CA1282353C - INTERFERON .alpha. 74 - Google Patents

Abstract

Abstract A new polypeptide, called IFN-.alpha.74, produced by E.coli transformed with a newly isolated and char-acterized human IFN-.alpha. gene is described. The polypep-tide exhibits interferon activities such as antiviral activity, cell growth regulation, and regulation of production of cell-produced substances.

Description

Description echnical 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,~pharma-ceutical~compositions~containing the~polypeptide, andtherapeuti~c methods empl~oying-the polypeptide.

Background Art ~ IFNs ar~e proteins~with~antiviral~,~immuno-modulatory,~ and~antlproliferative~activities produced~
15 by~mammallan~cells~in~re~spons~e~to a~variety ~f indu-cers (see~;Stewart,~W.E.,~The Interferon System, Springer-Verlag,~Nèw York, 197~9).~ The~a~tiv~ity~of IFN
is~ large~ly~spec~ies specific (Colby,~C., a~nd~Morgan,;M.
~ J.~ Ann`.~Rev.~Microbiol.~25~:333-360~;(19~71) and thus only~human~IFN~can~be~us~ed~for human~cl~lnical;stud;ies~
Human~IFNs ~are~classified into ~three groups~ a, ~, and~
y,~(Naturs,~2a6~:~110~ 1980)).~ hs~humsn~IFN-~ gene~s~
compose~a~ multlgene~family~shar~ing 85%-95% sequence homology~(Goed~del~, ~D.~V~ et~al~, Natur9 290~:20~2~7 (1981~ Nag~ata,~ S.,~ et~àl~ J~ Intersron'Rssearch~
333-336~ 98~ Several~of~the~IFN~ genes have~
been `cloned~and~'exprs~ssed~in~E~.c~oli~(Nagata, S., et~

~8X3~3 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 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 by an organism transformed with a newly isolated and newly characterized IFN-a gene.
This polypeptide is sometimes referred to herein as IFN-a74. Other objects of the invention are directed to providing the compositions 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 polypeptide having interferon activity and comprising the amino acid sequence~

CysAspLeuPreGln ThrHisSerLeuGly AsnArgArgAlaLeu IleLéuLeu~laGln MetGlyArgIleSer HisPheSerCysLeu LysAspArgHisAsp PheGlyPheProGlu GluGluPheAspGly"HisGlnPheGlnLys ThrGlnAlaIleSer ValLeuFiisGl~Met IleGlnGlnThrPhe AsnLeuPheSerThr GluAspSerSerAla AlaTrpGluGlnSer LeuLeuGluLysPhe SerThrGluLeuTyr GlnGlnLeuAsnAsp LeuGluAlaCysVal IleGlnGluValGly ValGluGluThrPro LeuMe~AsnValAsp SerIleLeuAlaVal ArcJLysTyrPheGln ArgIleThrLeuTyr LeuThrGluLysLys TyrSerProCysAla TrpGluValValArg AlaGluIleMetArg SerLeuSerPheSer ThrAsnLeuGlnLys ArgLeuArgArgLys:Asp :::: :: : :
``~ : : :

.: . . .
. : . , ;

. - , . . .
. ' ' . ., . . , !, ....
, lZ8Z3~

A second aspect of the invention is a DNA
unit or fragment comprising a nucleotide sequence that encodes the above described polypeptide.
A third aspect o 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 p~arma-ceutical composition having interferon activity com-prising an effective amount oE the above described polypeptide admixed with a p~armaceutically acceptable carrier.
St111 another aspect of the invention is a method of providing~interferon therapy to a human comprising administering a therapeutically effective amount of the above described polypeptide to the ~uman. ; ~
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Brief Description of the Draw1ngs~
Figure~ s~a partial r~èst~riction~map;which ~shows~the~two XhoII~restriction~sites~that;~produce a homologous ;260 base~pa~ir~DNA ~fragment;from the I~N-al and IFN-~a2 structura1 genes. Data~for this map;are ~rom~Str~eu~ M.~ et~al~Science,~;209:1343-1347 (1980).
~ Figure 2~depicts~the~ sequenaing strategy used~to obtain~the~complete DNA~sequénce of t~e IF~-a~74 gene~coding~region. ~Bacter~iophage~mp7:a74 ~8~3~3 DNA served as the template for sequences obtained with primers A, H and F and bacteriophage mp7:a74-2 DNA was the template for sequences obtained with primers E and G. The crosshatched area of the gene depicts the 5 region that encodes the 23 amino acid signal polypep-tide 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-74 including some of the flanking 5'- and 3'- noncoding regions of the gene. The region coding for preinterferon and the 15 mature polypeptide begins with the ATG codon at posi-tion 19 and terminates with the~TGA codon at posi-tion 586.
Figure 4 is a partial restriction~map of the coding region~of the IFN-a74 gene. The~crosshatching 20 represents the~region that encodes the~23 amino acid signal peptide and the open~box represents~the gene coding sequence~for the matur~e polypeptide.~ The scale, in base pairs, is~numb~er~ed wlth O representing the ATG start codon of preinterferon.
~Figure 5 shows the~amino~acid~ sequence of ~ ~
the 23 amino acid signal polypeptide and the~166 amino ~ -acid mature IFN-~a74~coded;for by~;the~g;ene~depicted in Figure~3~ The~189~amino~acid sequen~ce~is displayed ~above~the corresponding~nucleotide sequence.~ Amino 30 acid~24, cysteine, is the~first amino acid~of the mature~IFN-a74~;~protein.
Figure 6 is the DNA sequence of the E. coli trp~promoter~and the gene of~Figure 3 which was nserted between~the~EcoRI and H III sites of~the , ~3X3~3 plasmid pBR322. The amino acid sequence of Figure 5 is written above the corresponding DNA sequence and the location of the restriction si-tes used in the construction of the expression plasmid are indicated.
Figure 7 is a diagram of the expression plasmid, pGC7.

Modes for Carrying Out the Invention In general terms IFN-a74 was made by identi-fying and isolating the IFN-a74 gene by screening a library of human genomic DNA with an appropriate IFN-a DNA probe, constructing a vector containing the IFN-a74 gene, transforming microorganisms with the vector, cultivating transformants that express IFN-74 and collecting IFN-a74 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 pretreatment with 5-bromodeoxyuridine~(Tovey, M.G., et al, Nature 267:455-457 (1977)) and Newcastle Disease Virus (NDV). The polytA) tpolyadenylic acld)-containing messenger RNA (mRNA) was isolated from total~RNA by chromatography on oligo(dT)-cellulose~(type 3 from~
Collaborative Research; Aviv, H., and~Leder,~P., Proc Natl Acad Sci (USA), 69:1408-1412, (1972))~and enriched~for IFN mRNA by density gradient centrifu-gation on 5%-20;~ sucrose gradients. ~Fractions con-taining~IFN mRNA~wer~e identified~by translating the mRNA~by microinjecting aliquots of each~fraction into Xenopus~ oocytes~and~determi~ning the IFN activity of ~-the~products of~the translations according to a method ; :

~. : : ~. . ., , ,. , ,, :

:. :. :, : . ,. : ., :

~Z~3Z~3 described by Colman, A., and Morser, J., Cell, 17:517- -526 (1979).
The Namalwa cell human IFN 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 vector pBR322 (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.
The sequences of two IFN-a clones (IFN-al and IFN-a2) 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-al or the IFN-a2 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 DNA frag-ments were sèparated on a preparative 6% polyacryl-amide gel. DNA from the region of the gel correspon-ding to 260 bp was recovered by electroelution and recloned by ligation into t~e~BamHI site of the single strand bacteriophage ml3:mp7. T~irty-six clones were picked at random, the single stranded DNA i~solated therefrom, and the DNA was sequenced~. The DNA
sequences~of four of these clones were homologous to known IFN-a DNA~sequences. Clone mp7:a-260, with a DNA sequence identical to IFN-al DNA (Streuli, M. et al, Science, 209:1343-1347 (1980)) was chosen~as a highly~specific hybridization probe for identifying additional IF~-a DNA sequences. This clone is hereinafter referred to as~the "260 probe."

.

~X823S3 Screening of ~Jenomic DNA Library In order to isolate other IFN-a gene sequences, a 32P-labelled 260 probe was used to screen a library of human genomic DNA by in situ hybridiza-5 tion. The human gene bank, a gift from F. Blattner, University of Wisconsin (unpublished), was generated by partial cleavage of fetal human DNA with EcoRI and cloned into bacteriophage ~ Charon 4A. Approximately 200,000 clones were screened, of which about 30 10 hybridized with the 260 probe. Each individual clone was further characterized by restriction enzyme mapping and comparison with the ~published restriction maps of 10 chromosomal interferon genes (Nagata, S., et al, J. Interferon Research, 1:333-336 (1981)). One -15 of the clones, hybrid phage ~4Aa74 containing a 24.5 kb insert, was characterized as follows. A DNA
preparation of ~4A:a74 was~cleaved with H III, ~II, and EcoRI respectively, the fragments~separated on an agarose gel, transferred to a nitrocellulos~e 20 filter (Southern, E.M., J Mol Biol, 98:503-517 (1977)) and hybridized with 32P-labelled 260 probe.~ ~This procedure localized the IF~-a74;gene to a l.2 kb HindIII restriction fragment which was then~isolated and recloned, in both orientations,~by ligation of the 25 fragment into HlndIII cleaved~ml;3:mp7.~; The~two sub~
clones are~des~ignated mp7:a74-l~and mp7:a74-2. The ~
designation~indicates that the single-stranded bacter-iophage contains inse~rt~DNA complementary to the mRNA
(the~minus strand;) and the -2 designation;indlcates 30 that~the~inser~t;~DNA is the same ~sequence as the mRNA
~ (the plus strand).

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.: ~. , , . , :-~8Z3~3 Sequencing of the IFN-a74 5ene The Sanger dideoxy-technique was used to determine the DNA sequence of the IFN-a74 gene. The strategy employed is diagrammed in Figure 2, the DNA
sequence thus obtained is given in Figure 3, and a partial restriction enzyme map of the IFN-a74 gene is illustrated 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 sequence information from these known IFN-a genes made it possible to determine the correct translational reading frame and thus allowed the entire 166~amino acid sequence of IFN-a74 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-a74 gene and the amino acid~sequence predicted therefrom differ sub-stantially from the other Xnown IFN-a DNA and~IFN-a amino acid sequences.~ Nagata,~ S., et al, ~
(J Interferon Research, 1:333-336, (1981)) describe isolating two IFN-a genes~ IFN-a4a and IFN;a4b,~that ~ ~ ;
differ by ive nucleotides~which~entails 2~amino acid changes in the proteins expressed thereby. The sequence of IFN-~b is given~ln~European~patent Application No. 81300050.2 pubIished on;July~1~5, 1981~under the~Public-~at1on No.~ 032,134.~The IFN-d74 structura1 géne differs from~the~IFN-:~4b gene by 5~nucleotides which entails 4 30 amino acid~changes in the corresponding proteins: a `singl~e~nucl~eot~lde~ change;~creates ~an amin~o~ac1d suhstitution of alanine~for threonine at amino acid number 14 of th`e mature proteln; ~and a~doub~1e nuc1eotlde change;creates~an~
amino aci~d~substl~tut~on o~alanine for glutami~ne at amino acld~number l9 of the~mature~proteln.

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823~;3 Plasmid Preparation and Host Transformation Assembly of the plasmid for direct expres-sion of the IFN-a74 gene involved replacing the DNA
fragment encodiny the 23 amino acid signal polypeptide of preinterferon with a 120 bp EcoRI/Sau3A promoter fragment tE.coli trp promoter, operator, and trp leader ribosome binding site preceding an ATG initia-tion codon) and using the naturally occurring HindIII
site, 142 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 HindIII sites of pBR322 is shown in Figure 6 which also shows the exact location of relevant cloning sites. Details of the construction are described below.
The coding region for mature IFN-a74 encom-passes a Sau3A site between codons for amino acids 2 and 3 and an AvaI site between codons for amino acids 39 and 40. The 111 bp S 3A to AvaI fragment was iso-lated on a 6~ polyacrylamide gel following a~
Sau3A/AvaI double-digest of the 1.2 kb HindIII genomic fragment. Similarly, the~528 bp~fragment ~rom the AvaI site between codons for amino acids 39 and 40 and :
thè HindIII site 142 nucleotides 3'- of the transla-tional stop codon was i~olated on~a; 5~polyacrylamide gel. These two~ragments, together with a 120 bp EcoRI~to Sau3A~E.coli promoter fragment were ligated --together in a four way directed ligation into the EcoRI to HindIII~site of pBR322. The promoter frag-ment,~which contains a synthetic dindIII re~striction site,~ATG~inititation codon, the~initial cystelne~
codon (TGT) common to all known IFN-as, and Sau3A

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"sticky end", had been constructed previously. The ligation mixture was used to transform E.coli MM294 (Backman, K., et al, Proc Natl Acad Sci (USA) 73:~174-4178 (1974)). The desired correct transformation products, 23 out of 24 screened, were identified by restriction enzyme mapping of colonies which hybri-dized to a 32P-labelled IFN-a genomic fragment. Fig-ure 7 is a diagram of the final expression plasmid obtained, which is designated pGC7. Other prokaryotic hosts such as bacteria other than E.coli may, of course, be transformed with this or other suitable constructs to replicate the IFN-a74 gene and/or to produce IFN-a74-IFN-a74 produced in accordance with the invention is believed to be distinct rom the corres-ponding na~ive protein in several respects. Firstly, because the IFN-a74 gene was expressed by bacterial hosts that utilize N-formyl-methionine and/or methio-nine to initiate translation, some or aIl of the bac-terially produced IFN-a74 molecules are preceded by an N-formyl-methionine or methionine group. Some of the N-formyl-methionine or methionine groups could be removed by natural ln vivo bacterial cleavage mecha-nisms. 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-74 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. Secondly, the amino acid residues of the bacterially produced polypeptide are unsubstituted whereas the residues of the native protein may be substituted with sugar groups, ACTH or other moieties.

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A~so, native IF~-a extracts consist of mixtures of various IF~ molecules whereas the bacterially produced IFN-a74 is homogeneous; that is, bacterially produced IFN-a74 does not contain ~unctionally related polypeptides. Accordingly, the invention contemplates producing IFN-a74-containing compositions having biological activity that is attributable solely to IFN-a74 and/or said terminal N-formyl-methionine or methionine derivatives thereof.

Cultivation of Transformants Bacteria transformed with the IFN-a74 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-a74. If the bacteria are such that the protein is contained in their cytoplasm, the IFN-a74 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 chroma-tography, electrophoresis, or other protein purifi-cation techniques.

~iolo~ical ~esting of IFN-a74 IFN-a74 containing cell sonicates were tested ln vitro and found to have the following activities: (1) inhibition of viral replication of vesicular stomatitis virus (VSV) and herpes simplex virus-l (HSV-l); (2) inhibition of tumor cell growth ~3) inhibition of colony formation by tumor cells in soft agar; (4) activation of natural killer (NK) cells; (5) enhancement o~ the level of 2',5'-oligo-3X3~3 adenylate synthetase (2',5'-A); and (6) enhancement of the double-stranded RNA-dependent protein kinase. The sonicates were active in inhibiting viral infection in both human and other mammalian cells such as hamster, monkey, mouse, and rahbit cells.
The tests show that IFN-a74 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-a74 may be used to treat viral infections with a potential for interferon therapy such as chronic hepatitis B in~ection, ocular, local, or systemic herpes virus infections, influenza and other respira-tory tract virus infections, rabies and other viral~oonoses, arbovirus infections, and slow virus diseases such as Kuru and sclerosing panencephalitis.
It may also be useful for treating viral infections in immunocompromised patients such as herpes zoster and varicella, cytomegalovirus, Epstein-Barr virus infec-tion, herpes simplex infections, rubella, and progres-sive multifocal leukoencephalopathy. Its cell growth regulating activity makes it potentially useful for treating tumors and cancers such as osteogenic sar-coma, multiple myeloma, Hodgkin's disease, nodular,poorly differentiated lymphoma, acute lymphocytic leukemia, breast carcinoma, melanoma, and nasopharyn-geal carcinoma. The fact that IFN-a74 increases protein kinase and 2',5'-oligoadenylate synthetase indicates it may also increase synthesis of other enzymes or cell-produced substances commonly affected by IFNs such as histamine, hyaluronic acid, prosta-glandin E, tRNA methylase, and aryl hydrocarbon hydrolase. Similarly, it may be useful to inhibit . .
' ~ ' . ' .

~8;~3~3 enzymes commonly inhibited by IFNs such as tyrosine amino transferase, glycerol-3-phosphate dehydrogenase glutamine synthetase, ornithine decarboxylase, S-adenosyl-l-methionine decarboxylase, and UDP-N-acetylglucosamine-dolichol monophosphate transferase.
The ability of the IFN-74 to stimulate NK cell activity is indicative that it may also possess other activities such as the abilities to induce macrop~age activity and antibody 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-a74 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. Oral formulations, on the other hand, may be solid, eg tablet or capsule, or liquid solu-~5 tions or suspensions. IF~-~74 will usually be formu-lated as a unit dosage form that contains in the range of 104 to 107 international units, more usually 106 to 107 international units, per dose.
IFN-a74 may be administered to humans in various manners such as orally, intravenously, intra muscularly, intraperitoneally, intranasally, intra-dermally, and subcutaneously. The particular mode of administration and dosage regimen will be selected by the atten~ing physician taking into account the par-:

9 ~323~3 ticulars 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-a74 therapy may be combined with other treatments and may be combined with or used in association with other chemotherapeutic or chemo-preventive agents for providing therapy against viral infections, neoplasms, or other conditions against which it is effective. For instance, in the case of herpes virus keratitis treatment, therapy with IFN has been supplemented by thermocautery, debridement and trifluorothymidine therapy.
Modifications of the above described modes for carrying out the invention, such as, without limitation, use of alternative vectors, alternative expression control systems in the vector, and alter-native host microorganisms and other therapeutic or related uses of IFN-a74, that are o~vious to those of ordinary skill in the biotechnology, pharmaceutical, medical and/or related fields are intended to be within the scope of the following claims.

.

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Claims (6)

1. A process for preparing a polypeptide having interferon activity and comprising the amino acid sequence:

which process comprises:
(a) inserting into a cloning vector an IFN-.alpha. gene having the nucleotide sequence:

said vector comprising pGC7;

(b) transforming a host consisting of E. coli 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 at least some of the polypeptide produced has its initial cysteine residue preceded by an N-formylmethionine or methionine group.

3. A polypeptide having interferon activity and being non-glycosylated interferon alpha 74, and comprising the amino acid sequence:

whenever prepared or produced by or significantly derived from the process as defined in claim 1.

4. A DNA unit consisting of a nucleotide sequence which is:

that encodes the polypeptide comprising the amino acid sequence:

17 '

5. A cloning vehicle that includes the DNA
unit of claim 4, said cloning vehicle being the plasmid pGC7.

6. A host that is transformed with the cloning vehicle of claim 5 and produces IFN-.alpha. 74, wherein the host is E.-coli U