JPH0770194A - Human interleukin-2 protein - Google Patents

Abstract

PURPOSE:To obtain the subject protein being negative to Limulus test, hardly containing a contaminant protein and pyrogen and useful as a stock solution, etc., for injection. CONSTITUTION:This pure non-glycosylated human interleukin-2 protein is obtained by culture of Escherichia coli transformate and the protein has >=10<4>U/mg specific activity when measured by a method comprising floating a human peripheral blood lymphocyte onto 10% FCS-added PRM1164 medium so as to give 5X10<6>/ml concentration, adding 40mug/ml concanavalin A and 15ng/ml 12-O-tetradecanoylphorbol-13-acetate thereto and then culturing the bacterium in the presence of 5% CO2 at 37 deg.C for 48hr and adjusting a supernatant of the resultant cultured mixture to 1U/m$.

Description

Detailed Description of the Invention

The present invention relates to human interleukin-2 protein.

Interleukin-2 [hereinafter also referred to as IL-2 in some cases. IL-2 is also called T cell growth factor (TCGF). ] Is a lymphokine produced by T cells stimulated with lectin, alloantigen and the like [Science, 193, 1007 (197).
6 years); Immunological Review, Volume 51, 257
Page (1980)]. IL-2 proliferates T cells while maintaining their function in vitro and enables passage and maintenance for a long period of time, and also promotes mitogenic reaction of thymocytes until now (costemulator) and nude. It has been reported to have the activity of recovering the antibody-producing ability of mouse splenocytes against T cell-dependent antigens (T cell replancing factor) and promoting the differentiation and proliferation of killer cells (killer helper factor) [The Journal・
Of Immunology, 123, 2928 (197)
9), Immunological Review, Volume 51, 257
Page (1980)].

By using IL-2, the killer T has been used so far.
Many clones of cells, helper T cells, and natural killer cells have been obtained [eg, 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. Tumor-specific killer T cells thus proliferated can be transferred to an animal to suppress and prevent tumor growth [The Journal of Immunology, 125, 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 show that IL-2
Shows 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, Vol. 10, 719].
Page (1980)] or to restore the induction of killer T cells to allogeneic cells [Nature, 284, 2].
78 (1980)] and is expected to be applied to diseases with reduced immune function. However, human IL-
Since 2 is difficult to mass-produce, it cannot be applied clinically at present, and development of a technique capable of mass-producing human IL-2 with high purity easily and cheaply is desired. 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, IL-
Only the presence of 2-like substances was estimated, and in reality human I
There is no report that the human IL-2 protein produced by the transformant containing the 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 is (1) obtained by culturing an Escherichia coli transformant, wherein human peripheral blood lymphocytes are suspended in 10% FCS-added PRMI1640 medium at 5 × 10 ⁶ cells / ml, and concanavalin-A 40 μg / ml and 12
-O-Tetradecanoylphorbol-13-acetate (15 ng / ml) was added at 37 ° C. in the presence of 5% CO ₂ to give 4
Substantially pure non-glycosylated human interleukin-2 having a specific activity of 10 ⁴ U / mg or more measured by a method in which the supernatant of the culture cultivated for 8 hours is set to 1 U / ml.
It provides proteins. Examples of the DNA encoding human IL-2 used in the present invention include DNA (I) having a nucleotide sequence shown by codons 1 to 133 in [Fig. 2]. 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. Particularly preferred is TGA. DNA (I) is preferably linked to the downstream of a promoter, and examples of these promoters include tryptophan (trp) promoter, rec A (recA) promoter, λPL promoter, and the trp promoter is particularly preferable. In the present invention, for example, mRNA encoding human IL-2 from a culture solution of human peripheral leukocytes stimulated with concanavalin A etc.
Are separated, and single-stranded cDNA is synthesized using reverse transcriptase or the like, and then double-stranded DNA is synthesized. Then, by introducing it into a plasmid, transforming, for example, Escherichia coli or Bacillus subtilis, and isolating a cDNA-containing plasmid from this, a double-stranded DNA encoding human IL-2 can be produced.

The 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, page 363 (1982)]. it can. Using the obtained human IL-2 mRNA as a template and reverse transcriptase, a method known per se was used to prepare a cDNA.
Synthesize 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 coupling method [Nelson, T.
S., Methods in Enzymology, Volume 68, 41
Page (1979)], for example the plasmid pBR3
It is incorporated into the PstI restriction endonuclease cleavage site of 22. Furthermore, for example, after chemically synthesizing an oligonucleotide having a base sequence corresponding to a part of the amino acid sequence of human IL-2, it is labeled with ³² P and used as a probe to prepare a colony hybridization method by a method known per se [ Grun
stein, M., Hogness, DS, Proceedings of National Academy of Sciences US
A, Vol. 72, page 3961 (1975)]; Alwine,
JC et al., Methods in Enzymology, 68th
Vol. 220 (1979)], the desired clone is selected from among tetracycline-resistant or ampicillin-sensitive transformants. The nucleotide sequence of the DNA of the clone showing a positive result by the 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)] or a method for terminating a dinucleotide synthetic chain using phage M13 [Messing, J. et al., Nucleic.
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 excised from the obtained clone, and an appropriate promoter, SD (Shine and Dalgarno) in the plasmid was excised.
It can be connected downstream of the nucleotide sequence and introduced into an appropriate host.

A trp promoter and the like are preferably used as a promoter, and Escherichia coli (294 strain, DH 1 strain, etc.) and the like are advantageously used as a host. E. coli 294 strain [Be
ckman et al., Proceedings of the National Academy of Sciences USA, Vol. 73,
4174 (1976)] and DH 1 strain [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 Science USA, Vol. 69, page 2110 (1972)]. . The host thus obtained is a medium known per se, for example, M9 medium containing glucose and casamino acid [Miller, J., Experiments Molecular Genetex, 43.
Pp. 1-433 (Cold Spring Harbor Laboratory, New York, 1972)]. When the trp promoter is used, an agent such as 3-β-indolylacrylic acid can be added to make it work efficiently. Culture is usually 1
It may be carried out at 5 to 43 ° C. for 3 to 24 hours, and aeration or stirring may be carried out if 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 an IL-2 dependent human cell line but also a rat or mouse IL-2 dependent cell line [Journal of Immunology, vol. 130, page 981 and 988 pages (198
3 years)]. In particular, since the mouse IL-2-dependent cell line can be stably passaged for a long period of time, highly reproducible measurement results can be obtained.

When the human IL-2 of the present invention is extracted from cultured cells, the cells are collected by a known method after culturing, and the cells are suspended in a buffer solution containing a protein denaturant such as guanidine hydrochloride, After stirring in a cold place, centrifuge IL-2.
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 above-mentioned supernatant, it is possible to carry out an appropriate combination of separation and purification methods known per se. Known separation and purification methods for these include those utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, which are mainly used for measuring molecular weight. Method utilizing difference, method utilizing charge difference such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography , A method of utilizing a difference in isoelectric points such as an isoelectric focusing method. In particular, human IL
Since the -2 protein has a high hydrophobicity, hydrophobic column chromatography, especially high performance liquid chromatography using a reversed phase column, is extremely effective for the purification of the protein. Therefore, in the purification step of the present invention,
A process using reverse 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 denaturing agent such as guanidine hydrochloride (preferably used at a concentration of 2M to 8M) and stirred. The supernatant is collected by post 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. Then, the active fraction is concentrated by using an ultrafiltration device, and then, for example, N, N '
-Methylenebisacrylamide cross-linked allyl dextran such as Sephacryl S-200 (Pharmacia,
Perform gel filtration using a Swedish column or the like. The active fractions are collected and then subjected to the high performance liquid chromatography described above. By such a method, the non-glycosylated human IL-2 of the present invention can be obtained. The reversed-phase column used for high-performance liquid chromatography herein includes, for example, an alkylated (about C _1-18 ) silicon column. As the elution solvent, a C _1-6 lower alkanol (ethanol, propanol, etc.) or acetonitrile can be advantageously used, and a pH of 1.5 to 4 is preferable.
The elution rate is preferably 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 IL-2 activity measurement using 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, 2 × 10 ⁴ U / mg or more, it is possible to obtain a non-glycosylated human IL-2 protein purity showing a can reach specific activity 3 × 10 ⁴ U / mg. The unit (U) as the activity of IL-2 was calculated as follows. That is, a mouse cell line that proliferates depending on the IL-2 concentration was added to a suspension medium and cultured, and the proliferation of the cell line was determined using tritium thymidine incorporation 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 at 37 ° C. in the presence of 5% CO ₂ in RPMI 1640 medium containing 20% FCS containing conditioned medium containing human IL-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 put into the well of the first row of a 96-well flat bottom microtiter plate (Nunc, Denmark), and 50 μl
After making a 2-fold serial dilution series using the respective 20% FCS-supplemented RPMI 1640 medium until the 12th row,
Dispense 50 μl of KC3 cell suspension into each well, and
Incubate for 24 hours at 5 ° C in the presence of 5% CO ₂ . Culture 20
At the time point, 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). Uptake of tritium thymidine is measured using a liquid scintillation counter. At the time of measurement, perform the same operation as the data for the standard IL-2 standard, and measure the uptake of tritium thymidine.

Unit (U) calculation is journal of
Immunology, 120, 2027 (1978)
Probit conversion method according to That is, a standard IL-2 standard (human peripheral blood lymphocytes 5 × 10 ⁶ cells / ml)
Suspension in 10% FCS-supplemented RPMI 1640 medium to give concanavalin-A 40 μg and 12-O-
Tetradecanoylphorbol-13-acetate 15ng
/ Ml was added and the supernatant of the culture broth cultured for 48 hours in the presence of 5% CO ₂ at 37 ° C. was defined as 1 U / ml), and the maximum uptake was taken as 100% of the dilution series. Calculate percent uptake value for each dilution step. The obtained numerical values are plotted on a normal probability paper, and the dilution factor showing uptake of 50% is obtained from the plot. Similarly, for each material containing IL-2, the dilution factor showing 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, which showed almost the same specific activity as the non-glycosylated human IL-2 protein of the present invention.

The non-glycosylated human IL-2 protein of the present invention is preferably a poly-amino acid sequence shown in [FIG. 3] (SEQ ID NO: 2) [where X represents Met or hydrogen]. It contains the peptide (II).
The human IL-2 protein produced by the present invention has the following properties. 1) It was homogeneous by SDS-polyacrylamide gel electrophoresis, and the molecular weight measured by this method was 15,000 ± 100.
It is 0 Dalton. 2) It has alanine or methionine as the amino terminal amino acid. 3) It has threonine as the carboxy terminal amino acid. 4) It has an 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)) Negative and contains very few contaminating proteins and pyrogens, so it can be safely used as a drug substance for injections and the like. Non-glycosylated human IL-obtained according to the invention
The 2 protein has low toxicity and has an activity of proliferating normal T cells and natural killer cells while retaining 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,
Utilizing this property, the activity of human IL-2 can be measured. Furthermore, the human IL-2 protein of the present invention is, for example, an antigen-specific killer T cell that recognizes and destroys a tumor antigen, or a natural killer cell that has the ability to kill a tumor regardless of whether or not it has undergone antigen sensitization. Can be selectively proliferated in vitro, and when the killer T cells are transferred into a living body, the human IL-2 of the present invention is simultaneously inoculated to increase its antitumor effect. It can be used for the prevention and treatment of tumors of blood animals (eg, mice, rats, rabbits, dogs, cats, pigs, horses, sheep, cattle, humans, etc.) and treatment of immunocompromised 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. Furthermore, it can be used together with killer T cells or natural killer cells expanded in vitro as described above or alone.

The human IL-2 protein of the present invention has substantially the same biological activity as known human IL-2 isolated from nature, and thus can be used in the same manner as described above. Since the dissociation constant with the body is extremely small, a very small dose may be administered. For the purpose of expanding T cells in vitro, the human IL-2 of the present invention is used.
About 0.01-1 unit / ml, preferably about 0.1-
It can be used by adding it 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 with lymphocytes in the presence of 5% CO _{2 at} 37 ° C. for 3 days to obtain a cell suspension containing alloantigen-sensitized T cells. 1 to
Examples include a method of continuing the culture at a concentration of 0.5 unit / ml for about one month while changing the medium about every week.
The transformants disclosed in the following examples, E. coli
DH 1 / pTF4 is the Institute for Fermentation Research (Institute f
or Fermentation, Osaka) as IFO-14299, and this transformant was deposited under the deposit number FE at the Institute of Microbial Technology (FRI), Ministry of International Trade and Industry.
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 in the same research institute (FRI) as BP-628. In the specification and drawings of the application, when an abbreviation is used for a base, amino acid, etc., IUPAC-IUB is used.
It is based on the abbreviations by the Commission on Biochemical Nomenclature or the abbreviations commonly used in the field, examples of which are given in [Table 1]. When an amino acid may have optical isomers, L- unless otherwise specified.
It shall indicate the body.

[Table 1]

Example 1 (i) Isolation of mRNA Encoding Human IL-2 Lymphocytes prepared from human peripheral blood were treated with 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 destructive denaturation by a Teflon homogenizer in 50 mM Tris · HCl pH 7.6, 10 mM EDTA solution, sodium N-laurylyl sarcosinate was added to 4%, and the homogenized mixture was added to a 5.7 M cesium chloride solution (5 7M cesium chloride, 0.1M EDTA) 6ml and layered on a Beckman SW28 rotor for 15
An RNA precipitate was obtained by centrifugation at 24,000 rpm for 48 hours. 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 a cellulose column
NA in low salt solution [10 mM Tris-HCl, pH 7.6,1
300 μg of mRNA containing poly (A) was collected by elution with [mM EDTA, 0.3% SDS]. This mRNA was further precipitated with ethanol to give a 0.2 ml solution (10 mM Tris.HCl pH 7.6, 2 mM EDT.
A, 0.3% SDS), treated at 65 ° C. for 2 minutes and subjected to 10-35% sucrose density gradient centrifugation (Beckman S
2 at 20 ° C and 25000rpm using a W28 rotor
It was fractionated by centrifugation for 1 hour) to obtain 22 fractions. A portion of each of these fractions of RNA was injected into oocytes of Xenopus laevis, and I in the protein synthesized was injected.
L-2 activity was measured and fractions 11-15 (precipitation constant 8S-
The activity of IL-2 was detected in 15S). 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 reaction solution (5 μg mRNA, 50 μg oligo (dT),
100 units of reverse transcriptase, 1 mM each of dATP.dCT
P, dGTP and dTTP, 8 mM MgCl ₂ , 50 mM
KCl, 10 mM dithiothreitol, 50 mM Tris-
After incubating in HCI pH 8.3) at 42 ° C for 1 hour, the protein was deproteinized with phenol, and the mixture was washed with 0.1 N NaOH to 7%.
RNA was decomposed and removed by treatment at 0 ° 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,
After reacting in 0.25 M NaCl, 1.5 mM ZnSO ₄ , 60 units of S1 nuclease) at room temperature for 30 minutes, deproteinizing with phenol and precipitating DNA with ethanol, 50 μl of a reaction solution of terminal transferase ( Double-stranded DNA, 0.14M potassium cacodylate, 0.1.
3M Tris (base) pH 7.6, ₂ mM dithiothreitol, 1 mM CoCl ₂ , 0.15 mM dCTP, 30 unit 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 pBR322DNA
Restriction enzyme PstI in 50 μl reaction solution (10 μg DN
A, 50 mM NaCl, 6 mM Tris ・ HCl ・ pH7.4,
6 mM MgCl ₂ , 6 mM 2-mercaptoethanol, 1
00 μg / ml bovine serum albumin, 20 units Pst
PBR322 DNA by reacting in I) for 3 hours at 37 ° C.
Cleavage the PstI recognition site, which exists at one site, and deproteinize with phenol.
0 μl of 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 E. coli 0.1 μg of the synthetic double-stranded DNA thus obtained and 0.1 μg of the above plasmid pBR322, 0.5 μg were added to 0.1 M Na.
Cl, 50 mM Tris, HCl pH 7.6, 1 mMEDTA
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)] according to E. coli MM29.
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. Then IL was reported by Taniguchi et al. [Nature, 302 , 305 (1983)].
-Amino acid No. 74-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) corresponding nucleotide sequence (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)] was chemically synthesized. 50μ using T4 polynucleotide kinase against this oligonucleotide
l reaction solution (0.20 μg of oligonucleotide, 50 mM
Tris HCl pH 8.0, 10 mM MgCl ₂ , 10 mM mercaptoethanol, 50 μCi γ- ³² PATP, 3 unit T4 polynucleotide kinase) for 1 hour 3
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
1)] to associate with the DNA immobilized on the above nitrocellulose filter, and four strains that react with the above two kinds of 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 insertion part of the plasmid DNA was cut out with a restriction enzyme PstI, and the isolated plasmid containing the longest fragment of the insertion part was selected, and this plasmid was named pILOT135-8. The restriction map of this plasmid is shown in [Fig. 1]. Next, the primary structure (base sequence) of the cDNA sequence inserted into this pILOT-135-8 plasmid was determined by the dideoxynucleotide method and the Maxma-Gilbert method. Its primary structure is [Fig. 2] (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 terminus
This amino acid is considered the signal peptide. From the above primary structure, it was revealed that this plasmid has the entire nucleotide sequence encoding the human IL-2 protein.
Due to this fact, any gene of the IL-2 protein can be produced by incorporating the gene incorporated in the plasmid into another expression plasmid.

Example 2 The plasmid pILOT135-8 obtained in Example 1 was digested with the restriction enzyme HgiA1 to obtain a DNA fragment containing the 1294 bp IL-2 gene. After treating this DNA fragment with T4 polymerase, CGAT, a ClaI linker having alanine codon GCA and methionine codon ATG
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 transforming Escherichia coli DH 1 into the transformant containing the plasmid (Escherichia
coli DH 1 / pTF4) was obtained.

Example 4 250 ml of E. coli DH 1 / pTF4 obtained in Example 3
Bact trypton (Difco Laboratories, USA) 1% in a conical flask, Bact yeast extract (Difco Laboratories, USA) 0.5%,
50 ml of a liquid medium (pH 7.0) containing 0.5% of sodium chloride and 7 μg / ml of tetracycline was inoculated and cultivated at 37 ° C. under rotary shaking overnight. This culture solution 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 the mixture was incubated at 37 ° C. for 4 hours, followed by 3-β. -Indolylacrylic acid (25 μg / ml) was added, and the mixture was further cultivated with aeration and stirring for 4 hours to obtain 2.5 liters of the culture solution. The culture was centrifuged to collect the cells, which were frozen at -80 ° C and stored.

Example 5 12.1 g of the cryopreserved cells obtained in Example 4 was extracted with 7M guanidine hydrochloride and 0.1M Tris.HCl (pH 7.
0) Evenly suspended 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 0.01M Tris / HCl buffer (pH 8.5) and centrifuged at 19,000 × g for 10 minutes to give 94 ml of the dialyzed supernatant. Obtained. The dialysis supernatant was passed through a DE 52 (DEAE-cellulose, Whatman, UK) column (50 ml volume) equilibrated with 0.01 M Tris-HCl buffer (pH 8.5) to adsorb the protein, and the NaCl concentration was increased. Linear gradient (0-0.15M NaCl,
1 liter) was prepared 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 performed using a Sephacryl S-200 (Pharmacia, Sweden) column (500 ml volume) equilibrated with a 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 liquid was adsorbed on a Ultrapore RPSC (Altex, USA) column and subjected to high performance liquid chromatography 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. The active fractions with a retention time of about 39 minutes were collected under these conditions, and 0.53 mg of non-glycosylated human IL-2 protein [specific activity, 30,000 U / mg, activity recovery rate from starting material, 30.6%; 10 ml of a solution containing 99% of protein purity (according to densitometry) was obtained. The above 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)] and subjected to SDS-polyacrylamide slab gel electrophoresis followed by staining with Coomassie Brilliant Blue, the human IL-2 protein showed a single band (see FIG. 5). The band positions did not change under reducing or non-reducing conditions. (2) Molecular weight: The molecular weight of the human IL-2 protein is SD
About 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 does not substantially contain an 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 carried out using a Hitachi Model 835 amino acid analyzer. Cystine and cysteine are half-methods [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.
It was hydrolyzed over time and quantified as cysteic acid by an amino acid analyzer. The amino acid analysis value was determined by averaging the values obtained by hydrolysis for 24, 48 and 72 hours. 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]

(4) N-terminal amino acid sequence: the 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 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 then heated at 100 ° C. for 6 hours. . The obtained hydrazine decomposition product 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. This supernatant was freeze-dried and amino acid analysis was carried out using a Hitachi model 835 amino acid analyzer. As a result, only threonine was detected. (6) Peptide map for trypsin digestion: TPCK (L-1-tosylamido-2-phenylethylchloromethylketone) trypsin (Washington, USA) 0.4 μg and 0.02 M hydrogen carbonate were prepared using 15 μg of the IL-2 standard. The reaction was carried out in 120 µl of sodium at 37 ° C for 18 hours.
2-mercaptoethanol (5 μl) was added to the reaction solution, and the mixture was further reacted at 37 ° C. for 2 hours. Then, 75% of 1% trifluoroacetic acid was added to the reaction solution to stop the reaction. The obtained reaction liquid 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, USA). Column temperature: 30 ° C. Mobile phase: Liquid 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% solution B). 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
The activity of the non-glycosylated human IL-2 protein of the present invention was measured according to the method described in (1982).
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
RPMI-164 with 20% FCS so that it becomes 5U / 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 culturing, and the cells were repeatedly resuspended in the fresh culture medium. 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 Injectable preparation: The solution containing the non-glycosylated human IL-2 protein obtained in Example 6 was added to
Adsorbed under aseptic conditions on a CM Toyopearl (Toyo Soda Co., Ltd.) column equilibrated with 025 M ammonium acetate buffer (pH 5.0), and eluted with the above buffer containing 0.15 M NaCl. The eluate was diluted by adding 0.15 M NaCl as appropriate, HSA was added to 0.5% and filtered through a membrane filter (pore size: 0.22 μm), and the resulting filtrate was aseptically 1 ml. Each is dispensed into vials and lyophilized 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, its 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) 1.9
8 ml of 10 mM DTNB [5,5'-dithiobis (2-
Nitrobenzoic acid)] methanol solution 0.02 ml was added, and the reaction was allowed to proceed at room temperature for 30 minutes. The quantification was performed by measuring the yellow coloration of the reaction solution by the absorbance at 412 nm and using cysteine hydrochloride of known concentration as a standard substance. As a result, 0.8 mol / mol of human IL-2 protein
5 moles of thiol groups were detected and therefore human IL-2
It was found that among the three cysteine residues contained in the protein, 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 pp. 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.
Thiopropyl sepharose 6B (Pharmacia, Sweden) column (1. 3 ml) equilibrated with the same buffer.
0 cmφ × 5.1 cm, 4 ml) at a flow rate of 5 ml / h, and then 10 ml of the above buffer solution, 0.1 M sodium chloride-25 m
10 ml of M ammonium acetate buffer (pH 5.0) and 0.1.
The column was washed with 10 ml of 2M acetic acid (pH 3.0).

Next, 4 ml of this thiopropyl sepharose 6B gel was taken out 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 with stirring at 37 ° C. for 15 hours using 1 mg. After the reaction, the gel was packed in a column again, 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 20 mM 2-mercaptomethanol-25 mM ammonium acetate (pH 4. 5) The unreacted group in the gel was eluted as 2-thiopyridone using 30 ml. Continuing,
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 l, 20 mM 2-mercaptoethanol-25 mM ammonium acetate buffer (pH 8.
0) was used to elute 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 0.1% trifluoroacetic acid solution, and subjected to reverse phase high performance liquid chromatography under the following conditions. Column: Nucleosil 5C18 (0.8cmφ × 30cm,
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 ( 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 minutes (15% A + 85% B) Elution rate: 3 ml / min Detection wavelength: 230 nm As a result, four peaks were detected as shown in [Fig. 10]. , 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] 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-Thr131 and all four peaks were found to be 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 3 cysteine residues of the human IL-2 protein, Cys125 has a free thiol group, and Cys58 and Cys105.
It was found that and form a disulfide bond and do not include isomers having different thiol group bonding modes.

Experimental Example 2 The human IL-2 protein obtained in Example 6 was quantified for its endotoxin. The quantification method was performed according to the method of Iwanaga et al. [Haemostasis, Vol. 7, pp. 183-188 (1978)]. (1) First, 10 ^-2 ng / of standard endotoxin obtained from Escherichia coli 0111-B4 (attached to Pretogel for endotoxin detection reagent (manufactured by Teikoku Organ Pharmaceutical Co., Ltd.))
Aqueous solutions with concentrations of ml, 10 ⁻¹ ng / ml, 3 × 10 ⁻¹ ng / ml, 1 ng / ml and 3 ng / ml were prepared. LAL (Limulus
Amebocyte Lysate, horseshoe crab blood cell extract, Teikoku Organ Pharmaceutical Co., Ltd.) 25 μl, synthetic substrate Boc-Leu-Gly-Arg
-PNA (Seikagaku Corporation, Boc: butoxycarbonyl, p
NA: para-nitroanid) (1.6 mg / ml) (25 μl) and the standard endotoxin aqueous solution (50 μl) 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 prepare [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 an aqueous solution. These are the 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 the endotoxin amount per 1 mg protein of the human IL-2 protein obtained in Example 6 was calculated from this. The results are shown in [Table 6] below.

[Table 6]

Experimental Example 3 (1) Production of crude extract (i) Construction of expression plasmid Plasmid pILOT135-8 containing the human IL-2 gene obtained in Example 1 (vii) above was treated with restriction enzyme Hgi.
It was cut with AI. The obtained 1294 bp DNA fragment was added to T4
The ends were made blunt with DNA polymerase and the Eco RI linker dTGCCATGAATTCATGGCA was ligated using T4 DNA ligase. The obtained DNA was digested with Eco RI to obtain D having a translation initiation codon ATG and human IL-2 gene.
The NA fragment was obtained. This DNA fragment was previously
I- Pst I site-digested 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 a restriction enzyme
Treated with Bam HI and Eco RI, then Eco RI- B
plasmid p having a λPL promoter at the am HI site
It was inserted into TB281. The expression plasmid thus obtained was designated as pTB285. (ii) Production of Transformant Using the plasmid pTB285 obtained above, Escherichia coli N4830 was prepared according to the method of Cohen et al. [Procedures of National Academy of Science (Pr.
o. Natl. Acad. Sci USA), 69, 2110 (1972)], and a transformant containing the above plasmid, Escherichia coli N.
4830 / pTB285 was obtained. (iii) Culture and extraction of transformant Escherichia coli N4830 / p transformant obtained above
TB285 was cultured in the same manner as in Example 4. The E. coli transformant (8 g) thus obtained was added to 20 mM.
It was washed with Tris-hydrochloric acid (pH 7.5, containing 30 mM NaCl), suspended in a buffer solution (40 ml) having the same composition, and 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 three times, and the whole was centrifuged (28,000 x 2).
g, 30 minutes). The two crude extracts thus obtained are called lot 01 and lot 02.

(2) Culture, extraction and purification of transformant Escherichia coli N4830 transformant 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 recombinant human interleukin-2 (hereinafter, sometimes abbreviated as rIL-2) thus obtained are referred to as lot 51 and lot 52. (3), IL-2 activity The protein concentration and IL-2 activity of the crude extract and purified product obtained in (iii) and (iv) above were measured. The unit was measured by the same method 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 lot 01 and 0)
2 and purified product lots 51 and 52) were used. (B) Method and results: The quantification of endotoxin in the above products was determined by the method of T. Sakata et al. [Journal of Parenteral Sci.
ence and Technology, 39 , 197 (1985)]. First, Escherichia coli 055: B5-derived standard endotoxin Control 7D-4089 [Difco Laboratories] is dissolved in water, and the endotoxin concentration is 3.1 × 10 ⁻³ ng / ml, 6.3 × 10 ⁻³ ng / ml, 1.2
5 x 10 ^-2 ng / ml, 2.5 x 10 ^-2 ng / ml and 5.0 x 1
An aqueous solution of 0 ^-2 ng / ml was prepared. Toxinometer ET
The assay using -201 was performed in 100 μl LAL [Limulus amebocytelysate (Limbulus amebocyte lysate), horseshoe crab ( Limulu ) in a 10 mm diameter glass tube.
s polyphemus ) Blood cell extract; Associates of the
Cape Cod, Inc.] solution 100 μl
This was done by adding 100 μl of the test solution to.
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 [Table 8] below.

[Table 8] The above results were plotted to give graph A as a dose-dependent standard curve for endotoxin. log C = −3.07 × log T (G) +2.389 γ = −0.997 * Toxinometer ET-201 measures the change in turbidity of LAL / endotoxin solution during gelation at a wavelength of 660 nm. Automatically record the gel time of. This device uses the ratio R (t) of the continuously changing light quantity to the initial light quantity.
Are simultaneously measured every 12 seconds for up to 64 samples. 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% reduction in R (t), is displayed on a display device and printed or sent to an external computer to determine the endotoxin concentration. Used for calculation.

[0035]

[Table 9] Next, the rIL-2 product was dissolved in water to give 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 10] below.
Shown in.

[Table 10]

(C) Endotoxin Content As is apparent from [Table 10] in (b) above, the endotoxin content of the purified product is very small, 0.017 and 0.03.
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. Since the crude extract has a large amount of endotoxin in the sample, the crude extract cannot be used clinically. 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 lot 01 and 0)
2 and purified product lots 51 and 52) were used.
Rabbits were purchased from Ichikawaya (Tokyo). (B) Method and result: The pyrogen test is based on “Biological Standards, supervised by the Pharmaceutical Affairs Bureau of the Ministry of Health and Welfare, issued by the Society for Bacterial Preparations, 1
1985 ”pp. 223-224. The results are shown in [Table 11] below.

[Table 11] The buffer solution used did not contain a pyrogen. (C) Conclusion According to the criteria of the pyrogenic test method based on the criteria for biological products, purified products do not contain pyrogens. However, the crude extract appears to be pyrogenic under conditions of clinical dose administration to humans. More specifically, in this experiment, 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. Above [Table 11]
Although no pyrogenicity was detected in the purified product, as shown in Fig. 3, the crude extract contained a smaller amount of rIL-2 than the purified product.
Pyrogenicity was also detected by 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]

EFFECTS OF THE INVENTION The human interleukin-2 protein of the present invention is highly purified and contains very few contaminant 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)
Control of OT 135-8

[Equation 2] And the primary structure (base sequence) are shown.

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

[Equation 3] And the primary structure (base sequence) are shown.

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

FIG. 4 shows the expression plasmid pTF4 in Example 2.
A construction drawing 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 the uptake of tritium thymidine by the NKC3 cell line and human cell line of Example 7 (7).

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

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

FIG. 10 shows the results of reverse 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 Internal reference number FI Technical display location // A61K 38/00 ABD ADU (C12N 15/09 ZNA C12R 1:91) (C12P 21/02 C12R 1:19) A61K 37/02 ADU (C12N 15/00 ZNA A C12R 1:91)

Claims (1)

[Claims] 1. A human peripheral blood lymphocyte obtained by culturing an Escherichia coli transformant at a concentration of 5 × 10 ⁶ cells / ml.
% FCS-supplemented PRMI1640 medium, suspended in concanavalin-A 40 μg / ml and 12-O-tetradecanoylphorbol-13-acetate 15 ng / ml, and cultured at 37 ° C. in the presence of 5% CO ₂ for 48 hours. 10 ⁴ measured by the method of determining the supernatant of the liquid as 1 U / ml
A substantially pure non-glycosylated human interleukin-2 protein having a specific activity of U / mg or higher.