NO871590L - MICROBIAL EXPRESSION BEARER. - Google Patents

Description

The present invention relates to the area of recombinant DNA technology, i.e. methods used in recombinant DNA technology and products obtained by these methods.

More specifically, the present invention relates to polypeptides, in particular complete, human fibroblast interferon, pharmaceutical compositions containing them and a method for their production which consists in obtaining a culture of a microorganism transformed with a replicable microbial expression carrier capable of expressing these polypeptides to grow up and express these polypeptides. The present invention also includes the expression carriers used in this method and the new microorganisms that contain these expression carriers as well as the methods for their production. Finally, the invention relates to DNA sequences comprising sequences for coding for the amino acid sequence of complete human fibroblast interferon.

Human fibroblast interferon (FIF) is a protein with antiviral and a wide spectrum of other biological activities (for review see W.E. Stewart II, The Interferon System, Springer-Verlag, New York-Vienna, 1979). It is stated to be purified homogeneously as a single polypeptide with a molecular weight of 19,000-20,000 with a specific activity of 2-10

x 10 g units/mg (E. Knight, Proe. Nati. Acad. Sei. USA 73, 520-523 [1976]; W. Berthold et al., J. Biol. Chem. 253, 5206

-5212 [1978]). The sequence of the 13 NH2-terminal amino acids in FIF has been determined to be Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser- (E.Knight et al., Science 207 , 525-526

[1980]). Houghton et al. (Nucleic Acids Res. 8, 1913-1931

[1980]) have used synthetic deoxyoligonucleotides (predicted from this amino acid sequence) to determine the sequence of the 276 5'-terminal nucleotides of FIF mRNA. Taniguchi et al. (Nature 28J5, 547-549 [1980]; Gene 10, 11-15 [1980] and Derynck et al. (Nature 285, 542-547 [1980]) have recently been able to identify the nucleotide sequence of cloned cDNA copies of FIF mRNA in E. coli and have derived therefrom the complete amino acid sequence of human FIF including a 21 amino acid signal sequence. The complete peptide is 166 amino acids long. Finally, Taniguchi et al. (Proe. Nati. Acad. Sei. USA 77, 5230-5233 [1980]) constructed a plasmid that directs expression in E. coli of the human FIF gene yielding complete FIF.

Through the development of recombinant DNA technology, the controlled microbial production of an enormous variety of useful polypeptides has become possible. Bacteria modified through this technology to enable the production of such polypeptide products as somatostatin, the A and B chains of human insulin, human growth hormone are already available (Itakura et al., Science 198, 1056-1063 [1977] ; Goeddel et al., Nature 2_81, 544-548 [1979]). Even more recently, recombinant DNA techniques have been used to induce bacterial production of proinsulin, thymosin and leukocyte interferon.

The workhorse of recombinant DNA technology is the plasmid,

a non-chromosomal loop of double-stranded DNA found in bacteria and other microbes, often in multiple copies per cell. Contained in the information encoded in plasmid DNA is what is required to reproduce the plasmid in daughter cells (ie a "replicon") and normally one or more selection characteristics such as in the case of bacteria, resistance to antibiotics that allows recognition of clones by the host cells that containing the plasmid of interest and preferably growing in selective media. The utility of plasmids lies in the fact that they can be specifically cleaved by some restriction endonuclease or "restriction enzyme" each finding a different site on the plasmid DNA. Subsequently, heterologous genes or gene fragments can be inserted into the plasmid by end joining at the cleavage point or at reconstructed ends near the cleavage point. DNA recombination is carried out outside the cell, but the resulting "recombinant" plasmid can be introduced into it by a process known as transformation and large amounts of the heterologous gene-containing recombinant plasmid produced by culturing the transformant. Where the gene is inserted correctly

in relation to parts of the plasmid that direct transcription and translation of the encoded DNA message, the resulting expression carrier can further be used to actually produce polypeptide sequences that the inserted gene encodes, a process called expression.

Expression is initiated at a region known as the promoter found by and bound by RNA polymerase. In some cases, such as in the tryptophan or "trp" promoter preferred in the embodiment of the present invention, promoter regions are overlapped by the "operator" region forming a combined promoter-operator. Operators are DNA sequences that are recognized by so-called repressor proteins that serve to regulate the frequency of transcription initiation at a special promoter. The polymerase runs along the DNA, transcribing the information in the coding chain from its 5'- to 3<1->end into messenger RNA which is in turn transferred into a polypeptide with the amino acid sequence that the DNA codes for. Each amino acid is encoded by a nucleotide triplet or "codon" which for the following purposes is called the "structural gene", i.e. the part which encodes the amino acid sequence of the expressed product. After binding to the promoter, RNA polymerase first transcribes nucleotides encoding a ribosome binding site, then a translation initiation or "start" signal (normally ATG, which in the resulting messenger RNA becomes AUG), then nucleotide codons within the structural gene itself. So-called stop codons are transcribed at the end of the structural gene after which the polymerase can form an additional sequence of messenger RNA which, because of the present stop signal, will remain untranslated by the ribosomes. The ribosomes bind to the binding site provided on the messenger RNA, in bacteria normally as the mRNA is formed, and themselves produce the coded polypeptide by starting at the translation start signal and ending at the aforementioned stop signal. The desired product is produced if the sequences encoding the ribosome binding site are correctly positioned relative to the AUG initiation codon and if all remaining codons follow the initiation codon in phase. The resulting product can be obtained by cleaving the host cell and isolating the product by

appropriate purification from other bacterial protein.

Although isolation from donor fibroblasts has provided sufficient material for partial characterization and limited clinical studies with homogeneous leukocyte interferon, it is a completely unsatisfactory source for the quantities of interferon needed for large-scale clinical trials and for large-scale prophylactic and/or therapeutic use thereafter. Thus, clinical investigations to date that have used human fibroblast-derived interferons in anti-tumor and anti-virus testing have in principle been limited to raw

(i.e. ^1% pure) preparations of the material, and long lead times for the production of sufficient quantities, even at unrealistic price levels, have critically delayed investigations on an advanced front.

The applicant has found that the use of recombinant DNA technology would be the most efficient way to provide large quantities of fibroblast interferon which, despite the fact that the glycosylation properties found in human-derived material will not be present in the material thus obtained, will be able used clinically in the treatment of a wide range of viruses and neoplastic diseases and succeeded in producing complete human fibroblast interferon microbially by constructing a gene for this which could be inserted into microbial expression carriers and expressed under the control of microbial gene regulator-controllers.

The path to the production of a fibroblast gene entailed the following: 1. Partial amino acid sequences from human fibroblast interferon were used to construct sets of synthetic DNA samples whose codons in the aggregate represented all possible combinations capable of encoding the partial amino acid sequences. 2. Bacterial colony banks were prepared containing complementary DNA (cDNA) from induced messenger RNA. The samples from part (1) were used to initiate the synthesis of radioactively labeled single-stranded cDNA for use as hybridization samples. The synthetic samples would hybridize with induced mRNA as a template and be extended by reverse transcription to form induced, radioactively labeled cDNA. Clones from the colony bank which hybridized to radioactively labeled cDNA obtained in this way have been further examined to confirm the presence of a full-length interferon coding gene. Any partial-length putative gene fragment obtained was itself used as a sample of the full-length gene. 3. The full-length gene obtained above was spliced using synthetic DNA such that any leader sequence was removed that could prevent microbial expression of the full polypeptide and allowed correct placement in an expression vehicle relative to the initiation signals and ribosome binding site of a microbial promoter. Expressed interferon was purified to a point that allowed its character to be determined and its activity to be determined.

Using methods of recombinant DNA technology as described above, a variety of replicable plasmid expression vectors have been constructed that direct the high-level synthesis in transformant microorganisms of a complete polypeptide with the properties of authentic human fibroblast interferon. The polypeptide product exhibits the amino acid sequence of this interferon and is active in in vitro assays despite the lack of the glycosylation characteristic of the material isolated from humans. The expression "expression of complete fibroblast interferon" denotes here the bacterial or other microbial production of an interferon molecule which does not contain any glycosyl groups or a presequence which immediately participates in the mRNA translation of the human fibroblast interferon genome. Complete fibroblast interferon according to the present invention is expressed immediately from a translation initiation signal (ATG) which also encodes the first amino acid codon of the natural product. Presence or absence of the methionine, first amino acid

in the microbially expressed product, is controlled by a kinetic phen-

omen depending on fermentation growth conditions and/or degrees of expression in the transformant host. Complete fibroblast interferon could be co-expressed with a conjugated protein quite different from the conventional leader, the conjugate being specifically cleavable in an intra- or extracellular environment (see British Patent No. 2007676A). Finally, the complete interferon could be produced in conjunction with a microbial "signal" peptide that transports the conjugate to the cell wall where the signal is processed and the finished polypeptide is secreted.

The accompanying figures 1 to 5 are described in detail in the text later. Figure 6 schematically indicates the construction

of plasmids encoding direct expression of complete fibroblast interferon. Restriction points and residues are shown as ("Pst I", etc). "Ap<R>" and "Tc<R>" denote parts of the plasmid expressing ampicillin and tetracycline resistance, respectively. The term "p o" is short for 'promoter operator'.

DESCRIPTION OF PREFERRED EMBODIMENTS

A. Microorganisms used

The work described involved the use of the microorganism E. coli K-12 strain 294 (end A, thi , hsr , hsm£), as described in British patent no. 2055382 A. This strain is deposited with the American Type Culture Collection, ATCC Accession No. 3144 6. All recombinant DNA work was carried out in accordance with the stated guidelines from the National Institutes of Health.

Although the invention is described in its most preferred embodiments with reference to E. coli K-12 strain 294 as indicated above, it also includes other known E. coli strains such as E. coli B, E. coli x 1776 and E. coli W 3110, or other microbial strains, many of which are deposited and (potentially) available from recognized microorganism depositories such as the American Type Culture Collection (ATCC). See also German Offenlegungsschrift 2644432. These other microorganisms include e.g. bacilli such as Bacillus subtilis and other enterobacteriaceae of which Salmonella typhimurium and Serratia marcescens can be mentioned as examples using plasmids which can replicate and express heterologous gene sequences therein. Yeast, such as Saccharomyces cerevisiae can also advantageously be used as a host organism in the production of interferon protein by expressing the genes that code for this under the control of a yeast promoter.

B. General methods

Restriction enzymes were purchased from New England Biolabs and used as directed. Plasmid DNA was prepared by a standard clarified lysate procedure (D.B. Clewell, J. Bacteriol. 110, 667-676 [1972] and purified by column chromatography on Biogel A-50M. DNA sequencing was performed by the method of Maxam and Gilbert (Methods Enzymol. 65, 499-560 [1980]. DNA restriction fragments were isolated from polyacrylamide gels by electroelution. DNA fragments were radioactively labeled for use as hydridization probes by the statistical calf thymus DNA priming method of Taylor et al. (Biochim. Biophys. Acta 4_42, 324-330 [1976]).In situ colony hybridizations were performed by the Grunstein-Hogness method (Proe. Nati.Acad. Sei. USA 72, 3961-3965 [1975]).

C. Chemical synthesis of deoxyoligonucleotides

The deoxyoligonucleotides were synthesized by the modified phosphorus triester method in solution (Crea et al., Proe. Nati.Acad. Sei. USA 75, 5765-5769 [1978]), using trideoxynucleotides as building blocks (Hirose et al., Tetrahedron Letters 28, 2449-2452 [1978]). The materials and general methods were similar to those described by Crea et al., Nucleic Acids Res. 8, 2331-2348 [1980]. The 6 sets of primers (Fig. 1) containing 4 dodecanucleotides each were obtained by separately connecting 2 hexamer sets (from 2 different 5'-terminal sequences each) with 3 different hexamer sets (from 2 different 3'-terminal sequences each).

D. Induction of fibroblasts

Human fibroblasts (cell line GM-2504A) were cultured as previously described by Pestka et al., Proe. Nati. Acad. Pollock. USA 72, 3898-3901 [1975]. Growth medium (Eagle's mini-male essential medium containing 10% fetal calf serum) was removed from full bottles (850 cm 3 ) and replaced with 50 ml of growth medium containing 50 µg/ml poly(I):poly(C) and 10 µg/ml cycloheximide. This induction medium was removed after 4 hours at 37°C and the cell monolayer was washed with phosphate-buffered saline (PBS; 0.14M NaCl, 3mM KCl, 1.5mMKH2PO4, 8mM Na2HPC>4). Each bottle was incubated at 37°C

with 10 ml of a trypsin - EDTA solution (Gibco 610-5305) until the cells were detached and fetal calf serum was added to a concentration of 10%. Cells were rotated for 15 min. at 500 x g and the pellets were resuspended in PBS, pooled and resedimented. Cells were frozen in liquid nitrogen. About. 0.17 g of cells were obtained per roller bottle.

E. Preparation and measurement of interferon mRNA

Poly(A)-containing mRNA was prepared from human fibroblasts by phenol extractions and oligo(dT)-cellulose chromatography as described by Green et al. (Arch. Biochem. Biophys. 172, 74-89 [1975]). The poly(A)-containing RNA was enriched for interferon mRNA by centrifugation on a linear 5-20% (w/v) sucrose gradient. The RNA samples were heated to 80°C for 2 min., rapidly cooled, layered over the gradient, and centrifuged for 20 h at 30,000 rpm. at 4°C in a Beckman SW-40 rotor. Fractions were collected, ethanol precipitated and dissolved in H2O.

One microgram samples of mRNA were injected into Xenopus laevis oocytes as described by Cavalieri et al., Proe. Nati. Acad. Pollock. USA 74, 3287-3291 [1977]. The injected oocytes were incubated for 24 hours at 21°C, homogenized and centrifuged for 5 min. at 10,000 x g. The interferon in the supernatant was measured by the cytopathic effect (CPE) inhibition assay (Stewart, The Interferon System, Springer-Verlag, New-York-Wien, 1979) using Sindbis virus and human diploid cells ( WISH). Interferon strengths of 1,000 to 6,000 units recovered (NIH reference standard) per micrograms per RNA injected was obtained for the 12S species of mRNA.

F. Synthesis and cloning of cDNA

Single-stranded cDNA was prepared in 100 µl reactions containing 5 µg of 12S fraction mRNA, 20 mM Tris-HCl (pH 8.3), 20 mM

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KC1, 8 mM MgCl2, 30 mM 3-mercaptoethanol, 100 uCi of (a P)dCTP and 1 mM dATP, dCTP, dGTP, dTTP. The primer was the synthetic HindIII decamer dCCAAGCTTGG (Scheller et al., Science 196, 177-180 [1977]) which had been extended at the 3' end by ca. 20 to 30 deoxythymidine residues using terminal deoxynucleotidyl transferase (Chang et al., Nature 275, 617-624 [1978]). 100 units of reverse transcriptase were added and the reaction mixture was incubated at 42°C for 30 min. The second strand DNA synthesis was carried out as previously described (Goeddel et al., Nature 281, 544-548 [1979]). The double-stranded cDNA was treated with 1200 units of S1 nuclease for 2 hours at 37°C in 25 mM sodium acetate (pH 4.5), 1 mM ZnCl 2 , 0.3 M NaCl. After the phenol extraction, the mixture was separated by electrophoresis on an 8% polyacrylamide gel. cDNA (~0.5 pg) ranging from 550 - 1500 base pairs in size was recovered by electroelution. A 20 ng aliquot was extended with deoxy-syC residues using terminal deoxynucleotidyl transferase (Chang et al., supra) and annealed with 100 ng pBR322 that had been cleaved with Pstl and terminated with deoxyG residues (Chang et al., supra ). The cured mixture was used for

to transform E. coli K-12 strain 2 94 by a published method (Hershfield et al., Proe. Nati. Acad. Sei.

USA 71, 3455-3459 [1974]).

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G. Preparation of induced and non-induced P cDNA samples

5 µg 12S mRNA was. combined with either 2 µg oligo(dT)-j^-is or 5 µg of each synthetic primer set (Fig. 1) in 60 µl lOmM Tris-HCl (pH 8), 1 mM EDTA. The mixtures were boiled 3

my. and chilled on ice. 60 Jul 40 mM Tris-HCl (pH 8.3), 40

mM KCl, 16 mM MgCl^ 60 mM 3-mercaptoethanol, 1 mM dATP, dGTP, dTTP and 5 x 10~<7>M (α-<32>P) dCTP (2,000 - 3,000 Ci/mM) were added each template-primer mixture at 0°C. After the addition of 100 units of reverse transcriptase, the reactions were incubated at 42°C for 30 min. and purified by passing over 10 ml Sephadex G-50 columns. The products were treated with 0.3N NaOH for 30 min at 70 oC, neutralized and ethanol precipitated.

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The P cDNAs were combined with 100 µg poly(A) mRNA from uninduced fibroblasts in 50 µl 0.4 M sodium phosphate (pH 6.8), 0.1% sodium dodecyl sulfate (SDS). The mixtures were heated

OE OE

at 98 C for 5 min. and allowed to cure for 15 hours at 45 C. The DNA-RNA hybrids (containing non-induced cDNA sequences)

were separated from single-stranded DNA (induced cDNA sequences)

by chromatography on hydroxyapatite as described by Galau et al. (Proe. Nati. Acad. Sei. USA 74, 1020-1023 [1977]. The DNA-RNA hybrids were treated with alkali to remove RNA.

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H. Examination of recombinant plasmids with P cDNA samples

About. 1 µg samples of plasmid DNA were prepared from individual transformants by a published method (Birn-boim et al., Nucleic Acids Res. 7, 1513-1523 [1979]). The DNA samples were linearized by treatment with EcoRI, denatured in alkali and applied to each of 3 nitrocellulose filters by the dot hybridization method (Kafatos et al., Nucleic Acids Res.

7, 1541-1552 [1979]). The filters were hybridized with the<32>P-cDNA samples for 16 hours at 42°C in 50% formamide, 10x Denhardt's solution (Biochem. Biophys. Res. Comm. 2_3 , 641-646 [1966]), 6xSSC 40 mM Tris- HCl (pH 7.5), 2 mM EDTA, 40 µg/ml yeast RNA. Filters were washed with 0.1xSSC, 0.1% SDS twice for 30 min.

at 42°C, dried and exposed to Kodak XR-2 x-ray film using "Dupont Lightning-Plus intensifying screens" at -80°C. [SSC contains 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0].

I. Construction of plasmids for direct expression of FIF

The synthetic primers I (dATGAGCTACAAC) and II (dCATGAGCTACAAC) were phosphorylated using T4 polynucleotide kinase and

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(Y-P)ATP to a specific activity of 700 Ci/mM as described by Goeddel et al., Proe. Nati. Acad. Pollock. USA 76, 106-110 [1979]. Primer repair reactions were performed as follows: 250 pM of the<32>P primers were combined with 8 µg

(10 pM) of a 1200 bp Hhal restriction fragment containing the FIF cDNA sequence. The mixture was ethanol precipitated, resuspended in 50 µl 1 f 2 O, boiled 3 min. , quenched in a dry ice-ethanol bath and combined with a 50 µL solution of 20 mM Tris-HCl (pH 7.5), 14 mM MgCl , 120 mM NaCl, 0.5 mM dATP, dCTP, dGTP, dTTP at 0°C . 10 units of DNA polymerase I Klenow fragment were added and the mixture was incubated at 37°C for 4.5 hours. After extraction with phenol/CHCl 2 and restriction with PstI, the desired product was purified on a 6% polyacrylamide gel. Subsequent ligations were performed at room temperature (adhesive ends) or 4°C (rejecting ends) using previously stated conditions (Goeddel et al., supra).

J. Measurement of interferon expression in E. coli

Bacterial extracts were prepared for IF measurement as follows:

1 ml cultures were grown overnight in LB (Luria-Bertani) medium containing 5 µg/ml tetracycline, then diluted in 25 ml M9 medium supplemented with 0.2% glucose, 0.5% casamino acids and 5 µg/ml tetracycline. 10 ml samples were collected by centrifugation when the absorbance at 550 nm (Arr,.) reached 1.0.

DEAF

The cell clumps were quickly frozen in a dry ice-ethanol bath and clarified lysates were prepared as described by Clewell (supra). Interferon activity in the supernatants was determined by comparison with NIH FIF standards using CPE inhibition assays. Two different measurements were performed: (a) WISH (human amnion) cells were placed in microtiter dishes. Samples were added 16 to 20 hours later and diluted with serial 2-fold dilutions. Sindbis virus was added after at least 3 h of incubation. Plates were stained 20 to 24 hours later with crystal violet. (b) MDBK (stor-fenyr) cell line was co-seeded with 2-fold dilutions of samples. Vesicular stomatitis virus was added after 2 to 3 hours of incubation and plates were stained with crystal violet 16 to 18 hours later. To test pH 2 stability, bacterial extracts and standards were diluted in minimal essential medium to a concentration of 1000 units/ml. 1 ml aliquots were adjusted to pH 2 with 1N HCl, incubated at 4°C for 16 hours and neutralized by addition of NaOH. IF activity was measured by CPE inhibition assay using human amnion cells to establish antigen identity, 25 µl aliquots of 1000 units/ml interferon samples (untreated) were incubated with 25 µl rabbit anti-human leukocyte interferon for 60 min. at 37°C. Centrifuged at 12,000 x g for 5 min. and the supernatant measured. Fibroblast and leukocyte interferon standards were obtained from the National Institutes of Health. Rabbit antihuman leukocyte interferon was obtained from the National Institute of Allergy and Infectious Diseases.

K. Chemical synthesis of primer sets complementary to

FIF mRNA

The known amino-terminal protein sequence from human fibroblast interferon made it possible to deduce the 24 possible mRNA sequences that could code for the first 4 amino acids. The 24 complementary deoxyoligonucleotides were synthesized in

6 collections of 4 dode chambers each (fig. 1).

The 6 collections of 4 deoxyoligonucleotides were each synthesized by the modified phosphorous triester method in solution and on solid phase (Crea et al., supra). The basic strategy was to react 2 different 3<1->blocked trimers with an excess of a single 5'-protected trimer which gave a collection of 2 hexamers, all of which were equally represented. The coupling of the 2 collections that each contained 2 hexamers

resulted in a collection of 4 death chambers.

L. Identification of FIF cDNA clones

Using 12S mRNA from induced human fibroblasts (1,000 units of IF activity per pg in oocyte measurement), double-stranded cDNA was prepared and inserted into pBR32 2 at the Pstl point by the standard dG:dC assembly method described by Chang et al. ., above. A fibroblast cDNA pool consisting of 30,000 ampicillin-sensitive, tetracycline-resistant transformants of E. coli K-12 strain 294 was obtained from 20 ng of cDNA varying in size from 550 to 1300 base pairs. Plasmid DNA was prepared from 600 of the transformants and plated

3 sets of nitrocellulose filters as described above.

The principle followed in identifying hybrid plasmids containing fibroblast interferon cDNA sequences was similar to that used to identify human leukocyte interferon recombinant plasmids (Goeddel et al., Nature 287, 411-416 [1980]). Radiolabeled cDNA hybridization samples were prepared using either the 24 synthetic dodecamers or oligo(dT) 1 Z - 1 o0 as primers and 12S RNA from induced fibroblasts (5000 units/µg in oocytes) as template. 3 2 P-cDNAs (specific activity > 5 x 10<8>cpm/)jg) obtained were extensively hybridized to mRNA isolated from uninduced human fibroblasts, and the mRNA-cDNA hybrids were separated from unreacted cDNA by hydroxyapatite chromatography (Galau et al., supra). The single-stranded cDNA fractions should be enriched for sequences that are present in induced fibroblasts but not present in uninduced cells, and the mRNA-cDNA hybrids should represent sequences that are common to both induced and non-induced cells. About. 4 x 10 cpm single-stranded cDNA (hybridization sample A) and 8 x 10^ cpm cDNA-mRNA hybrids were obtained using oligo(dT) no primed cDNA; 1.5 x IO<6>cpm single chain 12-18 6

(hybridization sample B) and 1.5 x 10 cpm hybrids were obtained from cDNA primed using synthetic dodecamer assemblies 1-6. The cDNA-mRNA hybrids from both fractions were pooled, the RNA hydrolysed by treatment with alkali and

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P-cDNA used as hybridization sample C. Many of the 600 plasmid samples hybridized with both samples A and C indicating that the hybridization reactions between non-induced

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mRNA and P-cDNA (before the hydroxyapatite fractionation step) were not complete. However, only one of the 600 plasmids (pF526) hybridized strongly with the specifically primed, induced cDNA sample B (Fig. 2). Plasmid pF52 6 also hybridized with the total primed oligo(dT) 1Q, induced cDNA

iz —lo

sample A, and gave no detectable hybridization with the combined uninduced sample C.

Pstl processing of pF526 showed that the cloned cDNA insert was approx. 550 base pairs long, probably too short to contain the entire coding region for fibroblast interferon. Therefore became

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a P-labeled DNA sample prepared from this Pstl fragment by statistical priming with calf thymus DNA (Taylor et al., supra). This sample was used to screen 2000 individual colonies from a newly constructed fibroblast cDNA pool (the new cDNA pool was prepared using 12S mRNA from induced fibroblasts at a strength of 6000 units per ml in the oocyte assay system. 16 clones hybridized to the sample. The plasmid prepared from the bulk of these released 2 fragments when digested with Pstl which indicated that the cDNA contained an internal Pstl site. The clone pFIF3 contained the largest cDNA insert, approximately 800 base pairs. The DNA sequence of the insert was determined by Maxam-Gilbert<1>'s method (above) and is shown in Fig. 3. The amino acid sequence of human fibroblast interferon predicted from the nucleotide sequence is identical to that recently reported by Taniguchi et al. (Gene _10, 11-15 [1980 ] and by Derynck et al.

(above) from DNA sequencing of FIF cDNA clones. A per-cursor or signal peptide of 21 amino acids is followed by a sequence of 166 amino acids that constitutes the complete interferon, a stretch of 196 3<1-> untranslated nucleotides and a poly(A) tail. The NH 2 -terminal 20 amino acids of complete FIF have been directly determined by protein microsequencing and are the same as those predicted from the DNA sequence.

M. Direct expression of fibroblast interferon

To express large amounts of complete fibroblast interferon in E. coli, the initiation of protein synthesis must occur at the ATG codon of the complete polypeptide (amino acid 1) rather than at the ATG of the signal peptide (amino acid S1) (Fig. 3).

Present principle to remove the signal peptide coding regions

from pFIF3 is shown in fig. 4. A 1200 bp DNA fragment containing the entire cDNA insert was isolated from a polyacrylamide gel after treatment of pFIF3 with Hhal. 2 separate synthetic deoxyoligonucleotide primers, dATGAGCTACAAC(I) and dCATGAGCTACAAC(II) were prepared. Both primers contain the coding sequence for the first 4 amino acids of complete fibroblast interferon; primer II has an additional C at the 5' end. Primer repair reactions and subsequent ligation were performed separately for primers I and II and gave almost identical results. Therefore, only reactions using primer I are discussed in detail here. The primers were 5'-radiolabeled using (γ- 32 P)ATP and T4 polynucleotide kinase combined with the 1200 bp Hhal DNA fragment and the mixtures were denatured by boiling. After hybridization of the primer to the denatured Hhal DNA fragment, E. coli DNA polymerase I Kelnow fragment (Klenow et al., Proe. Nati. Acad. Sei. USA 6J5, 168-175 [1970]) was used to catalyze the repair synthesis of the plus (top) chain (Fig. 4). In addition, the accompanying 31 —> 51 exonuclease activity of the Klenow fragment removed the 3<1->protruding end from the minus (bottom) chain, leaving a direct end. Analyzes of samples of the reaction mixture by polyacrylamide gel electrophoresis indicated that the repair synthesis had not proceeded completely but stopped at several defined points. Therefore, the entire reaction mixture was treated with PstI and the desired 141 bp fragment (180,000 Cerenkow cpm; ^0.3 pM) was purified by polyacrylamide gel electrophoresis oresis (Fig. 5). Ligation of this fragment to 1 pg (~4 pM) of the 363 bp PstI-BglII fragment isolated from pFIF3 (Fig. 4), followed by Bglll treatment gave 50,000Cerenkov cpm (~0 .1 pM,/v30 ng) of 504 bp DNA fragment that contained the entire code the sequence for complete fibroblast inter-

feron. The same reactions using primer II yielded 83,000 cpm (~0.15 pM, ~50 ng) of 505 bp product.

The construction of plasmids directing the synthesis of human fibroblast interferon is shown in Fig. 6. Separate expression plasmids were constructed that placed FIF synthesis under the control of E. coli lac or trp promoter operator systems. Both of these systems have proven useful for the direct expression of eukaryotic genes in E. coli: human growth hormone has been efficiently synthesized using the lac system (Goeddel et al., Nature 281, 544-548 [1979] and human leukocyte interferon has been produced in large quantities using the trp system (Goeddel et al., Nature 287, 411 [1980]).

pBRH trp was treated with EcoRI restriction enzyme and the resulting fragment isolated by PAGE and electroelution. EcoRI-treated plasmid pSom 11 (Itakura et al., Science 198, 1056-1063 [1977]); GB patent publication no. 2 007 676 A) was combined with the above fragment. The mixture was ligated with T. DNA ligase and the resulting DNA transformed into E. coli K-12 strain 294 as previously described. Transformant bacteria were selected on ampicillin-containing plates. Resulting ampicillin-resistant colonies were examined by colony hybridization (Grunstein et al., supra) using as a probe the trp-promoter-operator-containing above-mentioned fragment isolated from pBRHtrp, which had been radioactively labeled 32

kert with P . Several colonies that proved positive by colony hybridization were selected, plasmid DNA was isolated and the orientation of the inserted fragments determined by restriction analysis using restriction enzymes BglII and BamHI in double treatment. E. coli 294 containing the plasmid named pSOM7A2, which had the trp promoter-operator fragment in the desired orientation was grown in LB medium containing 10 µg/ml ampicillin. The cells were grown to optical density 1 (at 550 nM), collected by centrifugation and resuspended in M9 media at a 10-fold dilution. Cells were grown for 2-3 hours, again to optical density 1, then lysed and total cell protein analyzed by SDS urea (15%) PAGE (Maizel et al., Methods Virol. 5, 180-246 [1971]).

Plasmid pBR322 was HindIII treated and the overhanging HindIII ends again treated with S1 nuclease. The Sl nuclease treatment involved treatment of 10 µg HindIII-cleaved pBR322 in 30 µl Sl buffer (0.3 M NaCl, 1 mM ZnCl 2 , 25 mM sodium acetate, pH 4.5) with 300 units of Sl nuclease for 30 min . at 15°C. The reaction was stopped by the addition of 1 µl 30

x Sl-nuclease stop solution (0.8M Tris base, 50 mM EDTA). The mixture was phenol extracted, chloroform extracted and ethanol precipitated, then EcoRI treated as previously described and the large fragment (1) obtained by the PAGE method followed by electroelution. The fragment obtained had a first EcoRI-binding end and a second rejecting end whose coding chain begins with the nucleotide thymidine.

Plasmid pSom7A2, as prepared above, was BglII-treated and the resulting BglII-binding ends double-stranded with Klenow polymerase I method using all 4 deoxynucleotide triphosphates. EcoRI digestion of the resulting product followed by PAGE and electroelution of the small fragment (2) yielded a linear piece of DNA containing the tryptophan promoter-operator and codons for the LE<1>"proximal" sequence upstream of the Bglll site ("LE' (p)"). The product had an EcoRI end and a rejecting end resulting from the filling in the Bglll point. However, the BglII site is reconstituted by ligation of the repulsive end of fragment (2) to the repulsive end of fragment (1). Thus, the 2 fragments were ligated in the presence of T^DNA ligase and formed the recycled plasmid pHKY 10 which was propagated by transformation in competent E. coli strain 294 cells.

Plasmid pGM1 has the E. coli tryptophan operon which involves the deletion ALE1413 (Miozzari et al., J. Bacteriology 133, 1457-1466 [1978]) and therefore expresses a fusion protein containing the first 6 amino acids of the trp leader and approximately the last third of the trp E polypeptide (hereafter in conjunction called LE'), as well as the trp D polypeptide in its entirety, all under the control of the trp promoter-operator system. The 20 pg plasmid was treated with the restriction enzyme PvuII, which cleaves the plasmid at 5 sites. The gene fragments were then combined with EcoRI linkers (consisting of a self-complementary oligonucleotide with the sequence: pCATGAATTCATG) which provides an EcoRI cleavage point for a later cloning into a plasmid containing an EcoRI point. The 20 µg DNA fragments obtained from pGM1 were treated with 10 units of T. DNA ligase in the presence of 200 picomol 5'-phosphorylated synthetic oligonucleotide pCATGAATTCATG and in 20 µl DNA ligase buffer (20 mM Tris, pH 7, 6, 0.5 mM ATP, 10 mM MgCl 2 , 5 mM dithiothreitol) at 4°C overnight. The solution was then heated for 10 min. at 70°C to stop ligation. The linkers were cleaved by EcoRI treatment and the fragments,

now with EcoRI ends were separated using 5% PAGE and the 3 largest fragments isolated from the gel by first treating with ethidium bromide, locating the fragments with ultra-violet light and cutting from the gel the parts that were interest. Each gel fragment with 300 microliters of 0.1xTBE was placed in a dialysis bag and subjected to electrophoresis at 100 V for one hour in 0.1xTBE buffer (TBE buffer contains: 10.8 gm Tris base, 5.5 gm boric acid, 0.09 gm Na2EDTA in 1 liter H20). The aqueous solution was collected from the dialysis bag, phenol extracted, chloroform extracted and made 0.2 M sodium chloride, and DNA extracted in water after ethanol precipitation. The trp promoter operator which contained the gene with EcoRI attaching ends was identified in the following described method which entails the insertion of fragments into a tetracycline sensitive plasmid which after promoter operator insertion becomes tetracycline resistant.

Plasmid pBRH1 (Rodriguez et al., Nucleic Acids Research 6, 3267-3287 [1979]) expressed ampicillin resistance and contains the gene for tetracycline resistance, but as there is no associated promoter, it does not express this resistance. The plasmid is consequently tetracycline resistant. By introducing a promoter-operator system into the EcoRI site, the plasmid can be made tetracycline resistant.

pBRH1 was treated with EcoRI and the enzyme removed by phenol extraction followed by chloroform extraction and recovered in water after ethanol precipitation. The resulting DNA molecule

were combined in separate reaction mixtures with each of the 3 DNA fragments obtained above and ligated with T DNA ligase as previously described. The DNA present in the reaction mixture was used to transform competent E. coli K-12 strain 294 by standard techniques (Hershfield et al., supra) and the bacteria plated on LB plates containing 20 µg/ml ampicillin and 5 µg/ml tetracycline. Several tetracycline-resistant colonies were selected, plasmid DNA isolated and the presence of the desired fragment confirmed by restriction enzyme analysis. The resulting plasmid is called pBRHtrp.

An EcoRI and BamHI treatment product from the viral genome

of hepatitis B was obtained by conventional means and cloned into the EcoRI and BamHI sites of plasmid pGH6 (Goeddel et al., Nature 281, 544-548 [1979] and formed plasmid pHS32. Plasmid pHS32 was cleaved with XbaI, phenol extracted, chloroform extracted and ethanol precipitated. It was then treated with 1 µl E. coli polymerase I, Klenow fragment in 30 µl polymerase buffer (50 mM potassium phosphate pH 7.4, 7 mM MgCl2>1 mM (3-mercaptoethanol) containing 0.1 mM dTTP and 0.lmll dCTP for 30 min at 0° C., then 2 h at 37° C. This treatment results in filling in 2 of the 4 nucleotides complementary to the 5' overhang of the XbaI cleavage site:

2 nucleotides, dC and dT, were inserted, giving an end with 2 5' overhanging nucleotides. This linear residue of plasmid pHS32 (after phenol and chloroform extraction and recovery in water after ethanol precipitation) was digested with EcoRI. The large plasmid fragment was separated from the small EcoRI-XbaI fragment by PAGE and isolated after electroelution. This DNA fragment from pHS32 (0.2 µg) was ligated under similar conditions to that described above to the EcoRI-Taq I fragment of the tryptophan operon (~0.01 pg), derived from pBRHtrp.

In the method for ligation of the fragment from pHS32 to the EcoRI-Taq1 fragment as described above, the Taq1 overhang is ligated to the XbaI remaining overhang even though it is not completely Watson-Crick base-paired:

A portion of this ligation reaction mixture was transformed into E. coli 294 cells, heat treated and plated on LB plates containing ampicillin. 24 colonies were selected, grown in 3 ml LB media and plasmid isolated. 6 of these were found to have XbaI sites regenerated through E. coli catalyzed DNA repair and replication.

These plasmids were also found to be cleaved with both EcoRI and HpaI and yield the expected restriction fragments. A plasmid called pTrpl4 was used for expression of heterologous polypeptides as discussed below.

The plasmid.pHGH 107 (Goeddel et al., Nature 281, 544-548

[1979]) contains a gene for human growth hormone made up of 23 amino acid codons prepared from synthetic DNA fragments and 163 amino acid codons obtained from complementary DNA prepared through reverse transcription of human growth hormone messenger RNA. This gene, although lacking the codons for the "pre" sequence of human growth hormone, contains an ATG translation initiation codon. This gene was isolated from 10 ug pHGH 107 after treatment with EcoRI followed by E. coli polymerase I Klenow fragment and dTTP and dATP as described above. After phenol and chloroform extraction and ethanol precipitation, the plasmid was treated with BamHI.

The human growth hormone (HGH) gene-containing fragment was isolated from PAGE followed by electroelution. The resulting DNA fragment also contains the first 350 nucleotides of the tetracycline-resistant structural gene, but lacks the tetracycline promoter-operator system so that, upon subsequent cloning into an expression plasmid, the plasmids containing the insert can be located upon restoration of tetracycline resistance. Because EcoRI -end of the fragment is filled in by the Klenow polymerase I method, the fragment has only one repulsive and one binding end which ensures correct orientation upon subsequent insertion into an expression plasmid.

The expression plasmid pTrpl4 was then prepared to receive the HGH gene containing fragment prepared above. Thus, pTrp14 was XbaI treated and the resulting binding ends filled in by the Klenow polymerase I method using dATP, dTTP, dGTP and dCTP. After phenol and chloroform extraction and ethanol precipitation, the resulting DNA was treated with BamHI and the resulting large plasmid fragment isolated by PAGE and electroelution. The pTrpl4-derived fragment had a repulsive and a binding end, allowing recombination in the correct orientation with the HGH gene containing the previously described fragment.

The HGH gene fragment and the pTRP14 AXba-BamHI fragment were combined and ligated together under similar conditions to those described above. The inserted XbaI and EcoRI ends were ligated together by repulsive termination while recreating both the XbaI and EcoRI sites:

Xbal filled in EcoRI filled in HGH gene initiation

This construct also reproduces the tetracycline resistance gene. As the plasmid pHGH 107 expresses tetracycline resistance from a promoter located upstream of the HGH gene (the lac promoter), this construct called pHGH 207 enables expression of the gene for tetracycline resistance under the control of the tryptophan promoter:: tor operator. Thus, the ligation mixture was transferred into E. coli 294 and colonies selected on LB plates containing 5 µg/ml tetracycline.

Plasmid pHGH 207 was EcoRI treated and the trp promoter-containing EcoRI fragment recovered by PAGE followed by electroelution. Plasmid pBRH1 was EcoRI-treated and the cleaved ends treated with bacterial alkaline phosphatase (BAP, 1 µg, in 50 mM Tris, pH 8, and 10 mM MgCl2 for 30 min at 65°C) to remove the phosphate groups on the overhanging EcoRI- ducks. Excess bacterial alkaline phosphatase was removed by phenol extraction, chloroform extraction and ethanol precipitation. The resulting linear DNA, because it lacks phosphates on the protruding ends during ligation, will only receive inserts whose complementary binding ends are phosphorylated but will not itself recycle, which enables an easier search for plasmids containing the inserts.

The EcoRI fragment derived from pHGH 207 and the linear DNA obtained from pBRH1 were combined in the presence of T₂ ligase as previously described and ligated. A portion of the resulting mixture was transformed into E. coli strain 294 as previously described, plated on LB media containing 5 µg/ml tetracycline and the 12 tetracycline-resistant colonies were selected. Plasmid was isolated from each colony and examined for the presence of a DNA insert by restriction endonuclease analysis using EcoRI and XbaI. A plasmid containing the insert was called pHKY1.

The above-described plasmid pHKY10 is a derivative of pBR322 which contains a Bglll point between the tetracycline resistance (Tc ) promoter and the structural gene. The large DNA fragment isolated after treatment of pHKYlO with Pstl and Bglll therefore contains parts of the ampicillin resistance (Ap ) gene and

R R

the entire Tc structural gene, but lacks the Tc promoter (Fig. 6). The plasmid pGH6 (Goeddel et al., Nature 281, 544-548 [1979]) was treated with EcoRI, the resulting single-stranded ends were filled in with DNA polymerase I, and the plasmid was digested with PstI. The small fragment, which contained a

part of the Ap gene, a double lac promoter and the lac ribosome binding site, but lacking an ATG initiation triplet^ were isolated. A similar trp promoter fragment containing the trp leader ribosome binding site but lacking an ATG sequence (Goeddel et al., Nature 287, 411-416[1980]) can be isolated from pHKY1 described above.

The trp fragment just mentioned is an analogue of the E. coli tryptophan operon from which the so-called trp attenuation term has been looped (Miozzari et al., J. Bact. 1_3_3 , 1457-1466 [1978]) to controllably raise expression levels. Expression plasmids containing the modified trp regulon can be grown to predetermined levels in nutrient media containing added tryptophan in amounts sufficient to repress the promoter-operator system, then stripped of tryptophan to derepress the system and cause expression of the intended product.

The expression plasmids can be assembled through 3 partial ligation reactions as shown in fig. 6. 15 ng (0.05 pM) of the composite FIF gene (504 or 505 bp), 0.5 µg (0.2 pM) of the large Pstl - BglII fragment of pHKY10 and 0.2 pg (0 .3 pM) of the appropriate promoter fragment was ligated and the mixture used to transform E. coli 294 (Goeddel et al., Nature 287, 411-416 [1980]). Plasmid DNA was prepared from individual transformants and analyzed by restriction mapping. Correct ligation of the assembled gene to the promoter fragment should restore the EcoRI (lac) or XbaI (trp) recognition sequences. The majority of plasmids gave the expected restriction enzyme treatment patterns. Individual clones (12 containing the trp promoter and 12 containing the lac promoter) were grown and the extract prepared for interferon measurement as described above.

When measured on human amnion (WISH) cells for antiviral activity in the CPE inhibition assay, 5 of the trp transformants were positive (each approximately equivalent); 11 of the lac transformants gave equivalent IF activities. Therefore, one transformant from each series (pFIFlac9 and pFIFtrp69) was selected for further studies (Table 1). DNA sequence analysis showed that the desired joining of the promoter to the FIF structural gene had taken place in both cases.

Cells were grown and extracts prepared as described above. The human amnion (WISH) cell line was used for the CPE inhibition assay. Indicated activities are the mean from 3 independent experiments. To determine the number of IF molecules per cell became a FIF specific activity on

4 x 10 g units/mg used (Knight, above).

The amounts of fibroblast interferon produced by pFIFlac9 and pFIFtrp69 are shown in Table 1. The trp promoter gave a FIF expression level measurably higher than the lac promoter did. In an: attempt to further increase FIF expression levels,

pFIFtrp69 was digested with EcoRI and 2300 bp EcoRI fragments containing the trp promoter (Goeddel et al., Nature 287, 411-416 [1980]) were inserted. The resulting plasmid, pFIFtrp 369, contains 3 consecutive trp promoters that read against the FIF gene. The amount of FIF synthesized by E. coli K-12 strain 294/pFIF trp 369 is 4-5 times that produced by pEIFtrp69 (Table 1). This is obviously due to the derepression of the trp promoter that occurs when trp repressor levels are titrated with the multiple copies of the trp operator.

FIF produced by E. coli K-12 strain 294/pFIFtrp69 behaves as authentic human FIF. As shown in Table 2, its antiviral activity is approx. 30 times greater on human cells than on bovine cells. In addition, the bacterially produced FIF is stable to treatment at pH 2 overnight and is not neutralized by rabbit anti-human leukocyte interferon antibodies (table 3).

LelF and FIF were NIH standard solutions at 20,000 units/ml and 10,000 units/ml, respectively. Measurements were performed as described above.

Experimental procedures described above. Measured with

CPE inhibition using WISH cells/Sindbis virus.

N. Cleansing

The purification method for bacterially derived fibroblast interferon is as follows: 1. Frozen cells are resuspended in 12 times the volume per unit weight of sucrose cleavage buffer (100 mM Tris-HCl, 10% sucrose, 0.2 M NaCl, 50 mM EDTA, 0.2 mM PMSF [phenylmethylsulfonyl chloride], pH 7.9) containing lysozyme at 1 mg/ml. The cell suspension is stirred for 1 hour at 4°C and centrifuged. Fibroblast interferon activity remains in the supernatant. 2. Polyethyleneimine (5%, v/v) is added to the sonicated supernatant to a final concentration of 0.5% (v/v). The solution is stirred for 1 hour at 4°C and centrifuged. Interferon activity remains in the supernatant. 3. Solid ammonium sulfate is added to the polyethyleneimine supernatant to a final concentration of 50% saturation, stirred for 30 min. at 4°C and centrifuged. Interferon activity is in the 50% lump. 4. The 50% ammonium sulfate lump is suspended in half the volume of the 50% ammonium sulfate suspension with PBS (20 mM sodium phosphate, 0.15 M NaCl, pH 7.4). Polyethylene glycol 6000 (50%, weight/volume, in PBS) is added until a final concentration of 12.5% (volume/volume), stirred at 4°C for 2 hours and centrifuged. Interferon activity is in the lump. The lump is resuspended in a minimal volume of sucrose cleavage buffer and clarified by centrifugation.

This first extraction procedure leads to a purification of fibroblast interferon from 0.001% of the total protein to 0.05% of the total protein. This material can further be purified to homogeneity by the following column chromatography steps.

5. Affinity chromatography on Amicon Blue B in sucrose cleavage buffer. 6. Anion exchange chromatography on QAE Sephadex in sucrose cleavage buffer in the absence of 0.2M NaCl. 7. Size exclusion chromatography on Sephadex G-75 in sucrose cleavage buffer.

8. Reversed-phase high-pressure liquid chromatography.

0. Parenteral administration

FIF can be given parenterally to patients who require antitumor or antiviral therapy. Dosages and dosage rates may parallel those used in clinical trials of human-derived materials, e.g. about. (1-10) x 10^ units daily, and in the case of materials with higher purity than 1%, probably up to approx. 15 x 10 7 units daily. Doses of bacterially produced FIF could be increased considerably for greater effect due to the almost non-existence of human proteins other than FIF, which proteins in fibroblast-derived materials can act as pyrogens, exhibiting deleterious effects, e.g. discomfort, temperature rise, etc.

As an example of a suitable dosage form for essentially homogeneous bacterial FIF in parenteral form, 3 mg of FIF with a specific activity of e.g. 2 x 10 g units/mg are dissolved in 2 5 ml of 5% human serum albumin, the solution is passed through a bacteriological filter and the filtered solution is divided aseptically into 100 bottles each containing 6 x 10 units of pure interferon suitable for parenteral administration. The glasses are preferably stored cold (-20 C) before use.

The compounds according to the present invention can be formulated according to known methods for producing pharmaceutically usable mixtures, whereby the polypeptide thereof is combined in mixture with a pharmaceutically acceptable carrier. Suitable carriers and their formulation are described in Remington's Pharmaceutical Sciences by E.W. Martin. Such compositions will contain an effective amount of the interferon protein together with an appropriate amount of carrier to provide pharmaceutically acceptable compositions suitable for effective administration to the host. A preferred method of administration is parenteral.

Claims (6)

1. Replicable microbial expression vector, characterized in that in a transformed microorganism it is capable of expressing developed human fibroblast interferon with the amino acid methionine freely chosen as the usual first amino acid of the interferon. 2. Expression vector according to claim 1, characterized in that it is a plasmid. 3. Expression vector according to claim 1, characterized in that it is selected from the group pFIFlac9, pFIFtrp69 and pFIFtrp <3> 69. 4. Method for producing a replicable microbial expression vector capable of expressing, in a transformed microorganism, developed human fibroblast interferon with the amino acid methionine freely chosen as the usual first amino acid of the interferon, characterized in that the method comprises forming a first DNA sequence that codes for developed human fibroblast interferon and operatively linked this first DNA sequence with a second sequence capable of providing microbial expression of the first DNA sequence. 5. Method according to claim 4, characterized in that the second DNA sequence comprises a multiple trp promoter-operator. 6. Use of a replicable microbial expression vector according to any one of claims 1-3 to transform a microorganism capable of expressing the encoded developed human fibroblast interferon.