WO1986006744A1

WO1986006744A1 - A cDNA MOLECULE CODING FOR THE EXPRESSION OF AN INTERFERON alpha TYPE POLYPEPTIDE, A BACTERIAL OR CELLULAR HOST TRANSFORMED WITH SUCH MOLECULE AND A POLYPEPTIDE SHOWING INTERFERON ACTIVITY PREPARED BY SUCH HOST

Info

Publication number: WO1986006744A1
Application number: PCT/SE1986/000228
Authority: WO
Inventors: Alexander Ulrich Von Gabain; Björn Olof LUND; Thomas Bernhard Edlund; Tor Erik Roger Ny; Erik Hugo Olaus Lundgren
Original assignee: Kabivitrum Ab
Priority date: 1985-05-15
Filing date: 1986-05-14
Publication date: 1986-11-20
Also published as: SE8502430L; SE8502430D0; EP0263102A1; JPS62502857A

Abstract

A cDNA molecule having the nucleotide sequence (alpha-88) (I) or sequences which hybridize to the foregoing sequence and which code for the expression of a polypeptide of the IFN-alpha type; or sequences which are degenerate as a result of the genetic code to the nucleotide sequence defined above and which code for the expression of a polypeptide of the IFN-alpha type; a bacterial or cellular host transformed with such molecule; a polypeptide having alpha-type interfering activity prepared by such host; and method of therapeutical treatment.

Description

A cDNA molecule coding for the expression of an interferon α type polypeptide, a bacterial or cellular host transformed with such molecule and a polypeptide showing interferon activity prepared by such host.

The present invention relates to a cDNA molecule having a nucleotide sequence coding for the expression of a polypeptide of the IFN-α type and sequences which hybridize or are degenerate as a result of the genetic code to such nucleotide sequence. The invention also covers a bacterial or cellular host transformed or transfected with such cDNA molecule or a biologically active fraction thereof. The invention also includes a polypeptide having α-88 type interferon activity prepared by such host . Finally, the invention covers a method of treating of disorders that are normally treated by using α type interferons.

Interferons have been used in therapy against viruses and tumors or cancers using different modes of administration. Thus, it is of general interest to find new interferons to enable broadening of interferon therapy. The present invention has for its object to provide DNA sequences that code for the expression of a polypeptide of the IFN-α-88 type in an appropriate host thereby transforming the host to produce a polypeptide having immunological or biological activity resembling that of human leukocyte interferon.

Another object of the invention is to provide a bacterial or cellular host transformed or transfected with such DNA sequence.

Yet another object is to provide for a polypeptide having α-88 type interferon activity, said polypeptide being prepared by such transformed or transfected host.

A further object of the invention is to provide a method of treating mammals including man for disorders which are normally treated by agents showing antiviral, antiproliferative and NK cell enhancing activity. For attaining these and other objects the invention provides for a cDNA molecule having the nucleotide sequence according to Fig. 2 enclosed hereto (α-88), or sequences which hybridize to the foregoing sequence and which code for the expression of a polypeptide of the IFN-α-88 type, or sequences which are degenerate as a result of the genetic code to the nucleotide sequence defined above and which code for the expression of a polypeptide of the IFN-α-88 type. The invention also covers a bacterial or cellular host transformed or transfected with a cDNA molecule of the type defined above or a biologically active fraction thereof. Such host is suitably selected from the species E.coli.

The cDNA sequences according to the invention are thus capable of controlling an appropriate host to prepare a polypeptide displaying IFN-α-88 activity, and the invention also covers the polypeptides thus prepared.

Finally, the invention also includes a method of treating mammals including man for disorders normally treated using interferon therapy, i.e. disorders that are subject to treatment using agents showing antiviral, antiproliferative and NK cell enhancing activity.

The invention will now be described by non-limiting examples with reference to the appended drawings. Fig. 1 is the physical map of the two cDNA clones (IFN-α-88 and IFN-β-89) subjected to sequence analysis. The underlying arrows indicate the sequencing strategy and on the left hand side of the figure the methods employed are marked. The nucleotides are numbered according to Goeddel et al (Goeddel, D.V., Leung, D.W., Dull, T.J., Gross, M., Lawn, R.M., McCandliss, R., Seeburg, P.H., Ullrich, A., Yelverton, E. and Gray, P.W. (1981) Nature 290, 20-26) for the first codon in the leader peptide. The nucleotides of the GC-tails are not connected; BstE II, Hae III, Hind II, Pst I and Sau 3A label the recognition sequence of the respective restriction endonucleases.

Fig. 2 shows the nucleotide sequence of clone 88 and the predicted aminoacid sequence. The differencies compared to λ2c₁ (Lawn, R.M., Adelman, J., Dull, T.J., Gross, M., Goeddel, D. and Ullrich, A. (1981) Science 212, 1159-1162) are given in the figure using the position designation of the authors of this article.

Several human IFN-α genes and one IFN-β gene have been identified in cDNA libraries derived from virus-induced primary cultures of blood leukocytes CNagata, S., Taira, H., Hall, A., Johnsrud, L., Streuli, M., Ecsödi, J., Boll, W., Cantell , K., and Weissmann, C. (1980) Nature 284, 316-320; Collins, J., (1983) In: "Interferons - From Molecular to Clinical Application". The Soc.Gen.Microbiol.Symp. vol 35, Eds. Burke, D.C. and Morris, A.G. Cambridge University Press) or individual cell lines (Goeddel, D.V., Leung, D.W., Dull, T.J., Gross, M., Lawn, R.M., McCandliss, R., Seeburg, P.H., Ullrich, A., Yelverton, E. and Gray, P.W. (1981) Nature 290, 20-26; Taniguchi, T., Sakai, M., Fujii-Kuriyama, Y., Muramatsu, M., Kobayashi, S. and Sudo, T. (1979) Proc.Japan Acad. 55, 464-469). So far, 8 major species of mRNA encoding IFN-α and one species encoding IFN-β have been isolated as cDNA clones, characterized and some expressed in E.coli. Subsequently, the isolation and mapping of chromosomal genes have been reported (Collins, J. (1983) In: "Interferons - From Molecular to Clinical Application". The Soc.Gen.Microbiol. Symp. vol. 35, Eds. Burke, D.C. and Morris, A.G. Cambridge University Press;Weissmann, C. (1981) In: "Interferon 1981", vol. 3. Ed. Gresser, I., pp. 101-.134, Academic Press (New York & London). Accordingly, the IFN-α genes span a multigene family of more than 15 members, whereas the IFN-β gene is represented by one unique copy per haploid genome. Previous reports indicate that the diversity of the IFN-α gene family is likely to be due to both genetic polymorphism (Lund, B., Edlund, T. , Lindenmeier, W . , Ny, T., Collins-., J., Lundgren, E. and von Gabain, A. (1984) Proc.Natl.Acad.Sci.USA, 81, pp 2435-39) and gene duplicators (Lawn, R.M. , Adelman, J., Dull, T.J.,Gross, M., Goeddel, D. and Ullrich, A. (1981) Science 212, 1159-1162; Ullrich, A., Gray, A., Goeddel, D.V. and Dull, T.J. (1982) J.Mol.Biol. 156, 467-486) Several lymphoid cell lines have been reported to produce IFN. Most T cell lines produce IFN-β, while B cell lines produce IFN-α and IFN-β in different proportions (Larsson, I., Lundgren, E., Nilssonl.,K. and Strannegard, D. (1979) Develop, biol Standard, 42, 193-197; Matsuyama, M., Hinuma, Y., Watanabe, Y. and Kawade, Y. (1982) J.geα. Virol. 60, 191-194). The Burkitt's lymphoma derived cell line Namalwaa has been used for large scale production of human IFN (Strander, H., Mogensen, K.E. and Cantell, K. (1975) J.Clin.Microbiol. 13, 116-117; Zoon, K.C., Buckler, C.E., Bridgen, P.J. and Gurari-Rotman, D. (1978) J.Clin. Microbiol. 7, 44-51; Johnston, M.D. (1981) J. gen.Virol. 56, 175- 184). Upon Sendai virus induction, several species of IFN-α are produced, comprising about 85. of the biological activity (Johnston, M.D. (1981) J. gen. Virol. 56, 175-184), the rest being IFN-β. The proportion of IFN-β is , however, variable, Shuttleworth et al. (Shuttleworth, J., Morser, J. and Burke, D.C. (1983) Eur.J.Biochem. 133, 399-404)described a Namalwa line producing no detectable IFN-β protein, although the gene was transcribed.

The present invention involves characterization of the dominating mRNA species encoding IFN proteins in this substrain of Namalwa cells in order to analyse the differential expression and characterization of the IFN genes expressed.

It has been found that the ratio of cDNA clones encoding IFN-α and IFN-β corresponds well to the ratio obtained as functional activity. By partial sequence analysis and by comparing results from physical mapping it was shown that the dominating mRNA species corresponded to that described as IFN-α A (Goeddel, D.V., Leung, D.W., Dull, T.J., Gross, M., Lawn, R.M. , McCandliss, R., Seeburg, P.H., Ullrich, A., Yelverton, E. and Gray, P.W. (1981) Nature 290, 20-26) or IFN-α₂ (Nagata, S., Taira, H., Hall, A., Johnsrud, L., Streuli, M., Ecsödi, J., Boll, W., Cantell, K. and Weissmann, C. (1980) Nature 284, 316-320) and to IFN-β ( Taniguchi, T., Sakai, M., Fujii-Kuriyama, Y., Muramatsu, M., Kobayashi, S. and Sudo, T. (1979) Proc,Japan Acad. 55, 464-469). One mRNA species was found representing an IFN-α not identified as a cDNA clone before, but likely to correspond to a genomic sequence reported previously (Lund, B., Edlund, T., Lindemeier, W., Ny, T., Collins, J., Lundgren, E. and von Gabain, A. (1984) Proc.Natl. Acad. Sci. USA, in press; Lawn, R.M., Adelman, J., Dull, T.J., Gross, M., Goeddel, D. and Ullrich, A. (1981) Science 212, 11591162). The sequence of this IFN-α clone encoding the major protein was determined, as well as of a full length IFN-β clone, confirming the conserved character of the IFN-β gene. Example General procedures.

Plasmid DNA was isolated by an alkaline lysis procedure (Birnboim, H.C. and Doly, J. (1979) Nucleic Acids Res. 7, 15131523) with the following modifications; 500 ml of bacteria culture were grown to an OD₆₀₀ of approximately 0.4, chlorampheni col was added to a final concentration of 170 μg/ml and the culture was incubated overnight. Cells were harvested and treated as described (Birnboim, H.C. and Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523), except that the volumes were scaled up to 32 ml of solution I, 64 ml of solution II and 48 ml of solution III, the lysate was precipitated with 0.6 volumes of isopropanol . Plasmid DNA was purified by two consecutive ethidium bromide/ CsCl equilibrium density gradient centrifugations.

Restriction enzymes were purchased from New England BioLabs, Beverly, Mich., USA, Boehringer Mannheim GmbH, Mannheim,

West Germany or from Bethesda Research Laboratories Inc., Gaithe burg, Md, USA,and were used according to the suppliers recommendations. Oligodeoxyribonucleotides were labelled at the 5' end by transfer or (γ^32P) ATP (Amersham, UK) by using T4 polynucleotide kinase (Boehringer Mannheim) as described (Sqaramella, V. and Khorana, H.G. (1972) J.Mol.Biol. 72, 427-444).

Nick-translation was carried out according to Maniatis et al. (Maniatis, T., Jeffrey , A. and Kleid, D.G. (1975) Proc. Natl.

Acad. Sci. USA 72, 1184-1188) modified as follows: approximately 500 ng of DNA was incubated for 1.5 h at 15°C in 20 μl of nicktranslation reaction mixture consisting of 50 mM Tris-HCl (pH

7.5), 50 mM NaCl, 10mM β-mercaptoethanol, 5 mM MgCl₂, 80 μCi of (α-^32p) dGTP, 500 μM dATP, 500 μM dCTP, 500 μM dTTP, 5 μ of DNA pol. I (New England BioLabs) and about 0.1 pg DNase I (Sigma Chemical Co., St. Louis, Mo, USA), titrated for optimal incorporation without excessive degradation of the DNA (analysed on denaturing polyacrylamide gels).

After addition of 30 μl of 0.1 % SDS and 10 mM EDTA, the labelled DNA was purified on a Sephadex G-150 column (Pharmacia Fine Chemicals, Uppsala, Sweden).

All chemicals used were of the highest purity commercially available.

Cell growth and IFN induction

Namalwa cells were obtained from Dr. A. Adams, Karolinska Institutet, Stockholm, Sweden. Cell line and karyotype, methods for cell preparation in fermentors, Sendai virus induction and IFN titration were in accordance. with conventional techniques. For preparation of a cDNA library, the cells were grown up to 100 litres collected by centrifugation of 1,000 g for 1 h after incubation with Sendai virus. A rabbit anti IFN-α serum was obtained from Dr. K. Cantell, Helsinki, Finland, with a neutralizat ion titer of 1/450,000 against IFN-α and 1/3,000 against IFN-β. A rabbit anti-IFN-β serum (EBAB 4640) was purchased from Enzo Biochem., New York, N.Y., USA, with a neutralization titer of ~30 U/ml against IFN-α and 300,000 U/ml against IFN-β. Neutralization titer was performed as described by Kawade (Kawade, Y. (1980) J. Interferon Res. 1, 61-70), the degree of neutralization being recalculated into per cen-t activity remaining after neutralization. RNA preparation.

Cytoplasmic RNA was extracted from Namalwa cells in the presence of Ribonucleoside-Vanadyl Complexes (BRL) (Berger, S.L. and Birkenmeier, C.S. (1979) Biochemistry 18, 5143-5149) and enriched for poly(A) RNA by passage over oligo(dT) cellulose,

(Collaborative Research, Waltham, Ma, USA) (Aviv, H. and Leder, P. (1972) Proc.Natl.Acad. Sci.USA 69, 1408-1412). Construction of a cDNA library. Partially purified IFN mRNA, giving a titer of approximate ly 2,000 units/,ug in the Xenopus laevis assay (Gurdon, J.B., Lane, CD., Woodland, H.R. and Marbaix, G. (1971) Nature 233, 177-182), was used as a template for cDNA synthesis, using the conditions described by Wickens et al. (Wickens, M.P. , Buell, G.N. and Schimke, R.T. (1978) J. Biol.Chem. 253,. 2483-2495). Oligo (dT_{1 2 - 1 8}) (Co l laborat ive Research) was used as a primer for reverse transcription, the reverse transcriptase was obtained from J.W. Beard, National Institutes of Health. Sl nuclease (Bethesda Research Laboratories) treatment of the cDNA and size fractionation on 5-231 sucrose gradient were as outlined by Hoeijmakers et al. (Hoeijmakers, J.H.J., Borst, P.,van der Burg, J., Weissman, C. and Cross, G.A.M. (1980) Gene 8, 391-417). cDNA molecules longer than 200 base pairs were tailed with deoxycytidine (Wu, R. and Deng, G. (1981) Nucleic Acids Res. 9, 41734188) and annealed (Peacock, S.L., Mclver, CM. and Monahan, J.J. (1981) Biochim.Biophys.Acta 655, 243-250) with Pst I digested, deoxyguanosine-elongated pBr 322 (Bolivar, F., Rodriquez, R.L., Greene, P.J., Betlach, M.C, Heyneker, H.L. and Boyer, H.W. (1977) Gene 2, 95-113). The resulting plasmid chimeras were used to transform E.coli strain 294 (Bochner, B.R., Huang, H-C., Schieven, G.L. and Ames, B.N. (1980)J. Bacteriol. 143, 926-933). Competent cells were prepared according to Dagert and Ehrlich (Dagert, M. and Ehrlich, S.D. (1979) Gene 6, 23-28). After transformation recombinant clones were selected on L-agar plates containing tetracycline at 20 μg/ml.

Synthesis of oligodeoxyribonucleotides.

The oligodeoxyribonucleotides in Table 1 as enclosed were synthesized and supplied by KabiGen AB (Stockholm, Sweden), using the solid-phase phosphite method (Chow, F., Kempe, T. and Palm, G. (1981) Nucleic Acids Res. 9, 2807-2817). Isolation and screening of cDNA clones. Individual clones were picked in an ordered array, allowed to grow overnight, transferred to Whatman 541 filter paper, amplified with chloramphenicol at 250 μg/ml, and prepared for hybridization as described by Gergen et al. (Gergen, J.P., Stern,

R.H. and Wensink, P.C (1979) Nucleic Acids Res. 7, 2115-2136). The filters were prehybridized, hybridized with ³²P labelled

DNA probes, and washed as described (Wallace, R.B., Johnson,

M.J., Hirose, T., Miyake, T., Kawashima, E.H. and Itakura, K. (1981) Nucleic Acids Res. 9, 879-894). The temperature for hybridization and final washing step was 37°C for the 13mer and 42°C for the 15mer.

DNA sequence determination.

Relevant fragments were cloned into phage M 13 cloning vector mp7, mp8, and mp9 (Messing, J., Crea, R. and Seeburg,

P.H. (1981) Nucleic Acids Res. 9, 309-321; Messing, J. and Vieira, J. (1982) Gene 19, 269-276), transformed into E.coli strain JM 103 (Messing, J., Cfea, R. and Seeburg, P.H. (1981) Nucleic Acids Res. 9, 309-321). Phages carrying IFN gene sequences were identified by plaque hybridization (Benton, W.D. and Davis, R. W. (1977) Science 196, 180-182) using nick-translated IFN-cDNA as a probe. Single stranded template DNA was isolated from the phage (Messing, J., Crea, R. and Seeburg, P.H. (1981) Nucleic Acids Res. 9, 309-321). The DNA sequences were determined by the dideoxy chain termination method (Sanger, F., Nicklen, S. and

Coulson, A.R. (1977) Proc.Natl .Acad. Sci. USA 74, 5463-5467), utilizing the primers outlined in Table 1 or the M 13 universal primer.

Minor parts of the sequences (Fig. 1) were analysed accor ding to Maxam and Gilbert (Maxam, A.M. and Gilbert, W. (1980) Methods Enzymol. 65, 499-560). Some of the clones carrying short inserts were sequenced using the double-stranded DNA as a template. The plasmids were purified over a Bio-Gel A5 m column (Bio-Rad Lab., Richmond, Ca, USA) before priming the dideoxy chain termination reactions with the 15mer (Wallace, R.B., Johnson, M.J., Suggs, S.V., Miyoshi, K., Bhutt, R. and Itakura, K. (1981) Gene 16, 21-26). Experimental results. Interferon production in Namalwa cells.

The proportion of IFN-α and IFN-β production induced by Sendai virus varied from run to run in the used cell line. The particular fermentation cycle, employed for preparing the cDNA library, was monitored for IFN production. The results are displayed in Table 2 as enclosed. The MDBK cells, only assaying IFN-α, revealed 55. of the activity determined with the Wish cell line assaying both IFN-α and IFN-β. An antibody neutralization experiment confirmed that the proportion of IFN-α and IFN-β was 55% to 45%. Construction of the cDNA library.

A bacterial cDNA library of about 6,000 colonies was obtained by synthesizing cDNA as described above, primed on RNA from the used Namalwa substrain. The cDNA was linked to the Pst I site of the vehicle pBR 322 following the cloning strategy by Chang et al. (Chang, A.C.Y., Nurnberg, J.H., Kaufman, R.J., Ehrlich, H.A., Schimke, R.T. and Cohen, S.N. (1978) Nature 257, 617-623). Two synthetic oligonucleotides (Table 1) were employed for screening the library by colony hybridization: one was designed to complement the 15 nucleotides between positions 486 to 500 (designation of nucleotides according to Goeddel.. (seep.12) - a region found to be conserved for the majority of IFN-α sequences characterized so far (Collins, J. (1983) In: "Interferons-From Molecular to Clinical Application". The Soc.Gen.Microbiol .Symp.vol. 35, Eds. Burke, D.C. and Morris, A.G. Cambridge University Press; Weissman, C. (1981) In: "Interferon 1981", vol. 3. Ed. Gresser, I., pp. 101-134, Academic Press (New York & London). The other was designed to complement a stretch of 13 nucleotides in positions 209 - 221, as it was identical for IFN-β and some of the IFN-α's (Taniguchi, T., Mantei, N., Schwarzstein, M., Nagata, S., Muramatsu, M. and Weissman, C. (1980) Nature 285, 547-549). Using the hybridization conditions described in Materials and Methods, 8 colonies were found to hybridize with the 15-mer and 6 with the 13-mer. It should be noticed that no colony of those hybridizing to the 15-mer gave a signal with the 13-mer as well. However, it was not possible to anticipate that all those cDNA clones carried were genuine IFN-α and IFN-β sequences, as oligonucleotide screening may give false positive signals (Goeddel, D.V., Leung, D.W., Dull, T.J., Gross, M., Lawn, R.M., .McCandliss, R., Seeburg,. P.H., Ullrich, A., Yelverton, E. and Gray, P.W. (1981) Nature 290, 20-26). Therefore, it was necessary to identify at least one clone each encoding IFN-α or IFN-β similar to the types published, in order to rescreen the library. Identification of cDNA clones encoding IFN-α' s.

The presence of IFN-α clones was explored by sequencing diagnostic stretches using the 15-mer as a primer. For this purpose, the cDNA inserts were either recloned in M 13 vectors, in order to obtain single-stranded template for the sequencing procedure, or double-stranded plasmid DNA was directly employed as a matrix for sequencing as described above. The length of the 8 inserts allowed for 3 of them to decode more than 200 nucleotides and 3 around 80 nucleotides. The remaining inserts were too short, to establish a diagnostic stretch of nucleotides. For the region being sequenced, 5 were identical to a previously introduced and sequenced cDNA designated as IFN-α₂ or IFN-α A (Streuli, M., Nagata, S. and Weissmann, C (1980) Science 209, 1343-1347; Goeddel, D.V., Leung, D.W., Dull, T.J., Gross, M., Lawn, R.M., McCandliss, R., Seeburg, P.H., Ullrich, A., Yelverton, E. and Gray, P.W. (1981) Nature 290, 20-26).

However, one insert designated 88, revealed remarkable differences compared to the homologous region of previously described IFN-α cDNA clones. Furthermore, the cDNA inserts were analysed according to their size by cleaving the chimeric plasmids with the endonuclease Pst I. The determination of the sizes of the inserts revealed that only two of them potentially could carry the entire coding region; whereas the rest of them were truncated cDNA inserts. Identification of cDNA clones encoding IFN-β. A number of candidates binding to the 13-mer were probed for the presence of an internal Pst I site characteristic for IFN-β (Taniguchi, T., Ohno, S., Fujii-Kuriyama, Y.and Muramatsu, M. (1980) Gene 10, 11-15). Two of the inserts carried an internal Pst I site. The size of these cDNA clones turned out to be sufficient to encode an entire IFN-β. The oligonucleotide used for their detection was employed for a partial sequence analysis as described above. The sequence confirmed that the two cDNA clones were identical with that of IFN-β described previously (Derynck, R., Content, J., DeClercq, E., Volckaert, G., Tavernier, J., Devos, R. and Fiers, W. (1980) Nature 285, 542-547).

Reprobing the cDNA library with identified cDNA clones encoding IFN-α and IFN-β.

The cDNA clone 2337 encoding an IFN-α similar to IFN-α₂ and the cDNA clone 99 encoding IFN-β were employed as a probe to rescreen the cDNA library. Out of the 6,000 colonies investigated, 14 bound to the IFN-α- probe and 21 bound to the IFN-β probe. The condition for hybridization were stringent (see above). The ratio of cDNA clones hybridizing to the IFN-α probe over those to the IFN-β probe was 2 over 3. The total frequency of IFN clones in the library was 0.6%.

The colonies identified with the two cDNA clones were not entirely identical with those identified with the two oligonucleotides (see Table 3 as enclosed). Out of the 14 colonies hybridizing to the IFN-α cDNA probe 6 were identified with the 15mer designed to be specific for IFN-α. Only two of the colonies hybridizing to the IFN-β cDNA were identified with the 13mer designed to be specific for α and β.

Sequence of the two cDNA clones encoding IFN-α 88 and IFN β.

The two IFN clones 89 and 88 were selected for sequencing, as they encoded potentially IFN-β and an unusual IFN-α respectively. For sequencing the cDNA inserts, two strategies were followed. i) recloning subfragments in M 13 mp8 or mp9 followed by sequencing procedure according to Sanger et al. (Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc.Natl.Acad. Sci. USA 74, 5463-5467). For this, the universal M 13 primers and. the two synthetic oligonucleotides were employed as primers. ii) Recloning subfragments in pBR 322 followed by sequencing the fragments of interest according to Maxam and Gilbert (Maxam, A.M. and Gilbert, W. (1980) Methods Enzymol. 65, 499-560).

The sequencing strategy is outlined in Figure 1. In Figure 2, the established sequences and the predicted amino acid sequence is compiled together with an IFN-α gene sequence recently found in a human genomic library (Lawn, R.M:, Adelman, J., Dull, T.J., Gross, M., Goeddel, D. and Ullrich, A. (1981) Science 212, 1159-1162). The IFN-βcDNA sequence showed complete identity with previously published sequences by Fiers and collaborators (Derynck, R., Content, J., DeClercq, E., Volckaert, C, Tavernier, J., Devos, R. and Fiers, W. (1980) Nature 285, 542-547), differing in only the 3rd base of codon 30, described by Taniguchi et al.

(Taniguchi, T., Ohno , S., Fuj ii-Kuriyama, Y. and Muramatsu, M. (1980) Gene 10, 11-15).

Biological activity 1. Antiviral activity The protein encoded by the sequence shown in Figure 2

(amino acids 1-166) was expressed in E.coli under the control of trp promoter, purified by an immune affinity column and assayed for antiviral activity on two test cells, bovine MDBK cells and human Wish amnion cells. Antiviral activity was tested on indica ted cell lines, using Vesicular Stomatitis Virus as challenge virus (Leanderson,T., Hillörn,V., Larsson,E-L. and Lundgren,E. J., Immunol. 129:490-494, 1982). The activity is given as International Units/mg protein, where International Units are determined by the 69/19 reference preparation and protein was measured by a sandwich type ELISA method using a polyvalent rabbit anti-IFN antibody specific for IFN-α (for reference see Clark, B.R. and Engvall, E. in Enzyme Immunoassay (Maggio, E.T., Ed.), pp. 167-179, CRC Press, 1980). Results: Specific activity of IFN-α88 (IU/mg protein) MDBK Wish

8,4 x 10⁸ U/mg prot. 9,7 x 10⁸ U/mg prot. 2 . Antiproliferative activity .

The antiproliferative effect was tested on the two B-lymphocytic cell lines Daudi of Burkitt's lymphoma origin and MN60 of acute lymphocytic leukemia origin. The cells were grown in their ordinary medium for 5 days, IFN added at 0,01, 0,1 and 1 ng/ml concentrations and viable cells counted daily(Leanderson, T. and Lundgren, E. Exp.Cell Res. 130: 421-426, 1980).

Daudi cells started to be inhibited after 24 hours and proliferation had ceased after 96 hours. MN60 cell lines, which are less sensitive to IFN showed growth inhibition after 48 hours and had ceased growing by 1 ng/ml at 96 hours and by 0,1 ng/ml at 120 hours.

3. NK-cell enhancing activity .

The effect on natural killing cells was assayed using K562 erythromyeloid cells as target cells in a 4 h ⁵¹Cr release to monitor cytolytic activity (Gustafsson, A and Lundgren, E. Cellular Immunol. 62: 367-376, 1981). Both at 0,1 ng/ml and 1 ng/ml a significant enhancement was seen compared to the controls. Thus, the assayed activity qualifies as an IFN as it has both antiviral, antiproliferative and NK cell enhancing activity. The E.coli cells containing the plasmid having inserted therein the cDNA molecule according to Figure 2 have been deposited with DSM, Deutsche Sammlung von Mikroorganismen, under the number DMS 3261, the receipt of deposit being dated March 11, 1985.

(Goeddel, D.V., Leung, D.W., Dull, T.J., Gross, M., Lawn, R.M., McCandliss, R., Seeburg, P.H., Ullrich, A., Yelverton, E. and Gray, P.W. (1981) Nature 290, 20-26) TABLE 1

Synthetic oligodeoxyribonucleotides.

The table lists the oligonucleotides employed to screen the cDNA library. IFN genes completely complementary to the oligonucleotides are indicated (2,7,8,42). Positions are indicated according to Goeddel et al. (2).

Sequence Complementary to

486 500

5 ' -CAACCTCCCAGGCAC-3 ' 1 5 -mer IFN-αA ; -αB ; -αC ; -αD ; -αH ; -αL ;

IFN-αJ;-α2c.-α2h;-α88

209 221

5'-CCTTCTGGAACTG-3' 13-mer IFN-αB; -αC; -αD; -αF; -αG;-αL; and IFN-β

TABLE · 2

Neutralization of Namalwa IFN

MDBK Wish

F 144 830^* 1 500

F 144 + anti-α < 1 150

F 144 + anti-β 830 750

F 144 + anti-α + anti-β NT < 1

The fermentation (F 144) was run in a 100 litre scale according to Materials and Methods. Anti-α was used in 1/100 dilution and anti-β in 1/10 dilution in the mixing experiment.

*

U/ml calibrated against the reference preparation 69/19.

TABLE 3

Screening of cDNA library with synthetic oligonucleotides and full length IFN-α and IFN-β probes. The table summarizes (Exp 1) the number of clones identified with the two oligonucleotides (15mer, 13mer) and the cDNA clone encoding IFN-αA (2337) or IFN-β (89). In addition (Exp 2) the table exhibits the number of colonies cross-hybridizing with the respective probes.

15-mer 13-mer clone clone

2337 89 Exp 1 primary screening 8 6 14 21

Exp 2 crosshybridizing probe

15-mer - 0 8 0

13-mer 0 - 0 2 clone 2337 1) 8 0 - 0 clone 89 2) 0 2 0 _

NOTES TO FORM PCT/RO/134

These Notes are intended to facilitate the use of the present form. For full information, see the text of the Patent Cooperation Treaty and the texts of the Regulations and the Administrative Instructions under that Treaty. In case of discrepancy between these Notes and the said texts, the latter are applicable. "Article" refers to Articles of the Treaty, "Rule" refers to Rules of the Regulations and "Section" refers to Sections of the Administrative Instructions.

(1) "To the extent that any indication with respect to the deposited microorganism is not contained in the description, it may be given on a separate sheet Where any such indication is so given it shall preferably be on the form provided in Annex F [of the Administrative Instructions] as form PCT/RO/134 and, if nimished at the time of filing, the said foπn shall, subject to paragraph (b), preferably be attached to the request and referred to in the Check List referred to in Rule 3.3(a)(ii))." (Section 209(a))

"For the purposes of the Japanese Patent Office when Japan is designated, paragraph (a) applies only to the extent that the said form or sheet is included as one of the sheets of the description of the international application at the time of filing." (Section 209(b))

When identifying the reference to the deposited microorganism in the description, indicate preferably the page and line on which the first such reference is made.

(2) "Any reference to a deposited microorganism shall be made in accordance with this Rule and, if so made, shall be considered as satisfying the requirements of the national law of each designated State." (Rule 13bis.2)

"A reference to a deposited microorganism shall indicate,

(i) the name and address of the depositary institution with which the deposit was made;

(ii) the date of deposit of the microorganism with that institution;

(iii) the accession number given to the deposit by that institution; and

(iv) any additional matter of which the International Bureau has been notified ...... provided that the requirement to indicate that matter was published in the Gazette at least two months before the filing of the international application." (Rule 13bis3(a))

(3) "A reference to a deposited microorganism shall be considered to be made for the purposes of all designated States, unless it is expressly made for the purposes of certain of the designated States only; the same applies to the indications included in the reference." (Rnle 13 bis5(a))

"References to different deposits of the microorganism may be made for different designated States." (Rule 13bis.5(b))

"Any designated Office shall be entitled to disregard a deposit made with a depositary institution other than one notified by it under Rule 13tbis.7(b)." (Rule 13bis.5(c))

(4) See Rule 13bis.3(a)(i) quoted in note (2) above.

(5) See Rule 13bis.3(a)(ii) quoted in note (2) above.

"Any date in the international application, or used in any correspondence emanating from International Authorities relating to the international application, shall be indicated by the Arabic number of the day, by the name of the month, and by the Arabic number of the year. The receiving Office, where the applicant has not done so, or the International Bureau, where the applicant has not done so and the receiving Office fails to do so, shall, after or below any date indicated by the applicant in the request, repeat the date, in parenthesis, by indicating it by two-dig-it Arabic numerals each for the number of the day, for the number of the month and for the last two numbers of the year, in that order and with a period after the digit pairs of the day and of the month (for example, "30 March 1978 (30.03.78)")." (Section 110)

(6) See Rule 13bis.3(a)(iii) quoted in note (2) above.

(7) See Rule 13bis.3(a)(iv) quoted in note (2) above.

For the purposes of the notification provided under Rule 28(4) of the Implementing Regulations to the Convention on the Grant of European Patents (Munich, October 5, 1973), a text in the following terms may be inserted in this box: "In respect of those designations in which a European patent is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a (8) Specify any indications concerning the deposit of the microorganism which will be furnished at a later date. (See also Section 209 (a) quoted in note (1) above).

(9) "If any of the indications referred to in Rule 13bis.3(a) is not included in a reference to a deposited microorganism in the international application as filed but is furnished by the applicant to the International Bureau within 16 months after the priority date, the indication shall be considered by any designated Office to have been furnished in time unless its national law requires the indication to be furnished at an earlier time in the case of a national application and the International Bureau has been notified of such requirement provided that the International Bureau has published such requirement in the Gazette at least two months before the filing of the international application. In the event that the applicant makes a request for early publication under Article 21(2)(b), however, any designated Office may consider any indication not furnished by the time such request is made as not having been furnished in time. Irrespective of whether the applicable time limit under the preceding sentences has been observed, the International Bureau shall notify the applicant and the designated Offices of the date on which it has received any indication not included in the international application as filed. The International Bureau shall indicate that date in the international publication of the international application if the indication has been furnished to it before the completion of technical preparations for international publication." (Rule 13 bis.4)

(10) See Rule 13 bis.4, last two sentences, quoted in note (9) above.

Claims

1. A cDNA molecule having the nucleotide sequence (α-88) according to Fig. 2; or sequences which hybridize to the foregoing sequence and which code for the expression of a polypeptide of the IFN-α type; or sequences which are degenerate as a result of the genetic code to the nucleotide sequence defined above and which code for the expression of a polypeptide of the IFN-α type.

2. A cDNA molecule having the nucleotide sequence (α-88) according to Fig. 2.

3. A bacterial or cellular host transformed with a cDNA molecule according to claim 1 or 2.

4. A host according to claim 3 selected from E.coli.

5. A polypeptide having α-type interferon activity prepared by a transformed host according to claim 3 or 4.

6. A polypeptide having α-type interferon activity according to Fig 2.

7. Method of treating mammals including man for disorders normally treated using antiviral, antiproliferative or NK cell activating agents, comprising administering a therapeutically effective amount of a polypeptide according to claim 5 or 6.