BE898473A - Gene encoded for interleukin-2 polypeptide, recombinant DNA carrying this gene, living cells containing recombinant DNA and production of interleukin-2 - Google Patents

Abstract

A gene encoded for a polypeptide which exhibits interleukin-2 activity is isolated and linked to a vector DNA which is capable of replicating in a prokaryotic or eurokaryotic cell at a position downstream of a d gene. a promoter sequence in the vector, obtaining a recombinant DNA, with which the cell is transformed to produce interleukin-2:

Description

       

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   PATENT OF INVENTION formed by
Ajinomoto Co., Inc. and Japanese Foundation for Cancer Research for:
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 "Gene encoded for interleukin-2 polypeptide, recombinant DNA carrying this gene, living cells containing recombinant DNA and production of interleukin-2"

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 "Gene encoded for interleukin-2 polypeptide, recombinant DNA carrying this gene, living cells containing the recombinant DNA and production of interleukin-211
The present invention relates to a gene, in particular to a cloned gene coding for an interleukin-2 polypeptide, to recombinant DNA carrying the gene, to a living cell line comprising the recombinant DNA as well as to a process for producing interleukin-2 using the cell line.



   Interleukin-2 (hereinafter referred to as "IL-2"), previously known as T cell growth factor, is a soluble protein (generally known as "lymphokine"), and is obtained from T cells activated by a lectin or an antigen [Morgan DA et al., Science, 193,1007-1008 (1976) and Gillis S. et al., J. Immunol., 120,2027-2033 (1978)]. Interleukin-2 (IL-2) is capable of modulating the reactivity of lymphocytes and of promoting the long-term, in vitro culture of T-lymphocytes, specific antigen effectors [Gillis S. et al., Nature 268,154- 156 (1977)]. It is also known that IL-2 exhibits other significant biological activities, such as an increase in thymocyte mitogenesis [Chen. B. M. et al., Cell. immunol., 22,211-224 (1977), Shaw J. et al., J.



  Immunol. 120, 1967-1973 (1978)], an induction of the reaction

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 the activity of cytotoxic T cells [Wagner H. et al., Nature, 284,278-280 (1980)] and the production of anti-CBRS plaque-forming cells [Gillis S. et al., J. Exp. Med., 149, 1960-1968 (1979)] in cultures of mouse spleen cells, naked. Consequently, this regulatory substance for lymphocytes proves to be interesting in potentiating the humoral and cellular immunodeficiency responses and in reducing the immunodeficient states to normal humoral and cellular immune states.

   These activities
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 identified immunological results of IL-2 clearly show that IL-2 is interesting in medical immunotherapy against immunological disorders, that neoplastic diseases, bacterial or viral infections, immunodeficiency diseases, autoimmune diseases, etc. [Papermaster B. et al., Adv. Immunopharm., 507 (1980)]. Like interferons, IL-2 has been shown to promote the activity of natural killer cells, which suggests potential use in the treatment of neoplastic diseases.

   In addition, IL-2 allows the conservation of monoclonal, functional T cell cultures and therefore appears to play an important role in the study of the molecular nature of T cell differentiation, of the mechanism of cell functions. T as well as the T cell antigen receptor mechanism



  It also turns out to be advantageous for producing, by a long-term culture of monoclonal T cells, a large number of other lymphokines derived from T cells, which are interesting in a wide range of fields. In addition, the production of IL-2 and the response of lym-

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 IL-2 cells may be important parameters of immunological functions, which are of interest in the clinical diagnosis of aberrant immunity.



   IL-2 has been obtained in the prior art by stimulating mouse, rat or human lymphocytes with a mitogen [Gillis S. et al., Nature, 268,154-156 (1977), Farrar J. et al., J. Immunol., 121, 1353-1360 (1978) and Gillis S. et al., J.



  Immunol., 120,2027-2033 (1978)]. By stimulation of human peripheral blood mononuclear lymphocytes with a mitogen {. Gillis S. et al., J. Immunol., 124, 1954-1962 (1980)], Gillis et al. obtained the preparation of murine IL-2 from a murine T cell lymphoma cell line [Gillis S. et al., J. Immunol., 125,2570-2578 (1980)] and the preparation of 'Human IL-2 from a human leukemia cell line [Gillis S. et al., J. Exp. Med., 152, 1709-1719, (1980)].



   The aforementioned articles by Gillis et al. relate a process for producing human IL-2 from a human T cell leukemia cell line stimulated by a mitogen by cell culture methods. However, a technique of this type leads to too low concentrations of human IL-2, and requires complex purification processes to obtain even small amounts of IL-2 from very large volumes of culture medium.

   In addition, since human T cell leukemia cell lines produce trace amounts of a large number of other biologically active substances which are analogous to human IL-2, significant difficulties are encountered. to isolate IL-2 from these other immunologically active molecules, or to isolate

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 IL-2 of the toxic lectins possibly present.



   As another technique, it would seem desirable
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 to use recombinant DNA techniques (DNA is the abbreviation for deoxyribonucleic acid) such as those used in the production of other biologically active human proteins, such as interferons [Gray EW et al., Nature, 295,503- 508 (1981), Nagata S. et al., Nature, 284,316-320 (1980), Taniguchi T. et al., Gene, 10,11-15 (1980)], for the production of IL-2. However, the trials used to date to produce IL-2 by recombinant DNA techniques have not been satisfactory.

   For example, it was reported in "Nikkei Biotechnology" (Japan), n 19, July 5, 1982, that attempts to construct IL-2 producing organisms by recombinant DNA have been unsuccessful, probably due to the fact that the gene encoding the IL-2 polypeptide had not yet been cloned.



   It is therefore always necessary
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 to make a cloned gene, coded for interleukin-2, as well as DNA produced by recombination, which carries the gene. It also appears necessary to produce a living cell line which comprises DNA produced by recombination, as well as a method for producing interleukin-2 using the cell line.



   These and other objects of the present invention, which will become more apparent from the description below, have been achieved by providing a cloned gene encoded for a polypeptide, which exhibits IL-2 activity, and by providing DNA produced by recombination which comprises a gene coded for a polypeptide possessing the activity of IL-

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 2, and a vector DNA capable of replicating in a prokaryotic or eukaryotic cell, the coding sequence of the gene being located at a position downstream of a promoter sequence.



   In addition, according to the present invention, prokaryotic or eukaryotic cell lines are provided which have been transformed to produce IL-2 by the above-mentioned DNA, vector DNA and coded gene, the DNA being capable of replicating in the cell, the coding sequence being located at a position downstream of a promoter sequence.



   According to the present invention, IL-2 is obtained by an aerobic culture of a medium containing a eukaryotic or prokaryotic cell line which has been transformed to produce IL-2 by DNA which has been modified by following recombination the invention by means of a gene encoded to produce a polypeptide which possesses the activity of IL-2, and by the introduction of vector DNA, which can replicate in the cell, the coding sequence of this gene being located at a position downstream of a promoter sequence.



   Other details and particularities of the invention will emerge from the description below, given by way of nonlimiting example and with reference to the appended drawings, in which:
FIG. 1 represents a map showing the cleavage points made by restriction endonucleases of a cloned gene coded to produce a polypeptide which possesses IL-2 activity (hereinafter called "IL polypeptide" -2 ").



   Figure 2 (a) shows the basic sequence of the

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 cloned gene. Figure 2 (b) shows the amino acid sequence I, as well as the amino acid sequences II and III, polypeptides that have IL-2 activity.



   FIG. 3 is a schematic representation showing the construction of a recombinant DNA (pCEIL-2), into which the coded gene is introduced.



   FIG. 4 represents the vector plasmid pTrS-3.



   Figures 5 (a), 5 (b) and 5 (c) are schematic representations showing the construction
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 of recombinant DNAs (pTIL 2-22, pTIL2-21, pTIL2-14 and pTIL2-15) using pTrS-3 as a vector.



   FIG. 6 is a schematic representation showing the construction of a recombinant DNA (pKIL2-21) using pKT218 as a vector.



   Figure 7 is a schematic representation showing the. construction of a recombinant DNA (pTuIL2-22) using pTUBlP-5 as a vector.



   Figure 8 shows vector DNAs that can replicate in a Saccharomyces cereviceae cell.



   In the figures, "A", "G", "C" and "T" respectively represent deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid and thymidylic acid.



   The cloned gene, coded for an IL-2 polypeptide, can be obtained by transcription of messenger RNA (mRNA; "RNA" is the abbreviation for ribonucleic acid) corresponding to IL-2 (hereinafter called " IL-2 "mRNA, and originating from a mammalian cell which is characterized

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 by the ability to produce a polypeptide which has the activity of IL-2, towards a complementary DNA (cDNA).



  The single stranded cDNA (ss-cDNA) obtained can be converted into a double stranded cDNA (ds-cDNA).



   The mRNA used as a template for the preparation of cDNA can be separated in the usual way from a mammalian cell capable of producing an IL-2 polypeptide. The separated RNA is polyadenylated [Gillis et al., Immunological Rev., 63,167-209 (1982)], and the polyadenylated RNA can be fractionated, for example, by centrifugation on a density gradient of sucrose as a sediment of 11 to 12 strands. Occasionally, a 13-strand mRNA will show IL-2 mRNA activity, and in this case it is assumed that the mRNA is
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 in an aggregated form of 11 to 12 strand mRNA.



  The mammalian cells capable of producing IL-2, which are the source of the mRNA of the present invention, can be T lymphocytes, such as peripheral blood mononuclear cells, tonsil cells, cells spleen, etc., which can be obtained from mammals. The cells can usually be treated beforehand, for example with a nylon column, an antiserum supplement, density gradient fractionation, multiple enzyme treatment, such as a combination of neuraminidase and galactose oxidase, radiation irradiation X or with trypsin to give cells the productivity of IL-2 or to increase the activity of IL-2.

   Likewise, it is also possible to use, as a source of mRNA, cloned T lymphocytes obtained from these mammalian cells after culture in the presence of a factor of

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 T cell growth, with T lymphocytes being preferred. Transformed lymphocyte cell lines, such as T lymphocytes derived from leukemia cell lines or lymphomas as such or from their derivatives obtained by pretreatment or mutation by the abovementioned methods, or alternatively transformed, cloned cell lines are preferable as sources of mRNA. Obviously, cloned cell lines ordinarily contain greater amounts of IL-2 mRNA compared to bulk, parenteral cell lines.

   T cell hybridomas, obtained by fusion of cells derived from lymphocytes mentioned above and from tumor cell lines, such as CEM, Molt 4F and BW5147, are also preferred mammalian cell lines which can be used in the context of present invention. In this case, the cell lines derived from lymphocytes include (1) the constitutive producers of IL-2 and (2) those which are producers of IL-2 only in the presence of a mitogen introduced into the culture, namely in the absence or in the presence of other cells stimulating jointly the production of IL-2.



   In order to produce IL-2 mRNA in constitutive IL-2 producer cells, the constitutive IL-2 producer cells are cultured under the conditions usually encountered in the field of cell culture. For mRNA production in cells producing IL-2 only in the presence of a mitogen, the cultured cells are washed thoroughly with culture medium and resuspended in medium of culture, such as

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 Rosewell Park Memorial Institute medium (called 1640 "below), Dulbecco-modified Eagle medium (hereinafter called" DMEM ") or in Click medium, which may or may not contain serum.

   These culture media can be supplemented with various additives, such as penicilin, streptomycin and other antibiotics, or with fresh L-glutamine, Hepes buffer and sodium bicarbonate in concentrations such as those generally used in the field of cell culture. The preferred cell density can be 0.5 to 4 x 10 6 cells / ml. To induce activation of mRNA and production of IL-2, appropriate stimulants are added.



  Suitable stimulants of this type are mitogens, neuraminidase, galactose oxidase, zinc derivatives, such as zinc chloride, or lymphocyte activating substances from microorganisms, such as protein A, streptolysin-O . The stimulated cells are recovered and washed. The simultaneous presence of macrophages or dendritic cells during mitogenic stimulation can also activate mRNA, or can increase the amount of activated mRNA. Likewise, the simultaneous presence of cell lines from T cells or B cell lines, such as Raji, Daudi, K562 and BALL-1, can activate mRNA or increase the amount of activated mRNA.



   To propagate mammalian cells, they are maintained in an in vitro cell culture or in histocompatibility compatible animals, under normal conditions. When kept in an in vitro culture to prepare

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 source of mRNA, cells can be grown in any of the culture media used to date to promote growth of T cells. These culture media may or may not be supplemented with mammalian serum, a serum component or serum albumin. The culture period for activation of mRNA will correspond to the period required for activation of cells to produce mRNA. This period usually coincides with the time required to initiate the excretion of IL-2 in the culture medium.

   The preferred period can range from 3 to 12 hours after the addition of a stimulant, such as a mitogen. An exaggerated extension of the culture period may occasionally cause the decomposition of the IL-2 mRNA produced. During activation of IL-2 producing cells, it may be preferable to use phorbol esters, such as PMA or TPA, in a concentration of 10 to 50 ng / ml to raise the level of activation.



   The IL-2 mRNA activation process described above can be carried out at temperatures of the order of 320 to 380C in a humidified atmosphere and at a pH of approximately 7.0 to 7.4.



   The methods of obtaining and culturing mammalian cells capable of producing IL-2 will now be explained.
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  (Acquisition of a cell line constitutively producing IL-2.



   (1) A Jurkat cell line formed of leukemic T cells, human (freely delivered by the Fred Hutchinson Cancer Institute Seattle, United States, the Salk Institute, San Diego, United States, is suspended.

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 the Cancer Center in Heidelberg, West Germany) in Click medium at a cell density of 1 x 106 cells / ml and are irradiated with X-rays of 8 x 103 R at an irradiation rate of 150 R / minute. Next, 0.1 cells of the cells thus irradiated are inoculated for 200 μl of medium in Click medium containing 5% of FCS in flat-bottomed microplates comprising 96 wells (Falcon 3072) and they are cultured for 3 weeks at 37 ° C. in a 5% CO 2 incubator (cloning by limiting dilution method).

   Viable growing cells are transferred to a 24-well culture plate (Nunc) before the cell layer becomes confluent and are cultured again for 5 days. The growing cells are further cultured in a serum-based synthetic culture medium free from serum albumin for about 2 days at an initial cell density of between 1 and 2 x 10 6 / ml. The culture supernatant is collected by centrifugation and filtered using a 0.22 micron millipore filter paper to separate debris and to sterilize the supernatant, mutants treated with X-rays capable of constitutively producing IL- 2 then being selected and cloned by measuring the activity of IL-2 present in the supernatant.



   (2) Acquisition of IL-2 producing cells from human peripheral blood mononuclear cells.



   Human peripheral blood is collected and peripheral blood lymphocytes (known as "PBL") are isolated using gradient centrifugation.

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 density on Ficoll-Hypaque. The PBLs are inoculated into 2 ml of Click medium containing 5% of FCS at a cell density of 1 × 10 6 cells / ml in a Nunc culture plate comprising 24 wells at the same time as 100 μl of phytohemaogLutinin-M (Gibco) ( pHA; 5 g / ml), and cultured for 48 hours under the conditions described above.



   The cells are washed and inoculated again in 1 ml of Click medium at a cell density of 1 x
105 cells / ml at the same time as with 1 ml of conditioned medium which has been prepared from human splenocytes stimulated with 2/5 g / ml of concanavalin A (hereinafter "Con A") for 48 hours , and the culture medium containing 50% of the conditioned medium is changed every 3 days so as to obtain a long-term culture of human T lymphocytes from PBL. The long-term cultured human T lymphocytes thus obtained are cloned by the limiting dilution method as described above, in the presence of a conditioned medium prepared from human splenocytes and the cell clones are propagated in a similar manner. .

   Next, the human T lymphocytes, cloned in 1 ml of RPMI 1640 are inoculated at a cell density of 1 × 10 6 cells / ml in a 24-well Nunc culture plate in the presence of 10 μg / ml of PHA and they are cultured for 24 hours at 37 C in a 7.5% CO incubator. The supernatants of the culture liquid are collected, centrifuged, filtered through a 0.22 micron millipore filter and examined for their IL-2 activity in order to detect clones of normal T lymphocytes, human IL-2 producers. .

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   (3) Acquisition of a malignant cell line derived from human lymphocytes which can produce IL-2 in the presence of a mitogen.



   The Jurkat cell line or cloned cell lines, such as Jurkat III, obtained by the limiting dilution method described above, are capable of producing from 10 to 4000 units / ml of IL-2, when cultured for 24 hours in the serum-free synthesis medium described above or in RPMI 1640 containing 1 to 2% of mammalian serum in the presence of a mitogen, such as 10 g / ml of Con A or 2.5 μg / ml of PHA. These malignant human cell lines also produce IL-2 when cultured in the presence of zinc chloride, protein A or picibanil.



   (4) Acquisition of cells capable of producing IL-2 in the simultaneous presence of a mitogen and other co-stimulatory cells or of soluble co-stimulatory factors.



   The Molt 4F human malignant cell line and certain cloned cell lines, such as Jurkat J99, obtained according to the limiting dilution method, do not produce IL-2, even when cultured for 24 to 72 hours in the presence lectins or mitogens in any concentration. However, these cells become susceptible to producing significant amounts of IL-2 (10 to 100 pg / ml) during a 24 hour culture period at 37 C, when grown in conjunction with 5

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 at 10 pg / ml of interleukin-1, one of the monokines, or with 50% of K562 or Raji cells.



   The extraction of IL-2 mRNA from cells activated in the aforementioned manner is carried out according to well known, usual methods, regardless of the difference in cell sources. For example, cells are partially or completely ruptured by the addition of a detergent, such as NP-40, SDS, Triton-X or deoxycholic acid, or by mechanical homogenization or freeze-liquefaction. To prevent degradation of RNA by ribonuclease during mRNA extraction, it is preferable to add RNase inhibitors, such as heparin, polyvinylsulfate, bentonite, macaroide, diethylpyrocarbonate or a vanadylated complex.

   IL-2 mRNA can be obtained from a polysome precipitated in the biosynthesis of IL-2, which is precipitated with an anti-IL-2 antibody by extraction with a detergent.



   The poly A-containing mRNA can be fractionated or concentrated by any usual method, such as by affinity chromatography or by discontinuous absorption on oligo dT-cellulose, poly U-sepharose or sepharose 2B, by centrifugation at sucrose density gradient or alternatively by agarose gel electrophoresis,
The mRNA fractions are then examined for their IL-2 mRNA activity by testing the biological activities of the proteins translated from the mRNA fractions or by identifying the translated protein using a monoclonal antibody against the IL- peptide. 2. For example, mRNA is commonly translated to

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 form the corresponding protein by microinjection into frog eggs (Xenopus laevis) [Gurdon J.

   B. et al., Nature, 233, 177-182 (1972)] or using the reticulocyte lysate dependent on mRNA or acellular wheat germ translation systems.



   IL-2 activity can be verified by the microassay process described in detail by Gillis et al., [Gillis S. et al., J. Immunol., 120,2027-2033 (1978)]. The assay controls IL-2 dependent cell proliferation of a cytotoxic T lymphocyte cell line (hereinafter called "CTLL") obtained according to the methods described by Gillis et al., That is to say that 4 x 10 CTLL cells are inoculated in 100 μl of RPMl 1640 medium containing 2% FCS in flat-bottom microplates comprising 96 wells at the same time as
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 100 μl of translation products diluted in series.

   After 20 hours of incubation at 370C in an incubator containing 5% of Cules cells are exposed for 4 3 hours to 0.5 pCi of H-TdR, collected on strips of glass fibers using a automated cell harvesting device and the incorporated radioactivity is then measured by liquid scintillation counting. By these test procedures, it was found that the CTLL cells cultured in the presence of IL-2 incorporated the H-TdR according to the dose used, which thus makes it possible to accurately calculate the amount of IL-2 contained in test samples.



   IL-2 has the activity to promote the proliferation of T lymphocytes, which makes it possible to measure the activity of IL-2 using an index of T cell growth activity. This is how the we transfer 5

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 CTLL cells in 100 μl of DMEM containing 2% FCS in flat-bottom microplates comprising 96 wells at the same time as 100 μl of the translation products diluted in series. After an incubation of 72 to 96 hours at 37 ° C. in an incubator containing 5% of CO, the number of cells produced and activated is counted under the microscope.

   As a positive external control group, 100 units / ml, 10 units / ml of IL-2 are added and the IL-2 activity of the test sample is calculated relative to the number of viable cells produced in these control groups.



     The IL-2 mRNA thus obtained from the most active fraction is used as a model for the synthesis of dsDNA and the ds-cDNA is linked to a vector DNA. CDNA synthesis is carried out by usual procedures.



   First, ss-cDNA is prepared which is complementary to mRNA in the presence of dATP, dGTP, dCTP, dTTP using reverse transcriptase and using mRNA as a template and oligodT as a primer. The mRNA model is then separated by alkaline treatment and ds-cDNA is obtained using reverse transcriptase or DNA polymerase and using the ss-cDNA synthesized above as a template.



   DNA obtained by recombination is prepared from the ds-cDNA thus obtained and from vector DNA containing a replicon capable of replicating in eukaryotic or prokaryotic cells. The recombinant DNA is then incorporated into the host cells.



   Ds-cDNA and a vector DNA capable of spreading in eukaryotic or pro-

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 caryotics are, before ligation, modified by various processes, such as exonuclease treatment, addition of chemically synthesized DNA fragments with attachment of G, C ends to give ligatable ends to the ends of dsDNA and vector DNA. Ligation DNA capable of ligation is carried out using, for example, phage T4 DNA ligase in the presence of ATP.



   With the recombinant DNA thus obtained, live cells are transformed to amplify the cloned cDNA or to produce IL-2 polypeptide.



   Carpatic hosts of interest, which are commonly used for the production of IL-2, are vertebrates, yeasts, etc. For example, one can use monkey cells, for example CV-1 cells, transformed by a defective mutant, of SV-40 origin and expressing the SV-40 T antigen (COS cells), as discussed by Y Gluzman (Cell, 23, 175-182,1981), cells derived from mice, as reported by Ohno S and Taniguchi T [Nucleic Acids Research, 10, 967-977 (1982)], and host systems (yeast ) -vector applied for the expression of IFN genes, reported by R. Hitzeman and Coll. [Nature, 293,717-722 (1981)]. Interesting prokaryotic host organisms are Escherichia coli, Bacillus subtilis, etc.

   For DNA amplification in host organisms, it may be preferable to use E. coli as the host; however, other hosts can be used.



   Interesting vectors used for E. coli are the EK type plasmids (tight type): pSC101, pRK353, pRK646, pRK248, pDF41, etc., the EK type vector plasmids (loose type): ColEl, pVH51,

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 pAC105, RSF2124, pCRi, pMB9, pBR313, pBR322, pBR324, pBR325, pBR327, pBR328, pKY2289, pKY2700, pKN80,
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 pKC7, pKB158, pMK2004, pACYCl, pACYC184, dul etc, the phage vectors:> gt. Ac, Àgt. B, ^ ZJvir., Ts622, J \. etc. In general, pBR322 has often been used as a vector for E. coli and, in this case, the best cloning sites are the Pst I and EcoRI sites.



   The transformation of the host cell with the recombinant DNA can be carried out in the usual manner as follows:
When the host is of the prokaryotic type, such as E. coli, competent cells are prepared which are capable of collecting DNA from cells collected after an exponential growth phase and then treated with the CaCl2 method. by well known processes. When MgCl or RbCl exists in the transformation reaction medium, the transformation efficiency increases. The transformation can also be carried out after having formed a protoplast of the host cell.



   When the host used is one. eukaryote, one can use a method of DNA transfection in the form of calcium phosphate precipitates, usual mechanical processes such as microinjection, introduction of a plasmid encapsulated in hosts constituted by red blood cells or in liposomes, treatment of cells with agents such as lysophosphatidylcholine, or virus vectors, etc.



   Cells with the IL-2 gene can be isolated after transformation by one of two methods.

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 following methods.



   (1) In the plus-minus method, partially purified IL-2 mRNA is obtained by a sucrose density gradient centrifugation of mRNA extracted from mammalian cells activated by a mitogen as a 12-stranded Il sediment and then synthesized 32s radioactive labeled ssDNA is synthesized using partially purified mRNA as a template. After separation of the mRNA model by alkaline treatment, the isolated cDNA is hybridized with 11 to 12 strand mRNA, partially purified from mammalian cells not activated by a mitogen. Then, the non-hybridized and hybridized cDNAs are fractionated by means of a chromatography on a hydroxylapatite column. The non-hybridized cDNA and the hybridized cDNA are called probe A and probe B respectively.

   The transformants are grown on two nitrocellulose filters in exactly the same way, and the DNA of the cells is fixed on the filter paper by an alkaline treatment. The probe A and the probe B are respectively hybridized by DNA on two different filter papers and an autoradiography test is then carried out to select the transformants which react positively to the probe A (plus), but which react weakly or which do not do not react at all to probe B (minus) (Taniguchi et al., Proc. Jpn. Acad., V 155B 464-469,1979).



   (2) The second method consists in dividing, for example, 1000 to 10000 transforming clones into several tens or several hundred groups of clones. The groups of divided clones are respectively cultivated by usual means to obtain DNA plasmids.

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  Then, these DNA plasmids are converted into ss-cDNA, for example by thermal denaturation, and the ss-cDNA obtained are fixed on nitrocellulose filter papers to carry out hybridization of mRNA complementary to the DNA fixed and prepared from cells. mammal incorporating activated IL-2 mRNA. Alternatively, the mRNAs containing IL-2 mRNA are hybridized by heat denatured DNA plasmids and then hybrid DNA-mRNA is fixed on nitrocellulose filter papers.



  These filter papers are then washed with a buffer with a low salt concentration, such as 1 mM HEPES, or else with 10 mM NaCl, and the mRNA adsorbed on the filter paper is extracted by treatment with a solution containing 0, 5 mM EDTA and 0.1% of a SDS solution, for example for 1 minute at 95 C. The purified ANRm is recovered by elution by means of chromatography on 00 l of oligo dT-cellulose . Then the mRNA is translated to form a protein by microinjection into Xenopus laevis eggs to ensure the activity of IL-2, or the mRNA is translated to form a protein using reticulocytes dependent on mRNA or an in vitro acellular wheat germ translation system, to analyze IL-2 activity using an anti-IL-2 antibody.

   According to these methods, the group in which the presence of IL-2 activity is detected is again repeatedly divided into groups formed by a smaller number of transforming clones until a single clone having IL-2 DNA is obtained.



   To obtain cDNA encoding IL-2 polypeptide from the IL-2 producing transformant, the recombinant DNA is separated in the transformant and cleaved using a restriction endonuclease. We se-

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 protects the insertion cDNA fraction of the DNA fragments formed by cleavage.



   The complete nucleotide sequence of the PstI DNA insertion coding for IL-2 polypeptides from the recombinant DNA of pIL2-50A was determined by the method of Maxam and Gilbert [Meth.



  Enzym. 65,499-560 (1980)] and by the dideoxynucleotide chain termination method [Smith A. J. M.



  Meth. Enzym. 65, 560-580 (1980)].



   The cleavage map made by endonuleases limiting the insertion of cDNA and the basic sequence of the insertion are represented respectively in FIG. 1 and FIG. 2 (a), the cDNA comprising sites cleaved by restriction endonucleases in the order of BstNI, XbaI and BstNI.



   The DNA sequence of the insert contains a single large open reading frame. We find the first ATG sequence, which usually serves as an initiation sequence in eukaryotes [Kozak M.



  Cell, 15, 1109-1123 (1978)], at nucleotides 48-50 from the 5 'end. This ATG is followed by 152 codons before meeting the TGA termination triplet at nucleotides 507 to 509. There is a succession of
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 residues A corresponding to the 3'-poly (A) end of the mRNA at the end of the DNA, this being preceded by the hexanucleotide AATAAA (position 771-776), which is usually found in most eukaryotic mRNAs [Proudfoot NJ and Brownlee CG, Nature 263, 211-214 (1976)].



   We could deduce the amino acid sequence, for which the cDNA codes, as shown in the figure

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 2 (b) (amino acid sequence I), the polypeptide of amino acid sequence I being formed of 153 amino acids and its molecular weight being 17631.7 daltons. Considered a common characteristic in most secretory proteins known so far [Blobel G. et al., Sym. Soc. exp. Med., 33,9-36 (1979)], the N-terminal region of the deduced IL-2 polypeptide is also quite hydrophobic and this region probably serves as a detection peptide which is cleaved during the secretion process of mature IL-2.

   This cleavage occurs either between Ser and Ala respectively at positions 20 and 21 or between Ala and Pro respectively at positions 21 and 22, by forming the polypeptide comprising the amino acid sequences II and III, since sites of amino acid have often been found. similar cleavage in other secretion proteins [Blobel G. et al., Symp. Soc., Exp. Med. 33.9-36 (1979)]. The mature IL-2 polypeptide would then contain 133 or 132 amino acids, the molecular weight calculated being 15420.5 daltons or 15349.4 daltons. This value is then compared to the value reported for human IL-2 protein from Jurkat cells (15,000 daltons) [Gillis S. et al., Immunological Rev., 63.67-209 (1982)].

   In addition, it has been confirmed that the DNA fragment starting from the codon CCT at position 111 to 113 in the base sequence, which, therefore, codes for a polypeptide starting from Pro at position 22 [amino acid sequence III on FIG. 2 (b)] expressed a polypeptide possessing the activity of IL-2, as represented in example 5. It was also confirmed that the DNA fragment starting from the sequence GCA in position 107 to 110 in the basic sequence, which,

  <Desc / Clms Page number 24>

 therefore, code for a polypeptide starting from Ala at position 21 [amino acid sequence II in FIG. 2 (b)] expresses a polypeptide possessing the activity of IL-2, as indicated in Example 8.



   It is known that eukaryotic genes often show a polymorphism, for example, in human interferon genes [Taniguchi et al., Gene 10, 11-15 (1980), Ohno Taniguchi, Proc. Natl. Acad. Sci USA, 77, 5305-5309 (1986), and Gray et al., Nature, 295.501-508 (1981)]. In some cases, the polymorphism is accompanied by a replacement of certain amino acids in protein products and, in other cases, the structure of the protein product remains unchanged. In the case of human IL-2 cDNA, another cDNA clone (pIL2-503) can be detected in which the residue A at position 503 of the pIL2-50A cDNA (FIG. 2) is replaced by a residue G. It can also be expected that other cDNA clones will have some base substitution compared to pIL2-50A cDNA.



   As can be seen from the above, the genes of the present invention include DNAs having the bam sequence shown in Figure 2 (a), DNAs starting from the ATG sequence at positions 48 to 50 and whose sequential bases follow the ATG sequence at least up to the ATC sequence in position 504-506, the DNAs starting from the GCA sequence in position 108-110 and whose sequential bases follow the GCA sequence at least up to the ATC codon and the DNA starting from the CCT sequence in position 111-113 and whose sequential bases follow the CCT sequence at least until the ACT sequence.

   The genes of the present invention also include

  <Desc / Clms Page number 25>

 the DNAs ending in the ACT sequence in position 504 to 506 and starting from A in position 1, the ATG sequence in position 48 to 50, the GCA sequence in position 108 to 110 or the CCT sequence in position 111 to 113.



  The genes of the present invention also include the DNAs ending in the TGA sequence in position 507 to 509 and starting from A in position 1, from the ATG sequence in position 48 to 50, from the GCA sequence in position 108 to 110 or of the CCT sequence at position 111 to 113. The genes of the present invention also include the DNAs ending in C at position 801 and starting from A at position 1, from the ATG sequence at position 48 to 50, from the GCA sequence in position 108 to 110 or of the CCT sequence in position 111 to 113. The genes of the present invention also include the DNAs ending in poly (A) and starting from the ATG codon in position 48 to 50, of the GCA sequence in position 108-110 or of the CCT sequence in position 111 to 113.

   The genes of the present invention also include those whose base sequences correspond to the amino acid sequences I, II and III. In addition, the polypeptides of which one or more amino acids of the amino acid sequence I are missing, or the polypeptides of which one or more amino acids of the amino acid sequence I are replaced by one or more amino acids, may have an IL- activity. 2.



  Consequently, the genes encoded for polypeptides of this type are genes of interest in the context of the present invention. Similarly, the genes comprising an additional bond of one or more base sequences, capable of expressing one or more amino acids to the amino acid sequences I, II or III, are suitable in the context of the present invention, for

  <Desc / Clms Page number 26>

 as far as the additionally linked amino acids do not interfere with the action of the polypeptides in the expression of IL-2 activity. We can use as part of
 EMI26.1
 the present invention an additionally modified amino acid region which impairs the function of the polypeptides as IL-2, as long as the additional linked region can be readily removed.



  This situation is completely the same for the additional binding of DNA to the 3 ′ end of genes corresponding to the amino acid sequences I, II and III, encoding additional amino acids at the C terminal of the sequences I, II and III having the amino acid sequences I, II and III respectively. Therefore, it can be considered that the use of genes encoded for such polypeptides is within the scope of the present invention.



   The recombinant DNAs that direct the production of IL-2 in living cells can be constructed by various methods. For example, the IL-2 cDNA coding sequence can be introduced into an expression vehicle downstream of the promoter sequence.



  Alternatively, a cDNA fragment carrying a promoter sequence can be introduced upstream of the IL-2 coding sequence, after or before the introduction of cDNA into the expression vehicle.



   The methods for constructing prokaryotic or eukaryotic cells that express IL-2 cDNA and produce IL-2 polypeptides are explained in more detail below: (1) Expression of IL- cDNA 2 in E. coli
In order to express the IL-2 cDNA in E. coli, the cDNA is welded to various bacterial promoters and obtained.

  <Desc / Clms Page number 27>

 holds hybrid plasmids containing the cDNA downstream of the promoters. The plasmids are transfected into, for example, a strain of E. coli HBlOl and bacteria synthesizing a protein product with human IL-2 activity are cloned. Essentially any type of bacterial promoter must direct expression of IL-2 cDNA when sequentially attached to the cDNA.

   Examples of this expression of cDNA are described in the present case.



   The cDNA cloned for IL-2 encodes a polypeptide formed of 153 amino acids, as illustrated in FIG. 2. The N-terminal region corresponding to approximately 20 amino acids of this polypeptide is completely hydrophobic and this is characteristic of the most secretory proteins. This hydrophobic sequence, also called detection sequence, is cleaved during the secretion process. Therefore, the IL-2 nature polypeptide must contain less than 153 amino acids. It is therefore desirable to express the portion of cDNA encoding the mature IL-2 polypeptide but not the portion corresponding to the IL-2 detection sequence.



   (i) Construction of an expression vehicle plasmid, pTrS-3, which comprises an E. coli Trp promoter. coli, its ribosome binding site (SD sequence) for the leader peptide having been previously reported [G.



  Miozzari and Yanofsky, C. J. Bacteriol. 133,1457-1466 (1978)] as well as an ATG codon located 13 bp downstream of the SD sequence [Nishi et al., Seikagaku, 54, nO 8,676 (1982)]. The vehicle plasmid also contains a single SphI site just downstream of the ATG initiation sequence (Figure 4).

  <Desc / Clms Page number 28>

 



   To express the IL-2 cDNA, the plasmid is first digested with SphI and treated with either DNA polymerase I. coli (Klenow fragment) or by bacteriophage T4 DNA polymerase I to separate the 3 ′ ends which protrude [FIG. 5 (a)]. Plasmid pIL2-50A is digested twice with PstI and HgiAI, and a larger fragment of cDNA is isolated. The DNA is then treated either with DNA polymerase I of E. coli (Klenow fragment) or by bacteriophage T4 DNA polymerase so that the 3 ′ ends which protrude are made flush. The cDNA treated above encodes an IL-2 polypeptide of 132 amino acids, as shown in Figure 5 (a).

   This cDNA is then ligated to the DNA of the pretreated plasmid pTrS-3 as above, so that the ATG initiation codon is attached to the CCT (Pro) sequence of the IL-2 cDNA.



  This is how a plasmid pTIL2-22 is obtained. The junction between the trp promoter sequence and the IL-2 cDNA sequence of pTIL2-22 is also illustrated in Figure 5 (a).



   The plasmid pTIL2-22 must direct the synthesis in E. coli of an IL-2 polypeptide formed of 132 amino acids starting from proline.



   (ii) Since it is also possible that IL-2 re contains analine (position 21) as an N-terminal amino acid in place of proline, the following plasmid which directs the synthesis of polypeptide will be discussed. IL-2, formed of 133 amino acids.



   Plasmid pTrS-3 contains a single ClaI site between the SD sequence and the ATG sequence (Figure 4). This plasmid is digested with Cla I and Sal Io The plasmid

  <Desc / Clms Page number 29>

 pIL2-50A is partially digested with PstI, treated with DNA polymerase I of E. coli and isolate the largest linear DNA. The DNA is then ligated using a synthetic DNA binder containing a restriction XhoI cleavage site and a clone containing the plasmid pIL2-50A (Xho) is isolated, which is first digested with HgiAI, treated either by a Klenow fragment from E. coli either with T4 DNA polymerase, and digested with XhoI and the cDNA fragment is isolated. The cDNA fragment is then ligated with pTrS-3 DNA pretreated with ClaI and SalI and with synthetic DNA, as indicated in FIG. 5 (b).

   Thus, it is possible to obtain a plasmid of pTIL2-21 which will direct the synthesis in E. coli of an IL-2 polypeptide formed of 133 amino acids starting from alanine, as illustrated in FIG. 5 ( b). A similar construction can also be carried out without using the XhoI binder.



   (iii) IL-2 polypeptides of different dimensions can be obtained with different N-terminal amino acids using the expression vehicle plasmid pTrS-3 by the following method. The IL-2 cDNA cloned into pIL2-50A contains a single DdeI site at the position of nucleotides 81-85. The plasmid pIL2-50A (Xho) is digested with DdeI and the DNA fragment contained is isolated.
 EMI29.1
 the largest portion of cDNA. The fragment will also contain approximately 3000 pairs of low $ of DNA from pBR322 [Figure 5 (c)]. The DNA fragment is treated with exonuclease Bal 31 and is then digested with XhoI.

   The DNA treated above is then ligated with pTrS-3, which is digested with SphI, treated either with the Klenow fragment or with T4 DNA polymerase

  <Desc / Clms Page number 30>

 and is then digested with SalI, as illustrated in Figure 5 (c). The ligated DNA is transfected into E. coli HB101 and the bacterial clones expressing human IL-2 are examined. These clones must express human IL-2 of different dimensions since the DNA corresponding to the N-terminal region of human IL-2 is
 EMI30.1
 so destroyed This is how pTIL2-14 and pTIL2-15 carrying IL-2 cDNA could be obtained.



   (iv) IL-2 cDNA can also be expressed by the use of pKT218 (obtained by K. Talmage) [Proc. Natl. Acad. Sci. USA, 77, pages 3369-3373 (1980)]. The plasmid pKT218 is digested with PstI and ligated by means of an insertion of IL-2 cDNA obtained by digestion of pIL2-50A DNA with HgiAI and PstI (FIG. 6).



  The plasmid pKIL2-21 resulting in the sequence at the beginning of the initiation of protein synthesis as shown in FIG. 6. This is how the plasmid pKIL2-21 must direct the synthesis in E. coli of a polypeptide condensate formed of 133 amino acids of IL-2 and an amino acid of P-lactamase (the first methionine is separated by cleavage in E. coli).



   (v) A pTuBlP-5 expression plasmid has previously been constructed in which the promoter sequence for tuf B is introduced in pBR322 [Taniguchi et al., Seikagaku, 53,966 (1981)]. The plasmid contains a single ClaI site and this site is located 2 bp downstream of the SD sequence, as shown in FIG. 7.



   Since pTrS-3 also contains a ClaI site between the SD sequence and the initiation sequence

  <Desc / Clms Page number 31>

 of ATG, and since this ClaI site is not destroyed during the construction of the expression plasmid using pTrS-3 and the IL-2 cDNA, as described above, it is very simple to replace the bacterial trp promoter with that of tufB, so that the human IL-2 cDNA is expressed under the control of the tufB promoter. For example, pTIL2-22 is digested with ClaI and Pvull and the DNA fragment containing the IL-2 cDNA is isolated. This fragment is then ligated with DNA from pTuBlP-5, predigested by ClaI and Pvull and a plasmid pTuIL2-22 is constructed, as illustrated in FIG. 7.

   IL-2 activity could be detected in the extract of E. coli HB101 harboring the plasmid pTuIL2-22.



   (vi) A similar construction can also be carried out using, for example, pTIL2-21 and essentially all of the expression plasmids which are constructed by the use of pTrS-3. It is also possible to optimize the distance between the SD and ATG sequences by digestion, for example, of pTuIL2-22 by ClaI, separation (or addition) of a few base pairs of DNA by Bal 31 or SI or by l DNA polymerase 1 (E. Coli) and then by religation of the plasmid.



   (2) Expression of IL-2 cDNA in yeast.



   IL-2 cDNA can also be expressed in yeast by inserting cDNA into appropriate expression vectors and introducing the product into host cells. Various transfer vectors for the expression of foreign genes in yeasts have been reported. [Heitzman, R. et al., Nature 293, 717-722 (1981), Valenzuela P. et al., Nature 298, 347-350 (1982), Miyanohara et al., Proc. Natl. Acad.

  <Desc / Clms Page number 32>

 Sci. USA 80,1-5 (1983)]. The vectors are capable of replication in both E. Coli and in host yeasts and they contain promoter sequences of yeast genes. Essentially all of these expression vectors can be used for expression of the IL-2 cDNA.

   It is possible to achieve higher levels of IL-2 production by using yeast compared to using bacteria or animal cells. We now describe an example of expression of human IL-2 cDNA in yeast.



   The yeast and E. coli transfer vectors pAT77 and pAM82 have been described by Miyanohara et al. [Proc. Natl. Acad. Sci. USA 80,1-5 (1983)]. The vector pAM82 is a derivative of pAT77 and both carry ar 1 markers [Stinchcomb D. T. et al., Nature 282,39-43, (1979), 2 pm ori (Broach J. R. et al.



  Gene 8, 121-133 (1979) and leu 2 (Ratzkin B. et al.



  Proc. Natl. Acad. Sci. USA 74, 474-491 (1979)] and the promoter for the yeast acid phosphatase (APase) gene. They also carry a 3.7 kb DNA segment of pBR322 which contains a marker for ampicillin resistance (Apr) and the origin of replication (Figure 8). The APase promoter is inducible from a high concentration of phosphate to a low concentration in the culture medium. In order to express the human IL-2 cDNA, pIL2-50A is digested with PstI after treatment with either the Klenow fragment of E. Coli either with T4 DNA polymerase, the cDNA is ligated using pAM82 previously digested with XhoI and incubated with the Klenow fragment from E. coli. Package to fill the ends.

   Hybrid plasmids are chosen in which the coding cDNA sequence is downstream from the yeast APase promoter sequence by cloning them into E. Coli. The plasmid obtained, pYIL-2a, is introduced,

  <Desc / Clms Page number 33>

 in yeast and, after introduction of the APase promoter, the activity of IL-2 in the yeast extract is measured. The plasmid pYIL-2a contains a series of GC residues between the yeast promoter and the IL-2 cDNA. It is possible that such a sequence inhibits the expression of the IL-2 cDNA. In order to overcome this problem, the following construction of a plasmid is carried out; the plasmid pIL2-50A is digested with PstI and the insertion of cDNA is isolated.

   This cDNA is then treated with T4 DNA polymerase in the presence of dATP, dGTP and DTTP so that the successions of residues C at the two ends of the cDNA are fragmented, and it is then treated with nuclease SI, so as to separate the successions of residues G. This DNA is ligated with a DNA binder from XhoI and the plasmid pBR322, the EcoRI site of which is cleaved and is made flush with EcoRI and the Klenow fragment, the plasmid resulting, pIL2-Xho, being digested with XhoI and insertion of isolated cDNA. The cDNA is then introduced into the unique XhoI site of pAM82 and a plasmid containing the coding sequence of IL-2 correctly oriented relative to the yeast APase promoter is cloned into E. Coli.

   The plasmid pYIL-2b is introduced into the yeast and, after introduction of the APase promoter, the activity of IL-2 in the yeast extract is measured.



   (3) Expression of cDNA in mammalian cells.



  A plasmid which would direct the synthesis of human IL-2 in mammalian cells can be constructed as follows. A plasmid pCE-1 is constructed from pKCR [O'Hare K. et al., Proc. Natl. Acad. Sci.



  USA., 78, 1527-1531, (1981)] and pBR328 [Soberon X. and
 EMI33.1
 coll., Gene, 9, 287-305 (1980)] by a series of modification processes as illustrated in FIG. 2,

  <Desc / Clms Page number 34>

 and an ATG initiation sequence of the IL-2 gene is linked downstream of the promoter of the SV40 early gene. This plasmid contains a single PstI site just downstream from the SV40 early promoter and upstream from the intron-containing portion of the rabbit p-globin chromosomal gene. The plasmid also contains the origin of the replication of SV40 as well as the polyadenylation site for the early gene. Thus, a plasmid pCEIL-2 is obtained, in which the structural gene for IL-2 must be transcribed from the SV40 early promoter into appropriate host cells (FIG. 2).



   This plasmid is digested with Hhal and is then introduced by DNA transfection into the COS-7 transformed monkey cell line, which allows replication of DNA containing the original sequences of SV40. It appears to be important to have the plasmid digested by Hhal before transfection for the effective expression of cDNA since the sequences which could hinder the replication of the transfected DNA in COS cells can be separated from the essential part of the plasmid for cDNA expression by this process. After one to three days of culture under ordinary culture conditions after transfection of this vector into cultured monkey cells COS-7 [Gluzman Y. Cell, vol. 23, 175-182 (1981)], IL-2 is ordinarily secreted and produced in the medium of cultured cells.

   In order to insert amplified DNA into other eukaryotic cells, a vector suitable for host organisms is similarly linked to an insertion of cleaved and isolated cDNA from prokaryotic cells and the eukaryotic cell can be transfected with the vector as well. synthesized and cultivated.



   Cells incorporating recombinant DNA are grown to amplify the recombinant DNA or

  <Desc / Clms Page number 35>

 to produce an IL-2 polypeptide. Cultivation is carried out by usual means. For example, a transformed yeast can be grown in a medium containing a carbon source, a nitrogen source, inorganic salts and, as necessary, organic nutrients, such as vitamins and amino acids, at a temperature of of the order of 200 to 370C and at a pH of the order of 4 to 7 under aerobic conditions. Transformed prokaryotic organisms such as E. coli or B. subtilis can also be grown under ordinary conditions.



   The IL-2 produced is recovered intracellularly or extracellularly by any known method, such as by precipitation with ammonium sulphate, dialysis to separate the salts (at ordinary pressure or under vacuum), gel filtration, chromatography, isoelectric focusing on preparatory flat bed, gel electrophoresis, liquid chromatography
 EMI35.1
 high yield (hereinafter called "CLHR") ion exchange chromatography, gel filtration and reverse phase, and affinity chromatography on a support bound to a dye, on activated Sepharose 4B coupled with a vis monoclonal antibody -in relation to IL-2 or on Sepharose 4B linked to lectin, etc. Methods for recovering and purifying IL-2 are described by Watson et al., J. Exp. Med., 150,848-861 (1979), Gillis et al., J.

   Immunol., 124, 1954-1962, (1980), Mochizuki et al., J. Immunol Methods 39, 185-201, (1980) and by Welte, K. et al., J. Exp. Med., 156, 454-464 (1982).



   The polypeptide thus obtained shows the same biochemical and biological behavior as that of IL-2 produced by mammalian cells by stimulation using a mitogen, and is endowed with the activity

  <Desc / Clms Page number 36>

 of IL-2. The molecular weight is around 15,000 daltons and the activity of IL-2 is completely neutralized or precipitated by monoclonal anti-IL-2 antibodies in the presence or in the absence of immunoadsorbents, such as Isgorb ( Enzyme Center). In immunoelectrophoresis, the IL-2 polypeptide shows only a single precipitate vis-à-vis the corresponding anti-IL-2 antibody. The activity of IL-2 remains stable after reduction with 2-mercaptoethanol, and is resistant to treatment with DNAse and RNAse and to heat treatment at 560C for 30 minutes.

   The activity is stable at a pH between 2 and 9. The IL-2 produced could promote the growth of functional monoclonal T cells (cytotoxic T lymphocytes), promote the mitogenesis of thymocytes, give rise to the production of T lymphocytes specific anti-tumor cytotoxics from a humoral state in the absence of antigen, and could be used to increase the activity of natural killer cells
 EMI36.1
 against YAC-1 and RL 1 cells.



   The present invention will now be described in more detail by means of certain specific examples which are given solely for the purpose of illustration and not for limiting purposes, unless otherwise indicated.



   Example 1.



   (1) A line of human T leukemia cells, namely Jurkat cells (freely available in Japan, the Federal Republic of Germany and the United States) is suspended in RPMI 1640 medium containing 10% by volume / volume of FCS and irradiated with X-rays up to 10,000 roentgens at room temperature for 50 seconds using an Exs 150 / 300-4 X-ray irradiator

  <Desc / Clms Page number 37>

 (Toshiba, Japan), and the irradiated cells are then cultured for 5 days at 37 ° C in a incubator.
 EMI37.1
 holding 5% CO 2 at an initial cell density of 15 × 10 5 ml cells in the above-mentioned culture medium.

   The mutated cells (0.2 cells / well) are placed in 10 flat-bottom microplates comprising 96 wells, and cultured at 37 ° C. in an incubator containing 5% CO 2 for 21 days.



   Clones obtained from wells showing growth are repeatedly transferred to fresh culture medium to propagate clone sizes, propagated clones are cultured for 24 hours at an initial cell density of 1 x
106 cells / ml in the presence of 50 g / ml of Con A and the activity of IL-2 is measured according to the methods described above. Therefore, a human T cell line called Jurkat-111 (hereinafter referred to as "J-III") (ATCC CRL8129) was selected, cloned from original Jurkat cells, whose IL productivity -2 was increased 40-fold over that of the base strain.

   The J-111 cloned cell line could grow under ordinary conditions and the growth rate is almost similar to that of ordinary Jurkat cells.



   (2) Cells (1 × 105 / mol) of J-111 are inoculated into 1,000 ml of synthetic culture medium without RITC 55-9 serum [Sato T. et al., Exp. Cell Res., 138, 127-134 (1982)] in culture bottles fixed in rotating drums (Falcon 3027) and they are cultivated for 4 days at 37 ° C., the cells which propagate being collected by centrifugation. The collected cells are again inoculated into the abovementioned medium to which 25 g / ml of Con A are added, so as to obtain 4 × 10 6 cells / ml. We carry out

  <Desc / Clms Page number 38>

 tre culture bottle tests on rolling drums (Falcon), placing in each test 1,000 ml of the inoculated culture medium.

   The culture is continued for 6 hours with rotation.



   (3) The Jurkat cells (1.2 x 10) thus stimulated with 25 g / ml of Con A for 6 hours are suspended in 8,000 ml of phosphate buffer balanced by a saline solution (hereinafter called "PBS" ). Cells are washed twice by centrifugation and are then resuspended in 800
 EMI38.1
 ml RSB solution (10 millimoles of Tris-HCl; pH 7.5, 10 millimoles of NaCl; 1.5 millimoles of mgCl) containing a ribonucleoside-vanadyl complex (10 millimoles), a nuclease inhibitor. Then add an NP-40 detergent so as to reach a final concentration of 0.05%, mix gently and separate the cell nuclei by centrifugation for 5 minutes
 EMI38.2
 at 3.000 rpm at 4 C.

   SDS (0.5%) and EDTA (5 millimoles) are added to the supernatant, and the cytoplasmic RNA is extracted by adding an equal volume of phenol. After three phenol extractions, the RNA is preciped with a double volume of ethanol and the precipitates are collected by centrifugation, which is dissolved in 10 millimoles of Tris-Hcl of pH 7.5. The amount of RNA obtained is 196 mg.



   An mRNA fractionation is carried out using affinity chromatography on oligo (dT) -cellulose (P. L. Biochemicals, Type 7). The adsorption solution is a pH 7.5 solution containing 20 millimoles of Tris-HCl, 0.5 moles of NaCl, 1 millimole of EDTA and 0.5% SDS and the elution is carried out successively with l and 10 millimoles of Tris-HCl (pH 7.5) after washing the column with the buffer (20 millimoles of Tris-HCl, pH 7.5; 0.5 moles of NaCl; 1

  <Desc / Clms Page number 39>

 
 EMI39.1
 millimole of EDTA). The resulting eluted mRNA is 3.6 mg.

   Then, 2.4 mg of the mRNA obtained is fractionated by a sucrose density gradient centrifugation (sucrose density gradient from 5 to 2.5%) in a solution at pH 7.5 containing 50 millimoles of Tris- HCl, 1 millimole of EDTA and 0.2 moles of NaCl, and centrifuged at 26,000 revolutions per minute for 24 hours at 4 C, the mRNA fractions comprising 11 to 12 strands being fractionated as fractions numbers 12,13 and 14 59 g, 46 g and 60 g respectively; (4) The mRNA obtained in the nr fraction. 13 is microinjected into the Xenopus laevis oocyte (50 ng of mRNA / egg) and the culture supernatant is used to test the activity of IL-2.

   As indicated in Table 1, the increase in the number of activated T lymphocytes is confirmed, which clearly shows that the mRNA in this fraction contains human IL-2 mRNA.

  <Desc / Clms Page number 40>

 TABLE 1 (a)
 EMI40.1
 
 <tb>
 <tb> Sample <SEP> Dilution <SEP> Fixation <SEP> from <SEP> Quantity <SEP> of IL-2
 <tb> 3H-TdR <SEP> (cmp) <SEP> (Units / ml)
 <tb> Control <SEP> 1-553 <SEP> 0 <SEP>
 <tb> (Middle <SEP> for
 <tb> test)
 <tb> Control <SEP> II <SEP> x <SEP> 2 <SEP> 590 <SEP> 0
 <tb> (Supernatant <SEP> from
 <tb> culture Egg <SEP>
 <tb> no <SEP> processed)

    <SEP> x <SEP> 32 <SEP> 572
 <tb> Product <SEP> from <SEP> translation <SEP> from <SEP> the <SEP> x <SEP> 8 <SEP> 14.683 <SEP> 32
 <tb> fraction <SEP> 13 <SEP> x <SEP> 32 <SEP> 10.165
 <tb>
 
 EMI40.2
 
 EMI40.3
 
 <tb>
 <tb> Dilution <SEP> Number <SEP> from <SEP> Quantity <SEP> of IL-2
 <tb> lymphocytes <SEP> (units / ml)
 <tb> T <SEP> (No. / well)
 <tb> Control <SEP> I <SEP> x <SEP> 2 <SEP> 0 <SEP> 0
 <tb> (Middle <SEP> for
 <tb> test) <SEP> x <SEP> 16 <SEP> 0 <SEP> 0
 <tb> Control <SEP> II <SEP> x <SEP> 2 <SEP> 0 <SEP> 0
 <tb> (Supernatant <SEP> from
 <tb> culture Egg <SEP> <SEP> x <SEP> 16 <SEP> 0
 <tb> no <SEP> processed)
 <tb> Product <SEP> from <SEP> translation <SEP> from <SEP> the <SEP> x <SEP> 2 <SEP> 115 <SEP> 40
 <tb> fraction <SEP> 13 <SEP> x <SEP> 16 <SEP> 55
 <tb>
 The unit is calculated by comparing the amount of 3H-TdR incorporated with that of standard IL-2 (10 units / ml)

   following a statistical analysis.



   (5) Then, we synthesize cDNA in

  <Desc / Clms Page number 41>

 
 EMI41.1
 in vitro from fraction nr. 13 of 11 to 12 strand mRNA containing IL-2 mRNA and recombinant DNA is reconstructed with the vector plasmid PBR 322. With recombinant DNA, Escherichia coli is transformed, and the clone is selected having acquired IL-2 cDNA clones in the following manner: (5-1) 50 millimoles of Tris-HCl buffer (pH 7.5), 30 millimoles of NaCl, 6 millimoles of MgCl2.5 millimoles of dithiothreitol (hereinafter referred to as "DTT"), 0.5 millimole of each of the compounds dATP, dGTP, dCTP and
 EMI41.2
 32 dTTP (dCTP is radioactively labeled with P, 0.7 pg of oligo (dT) 10 '10 pg of mRNA and 15 units of AMV reverse transcriptidase (J. W. Beard) are mixed and maintained for 90 minutes at 41 C.

   Once the reaction is complete, the DNA is recovered in the form of ethanolic precipitates after treatment with phenol, and the DNA is dissolved in a solution of pH 7.5 containing 20 millimoles of Tris-HCl and 1 millimole of EDTA . 2.5 μg of ss-cDNA are synthesized. To separate the mRNA present in this solution, NaOH is added to the solution until a 0.33 N NaOH solution is obtained, it is left to stand for 15 hours at room temperature, then neutralized the solution with an equal volume of 1M Tris-HCl of pH 7.5 and it is passed through a column of "Sephadex G-50". 1.8 g of cDNA are recovered.



   (5-2) 50 millimoles of phosphate buffer (pH 7.5) are mixed, 10 mM of MgCl 2 × 10 mM of DTT, 0.75 mM of each of the following compounds: dATP, dGTP,
 EMI41.3
 g dCTP and dTTP (dCTP is radioactively labeled with 3H), 1.8 g of ss-cDNA and 8 units of polymerase I (BRL, United States) and left to react for 15 hours at 15 C. Once the reaction finished, the DNA is recovered in the form of an ethanolic precipitate, after treatment

  <Desc / Clms Page number 42>

 with phenol and chloroform. 1.10 μg of ds-cDNA are obtained. A 50 mM a- mixture is incubated
 EMI42.1
 sodium acetate (pH 4.5), 0.2 M NaCl, 1 mM ZnCI- and 1.10 g ds-cDNA for 20 minutes at 35 C, 0.25 nuclease units are added S- (Sankyo, Japan), and incubate again for 15 minutes.

   Once the reaction is complete, the reaction product treated twice with phenol is applied to Sephadex G-50 to give 0.55 g of ds-cDNA.



   (5-3) A mixture of 0.14 M potassium cacodylate, 30 mM Tris-base, 0.1
 EMI42.2
 mM DTT, 1 mM COC12 ′, 64 mM P-dCTP fi (specific activity of 2.7 x cpm / n mole), 0.55 µg of ds-cDNA and 5 units of terminal transferase (BRL ) for 7 minutes at 350 ° C., and then it is applied to a column of Sephadex G-50 after treatment with phenol, which gives 0.50 g of DNA in the form of ethanolic precipitates. It is found that the DNA recovered has lengthened by approximately 50 residues of dCMP at the two 3 ′ ends.

   10 g of pBR 322 are cleaved by the restriction enzyme PstI, and the 3 ′ ends of the cleaved DNA are added to the dGMP chain, by the same method as that used in the addition of dCMP to ds-cDNA mentioned above, except that dGTP is used in place of dCTP.



   (5-4) A mixture of 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 5 mM EDTA, 0.05 μg of pBR 322 extended by residues of dGMP and 0.01 µg of dCMP-lengthened cDNA is first incubated for
 EMI42.3
 for 2 minutes at 65 ° C, then for 120 minutes at 46 ° C, for 60 minutes at 37 ° C and finally for 60 minutes at room temperature. Inoculate E. coli X 1776 [Curtiss III, R. et al., In Molecular Cloning o Recombinant DNA, edited by W. A. Scott & R.

   Werner, Aca-

  <Desc / Clms Page number 43>

 demic Press (1977)] in 50 ml of L broth containing 100 µg / ml diaminopimelic acid, 50 µg / ml thymidine, 1% tryptophan, 0.5% yeast extract, 0.5%
 EMI43.1
 of NaCl and 0.1% of glucose and shaking is carried out at 37 ° C. until the absorption of the culture liquid at 562 nm is around 0.3 optical density. After finishing the culture, the culture liquid is left at 0 ° C. for 30 minutes, and the bacterial cells are then collected by centrifugation followed by double washing with 25 ml of a solution containing 5 mM Tris-Hcl (pH 7.6), 0.1 M NaCl, 5 mM MgCl2 and 10 mM RbCl.

   The cells thus obtained are suspended in 20 ml of a solution containing 5 mM Tris-HCl (pH 7.6), 0.25 M KC1, 5 mM MgCI2'0, 1 M CaCl and 10 mM RbCl and left at 0 ° C. for 25 minutes, the cells are then collected to resuspend them in 1 ml of the same solution, the recombinant DNA described above is added in 0.2 ml of the cell suspension.
 EMI43.2
 and the suspension is left at 0 ° C. for 60 minutes.

   Then, 0.7 ml of broth L is added to the culture while shaking for 30 minutes at 37 C. The culture medium thus obtained (0.1 ml) is applied completely to the surface of a medium at 1.5 % agarose, formed of L broth containing 100 μg / ml diaminopimelic acid, 50 μg / ml of thymidine and 15 μg / ml of tetracycline, and is incubated at 37 ° C. for 2 days.



   (5-5) The four hundred and thirty two colonnies which appear are divided into 18 groups, each group containing 24 different bacterial clones, inoculated in 200 ml of broth L containing 100 μg / ml of diaminopimelic acid, 50 μg / ml of thymidine and 10 μg / ml of tetracycline and cultivated in shakes at 37 ° C. for 5 to 7 hours. Then we add again

  <Desc / Clms Page number 44>

 to culture overnight 200 ml of fresh L broth containing chloramphenicol at a final concentration of 170 pg / ml. The plasmid DNA thus amplified is purified by usual means. The clones comprising the IL-2 cDNA are detected by a hybridization-translation test of mRNA (hereinafter called "H-T" test).

   The HT test is used in the present case as follows: purified DNA (25 g) is cleaved by a restriction enzyme Hind III, treated three times with phenol, treated respectively with a mixture of phenol and chloroform and chloroform, precipitated with ethanol, washed with 80% ethanol and dissolved in 40; jl of 80% formamide. The reaction mixture is heated for denaturing
 EMI44.1
 ration at 90 ° C. for 5 minutes, and then diluted to 1.3 ml with 10 × SSC (1.5 M NaCl, 0.15 M sodium citrate).

   The DNA is then fixed on nitrocellulose filters, which filters are dried at 80 ° C. for 3 hours and incubated for 18 hours at 370 ° C. in a solution containing 50% formamide, 20 mM Pipes
 EMI44.2
 pH 6, 5, 0.75 M NaCl, 5 mM EDTA, 0.2% SDS and 250 μg of poly (A) mRNA from J-111 cells induced so as to hybridize the DNA fixed on filters with IL-2 mRNA. Then, the filters are washed at 650C three times with a solution containing 10 mM of Pipes of pH 6, 5 and 0.15 M of NaCl, a solution containing 1 mM of Pipes and 10 mM of NaCl and treated with a solution containing 0 , 5 mM EDTA and 0.1% SDS at 950C for 1 minute so as to recover the hybridized mRNA from the filters.

   The mRNA thus extracted is purified on a column of oligo dT-cellulose in the usual manner and is injected into Xenopus oocytes so as to determine the activity of IL-2 of the proteins translated. Only one of the 18 groups, each group comprising 24 clones, gives a positive IL-2 activity of 48 units / ml in

  <Desc / Clms Page number 45>

 
 EMI45.1
 3 the H-TdR incorporation test described above, all the other groups being clearly negative. Then, 24 isolated colonies belonging to the positive group are inoculated in 200 ml of broth L corresponding to the same composition as that described above, they are aerobically cultivated for 5 to 7 hours at 370C and they are again added, in a similar manner, fresh L broth containing chloramphenicol.

   After amplification of the plasmid DNA using overnight culture, the plasmid DNA is similarly purified according to standard procedures. After cleavage of approximately 5 µg of each plasmid DNA by Hind III, each plasmid DNA is similarly linked to nitrocellulose filters. The filters are hybridized with IL-2 mRNA and the hybridized mRNA is recovered for injection into Xenopus oocytes so as to determine the IL-2 activity of the translated proteins.



  As indicated in Table 2, only the plasmid DNA purified from a single colony, called p3-16, gives positive IL-2 activity. Therefore, this clone is identified as being the clone having the IL-2 cDNA [E. coli X 1776 / p3-16 AJ 11995 (FERM-BP-225)].
 EMI45.2
 



  Thus it is confirmed that the plasmid DNA, p3-16, exactly shares the DNA (IL-2 gene) capable of forming the specific hybrid with the IL-2 mRNA.

  <Desc / Clms Page number 46>

 TABLE 2 (a)
 EMI46.1
 
 <tb>
 <tb> *
 <tb> Sample <SEP> Dilution <SEP> Fixation <SEP> from <SEP> Quantity <SEP> of IL-2 <SEP>
 <tb> 3H-TdR <SEP> (cpm) <SEP> (Units / ml)
 <tb> Control <SEP> l <SEP> - <SEP> 2.

    <SEP> 010 <SEP> 0
 <tb> (Middle <SEP> for
 <tb> test)
 <tb> Control <SEP> II <SEP> x <SEP> 2 <SEP> 2.120 <SEP> 0
 <tb> (Supernatant <SEP> from
 <tb> liquid <SEP> from <SEP> cul-x <SEP> 32 <SEP> 2.482 <SEP> 0
 <tb> ture Egg <SEP>
 <tb> no <SEP> processed)
 <tb> Product <SEP> from <SEP> translation <SEP> of mRNA <SEP> x <SEP> 8 <SEP> 20.453 <SEP> 58
 <tb> x <SEP> 32 <SEP> 20.961
 <tb>
 
 EMI46.2
 (b)
 EMI46.3
 
 <tb>
 <tb> Sample <SEP> Dilution <SEP> Number <SEP> from <SEP> Quantity <SEP> of IL-2 *
 <tb> lymphocytes <SEP> T <SEP> (Units / ml)
 <tb> (Cells /
 <tb> well)
 <tb> Control <SEP> l <SEP> - <SEP> 0 <SEP> 0 <SEP>
 <tb> (Middle <SEP> for
 <tb> test)
 <tb> Control <SEP> II <SEP> x <SEP> 2 <SEP> 0 <SEP> 0
 <tb> (Supernatant <SEP> from
 <tb> liquid <SEP> from <SEP> cul-x <SEP> 32
 <tb> ture Egg <SEP>
 <tb> no <SEP> processed)

  
 <tb> Product <SEP> from <SEP> translation <SEP> of mRNA <SEP> x <SEP> 2 <SEP> 88 <SEP> 32
 <tb> x <SEP> 32 <SEP> 42
 <tb> mRNA <SEP> hybrid <SEP> by <SEP> cDNA <SEP> from <SEP> from <SEP> plasmid <SEP> p3-16.
 <tb>
 

  <Desc / Clms Page number 47>

 



  The insertion of cDNA from plasmid p3-16 shows characteristics which must be cleaved by the restriction enzyme XbaI at a single site and by BstNI at two sites (upstream and downstream of the XbaI cleavage site). However, plasmid p3-16 contains an insertion of cDNA formed of approximately 650 base pairs, which apparently corresponds to a part of the IL-2 mRNA of the size of 11 to 12 strands.



   Therefore, another cDNA batch is prepared according to the method of Land et al. [Land et al., Nucleic Acids Res., Vol. 9, p. 2551, (1981)] using IL-2 mRNA as a template or template. Single-stranded cDNA (1.6 µg) is synthesized using 4 µg of IL-2 mRNA extended by dCMP residues, and ds-cDNA is synthesized using oligo (dG) 12- 18 as an initiator for DNA polymerase 1 (Klenow fragment). We obtain a cDNA (0.6 pg) longer than a mar-
 EMI47.1
 DNA donor comprising 680 base pairs by a sucrose gradient centrifugation and it is introduced into the PstI site of pBR322 by the standard G-C end method.

   After transformation of E. coli X 1776 with recombinant DNA, approximately 2,000 colonies are detected by the Grunstein-Hogness in situ hybridization method with an insertion of p3-16 cDNA translated (by notch) as probe. and the colony containing the plasmid pIL 2-50A containing approximately 850 base pairs and the transformed clone [E. coli X 1776 pIL 2-50A, AJ 11996 (FERM-BP-226)]. The maps showing the cleavage points made by the endonucleases restricting the insertion of pIL 2-50A cDNA are represented in FIG. 1.



   To isolate a gene encoding an IL-2 peptide from pIL 2-50A of E. coli X 1776 transformed, plasmid DNA is digested by the enzyme

  <Desc / Clms Page number 48>

 PBS, and then 0.5 ml of PBS containing 20% glycerol is added, leaving it at room temperature for 3 minutes. Again, the medium is separated by aspiration and the cells are washed with 1 ml of PBS and cultured in 1 ml of DMEM containing 5% FCS. Every 24 hours, 500 μl of medium is replaced by fresh medium. Each medium, collected at appropriate time intervals, is maintained at 4 ° C until the time of use. Two to three days after transfection, the medium of cultured cells is examined for its activity of human IL-2.

   As indicated in Table 3, the culture supernatant resulting from COS-7 cells transfected with PCEIL-2 is endowed with IL-2 activity. No IL-2 activity was detected in the culture medium of cells transfected with pCE-1.



   TABLE 3
 EMI48.1
 
 <tb>
 <tb> DNA <SEP> transfected <SEP> by <SEP> Activity <SEP> of IL-2 <SEP> Growth <SEP> from
 <tb> measured <SEP> by <SEP> fi-lymphocites <SEP> T
 <tb> xation <SEP> from
 <tb> H-TdR <SEP> (p / ml)
 <tb> PCEIL-2 <SEP> 12 <SEP> ++++ <SEP>
 <tb> pCE-1 <SEP> 1
 <tb>
 
IL-2 activity found in cell culture medium after transfection of COS-7 cells with pCEIL-2 is neutralized from 12 units / ml to less than 1 unit / ml by anti-IL monoclonal antibodies -2-human mouse (BALB / c).

   The fact that the COS-7 cells transfected with pCEIL-2 secrete human IL-2 clearly shows that the eukaryotic cells transformed by a recombinant DNA comprising a gene coding for IL-2 polypeptide and a DNA from vector capable of replication in these cells, can prove to be very interesting for the production

  <Desc / Clms Page number 49>

 both contain a single PstI site just downstream from the SV40 early promoter.



   The insertion of cDNA from pIL 2-50 is excised by cleavage using PstI and ligated to pCE-1 cleaved by pCTI for the construction of pCEIL-2 in which the expression of the structural gene of IL-2 must be under the control of the SV40 early promoter. The plasmid pCE-1 is initially constructed for the cloning of cDNA by the GC extremity method [Chang ACY et al., Nature, 275,617-624, (1978)] in bacteria and direct expression in mammalian cells .



   This plasmid is digested with HhaI and is then introduced by DNA transfection (McCutchan et al., J. Natl. Cancer Inst. 41, 351-357, 1968) into a COS-7 transformed monkey cell line, which allows the DNA replication containing the original SV40 sequences, see Gluzman Y. (Cell 23, 175-182, 1981). It appears to be important to digest the plasmid by HhaI before transfection for the effective expression of cDNA since the sequences which could hinder the replication of the transfected DNA in COS cells can be separated from the essential part of the plasmid for cDNA expression by
 EMI49.1
 To this process.

   COS-7 cells (6 x are suspended in 0.5 ml of DMEM containing 5% FCS in a culture plate comprising 24 wells (Nunc) and incubated for 24 hours at 370C. Then, the cultures are added to the cultures a mixture of 1 pg of the vector
 EMI49.2
 above, 17.6 µl of 1mM Tris-HCl containing 0.1 mM EDTA, 2.4 µl of 2 M CaCl2 and 120 µl of 2 x HBS containing 50 mM Pipes, 280 mM of NaCl and 1.5 mM (pH 7.10). The cells are again incubated for 4 hours at 370C and the culture medium is suctioned off, washed with 1 ml of

  <Desc / Clms Page number 50>

 PstI restriction after isolating the DNA region from cells by conventional means. Thus, the smallest fragment obtained from the two DNA fragments produced is the DNA gene coding for the peptide of IL 2.

   The complete nucleotide sequence of the insertion of PstI from pIL 2-50A is determined by the method of Maxam and Gilbert [Maxam A. W. et al., Enzym. 65, 499-560 (1980)], the entire structure being shown in Figure 2.



   Example 2
The plasmid pKCR [O'Hare et al. Proc.



  Natl. Acad. Sci., USA, vol. 78, No. 3.1527-1531 (1981)] consists of (i) segments of DNA from SV40 (represented by the hatched sections in FIG. 3) containing an early gene promoter as well as an origin of replication ( 0.725-0.648 mu) and a polyadenylation site originating from the early gene (0.169-0.144 mu), (ii) part of the rabbit y-globin gene (represented by the open sections (fragment BamHI- PvuII) and (iii) a segment from pBT322 (EcoRI-BamHI fragment) containing an origin of replication and an ampicilin resistance gene. This plasmid is cleaved by BamHI and, after having completed the two ends of the 'DNA cleaved by DNA polymerase I (Klenow fragment), a PstI binder DNA is introduced to reconstruct pKCR (PstI).

   The plasmid pKCR (PstI) is cleaved by SalI, treated with the Klenow fragment to complete the ends and is then partially cleaved by EcoRI so as to obtain an EcoRI-SalI fragment which contains the total DNA originating from SV40 and the gene for globin. This fragment is then ligated to a piece of DNA from pBR328, which contains a tetracycline resistance gene and an origin of replication, as shown in FIG. 3. The plasmid pCE-1 results

  <Desc / Clms Page number 51>

 of IL-2.



   The plasmid PCEIL-2 (AJ 12008) incorporated into E. coli HB101 was deposited under the registration number FERM-BP 244.



   Example 3.



   Escherichia coli X 1776 / pIL 2-50A [AJ 11996 (FERM-BP-226)] prepared in Example 1 is inoculated into 250 ml of broth L, containing 100 μg / ml of acid
 EMI51.1
 diaminopimélique, 50 pg / ml of thymidine, 1% of tryptophan, 0.5% of yeast extract, 0.5% of NaCl and 0.1% of glucose, and it is cultivated in shakes at 37 ° C. that the optical density at 562 nm of the cultured medium is 0.5. Once the culture is complete, the cultured medium is left standing at 0 ° C. for 30 minutes, the cells are harvested by centrifugation, they are washed once with 20 mM Tris-HCl containing 30 mM NaCl and they are resuspended in 1.8 ml of the same buffer.



  A solution containing 0.2 μl of lysozyme (10 mg / ml) and 20 μl of 0.5 M EDTA is then added to the cells and the mixture is left to stand at 0 ° C. for 20 minutes, the mixture then being frozen. thawed three times successively. Then, cell extracts (1.5 ml) are obtained after centrifugation at 40,000 rpm for 30 minutes. The extract is subjected to salting out with 85% ammonium sulphates, it is applied to Sephadex G15 to separate the salts, it is then subjected to chromatography on a DEAE cellulose column and the fractions eluted with 0.06 M Tris-HCl buffer (pH 7.6).

   The fractions thus collected are dried by freezing and are then subjected to chromatography on glass beads with controlled pores (250Â, Funakoshi Pharmaceuticaly, Japan) so that the activity of IL-2 is imparted in the eluent with 0.3 M glycine-HCl buffer, frac-

  <Desc / Clms Page number 52>

 containing IL-2 exerting an activity of 12 units / ml of IL-2. The results clearly show that E. coli X 1776 / pIL 2-50A, AJ 11996 does indeed produce IL-2.



   Example 4.



   A cell line producing IL-2 in a constitutive manner J-Al886 (ATCC CRL8130), cloned from Jurkat cells is grown in a similar manner in culture bottles on rolling drums. example 1.



  The cells which have grown in fresh synthetic medium RITC-55-9 are resuspended at an initial cell density of 1 × 10 6 cells / ml and, 8 hours after the start of the culture, the cells are used for extraction of IL-2 Il-12-stranded mRNA fractions from 3 x 109 cells, following the steps described in Example 1.



   Double-stranded cDNA is synthesized in the same way as in Example 1 and a cDNA longer than 600 base pairs (2.4 μg) is obtained after fractionation on a sucrose density gradient. The cDNA is then extended with dCMP residues using a terminal deoxynucleotidyl transferase and a 50 ng portion is treated with 250 ng of pBR322 cleaved by PstI, extended by dGMP. The resulting hybrid plasmids are used to transform E. coli X 1776 and the transformants of approximately 4,000 clones are obtained. According to the Grunstein-Hogness method, three complementary clones are selected with the plasmid cDNA 3-16 used as probe. The clones thus selected are therefore transformed clones comprising the human IL-2 gene.



   Example 5.



   We build a plasmid which must direct

  <Desc / Clms Page number 53>

 synthesis of human IL-2 in E cells. coli as follows. A plasmid pTIL2-22 is constructed from pTrS-3 [Nishi T., Taniguchi T. et al., SEIKAGAKU 53.967 (1981)] and from pIL 2-50A containing IL-2 cDNA by a series modification processes as illustrated in Figure 5 (a). Plasmid pTrS-3 contains an insertion of the Trp promoter and Shine Dalgarno region (hereinafter called "SD") between the EcoRI site and the ClaI site of pBR322. The plasmid also contains an ATG 13 bp initiation codon downstream of the SD sequence as well as a single SphI site, as illustrated in FIG. 4.

   The vector proves to be very effective in producing this protein when the DNA sequence corresponding to said protein is introduced into the phase just downstream from the ATG codon, which is produced by digestion with SphI and by subsequent processing using Of T4 DNA polymerase from pTrS-3. Consequently, the plasmid pTrS-3 (30 μg) is cleaved by the restriction enzyme SphI in the usual manner and, after treatment with phenol and then with chloroform, the ethanolic precipitates are recovered, and then the two flush ends by treatment with T4 DNA polymerase. Then the DNA (21.4 pg) is recovered by a similar treatment with phenol and then with chloroform and precipitated with ethanol.

   On the other hand, 380 μg of pIL 2-50A containing IL-2 cDNA are cleaved by PstI and the insertion of IL-2 cDNA is isolated by agarose gel electrophoresis. The cDNA insertion (11 µg) is cleaved with HgiAI, treated with T4 DNA polymerase and 10 µg of DNA is isolated from the largest site by agarose gel electrophoresis. A cDNA (7.2 μg) encoding 132 amino acids is obtained in this way and this DNA fragment has truncated ends [FIG. 5

  <Desc / Clms Page number 54>

 (at)]. Next, the cDNA fragment thus obtained is ligated to a vector pTrS-3, previously digested with SphI and treated with T4 DNA polymerase just downstream from the ATG sequence. The plasmid ligated in this way is used to transform E. coli HB101 according to the usual procedures. The liguation is carried out as follows.



  The largest fragment of IL-2 cDNA (0.4 μg) and 0.2 μg of pTRS-3 vector DNA are mixed with 0.8 units of T4 DNA ligase in 66 mM Tris-HCl, pH 7.5, containing 6.6 mM MgCl 2, 1 mM ATP and 10 mM DTT, and the mixture is left to react at 40 ° C. overnight. Among the transformants appearing on the agar and broth plate L containing ampicilin, the colonies containing the portion of IL-2 cDNA, which encodes 132 amino acids, are selected by a colony hybridization test in if you. The colonies thus selected are cultured (10 ml) again to prepare plasmid DNA by means of lysozyme treatment and by freeze-liquefaction.

   Plasmid DNAs are cleaved by PstI and Xbal, and the resulting products are analyzed by agarose gel electrophoresis to identify pTIL 2-22 in which the cDNA is linked to the ATG sequence of pTrS-3 following a correct orientation.



  E. coli HB101 containing pTIL 2-22 is cultivated under the usual conditions known for the propagation of microorganisms. The cells are grown in 10 ml of X broth (2.5% Bactotrypton, 1.0% yeast extract, 0.1% glucose, 20 mM MgSO 4, 50 mM Tris-HCl, pH 7.5) containing 25 µg / ml streptomycin and 25 µg ampicillin at 370C overnight. 1 ml of the culture suspension is inoculated into the same broth X (100 ml) and cultured at 370C. When the optical density at 650 mp reaches around 1.5-2.0, 3-indole acrylic acid (IAA) is added.

  <Desc / Clms Page number 55>

 



  Three hours after the addition of the inducer, the cells are collected, washed with 20 mM Tris-HCl (pH 7.5; 30 mM NaCl) and they are resuspended in 8 ml of the same buffer . To obtain efficient functioning of the Trp promoter, inducers such as IAA are added at a final concentration of 50 μg / ml.



  The proteins thus obtained in the bacterial cells are extracted by sonication (0 C; 2 minutes) or by means of digestion with lysozyme (8 pg) (0 C; 20 minutes) followed by three successive freeze-liquefactions. Following these processes, the usual extraction of IL-2 from organisms is carried out. The activity of IL-2 extracted is between 10,000 and 120,000 units / ml.



   E. coli HB101 containing pTIL 2-22 (AJ 12009) was deposited under the identification number FERM-BP 245.



   Example 6.



   A plasmid pTuIL 2-22, carrying IL-2 cDNA, is constructed from pTuBlP-5 [Taniguchi, T. et al., Seikagaku, 53,966, (1981)] and pTIL 2-22 shown in Example 5, by the methods as illustrated in FIG. 7. The plasmid pTuBlP-5 comprises an insertion of the promoter sequence for tufB in pBR322. The plasmid also contains a single ClaI site and this site is arranged 2bp downstream of the SD sequence, as shown in FIG. 2.

   Since pTrS-3 also contains a ClaI site between the SD sequence and the ATG initiation codon, and since this ClaI site is not destroyed during the construction of the expression plasmid using pTrS-3 and IL-2 cDNA as described in Example 5, it is very simple to replace the bacterial trp promoter with that of tufB, so that the IL-2 cDNA is expressed under the tufB promoter control.

  <Desc / Clms Page number 56>

 



   Therefore, plasmid pTIL 2-22 (30 µg) is cleaved with the restriction enzymes Clal and PvuII in the usual manner. The fragment (about 2.2 kb) containing IL-2 cDNA is isolated and purified by agarose gel electrophoresis so as to recover 3 µg of DNA. On the other hand, 20 μg of pTuBIP-5 vector are similarly cleaved by ClaI and PvuII, and the largest fragment (approximately 3.4 kb) containing the ampicilin-resistant gene is isolated and isolated. purifies by agarose gel electrophoresis to recover 3.5 µg of DNA. Then, the two fragments obtained in this way, one (approximately 3.4 kb) containing the tufB promoter, the other (approximately 2.2 kb) containing the IL-2 cDNA, are ligated from the next way.

   The fragment containing the IL-2 cDNA (1.2 μg) and 0.3 μg of the fragment containing the tufB promoter is mixed with 0.8 units of T4 DNA ligase in 66 mM Tris-HCl pH 7.5 containing 6.6 mM MgCl2'1 mM ATP and 10 mM DTT, and the mixture is allowed to react at 40C overnight. The plasmid ligated in this way is then used to transform E. coli HB101 according to the usual procedures. Among the transformants appearing on the agar and broth L plate containing ampicilin, eight colonies containing the portion are selected.
 EMI56.1
 o IL-2 cDNA, such as pTuIL 2-22 in FIG. 7, and plasmid DNA is prepared as described in Example 5.

   E. coli HB101 containing pTuIL 2-22 is grown in L broth (100 ml) at 370C. When the optical density at 650 mp is around 0.5-1.0, the bacterial cells are harvested, washed with 20 mM Tris-HCl (pH 7.5; 30 mM NaCl) and resuspends them in 2 ml of the same buffer. The proteins thus obtained are extracted in the same way as in Example 5. The activity of IL-2 extracted is located

  <Desc / Clms Page number 57>

 between 6,000 and 56,000 units / ml.



   Escherichia coli HB101 containing pTuIL 2-22 (AJ 12010) has been deposited under the identification number FERM-BP 246.



   Example 7.



   A plasmid pGIL 2-22, carrying IL-2 cDNA, is constructed from pGL 101 [Roberts T. M. and Laucer G. D., Meth. Enzym., 68,473-483 (1979)] and pTIL 2-22 of Example 5.



   The plasmid pGL 101 (20 μg) containing a lac promoter is cleaved by the restriction enzyme PvuII in the usual manner so as to recover 17 μg of DNA by treatment with phenol, then with chloroform and by precipitation in ethanol. On the other hand, pTIL 2-22 (75 pg) is cleaved with ClaI and SalI so as to recover 2.2 pg of a DNA fragment containing IL-2 cDNA by gel electrophoresis of agarose. The fragment is made flush by treating it with DNA polymerase I (Klenow fragment), and the two fragments thus obtained (0.25 pg and 0.066 pg) are ligated with 1.0 unit of DNA ligase of T4 in the same manner as in Example 6. The plasmid thus ligated is then used to transform E. coli HB101 in the usual manner.

   Among the transformants, the transformants having the insertion of the ClaI-SalI fragment containing IL-2 cDNA as a probe are selected.



  These transformants are then cultivated in broth X (10 ml) containing 25 μg / ml of ampicilin and the plasmid DNA is prepared as described in Example 5. Thus, the plasmid DNA having the ATG initiation sequence of IL-2 cDNA just downstream of a lac promoter is obtained by cleavage with PstI and XbaI.



   The pGIL 2-22 thus obtained is inoculated into

  <Desc / Clms Page number 58>

 100 ml of broth L containing 25 μg / ml of ampicilin and 25 μg / ml of streptomycin and it is cultivated. When the optical density at 650 mp is around 0.5,
 EMI58.1
 isopropyl-ss-D-thiogalactopyrannoside (IPTG) in a concentration of 1 mM is added and one hour later, the bacterial cells are collected and the cell extracts are prepared as described in Example 6. Activity of extracted IL-2 is between 6,000 and 80,000 units / ml.



   Escherichia coli HB101 containing pGIL 2-22 (AJ 12011) has been deposited under the identification number FERM-BP 247.



   Example 8.



   Plasmid pTrS-3 (10 μg) was first cleaved with the restriction enzyme SalI and the SalI site was made flush with treatment with DNA polymerase (Klenow fragment) or with l Of T4 DNA polymerase. After cleavage with ClaI, a larger fragment, containing the region of the trp promoter, is isolated by electrophoresis on agarose gel in the usual manner to recover 3 μg of DNA.



   On the other hand, 11 μg of cDNA insertion obtained by the cleavage of PsTI from pIL2-50A are cleaved by HgiAI, treated with T4 DNA polymerase, a larger fragment is isolated and purified by agarose gel electrophoresis. Thus, a cDNA fragment coding for 132 amino acids of IL-2 is obtained in an amount of 7.2 μg. Next, 0.45 µg of the fragment containing a trp promoter (described above), 0.5 µg of the fragment of HgiAI-PstI containing IL-2 cDNA and oligonucleotides (5 ') CGATAAGCTATGGCA (3 ') and (3') TATTCGATACCGT (5 ') synthetic (each of 20 pmoles), both being phosphorylated at the 5' end, are ligated by one unit

  <Desc / Clms Page number 59>

 of T4 DNA ligase in the same manner as in Example 5 [Figure 5 (b)].



   The plasmid ligated in this way is then used to transform E. coli HB 101. Among the transformants which have appeared, the targeted transformants are chosen as follows.



   The transformant candidates capable of hybridization with the IL-2 cDNA and the synthetic oligonucleotides are first chosen by a colony hybridization method, then the transformants having the insertion of DNA fragment starting from the CCT sequence in position 111 to 113 in FIG. 2 (a) (CCTACT -----) just downstream of the ATG GCA sequence are chosen by cleavage with PstI, XbaI.



   The above transformants, which contain pTIL2-21a or pTIL2-21b, are grown in L-broth as described in Example 5, and high IL-2 activities can be found in cellular extracts of the transformants when examined as indicated in Example 5.



   Escherichia coli HB101 containing pTIL2-21a (AJ 12013) and Escherichia coli HB 101 containing pTIL2-21b (AJ 12014) have been deposited under the identification numbers FERM-BP 248 and FERM-BP 249 respectively.



   The hosts E. coli X 1776 and HB 101 [Boyer H. W. et al., J. Mol. Biol. 41.459 (1969)], used in the above examples are known and available to the public. In addition, hosts can be obtained from the deposited transformants by culturing the transformants in broth L at 37 ° C. so as to release the respective recombinant DNAs in the transformants and to separate the strains which become sensitive to tetracycline and to ampicillin as hosts.



   The vector plasmids pBR322 (which is de-

  <Desc / Clms Page number 60>

 commercially available, for example, by Bethesda Research Laboratory), pCE-1, PTrS-3 and pGL101 are known and available to the public. In addition, the vector plasmids can be obtained from the deposited transformants by separating the DNAs from recombinant plasmids in the transformants in the usual manner and by separating the vector plasmids by methods which appear obvious from the respective Examples. For example, pCE-1 can be obtained by digestion of pCEIL-2 with PstI and by separation of the largest DNA fragment formed. In addition, pTrS-3 and pTuBlP-5 were deposited respectively under the names E. coli FERM-P 6735 and E. coli ATCC 31871.



   It should be understood that the present invention is in no way limited to the above embodiments and that many modifications can be made thereto without departing from the scope of this patent.


    

Claims (128)

 CLAIMS 1. Cloned gene encoded for a polypeptide having the activity of interleukin-2.  2. Gene according to claim 1, which is prepared from a messenger RNA obtained by a mammalian cell line producing interleukin-2.  3. Gene according to claim 2, characterized in that the messenger RNA can be obtained as 11 to 12 strand sediment from a sucrose density gradient centrifugation.  4. Gene according to claim 2, characterized in that the mammalian cell line is constituted by human T lymphocytes, transformed human T lymphocytes or hybridomas of human T cells.  5. Gene according to claim 1, characterized in that it comprises sites cleaved by a restriction endonuclease in the order of Bst NI, Xba I and Bst NI from the 5 ′ end of the coding sequence.  6. Gene according to claim 1, characterized in that it comprises sites cleaved by a restriction endonuclease in the order of Dde I, Hinf I, BsT NI, Xba I, Bst NI and Sau 3A from 5 ′ end of the coding sequence.  7. Gene according to claim 1, characterized in that it comprises the sequence of bases represented by FIG. 2 (a).  8. Gene according to claim 1, the base sequence of which starts from the ATG sequence in position 48 to 50 and whose sequential bases follow the ATG sequence at least up to the ACT sequence in position 504 to 506 in FIG. 2 ( at).  <Desc / Clms Page number 62>    9. Gene according to claim 1, the base sequence of which starts from the GCA sequence in position 108 to 110 and whose sequential bases follow the GCA sequence at least up to the ACT sequence in position 504 to 506 in FIG. 2 ( at).  10. Gene according to claim 1, the base sequence of which starts from the CCT sequence in position 111 to 113 and whose sequential bases follow the CCT sequence at least up to the ACT sequence in position 504 to 506 in FIG. 2 ( at).  11. Gene according to claim 1, whose base sequence starts from A in position 1 and whose sequential bases which follow A end with the ACT sequence in position 504 to 506 in FIG. 2 (a).  12. Gene according to claim 8, the base sequence of which ends at the ACT sequence in position 504 to 506 in FIG. 2 (a).  13. The gene of claim 9, the base sequence of which ends at the ACT sequence in position 504 to 506 in Figure 2 (a).  14. The gene of claim 10, the base sequence of which ends at the ACT sequence in position 504 to 506 in Figure 2 (a).  15. The gene according to claim 1, in which the base sequence starts from A in position 1 and whose sequential bases which follow A end in the TGA sequence in position 507 to 509 in FIG. 2 (a).  16. The gene of claim 8, the base sequence of which ends at the TGA sequence in position 507 to 509 in Figure 2 (a).  17. The gene of claim 9, the base sequence of which ends at the TGA sequence in position 507 to 509 in Figure 2 (a).  18. The gene of claim 10, of which  <Desc / Clms Page number 63>  the base sequence ends at the TGA sequence at position 507 to 509 in Figure 2 (a).  19. Gene according to claim 1, whose base sequence starts from A in position 1 and whose sequential bases which follow A end in C in position 801 in FIG. 2 (a).  20. The gene of claim 8, the base sequence of which ends in C at position 801 in Figure 2 (a).  21. The gene of claim 9, the base sequence of which ends in C at position 801 in Figure 2 (a).  22. The gene of claim 10, the base sequence of which ends at C in position 801 in Figure 2 (a).  23. The gene of claim 8, the base sequence of which ends in the poly position (A) in Figure 2 (a).  24. The gene of claim 9, the base sequence of which ends in the poly position (A) in Figure 2 (a).  25. The gene of claim 10, the base sequence of which ends in the poly position (A) in Figure 2 (a).  26. The gene of claim 1, which shows the base sequence corresponding to the amino acid sequence I in Figure 2 (b).  27. The gene of claim 1 which shows the base sequence corresponding to the amino acid sequence II in Figure 2 (b).  28. The gene of claim 1 which shows the base sequence corresponding to the amino acid sequence III in Figure 2 (b).  29. DNA prepared by recombination, which  <Desc / Clms Page number 64>  comprises a gene coded for a polypeptide which has the activity of interleukin-2, and a vector DNA capable of propagating in a prokaryotic or eukaryotic cell, the coding sequence of this gene being located at a position downstream of a promoter sequence.  30. Recombinant DNA according to claim 29, characterized in that the gene is prepared with a messenger RNA produced by a mammalian cell line producing interleukin-2.  31. Recombinant DNA according to claim 30, characterized in that the mammalian cells are human T lymphocytes, transformed human T lymphocytes or human T cell hybridomas.  32. Recombinant DNA according to claim 30, characterized in that the messenger RNA can be obtained as 11 to 12 strand sediment from a sucrose density gradient centrifugation.  33. Recombinant DNA according to claim 29, characterized in that the gene comprises sites cleaved by a restriction endonuclease in the order of Bst NI, Xba I and Bst NI from the 5 ′ end of the coding sequence.  34. Recombinant DNA according to claim 29, characterized in that the gene comprises sites cleaved by a restriction endonuclease in the order of Dde I, Hinf I, Bst NI, Xba I, Bst NI and Sau 3A from l 'end 5' of the coding sequence.  35. Recombinant DNA according to claim 29, characterized in that the gene comprises the base sequence represented by FIG. 2 (a).  36. Recombinant DNA according to the claim  <Desc / Clms Page number 65>  cation 29, characterized in that the base sequence of the gene starts from the ATG sequence in position 48 to 50 and in that the sequential bases follow the ATG sequence at least up to the ACT sequence in position 504 to 506 in the figure 2 (a).  37. Recombinant DNA according to claim 29, characterized in that the base sequence of the gene starts from the GCA sequence in position 108 to 110 and in that the sequential bases follow the GCA sequence at least up to the ACT sequence in position 504 to 506 in Figure 2 (a).  38. Recombinant DNA according to claim 29, characterized in that the base sequence of the gene starts from the CCT sequence in position 111 to 113 and in that the sequential bases follow the CCT sequence at least up to the ACT sequence in position 504 to 506 in Figure 2 (a).  39. Recombinant DNA according to claim 29, characterized in that the base sequence of the gene starts from A in position 1 and in that the sequential bases which follow A end in the sequence ACT in position 504 to 506 in FIG. 2 (at).  40. Recombinant DNA according to claim 36, characterized in that the base sequence of the gene ends at the ACT sequence in position 504 to 506 in FIG. 2 (a).  41. Recombinant DNA according to claim 37, characterized in that the base sequence of the gene ends at the ACT sequence in position 504 to 506 in FIG. 2 (a).  42. Recombinant DNA according to claim 38, characterized in that the base sequence of the gene ends at the ACT sequence in position 504 to 506 in FIG. 2 (a).  <Desc / Clms Page number 66>    43. Recombinant DNA according to claim 29, characterized in that the base sequence of the gene starts from A in position 1 and in that the sequential bases which follow A end in the TGA sequence in position 507 to 509 in FIG. 2 .  44. Recombinant DNA according to claim 36, characterized in that the base sequence of the gene ends at the TGA sequence in position 507 to 509 in FIG. 2 (a).  45. Recombinant DNA according to claim 37, characterized in that the base sequence of the gene ends at the TGA sequence in position 507 to 509 in FIG. 2 (a).  46. Recombinant DNA according to claim 38, characterized in that the base sequence of the gene ends at the TGA sequence in position 507 to 509 in FIG. 2 (a).  47. Recombinant DNA according to claim 29, characterized in that the base sequence of the gene starts from A in position 1 and in that the sequential bases which follow A end in C in position 801 in FIG. 2 (a).  48. Recombinant DNA according to claim 36, characterized in that the base sequence of the gene ends in C at position 801 in Figure 2 (a).  49. Recombinant DNA according to claim 37, characterized in that the base sequence of the gene ends in C at position 801 in Figure 2 (a).  50. Recombinant DNA according to claim 38, characterized in that the base sequence of the gene ends in C at position 801 in Figure 2 (a).  <Desc / Clms Page number 67>    51. Recombinant DNA according to claim 36, characterized in that the base sequence of the gene ends in the poly (A) position in FIG. 2 (a).  52. Recombinant DNA according to claim 37, characterized in that the base sequence of the gene ends in the poly (A) position in Figure 2 (a).  53. Recombinant DNA according to claim 38, characterized in that the base sequence of the gene ends in the poly (A) position in Figure 2 (a).  54. Recombinant DNA according to claim 29, characterized in that the base sequence of the gene corresponding to the amino acid sequence I of FIG. II (b).  55. Recombinant DNA according to claim 29, characterized in that the base sequence of the gene corresponding to the amino acid sequence II of Figure II (b).  56. Recombinant DNA according to claim 29, characterized in that the base sequence of the gene corresponding to the amino acid sequence III of Figure II (b).  57. Recombinant DNA according to claim 29, characterized in that the prokaryotic cell line belongs to the genus Escherichia.  58. Recombinant DNA according to claim 29, characterized in that the prokaryotic cell line belongs to Escherichia coli.  59. Recombinant DNA according to claim 29, characterized in that the eukaryotic cell line belongs to the genus Saccharomyces.  60. Recombinant DNA according to the claim  <Desc / Clms Page number 68>  tion 29, characterized in that the eukaryotic cell line belongs to the genus Saccharomyces cereviceae.  61. Recombinant DNA according to claim 29, characterized in that the eukaryotic cell line are monkey cells transformed by SV-40 constitutively expressing the T antigen.  62. Eukaryotic or prokaryotic cell transformed by a recombinant DNA comprising a gene coding for a polypeptide having the activity of interleukin-2 and a vector DNA capable of propagating in said cell, the coding sequence of this gene being located at a position downstream of a promoter sequence.  63. Cell according to claim 62, characterized in that the gene is prepared with a messenger RNA obtained by a mammalian cell producing interleukin-2.  64. Cell according to claim 63, characterized in that the mammalian cell is a human T lymphocyte, a transformed human T lymphocyte or a T cell hybridoma.  65. Cell according to claim 63, characterized in that the messenger RNA can be obtained as 11 to 12 strand sediment from a sucrose density gradient centrifugation.  66. Cell according to claim 63, characterized in that the gene comprises sites cleaved by a restriction endonuclease in the order of Bst NI, Xba I and Bst NI from the 5 ′ end of the coding sequence.  67. Cell according to claim 62, characterized in that the gene comprises sites cleaved by a restriction endonuclease in the order of  <Desc / Clms Page number 69>  Dde I, Hinf I, Bst NI, Xba I, Bst NI and Sau 3A from the 5 'end of the coding sequence.  68. Cell according to claim 62, characterized in that the gene comprises the base sequence represented by FIG. 2 (a).  69. Cell according to claim 62, characterized in that the base sequence of the gene starts from the ATG sequence in position 48 to 50 and in that the sequential bases follow the ATG codon at least up to the ACT sequence in position 504 to 506 in Figure 2 (a).  70. Cell according to claim 62, characterized in that the base sequence of the gene starts from the GCA sequence in position 108 to 110 and in that the sequential bases follow the GCA sequence at least up to the ACT sequence in position 504 to 506 in Figure 2 (a).  71. Cell according to claim 62, characterized in that the base sequence of the gene starts from the CCT sequence in position 111 to 113 and in that the sequential bases follow the CCT sequence at least up to the ACT sequence in position 504 to 506 in Figure 2 (a).  72. Cell according to claim 62, characterized in that the base sequence of the gene starts from A in position 1 and in that the sequential bases which follow A terminate in the sequence ACT in position 504 to 506 in FIG. 2 ( at).  73. Cell according to claim 69, characterized in that the base sequence of the gene ends at the ACT sequence in position 504 to 506 in FIG. 2 (a).  74. Cell according to claim 70, characterized in that the base sequence of the gene is  <Desc / Clms Page number 70>  ends at the ACT sequence in position 504 to 506 in Figure 2 (a).  75. Cell according to claim 71, characterized in that the base sequence of the gene ends at the ACT sequence in position 504 to 506 in FIG. 2 (a).  76. Cell according to claim 62, characterized in that the base sequence of the gene starts from A in position 1 and in that the sequential bases which follow A end in the TGA sequence in position 507 to 509 in FIG. 2 ( at).  77. Cell according to claim 69, characterized in that the base sequence of the gene ends at the TGA sequence in position 507 to 509 in FIG. 2 (a).  78. Cell according to claim 70, characterized in that the base sequence of the gene ends at the TGA sequence in position 507 to 509 in FIG. 2 (a).  79. Cell according to claim 71, characterized in that the base sequence of the gene ends at the TGA sequence in position 507 to 509 in FIG. 2 (a).  80. Cell according to claim 62, characterized in that the base sequence of the gene starts from A in position 1 and in that the sequential bases which follow A end in C in position 801 in FIG. 2 (a).  81. Cell according to claim 69, characterized in that the base sequence of the gene ends in C at position 801 in FIG. 2 (a).  82. Cell according to claim 70, characterized in that the base sequence of the gene ends in C at position 801 in FIG. 2 (a).  <Desc / Clms Page number 71>    83. Cell according to claim 71, characterized in that the base sequence of the gene ends in C at position 801 in FIG. 2 (a).  84. Cell according to claim 69, characterized in that the base sequence of the gene ends in the poly (A) position in FIG. 2 (a).  85. Cell according to claim 70, characterized in that the base sequence of the gene ends in the poly (A) position in FIG. 2 (a).  86. Cell according to claim 71, characterized in that the base sequence of the gene ends in the poly (A) position in FIG. 2 (a).  87. Cell according to claim 65, characterized in that the base sequence of the gene corresponds to the amino acid sequence I of FIG. 2 (b).  88. Cell according to claim 65, characterized in that the base sequence of the gene corresponds to the amino acid sequence II of FIG. 2 (b).  89. Cell according to claim 65, characterized in that the base sequence of the gene corresponds to the amino acid sequence III of FIG. 2 (b).  90. Cell according to claim 62, characterized in that the prokaryotic cell belongs to the genus Escherichia.  91. Cell according to claim 62, characterized in that the prokaryotic cell belongs to Escherichia Coli.  92. Cell according to claim 62, characterized in that the eukaryotic cell belongs to the genus Saccharomyces.  93. Cell according to claim 62,  <Desc / Clms Page number 72>  characterized in that the eukaryotic cell belongs to the genus Saccharomyces cereviceae.  94. Cell according to claim 62, characterized in that the eukaryotic cell is a monkey cell transformed by SV-40 constitutively expressing the T antigen.  95. Process for the production of interleukin-2, characterized in that it comprises the culture of a eukaryotic or prokaryotic cell in a culture medium, transformed by a recombinant DNA to produce interleukin-2, and the recovery of interleukin-2 obtained, the recombinant DNA comprising a gene coded for a polypeptide which has the activity of interleukin-2 and a vector DNA which is capable of replicating in said cell, the coding sequence of the gene being located at a position downstream of a promoter sequence.  96. Method according to claim 95, characterized in that the gene is prepared with a messenger RNA obtained by a line of mammalian cells producing interleukin-2.  97. Method according to claim 96, characterized in that the mammalian cells are human T lymphocytes, transformed human lymphocytes or T cell hybridomas.  98. Method according to claim 96, characterized in that the messenger RNA can be obtained as 11 to 12 strand sediment from a sucrose density gradient centrifugation.  99. Cell according to claim 95, characterized in that the gene comprises sites cleaved by a restriction endonuclease in the order of Bst NI, Xba I and Bst NI from the 5 ′ end of the coding sequence.  <Desc / Clms Page number 73>      100. The method of claim 95, characterized in that the gene comprises sites cleaved by a restriction endonuclease in the order of Dde I, Hinf I, Bst NI, Xba I, Bst NI and Sau 3A from 5 ′ end of the coding sequence.  101. Method according to claim 95, characterized in that the gene comprises the base sequence represented by FIG. 2 (a).  102. Method according to claim 95, characterized in that the base sequence of the gene starts from the ATG sequence in position 48 to 50 and in that the sequential bases follow the ATG sequence at least up to the ACT sequence in position 504 to 506 in Figure 2 (a).  103. Method according to claim 95, characterized in that the base sequence of the gene starts from the GCA sequence in position 108 to 110 and in that the sequential bases follow the GCA sequence at least up to the ACT sequence in position 504 to 506 in Figure 2 (a).  104. Method according to claim 95, characterized in that the base sequence of the gene starts from the CCT sequence in position 111 to 113 and in that the sequential bases follow the CCT sequence at least up to the ACT sequence in position 504 to 506 in Figure 2 (a).  105. Method according to claim 95, characterized in that the base sequence of the gene starts from A in position 1 and in that the sequential bases which follow A end in the sequence ACT in position 504 to 506 in FIG. 2 ( at).  106. Method according to claim 102, characterized in that the base sequence of the gene ends at the ACT sequence in position 504 to 506 on the  <Desc / Clms Page number 74>  Figure 2 (a).  107. Method according to claim 103, characterized in that the base sequence of the gene ends at the ACT sequence in position 504 to 506 in FIG. 2 (a).  108. Method according to claim 104, characterized in that the base sequence of the gene ends at the ACT sequence in position 504 to 506 in FIG. 2 (a).  109. Process according to claim 95, characterized in that the base sequence of the gene starts from A in position 1 and in that the sequential bases which follow A end in the TGA sequence in position 507 to 509 in FIG. 2 ( at).  110. Method according to claim 102, characterized in that the base sequence of the gene ends at the TGA sequence in position 507 to 509 in FIG. 2 (a).  111. The method of claim 103, characterized in that the base sequence of the gene ends at the TGA sequence in position 507 to 509 in Figure 2 (a).  112. The method of claim 104, characterized in that the base sequence of the gene ends at the TGA sequence in position 507 to 509 in Figure 2 (a).  113. Method according to claim 95, characterized in that the base sequence of the gene starts from A in position 1 and in that the sequential bases which follow A end in C in position 801 in FIG. 2 (a).  114. The method of claim 102, characterized in that the base sequence of the gene ends in C at position 801 in Figure 2 (a).  <Desc / Clms Page number 75>    115. Method according to claim 103, characterized in that the base sequence of the gene ends in C at position 801 in FIG. 2 (a).  116. Method according to claim 104, characterized in that the base sequence of the gene ends in C at position 801 in FIG. 2 (a).  117. The method of claim 102, characterized in that the base sequence of the gene ends in the poly (A) position in Figure 2 (a).  118. Method according to claim 103, characterized in that the base sequence of the gene ends in the poly (A) position in FIG. 2 (a).  119. Method according to claim 104, characterized in that the base sequence of the gene ends in the poly (A) position in FIG. 2 (a).  120. The method according to claim 101, characterized in that the base sequence of the gene corresponds to the amino acid sequence I of FIG. 2 (b).  121. The method of claim 101, characterized in that the base sequence of the gene corresponds to the amino acid sequence II of Figure 2 (b).  122. The method of claim 101, characterized in that the base sequence of the gene corresponds to the amino acid sequence III of Figure 2 (b).  123. Process according to claim 95, characterized in that the prokaryotic cell belongs to the genus Escherichia.  124. Method according to claim 95, characterized in that the prokaryotic cell belongs to Escherichia coli.  125. Process according to claim 95,  <Desc / Clms Page number 76>  characterized in that the eukaryotic cell belongs to the genus Saccharomyces.  126. Method according to claim 95, characterized in that the eukaryotic cell belongs to the genus Saccharomyces cereviceae.  127. Method according to claim 95, characterized in that the eukaryotic cell is a monkey cell transformed by SV-40 constitutively expressing the T antigen.  128. Gene encoded for interleukin-2 polypeptide, recombinant DNA carrying this gene, living cell line containing the recombinant DNA and method for producing interleukin-2 using this cell, as described above, in particular in examples given.