JP2648860B2 - Human interferon-β2A and human interferon-β2B, vector containing the gene encoding the interferon, cell line producing the interferon, and use of the interferon as a pharmaceutical - Google Patents

Description

BACKGROUND OF THE INVENTION <FIELD OF THE INVENTION> The present invention is human Interferon-beta _2A and human Interferon-beta _2B, vectors containing genes which code them as cell lines and their pharmaceutical produced Related to uses.

<Background Art> Interferon is an important protein that has antiviral activity and antitumor activity and is produced by the human body. Because of its species specificity, clinical use requires human interferon. Interferons are classified into three types based on their reaction with antibodies. That is,
Interferon mainly produced by leukocytes
α, interferon-β produced mainly by fibroblasts, and interferon-γ produced mainly by T-lymphocytes.

Interferon produced by human fibroblasts in response to double-stranded (ds) RNA is primarily a 20Kd glycoprotein IF
An N-beta _1, which is Yotsute code to RNA of 0.9 kb, the RNA are those obtained from the genes of chromosomes 9 excluding the intron. However, when introduced into frog oocytes by microinjection, IFN
Another mRNA that produces activity is poly (γI) (γ
C) has been observed in human fibroblasts that have undergone IFN induction by cycloheximide (CHX) -actinomycin D treatment. Further, when the cloning of complementary (c) DNA of IFN-beta _1, was induced both 1.3kb
A cDNA clone corresponding to the other alternative RNA has been isolated, which is one of the reticulocyte lysates 23-2
It encodes a 6Kd polypeptide and has been tested to produce human interferon antiviral activity in oocytes [J. Weissenbach et al., (198
0) Proc. Nati. Acad. Sci. USA, 77, 7152-7156; UK Patent No. 2.063,882].

This activity, since it is by connexion blocking anti IFN-beta ₁ antibody, the polypeptide is referred to as IFN-β _2. I
The protein and IFN-beta ₁ obtained from RNA corresponding to the FN-beta _2, the cross-reaction with the same antibody, but their biological activity is prevented, both protein immunological distinction Is what is done. IFN-β ₂ RNA is formed by a mouse-human hybrid excluding chromosome 9,
1.3kb of RNA-beta ₂ is clearly derived from other genes other than IFN-beta ₁ gene [U.Nir, (1984) Ph.D.The
sis, Weizmann Institute of Science, Rehovot, Israe
l].

West German Patent Application No. P3,043,981 describes a method for producing interferon-β _{2B in} pure form. According to this production method, isolation of DNA containing interferons cultured human cells capable of producing, then the nucleotide sequence of human cells that encode interferons-beta _2B when stimulated by Interferon inducing agent Yotsute the production methods comprising, are getting Interferon-beta _2. In this production method, mRNA is isolated and purified from the induced cells, and then DNA is obtained by reverse transcription of the mRNA, and the DNA is cloned in an appropriate vector.

<Disclosure of the Invention> When the gene obtained as described above is isolated and transfected into hamster or mouse cells,
causing a human-specific IFN activity after dsRNA and cycloheximide stimulation, the presence of another alternative of the gene encoding IFN-beta ₂ was demonstrated. IFN produced here
Is that having the characteristics found to belong to the IFN-beta _2, however, does not have the same amino acid sequence and IFN-beta _2, which were designated as IFN-beta _2A and IFN-beta _2B .

Therefore, an object of the present invention is to provide a biologically active human IF
It _consists in isolating N-β _2A and human IFN-β _2B .

An object of the present invention is to produce biologically active human IFN-β _2A and IFN-β _2B .

CDNA is still another object of the present invention, cDNA encoding the human IFN-beta _2, it is more encoding human IFN-beta _2A
And it is to produce a cDNA encoding the human IFN-beta _2B.

Still another object of the present invention is to identify genes cDNA hybridizing IFN-beta _2.

Yet another object of the present invention is to provide a cDNA or IFN- _β2A cDNA.
It is to identify genes hybridizing to the cDNA of FN-beta _2B.

Still another object of the present invention, gate fleas poke clones containing IFN-beta ₂ gene, further isolating the gate fleas poke clone containing the gain fleas poke clones and IFN-beta _2B gene containing the IFN-beta _2A gene in detail It is in.

Still another object of the present invention is to provide IFN-β _2A and / or IFN-
beta _2B gene, and to provide a recombinant vector comprising a promoter sequence to control transcription of Interferon gene.

Yet another object of the present invention is to provide a biologically active human IFN
To provide cells producing-beta _2A and / or IFN-beta _2B.

Still another object of the present invention is to provide IFN-β _2A and / or IFN-
It is to provide cells which produce an exemplary amounts of beta _2B.

Still another object of the present invention is to provide a biologically active human IFN-β _2A and / or IFN-β
It is to provide a manufacturing method of _2B .

Yet another object of the present invention is to provide human IFN-β _2A and human IFN-β _2A having a substantial amount of biological activity when transfected into hamster or mouse cells by fusing to a strong transcription promoter of virus. And / or to provide a gene that produces IFN-β _2B .

Still another object of the present invention is to provide a cell growth or differentiation,
In particular, it has a desirable effect on the differentiation of terminal cancer cells.
It is in use of IFN-β _2A and / or IFN-β _{2B as} a pharmaceutical.

Yet another object of the present invention is to provide IFN-β _2A and / or IFN-β
_2B is for use in inhibiting fibroblast differentiation or preventing sclerosis after infection.

According to the present invention, the presence of biologically active interferon-β ₂ (IFN-β ₂ ) molecules has been identified, and these molecules are produced by recombinant DNA technology. Such interferons-beta ₂ are glycoproteins production by connexion Interferon in dsRNA or virus is secreted from induced human fibroblasts. IFN-beta ₂ corresponds to about 5% of the total IFN activity produced by fibroblasts. IFN
The amino acid sequence of-beta ₂ is determined by a method using a cDNA, the sequence of which other IFN produced by human cells
(IFN-β ₁ , IFN-α) is only about 20% homologous.

According to the present invention, a human cDNA of encoding IFN-beta _2A
Alternatively, the gene is fused to a strong transcription promoter of the virus. Hamster cells transformed with this fusion cDNA or gene produce human-specific interferon activity, which blocks viral replication and cytopathic effects, and reduces the biological potential of human cells for interferon. Proteins specific to the specific response are induced. When isolated and transfected into hamster or mouse cells, IFN produces human-specific IFN activity after induction with dsDNA and cycloheximide.
Another alternative genes encoding-beta ₂ were found. IFN produced by this gene has the characteristics found to belong to the IFN-beta _2, the amino acid sequence are not the same, they were designated as IFN-beta _2A and IFN-beta _2B.

Containing <preferable detailed description of embodiments of the invention> nucleotide sequence encoding the interferons, the gene for human fibroblasts when they are isolated, the presence of interferons-beta ₂ was confirmed. At this time, an appropriate exogenous factor is allowed to act on fibroblasts to induce the production of interferon mRNA.
mRNA was extracted. At the same time, mRNA was also extracted from fibroblasts after derivatization. CDNA probes were bound from mRNAs extracted from derivatized and untreated cells, respectively, using these as templates. A double-stranded cDNA was synthesized from mRNA obtained from the cells subjected to the derivatization treatment, then inserted into an appropriate vector, and transfected into a microorganism. Next, the transformant was cultured under conditions in which the transformant having the vector selectively proliferated, to obtain a first-generation colony. Next, a second generation colony was further formed from this colony, and DNA was extracted from both colonies. D from the second generation colony
NA was hybridized with a cDNA probe synthesized from mRNA obtained from the cells subjected to the derivatization treatment described above. On the other hand, DNA obtained from the colonies of the second generation was obtained by synthesizing cD
It was also hybridized to the NA probe. The cDNA probe obtained from the cells subjected to the derivatization treatment hybridized, but the cDNA probe obtained from the cells not subjected to the derivatization treatment selected a colony having a DNA that did not hybridize, and DNA was extracted from the colony. . This DNA was further examined to select for DNA that hybridizes to mRNA translated into interferon in lysates of frog oocytes or reticulocytes. Such DNA is essentially a DNA encoding interferon or a DNA containing a sequence sufficient to obtain a DNA encoding interferon.

During such studies, it was determined that two other alternative mRNAs encoding interferon were isolated from derivatized fibroblasts. By centrifuging these mRNAs using glycerol, a 11S fraction was obtained.
A small amount of mRNA precipitated. This was translated in a cell to obtain a protein. Such a protein is a protein having a molecular weight of 20,000 which precipitates in response to an antibody against one of the interferons purified from visiting fibroblasts. This protein is human Interferon-beta _1, is part of its amino acid sequence, Knight et al [(1980) Science, 207,525-52
6]. A large amount of mRNA precipitated in the 14S fraction, and a protein having a molecular weight of 23,000 was obtained from this mRNA. This protein was precipitated by reacting with an antibody against low-purity interferon obtained from fibroblasts. The protein was named human Interferon-beta _2. An mRNA high Zuburi soybean cDNA clones which are translated into Interferon-beta ₂ were prepared by the same method as described above,
It was prepared IFN-beta ₂ cDNA probes using the clone. Is a cDNA clone that mRNA with high Zuburi soybeans to be translated into Interferon -β _{2, IFN-β 2} clones A341 and E474 has a sequence in common, respectively, by fusing them, IFN-beta ₂ The AE20 cDNA was reconstructed (see FIG. 1). It was determined the IFN-beta ₂ cDNA nucleotide sequence. This revealed that it had an open reading frame of 212 amino acids expected to produce a 23,500 dalton protein. A transcription-translation test confirmed that this cDNA encodes the protein described above.

After partially digesting human adult blood cell DNA with EcoR I, λ Sharon 4
A [Y. Mory et al. (1981) Eur. J. Biochem. 120: 197-202].
From human gene library obtained by cloning in the middle, the gate fleas poke clone IFA-2 and IFA-11 hybridizing to the above-described IFN-beta ₂ DNA probes were isolated. Clone IFA-2 contains a gene with at least four exons that hybridizes to various segments of the cloned cDNA (FIG. 1). Since near the 5'-end of AE20cDNA has Xho ₁ site, gate fleas poke segment
Map conversion of A132 (Fig. 2) was performed. A132 is this Xh
o Has the first exon of IFA-2 ending 70 bp downstream from _one site. Using a DNA probe labeled with BstN1 site positions 40bp from Xho ₁ site, by performing the S1 nuclease analysis, human fibroblasts FS11 that have undergone induction treatment, by connexion distinction 20 pieces of nucleotide sequence Two having a 5'-terminal S-1 or S-2
It was found that it contained IFN-β ₂ RNA (FIG. 3).
These two starting points are located in the TATA upstream -30
It exists after the box (Fig. 4). RNA ending in S-2 was more abundant than other longer RNAs under various derivatization conditions (FIG. 3). Two RNAs with different 5'-ends were also observed for cytoplasmic RNA. Thus, it is clear that two RNAs are formed and show activity in human cells. Both of IFN-β ₂ RNA
Was induced in human fibroblasts by poly (γI) (γC). The induction in the case of IFN-β ₁ RNA [U.Nir et al,
(1984) Nucl. Acids. Res., 12: 6979-6993].
It is facilitated by adding FN itself. IFN-β ₂ RNA is also induced by prolonged treatment with CHX (6.5 h). If the processing time of CHX is short (3.5h),
Even poly (γI) (γC) alone is effective. When induced only with CHX without the addition of dsRNA, IFN-β
₂ RNA was only slightly induced, which caused IFN-β
₁ can be distinguished.

To express the IFN-2 gene, a DNA construct having the SV40 early promoter was created. IFN-2 DNA
4.8kb segment, p via synthetic oligonucleotide
SVE3 DNA [Y. Chernajovsky 5, (1984) DNA, 3,294-30
8] so that the ATG codon was located 150 bp downstream from the start of the immediate early RNA of SV40 (pSVIFA2-II, see FIG. 5). pSVIFA2-II DNA was transferred to cos7 monkey cells [Y.Glu
zman, (1981) Call, 23 : 175-182 introduced into], IFN-β ₂ c
Two RNAs of 1.35 and 2.2 kb were detected by the Northern blot method using DNA. These two RNAs are
N-beta ₂ by the transfer is finished RNA, or transcribed in SV40 polyadenylation site corresponded to end RNA (Figure 5). pSVIF
About the culture medium of Cos7 cells in which A2-II DNA is transiently expressed, human FS11 and Wish cells [D.
IFN activity was measured based on the inhibitory effect of VSV on cellular effects as provided in vick et al. (1983) J. Gen. Virol., 64: 905-910]. Antiviral activity was determined by pSVIFA2-
Two days after II DNA transfection, it was clearly detected (Table 1). 1 Antiviral specific activity of Yunitsuto per IFN-beta ₂ is 1 / 30-1 / 100 of the IFN-beta _1, IFN
Near Katsuta thereto (5-10 × 10 ⁶ Yunitsuto / mg) of-.alpha. _1.
Medium the expression of IFA-2 gene was performed IFN titer expected, about 3 titer when IFN-beta ₁ gene's write set downstream of the same SV40 promoter is expressed
% (Table 1). Furthermore, by exposing the FS11 cells to the medium of Cos7 cells into which pSVIFA2-II DNA has been introduced, (2'-5 ') oligo A synthetase [M.
Al, (1981), Meth.Enzymol.79: since 149-161] are derived, IFN-beta ₂ activity was demonstrated to occur in cells that are pSVIFA2-II DNA transflector Ekushi Yon (No. 6). The induction of (2'-5 ') oligo A synthetase is dose-dependent and quantitatively corresponds to antiviral activity.

Further, the IFN-2 gene, close to the Xho ₁ site of S-2 S
It was fused so that the V40 promoter was located (pSVIFA2-
I DNA, FIG. 5). Hamster CHO-K1DHFR ^- cells were treated with pSV
IFA2-1 and pSVDHFR [Y. Chernajovsky et al., (1984) DN
A, 3: 294-308] DNA was transfected together and a stable transformant SI-15 was isolated. For this cell line, when measured using human FS11 and Wish cells, 10
An IFN antiviral activity of -50 U / ml was observed. On the other hand, pSV
In the case of CHO cells transfected with only DHFR DNA, human IFN
No formation was observed (Table 1). SI
Yotsute to concentrating the medium of -15 culture, gamma IFN-beta ₂ solution is obtained having a titer of about 3000 U / ml. This γIFN−
beta ₂ has a characteristic with human IFN, also γIFN-
beta ₂ by at FS11 cells (2'-5 ') oligo A synthetase is induced even when a low concentration of 1U / ml (6
Figure). The CHOSi-15 cells, since the gamma IFN-beta ₂ is produced in the presence of cycloheximide (2'
5 ') It was demonstrated that the mRNA for producing oligo A synthetase C56 and HLA was specifically induced in humans. The gamma IFN-beta ₂ Antiviral activity, anti -IFN-beta ₁
It was found that the antibody was neutralized by a polyclonal or monoclonal antibody and was not neutralized by an anti-IFN-α or anti-IFN-γ antibody. The mouse - human hybridomas, only if it contains a chromosome 21 with a gene of type 1 IFN receptor, gamma IFN-beta _2, the mouse - it was divide acting against human hybridomas. From such biological characteristics of γIFN-β _2, γI
FN-beta ₂ an alien because it acts as IFN, IFN-beta
It proves that it is not acting as an inducer of No. ₂ .

DNA constructs obtained by fusing IFN-β _2A cDNA sequence from the SV40 early promoter (pSVCIFβ _2, Figure 5), and when the transflector Ekushi Yon with pSVDHFR into CHO cells in the same manner as described above, gamma IFN in high yield -β ₂ was obtained. Then Yotsute to selecting resistant cells, the yield of gamma IFN-beta ₂ were amplified for high concentrations of methotrexate.
Be filed in case of not amplified, CHO cells (e.g. clone B-132) transformed with PSVCIFbeta ₂ showed similar IFN-beta ₂ activity and SI-15 cells (Table 2). However, if you select the cells resistant to methotrexate showed high 800U / ml IFN-β ₂ activity (Table 2).

Using such a selected clone, secreted into the medium
The IFN-beta ₂ protein can be immunologically precipitation, also in a case where the cells are labeled with ³⁵ S- methionine, IFN-beta ₂ protein, analyzed by dodecylsulfate-polyacrylamide gel electrophoresis You can also. The size of the gamma IFN-beta ₂ were divide to be 21,000 daltons. According to actual experiments, in reticulocytes lysate, IFN-beta by ₂ RNA translation products of 23-26Kd is obtained which, in Vitr the dog pancreatin film
With o, it was found that it was converted to the shorter 21Kd protein. The human fibroblasts were treated derivatized addition, protein 21Kd is made, this protein is the product immunologically identical in 26Kd the product from IFN-beta ₂ RNA, however IFN-beta It was different from ₁ . 21Kd of mature IFN-β ₂
The N-terminus of the protein was not identified. The IFN-beta ₂ sequence, there are two glycosylation sites (FIG. 1), for this purpose, has difficulties summer to calculate the size of the area to be removed Te cowpea to Purosetsushingu. However, the area this removal is longer than that of IFN-beta _1, its size, maturation [W.Degrave et al, (1981) Gene, 10: 11-1
5]. IFN-β ₂ of 26Kd
Hydropathy of polypeptide
The plot), between the hydrophilic and hydrophobic regions of the IFN-beta ₂ and their mature IFN-beta _1, Part C the two proteins
-When arranged by terminus, it was found that there was significant commonality. The amino acid sequence of IFN-beta ₂ and other I
It was also found that the amino acid sequence of human IFN had a consensus sequence (FIG. 7). All known IFN-α and β sequences have 38 common amino acid sequences (starred in FIG. 7). When arranged IFN-beta ₂ with respect to the same manner C- terminus, of all the type I human IFN common 38 amino acids, 18 amino acids are also found in IFN-beta ₂ (7 Figure). Between the IFN-beta ₂ and other type I IFN is about 20% homology.

IFN-beta ₁ homology with the amino acid sequence and the IFN-beta ₂ amino acid sequences is low, the antiviral activity of IFN-beta ₁ and IFN-beta _2, for example anti -IFN-beta ₁ monoclonal antibodies [D.Nov
ick et, (1983) J.Gen.Virol, 64: . 905-910] from being neutralized by cross reaction with the same antibody, such as, those IFN-beta ₁ and IFN-beta ₂ of the active site It is possible that there is some homology. Activity of IFN-beta _2, since the connexion by the anti -IFN-alpha or -γ antibodies is not neutralized, this idea is supported. Gene encoding IFN-beta ₂ is chromosome 9 [U.Nir, (1984) Ph.D.Thesis , Weizma
nn Institute of Science, Rehovot, Israel] and is different from the type I IFN gene on chromosome 9 which takes a cluster structure due to the presence of introns. The IFA-2 gene is located on chromosome 7 [PBSehgal et al., (1986) Proc.
d.Su., USA]. Since the homology in the amino acid sequence is detected, IFN-beta ₂ gene is believed to be associated with genes underlying the type I IFN genes to take cluster structure.

To IFN-beta ₂ cDNA and high Zuburi soybean, another other Genomitsuku DNA clone IFA-11 has a different restriction map from the IFA-2 (Figure 8). From the transcript and its sequence, the exon at the 3'-end of IFA-2 and IFN-11
(Hbal-Hind ₁₁₁ segment)
% Homologous sequence), while no region cross-hybridizes with the exon at the 5 'end. The IFA-11 genomic clone (19 Kb) was ligated to the HSV-TK gene [F.
(1981) J. Mol. Biol., 150: 1-14] and transfected into L-TK-cells to obtain stable transformation. As shown in Table 1, the L cell clone LI-39 containing the human IFA-11 gene, when derivatized with poly (γI) (γC) and cycloheximide,
L-TK ⁺ cells did not show such activity while human IFN activity was shown. CHO clone IC40 containing IFN-11 gene
Also showed human IFN activity (Table 1). LI-39 or IC40 cells without derivatization did not show any activity. The IFN activity of the human IFA-11 transformant after the derivatization treatment was significantly higher than that of the case of the IFA-2 gene. ΓI produced by expression of the IFA-11 gene
FN-β _2B , as well as the product of the IFA-2 gene,
Its activity is neutralized Te cowpea to-beta ₁ antibody. A DNA probe specific to IFA-11, like the IFN-2 DNA probe,
It hybridized with RNA obtained from derivatized human fibroblasts, which indicates that both genes show activity in human cells.

Function of IFN-beta ₂ gene is believed to be due to other IFN genes are IFN-beta ₂ gene under conditions such not expressed is expressed. We when exposed fibroblasts to tumor necrosis factor (TNF) or Rinfuokain IL-1, IFN-β ₂ protein was found to be produced. It has also been found by others that antibodies neutralize IFN-β and consequently promote cell proliferation [M. Kohase et al., (1986) Cell Submission. ; Res
nitzky et al. (1986) Cell submission]. From this, IFN
-Beta ₂ can be seen to act to inhibit the growth of cells in response to growth factors. This means that IFN-γ mRNA
Together in peripheral blood mononuclear cells treated by connexion derivatized PHA such as Maitojien, IFN-β ₂ cDNA hybridized to RNA is also supported by the fact that is detected. Further, Rinfuokain such IL-1 is, in one diploid fibroblasts and other human cell lines, the IFN-beta ₂ cDNA hybridized to RNA, have been reported to induce strong [J.Content et al , (1985) Eur. J. Biochem., 152: 253-2.
57]. The promoter region of the IFN-2 gene consists of two TATs.
It contains the A box and two RNA start points (FIGS. 1 and 3). The IFN-beta ₂ promoter, poly (gamma
I) It has been shown to respond to either (γC) or cycloheximide [Y. Chernajovsky et al., (198
4), DNA, 3: 294-308]. In vivo, two IFN-β ₂
The gene promoter is activated by different inducers (dsRNA, protein synthesis inhibitors, IL-1, PHA). Various cells have been found to produce small amounts of autocrine IFN-β in growth arrest or differentiation [M. Revel (1983) Interferon, 5, pp. 205-23.
9, Acad. Press, London]. IFN-beta ₂ are those belonging to the small number of IFN species produced in accordance with the auto-regulated cell growth, other type I IFN is a specifically IFN species produced against viral infection is there.

IFN-beta is ₂ biologically important, conditions such as IFN-beta ₁ is not induced, for example under conditions such as metabolic mechanism is inhibited [A.Zilberstein et al, (1985) The Interferon
System, Serono Symposia Raven Press, pp. 73-8
3] is that IFN-beta ₂ is induced. And most importantly, IFN is expressed in fibroblasts
₂ that the proliferation of fibroblasts stimulated by IFN is further enhanced by the addition of anti-IFN-β antibodies. Many human cell lines have been shown to produce autocrine IFN-β upon differentiation, and these IFNs induce HLA antigens in cells [A. Yarden et al. (1984) Embo.J.,
3; 969-973]. In a mouse spinal cord leukemia cell line induced to differentiate by CSF-1, growth arrest characteristic of terminal differentiation is abolished by anti-IFN-β antibody. IL−
1 promotes the growth of several types of cells and has the growth factor PD
GF has been shown to induce IFN-β RNA in mouse cells [JNZullo et al., (1985) Cell 43: 793-80.
0]. There are many IFN-βs [PBSehgal, (9182), In
terferon4, Academic Press, pp. 122, and M. Revel (198
3), Interferon 5, Academic Press, pp. 205-239], IF
N-beta ₂ is believed to be one of autocrine automatic regulators of cell growth that occurs in response to growth factors. Cells producing autocrine IFN-β show only low antiviral activity [M. Revel (1983), Interferon 5, Academ.
ic Press, pp. 205-239].
Low specific activity of-beta ₂ (antiviral activity as a result of measurement by the immunoassay, it is 1 / 50-1 / 100 of the IFN-beta ₁₎
Matches. The hamster cells expressing IFN-beta ₂ cDNA is hamster CHO cell [Y.Chernajovsky et al, (1984), DNA3: 297-308 ] that holds the human IFN-beta ₁ gene why exhibit lower than IFN activity , Can be explained based on the above concept. Interestingly, IFN-α1 (D)
Account for a large proportion of leukocyte IFN [C. Weissmann
(1981), Interferon 3, Academic Press, NYpp. 101-1
34] Despite this, IFN-α1 (D) is also
[T. Goren et al., (1983), Vir.
ology 130: 273-280]. However, Yotsute the IFN-beta _2, some of the functions of many IFN, than for antiviral activity, can not be overlooked that is more effectively expressed. Despite low concentrations, growth regulator IF
N-β induces HLA and (2'-5 ') oligo A synthetase more potently and also inhibits the growth of the cells producing them longer and more potently. IFN by IL-1 and TNF
Since-beta ₂ is derived, IFN-beta ₂ can not only regulate the growth of cells, suggesting that plays the role of autocrine mediator of these cytokines in inflammation and acute-phase response.

IFN-beta ₁ cDNA instead IFN-β ₂ cDNA mRNA which hybridizes to a probe of the human cell line interleukin -1 (IL-I) [J.Content et al (1985), Eur.J.Bioc
hem., 152: 253-257] and TNF-α. IFN-β using R-antibody
Immunocompetition method specific to ₂
The assay), in response to these two cytokines, human fibroblasts FS11 cells, was examined actually whether secrete IFN-beta ₂ protein (Figure 9). γIL-α and TNF-
Both α induces the synthesis and secretion of IFN-beta ₂ protein, by immunoassay, IFN-beta ₂ of 10-20U / ml was secreted. When the concentration of γIL-1α is 4 U / ml (0.13 ng / ml),
IFN-beta ₂ optimal induction is observed, the concentration, prostaglandin E _2, the concentration of IL-1 in need of collagenase or hyaluronic acid allowed to induction in human fibroblasts [JHKorn (1985), Arthritis Rheum . , 28: 315-322]
Was the same as The IFN-beta _2-induced (400U / ml; 40ng / n
The optimal concentration of TNF-α for) is the concentration required to lyse the sensitive cells [0.1 ng / ml; AMWang et al. (1985) Sci.
ence, 228: 149-154] and higher than the concentration required for optimal growth promotion of diploid fibroblasts (2 ng / ml).
The start of the secretion of IFN-β ₂ is, is advanced by TNF-α,
In about 6 hours, half of the maximum secretion is reached. On the other hand,
In the case of IL-1, half the value is reached in 8-12 hours.

The documents cited in the preceding sections are hereby incorporated by reference in order to complete the description of the present invention.

Modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope of the invention. Further, the present invention is not limited to the description in the specification and the drawings.

[Brief description of the drawings]

FIG. 1 shows a restriction map and a nucleotide sequence of IFN-β _2A cDNA. This nucleotide sequence was obtained from the E474 and A341 cDNA clones [Weissenbach et al., (1980),
Proc. Natl. Acad. Sci. USA, 77: 7152-7156].
Determined from clone AE20 formed by site fusion. Its 5'-end sequence was revealed from the genomic clone IFN-2 shown in FIG. The numbers start with S-1 in FIGS. 3 and 4 as 1. IFN-β ₂
Was determined by a deductive method. FIG. 2 shows the genomic clone I containing the IFN-β _2A gene.
1 shows the structure of FA-2. A132 is a subclone obtained by cloning the EcoRI segment of IFA-2 with pBR322. The portions with scattered dots are cDNA clones A341 and E474 [We
Issenbach et al., (1980), Proc. Natl. Acad. Sci. USA, 77:
7152-7156] shows the hybridizing region.
The two starting points of RNA (Caps 1 and 2) and the TATA box are indicated. In the RNA, the open reading frame (ORF) of 212 amino acids (FIG. 1) is indicated by a thick black line. MC indicates a second ATG, and no other ORF was found in the IFN- _β2A cDNA. FIG. 3 shows S1 nuclease analysis of the 5′-end portion of IFN-β ₂ RNA. A DNA probe labeled with a BstN1 site, which is a fragment of the subclone A132 of the IFA-2 gene (FIG. 2), was hybridized with total RNA obtained from diploid fibroblasts treated by the following method. The treatment method was as follows: IFN-β ₁ 200 U / ml for 16 hours; pre-treatment and poly (γI) (γC) (pIC) 50 μg / ml for 3.5 hours; similar pre-treatment and cycloheximide (CHX) 50
μg / ml for 3.5 hours; same pretreatment and 3.5 times with pIC and CHX
Time treatment; same pretreatment and 6.5 hours treatment with CHX; 3.
5 hours treatment only; CHX 3.5 hours treatment only; pIC and CHX
Only 3.5 hours of processing. S-1 and S-2 are symbols indicating the start points of the two RNAs shown in FIG. FIG. 4 shows the promoter region of the IFA-2 gene encoding IFN-β _2A . Sequence portion of the subclones A 132 (FIG. 2) are presented as IFN-beta ₂ cDNA. S-1 and S
-2 indicates the starting point of the two RNAs shown in FIG. Initiator ATG is indicated by X. The first intron is i
Start with ntγl. FIG. 5 shows the fusion of the IFN-β _2A cDNA with the SV40 early promoter. The IFA-2 gene (5.5 in FIG. 2)
The 5'-terminal Xho ₁ -BamH ₁ (Xh-B) segment (7.5 kb to 7.5 kb) was converted to a 26 bp Cla ₁ -Xho ₁ synthetic adapter (reconstructing the 5'-end cDNA sequence) using Cla ₁ and BamH
_The plasmid was fused to the pBR plasmid cut in ₁ and subcloned to construct a plasmid pSVIFA-II. Hind
₁₁₁ and BamH 3 were subcloned in pBR plasmid cut with ₁ 'end of the _{BamH 1 -Hind 111 (B-H} ) gene segment (10.5 kb from 7.5kb of FIG. 2), Hin of pBR322
Cut on Cla ₁ site adjacent to the d ₁₁₁ site were ligated to the 5'-end segment to obtain the complete IFA-2 gene. H
pSVE3 vector cut with ind ₁₁₁ [Y.Chernajovsky et al (1984) DNA, 3: 294-308 ] was re-ligated with Cla ₁ linkers (forming PSVCla), then PSVCla the IFA-2 gene SV40 forming pSVIFA2-II at the Cla ₁ site of
It was introduced in the same direction as the early promoter. Xho for IFA-2
₁ site, except that fused directly to the Cla ₁ site of pBR before introduction into PSVE3, to construct a plasmid pSVIFA2-I in the same manner. IFN-β _2A cDNA fused to SV40 promoter
In order to obtain a pVCIFβ2 plasmid containing
The -II DNA, cut with Xb _1, then partially digested with Xmn _1, from Xho ₁ site of IFN-β ₂ cDNA sequence 60bp downstream Xm
n Open ₁ site (Fig. 1), and Xmn ₁ site located amp gene vectors has failed open. In this construct, IFN-
connecting the Xmn ₁ -Xba ₁ segment of beta ₂ cDNA clone AE20 (corresponding to 92-566 of FIG. 1), as shown in FIG, restoring the complete IFN-β _2A cDNA sequence. EES is RNA starting point of the SV-40T-ag gene, ATG start codon of the IFN-β _2, pA is I
₂ shows the polyadenylation sites of FN-β2 and SV40. pSVCIF
The beta ₂ DNA, with pDHER plasmid, CHO-K ₁ DHFR ^- transflector Ekushi and Yon the cell [Y.Chernajovsky et al (198
4), DNA, 3: 294-308].
Human IFN antiviral activity in FS11 cells was examined using sicular Stomatitis Virus (VSV). Gene amplification was achieved by selecting CHO clones resistant to methotrexate (MTX). CHO resistant to 250nM MTX
Atsusei of -SVCIFβ ₂ B132-5M cell culture medium is shown below in FIG. After the cytopathic effect of VSV appeared,
Cells in the microplate were stained with methylene blue. A series of 2-fold dilutions of the media are arranged from left to right. IF
By comparing the N standard (ST) and non-introduced CHO cells, it was calculated gamma IFN-beta ₂ titer of 300 U / ml. FIG. 6 shows poly (γI) (γC) -agarose bound to NP40 extract for human FS11 fibroblasts previously exposed to the cell culture medium shown below for 16 hours [M. Revel
Et al. (1981) Meth. Enzymol. 79: 149-161].
The results of the assay of (2'-5 ') oligo A synthetase are shown. (A) Cos7 cells two days after the introduction of pSVIFA2-II DNA (FIG. 5) (the results of the assays are shown in the leftmost four columns in FIG. 6),
(B) Hamster CHOSI-15 cells stably transformed with pSVIFA2-I DNA of FIG. 5 (next four rows), (c) IFN-β
CHOMEIF-5 cells expressing _one gene [Y. Chernajovsky
(1984), DNA, 3: 294-308] (next two columns), (d) D
CHO cells transformed with the HFR gene (next row), and (e) fresh medium only (none). The results of measurement of IFN antiviral activity per 1 ml of medium added to FS11 cells are shown. Phosphorylated 32P-α-ATP labeling (2 ′
-5 ') The results of electrophoresis of the oligo A product are shown. FIG. 7 compares the amino acid sequences of human type I IFN. Except for the IFN-β ₁ and a variety of IFN-α (α-E, α
-A, IFN-α represented by α-C, and IFN-α class II)
Amino acids that are common to bets have been marked with an asterisk on the IFN-beta ₁ sequence. The amino acids common to IFN-beta ₂ is that if an asterisk in the case of common that it all type I IFN, IFN-beta, which is marked with squares in the case of common with some IFN if _2A was aligned by comparing hydropathy plots and numbered from predicted processing sites. FIG. 8 shows a restriction map of _two different human IFN-β2s expressed in rodent cells. IFN-11 DNA (IFN-
_β2B gene) was transfected into either CHO or L cells and expressed IFN-β activity. Figure 9 is an illustration specific immunocompetent Atsusei method IFN-beta ₂ with R- antibodies, human fibroblasts FS11 cells, two cytokines, interleukin -1 (IL
-I) and IFN- in response to tumor necrosis factor (TNF-α).
It has been shown to secrete β ₂ protein. Both cytokines, low-level, i.e. the optimum induction of 10-20U / mlIFN-β ₂ is seen when the concentration of γIL-1α 4U / ml of (0.13ng / ml). IFN-beta ₂ optimal concentration of TNF-alpha for induction (400U / ml; 40ng / ml ) is higher than the concentration required for the cytopathic effect of sensitive cells (0.1 ng / ml), also double It is higher than the concentration required for optimal growth of somatic fibroblasts (2 ng / ml).

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location // (C12N 15/09 C12R 1:91) (72) Inventor Atsushiya Gilberstein Rehobot, Israel Epstein 50 (56) Reference JP-A-61-115024 (JP, A) Proc. Natl. Acad. Sc i. USA, 82 (16) P. 5490-5494 (1985) (54) [Title of the Invention] Human interferon-β2A and human interferon-β2B, a vector containing the gene encoding the interferon, a cell line producing the interferon, and the interferon Use of medicines as pharmaceuticals

Claims (1)

(57) [Claims] 1. A recombinant human interferon β _2A capable of inducing (2′-5 ′) oligo A synthetase, which is used to transform eukaryotic cells excluding human cells with a vector containing the following cDNA sequence: And a human recombinant interferon-β _2A obtainable by culturing the transformed cells and then isolating from the culture solution.