JP2530631B2 - Transformant - Google Patents

Description

TECHNICAL FIELD The present invention relates to a host human lung cancer-derived cell used in mass production of a glycosylated polypeptide by gene recombination technology using human cells.

[Prior Art] Most of physiologically active substances in vivo, such as lymphokines and hormones, which are expected to be used as pharmaceuticals, are glycosylated polypeptides, and sugar chains are required for expression of physiological activity and pharmacokinetics. There is something to do.

Chorionic gonadotropi
n), thyrotropin, follicular maturation hormone (follitropin) and luteinizing hormone (lutro)
pin) requires sugar chains for the expression of biological activity (TVRam
abhadran et al, Proc.Natl.Acad.Sci.USA, 81 , 6701,198
Four).

In addition, erythropoietin (erythropoietic)
oietin) loses its in vivo activity when the terminal sialic acid is removed (P. Lowy et al, Nature, 185 , 102, 1960).

On the other hand, tissue type plasminogen activato (tissue type plasminogen activato
Since r (TPA) has many cysteine residues in addition to sugar chains, it is thought that it cannot be produced unless it is an animal cell line that allows complex modification of polypeptides (RJKaufman et al, Mo.
I. Cell. Biol., 5, 1750, 1985).

The gene manipulation technology, which has been rapidly developing in recent years, is providing a new method capable of supplying a large amount of these trace physiologically active substances in the living body.

As a mass production method of glycosylated polypeptide, a system using Chinese hamster or (SWStanfield et al, Mol.
Cell.Biol., 4 , 173, 1984), a system using mouse cells (G.
N.Pavlakis et al, Proc.Natl.Acad.Sci.USA, 80 , 397,198
There is 3).

However, when a human-derived glycosylated polypeptide is produced using non-human cells, the structure of the sugar chain may differ from that of the natural type. desirable.

As a human cell expression system using gene manipulation technology, BK
System using virus (G. Milanesi et al, Mol.Cell.Bi
ol., 4 , 1551, 1984), a system using the EB virus (JLYea
tes et al, Nature, 313 , 812, 1985), a system utilizing an adenovirus (M. Yamada et al, Proc. Natl. Acad. Sci. USA,
82 , 3567, 1985) are known, but these are not reports that produced physiologically active substances that can be expected to be used as pharmaceuticals, and they have the following drawbacks.

The BK virus is thought to cause immune disorders such as Wiscott-Aldrich syndrome (J. Viro
L. 16 , 959, 1975), there is concern about contamination with virus-derived proteins. Cells such as Raji, which hosts the EB virus system, are difficult to transform, and EB virus is thought to cause Burkitt lymphoma (Zur Hausent.H.DN
A Tumor Virus, ed.Tooze.t, Cold Spring Harber Labora
(Tory, p747-795, 1982), there is a problem with safety.

Since the adenovirus system uses a virus infection system and is a transient expression, it is difficult to mass-produce it.

Further, as an example of production of human interferon γ by a human cell expression system, there is a system using WI · 26VA4 and FL (Japanese Patent Laid-Open No. 61-88875), but the productivity is 524 each.
Low as U / 24h / 10 ⁶ cells and 3U / 24h / 10 ⁶ cells.

Therefore, hitherto, there has been no human cell expression system suitable for producing a human-derived glycosylated polypeptide.

[Problems to be Solved by the Invention] An object of the present invention is to produce a naturally-occurring glycosylated polypeptide with high production efficiency, and to use a human cell line that is safe and has an unlimited number of passages by using genetic engineering techniques. The purpose is to provide a host for establishment.

[Means for Solving Problems] The present invention relates to a human interferon β gene and a human interferon γ under the control of a eukaryotic promoter.
The present invention relates to a transformant obtained by transforming human lung cancer-derived cell PC8 or human lung cancer-derived cell PC12 with a vector having a gene or human interleukin 2 gene.

A preferred production system using the host of the present invention, which is a system capable of producing human interferon constitutively or drug-induced, will be described below as an example.

The production of human interferon expression vector in human cells is carried out by using a gene sequence in which the human interferon gene is placed under the control of a promoter that functions in animal cells, and a gene sequence consisting of a replication origin in E. coli and a drug resistance gene. Can be achieved by combining The human interferon gene here means a gene encoding the amino acid sequence of a protein having biological activity exhibited by human interferon α, β or γ.

The eukaryotic promoter of the present invention may be any promoter of an animal virus or promoter of an animal cell gene, as long as it has a function of transcribing a human interferon gene downstream in human cells into mRNA.

As an example, SV40 early promoter, SV40 late promoter, HB virus gene promoter, MMTV promoter, human T cell leukemia virus promoter and the like can be mentioned as animal virus promoters. Examples of animal cell gene promoters include thymidine kinase gene promoters, metallothionein gene promoters, heat shock protein promoters, interferon gene promoters, and the like.

Among these, the promoter that is constitutively expressed is SV40.
Early promoter, SV40 late promoter, adenovirus gene promoter, HB virus gene promoter,
Examples include human T-cell leukemia virus promoter and thymidine kinase gene promoter. Inducible promoters include MMTV promoter, metallothionein gene promoter, heat shock protein promoter,
It is a promoter of interferon gene.

The eukaryotic promoter may be used alone or in combination of two or more. In addition, it is preferable to insert an enhancer sequence of 5'LTR of Harbey mouse sarcoma virus or an enhancer sequence of SV40, which is said to enhance transcription efficiency, upstream of the eukaryotic promoter.

Coupling of the replication origin and the drug resistance gene in Escherichia coli is useful for convenient, large-scale and pure preparation of the vector DNA. As the origin of replication, those derived from colicin E1 plasmid, which is known to cause amplification of replication with an Escherichia coli chromosomal DNA synthesis inhibitor, such as pBR322 and plasmids related thereto are preferable, but are not limited thereto. Absent.

As the drug resistance gene, ampicillin resistance gene,
Examples include tetracycline resistance gene, kanamycin resistance gene, streptomycin resistance gene, neomycin resistance gene and the like.

A human interferon expression vector in human cells can be prepared by ligating two DNA fragments having the above properties.

Moreover, as polypeptide genes other than the human interferon gene, genes of various hormones described above, erythropoietin, interleukin (1,2,
3), genes of physiologically active substances such as TPA. By introducing these genes, the target glycosylated polypeptide can be produced.

As a host human cell into which the vector DNA is introduced, a human cell that does not inhibit the growth of the produced glycosylated polypeptide is used. Preferably, cells derived from human lung cancer, especially PC8 and PC12 (M.Kinjo et al, Br.J.Cancer, 39 , 15, 197).
9) is mentioned.

Vector DNA can be prepared by a general method (T. Maniatis et al, Molecular Cloning, p86-96, 198).
2).

Vector DNA can be introduced into animal cells by the existing calcium phosphate method (FL Graham et al, Virolog
y, 54 , 536, 1973).

Human cells into which the glycosylated polypeptide expression vector has been introduced can be transformed with the G418 resistance gene expression vector pSV2ne.
o (PJSouthern et al, J.Mol.Appl.Genet., 1 , 327,198
2) or pNEO5 ′ (M.Lusky et al, Cell, 36 , 391, 19
When introduced together with 84), non-transformed cells can grow easily in a selection medium containing G418, which does not survive, so that they can be easily identified.

A cell transformed by a vector in which the human interferon gene is placed under the control of a constitutive promoter such as the SV40 early promoter constitutively secretes human interferon into the culture medium, and an inducible promoter such as the MMTV promoter leads to human expression. The cells transformed with the vector containing the interferon gene inducibly secrete human interferon into the culture medium by adding a drug to the culture medium.

Human interferon synthesized by the transformed cells can be purified from the culture medium by a general purification method. For example, by using an antibody column to which an anti-human interferon antibody is bound, human interferon that is almost pure can be obtained in one step. Various methods used for the purification of human interferon can be applied as they are.

[Examples] In the following, human interferon or human interleukin was introduced into human cells into which a human interferon gene or a human interleukin gene introduced under the control of the SV40 early promoter, the MMTV promoter, or the Drosophila heat shock protein promoter was introduced. Although the produced example is shown, the present invention is not limited to this.

Example 1 Construction of human interferon β expression vector pSVβ: pSVβ is human interferon β expression vector pSV2I
This is a vector obtained by removing a sequence (M.Lusky et al, Nature, 293 , 79, 1981) that inhibits replication in eukaryotic cells from FNβ (JP-A 61-52283). The manufacturing method is as follows.
(See Fig. 1) First, P located upstream of the SV40 early promoter of pSV2IFNβ
After replacing the vuII site with a SalI site using a SalI linker, it was cleaved with SalI and BamHI to isolate a 1.7 Kb DNA fragment required for expression of human interferon β.

Next, the vector pML2d (M.Lusky et al, Nature, 293 , 79,1) was prepared by removing the sequence that inhibits replication in eukaryotic cells from pBR322.
981) was digested with SalI and BamHI to separate a long chain fragment.

These two DNA fragments were ligated using T4 DNA ligase
pSVβ was obtained.

Example 2 Construction of Human Interferon β Expression Vector pSV (r) β: pSV (r) β is a CAAAG sequence upstream of the human interferon β signal peptide gene of pSVβ of Example 1 and said to enhance translation efficiency ( M. Kozak, Nature, 308 , 241,
1984) was inserted. The manufacturing method is as follows. (See FIG. 2) First, pSVβ was cleaved at the restriction enzyme XbaI site upstream of the human interferon β signal peptide gene, and S1 was cut.
After blunting with a nuclease, the sequence was digested with the restriction enzyme HindIII site located downstream of the SV40 early promoter to allow insertion of an arbitrary sequence upstream of the translation initiation codon ATG of the human interferon β gene.

Then, two chemically synthesized oligonucleotides were annealed to prepare an insertion sequence having a HindIII cleavage end at the 5'end and a blunt end at the 3'end.

The above two DNA fragments were ligated with T4 DNA ligase to obtain pSV (r) β.

Regarding the vector DNA introduced into Escherichia coli, the nucleotide sequence of the inserted portion was determined by the Maxam Gilbert method (Proc.Natl.Acad.Sci.USA, 7
4 , 560, 1977) and confirmed that the sequence was the intended one.

Example 3 Construction of human interferon β expression vector pSV (Ha, r) β: pSV (Ha, r) β is said to enhance transcription efficiency upstream of the SV40 early promoter of pSV (r) β of Example 2. Has Ha
This is a vector into which the 5'LTR enhancer sequence of rbey mouse sarcoma virus (hereinafter referred to as Ha enhancer) is inserted. The manufacturing method is as follows. (See Fig. 3) First, pSV (r) β was cleaved at the restriction enzyme SalI site upstream of the SV40 early promoter, and then DNA polymerase I was digested.
The Klenow fragment was treated to make it blunt-ended, and the terminal phosphorus was removed by treatment with BAP (Escherichia coli alkaline phosphatase).

Next, a 0.5 Kb fragment containing the Ha enhancer was isolated from the Ha enhancer-containing vector pNEO5 '(described above) by digestion with the restriction enzymes ClaI and SacI, followed by DNA polymerase.
The ends were made blunt by treating with IKlenow fragment.

The vector pSV (Ha, r) β was obtained by ligating the above two DNA fragments with T4 DNA ligase.

Example 4 Construction of human interferon β expression vector pMTVβ: pMTVβ is a vector in which the MMTV promoter was introduced in place of the SV40 early promoter of pSVβ of Example 1. The manufacturing method is as follows. (See Fig. 4) First, pSVβ was cleaved with the restriction enzyme SalI, the SalI site was replaced with the HindIII site using a HindIII linker, and then the fragment was cleaved with HindIII to exclude the SV40 initial promoter.
A 3.8 Kb DNA fragment was isolated. Furthermore, the terminal phosphorus was removed by BAP (described above) treatment.

Next, the vector pMTVdhfr (F.Le
e et al, Nature, 294 , 228, 1982) was digested with the restriction enzyme HindIII to isolate a 1.4 Kb DNA fragment containing the MMTV promoter.

By ligating these two DNA fragments with T4 DNA ligase, pMTVβ was obtained.

Example 5 Construction of human interferon β expression vector pMTV (r) β: pMTV (r) β is a vector in which an MMTV promoter was introduced in place of the SV40 early promoter of pSV (r) β of Example 2. The manufacturing method is as follows. (See Fig. 5) First, after replacing the SalI site upstream of the SV40 early promoter of pSV (r) β with a HindIII site, HindI
The fragment was cut with II and a 3.8 Kb DNA fragment containing no SV40 early promoter was isolated. Furthermore, the terminal phosphorus was removed by BAP (described above) treatment.

Next, pMTVdhfr (described above) was digested with the restriction enzyme HindIII to give M
A 1.4 Kb DNA fragment containing the MTV promoter was isolated.

The vector pMTV (r) β was obtained by ligating the above two DNA fragments with T4 DNA ligase.

Example 6 Construction of human interferon β expression vector pMTV (Ha) β: pMTV (Ha) β is a vector in which the Ha enhancer is introduced upstream of the MMTV promoter of pMTVβ of Example 4. The manufacturing method is as follows. (See Fig. 6) First, HindIII located upstream of the MMTV promoter of pMTVβ
The site was changed to a SalI site using a SalI linker. After that, it was cleaved with SalI, treated with a DNA polymerase IKlenow fragment to make it blunt-ended, and then treated with BAP (described above) to remove the terminal phosphorus.

Next, pNEO5 '(described above) was digested with ClaI and SacI to give 0.5 Kb.
Isolate the Ha enhancer fragment of DNA polymerase IKlen
The ow fragment was treated to make it blunt-ended.

These two DNA fragments were ligated with T4 DNA ligase to obtain pMTV (Ha) β.

Example 7 Construction of human interferon β expression vector pMTV (SV) β: pMTV (SV) β is a vector in which the MMTV promoter (described above) is introduced between the SV40 early promoter of pSVβ of Example 1 and the human interferon β gene. . In this vector, the human interferon β gene is placed under the control of the SV40 early promoter and the MMTV promoter. The manufacturing method is as follows.

(See Fig. 7) First, pSVβ was cleaved with the restriction enzyme HindIII before BAP (described above).
The terminal phosphorus was removed by the treatment.

Next, the vector MMTV promoter-containing vector pMTVdhfr (described above) was cleaved with the restriction enzyme HindIII to isolate a 1.4 Kb DNA fragment containing the MMTV promoter.

The vector pMTV (SV) β was obtained by ligating the above two DNA fragments with T4 DNA ligase.

Example 8 Construction of human interferon β expression vector pMTV (SV, r) β: pMTV (SV, r) β is MMTV between the SV40 early promoter of pSV (r) β of Example 2 and human interferon β gene.
This is a vector into which a promoter (described above) has been introduced. In this vector, the human interferon β gene is placed under the control of the SV40 early promoter and the MMTV promoter. The manufacturing method is as follows. (See FIG. 8) First, pSV (r) β was cleaved with the restriction enzyme HindIII and then BAP.
The terminal phosphorus was removed by the treatment (described above).

Next, the vector MMTV promoter-containing vector pMTVdhfr (described above) was cleaved with the restriction enzyme HindIII to isolate a 1.4 Kb DNA fragment containing the MMTV promoter.

The vector pMTV (SV, r) β was obtained by ligating the above two DNA fragments with T4 DNA ligase.

Example 9 Construction of Human Interferon β Expression Vector pMTV (Ha, r) β: pMTV (Ha, r) β is a vector in which the Ha enhancer is introduced upstream of the MMTV promoter of pMTV (r) β of Example 5. . The manufacturing method is as follows. (See FIG. 9) First, the restriction enzymes BamHI and Hin for pMTV (Ha) β of Example 6 were used.
Cleavage with dIII to separate long fragments before BAP (see above)
The terminal phosphorus was removed by the treatment.

Next, pMTV (r) β was digested with BamHI and HindIII to separate short chain fragments.

By ligating the above two DNA fragments with T4 DNA ligase, pMTV (Ha, r) β was obtained.

Example 10 Human interferon β expression vector pMTV (Ha, SV, r)
Preparation of β: pMTV (Ha, SV, r) β is the SV of pSV (Ha, r) β of Example 3.
40 This is a vector in which the MMTV promoter is introduced between the early promoter and the human interferon β gene. The manufacturing method is as follows. (See FIG. 10) First, pSV (Ha, r) β was cleaved with the restriction enzyme HindIII, and the terminal phosphorus was removed by BAP (described above) treatment.

Then the vector pMTVdhfr with the MMTV promoter (described above)
Digested with HindIII and 1.4 kb DNA containing MMTV promoter
The fragments were separated.

By ligating the above two DNA fragments with T4 DNA ligase, pMTV (Ha, SV, r) β was obtained.

Example 11 Human interferon β expression vector pBPV97SV (r) β
Preparation of pBPV97SV (r) β is a restriction enzyme AccII cleavage fragment (2Kb) required for expression of human interferon β gene in animal cells from pSV (r) β of Example 2 in BPV1 (bovine papillomavirus type 1). Vector containing the entire genome pdBPV1 (142-
6) (PMHowly, Proc.Natl.Acad.Sci.USA, 79 , 7147,198
This is the vector inserted into the PvuII site in 2). The manufacturing method is as follows. (See FIG. 11) First, pSV (r) β was cleaved with AccII to separate a 2 Kb DNA fragment containing the human interferon β gene.

Next, pdBPV1 (142-6) was digested with PvuII to separate a 10 Kb DNA fragment, and the terminal phosphorus was removed by BAP treatment.

By ligating these two DNA fragments with T4 DNA ligase, pBPV97SV (r) β was prepared.

Example 12 Construction of human interferon β expression vector pDH (r) β: pDH (r) β is a vector in which the MMTV promoter of pMTV (r) β of Example 5 was replaced with a Drosophila heat shock protein promoter. The manufacturing method is as follows. (See FIG. 12) First, pMTV (r) β was cleaved with HindIII to remove the MMTV promoter, and then blunt-ended by treatment with DNA polymerase IKlenow fragment. After that, the terminal phosphorus was removed by BAP treatment.

Next, after replacing the PstI site of the vector pSP6HS9 containing the Drosophila heat shock protein promoter with the HindIII site using the HindIII linker, it was cleaved with HindIII to isolate a 0.9 Kb DNA fragment containing the promoter (pSP6
HS9 is p232.3 (R. Holmgren et al, Cell, 18 , 1359,1979)
Drosophila heat shock protein promoter fragment of Hind
III / PstI 0.9 Kb is a vector incorporating pSP65 (Promega Biotech).

A vector pDH (r) β was obtained by ligating the above two DNA fragments with T4 DNA ligase.

Example 13 Human interferon β expression vector pBPVMTV (Ha) β
Preparation of pBPVMTV (Ha) β is a vector in which pMTV (Ha) β of Example 6 and the BPV1 whole genome (100%) are combined. The manufacturing method is as follows. (See FIG. 13) First, pMTV (Ha) β was cleaved at the BamHI site behind the SV40 poly A signal and then the terminal phosphorus was removed by BAP treatment.

Next, pdBPV1 (described above) was digested with BamHI to isolate an 8.0 Kb DNA fragment consisting of the entire BPV1 genome.

By ligating these two DNA fragments with T4 DNA ligase, pBPV100MTV (Ha) β was obtained.

Example 14 Human interferon β expression vector pBPV97MTV (Ha)
Preparation of β: pBPV97MTV (Ha) β is a restriction enzyme AccII cleavage fragment (3.1 Kb) required for expression of human interferon β gene in animal cells from pMTV (Ha) β of Example 6 in pdBPV1 (142-6).
This is a vector inserted into the PvuII site (described above). The manufacturing method is as follows. (See FIG. 14) First, pMTV (Ha) β was cleaved with AccII to separate a 3.1 Kb DNA fragment containing the human interferon β gene.

Next, pdBPV1 (142-6) was digested with PvuII to separate a 10 Kb DNA fragment, and then the terminal phosphorus was removed by BAP treatment.

By ligating these two DNA fragments with T4 DNA ligase, pBPV97MTV (Ha) β was prepared.

Example 15 Construction of human interferon γ expression vector pMTVγ: pMTVγ is a vector in which the human interferon γ gene is placed under the control of the MMTV promoter. The manufacturing method is as follows. (See FIG. 15) First, pMTVβ of Example 6 is located downstream of the MMTV promoter.
Cleavage at the HindIII site and the BglII site downstream of the human interferon gene, followed by blunting by treatment with the DNA polymerase I Klenow fragment, and then including the MMTV promoter 3.
A 9 Kb DNA fragment was isolated.

This DNA fragment and the human interferon γ gene obtained by cleaving pSVIFNγ (JP-A-61-252286) with DpnI.
The 8 Kb DNA fragment was ligated with T4 DNA ligase to obtain pMTVγ.

Example 16 Construction of human interferon γ expression vector pMTV (SV) γ: pMTV (SV) γ is a vector in which the SV40 early promoter is introduced upstream of the MMTV promoter of pMTVγ of Example 15. The manufacturing method is as follows. (See Fig. 16) First, pMTVγ was cleaved with SalI and DNA polymerase IKlenow
The fragment was treated to make it blunt-ended, and then BAP treatment to remove phosphorus at the end.

Next, pSV21FNβ (Japanese Patent Application No. 59-169388) was added to PvuII and Hind.
It was cut with III to isolate a 0.3 Kb DNA fragment containing the SV40 early promoter, and then blunt-ended by treatment with DNA polymerase I Klenow fragment.

These two DNA fragments were ligated with T4 DNA ligase to obtain pMTV (SV) γ.

Example 17 Transformation of PC12 with pSVβ: 2 μg of pSVβ of Example 1 and 0.2 μg of pSV2neo (described above) were introduced into about 10 ⁶ PC12 cells by the calcium phosphate method (described above). 400 μg of protein synthesis inhibitor G418 (GIBCO)
20 transformants were obtained by culturing in a selective medium (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical Co., Ltd.)) at a concentration of ml.

The antiviral activity (ie, human interferon β activity) of the culture supernatant was determined by the method of S. Rubinstein et al. (J. Virology, 37 , 75).
5, 1981), the activity of 100 to 900 units / ml was observed in 12 cells as measured by CPE inhibition method using human FL cells and VSV. (See FIG. 17) Example 18 Transformation of PC12 with pSV (r) β: 2 μg of pSV (r) β and pSV2neo (previously described) of Example 2
μg and about 10 ⁶ PC1 by the calcium phosphate method (described above)
2 cells were introduced. Protein synthesis inhibitor G418 (GIBCO) 40
Selection medium containing 0 μg / ml (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical))
When cultured in, 12 transformants were obtained.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (ie, human interferon β activity),
5 when measured by CPE inhibition method using human FL cells and VSV
The activity of 150 to 1800 units / ml was observed in each individual. (See FIG. 17) Example 19 Transformation of PC12 with pSV (Ha, r) β: 2 μg of pSV (Ha, r) β in Example 3 and pSV2neo (described above).
2 μg and about 10 PC1 by the calcium phosphate method (described above)
2 cells were introduced. Protein synthesis inhibitor G418 (GIBCO) 40
Selection medium containing 0 μg / ml (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical))
When cultured in, 19 transformants were obtained.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (that is, human interferon β activity),
8 when measured by CPE inhibition method using human FL cells and VSV
The activity of 100 to 4900 units / ml was observed in each individual. (See FIG. 17) Example 20 Transformation of PC12 with pMTVβ: 2 μg of pMTVβ of Example 4 and 0.2 μg of pSV2neo (described above) were introduced into about 10 ⁶ PC12 cells by the calcium phosphate method (described above). 400 μg of protein synthesis inhibitor G418 (GIBCO)
When cultured in a selective medium (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical)) containing 18 ml, 18 transformants were obtained.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (that is, human interferon β activity),
7 when measured by CPE inhibition method using human FL cells and VSV
The activity of 100 to 4300 units / ml was observed in each individual. (See FIG. 17) Example 21 Transformation of PC12 with pMTV (r) β: 2 μg of pMTV (r) β and pSV2neo (previously described) of Example 5
μg and about 10 ⁷ PC8 by the calcium phosphate method (described above)
Introduced into cells. Protein synthesis inhibitor G418 (GIBCO) 400
When cultured in a selective medium (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical)) containing 24 μg / ml, 24 transformants were obtained.

The antiviral activity of the culture supernatant (that is, human interferon β activity) was measured by the CPE inhibition method using human FL cells and VSV based on the method of S. Rubinstein et al. (Described above),
The activity of 100-5500 units / ml was observed in 19 cells. (See FIG. 17) Example 22 Transformation of PC12 with pMTV (Ha) β: 2 μg of pMTV (Ha) β and pSV2neo (described above) of Example 6
μg and about 10 ⁶ PC1 by the calcium phosphate method (described above)
2 cells were introduced. Protein synthesis inhibitor G418 (GIBCO) 40
Selection medium containing 0 μg / ml (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical))
When cultured in, 20 transformants were obtained.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (ie, human interferon β activity),
12 when measured by CPE inhibition method using human FL cells and VSV
The activity of 100 to 2800 units / ml was observed in each individual. (See FIG. 17) Example 23 Transformation of PC12 with pMTV (SV) β: pMTV (SV) β2 μg of Example 7 and pSV2neo (described above) 0.2
μg and about 10 ⁶ PC1 by the calcium phosphate method (described above)
2 cells were introduced. Protein synthesis inhibitor G418 (GIBCO) 40
Selection medium containing 0 μg / ml (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical))
When cultured in, 21 transformants were obtained.

The antiviral activity of the culture supernatant (that is, human interferon β activity) was measured by the CPE inhibition method using human FL cells and VSV based on the method of S. Rubinstein et al. (Described above),
The activity of 100 to 4500 units / ml was observed in 10 cells. (See FIG. 17) Example 24 Transformation of PC12 with pMTV (SV, r) β: pMTV (SV, r) β2 μg and pSV2neo of Example 8 (described above)
0.2 μg and about 10 ⁶ pieces by the calcium phosphate method (described above)
It was introduced into PC12 cells. Protein synthesis inhibitor G418 (GIBCO)
Was cultured in a selective medium (RPMI1640 medium containing 10% fetal calf serum and 100 µg / ml kanamycin (Nissui Pharmaceutical Co., Ltd.)) at a concentration of 400 µg / ml to obtain 22 transformants.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (ie, human interferon β activity),
20 when measured by CPE inhibition method using human FL cells and VSV
The activity of 800 to 48,000 units / ml was observed in each individual. (See FIG. 17) Example 25 Transformation of PC12 with pMTV (Ha, r) β: pMTV (Ha, r) β2 μg and pSV2neo of Example 9 (described above)
0.2 μg and about 10 ⁶ pieces by the calcium phosphate method (described above)
It was introduced into PC12 cells. Protein synthesis inhibitor G418 (GIBCO)
Selective medium containing 400 μg / ml of RPMI (RPMI medium containing 10% fetal calf serum and 100 μg / ml of kanamycin (Nissui Pharmaceutical))
When cultured in, 24 transformants were obtained.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (that is, human interferon β activity),
When measured by CPE inhibition method using human FL cells and VSV.
The activity of 100 to 30300 units / ml was observed in 20 cells. (17th
(See the figure) Example 26 Transformation of PC12 with pMTV (Ha, SV, r) β: 2 μg of pMTV (Ha, SV, r) β of Example 10 and 0.2 μg of pSV2neo (described above) were subjected to the calcium phosphate method (described above). About 10 ⁶
Were introduced into individual PC12 cells. Protein synthesis inhibitor G418 (GIBCO
Selective medium (fetal bovine serum 10) containing 400 μg / ml
When cultured in RPMI1640 medium (Nissui Pharmaceutical Co., Ltd.) containing 100% and kanamycin 100 μg / ml), 22 transformants were obtained.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (that is, human interferon β activity),
10 when measured by CPE inhibition method using human FL cells and VSV
The activity of 300 to 10300 units / ml was observed in each individual. (See FIG. 17) Example 27 Transformation of PC8 with pMTV (Ha, SV, r) β: 2 μg of pMTV (Ha, SV, r) β of Example 10 and 0.2 μg of pSV2neo (described above) were subjected to the calcium phosphate method (described above). ) At about 10 ⁷
Were introduced into individual PC8 cells. Protein synthesis inhibitor G418 (GIBCO
Selective medium (fetal bovine serum 10) containing 400 μg / ml
When cultured in RPMI1640 medium (Nissui Pharmaceutical Co., Ltd.) containing 100% and kanamycin 100 μg / ml, 27 transformants were obtained.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (ie, human interferon β activity),
18 when measured by CPE inhibition method using human FL cells and VSV
The activity of 150 to 25500 units / ml was observed in each individual. (See FIG. 17) Example 28 Transformation of PC12 with pDH (r) β: 2 μg of pDH (r) β of Example 11 and pSV2neo (described above) 0.2
μg and about 10 ⁶ PC1 by the calcium phosphate method (described above)
2 cells were introduced. Protein synthesis inhibitor G418 (GIBCO) 40
Selection medium containing 0 μg / ml (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical))
When cultured in, 24 transformants were obtained.

The antiviral activity of the culture supernatant (that is, human interferon β activity) was measured by the CPE inhibition method using human FL cells and VSV based on the method of S. Rubinstein et al. (Described above),
The activity of 63 to 402 units / ml was observed in 12 cells.

Transformation of Example 29 pBPV ₉₇ SV (r) PC12 by _{β: pBPV 97 SV (r)} β2μg and pSV2neo of Example 10 (above) 0.
2 μg and about 10 ⁶ PCs by the calcium phosphate method (described above)
12 cells were introduced. 4 protein synthesis inhibitors G418 (GIBCO)
Selection medium at a concentration of 00 μg / ml (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical))
When cultured in, 22 transformants were obtained.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (ie, human interferon β activity),
10 when measured by CPE inhibition method using human FL cells and VSV
The activity of 300 to 10300 units / ml was observed in each individual. (See FIG. 17) Example 30 Transformation of PC12 with pBPVMTV (Ha) β: 2 μg of pBPVMTV (Ha) β of Example 13 and pSV2neo (described above)
0.2 μg and about 10 ⁶ pieces by the calcium phosphate method (described above)
It was introduced into PC12 cells. Protein synthesis inhibitor G418 (GIBCO)
Was cultured in a selective medium (RPMI1640 medium containing 10% fetal calf serum and 100 µg / ml kanamycin (Nissui Pharmaceutical Co., Ltd.)) at a concentration of 400 µg / ml to obtain 22 transformants.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (that is, human interferon β activity),
When measured by CPE inhibition method using human FL cells and VSV 11
The activity of 100 to 22300 units / ml was observed in each individual. (See FIG. 17) Example 31 Transformation of PC8 with pBPVMTV (Ha) β: 2 μg of pBPVMTV (Ha) β of Example 13 and pSV2neo (described above)
0.2 μg and about 10 ⁷ by the calcium phosphate method (described above)
It was introduced into PC8 cells. Protein synthesis inhibitor G418 (GIBCO)
When cultured in a selective medium (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical Co., Ltd.)) at a concentration of 400 μg / ml, 25 transformants were obtained.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (ie, human interferon β activity),
When measured by CPE inhibition method using human FL cells and VSV 15
The activity of 100 to 20200 units / ml was observed in each individual. (17 see FIG.) Transformation of Example 32 pBPV ₉₇ MTV (Ha) PC12 by beta: pBPV ₉₇ MTV Example 13 (Ha) β2μg and pSV2neo (described above)
0.2 μg and about 10 ⁶ pieces by the calcium phosphate method (described above)
It was introduced into PC8 cells. Protein synthesis inhibitor G418 (GIBCO)
When cultured in a selective medium (RPMI1640 medium containing 10% fetal bovine serum and 100 μg / ml kanamycin (Nissui Pharmaceutical Co., Ltd.)) at a concentration of 400 μg / ml, 22 transformants were obtained.

Based on the method of S. Rubinstein et al. (Supra), the antiviral activity of the culture supernatant (that is, human interferon β activity),
12 when measured by CPE inhibition method using human FL cells and VSV
The activity of 100 to 20000 units / ml was observed in each individual. (See FIG. 17) Example 33 Transformation of PC12 with pSVIFNγ: 2 μg of pSVIFNγ (Japanese Patent Laid-Open No. 61-52286) and 0.2 μg of pSV2neo (described above) were subjected to about 10 ⁶ by the calcium phosphate method (described above).
Were introduced into individual PC12 cells. Protein synthesis inhibitor G418 (GIBCO
Selective medium (fetal bovine serum 10) containing 400 μg / ml
When cultured in RPMI1640 medium (Nissui Pharmaceutical) containing 100% and kanamycin 100 μg / ml, 9 transformants were obtained.

The antiviral activity (ie, human interferon γ activity) of the culture supernatant was measured by the CPE inhibition method using human FL cells and sindbis virus based on the method of S. Rubinstein et al. An activity of ~ 936 units / ml was observed. (Fig. 18 reference) transformation of PC12 according to Example _{34 pSVIFNγ / BPV 97 (P)} : pSVIFNγ / BPV 97 (P) ( JP 61-52286), the human was placed under the control of the SV40 early promoter This is a vector in which a 2.0 Kb gene sequence consisting of the interferon γ gene was introduced into the PvuII site of pdBPV1 (described above).

pSVIFNγ / BPV _{97 (P)} 2 μg and pSV2neo (above) 0.2 μg
And were introduced into about 10 ⁶ PC12 cells by the calcium phosphate method (described above). 400μ of protein synthesis inhibitor G418 (GIBCO)
The cells were cultured in a selective medium (RPMI1640 medium containing 10% fetal bovine serum and 100 μg / ml kanamycin (Nissui Pharmaceutical Co., Ltd.)) at a concentration of g / ml to obtain 23 transformants.

The antiviral activity of the culture supernatant (ie, human interferon γ activity) was measured by the CPE inhibition method using human FL cells and sindbis virus based on the method of S. Rubinstein et al. An activity of -21600 units / ml was observed. (See FIG. 18) Example 35 Transformation of PC12 with pMTVγ: 2 μg of pMTVγ of Example 15 and 0.2 μg of pSV2neo (described above) were introduced into about 10 ⁶ PC12 cells by the calcium phosphate method (described above). 400 μg of protein synthesis inhibitor G418 (GIBCO)
When cultured in a selective medium (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical)) containing 27 ml, 27 transformants were obtained.

The antiviral activity of the culture supernatant (ie, human interferon γ activity) was measured by the CPE inhibition method using human FL cells and sindbis virus based on the method of S. Rubinstein et al. An activity of -4110 units / ml was observed. (See FIG. 18) Example 36 Transformation of PC12 with pMTV (SV) γ: pMTV (SV) γ2 μg of Example 16 and pSV2neo (described above) 0.2
μg and about 10 ⁶ PC1 by the calcium phosphate method (described above)
2 cells were introduced. Protein synthesis inhibitor G418 (GIBCO) 40
Selection medium containing 0 μg / ml (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical))
When cultured in, 32 transformants were obtained.

The antiviral activity of the culture supernatant (ie, human interferon γ activity) was measured by the CPE inhibition method using human FL cells and sindbis virus based on the method of S. Rubinstein et al. An activity of ~ 46500 units / ml was observed. (See FIG. 18) Example 37 Antibody neutralization of pMTV (SV, r) β / PC12 clone-produced interferon: pMTV (SV, r) β / PC12 clones grown to a saturated cell density were induced with an induction medium (dexamethasone 10 ^−6). M and fetal calf serum 10
% Of RPMI1640 medium (Nissui Pharmaceutical Co., Ltd.) at 37 ° C for 48 hours, and the resulting culture broth was treated with anti-human interferon β antibody or anti-human interferon γ antibody and then treated with When the activity was measured, it was specifically neutralized with an anti-human interferon β antibody. The results are shown in Table 1.

From this result, it was confirmed that the pMTV (SV, r) β / PC12 clone produced human interferon β.

Example 38 pSVIFNγ / BPV97 (P) / PC12 clones producing interferon antibody neutralization: pSVIFNγ / BPV ₉₇ grown to saturation cell density _(P) / PC12
About the culture solution obtained by culturing the clone in RPMI1640 medium (Nissui Pharmaceutical) containing 10% fetal bovine serum at 37 ° C for 48 hours,
When the antiviral activity was measured by the method described above after treatment with the anti-human interferon β antibody or the anti-human interferon γ antibody, it was specifically neutralized with the anti-human interferon γ antibody. The results are shown in Table 2.

From this result, it was confirmed that the pSVIFNγ / BPV _{97 (P)} / PC12 clone produced human interferon γ.

Example 39 Human interferon β-producing ability of pMTV (Ha, SV, r) β / PC8 clone: pMTV (Ha, SV, r) β / PC8 clone N obtained in Example 27
After o19 was grown to a saturated cell density in a 24-well microplate (Corning), the amount of human interferon β production per day was measured for 25 days using the following four types of media. (See FIG. 19) In FIG. 19, 10% FCS + DEX is RPMI1640 medium containing 10% fetal bovine serum and 10 ⁻⁶ M dexamethasone, and 5% FCS + DEX is 5% fetal bovine serum and 10 ⁻⁶ dexamethasone.
RPMI1640 medium containing M, RPMI1640 medium containing 1% FCS + DEX and fetal bovine serum 1% and dexamethasone 10 ⁻⁶ M, RPMI1640 medium containing 10% FCS-DEX, respectively.

From this result, pMTV (Ha, SV, r) β / PC8 clone was
In order to continuously produce human interferon for 25 days in any medium, and to maintain high productivity of this clone, 5% or more of fetal bovine serum and 10 ^-6 M dexamethasone were added. It has been found that medium is preferred.

Example 40 Properties of pMTV (Ha, SV, r) β / PC8 clone-produced human interferon β: pMTV (Ha, SV, r) β / PC8 clone N obtained in Example 27
o19 RP containing 2.5% fetal bovine serum and 10 ^-6 M dexamethasone
Culture was performed in MI1640 (Nissui Pharmaceutical) medium to obtain a culture solution 32.5 showing human interferon β activity of 23800 units / ml.
Human interferon β in the culture solution was purified by a blue-sepharose column, an anti-human interferon β monoclonal antibody column, and reverse-phase HPLC, and the human fibroblast-derived natural human interferon β and SDS polyacrylamide gel had the same behavior. Met. (No. 20
(Refer to the figure) Native HuIFNβ in Fig. 20 is human interferon β produced by treating human normal diploid fibroblasts with a drug.
Is. HuIFNβ derived from PC8 is pMTV (Ha, SV, r) β / PC8
It is human interferon β produced by the clone. Escherichia coli-derived human interferon β is human interferon β having no sugar chain, which is produced by Escherichia coli transformed with the human interferon β Escherichia coli expression vector.

From this result, it was confirmed that the human interferon β produced by the pMTV (Ha, SV, r) β / PC8 clone is a polypeptide having a sugar chain added, as in the case of the natural type.

Example 41 Construction of human interleukin-2 expression vector pMTV (SV) IL2: pMTV (SV) IL2 is a pML322 part of the human interleukin-2 expression vector pSVhIL2 (Japanese Patent Application No. 60-171250).
The vector pSVIL2, which was replaced with
This is a vector in which the MMTV promoter is inserted. The manufacturing method is as follows. (See FIG. 21) i) Preparation of pSVIL2 First, pSVhIL2 (described above) was cleaved with HindIII and BamHI to isolate a short chain fragment containing the human interleukin 2 gene.

Next, pSBβ of Example 1 was digested with HindIII and BamHI to give p
A long fragment containing the ML2 sequence and the SV early promoter was isolated.

These two DNA fragments were ligated with T4 DNA ligase to obtain pSVIL2.

ii) Construction of pMTV (SV) IL2 First, pSVIL2 of () was cleaved with HindIII located downstream of the SV40 early promoter, and the terminal phosphorus was removed by treatment with BAP (described above).

Next, pMTVdhfr (described above) was cut with HindIII to obtain 1.4 Kb
The MMTV promoter fragment was isolated.

These two DNA fragments were ligated with T4 DNA ligase to obtain pMTV (SV) IL2.

Example 42 Transformation of PC12 with pMTV (SV) IL2: 2 μg of pMTV (SV) IL2 of Example 41 and pSV2neo (described above).
2 μg and about 10 PC1 by the calcium phosphate method (described above)
2 cells were introduced. Protein synthesis inhibitor G418 (GIBCO) 40
Selection medium containing 0 μg / ml (RPMI1640 medium containing 10% fetal calf serum and 100 μg / ml kanamycin (Nissui Pharmaceutical))
After culturing in, 25 transformants were obtained.

The interleukin 2 activity of the culture supernatant was determined by the method of T. Mosmann (J.Immuno.Methods., 65 , 55, 1983) based on the mouse cell line CTLL2 (S.Gillis et al, J. Immuno., 120 , 2027). (19
78)) was used to measure 10 to 25 out of 30 to 640
An activity of unit / ml was observed.

Transformation of PC12 according to Example _{43 pSVhIL2 / BPV 97c: pSVhIL2 /} BPV 97c ( Japanese Patent Application No. Sho 60-171250) is a gene sequence comprising human interleukin 2 gene placed under the control of the SV40 early promoter PdBPV1 (above The vector introduced into the PvuII site of

2 μg of pSVhIL / 2BPV _97c and 0.2 μg of pSV2neo (described above) were introduced into about 10 PC12 cells by the calcium phosphate method (described above). Selection medium containing protein synthesis inhibitor G418 (GIBCO) at a concentration of 400 μg / ml (10% fetal calf serum and 10 kanamycin)
When cultured in RPMI1640 medium containing 0 μg / ml (Nissui Pharmaceutical), 25 transformants were obtained.

The interleukin 2 activity of the culture supernatant was determined by the method of T. Mosmann (J.Immuno.Methods., 65 , 55, 1983) based on the mouse cell line CTLL2 (S.Gillis et al, J. Immuno., 120 , 2027). (19
78)) was used to measure 14 out of 25 to 6 to 1770
An activity of unit / ml was observed.

[Effects of the Invention] According to the present invention, a human cell line capable of producing natural human interferon with high efficiency and having an unlimited number of passages could be established.

[Brief description of drawings]

FIG. 1 shows a method for producing pSVβ, which is an example of the vector of the present invention. FIG. 2 shows a method for producing pSV (r) β, which is an example of the vector of the present invention. FIG. 3 shows an example of the vector of the present invention, pSV (Ha, r) β.
2 shows a manufacturing method of. FIG. 4 shows a method for producing pMTVβ, which is an example of the vector of the present invention. FIG. 5 shows a method for producing pMTV (r) β, which is an example of the vector of the present invention. FIG. 6 shows a method for producing pMTV (Ha) β, which is an example of the vector of the present invention. FIG. 7 shows a method for constructing pMTV (SV) β, which is an example of the vector of the present invention. FIG. 8 shows an example of the vector of the present invention, pMTV (SV, r) β
2 shows a manufacturing method of. FIG. 9 is an example of the vector of the present invention, pMTV (Ha, r) β
2 shows a manufacturing method of. FIG. 10 shows an example of pMTV (Ha, SV,
r) It shows a method for producing β. FIG. 11 shows an example of the vector of the present invention, pBPV97SV (r).
It shows a method for producing β. FIG. 12 shows a method for producing pDH (r) β, which is an example of the vector of the present invention. FIG. 13 shows an example of the vector of the present invention, pBPVMTV (Ha).
It shows a method for producing β. FIG. 14 shows an example of the vector of the present invention pBPV ₉₇ MTV (Ha)
It shows a method for producing β. FIG. 15 shows a method for producing pMTVγ, which is an example of the vector of the present invention. FIG. 16 shows a method for producing pMTV (SV) γ, which is an example of the vector of the present invention. FIG. 17 shows the activity of human interferon β produced by the host-vector system of the present invention. FIG. 18 shows the activity of human interferon γ produced by the host-vector system of the present invention. FIG. 19 shows continuous production of human interferon β of the pMTV (Ha, SV, r) β / PC8 clone of the present invention, and the effects of serum concentration and dexamethasone on the productivity. FIG. 20 shows the SDS polyacrylamide gel electrophoresis pattern of human interferon produced by the pMTV (Ha, SV, r) β / PC8 clone of the present invention. FIG. 21 shows pMTV (SV) IL2 which is an example of the vector of the present invention.
2 shows a manufacturing method of.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12P 21/02 9162-4B C12N 15/00 A C12R 1:91) (C12N 5/00 B C12R 1:91)

Claims (1)

(57) [Claims] 1. A human lung cancer-derived cell PC8 or human lung cancer-derived cell PC12 is transformed with a vector having the human interferon β gene, human interferon γ gene or human interleukin 2 gene under the control of a eukaryotic promoter. The transformant thus obtained.