresis was carried out for 7 hours at 25 milliamps and 4 ° C. The wires were then cut with a razor blade. The individual sections were softened at 70 ° C and extracted twice with phenol and once with chloroform. The fractions were then precipitated with ethanol and sequentially tested for IFN-? MRNA by injection into Xenopus laevis oocytes and antiviral activity. Only one activity peak was observed in the fractionated gel samples (Figure 2). This peak is due to microwaveting together with 18S RNA and has an activity of 600 units / ml per microgram of injected RNA. This activity is probably IFN-? Specific as it does not protect MDBK cells.

The discrepancy in the magnitude of the activity peaks observed for sucrose gradients (12S and 16S) and uric acid gels (18S) can be explained by the observation that these independent fractionation methods are not feasible under complete denaturation conditions.

D. Preparation of a library of colonies containing IFN-? Sequences

For the production of double stranded DNA by standard procedure, 3 pg of gel-fractionated mRNA is used (26, 39). The cDNA was fractionated on a 6 percent polyacrylamide gel. Two fractions were electroeluted, 800-1,500 bp (138 ng) and> 1500 bp (204 ng). Parts of 35 ng of cDNA were elongated with deoxyC residues using termination deoxynucleotidyl transferase (40) and renatured with 300 ng of pBR322 (41), which was similarly extended with deoxyC residues at the PstI site (40). Each renatured mixture was then transformed into E. coli K 12 strain 294. Approximately 8000 transformants with 800-1,500 bp cDNA and 400 transformants with> 1500 bp cDNA were obtained.

E. Colonial library screening for induced cDNA

Colonies were inoculated individually into wells of microtiter plates containing LB (58) + 5 mg / ml tetracycline and left at -20 ° C after DMSO was added to 7 percent. Two copies of the colonies were cultured on nitrocellulose filters, and DNA from each colony was fixed on the filter by the Grunstein-Hognes method (42).

P ³² -labeled cDNA probes were prepared using 18S gel gel fractionated mRNA from induced and non-induced PBL cultures. Oligo dT _{! 2} _ _{] g} is the primer used, the reaction conditions described earlier (1). filters containing 8000 transformants with cDNA size 6001500 bp and transformants with size> 1500 bp hybridize with 20 χ 10 ⁶ cpm of induced P ³² cDNA. A second set of filters was hybridized with 20 x 10 ⁶ cpm of the non-induced P ³² cDNA. Hybridization was performed for 16 hours under the conditions described by Fritsch et al. (43). the filters are vigorously flushed (43) and then exposed to UV-sensitive Kodak XR-5 film with Du-Pont Lightnis-Plus Reinforced Screen for 16 to 18 hours. Each hybridization of a sample of colonies is compared with the two probes. Approximately up to 40% of the colonies hybridize clearly with both probes, while about 50% of them fail to hybridize (shown in Fig. 3). 124 colonies hybridize significantly with the induction probe, but without being detectable or poorly registered with the non-inductive probe. These colonies were inoculated individually into the wells of the microtiter plates, cultured and hybridized with the same two probes as described above. Plasmid DNA isolated from each of these colonies by method (44) also binds to nitrocellulose filters and hybridizes (45) with an induction and non-induction probe. DNA from

colonies hybridize only with an induction probe, which is why these colonies are called induced colonies.

F. Characterization of induced colonies

Plasmid DNA was prepared from five of the induced colonies (46), which was used to characterize cDNA inserts. The restriction maps of five induced plasmids (p67, p68, p69, p71, and p72) show that four of them are similar. These are p68, p69, p71 and p72, each of which has four Ddel sites, 2 HifI sites and one Rsal site in cDNA. The fifth plasmid (p68) contains a common Ddel fragment and is apparently a short cDNA clone bound to the other four. The homology suggested by the restriction maps was also confirmed by hybridization. P ³² -labelled DNA probe is prepared (47) from the 600 bp Ddel fragment of the plasmid p67, and used for hybridization (42) to the other induced colonies. All five restriction endonuclease mapped colonies cross-hybridized with this probe as did 17 other induction / non-induction colonies. screening. The length of the insert cDNA in each of these cross-hybridized plasmids was determined by PstI and gel electrophoresis. The clone with the longest cDNA insert is apparently a clone 69 with an insert length of 1200 - 1400 bp. This DNA is c

used for all further experiments and its restriction map is shown in FIG. 4.

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

G. Sequential analysis of insert cDNA in p69

The complete nucleotide sequence of the p69 plasmid insert was determined by a dideoxynucleotide chain termination method (48) by subcloning the fragments in vector M13 tr7 (49) and the Mahat-Gilbert chemical procedure (52). The longest open reading frame encodes a protein of 166 amino acids shown in FIG. 5. The first encoded residue is the first met codon counted at the 5'-end of the cDNA. The first 20 residues at the amino terminus probably serve as a signal sequence for secretion of the remaining 146 amino acids. This signaling sequence probably has features common to other signal sequences, such as size and hydrophobicity. Moreover, the four amino acids found at the probable degradation site (ser- leucs) are identical to the four residues found at the degradation points of several leukocyte interferons (LelF B, C, D, F and H (2)). . The encoded mature amino acid sequence of 146 amino acids (hereinafter referred to as recombinant human immune interferon) has a molecular weight of 17,140.

There are two potential points of glycosylation (50) in the encoded protein sequence - for amino acids 28 to 30 (asn-gly-thr) and amino acids 100 - 102 (asn-tyr-ser). The existence of these positions is consistent with the observed glycosylation of human IFN-γ in the presence of reducing agents such as β-mercaptoethanol (51). The proposed mature amino acid sequence is too basic, with a total of 30 lysine, aspartic and histidine residues and only a total of 19 aspartic and glutamic acid residues.

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

N. Expression of recombinant human interferon in E. coli

In accordance with FIG. 7, 50 pg of plasmid p69 were digested using PstI and an insert of 1250 base pairs was isolated by gel electrophoresis in a 6 percent polyacrylamide gel. Approximately 10 pg of this insert was eluted from the gel. Then, 5 µg of this PstI fragment was digested into 3 units of BstNI (Bethesda Research Labs) for 15 minutes at 37 ° C and the reaction mixture was purified on a 6 percent polyacrylamide gel. Approximately 0.5 pg of the desired BstNI-PstI fragment consisting of

1100 base pairs are being restored. The two indicated in FIG. 7 deoxyoligonucleotides - 5'-dAATTCATGTGTTATTGTC and 5'dTGACAATAACACATG, were inserted by the phosphotriesterase method (53) and phosphorylated as follows: 100 pmol of each deoxy oligonucleotide were combined in 30 µM 60M, 60 µMC 60M, 60 µM 60M, 60M 60 MgCl ₂ , 15 mM β mercaptoethanol and 240 pCi (γ-P ³² ) ATF (Amersham, 5000 Ci / mmol). 12 units of T4 polynucleotide kinase were added and the reaction was allowed to continue for another 20 minutes. After--OH / CHClНС ₃ extraction, the oligomers were combined with the BstNI-PstI fragment having 1100 base pairs and precipitated with ethanol. These fractions were ligated at 20 ° C for two hours in 30 μl of 20 mM Tris - HCl (pH 7.5), 10 mM MgCl ₂ , 10 mM dithiothreitol, 0.5 mM ATf, and 10 units of T4 DNA ligase. The mixture was decomposed in one hour with 30 units of PstI and 30 units of EcoRI (to remove polymerization by ligation of the cohesive end) and subjected to electrophoresis at 6

percent polyacrylamide gel. The product of 1,115 base pairs (110,000 ct) is recovered by electroelution.

Plasmid pLelF A trp 103 (Figure 7) is derived from plasmid pLelF A 25 (1), in which the EcoRI site, distal to the LelF A gene, is removed (27). 3 pg of pLelF A trp 103 was digested with 20 units of EcoRI and 20 units of PstI for 90 min at 37 ° C and subjected to electrophoresis on a 6 percent polyacrylamide gel. The large vector fragment (~ 3900 base pairs) is recovered by electroelution. the fragment of size 1115 base pairs, EcoRI - PstI IFN- λ

DNA was included in 0.15 pg of the vector thus prepared. The transformation of

E. coli K-12 strain 294 (ATCC N 31446) yields 120 tetracycline resistant colonies. Plasmid DNA was obtained from 60 of these transformants and digested with EcoRI and PstI. Three of these plasmids contain degradation

EcoRI - PstI 1115 base fragment pairs. DNA sequence analysis confirms that these plasmids have the desired nucleotide sequence and the binding between the trp promoter, synthetic DNA and cDNA. One of these plasmids, pIFN- λ trp 48, was selected for further study. This plasmid was used to transform E. coli K-12 strain W3110 (ATCC N 27325).

I. Gene structure of the IFN- γ coding sequence The structure of the gene coding for IFN- y _k is analyzed by Southern hybridization. In this procedure (54), 5 micrograms of high molecular weight human lymphocyte DNA (obtained as described in 55) is digested with various restriction endonucleases, electrophoresed on 1.0 percent agarose gels (56) and stained with nitrocellulose filters (54). The P ³ -labeled DNA probe was prepared (47) from a 600 bp Ddel fragment of p69 insert cDNA and hybridized (43) with nitrocellulose DNA. An amount of 10 ⁷ per minute was hybridized for 16 hours and then washed as described in (43). Eight genomic DNA samples from different donors were digested with EcoRI restriction endonuclease and hybridized with the p69 ³² P - labeled probe. As shown in FIG. 9, two clear 8.8 kilobase pair (kbp) and 2.0 kbp hybridization signals are observed as obtained after comparing mobility with HindIII degraded CDNA. This could be the result of two IFN - γ genes or the division of one gene into an EcoRI site. Since p69 does not contain an EcoRI site, an intervention sequence (intron) with an internal EcoRI site will be required to explain the signal gene. To differentiate between these two options, another Southern hybridization with the same probe is performed against five other endonuclease degradation sites. one human DNA (Fig. 10). Two other hybridizing fragments were observed with two other endonuclease degradations, PvuII (6.7 kbp and 4.0 kbp) and HincII (2. 5 kbp and 2. 2 kbp). However, three other degradation endonuclease samples provide only a single DNA hybridization fragment: HindIII (9. 0 kbp), Bglll (11. 5 kbp) and BamHI (9. 5 kbp). Two IFN- γ genes would have to bind at an unusually close distance (less than 9.0 kbp) to be located within the same HindIII hybridization fragment. This result suggests that only one homologous IFN-γ gene (unlike many IFN-α related genes) is present in human genomic DNA and that this gene is separated by one or more introns containing EcoRI, PvuII and HincII sites. This assumption is confirmed by hybridization to P ³² - labeled (47) fragment prepared from just the 3'-untranslated region of the cDNA fragment of the cDNA p69 (130 bp Ddel fragment from 860 bp to 990 bp in FIG. 5) against an EcoRI cleavage of human genomic DNA. Only 2.0 kbp EcoRI fragments hybridize to this probe, suggesting that this fragment contains 3'-untranslated sequences, whereas the 8.8 kbp EcoRI fragment contains 5'-sequences. The gene structure of IFN-γ (one gene with at least one intron) is clearly distinguishable from IFN-α (multiple genes (2) without introns (56) or IFN-β (one gene without introns (57)).

J. Preparation of bacterial extracts

E. coli W3110 / pIFN-γ trp 48 overnight broth in Luna broth + 5 micrograms per ml tetracycline is used to inoculate M9 (58) medium containing 0.2 percent glucose, 0.5 percent casamic acids and 5 5 micrograms per ml tetracycline at a dilution of 1: 100. Indole acrylic acid is added to a final concentration of 20 micrograms per ml when the A ₅₅₀ is between 0. 1 and 0. 2. The yield of ten milliliters is collected by.

centrifugation at A ₅₅₀ = 1.0 and resuspended immediately in 1 ml of phosphate buffer solution containing 1 mg per ml of bovine serum albumin (PBS-BSA). The cells were opened by sonication and purified from debris by centrifugation. The supernatants were stored at 4 ° C until tested. Interferon activity in supernatants was determined at 250 units / ml by comparing IFN-α standards with the effect of the cytopathic inhibitory assay (3E).

K. Yeast Transformation / Strains and Environment

Yeast strains were transformed as described previously (59). E. coli strain JA300 (thr leuB6 thi thyA trpC 1117 hsdm-hsdR-str ^R ) (20) will be used to select plasmids containing a functional TRPI gene. The yeast strain RH128 with the genotype (a trpl gal2 SUC2 mal CPUI) (18) was used as a host for transformation. RH218 is deposited in ATCC under N 44076. M9 (minimum medium) with 0.25 percent casamic acids (CAA) and LB (rich medium) are as described by Miller (58) with 20 pg / ml ampicillin (Sigma) supplement after the media are autoclaved and cooled. Yeast is cultured on the following medium YEPD contains 1 percent yeast extract, 2 percent peptone and 2 percent glucose + 3 percent Difco arap. YNB + CAA contains 6. 7 grams of yeast nitrogen base (excluding amino acids) (YNB) (Difco), 10 mg of adenine, 10 mg of uracil, 5 grams of CAA, 20 grams of glucose and +30 grams of agar per liter.

L. Construction of a yeast expression vector

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

the opposite orientation.

pg of pFRW2 was linearized with HindIII and electrophoresed on 1 percent agarose gel. The linear molecules were eluted from the gel and 20 ng of them were then ligated with 500 ng of 3, 1kb HindIII insert of plasmid pB1 (13), which is a restriction fragment containing the yeast 3-phosphoglycerate kinesin gene. This ligator mixture was then used to transform E. coli strain 294 into a strain resistant to ampicillin and sensitive to tetracycline. The plasmid derived from one such recombinant possesses an intact TRP1 gene with a 3.1 kbp HindIII fragment of pB1 insertion DNA at the HindIII site of the tetracycline resistance gene. This plasmid is pFRM31. 5 pg of pFRM31 was completely digested with EcoRI, extracted twice with phenol and chloroform, then precipitated with ethanol. The cohesive ends of the molecule were filled using DNA polymerase I (Klenow fragment) in a reaction brought to 250 µM in each deoxynucleoside triphosphate. The reaction was then carried out for 20 minutes at 14 ° C, during which time the DNA was extracted twice with phenol-chloroform and then precipitated with ethanol. The resuspended DNA was then completely digested with Clal and electrophoresed on a 6 percent acrylamide gel. The vector fragment was eluted from the gel, extracted with phenol-chloroform and precipitated with ethanol.

ί i

The six N-terminal amino acids of the 3-phosphoglycerate enzyme, purified by humans, are the following:

and ^! 1- 2- 3- 4- 5- 6

SER - LEU - SER - HSM - LYS - LEU i

One of the translational reading frames generated by the DNA sequence of the 141 bp Sau3A-to-Sau3A restriction fragment (containing an internal Hindi site; see PGK restriction map in Fig. 11) produces the following amino acid sequence:

1. 2- 3- 4- 5- 6

V MET-SER-LEU-SER-SER-LYS-LEU After removal of the injectable methionine, it can be seen that the PGK N-terminal amino acid sequence has 5 of the 6 amino acids homologous to the N-terminal amino acid sequence. human PGK.

This concordance result suggests that the onset of the PGK structural gene is encoded by DNA in the 141 bp Sau3A restriction fragment of pB1. Earlier developments (20) suggested that the DNA sequence specific for PGK mRNA may be located in the region of the Hind fragment. Further alignment of the 141 bp Sau3A fragment further provided the DNA sequence of the PGK promoter (Fig. 12).

By standard methods (Crea et al., Nucleic Acids Res. 8, 2331 (1980)), a synthetic oligonucleotide with the sequence 5 'ATTTGTTGTAAA 3' was synthesized. 100 ng of this primer was labeled at the 5 'end using 10 units of III4 polynucleotide kinase in a 20 µl reaction also containing 200 pCi [γ ^{32 32} - P] ATf. This labeled primer solution is used in a primer recovery reaction created as a first step in a multi-step process to incorporate an EcoRI restriction site into PGK 5 'flanking DNA preceding the PGK sequence.

the structural gene.

100 µg of pB1 (20) was completely digested with NaCl, then passed through a 6 percent polyacrylamide gel. The upper band of ethidium stained gel (containing the PGK promoter region) was isolated by electroelution as described above. This portion of the DNA, consisting of 1200 bp NaCl, was restriction with Hindi and then passed through a 6 percent acrylamide gel. The 650 bp band was isolated by electroelution. 5 pg of DNA was isolated, then this 650 bp HaeIII-toHincII portion of the DNA was resuspended in 20 μΐ H ₂ O and mixed with 20 μΐ of the phosphorylated primer solution described above. This mixture was extracted once with phenol-chloroform and then precipitated with ethanol. The dry DNA was resuspended in 50 μΐ H ₂ O and heated in boiling water for seven minutes. This solution was then rapidly cooled in a dry ice-ethanol bath (10-20 seconds) and transferred to an ice water bath. To this solution were added 50 μΐ of 10 x DNA polymerase I buffer (Boeringer Mannheim), 10 μΐ of the up to 2.5 mM solution of each deoxynucleoside triphosphate (dATP, dTTP, dGTP and dCTP), 25 μΐ Η ₂ Ο and 5 units of DNA polymerase I, Klenow fragment. This 100 μΐ reaction was incubated at 37 ° C for 4 hours. The solution was then extracted 1x phenol-chloroform, precipitated with ethanol, dried by lyophilization and subjected to restriction to 10 units of Sau3A. This solution was then passed through a 6 percent acrylamide gel. The band corresponding to 39 bp in size was cut from the gel and isolated by electroelution described above. This 39 bp strip has one blind end and one Sau3A sticky end. This fragment was cloned into a modified pFIF trp 69 vector (5). 10 gg of pFIF trp 69 were linearized with Xbal, extracted 1 x with phenol-chloroform, and then precipitated with ethanol. The xbal adhesive end was filled with the help of DNA polymerase I Klenfow fragment in a 50 gl reaction containing 250 μΜ in each nucleoside triphosphate. This DNA was cut with BamHI, then passed through a 6 percent acrylamide gel. The vector fragment was isolated from the gel by electroelution and then resuspended in 20 gl H ₂ O. 20 ng of this vector was ligated with 20 ng of 39 bp fragment prepared as above for 4 hours at room temperature. One-fifth of the ligation mixture was used to transform E. coli strain 294 into ampicillin-resistant (on LB + 20 gg / ml ampicillin plaques). Transformants plasmids are tested by the rapid screening method (44). One plasmid pPGK-39 was selected for sequence analysis. 20 gg of this plasmid was digested with Xbal, precipitated with ethanol and then subjected to 100 units of bacterial alkaline phosphatase at 68 ° C for 48 min. The DNA was then extracted three times with phenol-chloroform and precipitated with ethanol. The dephosphorylated ends are then labeled in a 20gL reaction mixture containing 200gCi [γ ³² -Ρ] ATf and 10 units of T4 polynucleotide kinase. The plasmid was cut with Sall and passed through a 6 percent acrylamide gel.

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

2. Construction of the 321 bp PvuI-to-EcoRI PGK promoter fragment gg pPGK-39 (Fig. 13) was digested simultaneously with Sall and Xbal (5 units each) and then electrophoresed on a 6 percent polyacrylamide gel. The 390 bp band representing the 39 bp promoter is isolated by electroelution. The resuspended DNA was subjected to restriction with Sau3A, then electrophoresed on an 8 percent acrylamide gel. 39 bp The PGK promoter band is isolated by electroelution. This DNA contains 39 bp from the 5 'end of the PGK promoter of the Sau3A-to-XbaI fragment.

pg of pB1 was restricted with Pvul and ΚρηΙ and then electrophoresed on a 6 percent acrylamide gel. The 0.8 kbp strand 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. I) was isolated by electroelution.

The DNA was then ligated with the 39 bp promoter fragment above for 2 hours at room temperature. The ligator mixture was restricted with Xa1 and Pvul and then electrophoresed on a 6 percent acrylamide gel. The Xba-to-Pvul restriction fragment of size 312 bp was isolated by electroelution, then added to the ligator mixture containing 200 ng pBR322 (41) (isolated earlier, omitting 162 bp Pvul-to-PstI restriction fragment) and 200 ng Xbalto - PstI LelF A cDNA gene isolated from 20 pg pLelF trp A 25. This three-component ligator mixture was used to transform E. coli strain 294 into a tetracycline resistant strain. The transformed clones were subjected to mini-screening (44) and one of the colonies, PGK-300 was isolated as having 304 bp PGK 5'-flanking DNA fused to the LelF A gene in the pBR322 main vector. The 5 terminus of the LelF A gene has the following sequence: 5 '- CTAGAATTC-3'. Thus, the fusion of the XbdI site from the PGK promoter fragment in this sequence allows the addition of an EcoR1 site to the Xbal site. Thus, the pPGK - 300 contains part of the PGK promoter isolated in the Pvul - to EcoR1 fragment.

3. Construction of 1500 bp EcoRI-to-EcoRI PGK promoter fragment pg pBl was digested with Pvul and EcoRI and passed through a 6 percent acrylamide gel. The 1.3 kb Pvul-to-EcoRI strip of DNA from the PGK 5 'flanking DNA was isolated by electroelution. 10 pg pPGK300 was digested with EcoRI and Pvul and the 312 bp promoter fragment was isolated by electroelution after electrophoresis of the digested mixture in 6 percent acrylamide gel. 5 pg of pFRL4 was cut with EcoRI, precipitated with ethanol and treated with bacterial alkaline phosphatase at 68 ° C for 45 minutes. After three phenol / chloroform DNA extractions, ethanol precipitation and resuspension in 20 ml of H ₂ O, 200 ng of the vector were ligated with 100 ng of 312 bp EcoRI-to-Pvul DNA from pPGK-300 and 100 ng EcoRI-to - Pvul DNA from 'pBl. The ligator mixture was used to transform E. coli strain 294 into ampicillin resistant. One of the transformants obtained is pPGK 1500. This plasmid contains a 1500 bp PGK promoter fragment such as the EcoRI to-EcoRI or HindIII-to-EcoRI portion of DNA.

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

The yeast expression vector was isolated in a three-factor reaction by ligation together of the PGK promoter fragment (in the Clal-to-EcoRI moiety), the deleted pFRM-31 and the IFN-γ DNA isolated above. The reaction mixture was incubated at 14 ° C for 12 hours, then used to transmit E. coli strain 294 into ampicillin resistant. Transformants were assayed for the presence of a truly engineered expression plasmid, pPGK - IFN-γ (Fig. 16). Plasmids containing expression systems are used to transform spheroplasts of the yeast strain RH218 into tryptophan prototrophic when cultured on agar in the absence of tryptophan. These recombinant yeasts are then tested for the presence of recombinant human interferon.

Yeast extracts were obtained as follows: 10 ml cultures were grown in YNB + CAA to reach A ₆₆₀ = 1-2, collected by centrifugation and then resuspended in 500 μΐ PBS buffer (20 mM NaH ₂ PO ₄ , pH = 7.4, 150 mM NaCl). An equal amount of glass beads were added and the mixture was then stirred for 2 '. The extracts were rotated for 30 s. at 14,000 gr and the supernatants are removed. The interferon activity in the supernatant was determined to be 16,000 units / ml by comparison with the IFN-α standard using CPE inhibitory samples.

M. Construction of the cell culture vector pSVy69

The HindIII-PvuII fragment, consisting of 342 base pairs and encompassing the SV40 source, is converted into an EcoRI site restriction binding fragment. The HindIII site is transformed by the addition of a synthetic oligomer (5 'dAGCTGAATTC), and the PvuII site is transformed by binding the blind ends filled into the EcoRI site using polymerase I (Klenow fragment). The resulting EcoRI fragment was inserted into the EcoRI site by pML-1 (28). The SV40 late promoter plasmid upstream of the amp * gene was further modified by deletion of the EcoRI site closest to the pML-1 amp * gene (28).

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

The remaining EcoR1 site inserts the IFN-γ gene on 1250 base pairs of the PstI fragment of p69 after converting the PstI ends into EcoR1 ends. Clones were isolated in which the SV40 late promoter precedes the structural gene of IFN-γ. The resulting plasmids are then introduced into tissue culture cells (29) using the DEAExtran technique (61) modified so that transfection in the presence of DEAE-dextran is carried out for 8 hours. The cell medium was changed every 2-3 days. 200 microliters are taken daily for examination. Typical yields are 50-100 units / ml for samples taken three or four days after the start of transfection.

The transfection product lacks the CYS-TYR-CYS N-terminal portion of recombinant human immune interferon (cf. Fig. 5), undermining the appearance of signal peptide degradation at the CYS-GLN junction (amino acids 3 and 4 in Fig. 5), that the mature olipeptide actually consists of 143 amino acids.

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

To obtain more des-CYS-TYR-CYS recombinant human immune interferon, a fresh monolayer of COS-7 cells of ten 10 cm plates was transected with a total of 30 pg pDLIF at 110 mis

DEAE-Dextran 500,000 MW, 0.05M Tris pH 7.5 in DMEM). After 16 hours at 37 ° C, the plates were washed with DMEM. To each plate were added 15 mis fresh DMEM substituted with 10 percent f.b.s., 2mM glutamine, 50 μ / ml penicillin G and 50 mg / ml streptomycin. The next day, the medium was replaced with serum-free DMEM. Fresh medium is added daily. The collected medium was kept at 4 ° C until the next test or binding to CPG. It has been found that fractions of 3-4 day transfection samples contain essentially all the activity.

0.5 g of CPG (Electronucleics, CPG 350, pore size 120/200) were added to 100 ml of cell supernatant and the mixture was stirred for 3 hours at 4 ° C. After brief centrifugation in a desktop centrifuge, the beads were placed in a column and gently washed with 20 mM NaPO ₄ IM NaCl 0.1 percent β-mercaptoethanol pH 7.2. The activity was then eluted with the same buffer containing 30 percent ethylene glycol, followed by further elution with the above buffer containing 50 percent ethylene glycol. Essentially, all activity is associated with CPG. 75 percent of the eluted activity was detected in the fractions eluted with 30 percent ethylene glycol. These fractions were poured and diluted with 20 mM NaPO ₄ IM NaCl pH 7.2 to

a final concentration of 10 percent ethylene glycol and directly applied to a 10 ml Con A Sefarosa (Pharmacia) column. After thorough washing with 20 mM NaPO ₄ IM NaCl pH 7.2, the activity was eluted with 20 mM NaPO ₄ 1M NaCl 0.2M α-methyl-D-mannoside. A significant amount of activity (55 percent) did not bind to lectin. 45 percent of the activity was eluted with α-methyl-D-mannoside.

PHARMACEUTICAL COMPOSITIONS.: .- '.ίΛί «9ώα · ϊ -» »& 4 * Μ-> · .ζ

...

The compounds of the present invention can be formulated according to known methods for the preparation of pharmaceutically applicable compositions in which human immune interferon is combined with a mixture of pharmaceutically acceptable carriers. Suitable agents and their forms are described in Remington's Pharmaceutical Sciences by E.W. Martin, included in the reference. Such compositions contain an effective amount of interferon protein, together with an appropriate amount of agents for the preparation of pharmaceutically suitable compositions suitable for use by the patient.

A. Parenteral administration

The human immune interferon of the invention may be administered parenterally to patients requiring antitumor or antiviral treatment, as well as to those with immunosuppressive conditions. Doses and amounts thereof may be those used recently in clinical studies of other human interferons, such as e.g. about (ΙΙΟ) χ 10 ⁶ units per day, and in the case of materials with a purity greater than 1 percent, or up to 1 percent, up to 50 χ 10 ⁶ per day. Doses of IFN-γ can be increased for greater effect due to the lack of essentially human proteins other than ΙΙΝ-γ, which proteins in human-derived materials can induce a substantially opposite effect.

As an example of suitable doses of homogeneously substantially IFN-γ for parenteral administration, 3 mg IFN-γ with a specific activity of, e.g. 2 x ^{10 8} U / mg was dissolved in 25 ml 5N serum albumin (human) USP, the solution was passed through a bacteriological filter and the resulting filtered solution was aseptically distributed into 100 tubes, each containing 6 χ 10 ⁶ units of pure interferon suitable for parenteral introduction. The tubes are preferably stored cold (-20 ° C) until use.

DATA FROM BIOLOGICAL RESEARCH

A. Characterization of antiviral activity

To neutralize the antibodies, the samples were neutralized, if necessary, to a concentration of 500 - 1000 units / ml with PBS - BSA. Equal volumes of the sample were incubated for 2-12 hours at 4 ° at serial dilutions of rat anti-human lymphocytes, fibroblasts or immunointerferon antiserum. Anti-IFN-? And β were obtained from the National Institute of Allergy and Infectious Diseases. Anti-IFN-γ was obtained using authentic IFN-γ (520 percent purity) purified from stimulated peripheral blood lymphocytes. Samples were centrifuged for 3 minutes at 12 LLC x g for 3 minutes before testing. For pH stability testing, samples were brought to pH 2 by the addition of 1N HCl, incubated for 2-12 hours at 4 ° and neutralized by the addition of 1 N NaOH before testing. For sodium dodecyl sulfate (SDS) sensitivity testing, samples were incubated with an equal volume of 0.2 percent SDS for 2-12 hours at 4 ° C before testing.

B. Characterization of IFN-γ derived from E. coli and COS-7 cells Anti-viral activity (units / ml)

E. coli

W3110 / COS-7 cell /

Treatment IFN's IFN-β IFN -γ pIFN-ytrp48 extract pSVy69 the supernatant untreated 375 125 250 250 62.5 pH 2 375 125 <6 <12 <4

0.1% SDS 375 - <4 <8 - Rabbits anti- <8 125 250 250 187 IFN's Rabbits anti- 375 <8 187 250 125 IFN-β Rabbits anti- 375 125 <4 <8 <4

IFN-γ

The table shows the characteristic behavior of IFN-α, β and γ standards after various treatments. The interferon activity obtained from E. coli W3110 / pIFN-γ trp 48 and from COS-7 / pSVy69 is acid-sensitive, SDS-sensitive and neutralized by antibodies against IFN-α or β. These data confirm that the products obtained with these systems are immune interferons and that the cDNA inserts of plasmid p69 encode IFN-γ.

The immunointerferon protein of the invention was determined by determining the DNA gene and the deductive coordination of amino acids, see FIG. 5. It is clear that for this particular interferon there are natural allelic variants manifested from one individual to another. These variations can be demonstrated by (one) amino acid difference (s) in the entire sequence or by deletions, substitutions, insertions, inversions or additions of (one) amino acid (s) to said sequence. All such allelic variations are included within the scope of the invention.

The preferred embodiments presented should be construed as merely intended to illustrate the present invention, without limiting it beyond its legal scope, in accordance with the claims set forth below.

REFERENCES:

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Claims (2)

Patent Claims A method for producing a replicative expression agent capable of expressing a polypeptide in a transformed microorganism or cell culture comprising the amino acid sequence des-CYS-TYR-CYS of recombinant human immune interferon consisting of constructing the first DNA sequence, constructing the first DNA sequence this polypeptide and the operative binding of that first DNA sequence to a second DNA sequence capable of inducing expression of that first DNA sequence. 2. A method of producing a replicative expression agent capable of expressing a polypeptide in a transformed microorganism or cell culture comprising the amino acid sequence of recombinant human immune interferon, consisting of constructing the first DNA sequence encoding that polypeptide and operative a second DNA sequence capable of eliciting the expression of said first DNA sequence.