CA1341252C - Human interferon-.beta.2a and interferon-.beta.2b; vectors containing genes coding for said interferons: cell lines producing same and use of said interferons as pharmaceuticals - Google Patents

Abstract

Human interferon-beta2A and interferon-beta2B are produced in purified form by recombinant DNA techniques. Two separate human genes have been identified which code for the production of IFN-.beta.2A and IFN-.beta.2B, respectively. The sequence of IFN-.beta.2A cDNA is established. These genes and cDNA have been cloned into mammalian cells with an SV40 early promoter sequence and such genomic clones are capable of pro-ducing IFN-.beta.2A and IFN-.beta.2B. The antiviral activity of such recombinant IFN-.beta.2A and IFN-.beta.2B is demonstrated as well as other biological activity identifying them as human interferons. It has been shown that IFB-.beta.2 secretion is induced in human cells by growth-stimulatory cytokines.

Description

HUMAN INTERFERON-~3~~ AND INTERFERON-~3zg; VECTORS CONTAINING
GENES CODING FOR SAID INTERFERONS; CELL LINES PRODUCING SAME
AND USE OF SAID INTERFERONS AS PHARMACEUTICALS
This application is a divisional application of Canadian patent application 520,275 filed on 10 October 1986.
Interferon is an important antiviral and antitumor protein produced by the human body. Because of its species specificity, clinical use of interferon requires human interferon. Based on their neutralization by antibodies three types of interferon were defined which include interferon-alpha, produced mainly by leucocytes, interferon-beta, produced mainly by fibroblasts, and interferon-gamma, produced mainly by T-lymphocytes.
The major species of interferon produced by human fibroblasts in response to ds RNA is the 20 Kd glycoprotein IFN-(31, encoded by a 0.9 kb RNA which is transcribed from an intron-less gene on chromosome 9. However, additional mRNAs which yield IFN
activity when micro-injected into frog oocytes have been observed in human fibroblasts induced by poly (rI)(rC) and by a sequential cycloheximide (CHX)-actinomycin D treatment. While cloning the IFN-(31 cDNA, cDNA clones of another co-induced 1.3 kb RNA have been isolated which in recticulocyte lysates code for a 23-26 Kd polypeptide and in oocytes produce human IFN
antiviral activity (J. Weissenbach et al. 1980 Proc. Natl.
Acad. Sci. USA 77:7152-7156; British patent 2 063 882).
Since this activity was inhibited by anti-IFN-B1 antibodies, it was designated IFN-(32. The protein product of IFN-~i2 RNA is immunologically distinct from IFN-al, although the biological activities of the two proteins are cross-neutralized by the same antibodies. The IFN-(32 1.3 kb RNA clearly originates from another gene than IFN-(31, since IFN-~i2 RNA is formed in mouse-human hybrids lacking chromosome 9 (U. Nir (1984) Ph.D., Thesis, Weizmann Institute of Science, Rehovot, Israel).

In the German patent application P 30 43 981 published 1 October 1981 a process is disclosed for the preparation of interferon-Vii= in purified form. Said interferon-(3z is produced by isolating DNA containing the nucleotide sequence coding for interferon-(3z in human cells after cultivating those cells which are able to produce interferon-~3z when exposed to an inducer of interferon. mRNA from said induced cells is extracted and purified. Afterwards, the mRNA is transcribed into DNA and the DNA is cloned in a suitable vector.
It could be demonstrated that there is a second gene encoding IFN-(3z, which, when isolated and transfected into hamster or mouse cells produces human-specific IFN activity after induction by ds RNA and cycloheximide. The IFN produced has the properties ascribed to IFN-(3z but does not have identical amino acid sequences, the proteins produced by the two genes are denominated IFN-(3zA and IFN-~3zB respectively.
Accordingly, it is an aim of the present invention to isolate biologically active human IFN-(3zA and IFN-(3ze.
It is another aim of the present invention to enable the production of biologically active human IFN-(3zA and IFN-(3zB.
It is yet another aim of the present invention to prepare cDNA
which codes for human IFN-biz, and, more particularly, cDNA
which codes for human IFN-~izA and cDNA which codes for human IFN-~izB.
It is a further aim of the present invention to identify genes hybridizing to IFN-(3z cDNA.
It is still another aim of the present invention to identify genes hybridizing to IFN-(3zA cDNA and to IFN-(3zB cDNA.
It is yet a further aim of the present invention to isolate genomic clones containing an IFN-~3z gene, and, more particularly, a genomic clone containing an IFN-(32A gene and a genomic clone containing an IFN-~iza gene.
It is another aim of the present invention to provide a recombinant vector containing an IFN-~3~~A and/or IFN-(3~B gene and a promoter sequence capable of exerting transcription control over the interferon gene.
It is yet another aim of the present invention to provide cells which produce biologically active human IFN-(32A and/or IFN-(328.
It is another aim of the present invention to provide such cells which produce substantial quantities of such IFN-(3zA
and/or IFN-(328.
It is still another aim of the present invention to provide a process for the production of biologically active human IFN-(32A
and/or IFN-~i28 by recombinant DNA technology.
It is a further aim of the present invention to provide genes which, when fused to a strong viral transcriptional promoter and transfected into hamster or mouse cells, produce substantial quantities of biologically active human IFN-(32A
and/or I FN- X328 .
It is still another aim of the present invention to use IFN-(32A
and/or IFN-~i2B in pharmaceuticals which are useful wherever it is desired to influence cell growth and differentiation, especially during terminal differentiation of cancer cells.
An aim of the invention of the parent application is the use of IFN-(32A and/or IFN-(328 to inhibit fibroblast proliferation, to prevent sclerosis after infection.

In accordance with the present invention, the existence of biologic-ally active human interferon-betaz (IFN-(3z) molecules has been con-firmed and these molecules are produced by recombinant DNA technology.
IFN-(3z is a glycoprotein secreted by human fibroblasts which are induced by double-stranded (ds) RNA or viruses to produce interferon.
IFN-(3z amounts to about 5~ of the total IFN activity produced by fibroblasts. The amino acid sequence of IFN-~z, which was determined by cDNA sequencing, indicates only about 20~s overall homology with that of the other human IFNs produced by such human cells (IFN-(31 and IFN-~s) .
A human cDNA or gene encoding IFN-(3zA may be cloned in accordance with the present invention and fused to a strong viral transcriptional promoter. Hamster cells transformed by the fused cDNA or gene produce constitutively human-specific interferon activity which inhibits viral replication and cytopathic effect and induces the proteins typical of the biological response of human cells to interferons. A second gene encoding IFN-(3z is also disclosed which, when isolated and transfected into hamster or mouse cells, produces human-specific IFN activity after induction by ds RNA and cycloheximide. As the IFN produced by both genes have the properties ascribed to IFN-~3z, but do not have identical amino acid sequences, the proteins produced by the two genes are denominated IFN-(3zA and IFN-(3zB respectively.
Description of the drawings Figure 1 shows the restriction map, sequencing strategy and nucleotide sequence of IFN-azA cDNA determined from clone AE20, formed by fusing E474 cDNA and A341 cDNA clones (Weissenbach et al. (1980), supra) by their Xba-1 site. The 5' end sequence was completed from the genomic clone IFA-2 shown in Fig. 2. Numbering begins from the S-1 start of Fig. 3 and 4. The amino acid sequence of the IFN-azA protein is deduced.
Figure 2 shows the structure of genomic clone IFA-2 containing the IFN-[3zA gene. A132 is a subclone of an EcoRI segment of IFA-2 in X

s pBR322. Shaded areas are gene regions hybridizing to cDNA clones A341 and E474 (Weissenbach et al. (1980), supra). The positions of the two RNA starts (cap 1 and 2) and the TATA boxes are shown. In the RNA the longest open reading frame (ORF) of 212 amino acids (Fig.
1) is shown in black. M.C. indicates a second internal ATG in the same frame. No other long ORF are found in the entire IFN-(3zA cDNA
(Fig. 1) Figure 3 shows the S1 nuclease analysis of 5' starts in IFN-[3z RNA.
The DNA probe, a fragment from subclone A132 of IFA-2 gene (Figure 2 ) labelled at BstNl site was hybridized to total RNA from diploid fibroblasts treated (from left to right) by: priming with IFN-(31 200 U/ml, 16 hrs; priming and poly (rI) (rC) (pIC) 50 ~g/ml, 3.5 hours;
priming and cycloheximide (CHX) 50 ug/ml, 3.5 hours; priming and pIC
plus CHX 3.5 hours; priming and CHX for 6.5 hours; no priming and pIC
3.5 hours; same CHX 3.5 hours; same pIC plus CHX 3.5 hours. S-1 and S-2 are the signals generated by the 2 RNA starts of Figure 2.
Figure 4 shows the sequence of the promoter region of the IFA-2 gene encoding IFN-(32A. The sequence of part of subclone A132 (Figure 2) is shown compared to the IFN-(3~ cDNA. S1 and S2 are the 2 RNA starts determined in Figure 3. The initiator ATG is marked by X. The first intron starts at intr 1.
Figure 5 illustrates the fusion of IFN-a2A cDNA to the SV40 early promoter. A plasmid pSVIFA2-II was first constructed by subcloning the 5' Xhol-BamHl (Xh-B) segment of the IFA-2 gene (from 5.5 kb to 7.5 kb in Figure 2) fused with a Clal-Xhol synthetic adaptor of 26 by (restoring the 5' cDNA sequence), into a Clal and Bam H1-cut pBR plas-mid. The 3' BamHl-Hindu (B-H) gene segment (from 7.5 kb to 10.5 kb in Figure 2), subcloned in a a Hindlm. BamHi-cut pBR plasmid, was excised using the Clal site adjacent to Hindlll in pBR322 and ligated to the above 5' segment to obtain the complete IFA-2 gene. A pSVE3 vector cut by Hindlll as described in Y. Chernajovsky et al. (1984) DNA 3: 294-308, was reclosed with Clal linkers (forming pSVCla) and the above IFA-2 gene introduced in the Clal site of pSVCla in the same orientation as the SV40 early promoter forming pSVIFA2-II. A

plasmid pSVIFA2-I was similarly constructed but the Xhol site of IFA-2 was fused directly to the Clal site of pBR before introduction in pSVE3. To obtain the pSVCIFB2 plasmid containing the IFN-~zA
cDNA fused to the SV40 promoter, the above pSVIFA2-II DNA, cut by Xbl, was partially cut by Xmnl to open the Xmn, site located 60 by downstream from the Xhol site in the IFN-(3z cDNA sequence (Figure 1), and not an Xmnl site located in the amp gene of the vector. To this construct, the Xmnl-Xbal segment of IFN-(3z cDNA clone AE20 (co-ordinates 92-566 in Fig. 1) was ligated, restoring the complete IFN-(3zA cDNA sequence as shown in the Figure. EES is the early-early RNA start of SV40 T-ag gene; ATG is the IFN-(3z initiation codon; pA
are IFN-pz and SV40 polyadenylation sites, respectively. pSVCIF(~z DNA was contransfected with a pDHFR plasmid into CHO-K1 DHFR cells as in Y. Chernajovsky et al. (1984), (supra) and clones were tested for human IFN antiviral activity in FS11 cells using Vesicular Stomatitis Virus (VSV). Gene amplification was obtained by selecting CHO clones for methotrexate (MTX)-resistance. Assav of the cultural medium of CHO-SVCIF~iz B132-5M cells, resistant to 250 nM MTX, is shown in the lower part of the Figure. After VSV cytopathic effect developed, cells in the microplate were stained by methylene blue. Serial two-fold dilutions of the medium are from left to right. By com-parison to IFN standard (ST) and non-transfected CHO cells, a titer of 300 U/ml rIFN-[3z is calculated.
Figure 6 shows the results of an assay of (2'-5') oligo A synthetase with poly (rI)(rC)-agarose bound NP40 extracts (M. Revel et al.
(1981) Meth. Enzymol. 79:149-161) of human FS11 fibroblasts previously exposed for 16 hours to medium from a) Cos7 cells two days post-transfection with the pSVIFA2-II DNA (described in Figure 5) (leftmost 4 lanes), b) hamster CHO SI-15 cells stably transformed by the pSVIFA2-I
DNA of Fig. 5 (next 4 lanes), c) CHO MEIF-5 cells expressing IFN-(31 gene (Y. Chernajovsky et al. (1984), supra) (next 2 lanes); d) CHO
cells with DHFR gene only (next lane); e) fresh medium (Non Ind.).
The IFN antiviral activity measured per ml of the media added to FS11 cells as indicated. An electrophoresis of phosphatased, 32P-~-ATP
labelled (2'-5') oligo A product is shown.
x Figure 7 is a comparison of amino acid sequences between type I human IFNs. Amino acids that are conserved in IFN-(31 and the various IFN-«s (shown are «A, «C, replacements in other «s excluding «E, and IFN-« class II), are marked by stars over the IFN-[31 sequence. Amino acids conserved in IFN-(3z are shown above it's sequence by stars if conserved in all type I IFNs, or by squares if conserved in some IFNs. IFN-(3zA was aligned by comparing hydropathy plots (see text) and numbered from the presumed processing site.
Figure 8 shows restriction maps of the two different human IFN-(32 genes which are expressed in rodent cells. The IFA-11 DNA (IFN-(3zB
gene) was transfected into either CHO or L cells and expressed IFN-~i activity after induction (Table 1).
Figure 9 shows an IFN-(3Z specific immunocompetition assay with R-antibodies to show that human fibroblasts FS11 cells secrete the IFN-(32 protein in response to the two cytokines interleukin-I (I1-I) and Tumor Necrosis Factor (TNF-«). Both cytokines induce the synthesis and secretion of the IFN-(3z protein in two levels which have been estimated by the immunoassay to be 10 to 20 U/ml IFN-X32. Optimal IFN-induction was seen with the concentration of rIL-1« of 4 U/ml (0.13 ng/ml). The optimal concentration of TNF-« for IFN-(3z induction (400 U/ml; 40 ng/ml) was higher than that required for cytolytic effect on sensitive cells (0.1 ng/ml) and even higher than that giving optimal growth stimulation of diploid fibroblasts (2 ng/ml).
Detailed description of preferred embodiments The existence of interferon-betaz was discovered in the course of attempts to isolate genetic material containing the nucleotide sequence coding for interferon in human fibroblast cells. In this process, fibroblast cells were induced for the production of inducible interferon mRNA with a suitable exogenous factor and then the mRNAs were extracted. At the same time the mRNAs from a non-induced culture of the same host cells were extracted. cDNA probes were synthesized from the mRNAs of both the induced culture and the non-induced control s culture using the corresponding mRNAs as templates (induced cDNAs and non-induced cDNAs). Double-stranded cDNAs derived from the mRNA
extracted from the induced culture were then synthesized and such cDNAs were inserted in appropriate vectors and transfected into suitable microorganisms. The microorganisms were cultivated under conditions suitable to cause selective development of microorganism colonies of the modified vectors (initial colonies). Duplicate colonies of the initial colonies were formed and the DNAs of both the initial and duplicate colonies were freed in situ. The DNAs of one of the two sets of duplicate colonies were hybridized with the cDNA
probes synthesized from the mRNAs extracted from the induced culture and the other of the two sets of duplicate colonies were hybridized with the cDNA probes synthesized from the mRNAs extracted from the non-induced culture. Clones which selectively hybridized with the induced cDNA probes but not with the non-induced cDNA probes were selected and their DNAs recovered. These cloned DNAs were further tested to determine those DNAs capable of hybridizing with mRNA
translatable in frog oocytes or reticulocyte-lysates into interferon, which DNAs are essentially those which code for interferon, or contain enough of the sequence coding for interferon to be used to obtain a complete interferon DNA.
During this investigation, it was discovered that two different mRNAs coding for interferon could be isolated from human fibroblast cells after being appropriately induced. Upon glycerol gradient centri-fugation, the smallest mRNA sediments at 11S and yields by translation in a cell-free system a protein of molecular weight 20,000 which is selectively precipitated by antibodies prepared against one of the interferons that can be purified from these cells. This protein is human inteferon-betas of which the amino acid sequence was partially determined by E. Knight, et al. (1980) Science, 207, 525-526. The largest mRNA was found to sediment at 14S and yield a protein of molecular weight 23,000 which is precipitated by antibodies against a less purified preparation of fibroblast interferon. This protein is designated human interferon-beta2. cDNA clones hybridizable to mRNA
which translates to interferon-beta2 were prepared in such a manner and an IFN-(3Z cDNA probe was prepared therefrom.

Two such IFN-(3z clones A341 and E474 were found to represent over-lapping sequencing and were fused to reconstitute the IFN-(3z AE20 cDNA (Figure 1). The nucleotide sequence of this IFN-(3Z cDNA was determined and revealed an open reading frame of 212 amino acids predicting a protein of 23,500 daltons. Transcription-translation experiments confirmed that this cDNA encodes such a protein.
From a human gene library of human adult blood cell DNA cloned after partial EcoRI digestion. in 1~ charon 4A (Y. Mory et al. (1981) Eur. J.
Biochem. 120: 197-202), two genomic clones IFA-2 and IFA-11 hybrid-izing to such an IFN-(3z cDNA probe were isolated. Clone IFA-2 con-tains a gene with at least 4 exons hybridizing to various segments of the cloned cDNA (Figure 1). An Xhol site present near the 5' of the AE20 cDNA allowed to map the genomic segment A132 (Figure 2) which contains the first IFA-2 exon ending 70 by downstream from this Xhol site. By S1 nuclease analysis using DNA probes labelled at a BstNl site 40 by after the Zhol site, it was determined that induced FS11 human fibroblasts contain IFN-(3z RNA with two distinct 5' ends S-1 and S-2 seperated by 20 nucleotides (Figure 3). Each of these two starts is preceded in the IFA-2 gene by a potential TATA box at -30 (Figure 4). RNA ending as S-2 seems to be more abundant than the longer RNA under a variety of induction conditions (Figure 3 ). Both RNA 5' ends were also seen with cytoplasmic RNA instead of total cell RNA. Therefore, it is apparent that both RNAs are formed and active in human cells. Both IFN-~iZ RNAs are induced in human fibroblasts by poly (rI)(rC) and induction is stimulated by IFN priming as in the case of IFN-(31 RNA in these cells (U. Nir et al. (1984) Nucl. Acids Res. 12: 6979-6993). A prolonged (6.5 h) treatment with CHX also induces IFN-(3z RNA, but shorter (3.5 h) CHX treatment is effective only with poly (rI)(rC) (Figure 3). The low induction of IFN-(32 RNA
by CHX without ds RNA distinguishes this gene from IFN-(31.
DNA constructs were made to express the IFA-2 gene under the control of the Sv40 early gene promoter. A 4.8 kb segment of IFA-2 DNA was fused through a synthetic oligonucleotide to pSVE3 DNA (Y. Cherna-jovsky et al. (1984). supra) so that the ATG codon is 150 by down-stream from the SV40 early early RNA start (pSVIFA2-II, see legend, Figure 5). After transfection of pSVIFA2-II DNA into Cos7 monkey cells (Y. Gluzman (1981) Cell 23: 175-182), two RNAs of 1.35 and 2.2 kb were detected in Northern blots by IFN-(3Z cDNA; these two RNAs would correspond to transcripts ending at the IFN-~z and SV40 polyadenyl-ation sites respectively (Figure 5). Medium from the Cos7 cells during transient expression of the pSVIFA2-II DNA was assayed for IFN
activity by inhibition of the cytopathic effect of VSV on human FS11 and Wish cells as described in D. Novick et al. (1983) J.Gen.Virol.
64: 905-910. Antiviral activity could be clearly detected two days after transfection (Table 1). The specific antiviral activity of IFN-(32 per unit protein has been estimated to be 30 to 100 times lower than that of IFN-(31 and closer to that of IFN-«1 (5-10 x 106 units/mg). The IFN titers observed by transient ex-pression of the IFA-2 gene were as expected, about 3~ of those seen with the IFN-(31 gene under the same SV40 promoter control (Table 1).
Production of IFN-~z activity by pSVIFA2-II DNA cells was further demonstrated by induction of (2'-5') oligo A synthetase (M. Revel et al. (1981), supra) in FS11 cells exposed to the medium of the trans-fected Cos7 cells (Figure 6). The induction was dose-dependent and corresponded quantitatively to the antiviral activity.
The IFA-2 gene was also fused to the SV40 early promoter directly at the Xhol site near S-2 (pSVIFA2-I DNA, legend of Figure 5). Hamster CHO-Kl DHFR cells were cotransformed with pSVIFA2-I and pSVDHFR (Y.
Chernajovsky et al. (1984), supra) DNAs and a stably transformed line SI-15 was isolated. This cell line produces constitutively 10-50 U/ml of IFN antiviral activity measured on human FS11 and Wish cells, while CHO cells transformed only by pSVDHFR produce no human IFN
(Table 1). By concentration of the medium from SI-15 cultures, rIFN-(3z solutions titrating about 300 U/ml could regularly be ob-tained. This rIFN-~2 has the properties expected for a human IFN and induces the (2'-5') oligo A synthetase, in FS11 cells even at low concentrations of 1 U/ml (Figure 6). The rIFN-(3z constitutively produced by CHO SI-15 cells demonstrated human-specific induction of the mRNAs for (2'-5') oligo A synthetase C56 and HLA in the presence of cycloheximide, its antiviral activity was neutralized by anti-IFN-(31 (but not anti-IFN-« or anti-IFN-N) polyclonal and monoclonal x a antibodies and it acted on mouse-human hybrids only if they contain human chromosome 21 which carries the gene for the type 1 IFN receptor.
These further characterizations of the biological activity of rIFN-(3~
convincingly demonstrate that it acts directly as an IFN and is not an inducer of IFN-(31.
Improved yields of rIFN-a2 were obtained with DNA constructs contain-ing the IFN-(3zA cDNA sequence fused to the SV40 early promoter (pSVCIF(32 shown in Figure 5), when this DNA cotransfected with pSVDHFR into CHO cells as above, was amplified by selecting cells resistant to increasing concentrations of methotrexate. Without amplification, the pSVCIF(32-transformed CHO cells (e. g. clone B-132) produced levels of IFN-[3z activity similar to those produced by the SI-15 cells (Table 2). However, after selection for resistance to methotrexate, higher yields were obtained with up to 800 U/ml (Table 2).
From such clones it was possible to immunoprecipitate the IFN-az protein secreted into the culture medium and if the cells had been labelled by 35S-methionine to analyse the IFN-(32 protein by dodecyl sulphate polyacrylamide gel electrophoresis. The size of the secreted rIFN-(32 was found to be 21,000 daltons. Indeed, experiments have shown that the 23-26 Kd primary translation product of IFN-(32 RNA in reticulocyte lysates is processed in vitro by dog pancreatic membranes into a shortened 21 Kd protein, and that induced human fibroblasts produce a 21 Kd protein which is immunologically similar to the IFN-(3Z RNA 26 Kd product, but distinct from IFN-al. The N-terminus of the mature IFN-(32 21 Kd protein has not been determined and two potential glycosylation sites are present in the IFN-(3z sequence (Figure 1 )making it difficult to calculate the size of the region removed by processing. However, the decrease in size indicates that this processed region is longer than that in IFN-(31, the size of which increases by maturation (W. Degrave et al. (1981) Gene 10:
11-15). A hydropathy plot of the IFN-(3z 26 Kd polypetide has shown that there is a marked similarity between hydrophobic and hydrophilic regions of IFN-(32 and of mature IFN-(31, when the two proteins are aligned by their C-termini. The same alignment also reveals conserv-ation in the amino acid sequence of IFN-(3z with those of the other x type I human IFNs (Figure 7). Between all the known IFN-as and sequences, there are 38 amino acids which are conserved (marked by stars in Figure 7). When IFN-(3, is aligned as above, 18 of these 38 amino acids common to all type I human IFNs can be seen to be conserved (Figure 7). There is an overall homology of about 20% between IFN-(3~ and the other type I IFNs.
Although the sequence homology between IFN-(31 and X32 is low, the cross-neutralization of the antiviral activities of IFN-(31 and IFN-(3z by the same antibodies, including a monoclonal anti-IFN-ail (D. Novick et al. (1983) , supra) , could result from some conserved structure in the active site of IFN-(3. IFN-X32 activity is not neutralized by anti-IFN-a or y antibodies, justifying its designation as a (3-type IFN. The genes encoding IFN-X32 are not on chromosome 9 (U. Nir (1984), supra) and differ from the type I IFN genes in the chromosome 9 cluster by the presence of introns. The IFA-2 gene was determined to be on chromosome 7 ( P . B . Sehgal et al . ( 1986 ) Proc . Natl . Acad .
Sci. USA, published July 1986). The amino acid sequence homology detected is compatible with the notion that the IFN-(32 genes are related to the ancestral gene which gave rise to the type I IFN gene cluster.
The second genomic DNA clone IFA-11 hybridizing with the IFN-a2 cDNA has a restriction map which differs from that of IFA-2 (Figure 8). Transcript mapping and sequencing showed that the 3'exon of IFA-2 and IFA-11 (Xbal-HindIII segment) are identical (99% sequence homology), while there was no cross-hybridization under stringent conditions between more 5' exons. The IFA-11 genomic clone (19 Kb) was transfected into L-TK- cells together with a HSV-TK gene (F. Colbere-Garapin et al. (1981) J. Mol.
Biol. 150: 1-14) and stable transformants were isolated. Table 1 shows that L cell clone LI-39 containing the human IFA-11 gene, produces human IFN activity when induced by poly (rI) (rC) and cycloheximide, while L-TK+ cells produced no such activity.
A CHO clone 1C40 containing the IFA-11 gene also produced human IFN activity (Table 1). No activity was seen in non-induced ' 3~~.252 12a LI-39 or 1C40 cells. The IFN activity obtained with IFA-11 transformants after induction was significantly higher than with the IFA-2 gene. The rIFN-(32B produced by expression of the IFA-1 1 gene was neutralized with anti-IFN-(3, similarly to the IFA-2 gene product. IFA-11 specific DNA probes hybridized to RNA from induced human fibroblasts similarly to IFN-2 DNA probes, indicating that both genes are active in human cells The function of the IFN-~2 genes seems to result from their expression under conditions in which other IFN genes are not expressed. We find that fibroblasts exposed to Tumor Necrosis Factor (TNF) or to lymphokine IL-1 produce the IFN-~3z protein. Others found that neutralization of IFN-~i by antibodies results in a stimulation of cell proliferation (M. Kohase et al. (1986) Cell, published 6 June 1986; D. Resnitzky et al. (1986) Cell, published 4 July 1986), suggesting that the IFN-Vii, acts to limits cell growth in response to growth factors.
Supporting this conclusion is the fact that RNA hybridizing to IFN-J3z cDNA
was recently detected in peripheral blood mononuclear cells induced by mitogens such as PHA
along with IFN-Y mRIVA. It has also been reported that an IL-1 like lymphokine strongly induces RNA
hybridizing to IFN-~i2 cDNA in diploid fibroblasts and other human cell lines (J. Content et al. (1980 Eur. J. Biochem. 152: 243-257). The promoter region of the IFA-2 genes seems to contain rivo TATA boxes and rivo RNA starts (Figures 1 and 3). This IFN-(32 promoter was shown to respond to either poly (rI)(rC) or to cycloheximide (Y. Chernajovsky et al. (1984), supra). In vivo, the two IFN-(3Z gene promoters may be activated by different inducers (ds RNA, protein synthesis inhibitors, IL-1, PHA). A variety of cells have been found to produce autocrine IFN-~i in low amounts during growth arrest and differentiation (M.
Revel (1983) Interferon 5 pp. 205-239, Acad. press, London). IFN-(32 may belong to the same group of minor IFN species produced by cells to auto-regulate their growth, while the classical type I IFNs are probably more specifically involved in the response to viral infections.
The biological significance of IFN-~3z lies most probably in the fact that it is induced under conditions where IFN-(3, is not induced, as in metabolically stressed cells (A. Zilberstein et al., (1985) in The Interferon System Serono Symposia Raven Press, pp. 73-83).
Most important is the observation that TNF induces IFN-~iZ in fibroblasts and that the proliferation of these cells which is stimulated by TNF is further enhanced if anti-IFN-~
antibodies are added. A number of l4 1 3 41 252 human cell lines have been shown to produce autocrine IFN-(3 species when undergoing differentiation, and these IFNs are responsible for the induction of HLA antigens on these cells (A. Yarden et al.(1984) Embo .J.3:969-973). In a mouse myeloleukemic cell line induced to differentiate by CSF-1, the growth arrest of the cells which charact-erizes terminal differentiation, is abolished by anti-IFN-(3 antibodies.
IL-1 stimulates growth of several cell types and the classical growth factor PDGF was found to induce IFN-(3 RNA in mouse cells (J. N. Zullo et al. (1985) Cell 43: 793-800). Although multiple IFN-(3 species may be involved (P. B. Sehgal, (1982) in Interferon 4, Academic Press, pp.l-22, and M. Revel (1983) in Interferon 5, Academic Press, pp.205-239), IFN-[32 appears to be one of these autocrine autoregulators of cell growth produced in response to growth factors. This would be in line with the low specific activity of IFN-(3z (estimated by comparison of immunoassay and antiviral activity to be 50-100 times lower than for IFN-(31), since only very small antiviral activity is seen in cells producing the autocrine IFN-(3 species (M. Revel (1983) in Interferon 5, Academic Press, pp.205-239). This could also explain why the hamster cells expressing IFN-(3z cDNA produce less IFN activity than similar CHO
cells harbouring the human IFN-(31 gene (Y. Chernajovsky et al. (1984), DNA 3: 297-308), interestingly, the IFN-~ 1 (D) species has also a 100 times lower specific activity on human cells than other IFN-species (T. Goren et al. (1983), Viroloay 130: 273-280), although it represents a large proportion of the total mass of leukocyte IFN
(C. Weissmann (1981) in Interferon 3, Academic Press, N.Y. pp.101-134).
It is not excluded that some of the multiple IFN functions will be expressed more efficiently by IFN-(32 than the antiviral effect.
Despite their low concentration, growth-regulatory IFN-(3 induce HLA
and (2'-5') oliga A synthetase more strongly than expected, have more prolonged effects and markedly inhibit the growth of the cells which produce them. The induction of IFN-az by IL-1 and TNF suggests that it may play a role as a autocrine mediator of some effects of these cytokines in inflammation and acute-phase responses, as well as regulating cell proliferation.
,:
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Induction of mRNA which hybridizes with IFN-~i~ cDNA probes but not to IFN-Vii, cDNA has been observed in human cell lines after exposure to Interleukin-1 (IL-1) (J.
Content et al.
(1985), Eur. J. Biochem. 152: 253-257) and Tumor Necrosis Factor (TNF-a). The IFN-~32-specific immunocompetition assay with R-antibodies was used to examine if human fibroblasts FS 11 cells indeed secrete the IFN-biz protein in response to these two cytokines (Figure 9). Both rIL-1 a and TNF-a induced the synthesis and secretion of the IFN-(3z protein, to levels estimated by the immunoassay to be 10-20 U/ml IFN-(3Z. Optimal IFN-X32 induction was seen with concentration of rIL-la of 4 U/ml (0.13 ng/ml), similar to those required for IL-1 induction ofprostaglandin E,, collagenase and hyaluronic acid in human fibroblasts (J.H.
Korn ( 1985), Arthritis Rheum. 28: 315-322). The optimal concentration of TNF-a for IFN-biz induction (400 U/ml; 40 ng/ml) was higher than that required for the cytolytic effect, on sensitive cells (0.1 ng/ml; A.M. Wang et al. (1985) Science 228: 149-154) and even higher than that giving optimal growth stimulation of diploid fibroblasts (2 ng/ml).
The onset of IFN-~i2 secretion was somewhat faster with TNF-a reaching half maximal levels at about 6 hours, versus 8-12 hours with IL-1.
It will be obvious to those skilled in the art that various other changes and modifications may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown in the drawings and described in the specification.

Antiviral Activity of rIFN-P52 Produced in Trangfected Marrrcnalian Cells 6 _ I. IFA-2 GENE

A. Constitutive expression in CHO cells 10 pSVIFA2-I S1-15 cells . 10-50 U/ml#"

(control CHO cells . C ) H. Transient expression in Cos7 cells pSVIFA2-II DNA . 15 U/ml 15 (control IFN-F~1 pSVEIF DNA . 500 Ultnl) II. IFA-11 GENE

A, Induced eYpression"~''~in CH0 cells Clone 1C~IU . X100-500 Ulml (control CH0 cells . 100 Ulcnl) B. Induced expression L cells in Clone LI-39 . 250 U/ml (control L.-TK+ . < 4 Ulml) ce119 ~5 ._ ~ Assay with VSV in FS11 and WISH cells.
Neutralised by anti-IFN-131.
3000 U/ml after concentration.
Induction with poly IC and cyclohexirnide for 10 hrs.
Harvest at 28 hrs.

1341252_ m Antiviral Activity of Human rIFN-[3zA Produced in Hamster Cells Transfected and Amplified with IFN-(3zA cDNA
CHO-SVCIFB2 B132-lOM CLONE Human IFN activity*
U/ml B-131 (0 MTX)** 64 B-131-5M (250 nM MTX)** 300 B-131-lOM (500 nM MTX)** 800 * Medium from cultures was collected every 24 hours and assayed with VSV in FS11 human cells. The activity was neutralized by anti-IFN-(31, but not anti-IFN-~ or anti-IFN-Y.
** Cultures were selected for resistance to indicated concentrations of methotrextate.
i,.

Claims (87)

1. A process for producing recombinant human interferon-.beta.2A corresponding to an amino acid sequence substantially as shown in Figure 1, which comprises:
(a) preparing a recombinant vector comprising a DNA sequence which codes for human interferon-.beta.2A substantially as shown in Figure 1, and further comprising regulatory regions which are positioned in a way that expression of the human interferon-.beta.2A is possible;
(b) transforming cells with said recombinant vector; and (c) culturing the transformed cells, thereby producing the desired human interferon-.beta.2A.

2. A process according to claim 1 wherein the DNA sequence coding for human interferon-.beta.2A is a cDNA sequence.

3. A process according to claim 2 wherein the cDNA sequence is the sequence depicted in Figure 1.

4. A process according to claim 1 wherein the DNA sequence coding for human interferon-.beta.2A is a genomic DNA sequence.

5. A process according to any one of claim 1, 2, 3, or 4, wherein the regulatory DNA
sequences contain a promoter.

6. A process according to claim 5, wherein the promoter is an SV40 early promoter.

7. A process according to any one of claim 1, 2, 3, 4 or 6, wherein the vector is pSVIFA2-I.

8. A process according to claim 5, wherein the vector is pSVIFA2-I.

9. A process according to any one of claim 1, 2, 3, 4 or 6, wherein the vector is pSVIFA2-II.

10. A process according to claim 5, wherein the vector is pSVIFA2-II.

11. A process according to any one of claim 1, 2, 3, or 4, wherein prokaryotic cells are transformed by the expression vector and are cultured to produce recombinant human interferon-.beta.2A.

12. A process according to claim 5, wherein prokaryotic cells are transformed by the expression vector and are cultured to produce recombinant human interferon-.beta.2A.

13. A process according to any one of claim 1, 2, 3, 4, 6, 8 or 10, wherein eukaryotic cells are transformed by the expression vector and are cultured to produce recombinant human interferon-.beta.2A.

14. A process according to claim 5, wherein eukaryotic cells are transformed by the expression vector and are cultured to produce recombinant human interferon-.beta.2A.

15. A process according to claim 7, wherein eukaryotic cells are transformed by the expression vector and are cultured to produce recombinant human interferon-.beta.2A.

16. A process according to claim 9, wherein eukaryotic cells are transformed by the expression vector and are cultured to produce recombinant human interferon-.beta.2A.

17. A process according to claim 13, wherein eukaryotic cells are mammalian cells.

18. A process according to claim 14, wherein eukaryotic cells are mammalian cells.

19. A process according to claim 15, wherein eukaryotic cells are mammalian cells.

20. A process according to claim 16, wherein eukaryotic cells are mammalian cells.

21. A process according to any one of claims 17 or 18, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

22. A process according to any one of claim 19 or 20, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

23. A recombinant human interferon-.beta.2A whenever produced by a process according to any one of claim 1, 2, 3, 4, 6, 8, 10, 12, 14, 15, 16, 17, 18, 19 or 20.

24. A recombinant human interferon-.beta.2A whenever produced by a process according to claim 5.

25. A recombinant human interferon-.beta.2A whenever produced by a process according to claim 7.

26. A recombinant human interferon-.beta.2A whenever produced by a process according to claim 9.

27. A recombinant human interferon-.beta.2A whenever produced by a process according to claim 11.

28. A recombinant human interferon-.beta.2A whenever produced by a process according to claim 13.

29. A recombinant human interferon-.beta.2A whenever produced by a process according to claim 22.

30. A process for producing recombinant human interferon-.beta.2B, said interferon-.beta.2B
being encoded by a genomic DNA fragment having a restriction pattern substantially as shown in Figure 8 for the gene contained in the genomic clone having the designation IFA-11, which comprises:
(a) preparing a recombinant vector comprising a DNA sequence which codes for said human interferon-.beta.2B substantially as shown in Figure 8, and further comprising regulatory regions which are positioned in a way that expression of the human interferon-.beta.2B is possible;
(b) transforming cells with said recombinant vector; and (c) culturing the transformed cell, thereby producing the desired human interferon-.beta.2B.

31. A process according to claim 30, wherein the DNA sequence is a genomic DNA
sequence.

32. A recombinant human interferon-.beta.2B whenever produced by a process according to any one of claims 30 or 31.

33. A DNA consisting essentially of a nucleotide sequence encoding the 212 amino acid sequence shown in Figure 1.

34. A DNA consisting essentially of cDNA which codes for human interferon-.beta.2A
corresponding to an amino acid sequence or nucleotide sequence substantially as shown in Figure 1.

35. A DNA consisting essentially of a nucleotide sequence encoding the amino acid sequence of a biologically active interferon-.beta.2A shown in Figure 1, wherein said amino acid sequence has the N-terminus of mature interferon-.beta.2A.

36. A DNA according to claim 35 which is a cDNA.

37. A DNA capable of hybridizing to a DNA according to claim 34 and which is capable of expressing human interferon-.beta.2A.

38. A DNA capable of hybridizing to a DNA according to claim 35 and which is capable of expressing human interferon-.beta.2A.

39. A recombinant vector comprising a DNA sequence according to claim 33, 34, 35, 36, 37 or 38, further comprising regulatory regions which are positioned in such a way that expression of the human interferon-.beta.2A is possible.

40. A recombinant vector comprising a genomic DNA sequence according to claim 33, 34, 35, 36, 37 or 38, further comprising regulatory regions which are positioned in such a way that expression of the human interferon-.beta.2A is possible.

41. A recombinant vector according to claim 39, wherein said DNA sequence is a cDNA
sequence.

42. A recombinant vector according to claim 41, wherein the cDNA sequence is the sequence as depicted in Figure 1.

43. A recombinant vector comprising a DNA sequence substantially as shown in Figure 8 which codes for a human interferon-.beta.2B, said interferon-.beta.2B being encoded by a genomic DNA fragment having a restriction pattern substantially as shown in Figure 8 for the gene contained in the genomic clone having the designation IFA-11, and further comprises regulatory regions which are positioned in a way that expression of the human interferon-.beta.2B is possible.

44. A recombinant vector according to claim 43, wherein said DNA sequence is a genomic DNA sequence.

45. A recombinant vector according to claim 43, wherein said DNA sequence is a cDNA sequence.

46. A recombinant vector according to any one of claims 41, 42, 43, 44 or 45, wherein the regulatory DNA sequences contain a promoter.

47. A recombinant vector according to claim 46, wherein the promoter is an SV40 early promoter.

48. A recombinant vector according to any one of claim 41 or 42, wherein the vector is pSVIFA2-I comprising the 2kb XhoI-BamHI fragment and the 3kb BamHi-HindIII
fragment of the gene encoding interferon-.beta.2A corresponding to an amino acid sequence substantially as shown in Figure 1 being inserted into plasmid pSVE3.

49. A recombinant vector according to any one of claim 41 or 42, wherein the vector is pSVIFA2-II which comprises the 2kb XhoI-BamHI fragment and the 3kb BamHi-HindIII
fragment of the gene encoding interferon-2A corresponding to an amino acid sequence substantially as shown in Figure 1 being inserted into plasmid pSVE3 and a 26 bp synthetic ClaI-XhoI oligonucleotide restoring the 5' cDNA sequence.

50. A recombinant vector according to claim 42, wherein the vector is pSVCIF.beta.2 being shown in Figure 5.

51. A cell line transformed by a recombinant vector according to any one of claim 41, 42, 43, 44, 45, 47 or 49.

52. A cell line transformed by a recombinant vector according to claim 46.

53. A cell line transformed by a recombinant vector according to claim 48.

54. A cell line transformed by a recombinant vector according to claim 49.

55. A cell line according to claim 51, wherein said cell line is eukaryotic.

56. A cell line according to claim 52, wherein said cell line is eukaryotic.

57. A cell line according to claim 53, wherein said cell line is eukaryotic.

58. A cell line according to claim 54, wherein said cell line is eukaryotic.

59. A cell line according to claim 55, 56, 57 or 58, wherein said cell line is a hamster cell line.

60. A cell line according to claim 55, 56, 57 or 58, wherein said cell line is a CHO cell line.

61. A cell line according to claim 55, 56, 57 or 58, wherein said cell line is an L-TK cell line.

62. A cell line according to any one of claim 52, 53, 54, 55, 56, 57 or 58, which is capable of producing one of human interferon-.beta.2A and human interferon-.beta.2B.

63. A cell line according to claim 51, which is capable of producing one of human interferon-.beta.2A and human interferon-.beta.2B.

64. A cell line according to claim 59, which is capable of producing one of human interferon-.beta.2A and human interferon-.beta.2B.

65. A cell line according to claim 60, which is capable of producing one of human interferon-.beta.2A and human interferon-.beta.2B.

66. A cell line according to claim 61, which is capable of producing one of human interferon-.beta.2A and human interferon-.beta.2B.

67. A cell line according to claim 55, 56, 57 or 58, wherein said cell line is a CHO cell line and wherein the recombinant vector used for transformation is pSVCIF.beta.2 and wherein the cell line is cotransfected with pSVDHFR and has been subjected to methotrexate-induced amplification.

68. A host cell transformed by a recombinant vector according to claim 39 which is capable of producing a biologically active human interferon-.beta.2A.

69. A host cell according to claim 68, wherein the host cell is a prokaryotic host cell.

70. A process for producing a biologically active human interferon-.beta.2A, comprising the steps of cultivating the host cell according to claim 68 or 69 and recovering the produced human interferon-.beta.2A.

71. A pharmaceutical composition containing human interferon-.beta.2B whenever produced by a process according to any one of claim 30 or 31 and a pharmaceutically acceptable carrier.

72. The use of a cell line transformed by a recombinant vector according to claim 41, 42 or 50 for producing human interferon-.beta.2A.

73. The use of a cell line transformed by a recombinant vector according to claim 41, 42 or 50 for producing human interferon-.beta.2A, the cell line being a hamster cell line.

74. The use of a cell line transformed by a recombinant vector according to claim 41, 42 or 50 for producing human interferon-.beta.2A, the cell line being a CHO cell line.

75. The use of a cell line transformed by a recombinant vector according to claim 41, 42 or 50 for producing human interferon-.beta.2A, the cell line being an L-TK cell line.

76. The use of a cell line transformed by a recombinant vector according to claim 41, 42 or 50 for producing human interferon-.beta.2A, the cell line being capable of producing human interferon-B2A.

77. A pharmaceutical composition containing human interferon-.beta.2B encoded by a genomic DNA fragment having a restriction pattern substantially as shown in Figure 8 for the gene contained in the genomic clone having the designation IFA-11 and a pharmaceutically acceptable carrier.

78. An unglycosylated polypeptide characterized by the amino acid sequence Pro-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-Pro-His-Arg-Gln-Pro-Leu-Thr-Ser-Ser-Glu-Arg-Ile-Asp-Lys-Gln-Ile-Arg-Tyr-Ile-Leu-Asp-Gly-Ile-Ser-Ala-Leu-Arg-Lys-Glu-Thr-Cys-Asn-Lys-Ser-Asn-Met-Cys-Glu-Ser-Ser-Lys-Glu-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Asn-Leu-Pro-Lys-Met-Ala-Glu-Lys-Asp-Gly-Cys-Phe-Gln-Ser-Gly-Phe-Asn-Glu-Glu-Thr-Cys-Leu-Val-Lys-Ile-Ile-Thr-Gly-Leu-Leu-Glu-Phe-Glu-Val-Tyr-Leu-Glu-Tyr-Leu-Gln-Asn-Arg-Phe-Glu-Ser-Ser-Glu-Glu-Gln-Ala-Arg-Ala-Val-Gln-Met-Ser-Thr-Lys-Val-Leu-Ile-Gln-Phe-Leu-Gln-Lys-Lys-Ala-Lys-Asn-Leu-Asp-Ala-Ile-Thr-Thr-Pro-Asp-Pro-Thr-Thr-Asn-Ala-Ser-Leu-Leu-Thr-Lys-Leu-Gln-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Asp-Met-Thr-Thr-His-Leu-Ile-Leu-Arg-Ser-Phe-Lys-Glu-Phe-Leu-Gln-Ser-Ser-Leu-Arg-Ala-Leu-Arg-Gln-Met.

79. A nucleotide sequence which encodes a polypeptide characterized by the amino acid sequence Pro-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-Pro-His-Arg-Gln-Pro-Leu-Thr-Ser-Ser-Glu-Arg-Ile-Asp-Lys-Gln-Ile-Arg-Tyr-Ile-Leu-Asp-Gly-Ile-Ser-Ala-Leu-Arg-Lys-Glu-Thr-Cys-Asn-Lys-Ser-Asn-Met-Cys-Glu-Ser-Ser-Lys-Glu-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Asn-Leu-Pro-Lys-Met-Ala-Glu-Lys-Asp-Gly-Cys-Phe-Gln-Ser-Gly-Phe-Asn-Glu-Glu-Thr-Cys-Leu-Val-Lys-Ile-Ile-Thr-Gly-Leu-Leu-Glu-Phe-Glu-Val-Tyr-Leu-Glu-Tyr-Leu-Gln-Asn-Arg-Phe-Glu-Ser-Ser-Glu-Glu-Gln-Ala-Arg-Ala-Val-Gln-Met-Ser-Thr-Lys-Val-Leu-Ile-Gln-Phe-Leu-Gln-Lys-Lys-Ala-Lys-Asn-Leu-Asp-Ala-Ile-Thr-Thr-Pro-Asp-Pro-Thr-Thr-Asn-Ala-Ser-Leu-Leu-Thr-Lys-Leu-Gln-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Asp-Met-Thr-Thr-His-Leu-Ile-Leu-Arg-Ser-Phe-Lys-Glu-Phe-Leu-Gln-Ser-Ser-Leu-Arg-Ala-Leu-Arg-Gln-Met.

80. A recombinant vector comprising bacterial cell expression control sequences operably asociated with a nucleotide sequence which encodes a polypeptide characterized by the amino acid sequence Pro-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-Pro-His-Arg-Gln-Pro-Leu-Thr-Ser-Ser-Glu-Arg-Ile-Asp-Lys-Gln-Ile-Arg-Tyr-Ile-Leu-Asp-Gly-Ile-Ser-Ala-Leu-Arg-Lys-Glu-Thr-Cys-Asn-Lys-Ser-Asn-Met-Cys-Glu-Ser-Ser-Lys-Glu-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Asn-Leu-Pro-Lys-Met-Ala-Glu-Lys-Asp-Gly-Cys-Phe-Gln-Ser-Gly-Phe-Asn-Glu-Glu-Thr-Cys-Leu-Val-Lys-Ile-Ile-Thr-Gly-Leu-Leu-Glu-Phe-Glu-Val-Tyr-Leu-Glu-Tyr-Leu-Gln-Asn-Arg-Phe-Glu-Ser-Ser-Glu-Glu-Gln-Ala-Arg-Ala-Val-Gln-Met-Ser-Thr-Lys-Val-Leu-Ile-Gln-Phe-Leu-Gln-Lys-Lys-Ala-Lys-Asn-Leu-Asp-Ala-Ile-Thr-Thr-Pro-Asp-Pro-Thr-Thr-Asn-Ala-Ser-Leu-Leu-Thr-Lys-Leu-Gln-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Asp-Met-Thr-Thr-His-Leu-Ile-Leu-Arg-Ser-Phe-Lys-Glu-Phe-Leu-Gln-Ser-Ser-Leu-Arg-Ala-Leu-Arg-Gln-Met.

81. A recombinant vector comprising mammalian cell expression control sequences operably associated with a nucleotide sequence which encodes a polypeptide characterized by the amino acid sequence Pro-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-Pro-His-Arg-Gln-Pro-Leu-Thr-Ser-Ser-Glu-Arg-Ile-Asp-Lys-Gln-Ile-Arg-Tyr-Ile-Leu-Asp-Gly-Ile-Ser-Ala-Leu-Arg-Lys-Glu-Thr-Cys-Asn-Lys-Ser-Asn-Met-Cys-Glu-Ser-Ser-Lys-Glu-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Asn-Leu-Pro-Lys-Met-Ala-Glu-Lys-Asp-Gly-Cys-Phe-Gln-Ser-Gly-Phe-Asn-Glu-Glu-Thr-Cys-Leu-Val-Lys-Ile-Ile-Thr-Gly-Leu-Leu-Glu-Phe-Glu-Val-Tyr-Leu-Glu-Tyr-Leu-Gln-Asn-Arg-Phe-Glu-Ser-Ser-Glu-Glu-Gln-Ala-Arg-Ala-Val-Gln-Met-Ser-Thr-Lys-Val-Leu-Ile-Gln-Phe-Leu-Gln-Lys-Lys-Ala-Lys-Asn-Leu-Asp-Ala-Ile-Thr-Thr-Pro-Asp-Pro-Thr-Thr-Asn-Ala-Ser-Leu-Leu-Thr-Lys-Leu-Gln-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Asp-Met-Thr-Thr-His-Leu-Ile-Leu-Arg-Ser-Phe-Lys-Glu-Phe-Leu-Gln-Ser-Ser-Leu-Arg-Ala-Leu-Arg-Gln-Met.

82. A bacterial cell transformed with a recombinant vector comprising bacterial cell expression control sequences operably associated with a nucleotide sequence which encodes a polypeptide characterized by the amino acid sequence Pro-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-Pro-His-Arg-Gln-Pro-Leu-Thr-Ser-Ser-Glu-Arg-Ile-Asp-Lys-Gln-Ile-Arg-Tyr-Ile-Leu-Asp-Gly-Ile-Ser-Ala-Leu-Arg-Lys-Glu-Thr-Cys-Asn-Lys-Ser-Asn-Met-Cys-Glu-Ser-Ser-Lys-Glu-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Asn-Leu-Pro-Lys-Met-Ala-Glu-Lys-Asp-Gly-Cys-Phe-Gln-Ser-Gly-Phe-Asn-Glu-Glu-Thr-Cys-Leu-Val-Lys-Ile-Ile-Thr-Gly-Leu-Leu-Glu-Phe-Glu-Val-Tyr-Leu-Glu-Tyr-Leu-Gln-Asn-Arg-Phe-Glu-Ser-Ser-Glu-Glu-Gln-Ala-Arg-Ala-Val-Gln-Met-Ser-Thr-Lys-Val-Leu-Ile-Gln-Phe-Leu-Gln-Lys-Lys-Ala-Lys-Asn-Leu-Asp-Ala-Ile-Thr-Thr-Pro-Asp-Pro-Thr-Thr-Asn-Ala-Ser-Leu-Leu-Thr-Lys-Leu-Gln-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Asp-Met-Thr-Thr-His-Leu-Ile-Leu-Arg-Ser-Phe-Lys-Glu-Phe-Leu-Gln-Ser-Ser-Leu-Arg-Ala-Leu-Arg-Gln-Met.

83. A mammalian cell transformed with a recombinant vector comprising mammalian cell expression control sequences operably associated with a nucleotide sequence which encodes a polypeptide characterized by the amino acid sequence Pro-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-Pro-His-Arg-Gln-Pro-Leu-Thr-Ser-Ser-Glu-Arg-Ile-Asp-Lys-Gln-Ile-Arg-Tyr-Ile-Leu-Asp-Gly-Ile-Ser-Ala-Leu-Arg-Lys-Glu-Thr-Cys-Asn-Lys-Ser-Asn-Met-Cys-Glu-Ser-Ser-Lys-Glu-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Asn-Leu-Pro-Lys-Met-Ala-Glu-Lys-Asp-Gly-Cys-Phe-Gln-Ser-Gly-Phe-Asn-Glu-Glu-Thr-Cys-Leu-Val-Lys-Ile-Ile-Thr-Gly-Leu-Leu-Glu-Phe-Glu-Val-Tyr-Leu-Glu-Tyr-Leu-Gln-Asn-Arg-Phe-Glu-Ser-Ser-Glu-Glu-Gln-Ala-Arg-Ala-Val-Gln-Met-Ser-Thr-Lys-Val-Leu-Ile-Gln-Phe-Leu-Gln-Lys-Lys-Ala-Lys-Asn-Leu-Asp-Ala-Ile-Thr-Thr-Pro-Asp-Pro-Thr-Thr-Asn-Ala-Ser-Leu-Leu-Thr-Lys-Leu-Gln-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Asp-Met-Thr-Thr-His-Leu-Ile-Leu-Arg-Ser-Phe-Lys-Glu-Phe-Leu-Gln-Ser-Ser-Leu-Arg-Ala-Leu-Arg-Gln-Met.

84. A process for producing an unglycosylated polypeptide characterized by the amino acid sequence Pro-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-Pro-His-Arg-Gln-Pro-Leu-Thr-Ser-Ser-Glu-Arg-Ile-Asp-Lys-Gln-Ile-Arg-Tyr-Ile-Leu-Asp-Gly-Ile-Ser-Ala-Leu-Arg-Lys-Glu-Thr-Cys-Asn-Lys-Ser-Asn-Met-Cys-Glu-Ser-Ser-Lys-Glu-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Asn-Leu-Pro-Lys-Met-Ala-Glu-Lys-Asp-Gly-Cys-Phe-Gln-Ser-Gly-Phe-Asn-Glu-Glu-Thr-Cys-Leu-Val-Lys-Ile-Ile-Thr-Gly-Leu-Leu-Glu-Phe-Glu-Val-Tyr-Leu-Glu-Tyr-Leu-Gln-Asn-Arg-Phe-Glu-Ser-Ser-Glu-Glu-Gln-Ala-Arg-Ala-Val-Gln-Met-Ser-Thr-Lys-Val-Leu-Ile-Gln-Phe-Leu-Gln-Lys-Lys-Ala-Lys-Asn-Leu-Asp-Ala-Ile-Thr-Thr-Pro-Asp-Pro-Thr-Thr-Asn-Ala-Ser-Leu-Leu-Thr-Lys-Leu-Gln-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Asp-Met-Thr-Thr-His-Leu-Ile-Leu-Arg-Ser-Phe-Lys-Glu-Phe-Leu-Gln-Ser-Ser-Leu-Arg-Ala-Leu-Arg-Gln-Met which process comprises (a) preparing a recombinant vector comprising bacterial cell expression control sequences operably associated with a nucleotide sequence which encodes said polypeptide, (b) transforming bacterial cells with said recombinant vector to produce transformed cells, and (c) culturing said transformed cells such that said polypeptide is produced.

85. A process for producing a glycosylated polypeptide characterized by the amino acid sequence Pro-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-Pro-His-Arg-Gln-Pro-Leu-Thr-Ser-Ser-Glu-Arg-Ile-Asp-Lys-Gln-Ile-Arg-Tyr-Ile-Leu-Asp-Gly-Ile-Ser-Ala-Leu-Arg-Lys-Glu-Thr-Cys-Asn-Lys-Ser-Asn-Met-Cys-Glu-Ser-Ser-Lys-Glu-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Asn-Leu-Pro-Lys-Met-Ala-Glu-Lys-Asp-Gly-Cys-Phe-Gln-Ser-Gly-Phe-Asn-Glu-Glu-Thr-Cys-Leu-Val-Lys-Ile-Ile-Thr-Gly-Leu-Leu-Glu-Phe-Glu-Val-Tyr-Leu-Glu-Tyr-Leu-Gln-Asn-Arg-Phe-Glu-Ser-Ser-Glu-Glu-Gln-Ala-Arg-Ala-Val-Gln-Met-Ser-Thr-Lys-Val-Leu-Ile-Gln-Phe-Leu-Gln-Lys-Lys-Ala-Lys-Asn-Leu-Asp-Ala-Ile-Thr-Thr-Pro-Asp-Pro-Thr-Thr-Asn-Ala-Ser-Leu-Leu-Thr-Lys-Leu-Gln-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Asp-Met-Thr-Thr-His-Leu-Ile-Leu-Arg-Ser-Phe-Lys-Glu-Phe-Leu-Gln-Ser-Ser-Leu-Arg-Ala-Leu-Arg-Gln-Met which process comprises (a) preparing a recombinant vector comprising mammalian cell expression control sequences operably associated with a nucleotide sequence which encodes said polypeptide, (b) transforming mammalian cells with said recombinant vector to produce transformed cells, and (c) culturing said transformed cells such that said polypeptide is produced.

86. Use of a transformed bacterial cell for producing a polypeptide characterized by the amino acid sequence Pro-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-Pro-His-Arg-Gln-Pro-Leu-Thr-Ser-Ser-Glu-Arg-Ile-Asp-Lys-Gln-Ile-Arg-Tyr-Ile-Leu-Asp-Gly-Ile-Ser-Ala-Leu-Arg-Lys-Glu-Thr-Cys-Asn-Lys-Ser-Asn-Met-Cys-Glu-Ser-Ser-Lys-Glu-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Asn-Leu-Pro-Lys-Met-Ala-Glu-Lys-Asp-Gly-Cys-Phe-Gln-Ser-Gly-Phe-Asn-Glu-Glu-Thr-Cys-Leu-Val-Lys-Ile-Ile-Thr-Gly-Leu-Leu-Glu-Phe-Glu-Val-Tyr-Leu-Glu-Tyr-Leu-Gln-Asn-Arg-Phe-Glu-Ser-Ser-Glu-Glu-Gln-Ala-Arg-Ala-Val-Gln-Met-Ser-Thr-Lys-Val-Leu-Ile-Gln-Phe-Leu-Gln-Lys-Lys-Ala-Lys-Asn-Leu-Asp-Ala-Ile-Thr-Thr-Pro-Asp-Pro-Thr-Thr-Asn-Ala-Ser-Leu-Leu-Thr-Lys-Leu-Gln-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Asp-Met-Thr-Thr-His-Leu-Ile-Leu-Arg-Ser-Phe-Lys-Glu-Phe-Leu-Gln-Ser-Ser-Leu-Arg-Ala-Leu-Arg-Gln-Met.

87. Use of a transformed mammalian cell for producing a polypeptide characterized by the amino acid sequence Pro-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-Pro-His-Arg-Gln-Pro-Leu-Thr-Ser-Ser-Glu-Arg-Ile-Asp-Lys-Gln-Ile-Arg-Tyr-Ile-Leu-Asp-Gly-Ile-Ser-Ala-Leu-Arg-Lys-Glu-Thr-Cys-Asn-Lys-Ser-Asn-Met-Cys-Glu-Ser-Ser-Lys-Glu-Ala-Leu-Ala-Glu-Asn-Asn-Leu-Asn-Leu-Pro-Lys-Met-Ala-Glu-Lys-Asp-Gly-Cys-Phe-Gln-Ser-Gly-Phe-Asn-Glu-Glu-Thr-Cys-Leu-Val-Lys-Ile-Ile-Thr-Gly-Leu-Leu-Glu-Phe-Glu-Val-Tyr-Leu-Glu-Tyr-Leu-Gln-Asn-Arg-Phe-Glu-Ser-Ser-Glu-Glu-Gln-Ala-Arg-Ala-Val-Gln-Met-Ser-Thr-Lys-Val-Leu-Ile-Gln-Phe-Leu-Gln-Lys-Lys-Ala-Lys-Asn-Leu-Asp-Ala-Ile-Thr-Thr-Pro-Asp-Pro-Thr-Thr-Asn-Ala-Ser-Leu-Leu-Thr-Lys-Leu-Gln-Ala-Gln-Asn-Gln-Trp-Leu-Gln-Asp-Met-Thr-Thr-His-Leu-Ile-Leu-Arg-Ser-Phe-Lys-Glu-Phe-Leu-Gln-Ser-Ser-Leu-Arg-Ala-Leu-Arg-Gln-Met.