CA1217440A - INTERFERON .alpha. 6L - Google Patents

Abstract

Abstract A new polypeptide, called IFN-.alpha.6L, produced by E.coli transformed with a newly isolated and char-acterized human IFN-.alpha. pseudogene is described. The polypeptide exhibits interferon activities such as antiviral activity, cell growth regulation, and regu-lation of production of cell-produced substances.

Description

f~ r3 INTERFER0~-ALPHA 6L

Description Technical Field The invention is in the field of biotech-5 nology. More particularly it relates to a polypeptidehaving interferon (IF~) activity, DNA that codes for the polypeptide, a recombinant vector t~at includes the DNA, a host organism transformed with the recom-binant vector that produces the polypeptide and phar-10 maceutical compositions containing the polypeptide.

Bac~ground Art IF~s are proteins with antiviral, immuno-modulatory, and antiproliferative activities produced by mammalian cells in response to a variety of indu-15 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.
20 Human IFNs are classified into three groups, a, ~, and y, (~ature, 286:110, (1980))~ The human IFN-a genes ccmpose a multigene family sharing 85~-95% sequence homology (Goeddel, D. V., et al, ~ature 290:20-27 (1981) ~agata, S., et al, J. Intereron Research 1:333-336 (1981)). Several of the IFN-a genes have been cloned and expressed in E.coli (Nagata, S., et al, Nature 284:316-320 (1980); Goeddel, D. V., et a1, Nature 287:411-415 (1980); Yelverton, E., et al, Nucleic Acids Research, 9:731-741, (1981); Streuli, 30 M., et al, Proc Nat Acad Sci (USA), 78:2848-2852. The -resulting polypeptides have been purified and tested or biological activities associated with partially `:

purified native human IFNs and found to possess simi-lar activities. Accordingly such polypeptides are potentially useful as antiviral, immunomodulatory, or antiproliferative agents.
A principal object of the present invention is to provide a polypeptide having interferon activity that is produced hy an organism transformed with a newly isolated and newly characterized IFN-~ gene that is not expressed naturall~. This polypeptide is some-times referred to herein as "IFN-a6L". Other objects of the invention are directed to providing the compo-sitions and organisms that are used to produce this polypeptide and to therapeutic compositions and methods that use this polypeptide as an active ingredient.

Disclosure of the Invention _ One aspect of the invention is a recombinantly produced polYpeptide having inter~eron activity and com-prising the amino acid sequence:

~s~spLeuProGln ThrHisThrLeuArg AsnArgArgAlaLeu IleLeuLeuGlyGln ~etGlyArgIleSer ProPheSerCysLeu LysAspArgHisAsp PheArgIleProGln GluGluPheAsp~ly AsnGlnPheGlnLys AlaGlnAlaIleSer ValLeuHisGl~et IleGlnGlnThrPhe AsnLeuPheSerThr Glu~pSerSerAla AlaTrpGluG~nSer LeuLeuGluLysPhe SerThrGluIleTyr Gln51nLeu~snAsp LeuGluAlaCysVal IleGlnGluValGly ValGluGluThrPro LeuMetAsnGluAsp SerIleLeuAlaVal ArgLysTyrPheGln ArgIleThrLeuTyr LeuIleGluArgLys TyrSerPro~sAla TrpGluValValArg AlaGluIleMetArq SerLeuSerPheSer ThrAsnLeuGlnLys ArgLeuArgArgLys Asp.
A second aspect of the invention is a DNA
unit or fragment comprising a nucleotide s~quence that encodes the above described polypeptide.
A third aspect of the invention is a cloning

2~ vehicle or vector that includes the above described DNA.

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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 efective amount of ~he above described polypeptide admixed with a pharmaceutically acceptable carrier.

Brief Descri~ n of the Drawings Figure 1 is a partial restriction map ~hich shows the two XhoII restriction sites that produce a homologous 260 base pair DNA fragment from the IFN-al and IFN-a2 structural genes. This fragment is used as a probe in identifying and isolating the IFN-a6L
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 D~A sequence of the IFN-a6L gene coding region. Bacteriophage mp7:a6L-l D~A served as the template ~or sequen~es obtained with primers A, H and F and bacteriophage mp7:a6L-2 D~A was the template for sequences obtained with primers E 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-a6L 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 IF~-a6L 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 mat1lre polypeptide. The scale, in base pairs, is numbered with 0 representing the ATG start codon of prein~erferon.
Figure 5 shows the amino acid sequence of the 23 amino acid signal peptide and the 166 amino acid mature IFN-6L coded for by the gene depicted in ~0 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-a6L protein.
Figure 6 is the D~A sequence of the E. 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 corresponding 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 expressi~n plasmid, pGW21, used to transform bacteria with the IF~-a6L gene.

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Modes for Carrying Out the Invention In general terms IFN-~6L was made by identi-fying and isolating the IF~-6L gene by screening a library of human genomic DNA with an appropriate IFN-a DNA probe, constructing a vector containing the IFN-a6L gene, transforming microorganisms with the vector, cultivating transformants that produce IFN-6L
and collecting IFN-~6L from the culture. A preferred embodiment of this procedure is described below.

D~A Probe Preparation Total cytoplasmic RNA was extracted from human lymphoblastoid cells, Namalwa, which had been induced for IF~ production by pretreatment with 5-bromodeoxyuridine and Newcastle Disease Virus l~DV). The poly(A) (polyadenylic acid)-containing messenger RNA (mR~A) was isolated from total RNA by chromatography on oligo(dT)-cellulose (type 3 from Collaborative Research; Aviv, H., and Leder, P., Proc ~atl Acad Sci (USA), 69:1408-1412, ~1972)) and enriched for IF~ mRNA by density gradient centrifuga-tion on 5~-20% sucrose gradients. Fractions contain-ing IEN mR~A were identified by translating the mR~A
by microinjecting aliquots of each fraction int~
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 ~amalwa cell IF~ enriched mRNA was used to construct complementary DNA (cDNA) clones in E. coli by the G/C tailing method using the PstI site of the cloning vec or pBR322 (Bolivarr F., et al, Gene, 2:95-113 (1977)). A population of transformants containing approximately 50,000 individual cDNA clones 7 4 ~ ~

was grown in one liter o medium overnight and the total plasmid DNA was isolated therefrom.
The sequences of two IFN-a clones (IFN-al and IFN 2) have been published (Streuli, M., et al, 5 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-al or the IFN-~2 gene ~see Figure 1~. XhoII was prepared in accordance with the 10 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 ~as digested with XholI and the resul~ing DNA fragments were separated on a preparative 6% poly-15 acrylamide gel. DNA from the region of the gel cor-responding to 260 bp was recovered by electroelution and recloned by ligation into the BamHI site of the single strand bacteriophage M13:mp7. Thirty-six clones were picked at random and single stranded DNA
20 was isolated therefrom, and sequenced. The DNA
sequences of four of these clones were homologous to known IFN-~ DNA sequences. Clone mp7:a-260, with a DNA sequence homologous to IF~-al DNA (Streuli, M. et al, Science, 209:1343-1347 ~1980)) was chosen as a 25 highly specific hybridization probe for identifying additional IFN-a DNA sequences. This clone is herein-after referred to as the "260 probe."

Screening of Genomic DNA Librar~
In order to isolate other IFN-a gene 30 sequences, a 32P-labelled 260 probe was u~ed 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 ~NA with HaeIII and AluIand cloned into bacteriophage ~ Charon 4A with syn-thetic EcoRI linkers. Approximately 800,000 clones were screened, of which about 160 hybridizea 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 ~4A:a6L containing a 13.9 kb insert, was characterized as follows. A DNA preparation of ~4A:5L was cleaved with HindIII, BglII, and EcoRI respectively, the frag-ments separated on an agarose gel, transferred to a nitrocellulose filter, and hybridized with 32p_ labelled 260 probe. This procedure localized the IF~-6L 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:a6L-l and mp7:6L-2. The -1 designation indicates that the single-stranded bacteriophage contains insert D~A
ccmplementary to the mRNA (the minus strand) and the -2 designation indicates that the insert DNA is the same sequence as the mR~A (the plus strand).

Sequencing of the IF~-a6L Gene The Sanger dideoxy-technique was used to determine the DNA sequence of the IFN-a6L gene. The strategy employed is diagrammed in Figure 2, the D~A
sequence thus obtained is given in Figure 3, and a 30 partial restriction map of the IFN-~6L gene i8 illus-trated in Figure 4. Unlike many genes from euXaryotic organisms, but analogous to other IFN chromosomal genes which have been characterised, the D~A sequence ~ -8-of this gene demonstrates that it lacks introns.
Homology to protein sequences of the publi~hed IFN-a genes made it possible to determine the correct trans-lational reading frame and thus allowed the entire 166 amino acid sequence of IFN-a6L to be predicted from the D~A sequence as well as a precursor segment, or signal polypeptide, of 23 amino acids ~Figure 5).
The DNA sequence of the IFN-a6L gene (Fig 3) and the amino acid sequence predicted therefrom (Fig 5) differ from the other known IF~-a DNA and IF~-a amino acid sequences. Translation of the DNA sequence of the IFN-~6L 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-15 wise the coding region of the gene is intact and it can be expressed in transformed microorganisms as a mature polypeptide. The IF~-6L 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-a gene, IF~ C, that differs from the IF~-~6L gene by six nucleotides that result in the stop codon in the leader sequence and three amino acid changes in the rnature polypeptide.
25 The nucleotide change that causes the stop codon occurs at position 101 and is a change from T to A.
The three substitutions that caus2 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 30 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 Ile. 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 regionO

Plasmid Preparation and Host Transformation Assembly of the plasmid for direct expres-sion of the IFN-a6L gene involved replacing the DNA
fragment encoding the 23 amino acid signal peptide with a 120 bp EcoRI/Sau3A promoter fragment E.coli trp promoter, operator, and trp leader ribosome binding site preceding an ATG initiation codon and using the naturally occurring AccI 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 AccI 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-a6L 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 AccI site on the 3' flanking region (at nucleotide 760 in Fig 3) and a second A I site approximately 240 nucleotides 5'- of the sequence sho~ in Fig 3. The mp7:a6L 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 parte 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 ~ l;3 --10-- `

acrylamide gel. The resulting 646 bp fragmentæ in the digests were eluted from the gel, precipitated with ethanol, and combinsd. The precipitates were spun down, resuspended in Tris-EDTA containing 0.1 M NaCl, 5 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 _coRI-AccI vector fragment derived by diges-ting p~R322 with EcoRI and AccI. The ligation was carried out at 4C. 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 32p labelled IFN-a geno~ic 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-a6L gene and/or to produce IFN-a6L.
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
~o. 39409 Cultivation of Transformants Bacteria transformed with the IFN-a6L 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 the bacteria to grow and produce IFN-a6L. If the bacteria are such that the protein is contained in their cytoplasm, the IFN-~6L 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 sulfate. Further purification of the extract may be achieved by affinity chromatog-raphy, electrophoresis, or other protein purification techniques.
Expression of the IFN-a6L gene by bacterial hosts such as E.coli that utilize N-formyl-methionine and/or methionine to initiate translation produces IFN-a6L 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 bac~erial claavage 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-a6L molecules, those containing an initial ~-formyl-methionine or methionine, khose not containing an N-formyl-methionine or methionine and any mixture thereof, are encompassed by the present in~ention. Accordingly, the invention contemplate~
producing IFN-a~L-containing compositions having IFN
activity that is attributable solely to IFN-a6L and/or said terminal N-formyl-mekhionine or methionine derivative khereof.

Biological Testing of IFN-6L
IFN-6L-containing cell sonicates were tested _ vitro and found to have the following activities: (1) inhibition of viral replication of vesicular stomatitis virus (VSV) and herpes simplex C!~

virus-l (HSV-l); t2) 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',S'-oligoadenylate synthetase (2',5'-A); and (6) enhance-ment of the double-stranded RNA-dependent protein kinase. IF~ a6L was active in inhibiting viral replication in both human and other mammalian cell~, such as hamster, monkey, bovine, and rabbit cells.
The tests show that IFN-a6L exhibits anti-viral activity against D~A and R~A 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-a6L may be used to treat viral infections with a potential for interferon therapy such as chronic hepa-titis B inEection, ocular, local, or systemic herpes virus infections, influenza and other respiratory tract virus infections, rabies and other ~iral zoonoses, arbovirus infections, and slow virus diseases such as Kuru and sclerosing panencepha-litis. It may also be useful for treating viral infections in immunocompromised patients such as herpes zoster and varicella, cytomegalovirus, Epstein-25 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, 30 HodgXin's disease, nodular, poorly differentiated lymphoma, acute lymphocytic leukemia, breast carci-noma, melanoma, and nasopharyngeal carcinoma. The fact that IF~-a6L increases protein kinase and 2',5'-oligoadenylate sythetase indicates it may also J

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-phosp~ate dehydrogenase glutamine synthetase, orni-thine decarboxylase, S-adenosyl-l-methionine decarboxylase, and UDP-N-acetylglucosamine-dolichol monophosphate transerase. The ability of the IF~-a6L
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 ~urface charge, altered capacity to bind substances such as cholera toxin, concanavalin A and thyroid-stimulating hormone, and change in the exposur~ of surface gangliosides.
Pharmaceutical compositions that contain IF~-a6L as an active ingredient will normally 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-~6L will usually be formulated as a unit dosage form that contains in the range of 104 to 107 in~ernational units, more usually 106 to 107 international units per dose.
IFN-6L may be administered to humans or other mammals in various manners such as orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, ancl subcutaneously. The particular mode of administration and dosage regimen will be selected by the attending physician taking into account the particulars o~ 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 ~o a few weeks, whereas tumor or cancer treatment involves daily or multidaily doses over months or years. IF~-~6L therapy may be combined with other treatmentsO 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 i5 effective. For instance, in the case of herpes virus keratitis treat-ment therapy with native IF~ has been supplemented by thermocautery, debridement and trifluorothymidine therapy.

Claims (19)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a polypeptide having interferon activity and comprising the amino acid sequence:
which comprises:
a) inserting into a cloning vehicle an IFN-.alpha. gene having the nucleotide sequence:
b) transforming a host with the vector;
c) cultivating transformants which express the IFN-.alpha. gene; and d) collecting the polypeptide from the resulting culture.

2. The process of claim 1, wherein the cloning is a plasmid.

3. The process of claim 2, wherein the cloning is the plasmid pGW 21.

4. The process of claim 1, wherein the host is a prokaryote.

5. The process of claim 4, wherein the host is E. coli.

6. The process of claim 5, wherein at least some of the polypeptide produced has its initial cysteine residue preceded by an N-formylmethionine or methionine group.

7. A polypeptide as defined in claim l whenever prepared by the process of claim 1, 2 or 3, or its obvious chemical equivalents.

8. A polypeptide as defined in claim 1 whenever prepared by the process of claim 4, 5 or 6, or its obvious chemical equivalents.

9. A DNA consisting of a nucleotide sequence that encodes the polypeptide of claim 1.

10. A DNA unit consisting of a nucleotide sequence that encodes the polypeptide of claim 6.

11. The DNA unit fo claim 9 wherein the nucleotide sequence is:

12. A cloning vehicle that includes the DNA
unit of claim 9.

13. A cloning vehicle that includes the DNA
unit of claim 11.

14. The cloning vehicle of claim 12, wherein the cloning vehicle is a plasmid.

15. The cloning vehicle of claim 12, wherein the cloning vehicle is the plasmid pGW 21.

16. A host that is transformed with the cloning vehicle of claim 12 and produces IFN-?6L.

17. The host of claim 15, wherein the host is a prokaryote.

18. The host of claim 16, wherein the host organism is E. coli.

19. A host that is transformed with the cloning vehicle of claim 15 and produces IFN-? 6L, wherein the host is E. coli.