AT392082B - The gene encoding polypeptide Interleukin-2, recombinant, DNA containing this gene, cell recombinant, and method for producing interleukin-2 using the named cells - Google Patents

Abstract

A DNA sequence which codes for a polypeptide having the activity of human interleukin-2 is provided. This DNA sequence can be inserted into a vector DNA which is capable of replication in a prokaryotic or eukaryotic cell, and the DNA obtained by recombination is suitable for transforming a cell line through cultivation of which interleukin-2 is then produced. The invention also relates to a process for the preparation of interleukin-2 using a cell transformed in this way.

Description

AT 392 082 B

The invention relates to a DNA sequence which has a coding sequence which codes for human interleukin-2 polypeptide, recombinant DNA which contains this DNA sequence, a living cell line which has the recombinant DNA, and a method for the production of interleukin-2 using the cell line.

Interleukin-2 (hereinafter also abbreviated as " IL-2 "), formerly referred to as T cell growth factor, is a soluble protein (commonly known as " Lymphokin ") which is derived from T activated with a lectin or an antigen - cells are formed; D. A. Morgan et al., Science, Vol. 193 (1976), pp. 1007-1008, S. Gillis et al., J. Immunol., Vol. 120 (1978), pp. 2027-2033. Interleukin 2 (IL-2) is able to regulate the lymphocyte reactivity and to promote the in vitro long-term culture of antigen-specific effector T lymphocytes; S. Gillis et al., Nature, Vol. 268 (1977), pp. 154-156. Furthermore, IL-2 also has other important biological effects, such as an enhancement of thymocyte mitogenesis (BM Chen et al., Cell. Immunol., Vol. 22 (1977), pp. 211-224, J. Shaw et al., J Immunol., Vol. 120 (1978), pp. 1967-1973), induction of cytotoxic T cell reactivity (Wagner et al., Nature, vol. 284 (1980), pp. 278-280) and anti-SRBC- plaque-forming cell reactions (S. Gillis et al., J. Exp. Med. Vol. 149 (1979), pp. 1960-1968) in cultures of bare mouse spleen cells. As a result, this lymphocyte regulatory substance is suitable for strengthening the humoral and cellular immune reactions and for remedying immunodeficient states to a normal humoral and cellular immune state. These known immunological activities of IL-2 strongly suggest that IL-2 is a valuable drug for immunotherapy against immunological disorders including neoplastic diseases, bacterial or viral infections, immunodeficiency diseases, autoimmune diseases and the like; B. Papermaster et al., Adv. Immunopharm., (1980), p. 507. As with interferons, IL-2 has been shown to increase natural killer cell activity, suggesting potential use in the treatment of neoplastic diseases. Furthermore, IL-2 enables the maintenance of cultures of functional, monoclonal T cells and thus seems to play a key role in the investigation of the molecular nature of T cell differentiation and the mechanism of differentiated T cell function and the mechanism of T cell antigen receptors. It is also useful in the long-term cultivation of monoclonal T cells to form many other T cell-derived lymphokines, which have proven valuable in a number of areas. In addition, IL-2 formation and lymphocyte response to IL-2 can be important parameters of immunological functions that are valuable in the clinical diagnosis of immunity abnormalities. IL-2 has so far been formed by stimulating mouse, rat or human lymphocytes with a mitogen; S. Gillis et al., Nature, Vol. 268 (1977), pp. 154-156, J. Farrar et al., J. Immunol., Vol. 121 (1978), pp. 1353-1360, S. Gillis et al., J. Immunol, Vol. 120 (1978), pp. 2027-2033. S. Gillis et al., J. Immunol, Vol. 124 (1980), pp. 1954-1962 stimulated human, peripheral, mononuclear blood lymphocytes with a mitogen. S. Gillis et al., 1980, J. Immunol, 125: 2570-2578, also reported the formation of mouse IL-2 from mouse T cell lymphoma cell lines and the formation of human IL- 2 from human leukemia cell lines (S. Gillis et al., J. Exp. Med., Vol. 152 (1980), pp. 1709-1719).

The aforementioned work by Gillis et al. discuss the process of forming human IL-2 from mitogen-stimulated human T cell leukemia cell lines by cell culture techniques. However, this technique results in undesirably low levels of human IL-2 and even requires complicated purification procedures to form small amounts of IL-2 from large volumes of culture media. In addition, since human T cell leukemia cell lines form trace amounts of many other biologically active substances that are analogous to human IL-2, when IL-2 is separated from these other immunologically active molecules or when IL- 2 of occasional toxic lectins present considerable difficulties.

By stimulating a mouse T-lymphoma cell lumen, variant IL-4, and extracting the stimulated cells, RNA was obtained which could be translated into Xenopus laevis oocytes. Due to the injection, the oocytes synthesize a material with the biological and biochemical properties of mouse IL-2 (R.C. Bleackley et al The Journal of Immunology (1981), volume 127, pages 2432-2435).

It would be desirable to use recombinant DNA techniques (DNA is an abbreviation for deoxyribonucleic acid) as used in the formation of other biologically active human proteins, e.g. B. with interferons (PW Gray et al., Nature, vol. 295 (1981), pp. 503-508, S. Nagata et al., Nature, vol. 284 (1980), pp. 316-320, T. Taniguchi et al., Gene, Vol. 10 (1980), pages 11-15) for the production of IL-2. However, previous attempts to form IL-2 by recombinant DNA techniques have not been successful. For example, NIKKEI BIOTECHNOLOGY (Japan), No. 19.5. July 1982 reports that attempts to construct IL-2-forming organisms by recombinant DNA techniques have been unsuccessful, presumably due to the fact that the gene encoding the IL-2 polypeptide had not yet been cloned.

European patent application EP-A-0 088 195 relates to an m-RNA with a sedimentation coefficient of 8 s to 15 s, which is translated in the Xenopus laevis oocyte system to a polypeptide with the activity of human interleukin-2.

The problem to be solved by the present invention is to provide a DNA sequence coding for interleukin-2 as well as a recombinant DNA which contains this gene.

AT 392 082 B are also intended to provide living cell lines which contain this recombinantly generated DNA and a method for producing interleukin-2 using these cell lines.

The invention accordingly relates to a DNA sequence which comprises a DNA sequence which codes for a polypeptide with the activity of human interleukin-2, this polypeptide being selected from the group below: a) a polypeptide which has the amino acid sequence below has: 1

Met-Tyr-Arg-Met-Gln-Leu-Leu-Ser-Cys-Ile-Ala-Leu-Ser-Leu- 20

Ala-Leu-Val-Thr-Asn-Ser-Ala-Pro-Thr-Ser-Ser-Ser-Thr-Lys-

Lys-Thr-Gln-Leu-Gln-Leu-Glu-His-Leu-Leu-Leu-Asp-Leu-Gln-

Met-Ile-Leu-Asn-Gly-He-Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu

Thr-Arg-Met-Leu-Thr-Phe-Lys-Phe-Tyr-Met-Pro-Lys-Lys-Ala-

Thr-Glu-Leu-Lys-His-Leu-Gln-Cys-Leu-Glu-Glu-Glu-Leu-Lys-

Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-Ala-Gln-Ser-Lys-Asn-Phe-

His-Leu-Arg-Pro-Arg-Asp-Leu-Ile-Ser-Asn-Ile-Asn-Val-Ile-

Val-Leu-Glu-Leu-Lys-Gly-Ser-Gl-Thr-Thr-Phe-Met-Cys-Glu-

Tyr-Ala-Asp-Glu-Thr-Ala-Thr-Ile-Val-Glu-Phe-Leu-Asn-Arg-153

Trp-He-Thr-Phe-Cys-Gln-Ser-Ile-Ile-Ser-Thr-Leu-Thr b) a polypeptide that lacks (a) one or more amino acids; c) a polypeptide in which one or more amino acids are exchanged for (a); d) a fusion polypeptide which contains a polypeptide according to (a), (b) or (c) in which the additionally linked amino acids do not interfere with the biological interleukin-2 activity or can be easily eliminated, e) a polypeptide, which is an allelic derivative of a polypeptide according to (a).

The invention also relates to a DNA sequence comprising a DNA sequence encoding a polypeptide with the activity of human interleukin-2, the encoded polypeptide being selected from (a) a polypeptide having the following amino acid sequence: X-Thr -Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu-Gln-Leu-Glu-

His-Leu-Leu-Leu-Asp-Leu-Gln-Met-Ile-ljeu-Asn-Gly-Ile-Asn-

Asn-Tyr-Lys-Asn-Pro-Lys-Leu-Thr-Arg-Met-Leu-Thr-Phe-Lys-

Hie-Tyr-Met-Pro-Lys-Lys-Ala-Thr-Glu-Leu-Lys-His-Leu-Gln-

Cys-Leu-Glu-Glu-Glu-Leu-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-

Leu-Ala-Gln-Ser-Lys-Asn-Phe-His-Leu-Arg-Ero-Arg-Asp-Leu- Üe-Ser-Asn-Ile-Asn-Val-Ile-Val-Leu-Glu-Leu- Lys-Gly-Ser-

Glu-Thr-Thr-Phe-Met-Cys-Glu-Tyr-Ala-Asp-Glu-Thr-Ala-Thr-

Ile-Val-Glu-Phe-Leu-Asn-Arg-Trp-Ile-Thr-Phe-Cys-Gln-Ser-

Ile-Ile-Ser-Thr-Leu-Thr, where X stands for Ala-Pro, Pro, Met-Ala-Pro or Met-Pro, b) a polypeptide which is an allelic derivative of polypeptide (a), c) a fusion polypeptide which contains a polypeptide according to (a) in which the additionally linked amino acids do not interfere with the biological activity or can be removed easily.

In addition, the present invention relates to a DNA produced by recombination, comprising a DNA sequence according to one of Claims 1 to 13 in a vector DNA which is capable of replication in a prokaryote or eukaryote cell in which the coding DNA sequence is located in a position downstream of a promoter sequence.

The present invention furthermore relates to a eukaryotic or prokaryotic cell line which is transformed with this recombinant DNA, and to a method for producing human interleukin-2, in which a eukaryotic or prokaryotic cell which is associated with a recombinant DNA is transformed, cultured to produce interleukin-2, and the interleukin-2 formed is recovered, the recombinant DNA containing a DNA sequence according to any one of claims 1 to 13 in a vector DNA which is for replication in is capable of this cell, the coding sequence of this DNA sequence being arranged in a position downstream of a promoter sequence.

Special embodiments of the invention and the advantages achieved according to the invention have been explained in more detail below with reference to the drawings. Show it:

Figure 1 is a restriction endonuclease cleavage map of a cloned gene encoded to produce a polypeptide with the activity of IL-2 (hereinafter referred to as vIL-2 polypeptides); -3-

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Figure 2 (a) shows the base sequence of the cloned gene;

Fig. 2 (b) the amino acid sequence I and the amino acid sequences II and ΙΠ of the polypeptides with IL-2 activity;

Fig. 3 is a flow chart showing the construction of a recombinant DNA (pCEIL-2) in which the encoded gene is inserted;

4 shows the plasmid vector pTrS-3;

Figures 5 (a), 5 (b) and 5 (c) are flow charts illustrating the construction of recombinant DNAs (pTIL2-22, pTIL2-21, pHL2-14 and pTIL2-15) using pTrS-3 as a vector;

Fig. 6 is a flow diagram explaining the construction of a recombinant DNA (pKIL2-21) using pKT218 as a vector;

Fig. 7 is a flow diagram explaining the construction of a recombinant DNA (pTuIL2-22) using pTUBlP-5 as a vector; and

Fig. 8 vector DNAs that are capable of replication in cells of Saccharomyces cereviseae.

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

The cloned gene coding for an IL-2 polypeptide can be transcribed by transcription of a messenger RNA corresponding to IL-2 (mRNA, "RNA" is an abbreviation for ribonucleic acid) (hereinafter abbreviated as "IL-2 mRNA"), derived from mammalian cells capable of forming a polypeptide with IL-2 activity to form 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 production of cDNA can be separated in a conventional manner from mammalian cells which are able to form the IL-2 polypeptide. The separated RNA is subjected to polyadenylation (Gillis et al “Immunological Rev., Vol. 63 (1982), pp. 167-209). The polyadenylated RNA can be fractionated as sediment from 11 to 12 S, for example by centrifugation on a sucrose density gradient. Occasionally, mRNA shows 13S IL-2 mRNA activity. In these cases it is assumed that mRNA is present in an aggregated form of 11 to 12 S mRNA.

As mammalian egg cells capable of producing IL-2, which are the mRNA source of the present invention, T-lymphocytes such as peripheral blood mononuclear cells, tonsil cells, spleen cells or the like, which are obtainable from mammals, can be used. The cells can be pretreated in a conventional manner, for example with nylon columns, antiserum complement, density gradient fractionation, multiple enzyme treatment, e.g. B. with a combination of neuraminidase and galactose oxidase, X-rays or trypsin to give the cells DL-2 productivity or to increase IL-2 activity. Cloned T lymphocytes obtained from the mammalian cells mentioned after culturing in the presence of T cell growth factor can also be used as mRNA cells. These are preferred as T lymphocytes. Transformed lymphocyte cell lines, such as those from leukemia or lymphoma cell lines themselves or their derivatives obtained by the above pretreatment or mutation methods, or cloned transformed cell lines are preferred mRNA sources. Obviously, cloned cells generally contain larger amounts IL-2 mRNA than is the case with original cell lines. T cell hybridomas obtained by fusion of the aforementioned lymphocyte derivative cells and tumor cell lines such as CEM, Molt 4 F and BW 5 147 are also preferred mammalian cell lines according to the invention. In this case, the cell lines derived from lymphocytes comprise 1 constitutive formers of IL- 2 and 2 are those that form IL-2 only in the presence of a mitogen introduced into the culture, either in the absence or in the presence of other cells that stimulate IL-2 formation.

In order to generate IL-2 mRNA in constitutive IL-2 generator cells, the constitutive IL-2 image neurons are grown under conditions which are generally customary in the field of cell culture. For the generation of the mRNA in cells which contain IL-2 only in the presence of a Mitogens form, the cultured cells are thoroughly washed with culture medium and in a culture medium such as Rosewell Park Memorial Institute 1640 (hereinafter referred to as " RPMI1640 "), Dulbecco Modified Eagle Medium (short " DMEM ") or in click medium optionally contain serum, resuspended. These culture media can be supplemented with various additives, such as penicillin, streptomycin or other antibiotics, or with fresh L-glutamine, Hepes buffer and sodium hydrogen carbonate in concentrations which are customary in the field of cell culture technology. The preferred cell density is 0.5 to 4 x 10 ^ cells / ml. Suitable stimulants are added to induce the activation of mRNA and the formation of IL-2. Examples of corresponding stimulants are mitogens, neuraminidase, galactose oxidase, zinc derivatives, such as zinc chloride, or lymphocyte-activating substances from microorganisms, such as protein A and streptolysin-O. The stimulated cells are obtained and washed. The presence of macrophages or dendritic cells during mitogen stimulation can also activate the mRNA or increase the amount of activated mRNA. Similarly, the presence of cell lines derived from B lymphocytes or from B lymphocyte lines such as Raji, Daudi, K562 and Ball-1 can activate the mRNA or increase the amount of activated mRNA.

In order to proliferate the mammalian cells, they are maintained under normal conditions in an in vitro cell culture or in animals that are appropriate for their tissue tolerance. When using the in vitro -4-

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Culture to produce the mRNA source, the cells are grown in any growth media previously determined to allow growth of T cells. These growth media may optionally be supplemented with mammalian serum, serum components or serum albumin. The growing time for activating the mRNA corresponds to the time required for the activation of the cells to form mRNA. This time period generally corresponds to the time required for the IL-2 to start being secreted into the growth medium. A preferred period is 3 to 12 hours after the addition of a stimulant, such as a mitogen. An unnecessary extension of the breeding time can occasionally lead to a decomposition of the IL-2 mRNA formed. During the course of activation of IL-2 forming cells, phorbol esters such as PMA or ΊΡΑ, preferably at a concentration of 10 to 50 ng / ml, can be used to promote the degree of activation.

The above-mentioned method for activating IL-2 tnRNA can be carried out at temperatures in the range from 32 to 38 ° C. in a humidified atmosphere and at a pH of about 7.0 to 7.4.

The methods for obtaining and growing mammalian cells capable of producing IL-2 are explained below. f 11 Provision of a cell line with constitutive IL-2 formation

A Jurkat cell line of a human, leukemic T cell (freely available from the Fred Hutchinson Cancer Institute, Seattle, V. St. A., Salk Institute, San Diego, V. St. A., German Cancer Research Center, Heidelberg) is clicked -Medium suspended in a cell density of 1 x 10 ^ cells / ml and irradiated with 8 x Hp R X-rays with an irradiation rate of 150 R / min. Then 0.1 cells per 200 μΐ medium are clicked from the cells irradiated in this way. Medium containing 5 percent FCS (fetal calf serum) was inoculated into flat-bottomed microplates with 96 wells (Falcon 3072) and grown for 3 weeks at 37 ° C in an incubator with 5 percent CO2 (cloning with limited dilution). The cultured living cells are transferred to 24-well growth plates (Nunc) and grown for a further 5 days prior to the formation of confluent cell layers. These cells are then cultured in a synthetic growth medium for approximately 2 days at an original cell density of approximately 1 to 2 x 10 ^ / ml that is free of serum and serum albumin. The culture supernatant is harvested by centrifugation and filtered through 0.22 millipore filter paper to separate cell debris and sterilize the supernatant. Then mutants produced by X-ray radiation, which are capable of constituting IL-2, are selected and cloned, measuring the IL-2 activity present in the supernatant (2) providing IL-2 donor cells from human, peripheral , monocular blood cells

Peripheral human blood is obtained. Peripheral blood lymphocytes (TBL 'for short) are isolated from this by density gradient centrifugation on a Ficoll-Hypaque device. The PBL are in 2 ml click

Inoculated medium containing 5 percent FCS with a cell density of 1 x 10 ^ cells / ml in a 24-well Nunc growth plate together with 100 μl of a 5 pg / ml solution of phytohemaglutinin-M (Gibco) (pHA) and Bred for 48 hours under the above conditions. The cells are then washed and again on 1 ml of click medium in a cell density of 1 × 10 ^ cells / ml together with 1 ml of a conditioned medium which has been prepared from human splenocytes, with stimulation with 2.5 μg / ml of concanavalin ( short " Con A ") vaccinated for 48 hours. The culture medium containing 50 percent of the conditioned medium is exchanged every third day to obtain a long-term culture of human T-lymphocytes from PBL. The long-term cultures of human T lymphocytes produced in this way are, as explained above, cloned by the limited dilution method in the presence of human splenocytes derived from conditioned medium. The cell clones are multiplied accordingly. Cloned human T lymphocytes are then inoculated onto 1 ml RPMI1640 in a cell density of 1 × 10 ^ cells / ml in 24-well Nunc culture plates in the presence of 10 μg / ml PHA and in the presence in an incubator at 37 ° C. for 24 hours grown from 7.5 percent CO2. The supernatants of the culture liquid are harvested, centrifuged, filtered through a 0.22 μτη Millipore filter and tested for their IL-2 activity in order to identify the human, normal T-lymphocyte clones with IL-2 formation. (3) Provision of malignant cell lines. derived from human cells capable of producing IL-2 in the presence of a mitogen

Jurkat cell lines or cloned cell lines, such as Jurkat 111, obtained by the limited dilution method described above are able to produce 10 to 4000 units / ml IL-2 when they are in a aforesaid serum-free synthetic medium or in RPMI 1640 containing 1 to 2 percent mammalian serum in the presence of a mitogen, such as 10 pg / ml Con A or 2.5 pg / ml PHA. These malignant human cell lines also form IL-2 in the presence of zinc chloride, protein A or Prcibanil. -5-

AT 392 082 B (4) Providing cells capable of IL-2 formation in the presence of a mitogen and other costimulatory cells or costimulatory soluble factors

The human malignant cell line Molt F4 and some cloned cell lines, such as Jurkat J99, obtained by the limited evaporation method do not form IL-2, even if they are grown for 24 to 72 hours in the presence of lectins or mitogens in any concentration . However, these cells are capable of producing IL-2 in substantial amounts (10 to 100 µg / ml) over a 24 hour culture period at 37 ° C when taken with 5 to 10 pg / ml of Interleukin-1, one of the monokines or grown with a 50 percent number of K562 or Raji cells.

The extraction of IL-2 mRNA from cells activated in the above manner is carried out according to conventional methods, regardless of the 10 different cell sources. For example, cells are partially or completely broken up by adding a detergent, such as sodium dodecyl sulfate and deoxycholic acid, or by mechanical homogenization or freezing / thawing. In order to prevent degradation of RNA by ribonuclease during mENA extraction, RNase inhibitors such as heparin, polyvinyl sulfate, bentonite, macaroid, diethyl pyrocarbonate or vanadyl complex are preferably added. IL-2-15 mRNA can be obtained from precipitated polysome in IL-2 biosynthesis, which is precipitated with anti-IL-2 antibody by extraction with a detergent.

The poly-A containing mRNA can be fractionated or concentrated in a conventional manner, for example by affinity chromatography or by batch absorption on oligo dT cellulose, poly U-Sepharose by Sephaiose 2B, sucrose density gradient centrifugation or agarose gel electrophoresis. The mRNA fractions are then tested for their Π, -2 mRNA activity by testing the biological activities of proteins obtained by translation from the mRNA fractions or the protein obtained by translation using monoclonal antibody against the IL- 2 peptide identified. For example, mRNA is generally translated by microinjections into frog eggs (Xenopus laevis) (J.B. Gurdon et al., Nature, Vol. 233 (1972), pp. 177-182) or using the mRNA-dependent reticulolysate or cell-free wheat germ translation systems.

EL-2 activity can be determined by the microassay method fundamentally discussed by Gillis et al., (J. Immunol., Vol. 120 (1978), pp. 2027-2033). In these tests, IL-2-dependent cellular proliferation of cytotoxic T-lymphocyte cell lines ("CTLL" for short) was carried out according to the methods of

Gillis et al. have been generated. 4 x 10 ^ CTLL cells are inoculated onto 100 μΐ RPMI 30 1640 medium containing 2 percent FCS in flat-bottomed microplates with 96 wells together with 100 μΐ serially diluted translation products. After 20 hours of incubation at 37 ° C in an incubator with 5 percent CO2, the cells are pulsed with 0.5 μΟι% -TdR for 4 hours and harvested on glass fiber strips using an automatic cell meter. Then the built-in radioactivity is measured by liquid scintillation counting. According to this test procedure, it is found that the CTLL cells grown in the presence of 35 IL-2 incorporate% -TdR in a dose-dependent manner, which enables calculation of the amount of IL-2 incorporated in the test samples that IL-2 is capable of To promote proliferation of T lymphocytes, which enables the measurement of EL-2 activity using an index of C cell growth activity. 5 CTLL cells are put together on 100 μΐ DMEM containing 2 percent FCS in flat-bottomed microplates with 96 wells with 40 100 μΐ of the serial diluted translation products. After 72 to 96 hours of incubation at 37 ° C in an incubator at 5 percent CO2, the number of cultured and activated cells below the

Counted microscope. As a positive external control, 100 units / ml or 10 units / ml of IL-2 are added. The IL-2 activity of the test sample is calculated in comparison with the number of viable cells grown in these control groups. 45 The EL-2 mRNA obtained in this way from the most active fraction is used as a template for the synthesis of ds-cDNA. The ds-cDNA is linked to a vector DNA. The synthesis of cDNA is carried out according to customary methods

First, ss-cDNA, which is complementary to mRNA, is produced in the presence of dATP, dGTP, dCTP, dTTP using reverse transcriptase and using mRNA as a template (template) and oligo-dT 50 as an initiator (primer). The template mRNA is then removed by alkaline treatment. Ds-cDNA is obtained using reverse transcriptase or DNA polymerase and using the ss-cDNA synthesized above as template.

DNA formed by recombinant technology is obtained from the ds cDNA obtained in this way and a vector DNA with a replicon which is capable of replication in eukaryotic or prokaryotic cells. The recombinant DNA is then incorporated into the host cells

The ds cDNA and a vector DNA capable of propagation in eukaryotic or prokaryotic cells are pre-ligated by various methods such as treatment with exonuclease, additions of chemically synthesized pieces of DNA and G, C-tailing to form ends the ds-cDNA and the vector-DNA linkable terms, modified. The linkage of the linkable DNA is carried out, for example, by T ^ phage DNA ligase in the presence of ATP -6-

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Living cells are transformed with the resulting eccombinant DNA to amplify the cloned cDNA or to form IL-2 polypeptide.

Examples of eukaryotic host organisms that can be used to form IL-2 are vertebrates, yeasts and the like. For example, monkey cells, such as CV-1 cells, transformed by an original defective mutant of SV-40 and expressing the SV-40-sized T antigen (COS cells according to Y. Gluzman (Cell, Vol. 23, ( 1981), pp. 175-182), cells derived from mice according to S. Ohno and T. Taniguchi (Nucleic Acids Research, Vol. 10 (1982), pp. 967-977) and yeast host vector systems used for the expression of IFN gene according to R. Hitzeman et al. (Nature, Vol. 293 (1981), pp. 717-722). Suitable prokaryotic host organisms are Escherichia coli and the like. E. coli is preferred as the host for amplification of DNA in host organisms, however other hosts can also be used.

Suitable vectors for E. coli are plasmid vectors of the EK type (stringent type), such as pSClOl, pRK353, pRK646, pRK248, pDF41 and the like, plasmid vectors of the EK type ("relaxed" type), such as ColEl, pVH51, pAC105 , RSF2124, pCRl, pMB9, pBR313, pBR322, pBR324, pBR325, pBR327, pBR328, pKY2289, pKY2700, pKN80, pKC7, pKB158, pMK2004, pACYCl, pACYC184, Xdu 1, and the same as X PhD and Xdu 1, and the type : Xgt. Xc, λgL λΒ, λ WES, XC, XWES. λΒ, XZJvir., λΒ ’, XALO, λΒ, λ WES. Ts622, XDam and the like. In general, pBR322 is often used as a vector for E. coli. In this case, the best monifying sites are the Pstl and EcoRI sites.

The transformation of the host cell with the recombinant DNA can be carried out in the usual way as follows:

If the host is a prokaryote, such as E. coli, corresponding cells which are suitable for DNA uptake are produced from cells which have been harvested according to known methods after the exponential growth phase and have subsequently been treated in accordance with the CaC ^ method . If MgC ^ or RbCl is present in the reaction reaction medium, the transformation efficiency increases. The transformation can also be carried out after formation of a protoplast of the host cell.

If the host is a eukaryote, a transfection method of DNA, such as calcium phosphate precipitate formation, conventional mechanical methods, e.g. B. microinjection, insertion of a plasmid encapsulated in red blood cell hosts or in liposomes, treatment of cells with agents such as lysophosphatidylcholine or use of virus vectors or the like.

Cells with the IL-2 gene can be isolated after transformation by either of the following two methods. (1) In the plus-minus method, partially purified IL-2 mRNA is obtained by sucrose density gradient centrifugation of mRNAs extracted from mitogen-activated mammalian cells as 11 or 12s sediment, and then 32P-radioactively labeled ss -cDNA synthesized using partially purified mRNA as a template. After removal of the template mRNA by alkaline treatment, the isolated cDNA is hybridized with partially purified 11- or 12s-mRNA which has been extracted from mammalian cells not activated with a mitogen. Subsequently, non-hybridized and hybrid-forming cDNA are chromatographically fractionated on a hydroxylapatite column. The non-hybridized cDNA and the hybridized cDNA are provisionally referred to as " probe " A or " probe " B designated. Transformants are grown on 2 nitrocellulose filters practically in the same way. The DNA of the cells is fixed on the filter paper by alkali treatment. Probe A and probe B are hybridized with the DNA on 2 different filter papers. An autoradiographic determination is then carried out in order to select the transformants which react positively with probe A (+) but react weakly or not at all with probe B (-) (Taniguchi et al., Proc. Jpn. Acad., Vol 155B (1979), pp. 464-469). (2) The second method is to split, for example, 1000 to 10,000 transformant clones into groups of a few 10 to a few 100 clones each. The divided clone groups are each grown in a conventional manner to form plasmid DNAs. These plasmid DNAs are then converted into ss-cDNAs, for example by heat denaturation. The resulting ss-cDNAs are fixed on nitrocellulose filter paper in order to achieve the hybridization of mRNA, which is complementary to the fixed DNAs and has been produced from mammalian cells, including activated IL-2 mRNA. Another possibility is to hybridize mRNAs containing IL-2 mRNA with heat-denatured plasmid DNAs and then to fix the DNA-mRNA hybrid on nitrocellulose filter papers. These filter papers are then washed with a low salt concentration buffer such as 1 millimolar N-Z-hydroxyethylpiperazine-n-2-ethanesulfonic acid or with 10 millimolar NaCl. The mRNA adsorbed on the filter paper is extracted by treatment for example for 1 minute at 95 ° C. with a solution containing 0.5 millimolar EDTA and 0.1 percent SDS. Purified mRNA is obtained by column chromatography using oligo-dT cellulose. The mRNA is then translated into protein by microinjection into Xenopus laevis eggs to determine IL-2 activity, or the mRNA is translated into a protein using the mRNA-dependent reticulocyte system or the in vitro cell-free wheat germ translation system to analyze IL-2 activity using anti-IL-2 antibody. According to these procedures, the group, -7-

AT 392 082 B, in which the presence of IL-2 activity is determined, is repeatedly divided into further groups with a smaller number of transformant clones until a single clone with IL-2 DNA is specified.

In order to obtain cDNA encoding IL-2 polypeptide from the IL-2-forming transformant, the recombinant DNA in the transformant is separated and cleaved with a restriction endonuclease. The insert cDNA fraction is separated from the 5 DNA fragments formed by the cleavage.

The complete nucleotide sequence of the PstI DNA insert, which codes for the IL-2 polypeptide, from the recombinant DNA of pIL2-50A was determined according to the method of Maxam and Gilbert (Meth. Enzym ,, Vol. 65 (1980), S . 499-560) and by the dideoxynucleotide chain termination method (AJM Smith, Meth. Enzym., Vol. 65 (1980), pp. 560-580). 10 The restriction endonuclease cleavage map of the cDNA insert and the base sequence of the insert are given in Figures 1 and 2 (a), the cDNA cleavage sites with restriction endonculease from BstNI, Xbal and BstNI in the order given.

The DNA sequence of the insert contains a single large open reading grid. The first ATG sequence, which usually serves as an initiation sequence in eukaryotes (M. Kozak, Cell, Vol. 15 (1978), pp. 1109-1123), is found 15 at nucleotides 48 and 50 from the 5-end. This ATG is followed by 152 codons, followed by the termination triplet TGA at nucleotides 507 to 509.

A region of A residues corresponding to the 3-poly (A) terminus of the mRNA is found at the end of the cDNA. This is preceded by the hexanucleotide AATAAA (positions 771 to 776), which is usually found in most eukaryotic mRNAs (N.J. Proudfoot and C.G. Brownlee, Nature, Vol. 263 (1976), 20 pp. 211-214).

The amino acid sequence encoded by the cDNA could be derived as shown in Fig. 2 (b) (amino acid sequence I). The polypeptide of amino acid sequence I consists of 153 amino acids, the molecular weight of which is calculated to be 17 631.7 daltons. According to a common feature of most secretion proteins obtained to date (G. Blobel et al “Symp. Soc. Exp. Med., Vol. 33 (1979), pp. 9-36) is the N-25 terminal region of the derived IL-2 Polypeptide also quite hydrophobic. This region is believed to serve as a signal peptide that is cleaved from mature IL-2 during the secretion process. This cleavage takes place either between Ser and Ala in positions 20 and 21 or between Ala and Pro in positions 21 and 22, which results in the polypeptide with amino acid sequences II and III. Similar cleavage sites are often found in other secretion proteins (G. Blobel et al ., Symp. Soc. Exp. Med., Vol. 33 (1979), pp. 9-36). 30 The mature IL-2 polypeptide thus contains 133 or 132 amino acids, which results in a calculated molecular weight of 15,420.5 and 15,349.4 daltons, respectively. This value is compared with the known value for human IL-2 protein from Jurkat cells (15,000 Daltons) (S. Gillis et al., Immunological Rev., Vol. 63 (1982), pp. 67-209). It was further confirmed that the DNA fragment from the CCT codon in positions 111 to 113 in the base sequence, which thus codes for a polypeptide from Pro in position 22 (amino acid sequence III 35 in FIG. 2 (b)) Expressed polypeptide with IL-2 activity as shown in Example 4. It was also confirmed that the DNA fragment from the GCA sequence at positions 107 to 110 in the base sequence, thus encoding a polypeptide from Ala at position 21 (amino acid sequence II in Fig. 2 (b)) Expressed polypeptide with IL-2 activity as shown in Example 7.

As is known, polymorphism frequently occurs in eukaryotic genes, e.g. In human interferon genes 40 (Taniguchi et al., Gene, Vol. 10 (1980), pp. 11-15, Ohno and Taniguchi, Proc. Natl. Acad. Sci. USA, Vol. 77 (1980), p. 5305 -5309, Gray et al., Nature, Vol. 295 (1981), pp. 501-508). In some cases the polymorphism is accompanied by a replacement of certain amino acids of the protein products, while in other cases the structure of the protein product remains unchanged. In the case of human DL-2 cDNA, another cDNA clone (pIL2-503), in which the A residue is in position 503 of pIL2-50A cDNA (Fig. 2) by a G-45 residue is replaced. More cDNA clones with some base substitutions compared to pIL2-50A cDNA can be »waited.

It follows from the above statements that the genes according to the invention include the following: DNA with the base sequence shown in FIG. 2 (a), DNAs from the ATG sequence in positions 48 to 50 with the sequential bases following the ATG sequence up to at least the ATC sequence in positions 50 504 to 506, DNAs from the GCA sequence in positions 108 to 110 with the sequential bases in

Connection to the GCA sequence up to at least the ATC codon and DNAs from the CCT sequence in positions 111 to 113 with the sequential bases following the CCT sequence up to at least the ACT sequence. The genes according to the invention also comprise DNAs which end in the ACT sequence in positions 504 to 506 and with A in position 1, the ATG sequence in positions 48 to 50, the GCA sequence in 55 positions 108 to 110 or the CCT sequence begin in positions 111 to 113. The invention

Genes also include DNAs that end in the TGA sequence in positions 507 to 509 and end with A in position 1, the ATG sequence in positions 48 to 50, the GCA sequence in positions 108 to 110 or the Start the CCT sequence in positions 111 to 113. Furthermore comprehensive »! the genes of the invention DNAs ending at C in position 801 and with A in position 1, the ATG sequence in positions 48 to 50, the GCA-60 sequence in positions 108 to 110 or the CCT sequence in positions 111 to 113 begin. Furthermore, the genes according to the invention comprise DNAs which end at poly (A) and with the ATG codon in positions 48 to 50, the GCA sequence in positions 108 to 110 or the CCT sequence in positions -8-

AT 392 082 B

Positions 111 through 113 begin. Furthermore, the genes of the invention include base sequences corresponding to amino acid sequences I, II and III. Furthermore, polypeptides that lack one or more amino acids in amino acid sequence I or polypeptides in which one or more of the amino acids of amino acid sequence I are replaced by one or more other amino acids can have IL-2 activity. Therefore, genes that code for these polypeptides are also suitable genes for the purposes of the invention. Similarly, according to the invention, genes with an additive connection of one or more base sequences are also suitable which are capable of expressing one or more amino acids in addition to amino acid sequences I, II or III, provided that the additionally linked amino acids affect the effect of the polypeptides do not interfere with their EL-2 activity. Modified, additionally linked amino acid regions which interfere with the polypeptide function with regard to IL-2 can also be used according to the invention, provided that the additionally linked regions are easily removable. The same applies to the additive linking of DNA at the 3'-terminus of genes corresponding to the amino acid sequences I, II and ΠΙ, which code for additional amino acids at the C-terminus of I, II or III with the amino acid sequences I, II or III. The use of genes which are encoded for such polypeptides is therefore possible according to the invention.

Recombinant DNAs that direct the formation of IL-2 in living cells can be constructed using various methods. For example, the coding sequence of IL-2 cDNA can be inserted into an expression vehicle down the promoter sequence. Another possibility is to insert a piece of cDNA with a promoter sequence up to the IL-2 coding sequence after or before the insertion of cDNA into the expression vehicle.

Methods for constructing prokaryotic or eukaryotic cells that express IL-2 cDNA and that form the IL-2 polypeptide are discussed in more detail below. (11 Expression of IL-2 cDNA in E. coli

In order to express IL-2 cDNA in E. coli, cDNA is fused with various bacterial promoters. Hybrid plasmids containing cDNA down from the promoters are obtained. The plasmids are the transfection, e.g. B. in the E. coli strain HB101 and bacteria that synthesize a protein product with human IL-2 activity are cloned. In essence, any type of bacterial promoter should direct the expression of IL-2 cDNA if they do so attached to the cDNA. Examples of this cDNA expression are described here.

The cloned cDNA for IL-2 encodes a polypeptide consisting of 153 amino acids, as shown in FIG. 2. The N-terminal region corresponding to about 20 amino acids of this polypeptide is quite hydrophobic, which is a characteristic of most secretion proteins. Such a hydrophobic sequence, the so-called signal sequence, is split off during the secretion process. Therefore, the mature IL-2 polypeptide is said to contain less than 153 amino acids. Thus, it is desirable to express the portion of the cDNA encoding the mature IL-2 polypeptide but not the portion corresponding to the IL-2 signal sequence. (i) Construction of an expression plasmid vehicle, pTrS-3, the E. coli trp promoter whose ribosome binding site (SD sequence) for the lead peptide has recently been described (G. Miozzari and C. Yanofsky, J. Bacteriol., Vol. 133 (1978), pp. 1457-1466) and an ATG codon at a distance of 13 bp down to the SD sequence (Nishi et al., SEIKAGAKU, Vol. 54, No. 8 (1982), p. 676). The plasmid vehicle also contains a single Sphl site immediately down from the ATG initiation sequence (Fig. 4).

To express IL-2 cDNA, the plasmid is first cleaved with SphI and treated either with E. coli DNA polymerase I (Klenow fragment) or with bacteriophage T4 DNA polymerase I to the 3 'protruding ends (Fig. 5 (a)) to remove. The plasmid pIL2-50A is digested twice with Pstl and HgiAI and a larger cDNA fragment is isolated. The DNA is then treated with either E. Coli DNA polymerase I (Klenow fragment) or with bacteriophage T4 DNA polymerase so that the 3 'protruding ends are smoothed. The cDNA treated in this manner encodes the 132 amino acid IL-2 polypeptide as shown in Fig. 5 (a). This cDNA is then linked to the pTrS-3 plasmid DNA pretreated in the above manner such that the ATG initiation codon is present on the CCT (Pro) sequence of the IL-2 cDNA. A plasmid pHL2-22 is thus obtained. The connection between the trp promoter sequence and the IL-2 cDNA sequence of pTIL2-22 is also shown in Fig. 5 (a).

The plasmid pTIL2-22 should direct the synthesis of an IL-2 polypeptide with 132 amino acids in E. coli starting from proline. (ii) Since it is also possible for the mature IL-2 to contain alanine (position 21) as the N-terminal amino acid instead of proline, the following plasmid, which directs the synthesis of 133 amino acid EL-2 polypeptide, is discussed .

The plasmid pTrS-3 contains a single Clal site between the SD sequence and the ATG sequence (Fig. 4). This plasmid is cleaved by Clal and Sali. The plasmid pIL2-50A is partially digested with pstl, treated with E. coli DNA polymerase I, and the largest linear DNA is isolated. The DNA is then linked to a synthetic DNA linker containing a restriction Xhol cleavage site and a clone containing plasmid pIL2-50A (Xho) in which the linker DNA is down in the 3 'position from the decoding sequence is isolated. The plasmid pIL2-50A (Xho) is first digested with HgiAI, either with E. Coli Klenow fragment or treated with T4 DNA polymerase, cleaved with Xhol, -9-

AT 392 082 B and the cDNA fragment is isolated. This cDNA fragment is then linked to pTrS-3 DNA pretreated with Clal and Sali and with a synthetic DNA as shown in Figure 5 (b). A plasmid pTIL2-21, which should direct the synthesis of an IL-2 polypeptide with 133 amino acids, starting from alanine, in E. coli can thus be produced, as explained in FIG. 5 (b). A similar construction can also be carried out without using Xhol linker. (iii) Different size EL-2 polypeptides with different N-terminal amino acid can be prepared using the pTrS-3 expression plasmid vehicle by the following methods. The cloned IL-2 cDNA in pIL2-50A contains a single Ddel site at nucleotide positions 81 to 85. The plasmid pIL2-50A (Xho) is cleaved by Ddel and the DNA fragment containing the major part of the cDNA is isolated. The fragment should also contain DNA with about 3000 base pairs of pBR322 (Fig. 5 (c)). The DNA fragment is treated with exonuclease Bal31 and then digested with Xhol. The DNA so treated is then linked to pTrS-3 digested with SphI, treated either with Klenow fragment or with T4 DNA polymerase and then kept with Sali, as shown in Fig. 5 (c). The linked DNA is then subjected to transfection in E. coli HB 101, and bacterial clones expressing human IL-2 are selected (screening). These clones should express human IL-2 of different sizes because the DNA is removed in different ways according to the N-terminal region of human IL-2. Thus pTIL2-14 and pTIL2-15 carrying IL-2 DNA can be obtained. (iv) The IL-2 cDNA can also be expressed using pKT218 (provided by K. Talmage, Proc. Natl. Acad. Sci. USA, 1980, 77: 3369-3373). Plasmid pKT218 is digested with psu and linked to an IL-2 cDNA insert obtained by cleaving pIL2-50A DNA with HgiAI and pstI (Fig. 6). The plasmid pKIL2-21 obtained has the sequence at the beginning of protein synthesis initiation, as shown in FIG. 6. Thus, the plasmid pKIL2-21 in E. coli should direct the synthesis of a fused polypeptide with 133 amino acids from IL-2 and the amino acids from ß-lactamase (the first methionine has been split off in E. coli). (v) An expression plasmid pTuBlP-5, in which the promoter sequence for tufB in pBT322 is used, is first constructed (Taniguchi et al., SEIKAGAKU, Vol. 53 (1981), p. 966). The plasmid contains a single clal site located 2 base pairs down from the SD sequence, as shown in Figure 7.

Because pTrS-3 also contains a Clal site between the SD sequence and the ATG initiation sequence and because this Clal site does not destroy during the construction of the expression plasmid using pTrS-3 and IL-2 cDNA in the manner described above , it is very easy to replace the bacterial tip promoter with that of tufB so that human IL-2 cDNA is expressed under the control of the tufB promoter. For example, pTIL-2-22 is digested with Clal and PvuII and the DNA fragment containing IL-2 cDNA is isolated. This fragment is then linked to pTUBlP-5 DNA which has previously been cleaved with Clal and PvuII. A plasmid pTuIL2-22 is thus constructed, as shown in FIG. 7. The IL-2 activity could be detected in the extract of E. coli HB 101 containing the plasmid pTuIL2-22. (vi) A similar construction can also be carried out using pTIL2-21 and essentially all expression plasmids constructed using pTrS-3. It is also possible to determine the distance between the SD and ATG sequence by columns, e.g. B. pTuIL2-22 with Clal, removal (or addition) of some base pairs of DNA by Bal31 or S1 or DNA polymerase I (E. coli) and subsequent relinking of the plasmid. (1) Expression of the IL-2 cDNA in yeast IL-2 cDNA can also be expressed in yeast by inserting the cDNA into suitable expression vectors and introducing the product into host cells. Various shuttle vectors were used to express foreign genes in Yeast reports (R. Heitzman et al., Nature, Vol. 293 (1981), pp. 717-722, P. Valenzuela et al., Nature, Vol. 298 (1982), pp. 347-350, Miyanohara et al ., Proc. Natl. Acad. Sci. USA, Vol. 80 (1983), pp. 1-5). The vectors are capable of replication in E. coli and in yeast hosts and contain promoter sequences from yeast genes. Essentially all such expression vectors can be used to express IL-2 cDNA. When using yeast, IL-2 concentrations may be higher than when using animal cells or bacteria. An example of the expression of human IL-2 cDNA in yeast is described below.

The yeast-E. coli shuttle vectors pAT77 and pAM82 have been described by Miyanaohara et al., (Proc. Natl. Acad. Sci. USA, Vol. 80 (1983), pp. 1-5). The vector pAM82 is a derivative of pAT77. Both carry markers from ars 1 (DT Stinchcomb et al., Nature, Vol. 282 (1979), pp. 39-43), 2 μιη ori (JR Broach et al., Gene, Vol. 8 (1979), p. 121-133) and leu2 (B. Ratzkin et al., Proc. Natl. Acad. Sci. USA, Vol. 74 (1979), pp. 474-491) and the promoter for the acid phosphatase (APase) gene from Yeast. They also carry the 3.7 kb DNA segment of pBR322, which contains a marker for ampicillin resistance (Apr) and the origin of replication (Fig. 8). The APase promoter can be induced by shifting a high phosphate concentration to a low concentration in the culture media. In order to express human IL-2 cDNA, pIL2-50A is digested with PstI after treatment with E. coli Klenow fragment or with T4 DNA polymerase, the cDNA is linked with pAM82, which has previously been digested with Xhol and -10-

AT 392 082 B incubated with the E. coli Klenow fragment to notice the ends. Hybrid plasmids in which the cDNA coding sequence is located downstream of the yeast APase promoter sequence are selected by cloning in E. coli. The resulting plasmid, pYIL-2a, is introduced into yeast. After induction of the APase promoter, the IL-2 activity in the yeast extract is measured. The plasmid pYIL-2A contains a region of the GC residues between the yeast promoter and the IL-2 cDNA. It is possible that such a sequence inhibits the expression of IL-2 cDNA. To overcome this difficulty, a plasmid can be constructed as follows: The plasmid pIL2-50A is digested with PstI and the cDNA insert is isolated. This cDNA is then treated with T4-DNA-Folymase in the presence of dATP, dGTP and dTTP , so that the regions of the C residues at both ends of the cDNA are separated and then treated with nuclease S1 to remove the regions of the G residues. This DNA is linked to XhoI DNA linker and plasmid pBR322, the EcoRI site of which has been cleaved and blunted by EcoRI and the Klenow fragment. The plasmid pIL2-Xho formed is cleaved with Xhol and the cDNA insert is isolated. The cDNA is then introduced into the unique Xhol site of pAM82 and a plasmid containing the sequence coding for IL-2, which is correctly oriented with respect to the yeast APase promoter, is cloned into E. coli. The plasmid pYIL-2b is introduced into yeast. After induction of the APase promoter, the IL-2 activity in the yeast extract is measured. (3) Expression of da cDNA in mammalian cells

A plasmid that should control the synthesis of human IL-2 in mammalian cells can be constructed as follows: A plasmid pCE-1 is made from pKCR (K. OHare et al., Proc. Natl. Acad. Sci. USA, Vol. 78 (1981), pp. 1527-1531) and from pBR328 (X. Soberon et al., Gene, Vol. 9 (1980), pp. 287-305) according to a series of modification methods according to FIG. 3. The initiation sequence ATG of the IL-2 gene is linked down the promoter for the SV40 early gene. The plasmid contains a single PstI site immediately down from the SV40 early promoter and up from that part of the rabbit β-globin chiomosomal gene that contains an intron. The plasmid also contains the origin of replication of SV40 and the polyadenylation site for the early gene. A plasmid pCEIL-2 is thus obtained, in which the IL-2 structural gene from the early promoter of S V40 should be transcribed in corresponding host cells (FIG. 3).

This plasmid is cleaved with Hhal and then transformed by DNA transfection into the transformed monkey cell line COS-7, which allows the replication of DNA with SV40 origin sequences. The cleavage of the plasmid with Hhal before transfection appears important for an effective expression of cDNA to be carried out, since sequences which could hinder replication of the transfected DNA in COS cells can be removed by this procedure from the part of the plasmid which is essential for cDNA expression. After transfection of the vector on monkey culture cells COS-7 (Y. Gluzman, Cell, Vol. 23 (1981), pp. 175-182), after 1 to 3 days of cultivation under normal breeding conditions, IL-2 is generally secreted and in the bred Cell medium formed To insert amplified DNA into other eukaryotic cells, a vector suitable for the host organism, which is linked to the cDNA insert, is cleaved and isolated from prokaryotic cells. The eukaryotic cells can be transfected with the vector synthesized in this way and be bred.

Cells with the recombinant DNA are grown to amplify the recombinant DNA or to form IL-2 polypeptide. The cultivation is carried out using conventional means. For example, transformed yeast can be grown in a medium containing carbon sources, nitrogen sources, inorganic salts and optionally organic nutrients such as vitamins and amino acids at temperatures in the range from 20 to 37 ° C. and a pH of 4 to 7 are grown under aerobic conditions. Transformed prokaryotic organisms such as E. coli can also be grown under conventional conditions.

The intracellularly or extracellularly formed IL-2 is obtained by known methods, for example by precipitation with ammonium sulfate, dialysis to remove salts (under normal pressure or under reduced pressure), gel filtration, chromatography, preparative isoelectric flatbed focusing, gel electrophoresis, high-performance liquid chromatography (HPLC) (Ion exchange, gel filtration and reverse phase chromatography), and affinity chromatography on a support linked with a dye, on Sepharose 4B coupled with monoclonal antibodies against IL-2 or on Sepharose 4B with bound lectin and the like. Methods for obtaining and purifying IL-2 are described in the following references: Watson et al., J. Exp. Med., Vol. 150 (1979), pp. 849-861, Gillis et al., J. Immunol., Vol. 124 (1980), pp. 1954-1962, Mochizuku et al., J. Immunol. Methods, Vol. 39 (1980), pp. 185-201 and K. Welte et al., J. Exp. Med., Vol. 156 (1982), pp. 454-464.

The polypeptide obtained in this way shows the same biochemical and biological behavior as IL-2 produced by mammalian cells under mitogen stimulation and has IL-2 activity. The molecular weight is approximately 15,000 daltons. The IL-2 activity is completely neutralized or precipitated with monoclonal anti-IL-2 antibodies in the presence or absence of immunoadsorbents, such as Igsorb (Enzyme Center). In immunoelectrophoresis, the IL-2 polypeptide shows only a single precipitate against the corresponding anti-IL-2 antibody. The IL-2 activity remains stable after reduction with 2-mercaptoethanol and is resistant to treatment with DNase and RNase as well as to a 30-minute heat treatment at 56 ° C. The -11-

AT 392 082 B

Activity is stable at a pH of 2 to 9. The IL-2 formed promotes the growth of monoclonal functional T cells (cytotoxic T lymphocytes), enhances hiymocyte mitogenesis, leads to the formation of anti-tumor-specific cytotoxic T lymphocytes from the memory in the absence of the antigen and can increase the natural kiUeizellen activity against YAC-1 and RLS 1 cells.

Examples:

Example 1 (1) A human T-leukemia cell line, Jurkat cells (freely available in Japan, the Federal Republic of Germany and the United States) was suspended in RPMI 1640 medium containing 10 percent (VoL / Vol.) FCS and at Room temperature 50 seconds irradiated with a Roentgen apparatus Exs 150 / 300-4 (Toshiba / Japan) to 10,000 Roentgen. The irradiated cells were then 5 days at 37 ° C in an incubator at 5 percent CO2 with an original cell density of 1 x 10 ~ * Cells / ml grown in the aforementioned culture medium The mutant cells (0.2 cells / well) were placed in wells of 10-part, flat-bottomed microplates with 96 wells and grown for 21 days at 37 ° C. in an incubator at 5 percent CO2

Clones obtained from these wells were repeatedly transferred to fresh growth media to increase clone sizes. The propagated clones were grown for 24 hours at an original cell density of 1 x 10 ^ cells / ml in the presence of 50 pg / ml ConA. IL-2 activity was measured according to the methods described above. Then, a human T cell line called Jurkat-111 (J-III for short) (ATW CRL8129), which had been cloned from the parent Jurkat cells, was selected, the productivity of which in relation to IL-2 compared to the parent strain was that Had been increased 40 times. The cloned cell line J-III grew under conventional conditions. The growth rate was practically the same as that of conventional Jurkat cells.

(2) J-III cells (1 x 10 ^ / ml) were grown in 1000 ml serum-free synthetic growth medium RITC 55-9 (T. Sato et al., Exp. Cell. Res., Vol. 138 (1982), P. 127-134) in roller cultivation bottles (Falcon 3027) and inoculated for 4 days at 37 ° C. The propagated cells were harvested by centrifugation. The harvested cells were again inoculated into the aforementioned medium to which 25 μg / ml ConA. (Cell content 4 × 10 ^ cells / ml) had been added. 1000 ml of the inoculated growth medium were brought into 4 batches of roll culture bottles (Falcon). Cultivation was continued for 6 hours while rotating (3) Jurkat cells (1.2 x 10 ^) stimulated in this way with 25 pg / ml ConA were in 8000 ml saline-phosphate buffer (PBS) for 6 hours The cells were washed twice by centrifugation and in 800 ml of RSB solution (10 millimolar Tris-HCl, pH 7.5, 10 millimolar NaCl, 1.5 millimolar MgC ^) containing a ribonucleoside-vanadyl complex (10 millimolar), a nuclease inhibitor. Detergent was then added to a final level of 0.05 percent. The mixture was mixed moderately and the cell culture was removed by centrifugation at 3000 rpm and 4 ° C. for 5 minutes. SDS (0.5%) and EDTA (5 millimolar) were added to the supernatant and the cytoplasmic RNA was added by adding the same Volume of phenol extracted After extraction three times with phenol, the RNA was precipitated with twice the volume of ethanol. The precipitates were obtained by centrifugation and dissolved in 10 molar molar Tris-HCl of pH 7.5. The amount of RNA obtained was 196 mg.

Fractionation of mRNA was performed using affinity chromatography on oligo- (dT) -cellulose (P.L. Biochemicals, type 7). A solution with a pH of 7.5 and containing 20 millimolar Tris-HCl, 0.5 m NaCl, 1 millimolar EDTA and 0.5 percent SDS was used as the adsorption solution. Elution was carried out with water and 10 millimolar Tris-HCl (pH 7.5) alternately after washing the column with the buffer (20 millimolar Tris-HCl, pH 7.5.0.5 M NaCl, 1 millimolar EDTA). 3.6 mg of mRNA were eluted. Then 2.4 mg of the mRNA obtained were fractionated by sucrose density gradient centrifugation (5 to 2.5 percent sucrose density gradient in a solution of pH 7.5 containing 50 millimolar Tris-HCl, 1 millimolar EDTA and 0.2 m NaCl) . Centrifugation was carried out at 26,000 rpm and 4 ° C. for 24 hours. The 11-12S fraction of mRNA was fractionated into fractions # 12, 13 and 14 in amounts of 59.46 and 60 pg, respectively.

(4) The mRNA obtained in fraction No. 13 was microinjected into Xenopus laevis oocytes (50 ng mRNA / egg). The culture supernatant was used to determine the Π-2 activity. As shown in Table I, an increase in incorporation was observed % -TdR and an increase in the number of activated T-lymophocytes, which clearly confirms that the mRNA in this fraction contains human IL-2 mRNA -12-

AT 392 082 B

Table I (a)

Sample dilution Uptake of% -TdR (cpm) amount of IL-2 * (units / ml) control I 533 0 (test medium) control Π x2 590 0 (supernatant from untreated egg culture) x 32 572 translation product x 8 14 683 32 from Fraction 13 x 32 10 165

Dilution (b) T-lymphocyte count (number per well) Amount of IL-2 * (units / ml) control I x 2 0 0 (test medium) x 16 0 control II x2 0 0 (supernatant from untreated egg culture) x 16 0 translation product x 2 115 40 from fraction 13 x 16 55 * The units were calculated by comparing the amount of% -TdR incorporated according to the probit analysis with standard IL-2 (10 units / ml). (5) Subsequently, cDNA was synthesized in vitro from fraction No. 13 of 11 to 12S mRNA containing IL-2 mRNA. Recombinant DNA was constructed with the plasmid vector PBR322. Escherichia coli was transformed with the recombinant DNA. Clones with acquired IL-2 cDNA were selected as follows: (5-1) 50 millimolar Tris-HCl buffer (pH 7.5, 30 millimolar NaCl, 6 millimolar MgC ^, 5 millimolar dithiothreitol (briefly " DTT " ), each 0.5 millimolar dATP, dGTB, dCTP, dTTP (dCTP contained 32P- radioactively labeled product), 0.7 pg oligo (dT) ^, 10 μg mRNA and 15 units AMV-reverse

Transcriptidase (J.W. Beard) were mixed and left at 41 ° C for 90 minutes.

After the reaction, DNA was obtained as ethanol precipitate after phenol treatment. The DNA was dissolved in a solution of pH 7.5 containing 20 millimolar Tris and 1 millimolar EDTA. 2.5 μg ss-cDNA were synthesized. To remove the mRNA present in this solution, the solution was brought to an NaOH concentration of 0.33 by adding NaOH and left to stand for 15 hours at room temperature. The solution was then neutralized with an equal volume of 1 m Tris-HCl of pH 7.5 and passed over a column packed with Sephadex G-50. 1.8 μg of cDNA were obtained. -13-

AT 392 082 B (5-2) 50 millimolar phosphate buffer with a pH value of 7.5.10 millimolar MgClj, 10 millimolar DTT, each 0.75 millimolar dATP, dGTP, dCTP, (dCTP contained ^ H-radioactive labeled product), 1.8 pg ss cDNA and 8 units of polymerase I (BRL, V. ST. A.) were mixed and reacted at 15 ° C. for 15 hours. After completion of the reaction, DNA was obtained as ethanol precipitate after treatment with phenol and chloroform. 1.10 pg ds cDNA were formed. A mixture of 50 millimolar sodium acetate (pH 4.5.0.2 M NaCl, 1 millimolar ZnC ^ and 1.10 pg ds-cDNA was incubated for 20 minutes at 37 ° C. with 0.25 units of nuclease

Sj (Sankyo, Japan) was added and incubated for 15 minutes.

After the reaction had ended, the reaction product treated twice with phenol was applied to a gel filtration medium from "Pharmacia Fine Chemicals Ine." packed column, giving 0.55 pg ds cDNA.

(5-3) A mixture of 0.14 M potassium cocodylate, 30 millimolar Tris base, 0.1 millimolar DTT, 1 millimolar COC ^, 0.64 millimolar 32P-dC'IP (specific activity 2.7 x 10 ^ cpm / nmol), 0.55 pg ds-cDNA and 5 units of terminal transferase (BRL) were incubated for 7 minutes at 37 ° C. and then, after phenol treatment, placed on a column packed with the above-mentioned gel filtration medium, whereby 0.50 pg DNA as Received ethanol precipitate. The DNA obtained was extended at both 3 ends with about 50 dCmP residues. 10 pg pBR322-DNA were digested with the restriction enzyme Pstl. The 3 'ends of the digested DNA were added using the same procedure used above for adding dCMP to ds-cDNA, except that dGTP was used instead of dCTP. (5-4) A mixture of 50 millimolar Tris-HCl (pH 7.5), 0.1 m NaCl, 5 millimolar EDTA, 0.05 pg pBR322 (extended with dGMP residues) and 0.01 pg cDNA (extended with dCMP) was first incubated at 65 ° C. for 2 minutes, then at 46 ° C. for 120 minutes, at 37 ° C. for 60 minutes and finally at room temperature for 60 minutes. E. coli X 1776 (R. Curtiss III et al., &Quot; Molecular Cloning of Recombinant DNA ", ed. WA Scott & R. Werner, Academic Press (1977)) was in 50 ml L-broth containing 100 pg / ml diaminopimelic acid, 50 pg / ml thymidine, 1 percent tryptophan, 0.5 percent yeast extract, 0.5 percent NaCl and 0.1 percent glucose inoculated. The cultivation was carried out with shaking at 37 ° C. until the cultivation liquid had an absorption of 0.3 at 562 nm. After the cultivation had ended, the culture liquid was left to stand at 0 ° C. for 30 minutes. The bacterial cells were then collected by centrifugation and washed twice with 25 ml of a solution containing 5 millimolar Tris-HCl (pH 7.6), 0.1 M NaCl, 5 millimolar MgC ^ and 10 millimolar RbCl.

The cells thus obtained were in 20 ml of a solution containing 5 molar Tris-HCl (pH 7.6), 0.25 M KCl, 5 millimolar MgClj, 0.1 M CaC ^ and 10 millimolar RbCl suspended and left at 0 ° C for 25 minutes. The cells were then recovered and resuspended in 1 ml of the same solution. The recombinant DNA described above was placed in 0.2 ml of the cell suspension, and the suspension was allowed to stand at 0 ° C for 60 minutes. Then 0.7 ml of the L broth was added to the culture. It was shaken at 37 ° C for 30 minutes. 0.1 ml of the growth medium obtained was thoroughly spread onto the surface of 1.5 percent agarose medium consisting of L broth containing 100 pg / ml diaminopimelic acid, 50 pg / ml thymidine and 15 pg / ml tetracycline, and 2 Incubated for days at 37 ° C. (5-5) 432 colonies which occurred were divided into 18 groups of 24 different bacterial clones each, inoculated into 200 ml L broth containing 100 pg / ml diaminopimelic acid, 50 pg / ml thymidine and 10 pg / ml tetracycline and 5 to Cultivated for 7 hours with shaking at 37 ° C. Then 200 ml of a fresh L broth containing chloramphenicol at a final concentration of 170 pg / ml was added, after which the cultivation was continued overnight. The plasmid DNA amplified in this way was purified in a conventional manner. Clones containing IL-2 cDNA were selected by means of an mRNA hybridization translation test (hereinafter "HT test"). The HT test was carried out in the following manner : 25 pg of purified DNA were digested with the restriction enzyme, treated 3 times with phenol, treated with phenol-chloroform and with chloroform, precipitated with ethanol, washed with 80 percent ethanol and dissolved in 40 μl of 80 percent formamide.

The reaction mixture was warmed to 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 was then fixed on nitrocellulose filters. These filters were operated for 3 hours Dried 80 ° C and 18 hours at 37 ° C in a solution containing 50 percent formamide, 20 millimolar pipes with pH 6.5, 0.5 m NaCl, 5 millimolar EDTA, 0.2 percent SDS and 250 pg poly (A) mRNA from induced J11 cells for hybridization of the DNA fixed to the filters with IL-2 mRNA. The filters were then washed 3 times at 65 ° C. with a solution of 10 millimolar pipes with a pH of 6.5, 0.15 m NaCl, 1 millimolar pipes, 10 millimolar NaCl and for 1 minute at 95 ° C. with a solution 0.5 millimolar EDTA and 0.1 percent SDS treated to extract the hybridized mRNA from the filters. The mRNA extracted in this way was purified on an oligo dT cellulose column in a conventional manner and used to determine the IL-2- -14-

AT 392 082 B

Activity of the translated proteins injected into Xenopus oocytes. One of 18 groups, each consisting of 24 clones, gave a positive reaction with 48 units / ml of Il-2 activity in the above-described test for incorporation of% -TdR, while the others were clearly negative. The 24 individual colonies belonging to the positive group were inoculated into 200 ml of L broth of the composition given above and grown under aerobic conditions for 5 to 7 hours at 37 ° C. In a corresponding manner, fresh L broth containing chloramphenicol was added. After amplification of the plasmid DNA by culturing overnight, the plasmid DNA was purified by the usual methods. After cleaving about 5 μg of the individual plasmid DNAs with Hind, the plasmid DNAs were bound to nitrocellulose filters in the same way. The filters were hybridized with IL-2 mRNA. The hybridized mRNA was collected and injected into Xenopus oocytes to determine the IL-2 activity of the translated proteins.

As shown in Table II, only the plasmid DNA purified from a single colony named p3-16 gave positive IL-2 activity. This clone is thus identified as a clone containing IL-2 cDNA (E. coli X 1776 / p3-16 AJ 11995 (EERM-BP-225)). This plasmid DNA p3-16 has exactly the DNA (IL-2 gene) which is capable of binding the special hybrid with IL-2 mRNA.

Tables (a)

Sample dilution Uptake of 3H-TdR (cDm) amount of IL-2 (units / ml) control I 2010 0 (test medium) control II x 2 2120 (supernatant from untreated egg culture) x 32 2482 0 translation product x 2 20453 58 of mRNA x 32 20961 (b)

Sample dilution T-lymphocyte cell count (cells per well) Amount of IL-2 (units / ml) Control I. 0 0 (test medium) control II x 2 0 0 (supernatant from untreated (egg culture) x 32 translation product x 2 88 32 con mRNA1 x 32 42 -15-1 mRNA, hybridized with cDNA from plasmid p3-16.

The cDNA insert of the plasmid p3-16 is cleaved by the restriction enzyme Xbal at a single site and by BstNI at 2 sites (up and down to the Xbal cleavage site). However, plasmid p3-16 contains a cDNA insert containing approximately 650 base pairs, which apparently corresponds to a portion of IL-2 mRNA size 11 to 12S.

AT 392 082 B

Another cDNA library was prepared according to the method of Land et al., (Nucleic Acids Res., Vol. 9 (1981), p. 2551) using IL-2 mRNA as a template. Single-stranded cDNA (1.6 μg) was synthesized using 4 μg IL-2 mRNA (extended with dCMP residues). ds-cDNA was synthesized using oligo- (dG) J2-18 as ^ primer for DNA polymerase I (Klenow fragment). The cDNA (0.6 μg) with more than 680 base pairs as a DNA size marker was obtained by sucrose gradient centrifugation and was inserted into the PstI site of pBR322 according to the standard GC trimming method. After transformation of E. coli χ 1776 by the recombinant DNA about 2000 colonies were found according to the in situ hybridization method of Grunstein-Hogness with nick translated p3-16 cDNA insert as a probe. The plasmid plL-250A containing approximately 850 base pairs and the transformed clone (E. coli X 1776 / pIL-2-50A 11996 (FERM-BP-226)) were identified. A restriction endonuclease cleavage map of the pIL-2-50A cDNA insert is shown in FIG. 1

In order to isolate a gene coding for the IL-2 peptide from transformed E. coli X 1776 pIL-2-50A, after isolation of the DNA region from the cells, plasmid DNA was cleaved with the restriction enzyme Pstl by conventional methods. The smaller fragment formed in this way from two DNA fragments formed was DNA which encoded the IL-2 peptide. The complete nucleotide sequence of the PstI insert of pIL-2-50A was determined according to the method of Maxam and Gilbert (Enzym., Vol. 65 (1980), pp. 499-560). The complete structure is shown in FIG. 2.

Example 2

The plasmid pKCR (O'Hare et al., Proc. Naü. Acad. Sei. USA, Vol. 78, No. 3 (1981), S 1527-1531) consists of (i) segments of SV40-DNA (in FIG 3 represented by hatched blocks) containing an early gene promoter and an origin of replication (0.725 to 0.648 mu) and a polyadenylation site from the early gene (0.169 to 0.144 mu), (ii) part of the rabbit-ß-globin gene ( shown as open blocks) (BamHI-PvuII fragment) and (iii) a segment of pBR322 (EcoRi-BamHI fragment) containing an origin of replication and the ampicillin resistance gene. This plasmid was digested with BamHI. After both ends of the cleaved DNA had been filled in by DNA polymerase I (Klenow fragment), synthetic PstI linker DNA was introduced to construct pKCR (PstI). Plasmid pKCR (Pstl) was digested with Sali, treated with Klenow fragment to fill in the ends, and then partially digested with EcoRI to obtain an EcoRI-SalI fragment containing all SV40-derived DNA and globingen. This fragment was then linked to a piece of pBR328 DNA containing the tetracycline resistance gene and an origin of replication, as shown in FIG. 3. The plasmid pCE-1 obtained contains a single Psu site immediately downstream of the SV 40 early promoter.

The cDNA insert from pIL-2-50A was excised by PstI cleavage and linked to PstI-cleaved pCE-1 to construct pCEIL-2 to express the IL-2 structural gene under the control of the SV40 early promoter . Plasmid pCE-1 was originally constructed for cDNA cloning by the G-C truncation method (A.C. A. Chang et al., Nature, Vol. 275 (1978), pp. 617-624) in bacteria and direct expression in mammalian cells.

The plasmid was digested with Hhal and then introduced into the transformed monkey cell line COS-7 by DNA transfection (McCutchan et al., J. Natl. Cancer Inst., Vol. 41 (1968), pp. 351-357), the one Replication of the DNA containing SV40 source sequences contains (available from Y. Gluzman (Cell, Vol. 23 (1981), pp. 175-182). It is important for an effective expression of cDNA, the plasmid before transfection with Hhal, since sequences which could prevent replication of the transfected DNA in COS cells can be removed from the part of the plasmid which is essential for cDNA expression in this method. COS-7 cells (6 × 10 ^ / ml) were suspended in 0.5 ml of DMEM containing 5 percent FCS in 24-well growth plates (Nunc) and incubated for 4 hours at 37 ° C. The grown cells were then mixed with a mixture of 1 pg of the vector described above, 17.6 μΐ 1 millimolar Tris-HCl containing 0.1 millimole ar EDTA, 2.4 μΐ 2 m CaCl2 and 120 μΐ 2 x HBS containing 50 millimolar pipes, 280 millimolar NaCl and 1.5 millimolar Ν32ΗΡ04.12Η20 (pH 7.10).

The cells were incubated for a further 4 hours at 37 ° C. The growth medium was then aspirated. After washing with 1 ml PBS, 0.5 ml PBS containing 20 percent glycerin was added. After standing for 3 minutes at room temperature, the medium was suctioned off again. The cells were washed with 1 ml PBS and grown in 1 ml DMEM containing 5 percent FCS. After 24 hours, 500 μΐ medium were replaced by fresh medium. The individual media obtained at the appropriate times were left at 4 ° C. until used. The cultured cell medium was tested for its activity on human IL-2 2 to 3 days after the transfection. As can be seen from Table III, the culture supernatant of COS-7 cells transfected with pCEIL-2 contained the IL-2 activity. No IL-2 activity could be detected in the culture media of cells transfected with pCE-1. -16-

AT 392 082 B

Tables!

Transfection of DNA with iL-2 activity, measured by% -TdR uptake (M / ml)

T lymphocyte growth pCEIL-2 12 ++++ pCE-1 1

The IL-2 activity in the cell culture medium after transfection of COS-7 with pCEIL-2 was neutralized with mouse (B ALB / c) -antihuman-IL-2 monoclonal antibody from 12 units / ml to below 1 unit / ml. The result that COS-7 cells transfected with pCEIL-2 secreted human * IL-2 clearly shows that eukaryotic cells transformed with a recombinant DNA, which have a gene coding for IL-2 polypeptide and a gene capable of replication in these cells Containing vector DNA can be used to form IL-2.

The plasmid pCEIL-2 built into E. coli HB 101 (AJ / 2008) received the accession number FERM-BP 244.

Example 3

The constitutive IL-2-forming cell line J-A1886 (ATCC CRL8130), which had been cloned from Jurkat cells according to Example 1, was grown in a corresponding manner in roller culture bottles. The cultured cells were resuspended in fresh synthetic medium RITC-55-9 with an original cell density of 1 x 106 cells / ml. 8 hours after the start of cultivation, extraction of IL-2 mRNA in the form of an 11 to 12S fraction from 3 × 10 ^ cells was carried out in accordance with the steps described in Example 1

Double-stranded cDNA was synthesized according to Example 1, and the cDNA with more than 600 base pairs (2.4 pg) was kept on a sucrose density gradient after fractionation. The cDNA was then expanded with dCMP residues using the terminal deoxynucleotidyl transferase. A 50 ng aliquot was welded with 250 ng dGMP-extended, PstI-cleaved pBR322. The hybrid plasmids obtained were used to transform E. coli X1776. The transformants were used obtained from about 4000 clones. According to the Grunstein-Hogness method, 3 clones complementary to the plasmid 3-16 cDNA used as the probe were selected. The clones selected in this way are transformed clones which have human IL-2 gene.

Example 4

A plasmid that directs the synthesis of human IL-2 in E. coli cells was constructed as follows. A plasmid pTIL2-22 was constructed from pTrS-3 (T. Nishi, T. Taniguchi et al., SEKAGAKU, Vol. 53 (1981), p. 967)). pIL-2-50A containing IL-2 cDNA was constructed using a sequence of modified procedures as shown in Fig. 5 (a). A plasmid pTrS-3 comprises the insertion of the region of the Trp promoter and of Shine Dalgamo (SD for short) between the EcoRI site and the Clal site of pBR322.

The plasmid also contains an ATG initiation codon 13 base pairs down from the SD sequence and a single Sphl site, as illustrated in Figure 4. The vector is a very effective generator of this protein when the DNA sequence corresponding to this protein is immediately down of the ATG codon, which was generated by Sphl cleavage and subsequent treatment with T4-DNÄ polymerase from pTrS-3. Therefore, the plasmid pTrS-3 (30 pg) was cleaved with the restriction enzyme SphI in the usual way and then treated successively with phenol and chloroform. The ethanol precipitates were obtained and both ends were blunted by treatment with T4-DNA polymerase. Then the DNA (21 , 4 pg) by appropriate successive treatment with phenol and chloroform and precipitation with ethanol. On the other hand, 380 pg pIL-2-50A containing IL-2 cDNA was digested with Pstl. The IL-2 cDNA insert was isolated by agarose gel electrophoresis. The cDNA insert (11 pg) was digested with HgiAI and treated with T4 DNA polymerase. 10 pg of the larger DNA fragment was isolated by agarose gel electrophoresis according to this procedure a cDNA (7.2 pg) was obtained which codes for 132 amino acids. This DNA fragment had blunt ends (FIG. 5 (a)). This cDNA fragment was then linked to a pTrS-3 vector which had previously been cleaved with SphI and treated with T4 DNA polymerase immediately downstream of the ATG sequence. This linked plasmid was then transformed into E. coli HB 101 by conventional methods. The ligation was carried out as follows. The larger fragment of IL-2 cDNA (0.4 pg) and 0.2 pg pTrS-3 vector DNA were mixed with 0.8 units of T4 DNA ligase in 66 millimolar Tris-HCl, pH 7 , 5 containing -17-

AT 392 082 B 6.6 millimolar MgC ^, 1 millimolar ATB and 10 millimolar DTT mixed. The mixture was allowed to stand at 4 ° C overnight. From among the transformants appearing on L-broth agai plates containing ampicillin, colonies containing the EL-2 cDNA region encoding 132 amino acids were selected by an in situ colony hybridization test. The selected colonies were grown again (10 ml), 5 after which plasmid DNA was prepared by treatment with lysozyme and by freezing / thawing. The plasmid DNAs were digested with Pstl and Xbal. The products obtained were analyzed by agarose gel electrophoresis to identify pTIL-2-22 in which the cDNA of the ATG sequence of pTrS-3 was linked in the correct orientation. E. coli HB 101 containing pTIL-2-22 was grown under conventional microorganism growth conditions. The cells were in 10 ml of 10% broth (2.5 percent bactotryptone, 1.0 percent yeast extract, 0.1 percent glucose, 20 millimolar MgS04, 50 millimolar Tris-HCl, pH 7.5) containing 25 pg / ml streptomycin and 25 μg ampicillin grown overnight at 37 ° C. 1 ml of the culture suspension was inoculated onto the same% broth (100 ml) and grown at 37 ° C. As soon as the optical density at 650 μm reached a value of 1.5 to 2.0, 3-indolacrylic acid (IAA) was added 3 hours after the addition of the inductor, the cells were obtained, with 15 20 millimolar Tris-HCl (pH value 7.5, 30 millimolar NaCl) and resuspended in 8 ml of the same buffer. For the Trp promoter to function effectively, inducers such as IAA were added in a final concentration of 50 μg / ml. The proteins thus formed in the bacterial cells were extracted by ultrasound treatment (0 ° C., 2 min) or by digestion (0 ° C., 20 min) with lysozyme (8 μg) with subsequent freezing / thawing 3 times. This method is used IL-2 20 usually extracted from organisms The extracted IL-2 activity was 10,000 to 120,000 units / ml. E. coli HB 101 containing pTTL-2-22 (AJ / 2009) was filed under the number FERM-BP 245.

Example 5

A plasmid pTuIL-2-22, which carries IL-2 cDNA was from pTu-BIP-5 (T. Taniguchi et al, Seikagaku, Vol. 53 25 (1981), p. 966) and pTIL-2-22 from Example 4, constructed according to the methods illustrated in FIG. 7

Plasmid pTuBIP-5 involves inserting the promoter sequence for tufB into pBR322. The plasmid also contains a single clal site located 2 base pairs down from the SD sequence as shown in Fig. 7 because pTrS-3 also contains a clal site between the SD sequence and the ATG initiation codon and there this Clal site is not destroyed during construction of the expression plasmid using pTrS-3 and IL-2 cDNA according to Example 4, it is very easy to replace the bacterial trp promoter with that of tufB so that IL- 2-cDNA is expressed under the control of the tufB promoter.

Therefore, the plasmid pTIL-2-22 (30 pg) was cleaved in a conventional manner with the restriction enzymes Clal and PvuII. The fragment (about 2.2 kb) containing IL-2 cDNA was isolated and purified by agarose gel electrophoresis. 3 pg of DNA were obtained. On the other hand, 20 pg pTuBIP-5 vector was cleaved in 35 corresponding ways by Clal and PvuH. The larger fragment (about 3.4 kb) containing the ampicillin-resistant gene was isolated and purified by agarose gel electrophoresis, giving 3.5 pg of DNA. Then these two fragments, one (about 3.4 kb) containing tufB promoter and the other (about 2.2 kb) containing IL-2 cDNA, were linked in the following manner. The fragment containing EL-2 cDNA (1.2 pg) and 0.3 of the fragment containing tufB promoter 40 were pH-adjusted with 0.8 units of T4 DNA ligase in 66 millimolar Tris-HCl -Value 7.5 mixed with 6.6 millimolar MgC ^, 1 millimolar ATP and 10 millimolar DTT. The mixture was allowed to react at 4 ° C overnight. The plasmid linked in this way was then used for transformation into E. coli HB 101 by conventional methods. Among the transformants appearing on L-broth agar plates containing ampicillin, 8 colonies containing the IL-2 cDNA region, such as pTuIL-2-22 45 in Fig. 7, were selected. Plasmid DNA was prepared according to example 4. E. coli HB 101 containing pTuIL-2-22 was grown in 37 ml L-broth at 37 ° C. When an optical density at 650 mp of about 0.5 to 1.0 was reached, the bacterial cells were obtained, washed with 20 millimolar Tris-HCl (pH 7.5.30 millimolar NaCl) and resuspended in 2 ml of the same buffer. The proteins obtained in this way were extracted in accordance with Example 4. The extracted IL-2 activity was 6,000 to 56,000 units / ml. 50 Escherichia coli HB101 containing pTuIL-2-22 (AJ12010) was deposited under the number FERM-BP 246.

Example 6

A plasmid pGIL-2-22, which carries IL-2 cDNA, was from pGL 101 (TM Roberts and G. D, Laucer, Meth. 55 Enzym., Vol. 68 (1979), pp. 473-483) and pTIL-2-22 constructed from Example 4.

The plasmid pGL 101 (20 pg) containing a lac promoter was digested with the restriction enzyme PvuII in a conventional manner. Subsequent treatment with phenol, chloroform and ethanol precipitation gave 17 pg of DNA. Furthermore 75 pg pTIL-2-22 were cleaved with Clal and Sali. After agarose gel electrophoresis, 2.2 pg of a DNA fragment containing IL-2 cDNA was obtained. This 60 fragment was blunted by treatment with DNA polymerase I (Klenow fragment). The two fragments obtained (0.25 pg and 0.66 pg) were treated with 1.0 unit of T4 DNA ligase according to Example 5 -18-

AT 392 082 B linked. The linked plasmid was used in a conventional manner for transformation in E. coli HB 101. Among the transformants, the transformants which had the insertion of the Clal-Sall fragment containing IL-2 cDNA as a probe were in% broth ( 10 ml) containing 25 pg / ml ampicillin. Plasmid DNA was prepared according to Example 4. The plasmid DNA with the initiation sequence ATG of IL-2 cDNA immediately down from the lac promoter was obtained by cleavage with Pstl and Xbal.

The pGIL-2-22 obtained in this way was inoculated onto 100 ml of L broth containing 25 μg / ml ampicillin and 25 μg / ml streptomycin and cultured. After reaching an optical density at 650 μm of about 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG) was added in a concentration of 1 millimolar. The bacterial cells were collected 1 hour later. The cell extracts were prepared according to example 5. The extracted IL-2 activity was 6000 to 80,000 units / ml.

Escherichia coli HB101 containing pGIL-2-22 (AJ12011) was deposited under the number FERM-BP 247.

Example 7 10 μg of the plasmid pTrS-3 were first cleaved with the restriction enzyme Sali. The Sall site was blunted by treatment with DNA polymeiase (Klenow fragment) or with T4 DNA polymerase. After clal digestion, a larger fragment containing the trp promoter region was isolated in a conventional manner by agarose gel electrophoresis. 3 pg of DNA was obtained.

Furthermore, 11 pg of a cDNA insert obtained by PstI digestion of pIL2-50A was digested with HgiAI and treated with T4 DNA polymerase. The larger fragment was isolated and purified by agarose gel electrophoresis. Thus, a dDNA fragment coding for 132 amino acids of IL-2 was obtained in an amount of 7.2 pg. Then 0.45 pg of the fragment containing a trp promoter (as described above), 0.5 pg HgiAI-PstI fragment containing IL-2 cDNA and synthetic oligonucleotides (5> CGATAAGCTATGGCA- (3 ') and (3> TATTCGATACCGT- (5') (20 pmol each), both of which were phosphorylated at the 5 'end, were linked to 1 unit of T4 DNA ligase according to Example 4 (see FIG. 5 (b)) .

The plasmid linked in this way was then used to transform E. coli HB 101. From the transformants that occurred, the desired transformants were selected as follows

The desired transformants capable of hybridization with IL-2 cDNA and synthetic oligonucleotides were first selected according to the colony hybridization method. The transformants which had the insertion of the DNA fragment starting from the CCT sequence in position 111 to 113 in FIG. 2 (a) (CCTACT-) immediately downstream of the ATG GCA sequence were then by Pstl and Xbal cleavage selected.

The above transformant, containing pTIL2-21a or pTTL2-21b, was grown in L broth according to Example 4. High activities of IL-2 were found in the cell extracts of the transformants when tested according to Example 4

Escherichia coli HB 101 with pTIL2-21a (AJ 12013) and Escherichia coli HB 101 with pTIL2-21b (AJ 12014) were filed under the numbers FERM-BP 248 and FERM-BP 249.

The hosts, E. coli X 1776 and HB101 (HW Boyer et al., J. Mol. Biol., Vol. 41 (1969), p. 459, which were used in the above examples, are known and freely accessible. Furthermore the hosts can be obtained from the deposited transformants by culturing these transformants in L broth at 37 ° C. in order to release the corresponding recombinant DNAs in the transformants, and strains which are sensitive to tetracycline and ampicillin are then selected as hosts.

The plasmid vectors pBR322 (commercial product of the Bethesda research Laboratory), pCE-1, pTrS-3 and pGLlOl are known and freely accessible. Furthermore, the plasmid vectors can be obtained from the deposited transformants by separating the recombinant plasmid DNAs in the transformants in a conventional manner and separating the plasmid vectors in a corresponding manner as described in the examples. For example, pCE-1 can be obtained by cleaving pCEIL-2 by pstl and separating the larger DNA fragment formed. PTrS-3 and pTuBIP-5 were also deposited as E. coli FERM-BP 328 (= FERM-P6735) and E. coli ATCC 31871 (see EP-A 64681) -19-

Claims (25)

AT 392 082 B PATENT CLAIMS 1. DNA sequence which comprises a DNA sequence which codes for a polypeptide with the activity of human interleukin-2, this polymer peptide being selected from the group below: a) a polymer peptide which comprises the following Amino acid sequence has: 1 Met-Tyr-Arg-Met-Gln-Leu-Leu-Ser-Cys-He-Ala-Leu-Ser-Leu- 20 Ala-Leu-Val-Thr-Asn-Ser-Ala-Pro-Thr -Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu-Gln-Leu-Glu-His-Leu-Leu-Leu-Asp-Leu-Gln-Met-Üe-Leu-Asn-Gly-Ile -Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu- Thr-Arg-Met-Leu-Tlu - Phe-Lys-Phe-Tyr-Met-Pro-Lys-Lys-Ala · Thr-Glu- Leu-Lys-His-Leu-Gbi-Cys-Leu-Glu-Glu-Leu-Lys- Pio-Leu-Glu-Glu-Val-Leu-Asn-Leu-Ala-Gln-Ser-Lys-Asn-Phe- His-Leu-Arg-Pro-Arg-Asp-Leu-Ile-Ser-Asn-Ile-Asn-Val-Ile- Val-Leu-Glu-Leu-Lys-Gly-Ser-Glu-Thr-Thr-Phe- Met-Cys-Glu- Tyr-Ala-Asp-Ghi-Thr-Ala-Thr-Ile-Val-Glu-Phe-Leu-Asn-Arg-153 Tip-Üe-Thr-Phe-Cys-Gln-Ser-Ile -Ile-Ser-Thr-Leu-Thr b) a polypeptide against which (a) one or more Amino acids are missing; c) a polypeptide in which one or more amino acids are exchanged for (a); d) a fusion polypeptide which contains a polypeptide according to (a), (b) or (c) in which the additionally linked amino acids do not interfere with the biological interleukin-2 activity or can be easily eliminated, e) a polypeptide, which is an allelic derivative of a polypeptide according to (a). 2. DNA sequence which comprises a DNA sequence which codes for a polypeptide with the activity of human interleukin-2, the coded polypeptide being selected from a) a polypeptide having the following amino acid sequence: X-Thr-Ser-Ser -Ser-Thr-Lys-Lys-Thr-Gln-Leu-Gln-Leu-Glu- His-Leu-Leu-Leu-Asp-Leu-Gln-Met-De-Leu-Asn-Gly-Ile-Asn-Asn -Tyr-Lys-Asn-Pro-Lys-Leu-Thr-Arg-Met-Leu-Thr-Phe-Lys-Phe-Tyr-Met-Pro-Lys-Lys-Ala-Thr-Glu-Leu-Lys-His -Leu-Ghi- Cys-Leu-Glu-Glu-Glu-Leu-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-Ala-Gln-Ser-Lys-Asn-Phe-His-Leu -Arg-Pro-Arg-Asp-Leu- De-Ser-Asn-Ile-Asn-Val-Ile-Val-Leu-Glu-Leu-Lys-Gly-Ser-Glu-Thr-Thr-Phe-Met-Cys -Glu-Tyr-Ala-Asp-Glu-Thr-Ala-Thr- De-Val-Glu-Phe-Leu-Asn-Arg-Trp-Ile-Thr-Phe-Cys-Gln-Ser-He-Ile-Ser -Thr-Leu-Thr, where X is Ala-Pro, Pro, Met-Ala-Pro or Met-Pro, b) a polypeptide, which is an allelic derivative of polypeptide (a), c) a fusion polypeptide, which is a Contains polypeptide according to (a), in which the additionally linked amino acids do not interfere with the biological activity or can be easily removed. -20- AT 392 082 B 3. DNA sequence according to claim 1 or 2, which has restriction endonuclease cleavage sites which, from the 5 'terminus of the coding sequence, are in the order Bst NI, Xba I and Bst NI. 4. DNA sequence according to claim 1 or 2, which has restriction endonuclease cleavage sites which, from the 5 'terminus of the coding sequence, are in the order Dde I, Hinf I, Bst NI, Xba I, Bst NI and Sau 3A . 5. DNA sequence according to claim 1, whose base sequence starts at A in position 1 and has the successive bases up to at least the ACT sequence in position 504 to 506 in Fig. 2 (a) 6. DNA sequence according to claim 1, the base sequence of which begins at the ATG sequence in positions 48 to 50 and the successive bases following the ATG sequence up to at least the ACT sequence in positions 504 to 506 in FIG. 2 ( a) has. 7. DNA sequence according to claim 1, the base sequence of which begins at the GCA sequence in positions 108 to 110 and the bases which follow one another following the GCA sequence at least up to the ACT sequence in positions 504 to 506 in FIG. 2 ( a) has. 8. DNA sequence according to claim 1, the base sequence of which begins at the CCT sequence in positions 111 to 113 and the bases which follow one another following the CCT sequence up to at least the ACT sequence in positions 504 to 506 in FIG. 2 ( a) has. 9. DNA sequence according to one of claims 5 to 8, whose base sequence ends at the ACT sequence in position 504 to 506 in Fig. 2 (a). 10. DNA sequence according to one of claims 5 to 8, whose base sequence ends at the TGA sequence in position 507 to 509 in Fig. 2 (a). 11. DNA sequence according to one of claims 5 to 8, whose base sequence ends at C in position 801 in Fig. 2 (a) 12. DNA sequence according to one of claims 5 to 8, the base sequence of which ends at poly (A) in FIG. 2 (a) 13. A DNA sequence according to claim 1, which has the base sequence shown in Fig. 2 (a) 14. Recombinantly produced DNA containing a DNA sequence according to one of claims 1 to 13 in a vector DNA which is capable of replication in a prokaryote or eukaryote cell in which the coding DNA sequence is in a position downstream is arranged by a promoter sequence 15. Recombinant DNA according to claim 14, in which the prokaryote cell line belongs to the genus Escherichia, preferably Escherichia coli 16. Recombinant DNA according to claim 14, in which the eukaryotic cell line belongs to the genus Saccharomyces and preferably belongs to Saccharomyces cerevisiae. 17. Recombinant DNA according to claim 14, in which the eukaryote cell line is an SV-40 transformed monkey cell which constitutively expresses Large T antigen. 18. Recombinant DNA as claimed in any one of claims 14 to 16, which is capable of expressing, in a suitable host, the cDNA section encoding mature human interleukin-2 polypeptide but not the section corresponding to the signal sequence . 19. Eukaryotic or prokaryotic cells which are transformed with a recombinant DNA according to one of claims 14 to 16 or 18. 20. Cell according to claim 19, wherein the prokaryote cell belongs to the genus Escherichia, preferably Escherichia coli, and is transformed with the recombinant DNA according to claim 14, 15 or 18. 21. Cell according to claim 19, wherein the eukaryotic cells belong to the genus Saccharomyces, preferably Saccharomyces cerevisiae, and are transformed with the recombinant DNA according to claim 14, 16 or 18. -21- AT 392 082 B 22. The cell of claim 19, wherein the eukaryotic cell is a mammalian cell. 23. The cell of claim 22, wherein the mammalian cell is an SV-40 transformed monkey cell that constitutively expresses Large T antigen. 24. A method for producing human interleukin-2, in which a eukaryote or prokaryote cell transformed with a recombinant DNA is cultivated in a culture medium to produce interleukin-2, and the interleukin-2 formed is obtained characterized in that the recombinant DNA contains a DNA sequence according to any one of claims 1 to 13 in a vector DNA which is capable of replication in this cell, the coding sequence of this DNA sequence being arranged in a position downstream of a promoter sequence is. 25. The method according to claim 24, characterized in that the eukaryotic or prokaryotic cell, which is transformed with a recombinant DNA to form interleukin-2, is a cell or a cell line according to any one of claims 19 to 23. Including 12 sheets of drawings -22-