Description Technical Field

The invention is in the field of biotech- nology. More particularly it relates to a polypeptide having interferon (IFN) activity, DNA that codes for the polypeptide, a recombinant vector that includes the DNA, a host organism transformed with the recom¬ binant vector that produces the polypeptide and phar- maceutical compositions containing the polypeptide.

Background Art

IFNs are proteins with antiviral, immuno- modulatory, ancϊ antiproliferative activities produced by mammalian cells in response to a variety of indu- cers (see Stewart, W.E., The Interferon System,

Springer-Verlag, New York, 1979). The activity of IFN is largely species specific (Colby, C, and Morgan, M. J., Ann. Rev. Microbiol. 25:333-360 (1971) and thus only human IFN can be used for human clinical studies. Human IFNs are classified into three groups, α, β, and γ, (Nature, 286:110, (1980)). The human I -α genes compose a multigene ^' family sharing 85%-95% sequence ho ology (Goeddel, D. V., et al, Nature 290:20-27 (1981) Nagata, S. , et al, J. Interferon Research 1:333-336 (1981)). Several of the IFN-α genes have been cloned and expressed in E.c-o-1i (Nagata, S. , et al, Nature 284:316-320 (1980); Goeddel, D. V., et al, Nature 287:411-415 (1980); Yelverton, E. , et al. Nucleic Acids Research, 9:731-741, (1981); Streuli, M., et al, Proc Nat Acad Sci (USA), 78:2848-2852. The resulting polypeptides have been purified and tested for biological activities associated with partially

purified native human IFNs and found to possess s imi¬ lar activities . Accordingly such polypeotides are potentially useful as antiviral , immunomodul ory, or antiproli ferative agents . A principal object of the present invention is to provide a polypeptide having interferon activity that is produced by an organism transformed with a newly isolated and newly characterized IFN-α gene that is not expressed natural ly . This polypeptide is some- times referred to herein as " IFN-α6L" . Other objects o f the invention are directed to providing the compo¬ sitions and organisms that are used to produce this polypeptide and to therapeutic compos itions and methods that use this polypeptide as an active ingredient .

Disc losure of the Invention

One aspect o f the invention is a polypeptide having interferon activity and compris ing the amino acid sequence :

CysAspLeuProGln ThrHisThrLeuArg AsnArgArgAlaLeu IleLeuLeuGlyGln MetGlyArglleSer ProPheSerCysLeu ysAspArgHis sp PheArglleProGln GluGluPhe s Gly AsnGlnPheGlnLys AlaGlnAlalLeSer ValLeuHisGLuMet IleGlnGlnThrPhe AsnLeuPheSerThr GluAspSerSerAla AlaTrpGIuGlnSer LeuLeuGluLysPhe SerThrGluIleTyr GlnGlnLeuAsnAsp LeuGluAlaCysVal IleGlnGluValGly ValGluGluThrPro LeuMetAsnGluAsp SerlleLeuAlaVal ArgLysTyrPheGln ArglleThrLeuTyr LeuIleGluArgLys TyrSerProCysAla TrpGluValVal ^' Arg AlaGluIleMetArg SerLeuSerPheSer ThrAsnLeuGlnLys ArgLeuArgArgLys Asp.

A second aspect of the inven tion is a DNA unit or fragment comprising a nuc leotide sequence that encodes the above desc ribed po lypeptide .

A third aspect of the invention is a cloning vehicle or vector that includes the above described DNA.

A fourth aspect of the invention is a host organism that is transformed with the above described cloning vehicle and that produces the above described polypeptide. A fifth aspect of the invention is a process for producing the above described polypeptide compri¬ sing cultivating said transformed host organism and collecting the polypeptide from the resulting culture. Another aspect of the invention is a pharma- ceutical composition having interferon activity com¬ prising an effective amount of the above described polypeptide admixed with a pharmaceutically acceptable carrier.

Brief Description of the Drawings Figure 1 is a partial restriction map which shows the two XhoII restriction sites that produce a homologous 260 base pair DNA fragment from the IFN-αl and IFN-α.2 structural genes. This fragment is used as a probe in identifying and isolating the IFN-α6L gene. Data for this map are from Streuli, M . , et al Science, 209:1343-1347 (1980).

Figure 2 depicts the sequencing strategy used to obtain the complete DNA sequence of the IFN-α6L gene coding region. Bacteriophage mp7:α6L-l DNA served as the template for sequences obtained with primers A, H and F and bacteriophage mp7:α6L-2 DNA was the template for sequences obtained with primers Ξ and G. The crosshatched area of the gene depicts the region that encodes the 23 amino acid signal peptide (preinterferon) and the open box depicts the region that encodes the mature polypeptide. The scale, in base pairs, is numbered with 0 representing the ATG

start codon of preinterferon. The arrows indicate the direction and extent of sequencing with each primer. Figure 3 is the nucleotide sequence of the structural gene coding for IFN-α6L including some of the flanking 5 '- and 3 '- noncoding regions of the gene. The region coding for preinterferon .and the mature polypeptide begins with the ATG codon at posi¬ tion 42 and terminates with the TGA codon at posi¬ tion 609. Figure 4 is a partial restriction map of the coding region of the IFN-α6L gene. The crosshatching represents the region that encodes the 23 amino acid signal peptide and the open box represents the gene coding sequence for the mature polypeptide. The scale, in base pairs, is numbered with 0 representing the ATG start codon of preinterferon.

Figure 5 shows the amino acid sequence of the 23 amino acid signal peptide and the 166 amino acid mature IFN-α6L coded for by the gene depicted in Figure 3. The 189 amino acid sequence is displayed above the corresponding nucleotide sequence. Amino acid 24, cysteine, is the first amino acid of the mature IFN-α _. 6L protein.

Figure 6 is the DNA sequence of the 5. coli trp promoter and the gene of Figure 3 which was inserted between the EcoRI and AccI sites of the plasmid pBR322. The amino acid sequence of Figure 5 is written above the correspon ing DNA sequence and the location of the restriction sites used in the construction of the expression plasmid are indicated. Figure 7 is a diagram of the expression plasmid, pGW21, used to trans orm bacteria with the IFN-α6L gene.

Modes for Carrying Out the Invention

In general terms IFN-α6L was made by identi¬ fying and isolating the IFN-α6L gene by screening a library of human genomic DNA with an appropriate IFN-α DNA probe, constructing a vector containing the IFN-α6L gene, transforming microorganisms with the vector, cultivating transformants that produce IFN-α6L and collecting IFN-α6L from the culture. A preferred embodiment of this procedure is described below.

DNA Probe Preparation

Total cytoplasmic RNA was extracted from human lymphoblastoid cells, Namalwa, which had been induced for IFN production by pretreat ent with 5-bromodeoxyuridine and Newcastle Disease Virus (NDV). The ρoly(A) (polyadenylic acid)-containing messenger RNA (mRNA) was isolated from total RNA by chromatography on oligo(d )-cellulose (type 3 from Collaborative Research; Aviv, H., and Leder, P., Proc Natl Acad Sci (USA) , 69:1408-1 12, (1972)) and enriched for IFN mRNA by density gradient centrifuga- tion on 5%-20% sucrose gradients. Fractions contain¬ ing IFN mRNA were identified by translating the mRNA by microinjecting aliquots of each fraction into Xenopus oocytes and determining the IFN activity of the products of the translations according to a method described by Colman, A., and Morser, J., Cell, 17:517- 526 (1979).

The Namalwa cell IFN enriched mRNA was used to construct complementary DNA (cDNA) clones in E. coli by the G/C tailing method using the Pstl site of the cloning vector ρBR322 (Bolivar, F., et al, Gene, 2:95-113 (1977)). A population of transformants containing approximately 50,000 individual cDNA clones

was grown in one liter of medium overnight and the total plasmid DNA was isolated therefrom.

The sequences of two IFN-α clones (iFN-αl and IFN-α2) have been published (Streuli, M., et al, Science, 209:1343-1347 (1980)). Examination of the DNA sequences of these two clones revealed that the restriction enzyme XhoII would excise a 260 bp frag¬ ment from either the IFN-αl or the IFN-α2 gene (see Figure 1). XhoII was prepared in accordance with the process described by Gingeras, T.R., and Roberts, R.J., J Mol Biol, 118:113-122 (1978).

One mg of the purified total plasmid DNA preparation was digested with XhoII and the resulting DNA fragments were separated on a preparative 6% poly- acrylamide gel. DNA from the region of the gel cor¬ responding to 260 bp was recovered by electroelution and recloned by ligation into the Ba HI site of the single strand bacteriophage M13:mp7. Thirty-six clones were picked at random and single stranded DNA was isolated therefrom, and sequenced. The DNA sequences of four of these clones were homologous to known IFN-α DNA sequences. Clone mp7:α-260, with a DNA sequence homologous to IFN-αl DNA (Streuli, M. et al, Science, 209:1343-1347 (1980)) was chosen as a highly specific hybridization probe for identifying additional IFN-α DNA sequences. This clone is herein¬ after referred to as the "260 probe."

Screening of Genomic DNA Library

In order to isolate other IFN-α gene sequences, a ³ P-labelled 260 probe was used to screen a library of human genomic DNA by _in_ situ hybridiza¬ tion. The human gene bank, prepared by Lawn, R.M., et al, Cell, 15:1157-1174 (1978), was generated by par-

tial cleavage of fetal human DNA with Haelll and Alul and cloned into bacteriophage λ. Charon 4A with syn¬ thetic _j EcoRI linkers. Approximately 800,000 clones were screened, of which about 160 hybridized with the 260 probe. Each of the 160 clones was further charac¬ terized by restriction enzyme mapping and comparison with the published restriction maps of 10 chromosomal IFN genes (Nagata, S., et al, J Interferon Research, 1:333-336 (1981)). One of the clones, hybrid phage λ4A:α6L containing a 13.9 kb insert, was characterized as follows. A DNA preparation of λ4A:α6,L was cleaved with Hindlll, Bglll, and EcoRI respectively, the frag¬ ments separated on an agarose gel, transferred to a nitrocellulose filter, and hybridized with -. ^** _*-P- labelled 260 probe. This procedure localized the IFN-α6L gene to a 2.0 kb EcoRI restriction fragment which was then isolated and recloned, in both orienta¬ tions, by ligation of the fragment into EcoRI -cleaved M13:mp7. The two subclones are designated mp7:α6L-l and mp7:α6L-2. The -1 designation indicates that the single-stranded bacteriophage contains insert DNA complementary to the mRNA (the minus strand) and the -2 designation indicates that the insert DNA is the same sequence as the mRNA (the plus strand).

Sequencing of the IFN-g6L Gene

The Sanger dideoxy-technique was used to determine the DNA sequence of the IFN-α6L gene. The strategy employed is diagrammed in Figure 2, the DNA sequence thus obtained is given in Figure 3, and a partial restriction map of the IFN-α6L gene is illus¬ trated in Figure 4. Unlike many genes from eukaryotic organisms, but analogous to other IFN chromosomal genes which have been characterised, the DNA sequence

of this gene demonstrates that it lacks introns. Homology to protein sequences of the published IFN-α genes made it possible to determine the correct trans- lational reading frame and thus allowed the entire 166 amino acid sequence of IFN-α6L to be predicted from the DNA sequence as well as a precursor segment, or signal polypeptide, of 23 amino acids (Figure 5).

The DNA sequence of the IFN-α6L gene (Fig 3) and the amino acid sequence predicted therefrom (Fig 5) differ from the other known IFN-α DNA and IFN-α amino acid sequences. Translation of the DNA sequence of the IFN-α6L gene reveals that the gene is a pseudo- gene that cannot be expressed naturally because of a stop codon in the leader polypeptide sequence. Other- wise the coding region of the gene is intact and it can be expressed in transformed microorganisms as a mature polypeptide. The IFN-α6L is, therefore, a truly novel polypeptide in that it has never been produced by or isolated from human cells. Goeddel, D.V., et al, Nature, 290:20-27

(1981) describes isolating an IFN-α gene, .IFN-C, that differs from the IFN-α6L gene by six nucleotides that result in the stop codon in the leader sequence and three amino acid changes in the mature polypeptide. The nucleotide change that causes the stop codon occurs at position 101 and is a change from T to A. The three substitutions that cause the amino acid changes are: (1) a change from G to C at nucleotide 133 resulting in a change at amino acid 8 from Ser to Thr, (2) a change from G to C at position 138 resul¬ ting in a change at amino acid 10 from Gly to Arg, and (3) a change from C to A at position 375 resulting in a change- at amino acid 89 from Leu to lie. As regards the other two changes one is a neutral change from C

to G at position 137 and the other is a change from C to T at position 711 well outside the coding region.

Plasmid Preparation and Host Transformation ^'

Assembly of the plasmid for direct expres- sion of the IFN-<∑6L gene involved replacing the DNA fragment encoding the 23 amino acid signal peptide with a 120 bp EcoRl/Sau3A promoter fragment E.coli trp promoter, op-erator, and trp leader ribosome binding site preceding an ATG initiation codon and using the naturally occurring AceI site, 153 bp 3 ' - of the TGA translational stop codon, to insert the gene into a vector derived from the plasmid pBR322. The complete DNA sequence of the promoter and gene fragments inserted between the EcoRI and AceI sites of pBR322 is shown in Figure 6 which also shows the exact location of relevant cloning sites. Details of the construc¬ tion are described below.

The IFN-α6L gene has a Sau3A restriction site following the codon for the initial cysteine of the mature protein, a second Sau3A site in the coding region, and a third Sau3A site in the 3'- flanking region. It also contains an AceI site on the 3' flanking region (at nucleotide 760 in Fig 3) and a second AccI site approximately 240 nucleotides 5'- of the sequence shown in Fig 3. The mp7:α6L clone was digested with AccI and the -one kb AccI fragment was isolated on polyacrylamide gel. The AccI fragment was then subject to partial digestion with Sau3A. Several partial digestions of fragment were carried out using a digestion mixture of 10 parts DNA, 12.5 parts buf¬ fer, 1.25 parts Sau3A and 100 parts water (parts are by volume). The digestions were made at 30°C for varying times. The digests were resolved on 5% poly-

acrylamide gel. The resulting 646 bp fragments in the digests were eluted from the gel, precipitated with ethanol, and combined. The precipitates were spun down, resuspended in Tris-EDTA containing 0.1 M NaCl, filtered, reprecipitated and spun down, washed with 70% ethanol and resuspended in water.

The 646 bp Sau3A-AccI fragment was ligated in a three-fragment, sticky end ligation with the pre¬ viously described 120 bp promoter fragment and a 2116 bp EcoRI-AccI vector fragment derived by diges¬ ting pBR322 with EcoRI and AccI. The ligation was carried out at 4°C. The ligation mixture was used to transform E.coli MM 294. The correct transformants were identified by restriction enzyme mapping of colonies that hybridized to a ³² P labelled IFN-α genomic fragment and by cytopathic effect activity on human cells. Four out of 18 clones screened contained the correct construction. Fig ^' 7 is a diagram of the correct expression construct, designated pGW21. Other prokarytic hosts such as bacteria other than E. coli may, of course, be transformed with this construct or other suitable constructs either to replicate the IFN-α6L gene and/or to produce IFN-α6L.

A sample of one of the correct transformants was deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. on August 12 , 1983. The sample was assigned ATCC No. 39409

Cultivation of Transformants Bacteria transformed with the IFN-α6L gene may be cultivated in an appropriate growth medium, such as a minimum essential medium, that satisfies the nutritional and other requirements needed to permit

-li¬

the bacteria to grow and produce IFN-α6L. If the bacteria are such that the protein is contained in their cytoplasm, the IFN-α6L may be extracted from the cells by lysing the cells such as by sonication and/or treatment with a strong anionic solubilizing agent such as sodium dodecyl sulf te. Further purification of the extract may be achieved by affinity chromatog- raphy, electrophoresis, or other protein purification techniques. Expression of the IFN-α6L gene by bacterial hosts such as E.coli that utilize N-formyi-methionine and/or methionine to initiate translation produces IFN-α6L "molecules that are preceded by an N-formyl- methionine or a methionine group. Some of the N- formyl-methionine or methionine groups could be removed by natural in vivo bacterial cleavage mech¬ anisms. This would result in a mixture of molecules, some of which would include an initial N-formyl- methionine or methionine and others that would not. All such IFN-α6L molecules, those containing an initial N-formyl-methionine or methionine, those not containing an N-formyl-methionine or methionine and any mixture thereof, are encompassed by the present invention. Accordingly, the invention contemplates producing IFN-α6L-containing compositions having IFN activity that is attributable solely to IFN-α6L and/or said terminal N-formyl-methionine or methionine derivative thereof.

Biological Testing of IFN-α6L IFN-α6L-containing cell sonicates were tested in vitro and found to have the following activities: (1) inhibition of viral replication of vesicular stomatitis virus (VSV) and herpes simplex

^■ ^V ZEATΓ

virus-1 (HSV-1 ) ; (2) inhibition of human tumor cell growth; (3) inhibition of colony formation by tumor cells in soft agar; (4) activation of natural killer (NK) cells; (5) enhancement of the level of 2 ',5'- oligoadenylate synthetase (2',5'-A); and (6) enhance¬ ment of the double-stranded RNA-dependent protein kinase. IFN-α6L was active in inhibiting viral replication in both human and other mammalian cells, such as hamster, monkey, bovine, and rabbit cells. The tests show that IFN-α6L exhibits anti¬ viral activity against DNA and RNA viruses, cell growth regulating activity, and an ability to regulate the production of intracellular enzymes and other cell-produced substances. Accordingly, it is expected IFN-α6L may be used to treat viral infections with a potential for interferon therapy such as chronic hepa¬ titis 3 infection, ocular, local, or systemic herpes virus infections, influenza and other respiratory tract virus infections, rabies and other viral zoonoses, arbovirus infections, and slow virus diseases such as Kuru and sclerosing panen.cepha- litis. It may also be useful for treating viral infections in immunocompromised patients such as herpes zoster and varicella, cytomegalovirus, Epstein- Barr virus infection, herpes simplex infections, rubella, and progressive multifocal leukoencephalo- pathy. Its cell growth regulating activity makes it potentially useful for treating tumors and cancers such as osteogenic sarcoma, multiple myeloma, Hodgkin's disease, nodular, poorly di ferentiated lymphoma, acute lymphocytic leukemia, breast carci¬ noma, melanoma, and nasopharyngeal carcinoma. The fact that IFN-α6L increases protein kinase and 2' , 5'- oligoadenylate sythetase indicates it may also

increase synthesis of other enzymes or cell-produced substances commonly affected by IFNs such as hista- mine, hyaluronic acid, prostaglandin E, tRNA methyl- ase, and aryl hydrocarbon hydrolase. Similarly, it may be useful to inhibit enzymes commonly inhibited by IFNs such as tyrosine amino transferase, glycerol-3- phosphate dehydrogenase glutamine synthetase, orni- thine decarboxylase, S-adenosyl-1-methionine decarboxylase, and UDP-N-acetylglucosamine-dolichol raonophosphate transferase. The ability of the IFN-α6L to stimulate NK cell activity is indicative that it may also possess other interferon activities such as the abilities to induce macrophage activity and anti¬ body production and to effect cell surface alterations such as changes in plasma membrane density or cell surface charge, altered capacity to bind substances such as cholera toxin, concanavalin A and thyroid- stimulating hormone, and change in the exposure of surface- gangliosides. Pharmaceutical compositions that contain

IFN-α6L as an active ingredient will norma'lly be for¬ mulated with an appropriate solid or liquid carrier depending upon the particular mode of administration being used. For instance, parenteral formulations are usually injectable fluids that use pharmaceutically and physiologically acceptable fluids such as physio¬ logical saline, balanced salt solutions, or the like as a vehicle. IFN-α6L will usually be formulated as a

4 unit dosage form that contains in the range of 10 to 10 7 international units, more usually 106 to 107 international units per dose.

IFN-α6L may be administered to humans or other mammals in various manners such as orally, intravenously, intramuscularly, intraperitoneally,

intranasally, intradermally, and subcutaneously. The particular mode of administration and dosage regimen will be selected by the attending physician taking into account the particulars of the patient, the disease and the disease state involved. For instance, viral infections are usually treated by daily or twice daily doses over a few days to a few weeks ; whereas tumor or cancer treatment involves daily or multidaily doses over months or years. IFN-α6L therapy may be combined with other treatments. In this regard it may be combined with or used in association with other chemotherapeutic or chemopreventive agents for providing therapy against viral diseases, cancer and other conditions against which it is effective. For instance, in the case of herpes virus keratitis treat¬ ment therapy with native IFN has been supplemented by thermocautery, debridement and tri luorothymidine therapy.