JP2555816B2 - Method for producing human interleukin-2 protein - Google Patents

Description

Detailed Description of the Invention

The present invention relates to a method for producing human interleukin-2 protein by a genetic engineering technique.

[0002] Interleukin-2 [hereinafter sometimes abbreviated as IL-2. IL-2 is also called T cell growth factor (TCGF). ] Is a lymphokine produced by T cells stimulated with lectin, alloantigen, etc. [Science, 193, 1007 (197)
6 years); Immunological Review, Vol. 51, 257
P. (1980)]. In addition to allowing T cells to proliferate while retaining their functions in vitro and maintaining long-term passage, IL-2 has also promoted the mitogen response of thymocytes (costimulator), It has been reported that mouse spleen cells have an activity of restoring the ability to produce antibodies against a T cell-dependent antigen (T cell replacing factor) and promoting the differentiation and proliferation of killer cells (killer helper factor) [The Journal]・
Of Immunology, Vol. 123, p. 2928 (197
9), Immunological Review, Volume 51, 257
P. (1980)].

[0003] By using IL-2, killer T
Many clones such as cells, helper T cells, and natural killer cells have been obtained [for example, Nature, 268, 154 (1977); The Journal of Immunology, 130, 981]. (1983)]. In addition to such direct use as cloning of T cells and natural killer cells, IL-
2 can be used to selectively proliferate in vitro antigen-specific killer T cells that recognize and destroy certain specialized antigens, such as tumor antigens. The tumor-specific killer T cells grown in this way can be transferred to animals to suppress and inhibit tumor growth [The Journal of Immunology, Vol. 125, p. 1904 (1980).
Year)〕. In addition, IL-2 induces interferon γ production [(The Journal of Immunology, Vol. 130, p. 1784 (1983)] and activates natural killer cells [The Journal
Of Immunology, 130, 1970 (198
3 years)] is known. These experimental facts indicate that IL-2
Indicates the possibility of being used as an antitumor agent.
IL-2 also restores helper T cell function in nude mice lacking thymic function [European Journal of Immunology, 10, 719].
(1980)] and restoring the induction of killer T cells to allogeneic cells [Nature, 284, 2].
78 (1980)], and can be expected to be applied to diseases with reduced immune function. However, human IL-
No. 2 cannot be clinically applied at present because of its difficulty in mass production, and there is a demand for the development of a technology that enables easy and inexpensive mass production of high-purity human IL-2. Tani
guchi et al., IL-2 m of human T leukemia cell line Jurkat
Cloning the human IL-2 gene from RNA,
The amino acid sequence of the human IL-2 protein deduced therefrom was reported [Nature, 302, 305 (19).
1983)]. Subsequently, Devos et al. Reported that the IL-2 gene derived from human spleen cells was cloned and expressed in Escherichia coli [The Nucleic Acid Research, Vol. 11, p. 4307 (1983)]. . However, until now, it has IL-
Only the presence of 2-like substances was estimated, and in reality human I
There is no report that a human IL-2 protein produced by a transformant containing DNA encoding L-2 was purified and obtained.

The present inventors cloned the human IL-2 gene using gene manipulation technology and transferred the obtained recombinant DNA molecule into a host to express the human IL-2 gene. As a result of research and development of a technique capable of obtaining an IL-2 protein, a method for producing a substantially pure non-glycosylated human IL-2 protein was established and the present invention was completed. That is, the present invention provides ( 1 ) a human interleukin-2 obtained by culturing an Escherichia coli transformant containing a DNA having a nucleotide sequence encoding human interleukin-2, allowing human interleukin-2 to be produced and accumulated in the culture. 5 × 1 human peripheral blood lymphocytes characterized in that an interleukin-2 containing solution is purified by a purification step including a guanidine hydrochloride extraction step and a reverse phase high performance liquid chromatography treatment.
0 ⁶ cells / ml and made as 10% FCS added RPMI1
Suspended in 640 medium and concanavalin-A 40 μg / ml
And 12-O-tetradecanoylphorbol-13-
Add 15 ng / ml of acetate and add 5% CO at 37 ℃.
The supernatant of the culture cultivated for 48 hours in the presence of ₂ was added at 1 U / m.
A method for producing a substantially pure non-glycosylated human interleukin-2 protein having a specific activity of 10 ⁴ U / mg or more measured by the method defined as 1 and having an extremely low content of endotoxin and pyrogen. It is provided. D encoding human IL-2 used in the present invention
Examples of NA include DNA (I) having a nucleotide sequence represented by codons 1 to 133 in FIG. This DNA
(I) may have ATG or a signal codon represented by codons S1 to S20 in FIG. 2 at its 5'end, and preferably has TAA, TGA or TAG at its 3'end, and particularly TGA is preferred. DNA
(I) is preferably linked downstream of a promoter, and tryptophan (t
rp) promoter, recA (recA) promoter, λPL promoter and the like, and particularly tr
The p promoter is preferred. In the present invention, for example, mRNA encoding human IL-2 is isolated from a culture solution of human peripheral leukocytes stimulated with concanavalin A or the like, and single-stranded cDNA is synthesized using reverse transcriptase or the like, and then double-stranded. Synthesize strand DNA. Then, a double-stranded DNA encoding human IL-2 can be produced by introducing the plasmid into a plasmid, transforming, for example, Escherichia coli or Bacillus subtilis, and isolating the cDNA-containing plasmid.

[0005] mRNA encoding human IL-2 used in the present invention can be obtained by the method of Hinuma et al. [Biochemical and Biophysical Research Communications, Vol. 109, p. 363 (1982)]. it can. Using the obtained human IL-2 mRNA as a template, cD
Synthesize an NA chain and convert cDNA to double-stranded DNA [M
aniatis, T. et al., Cell, Vol. 8, p. 163 (1976).
Land, H. et al., Nucleic Acid Research, Vol. 9, p. 2251 (1981)]. This DNA
By the dG-dC homopolymer binding method [Nelson, T .;
S., Methods in Engineering, 68, 41
(1979)], for example, the plasmid pBR3
Incorporation into 22 PstI restriction endonuclease cleavage sites. Further, for example, after chemically synthesizing an oligonucleotide having a nucleotide sequence corresponding to a part of the amino acid sequence of human IL-2, labeling it with ³² P and using it as a probe, a colony hybridization method [known in the art] Grun
stein, M., Hogness, DS, Proceedings of National Academy of Sciences US
A, 72, 3961 (1975)]; Alwine,
JC et al., Methods in Engineering, No. 68
Vol., P. 220 (1979)], a clone to be sought is selected from tetracycline-resistant or ampicillin-sensitive transformants. The nucleotide sequence of the DNA of a clone that showed a positive result in the above hybridization method was determined by the Maxam-Gilbert method [Maxam, AM et al., Proceedings of the National Academy of Sciences USA, Vol. 74, 560]. Page (197
7 years)] or a method for terminating the dinucleotide synthesis chain using phage M13 [Messing, J. et al.
Acid Research, Vol. 9, p. 309 (1981)
Year)] to confirm the presence of the human IL-2 gene. Next, all or part of the human IL-2 gene was cut out from the obtained clone, and an appropriate promoter in the plasmid, SD (Shine and Dalgarno) was used.
It can be introduced into an appropriate host by being linked downstream of the nucleotide sequence.

As a promoter, a trp promoter or the like is advantageously used, and as a host, Escherichia coli (294 strain, DH1 strain, etc.) is advantageously used. E. coli 294 strain [Be
ckman et al., Proceedings of the National Academy of Sciences USA, Vol. 73,
4174 (1976)] and one strain of DH [Selson,
ME et al., Nature, 217, 1110 (1
968)] are all known strains. Transformation of a host with DNA can be carried out by a known method [Cohen, SN et al., Proceedings of the National Academy of Sciences, USA, 69, 2110 (1972)]. . The host thus obtained is used in a medium known per se, for example, an M9 medium containing glucose and casamino acid [Miller, J., Experiments Molecular Genetex, 43
Pages 1-433 (Cold Spring Harbor Laboratory, New York, 1972)]. When using the trp promoter, an agent such as 3-β-indolylacrylic acid can be added to make it work efficiently. Culture is usually 1
The reaction can be performed at 5 to 43 ° C. for 3 to 24 hours, and aeration or stirring can be performed as necessary. Human I thus produced
Although an IL-2 dependent cell line can be used for the measurement of L-2, human IL-2 is known to promote proliferation of IL-2 dependent cells such as rat and mouse in addition to human. There [Immunological Review, Vol. 51,
257 (1980)], it is possible to use not only IL-2-dependent human cell lines but also rat or mouse IL-2-dependent cell lines [Journal of Immunology, Vol. 130, p. 988 pages (198
3 years)]. In particular, a mouse IL-2-dependent cell line can be stably maintained for a long period of time, so that highly reproducible measurement results can be obtained.

In extracting human IL-2 of the present invention from cultured cells, after culturing, the cells are collected by a known method, and the cells are suspended in a buffer containing a protein denaturant such as guanidine hydrochloride. After stirring in a cold place, IL-2 was centrifuged.
Or a method of obtaining a supernatant containing IL-2 by centrifugation after disrupting the bacterial cells by sonication, lysozyme and / or freeze-thawing, etc. Can be used as appropriate. In order to separate and purify IL-2 from the supernatant, known separation and purification methods can be appropriately combined. These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and the like. Method using difference, method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity chromatography, method using difference in hydrophobicity such as reversed phase high performance liquid chromatography And a method utilizing the difference in isoelectric point, such as isoelectric point electrophoresis. In particular, human IL
Since -2 protein has high hydrophobicity, hydrophobic column chromatography, particularly high-performance liquid chromatography using a reversed-phase column, is extremely effective in purifying the protein. Therefore, in the purification step of the present invention,
A step using reversed phase high performance liquid chromatography is employed.

For example, cells obtained by culturing Escherichia coli containing a gene encoding human IL-2 are suspended in a protein denaturant such as guanidine hydrochloride (preferably used at a concentration of 2M to 8M) and stirred. The supernatant is then collected by centrifugation. The supernatant liquid is dialyzed as it is or after being concentrated by an ultrafiltration device or the like, the resulting precipitate is removed by centrifugation, and the resulting supernatant liquid is, for example, diethylaminoethyl cellulose [DE52 cellulose (manufactured by Whatman, USA) column etc.] And perform anion exchange chromatography to collect active fractions. Next, after the active fraction was concentrated using an ultrafiltration apparatus, for example, N, N ′
-Methylenebisacrylamide-bridged allyl dextran such as Sephacryl S-200 (manufactured by Pharmacia,
Perform gel filtration using a Swedish column or the like. The active fractions are collected and then subjected to the above-described high performance liquid chromatography. By such a method, the non-glycosylated human IL-2 of the present invention can be obtained. The reversed phase column used in the high performance liquid chromatography includes, for example, an alkylated (about C _1-18 ) silicon column. As the elution solvent, C _1-6 lower alkanols (ethanol, propanol, etc.) and acetonitrile can be advantageously used, and those having a pH of 1.5 to 4 are preferable.
The elution rate is preferably from 0.1 to 100 ml / min. If necessary, the human IL-2 protein solution obtained here can be lyophilized into a powder. For lyophilization, sorbitol, mannitol, dextrose,
Maltose, glycerol, human serum albumin (HS
Stabilizers such as A) can be added.

The human IL-2 protein obtained by the present invention is I
In the measurement of IL-2 activity using the radioactive thymidine incorporation as an index using L-2-dependent mouse cells, 1 × 10 ⁴
It shows a specific activity of U / mg or more. According to the present invention, a high-purity non-glycosylated human IL-2 protein having a specific activity of 2 × 10 ⁴ U / mg or more and reaching 3 × 10 ⁴ U / mg can be obtained. The unit (U) as the activity of IL-2 was calculated as follows. That is, a sample containing IL-2 was added to a medium in which a mouse cell line that proliferated depending on the IL-2 concentration was suspended, and cultured, and the growth of the cell line was determined using tritium thymidine uptake as an index. In order to calculate the unit (U) in the target sample, the standard IL-2 (1 U / ml) was always arranged and assayed, and the unit was calculated from the ratio.
Specifically, I was subcultured and maintained in RPMI 1640 medium supplemented with 20% FCS containing conditioned medium containing human IL-2 at 37 ° C. in the presence of 5% CO _2.
L-2 dependent mouse cell line (NKC3), Hinuma et al.
Biochemical Biophysical Research Communications, Vol. 109, p.363 (1982)
)] Was washed twice with serum-free RPMI 1640 medium, and 6 × 1 was added to RPMI 1640 medium supplemented with 20% FCS.
0 to ⁵ cells / ml as the re-floating. 50 μl of the material containing IL-2 was placed in the well of the first row of a 96-well flat bottom microtiter plate (Nunc, Denmark), and 50 μl was added.
Using a 20% FCS-supplemented RPMI 1640 medium, a two-fold serial dilution series was sequentially prepared up to the twelfth column.
Dispense 50 μl of KC3 cell suspension into each well and incubate at 37 ℃
Culture for 24 hours in the presence of 5% CO ₂ . After 20 hours of culture, 1 μCi of tritium thymidine (Amersham, UK) was added to each well and the culture was continued for another 4 hours. Then, cells were collected on a glass filter using a cell harvester (Flow, USA). Then, the uptake of tritium thymidine is measured using a liquid scintillation counter. At the time of measurement, the same operation as that of the sample is performed for a standard IL-2 sample, and the incorporation of tritium thymidine is measured.

The calculation of the unit (U) is based on the journal of
Immunology, Vol. 120, p. 2027 (1978)
Probit conversion method according to That is, a standard IL-2 sample (5 × 10 ⁶ human peripheral blood lymphocytes / ml)
Suspension in 10% FCS-supplemented RPMI 1640 medium to give concanavalin-A 40 μg and 12-O-
Tetradecanoylphorbol-13-acetate 15ng
/ U, and the centrifuged supernatant of the culture solution cultured at 37 ° C in the presence of 5% CO ₂ for 48 hours is defined as 1 U / ml). Calculate the percentage (%) of the uptake value for each dilution step. The obtained numerical values are plotted on a normal probability paper, and a dilution factor indicating 50% uptake is obtained from the drawing. Similarly, for each sample containing IL-2, a dilution factor indicating 50% uptake is determined. Material IL
-2 concentration (U / ml) is calculated according to the following formula:

[Equation 1] The natural IL-2 specific activity obtained from human peripheral blood determined by this quantification method is 20,000 to 70,000 U.
/ Mg, indicating a specific activity almost equivalent to that of the non-glycosylated human IL-2 protein of the present invention.

[0011] The non-glycosylated human IL-2 protein of the present invention preferably has a polymorphism comprising the amino acid sequence shown in FIG. 3 (SEQ ID NO: 2), wherein X represents Met or hydrogen. It contains peptide (II).
The human IL-2 protein produced according to the present invention has the following properties. 1) Uniform by SDS-polyacrylamide gel electrophoresis. The molecular weight measured by this method is 15,000 ± 100.
It is 0 Dalton. 2) The amino terminal amino acid has alanine or methionine. 3) Has threonine as the carboxy terminal amino acid. 4) It has the activity of proliferating normal T cells and natural killer cells while maintaining their functions.

The human IL-2 protein produced according to the present invention has a limulus test [hemostasis, Vol. 7, 183].
Page (1978)] is negative and contains very few contaminating proteins and pyrogens, so that it can be safely used as an injection drug substance. Non-glycosylated human IL- obtained according to the present invention
The two proteins have low toxicity and have the activity of proliferating normal T cells and natural killer cells while maintaining their functions. Therefore, the IL-2 protein of the present invention can be used to proliferate, passage or clone T cells and natural killer cells in vitro for a long period of time. In addition,
Using this property, the activity of human IL-2 can be measured. Further, the human IL-2 protein of the present invention may be, for example, an antigen-specific killer T cell that recognizes and destroys a tumor antigen, or a natural killer cell capable of killing a tumor regardless of whether or not it has undergone antigen sensitization. Can be selectively expanded in vitro, and when the killer T cells are transferred to a living body, their antitumor effect is increased by co-inoculation with the human IL-2 of the present invention. It can be used for the prevention and treatment of tumors in blood animals (eg, mice, rats, rabbits, dogs, cats, pigs, horses, sheep, cows, humans, etc.) and for the treatment of immune-compromised diseases. Since the human IL-2 protein of the present invention is purified to high purity, it has no antigenicity and low toxicity. To use the human IL-2 protein of the present invention as a prophylactic or therapeutic agent for tumors, the protein is mixed and diluted with a carrier known per se and administered parenterally or orally, for example, as an injection or capsule. can do. In addition, it can be used with or without killer T cells or natural killer cells grown in vitro as described above.

The human IL-2 protein of the present invention has substantially the same biological activity as that of known human IL-2 isolated from nature and can be used in the same manner. Since the dissociation constant with the body is extremely small, a very small dose is sufficient. In order to use T cells for the purpose of expanding in vitro, the human IL-2 of the invention
From about 0.01 to 1 unit / ml, preferably from about 0.1 to 1 unit / ml.
It can be used after being added to the medium at a concentration of 0.5 unit / ml. Specific examples of the use for the purpose of proliferating T cells in vitro include, for example, T cells (1 × 10 ⁶ cells / ml) separated from human peripheral blood in RPMI 1640 medium containing 20% fetal bovine serum and X-ray ( B cell transformants (1 x 10 ⁶ ) irradiated with 1500 rads
Cells / ml) and mixed culture of lymphocytes at 37 ° C. in the presence of 5% CO ₂ for 3 days. The IL-2 protein of the present invention was added to a cell suspension containing alloantigen-sensitized T cells in an amount of 0.1%. 1 to
A method of continuing culture for about one month while changing the medium at a concentration of 0.5 unit / ml about once a week, and the like.
The transformant disclosed in the Examples below, E. coli
DH1 / pTF4 is the Institute of Fermentation (Institute f)
or Fermentation, Osaka) as IFO-14299, and this transformant was deposited with the Research Institute of Microorganisms and Technology (FRI) of the Ministry of International Trade and Industry of Japan under the accession number FE.
Deposited as RM P-7578, which has been converted to a deposit under the Budapest Treaty and has been deposited with accession number FERM.
It is stored at the same research institute (FRI) as BP-628. In the specification and the drawings of the present application, when bases, amino acids, and the like are indicated by abbreviations, IUPAC-IUB
It is based on the abbreviation by the Commission on Biochemical Nomenclature or the abbreviation commonly used in this field, and examples thereof are shown in [Table 1]. When amino acids may have optical isomers, L-
The body shall be indicated.

[Table 1] ──────────────────────── DNA: Deoxyribonucleic acid cDNA: Complementary deoxyribonucleic acid A: Adenine T: Thymine G: Guanine C: Cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate dCTP: deoxycytidine triphosphate ATP: adenosine triphosphate EDTA: ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulphate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine The: Threonine Cys: Cysteine 1 / 2Cys: Half cystine Met: Methionine Glu: Glutamic acid asparasic acid Asparagine: Asparaginate : Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine ─────────────────────────

Example 1 (i) Isolation of mRNA Encoding Human IL-2 Lymphocytes prepared from human peripheral blood were isolated from 12-O-tetradecanoylphorbol-13-acetate (TPA) (1
R containing 5 ng / ml) and concanavalin A (40 μg / ml)
PMI 1640 medium (containing 10% fetal calf serum)
The medium was cultured at 37 ° C. to induce IL-2. After 24 hours, the induced 1 × 10 ¹⁰ human lymphocytes were treated with 5M guanidine thiocyanate, 5% mercaptoethanol,
After destruction and denaturation in a 50 mM Tris.HCl pH 7.6, 10 mM EDTA solution using a Teflon homogenizer, sodium N-lauroylylsarcosinate was added to a concentration of 4%, and the homogenized mixture was added to a 5.7 M cesium chloride solution (5%). (0.7 M cesium chloride, 0.1 M EDTA) and 6 ml of Beckman SW28 rotor.
Centrifugation was performed at 24,000 rpm for 48 hours at ℃ to obtain an RNA precipitate. This RNA precipitate was dissolved in a 0.25% sodium N-lauroyl sarcosinate solution and then precipitated with ethanol to obtain 10 mg of RNA. This RNA was added to a high salt solution [0.5 M NaCl, 10 mM Tris.HCl pH 7.
6,1 mM EDTA, 0.3% SDS] in oligo (d
T) mR containing poly (A) adsorbed on cellulose column
NA was adjusted to a low salt solution [10 mM Tris-HCl pH 7.6, 1
By eluting with [mM EDTA, 0.3% SDS], 300 μg of mRNA containing poly (A) was collected. This mRNA was further precipitated with ethanol, and 0.2 ml of a solution (10 mM Tris · HCl pH 7.6, 2 mM EDT) was added.
A, 0.3% SDS), treated at 65 ° C. for 2 minutes, and centrifuged at 10-35% sucrose density gradient (Beckman S)
2 at 20 ° C and 25000rpm using a W28 rotor
(1 hour centrifugation) to obtain 22 fractions. A portion of the RNA for each of these fractions was injected into Xenopus oocytes and the I
The L-2 activity was measured and fractions 11 to 15 (precipitation constant 8S
15S), the activity of IL-2 was detected. IL of this fraction
-2 mRNA was about 25 µg.

(Ii) Synthesis of single-stranded DNA Using the mRNA and reverse transcriptase obtained above, 100 μm
l of the reaction mixture (5 μg mRNA, 50 μg oligo (dT),
100 units of reverse transcriptase, 1 mM dATP. d
CTP, dGTP and dTTP, 8 mM MgCl ₂ , 50
mM KCl, 10 mM dithiothreitol, 50 mM Tri
s-HCl pH 8.3) at 42 ° C. for 1 hour and then deproteinized with phenol to give 0.1 N NaOH.
RNA was decomposed and removed by treatment at 70 ° C. for 20 minutes. (iii) Synthesis of double-stranded DNA By reacting the single-stranded complementary DNA synthesized here in 50 μl of reaction solution (the same reaction solution as above except that mRNA and oligo dT are not included) at 42 ° C. for 2 hours. Double-stranded DN
A was synthesized. (iv) Addition of dC tail To this double-stranded DNA, 50 μl of reaction solution of nuclease S1 (double-stranded DNA 0.1 M sodium acetate pH 4.5,
The mixture was allowed to act in 0.25 M NaCl, 1.5 mM ZnSO ₄ , 60 units of S1 nuclease) at room temperature for 30 minutes, deproteinized with phenol, precipitated with DNA in ethanol, and then mixed with 50 μl of a terminal transferase reaction solution (50 μl). Double stranded DNA, 0.14 M potassium cacodylate, 0.1
3M Tris (base) pH 7.6, ₂ mM dithiothreitol, 1 mM CoCl ₂ , 0.15 mM dCTP, 30 units terminal transferase) for 3 minutes at 37 ° C. The deoxycytidine chain was extended. About 30 in these series of reactions
A double-stranded DNA having 0 ng of deoxycytidine chain was obtained. (v) Cleavage of E. coli plasmid and addition of dG tail On the other hand, 10 μg of E. coli plasmid pBR322 DNA
And 50 μl of the reaction solution (10 μg DN)
A, 50 mM NaCl, 6 mM Tris.HCl, pH 7.4
6 mM MgCl ₂ , 6 mM 2-mercaptoethanol, 1
00 μg / ml bovine serum albumin, 20 units Pst
PBR322 DNA by working in I) for 3 hours at 37 ° C
One PstI recognition site is cleaved and the protein is removed with phenol.
0 μl of the reaction solution (DNA 10 μg, 0.14 M potassium cacodylate, 0.3 M Tris, base pH 7.6, ₂ mM dithiothreitol, 1 mM CoCl ₂ , 0.15 mM GTP, 30
3 minutes in unit terminal transferase)
It was allowed to act at 7 ° C. to extend about 17 deoxyguanine chains at both 3 ′ ends of the above plasmid pBR322 DNA.

(Vi) Association of cDNA and transformation of Escherichia coli 0.1 μg of the synthetic double-stranded DNA thus obtained and 0.1 μN of the above plasmid pBR322, 0.5 μg were added.
Cl, 50 mM Tris · HCl pH 7.6, 1 mM EDTA
In a solution consisting of 65 ° C. for 2 minutes and 45 ° C. for 2 hours, and then slowly cooled to associate them with the method of Enaa et al. [J. Mol. Biol.,
96 , 495 (1975)].
4 was transformed. (vii) Isolation of cDNA-Containing Plasmid In this way, about 20,000 tetracycline-resistant strains were isolated, and the DNA of each of them was immobilized on a nitrocellulose filter. The IL reported by Taniguchi et al. [Nature, 302 , 305 (1983)]
-2 based on the amino acid sequence of -2
^{78 (Lys 74 -His-Leu-} Gln-Cys) and amino acid ^{No. 122~126 (Thr 122 -Phe-Met} -Cys-
Glu) (5 'AAA CAT CT
T CAG TGT 3 'and 5' ACA TTC A
Oligonucleotides complementary to TG TGT GAA 3 ') were prepared by the triester method [Crsa, R. et al. Proc, Na.
tl. Acad. Sci. USA, 7 5, 5765 (197
8)]. 50μ using T4 polynucleotide kinase against this oligonucleotide
l of the reaction mixture (0.20 μg of oligonucleotide, 50 mM
Tris · HCl pH 8.0, 10 mM MgCl ₂ , 10 mM mercaptoethanol, 50 μCiγ- ³² PATP, 3 units T4 polynucleotide kinase for 1 hour
The reaction was carried out at 7 ° C. and the 5 ′ end was labeled with ³² P. Using this labeled oligonucleotide as a probe, the method of Lawn et al. [Nucleic Acids Res., 9 , 6103 (198
According to 1)], the DNA was associated with the DNA immobilized on the nitrocellulose filter, and four strains which reacted with the two oligonucleotide probes were isolated by autoradiography. Plasmid DNA was prepared from each of these strains by the alkaline method [Birncoim HC &
Doly, J. Nucleic Acids Res., 7 , 1513 (197
9)]. Next, the inserted portion of the plasmid DNA was cut out with the restriction enzyme PstI, and among the separated plasmids, those containing the fragment having the longest inserted portion were selected, and this plasmid was named pILOT135-8. A restriction map of this plasmid is shown in FIG. Next, the primary structure (base sequence) of the cDNA sequence inserted into the pILOT-135-8 plasmid was determined by the dideoxynucleotide method and the Maxma-Gilbert method. Its primary structure is [Figure 2] (sequence list: SEQ ID NO: 1).
It was shown to. The peptide defined by this base sequence consists of 153 amino acids starting from its synthesis initiation signal (ATG of Nos. 64-66). 20 from the N-terminal
This amino acid is considered the signal peptide. From the above primary structure, it was found that this plasmid had the entire nucleotide sequence encoding the human IL-2 protein.
By this fact, any polypeptide of the IL-2 protein can be produced by incorporating the gene integrated in the plasmid into another expression plasmid.

Example 2 Plasmid pILOT135-8 obtained in Example 1 was digested with restriction enzyme HgiA1 to obtain a DNA fragment containing 1294 bp IL-2 gene. After treating this DNA fragment with T4 polymerase, a ClaI linker having a codon GCA of alanine and a codon ATG of methionine, CGAT
After AATGGCA was bound and treated with ClaI and PstI, it was incorporated into ClaI and PstI sites of ptrp 771 and the resulting expression plasmid was designated as pTF4 (FIG. 4).

Example 3 Using the plasmid pTF4 obtained in Example 2, the method of Cohen et al. [Proc. Natl. Acad. Sci. USA, 69 , 21].
10 (1972)], and transformants containing the plasmid (Escherichia
coli DH 1 / pTF4) was obtained.

Example 4 250 ml of E. coli DH1 / pTF4 obtained in Example 3
Bacto Tripton (Difco Laboratories, USA) 1% in a Erlenmeyer flask, Bacto Yeast Extract (Difco Laboratories, USA) 0.5%,
The cells were inoculated into 50 ml of a liquid medium (pH 7.0) containing 0.5% of sodium chloride and 7 μg / ml of tetracycline, and cultured with shaking at 37 ° C. overnight. The culture was transferred to a 5 liter jar fermenter containing 2.5 liters of M9 medium containing 0.5% casamino acid, 0.5% glucose and 7 μg / ml tetracycline, and was placed at 37 ° C. for 4 hours, then 3-β -Indolylacrylic acid (25 µg / ml) was added, and the mixture was further aerated and stirred for 4 hours to obtain 2.5 liters of a culture solution. The culture was centrifuged to collect the cells, frozen at -80 ° C and stored.

Example 5 12.1 g of the cryopreserved bacterial cell obtained in Example 4 was extracted with 7 M guanidine hydrochloride and 0.1 M Tris · HCl (pH 7.0.
0) Suspended uniformly in 100 ml and stirred at 4 ° C for 1 hour.
This lysate was centrifuged at 28,000 xg for 20 minutes to obtain 93 ml of supernatant.

Example 6 The supernatant obtained in Example 5 was dialyzed against a 0.01 M Tris / HCl buffer (pH 8.5), and then centrifuged at 19,000 × g for 10 minutes to obtain 94 ml of the dialyzed supernatant. Obtained. The dialysis supernatant was passed through a DE 52 (DEAE-cellulose, Whatman, UK) column (50 ml) equilibrated with 0.01 M Tris-HCl buffer (pH 8.5) to adsorb the protein, and the NaCl concentration was adjusted. Linear gradient (0 to 0.15M NaCl,
(1 liter) to elute IL-2. 53 ml of the active fraction was concentrated to 4.8 ml using a YM-5 membrane (Amicon, USA) to obtain 0.1M Tris HC.
Gel filtration was carried out using a Sephacryl S-200 (Pharmacia, Sweden) column (500 ml) equilibrated with l (pH 8.0) -1M NaCl buffer.
28 ml of the active fraction was concentrated to 2.5 ml with a YM-5 membrane. The obtained concentrated solution was adsorbed on a column of Ultrapore RPSC (manufactured by Artex, USA), and high-performance liquid chromatography was performed using a trifluoroacetic acid-acetonitrile system as an elution solvent. Column, Ultrapore R
PSC (4.6 × 75 mm); column temperature, 30 ° C .; elution solvent A, 0.1% trifluoroacetic acid-99.9% water; elution solvent B, 0.1% trifluoroacetic acid-99.9% acetonitrile. Elution program, 0 minutes (68% A + 32% B)
-25 minutes (55% A + 45% B) -35 minutes (45% A +
55% B) -45 minutes (30% A + 70% B) -48 minutes (100% B); elution rate, 0.8 ml / min; detection wavelength,
230 nm. Under these conditions, an active fraction having a retention time of about 39 minutes was collected, and 0.53 mg of non-glycosylated human IL-2 protein [specific activity, 30,000 U / mg, activity recovery from starting material, 30.6%; 10 ml of a solution containing 99% of protein purity (by densitometry) was obtained. The solution was freeze-dried to obtain a white powder. The specific activity of this powder is 2
It was 6,000 U / mg.

Example 7 The following properties of the human IL-2 protein obtained in Example 6 were examined. (1) Unity: Laemli's method [Nature, 227 , 68
0 (1970)], followed by SDS-polyacrylamide slab gel electrophoresis followed by staining with Coomassie brilliant blue. As a result, the human IL-2 protein showed a single band (see FIG. 5). The position of the band did not change under reducing or non-reducing conditions. (2) Molecular weight: The molecular weight of the human IL-2 protein is SD
Approximately 1 from S-polyacrylamide slab gel electrophoresis
It was calculated to be 5,000 daltons (see FIG. 5). From the results of the above items (1) and (2), it is clear that the human IL-2 protein obtained in Example 6 contains substantially no oligomer.

(3) Amino acid composition: 20 μg of the human IL-2 protein was placed in a glass hydrolysis test tube, constant boiling hydrochloric acid containing 4% thioglycolic acid was added, and the tube was sealed under reduced pressure and then at 110 ° C. Hydrolysis for 24, 48, 72 hours. After hydrolysis, the tube was opened, hydrochloric acid was removed under reduced pressure, the residue was dissolved in 0.02N hydrochloric acid, and amino acid analysis was performed using a 835 type amino acid analyzer manufactured by Hitachi. Cystine and cysteine were obtained by the half method [Methods in Enzymol., 1] .
1 , 197 (1967)], the human IL-2 protein is subjected to peroxidic acid oxidation, and then, in a constant boiling point hydrochloric acid under reduced pressure.
After hydrolyzing for a time, it was quantified as cysteinic acid by an amino acid analyzer. Amino acid analysis values were determined by averaging the values obtained after 24, 48 and 72 hours of hydrolysis. However, the values of serine and threonine were obtained by extrapolating the hydrolysis time to 0 hours. The results are shown in [Table 2].

[Table 2] ──────────────────── Amino acid mol% ──────────────────── Asp / Asn 8.8 Thr 9.3 Ser 5.7 Glu / Gln 13.7 Pro 3.4 Gly 1.7 Ala 3.8 1/2 Cys 2.3 Val Val 3.1 Met 3.7 Ile 6.3 Leu 16.3 Tyr 2.3 Phe 4.5 Lys 8.3 His 2.5 Arg 3.1 Trp 1.1 ───────────────────────

(4) N-terminal amino acid sequence: said human IL-
The N-terminal amino acid sequence was analyzed by applying an automatic Edman degradation method using a gas phase protein sequenator (Model 470A manufactured by Applied Biosystems, USA) to 34 μg of 2 proteins. Phenylthiohydantoin amino acid (PTH-amino acid) is available in Micropack SP-OD
It was identified by high performance liquid chromatography using an S column (Varian, USA). The PTH-amino acids detected in each step are shown in [Table 3].

[Table 3] Y in the table is undecided.

(5) C-terminal amino acid: 30 μg of the human IL-2 protein was placed in a glass hydrazine decomposition test tube, 0.05 ml of anhydrous hydrazine was added, the tube was sealed under reduced pressure, and the mixture was heated at 100 ° C. for 6 hours. . The resulting hydrazine hydrolyzate was freeze-dried and then dissolved in distilled water. Benzaldehyde was added to this solution, stirred at room temperature for 1 hour, and centrifuged to obtain a supernatant. The supernatant was freeze-dried and subjected to amino acid analysis using a Hitachi 835 amino acid analyzer. As a result, only threonine was detected. (6) Peptide map of trypsin digestion: the IL-2
15 μg of the standard was added to TPCK (L-1-tosylamide-2-
Phenylethyl chloromethyl ketone) trypsin (Washington, USA) 0.4 μg was reacted with 120 μl of 0.02 M sodium hydrogen carbonate at 37 ° C. for 18 hours. After adding 5 μl of 2-mercaptoethanol to the reaction solution and further reacting at 37 ° C. for 2 hours, 75 μl of 1% trifluoroacetic acid was added to the reaction solution to stop the reaction. The resulting reaction solution was analyzed by high performance liquid chromatography under the following conditions to obtain a map shown in FIG. Column: Ultrasphere Octyl (5μm, 4.6x
250 mm; manufactured by Altex Corporation, USA). Column temperature: 30 ° C Mobile phase: solution A, 0.02% trifluoroacetic acid-99.98
% Water B solution, 0.02% trifluoroacetic acid-99.98% acetonitrile 0 minutes (95% solution A + 5% solution B) -40 minutes (30% solution A +
70% B solution). Elution rate: 1.0 ml / min. Detection method: Fluorescence method using Florescamine (Roche, USA) [Analytical Biochem., 67 , 438]
(1975)].

(7) Activity against IL-2 dependent cell line: Biochem. Biophys. Res. Commun., 109 , 363.
(1982), the activity of the non-glycosylated human IL-2 protein of the present invention was measured.
The L-2 protein is an IL-2-dependent mouse cell line (NKC
3, [see FIG. 7]) and human cell lines (see [FIG. 8])
It had the activity of promoting the uptake of tritium thymidine for both of these. In addition, the IL-2 protein was used in an amount of 0.
RPMI-164 with 20% FCS to 5 U / ml
The NKC3 strain was suspended in 0 medium and suspended at 2 × 10 ⁵ cells / ml, and subcultured at 37 ° C. in the presence of 5% CO ₂ in a Limbro Multi Dish (Flow Co., USA). Cultured. The number of viable cells was counted every 2 to 3 days of culture, and the cells were repeatedly resuspended in the above fresh culture solution. As a result, as shown in FIG.
It had an activity capable of maintaining the growth of the KC3 strain for a long period of time.

Example 8 Formulation for Injection: The solution containing the non-glycosylated human IL-2 protein obtained in Example 6 was added to 0.1%
It is adsorbed under aseptic conditions onto a CM Toyopearl (Toyo Soda) column equilibrated with a 025 M ammonium acetate buffer (pH 5.0), and eluted with the above buffer containing 0.15 M NaCl. The eluate was diluted appropriately with 0.15M NaCl, HSA was added to a concentration of 0.5%, and the mixture was filtered through a membrane filter (0.22 μm in diameter). Each is dispensed into a vial and freeze-dried to prepare human IL-2 for injection. The injectable preparation is dissolved in 1 ml of distilled water for injection before use.

Experimental Example 1 With respect to the human IL-2 protein obtained in Example 6, the thiol group was quantified and the position of the thiol group was determined. (1) First, for quantification of thiol groups, Ellman's method by the DTNB method [Archives of Biochemistry and Biophysics (Archives of Bio
Chemistry and Biophysics) Vol. 32, p. 70 (1959)
Year)). That is, 6M guanidine hydrochloride-10 mM ethylenediaminetetraacetic acid containing 0.4 mg (26.7 nmol) of human IL-2 protein obtained in Example 6
Disodium-75 mM Tris-HCl buffer (pH 8.3)
To 1.98 ml, 0.02 ml of a 10 mM DTNB [5,5'-dithiobis (2-nitrobenzoic acid)] methanol solution was added, and the reaction was carried out at room temperature for 30 minutes. The quantification was performed by measuring the yellow coloration of the reaction solution by absorbance at 412 nm, and using cysteine hydrochloride of a known concentration as a standard substance. As a result, the amount of human IL-2 protein was 0.1 per mole.
85 moles of thiol groups were detected and therefore human IL-
It was found that among the three cysteine residues of the two proteins, only one residue has a free thiol group and the other two residues form a disulfide bond with each other. (2) Next, the method for determining the position of the thiol group was reported by Egorov et al. [Proceedings of the National Academy of Sciences USA, No. 7].
2, 3029-3033 (1975)]. That is, 6 M guanidine hydrochloride-0.1 M sodium chloride-25 mM ammonium acetate buffer (pH 5.0) containing 6.3 mg of the human IL-2 protein obtained in Example 6.
3 ml of a thiopropyl Sepharose 6B (Pharmacia, Sweden) column (1.
0 cmφ × 5.1 cm, 4 ml) at a flow rate of 5 ml / h, and then 10 ml of the above buffer, 0.1 M sodium chloride-25 m
10 ml of M ammonium acetate buffer (pH 5.0) and 0.1 ml.
The column was washed with 10 ml of 2M acetic acid (pH 3.0).

Next, 4 ml of the thiopropyl sepharose 6B gel was removed from the column, and 0.2 M acetic acid (pH 3.
0) 6 ml was added and pepsin (Sigma, USA) was added.
The hydrolysis reaction was carried out while stirring at 37 ° C. for 15 hours using 1 mg. After the reaction, this gel was packed again in a column, washed with 10 ml of 0.2 M acetic acid (pH 3.0) and 10 ml of 25 mM ammonium acetate buffer (pH 4.5), and then washed with 20 mM 2-mercaptomethanol-25 mM ammonium acetate (pH 4.0. 5) Unreacted groups in the gel were eluted as 2-thiopyridone using 30 ml. Following on,
10 ml of 25 mM ammonium acetate buffer (pH 4.5) and 10 mM of 25 mM ammonium acetate buffer (pH 8.0)
After washing the column with 1 l of 20 mM 2-mercaptoethanol-25 mM ammonium acetate buffer (pH 8.
Elution was carried out with 0) at a flow rate of 5 ml / h to obtain 10 ml of a fraction containing a peptide fragment derived from a human IL-2 protein having a free thiol group. Further, this fraction was concentrated to dryness using a surband, dissolved in 0.5 ml of a 0.1% trifluoroacetic acid solution, and subjected to reversed-phase high-performance liquid chromatography under the following conditions. Column: Nucleosil 5C18 (0.8cmφ × 30cm,
(Nagel, West Germany) Column temperature: 30 ° C. Elution solvent A: 0.1% trifluoroacetic acid-99.9% water Elution solvent B: 0.1% trifluoroacetic acid-99.9% acetonitrile Elution program: 0 min ( 95% A + 5% B) -60 minutes (70% A + 30% B) -75 minutes (50% A + 50%
B) -85 minutes (40% A + 60% B) -95 minutes (25%
A + 75% B) -98 min (15% A + 85% B) Elution rate: 3 ml / min Detection wavelength: 230 nm As a result, four peaks were detected as shown in FIG. And concentrated to dryness with a servant, and amino acid composition analysis was performed for each. The results are shown in [Table 4].

[Table 4] ────────────────────────────── Amino acid composition ratio ───────────── ──────────── Peak 1 Peak 2 Peak 3 Peak 4 ─────────────────────────────── Asp / Asn 0.0 0.0 0.0 0.0 Thr 0.0 0.9 (1) 0.1 1.7 (2) Ser 1.5 (2) 1.6 (2) 0.9 (1) 1.6 (2) Glu / Gln 1.0 (1) 1.0 (1) 1.0 (1) 1.0 (1) Pro 0.0 0.0 0.0 0.0 Gly 0.0 0.0 0.1 0.0 Ala 0.0 0.0 0.0 0.0 1/2 Cys 0.7 (1) 0.8 (1) 0.8 (1) 0.8 (1) Val 0.0 0.0 0.0 0.0 Met 0.0 0.0 0.0 0.0 Ile 1.5 (2) 1.5 (2) 1.4 (2) 1.5 (2) Leu 0.0 0.0 0.0 0.9 (1) Tyr 0.0 0.0 0.0 0.0 Phe 0.0 0.0 0.0 0.0 Lys 0.0 0.0 0.0 0.0 His 0.0 0.0 0.1 0.0 Arg 0.0 0.0 0.0 0.0 Trp 0.1 0.0 0.1 0.0 Identified Cys125- Cys125- Cys125- Cys125- Fragment Ser130 Thr131 Ile129 Thr133 ─────────────────────────────── Numbers in parentheses indicate theoretical values From the above results, peak 1 is Cys125-Ser130, peak 2 is Cys125-Thr131, and peak 3 is Cys12.
5-Ile129, peak 4 was easily identified as Cys125-Thl131, indicating that all four peaks were fragments containing Cys125. In addition, the recovery rate was 32.6% through the whole processes of thiopropyl sepharose column chromatography, protease reaction, and reverse phase high performance liquid chromatography. Therefore, in the purified human IL-2 protein obtained in Example 6, among the three cysteine residues of the human IL-2 protein, Cys125 has a free thiol group, and Cys58 and Cys105.
Formed a disulfide bond, and did not include isomers having different thiol group bonding modes.

Experimental Example 2 The endotoxin of the human IL-2 protein obtained in Example 6 was quantified. The quantitative method was performed according to the method of Iwanaga et al. [Haemostasis, Vol. 7, pp. 183-188 (1978)]. (1) First, a standard endotoxin obtained from E. coli 0111-B4 (attached to an endotoxin detection reagent Pregel (manufactured by Teikoku Organ Pharmaceutical Co., Ltd.)) at 10 ⁻² ng /
Aqueous solutions having concentrations of ml, 10 ^-1 ng / ml, 3 x 10 ^-1 ng / ml, 1 ng / ml and 3 ng / ml were prepared. LAL (Limulus
Amebocyte Lysate, horseshoe crab blood cell extract component, Teikoku Organs Pharmaceutical Co., Ltd.) 25 μl, synthetic substrate Boc-Leu-Gly-Arg
-PNA (manufactured by Seikagaku Corporation, Boc: butoxycarbonyl, p
NA: paranitroanid) (1.6 mg / ml) and 50 μl of the above standard endotoxin aqueous solution were mixed,
After reacting at 37 ° C. for 60 minutes, 12.5% acetic acid was added,
The absorbance at 405 nm was measured twice, and the following results were obtained.

[Table 5] The above results were plotted to create [FIG. 11].

(2) Next, the human IL-2 protein obtained in Example 6 was dissolved in water to give 1.10 mg / ml and 1.16 mg / ml, respectively, to give aqueous solutions. These are referred to above (1)
The reaction was carried out in the same manner as in the above section, and the absorbance at 405 nm was measured.
From the obtained absorbance, the endotoxin concentration of the aqueous solution was calculated based on FIG. 11, and from this, the endotoxin amount per 1 mg protein of the human IL-2 protein obtained in Example 6 was calculated. The results are shown in the following [Table 6].

[Table 6]

Experimental Example 3 (1) Preparation of Crude Extract (i) Construction of Expression Plasmid Plasmid pILOT135-8 having the human IL-2 gene obtained in Example 1 (vii) was replaced with the restriction enzyme Hgi.
Cut with AI. The obtained 1294 bp DNA fragment was converted to T4
The ends were made blunt with DNA polymerase and EcoRI linker dTGCCATGAATTCATGGCA was ligated using T4 DNA ligase. The resulting DNA was digested with EcoRI, and the DNA containing the translation initiation codon ATG and the human IL-2 gene was digested.
An NA fragment was obtained. This DNA fragment was previously prepared with Eco R
I- Pst was digested with I site ptrp781 [Nucleic
Acids Research, Vol. 11, pp. 3077 (1983)]
Was inserted using T4 DNA ligase. The expression plasmid pTF1 thus obtained has a translation initiation codon and the human IL-2 gene downstream of the trp promoter. The plasmid pTF1 was cut with the restriction enzyme Stu I and ligated with the Bam HI linker. This plasmid DNA is
Treated with Bam HI and Eco RI, then Eco RI- B
plasmid p with λPL promoter at the am HI site
Inserted into TB281. The expression plasmid thus obtained was named pTB285. (ii) Production of transformant Escherichia coli N4830 was obtained using the plasmid pTB285 obtained above by the method of Cohen et al. [Procedures of National Academy of Science (Pro.
Natl. Acad. Sci USA), 69, 2110 (19).
72)], and a transformant Escherichia coli N4830 / pTB285 containing the above plasmid.
I got (iii) Culture and extraction of the transformant Escherichia coli N4830 / p of the transformant obtained above
TB285 was cultured in the same manner as in Example 4. E. coli transformant (8 g) thus obtained was treated with 20 m
M Tris-hydrochloric acid (pH 7.5, containing 30 mM NaCl)
It was washed with, and suspended in a buffer solution (40 ml) of the same composition, and this was subjected to sonication (0 ° C, 2 minutes). Furthermore, the digest was digested with lysozyme (1 μg / ml) (0 ° C, 20 minutes), and then freeze-thawed 3 times, and the whole was centrifuged (28,000).
Xg, 30 minutes). The two crude extracts thus obtained are referred to as Lot 01 and Lot 02.

(2) Culture, extraction and purification of the transformant The transformant Escherichia coli N4830 obtained above
/ PTB285 was cultured in the same manner as in Example 4, and extracted and purified in the same manner as in Examples 5 and 6. The two purified products of the recombinant human interleukin-2 (hereinafter sometimes abbreviated as rIL-2) thus obtained are referred to as a lot 51 and a lot 52. (3), IL-2 activity The protein concentration and IL-2 activity of the crude extract and purified product obtained in the above (iii) and (iv) were measured. The unit was measured in the same manner as described above. The results are shown in Table 7
Shown in

[Table 7]

Experimental Example 4 (1) Measurement of endotoxin content in rIL-2 preparation (a) Material: rIL-2 product (crude extract lots 01 and 0)
2 and refined product lots 51 and 52) were used. (B) Method and results: The amount of endotoxin in the above product was determined by the method of T. Sakata et al. [Journal of Parenteral Sci.
ence and Technology, 39 , 197 (1985)]. First, standard endotoxin Control 7D @ 4089 [Difco Laboratories] derived from Escherichia coli 055: B5 was dissolved in water, and the endotoxin concentration was 3.1 × 10 ⁻³ ng / ml, 6.3 × 10 ⁻³ ng / ml, 1.2
5 × 10 ⁻² ng / ml, 2.5 × 10 ⁻² ng / ml and 5.0 × 1
An aqueous solution of 0 ^-2 ng / ml was prepared. Toxinometer ET
The assay using -201 was performed in 100 μl of LAL [Limulus amebocytelysate (Limbulus amebocyte lysate), horseshoe crab ( Limul u ) in a glass tube with a diameter of 10 mm.
s polyphemus ) blood cell extract; Associates of
Cape Cod, Inc.] 100 μl solution
Was performed by adding 100 μl of the test solution.
After mixing the reaction solution for several seconds with a vortex mixer, the test tube was inserted into the optical unit of Toxinometer ET-201, and turbidity measurement was started. The reaction time of each sample was measured individually until gelation was confirmed. The results are shown in the following [Table 8].

[Table 8] The above results were plotted to give graph A as a dose-dependent standard curve for endotoxin. logC = −3.07 × log T (G) +2.389 γ = −0.997 * Toxinometer ET-201 measures the change in turbidity during gelation of the LAL / endotoxin solution at a wavelength of 660 nm, and samples The gel time of the gel is automatically recorded. This apparatus uses a ratio R (t) of a continuously changing light amount to an initial light amount.
Is measured every 12 seconds up to 64 samples simultaneously. When the sample is incubated statically at a constant temperature of 37 ± 0.5 ° C., gelation can be objectively detected without interference from the vibration of the sample. The gel time, T (G), defined as the reaction time required to obtain a 5% decrease in R (t), is displayed on a display and printed or sent to an external computer to determine the endotoxin concentration. Used for calculations.

[0035]

[Table 9] Next, the rIL-2 product was dissolved in water to obtain an aqueous solution.
These solutions were subjected to the same operation as above, and the gelation time was measured. The gelling time thus obtained was compared with the curve shown in [Table 9] to calculate the endotoxin concentration in the aqueous solution and converted into the amount of endotoxin per mg of rIL-2 protein. The results thus obtained are shown in Table 1 below.
0].

[Table 10]

(C) Endotoxin content As is clear from [Table 10] in (b) above, the purified product has an extremely small endotoxin content of 0.017 and 0.0.
14 ng / mg protein. In contrast, the endotoxin content of the crude extract is 2 × 10 ³ and 2.9 × 10 ³ ng / mg protein, which is about 1.6 × 10 ⁵ times that of the purified product. The crude extract cannot be used for clinical use because the crude extract has a large amount of endotoxin in the sample. On the other hand, since the endotoxin content of the purified product is extremely small, the purified product can be used clinically as it is. (2) Measurement of pyrogen in rIL-2 product (a) Material: rIL-2 product (crude extract lots 01 and 0)
2 and refined product lots 51 and 52) were used.
Rabbits were purchased from Ichikawaya (Tokyo). (B) Method and results: The pyrogen test was conducted in accordance with "Biological Product Standards, supervised by the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, published by
985 ", pp. 223-224. The results are shown in the following [Table 11].

[Table 11] The buffer solution used did not contain a pyrogen. (C) Conclusion Purified products do not contain pyrogens according to the criteria for the fever test method based on biological products. However, it appears that the crude extract is pyrogenic under conditions of clinical dose administration to humans. More specifically, this experiment was performed with a planned clinical dose of 1 × 10 ³ U / person (20 U / kg body weight;
Based on the amount of 0 kg), both the crude extract and the purified product were administered with a 10-fold amount of 200 U / kg to conduct a pyrogenicity test. However, in the dose determination experiment, the rabbits to which the crude extract was administered died due to an increase in body temperature. Therefore, in the crude extract group, the dose was reduced to 20 U / kg. [Table 11]
As shown in Table 2, no pyrogenicity of the purified product was detected, but the crude extract contained a smaller amount of rIL-2 than the purified product.
The fever was also detected by the administration of. Therefore, the crude extract cannot be used clinically because of its febrile nature. On the other hand, since the purified product has no pyrogenicity, it can be used clinically.

[0037]

EFFECT OF THE INVENTION The human interleukin-2 protein produced by the method of the present invention is highly purified and contains very few contaminating proteins and pyrogens, and thus can be safely used as an injectable drug substance.

[0038]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 604 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Origin organism name: Human strain name: Tissue sequence: GGGGGGGGGG GGGGGGGATC ACTCTCTTTA ATCACTACTC ACAGTAACCT CAACTCCTGC 60 CACAATGTAC AGGATGCAAC TCCTGTCTTG CATTGCACTA AGTCTTGCAC TTGTCACAAA 120 CAGTGCACCT ACTTCAAGTT CTACAAAGAA AACACAGCTA CAACTGGAGC ATTTACTGCT 180 GGATTTACAG ATGATTTTGA ATGGAATTAA TAATTACAAG AATCCCAAAC TCACCAGGAT 240 GCTCACATTT AAGTTTTACA TGCCCAAGAA GGCCACAGAA CTGAAACATC TTCAGTGTCT 300 AGAAGAAGAA CTCAAACCTC TGGAGGAAGT GCTAAATTTA GCTCAAAGCA AAAACTTTCA 360 CTTAAGACCC AGGGACTTAA TCAGCAATAT CAACGTAATA GTTCTGGAAC TAAAGGGATC 420 TGAAACAACA TTCATGTGTG AATATGCTGA TGAGACAGCA ACCATTGTAG AATTTCTGAA 480 CAGATGGATT ACCTTTTGTC AAAGCATCAT CTCAACACTG ACTTGATAAT TAAGTGCTTC 540 CCACTTAAAA CATATCAGGC CTTCTATTTA TTTAAATATT TAAATTTTAC CCCCCCCCCC 600 CCCC 604

SEQ ID NO: 2 Sequence length: 133 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: X-Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 5 10 15 His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr 20 25 30 Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro 35 40 45 Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu 50 55 60 Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His 65 70 75 Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu 80 85 90 95 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 100 105 110 Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser 115 120 125 Ile Ile Ser Thr Leu Thr 130

[Brief description of drawings]

FIG. 1 shows the plasmid pIL obtained in Example 1 (vii).
OT 135-8

[Equation 2] And a primary structure (base sequence).

FIG. 2 shows the plasmid pILOT obtained in Example 1 (vii).
135-8 system

(Equation 3) And a primary structure (base sequence).

FIG. 3 shows the amino acid sequence of the non-glycosylated human IL-2 protein of the present invention (in the figure, X represents Met or hydrogen)
Indicates.

FIG. 4 shows the expression plasmid pTF4 in Example 2.
The construction diagram is shown.

FIG. 5 shows the results of SDS-polyacrylamide slab gel electrophoresis of Examples 7 (1) and (2).

FIG. 6 shows a tryptic peptide map of Example 7 (6).

FIG. 7 shows the effect of the human IL-2 protein of the present invention on tritiated thymidine uptake of the NKC3 cell line and the human cell line of Example 7 (7).

FIG. 8 shows the effect of the human IL-2 protein of the present invention on tritiated thymidine uptake of the NKC3 cell line and the human cell line of Example 7 (7).

FIG. 9 shows the results of long-term subculture of the NKC3 cell line of Example 7 (7).

FIG. 10 shows the results of reversed-phase high performance liquid chromatography obtained in Experimental Example 1.

FIG. 11 shows the relationship between the concentration of standard endotoxin and the absorbance at 405 nm in Experimental Example 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12P 21/02 C12R 1:19) (56) References JP-A-59-70620 (JP, A) ) JP-A-60-12986 (JP, A) European Patent 91539 (EP, A) PROGRAMME AND ABS TRACTS 3RD INTERNA TIONAL SYMPOSIUM O N HPLC OF PROTEIN S, PEPTIDES AND POL YNUCLEO VOLOMER 1983, EP SESSIONS, PR EPARATIVE ISOLATIO N OF BIOMOLECULES 207

Claims (1)

(57) [Claims] 1. A human interleukin-2 obtained by culturing an Escherichia coli transformant containing a DNA having a nucleotide sequence encoding human interleukin-2, and allowing human interleukin-2 to be produced and accumulated in the culture. Containing liquid,
Human peripheral blood lymphocytes characterized by being purified by a purification step including a guanidine hydrochloride extraction step and a reverse phase high performance liquid chromatography treatment to give 5 × 10 ⁶ cells / ml
Suspended in RPMI1640 medium with 10% FCS
Canavalin-A 40 μg / ml and 12-O-tetra
Decanoyl phorbol-13-acetate 15 ng / m
and culturing for 48 hours at 37 ° C in the presence of 5% CO _2.
Measure the supernatant of the cultivated culture solution by 1U / ml
A method for producing a substantially pure non-glycosylated human interleukin-2 protein, which has a specific activity of 10 ⁴ U / mg or more and has a very low content of endotoxin and pyrogen.