IE57996B1 - A genetic engineering process for the preparation of human interleukin-2 and means for carrying out this process - Google Patents

Abstract

A synthetic DNA sequence can be used to obtain human interleukin-2 in a genetic engineering process. The gene is advantageously synthesised in the form of a plurality of fragments which are ligated enzymatically to give larger part-sequences which are incorporated into hybrid plasmids and amplified in host organisms. The part-sequences are reisolated and then combined to give the complete gene and incorporated into a hybrid plasmid, and the latter is expressed in a host organism.

Description

The invention is defined in the patent claims. It relates in particular to a process for the preparation of human interleukin-2 and polypeptides which are derived from it and have the biological and immunological activity of human interleukin-2, to chemically synthesized genes which code for these peptides and to suitable vector constructions and host organisms for the expression of these polypeptides.

Human interleukin-2, called IL-2* in the following text, is a polypeptide comprising 133 amino acids. The DNA sequence of human IL-2 and its genetic engineering synthesis are described in the European Patent Application with Publication No. EP 0,091,539 Al (amino acid sequence II of Fig. 2b). This synthesis is based on a mRNA which was isolated from mammalian cells and converted into cDNA, incorporated into vectors and introduced into host cells, including E. coli, for expression.

For a number of reasons, bacteria, in particular E. coli, are preferred host cells for the genetic engineering production of polypeptides. Genes of eukaryotic cells, which have been obtained, as described in the patent application mentioned, from mRNA by reverse transcriptase, are frequently expressed only unsatisfactorily after incorporation into plasmids and transformation into bacteria. The invention thus relates to a synthetic DNA sequence which codes for IL-2 and which leads particularly advantageously in E. coli to the expression of a polypeptide having IL-2 activity. The invention also relates to those DNA sequences which hybridize with the synthetic DNA sequence and which are derived from the latter by single or multiple substitution, deletion or insertion of bases. The exchange, the insertion or the deletion of codons or a combination thereof leads to peptides having one or more exchanged amino acid(s) or to longer or shorter IL-2 derivatives, to which the invention likewise relates. These polypeptides having the biological or immunological activity of IL-2 may have had their properties modified in such a manner that the stability of the peptide is increased, the lipophilicity of the polypeptide has been modified in favor of improved solubility, the administration as a medicament is facilitated or the biological activity is increased.

It is well known that the genetic code is degenerate, that is to say only two amino acids are coded for by a single nucleotide sequence, while the remaining 18 genetically codable amino acids are assignable to 2 to 6 triplets. However, the host cells of different species do not always make the same use of the possibilities of variation arising from this. Thus, there is an immense multiplicity of codon possibilities for the synthesis of genes.

It has now been found that the DNA sequence I (appendix), which codes for the entire amino acid sequence 1-133 of IL-2, and the DNA part sequences used for the synthesis of sequence I (sequence II, appendix, with the part sequences Ila (IL 2-1) to lid (IL 2-IV) are particularly advantageous for the genetic engineering synthesis of IL-2. A protruding DNA sequence, for example corresponding to the restriction endonuclease Eco RI, is located at the 5' end of the coding strand of DNA sequence I, whereas another single-stranded protruding sequence, for example corresponding to the restriction enzyme Sal I, is located at the 3' end of the coding strand. These two different recognition sequences ensure the insertion of the DNA into plasmids in the desired orientation. Of course, it is also possible to select other protruding sequences which correspond to restriction sites in the intended vector.

These recognition sequences permit not only the reisolation of the DNA from a vector by restriction with the appropriate enzymes, for example Eco RI and Sal I, but also numerous modifications of the DNA, such as removal of the single-stranded regions, for example using mung bean nuclease, partial or complete filling in of the shorter end, for example with Klenow polymerase, or the addition of suitable adaptors or linkers. The overlapping ends are thus extremely advantageous for the incorporation into expression vectors of the DNA according to the invention.

Between these recognition sequences and the codons for the amino acid sequence, there is located, at the 5' end of the coding strand, the codon for the amino acid methionine (which is numbered 0 in the DNA sequence I). Alternatively, this can be replaced by a presequence (also called a signal or leader sequence) of a bacterial or other host-intrinsic protein (review article: Perlman and Halvorson; J. Mol. Biol. 167 (1983), 391) which brings about the secretion of the desired polypeptide from the cytoplasm and, in this excretion process, is cleaved off by a signal peptidase occurring naturally in the host cell. At the end of the coding strand, one, or preferably two, stop triplet(s) follow(s) triplet 133 which codes for threonine. In the appendix, DNA sequence I, which comprises nucleotides 9 to 407 (amino acids 1-133), is shown together with nucleotide sequence 1 to 8 (protruding sequence corresponding to Eco RI and Met codon) and two stop codons (nucleotides 408 to 413) and the protruding sequence corresponding to Sal I. The two stop codons represent a preferred embodiment of the invention. They ensure that even at high rates of protein synthesis the molecules are cut off at the desired end and no fusion proteins are formed.

Three unique internal restriction sites for the restriction enzymes Pst I, Xba I and Sac I (nucleotides 69-74, 182-187 and 291-296 of the coding strand of DNA sequence I) permit the subcloning of four gene fragments IL 2-1 to IL 2-IV, which can be incorporated into cloning vectors which have been thoroughly investigated, such as, for example, pUC 12. In addition, a number of other unique recognition sequences for restriction enzymes are incorporated inside the structural gene, and these, on the one hand, provide access to part sequences of IL-2 and, on the other hand, permit variations to be carried out: Restriction Recognition Position of the first enzyme sequence nucleotide in the recognition sequence (coding strand) ’ 3’ Aha II GGCGCC 8 Ava II GGACC 65 Ban I GGCGCC 8 Ban II GAGCTC 291 3bv I GCTGC 59 Bst NI CCTGG 221 Dde I CTCAG 226 Fnu 4HI GCTGC 59 Hae II GGCGCC 8 Hha I GCGC 9 Hinf I GACTC 35 Hph I TCACC 373 Mlu I ACGCGT 117 Nar I GGCGCC 8 Pvu I CGATCG 346 Sau 96 I GGACC 65 Scr FI CCTGG 221 DNA sequence I can be constructed from 38 oligonucleotides of various lengths (see DNA sequence II) by first synthesizing them chemically and then linking them enzymatically via sticky ends of 4 to 9 nucleotides.

Furthermore, account was taken in DNA sequence I of the fact that for those amino acids to which several codons are assignable the latter are not equivalent but, on the contrary, show different preferences in each particular host cell, such as E. coli. In addition, palindromic sequences were reduced to a minimum.

Thus the gene structure of the DNA sequence I is readily accessible from relatively small structural units, permits the subcloning of four gene fragments into wellknown vectors and permits their combination to produce the total gene. The unique recognition sequences for restriction enzymes very greatly facilitate the formation of extensions, modifications and curtailments of the protein molecule.

Extensions are obtained, after reaction with the particular restriction enzyme, by addition of suitable chemically synthesized DNA molecules. Modifications are obtained by cutting out individual sections of the DNA using suitable restriction enzymes and replacing them by other DNA sequences obtained chemically. Curtailments can be obtained, after cleavage with the particular restriction enzyme, by reaction with nucleases.

The biological activity of the extended, modified or curtailed molecules can be tested in a biological assay system, for example by induction of cell growth in a T cell population which is strictly dependent for propagation on the presence of IL-2 in the medium.

The incorporation of the synthesized genes or gene fragments into cloning vectors, for example into commercially available plasmids such as pUC 12 or other generally accessible plasmids such as ptac 11, ptrp Hl and pKK 177.3, is carried out in a manner known per se. Moreover, the chemically synthesized genes can be provided beforehand with suitable chemically synthesized control regions which permit expression of the proteins. In this context, reference may be made to the textbook by Maniatis (Molecular Cloning, Maniatis et al., Cold Spring Harbor, 1982). The transformation into suitable host organisms, advantageously E. coli, of the hybrid plasmids thus obtained is likewise known per se and is described in detail in the abovementioned textbook.

The gene fragments IL 2-1 to IL 2-IV obtained according to the invention, the hybrid plasmids obtained with them, and the transformed host organisms are likewise new and the invention relates to them. The same applies to the new DNA sequences which are modifications of DNA sequence I. Further embodiments of the invention are set out in the patent claims.

In the examples which follow, some embodiments of the invention are also illustrated in detail, from which the multiplicity of possible modifications and combinations are evident to those skilled in the art. In these examples , percentage data relate to weight unless otherwise specified.

Examples 1.Chemical synthesis of a single-stranded oligonucleotide The synthesis of the structural units of the gene is illustrated using the example of structural unit Ia of the gene, which comprises nucleotides 1-17 of the coding strand. The nucleoside located at the 3' end, i.e. cytidine in the present case (nucleotide no. 17), is covalently bonded by known methods (M.J. Gait et al., Nucleic Acids Res. 8 (1980) 1081-1096)) to silica gel ( *FRACTOSIL, Merck) via the 3'-hydroxyl group. For this purpose, first the silica gel Is reacted with 3-(triethoxysilyl)propyl amine, with elimination of ethanol, this producing a Si-O-Si bond. The cytidine is reacted as the N*-benzoyl-3'-0-succinoyl-5'-dimethoxytrityl ether with the modified carrier, in the presence of paranitrophenol and Ν,Ν'-dicyclohexylcarbodiimide, the free carboxyl group of the succinoyl group acylating the amino radical of the propylamino group.

* Trade Mark In the synthetic steps which follow, the base component is used as the dialkylamide or chloride of the monomethyl ester of the 5'-0-dimethoxytritylnucleoside-3'-phosphorous acid, the adenine being in the form of the N6-benzoyl compound, the cytosine being in the form of the N*-benzoyl compound, the guanine being in the form of the N2-isobutryl compound and the thymine, which contains no amino group, being without a protective group. mg of the polymeric carrier, which contains 2 μmol of bound cytosine, are treated successively with the following agents: a) nitromethane, b) saturated zinc bromide solution in nitromethane containing 1% water, c) methanol, d) tetrahydrofuran, e) acetonitrile, f) 40 μ mol of the appropriate nucleoside phosphite and 200 pmol of tetrazole in 0.5 ml of anhydrous acetonitrile (5 minutes), g) 20% acetic anhydride in tetrahydrofuran containing 40% lutidine and 10% dimethylaminopyridine (2 minutes), h) tetrahydrofuran, i) tetrahydrofuran containing 20% water and 40% lutadine, j) 3% iodine in collidine/water/tetrahydrofuran in the ratio by volume 5:4:1, k) tetrahydrofuran and l) methanol.

In this context, the term -phosphite is to be understood to be the monomethyl ester of the deoxyribose-3'-monophosphorous acid, the third valency being saturated by chlorine or a tertiary amino group, for example a mor35 pholino radical. The yields in the individual synthetic steps can in each case be determined after the detritylation reaction b) by spectrophotometry, measuring the absorption of the dimethoxytrityl cation at a wavelength of 496 nm.

When synthesis of the oligonucleotide is complete, the methyl phosphate protective groups of the oligomer are eliminated using p-thiocresol and triethylamine.

Then the oligonucleotide is separated from the solid carrier by treatment with ammonia for 3 hours. Treatment of the oligomers with concentrated ammonia for 2 to 3 days quantitatively eliminates the amino protective groups on the bases. The crude product thus obtained is purified by high-pressure liquid chromatography (HPLC) or by polyacrylamide gel electrophoresis.

The other structural units Ib-IVj of the gene are synthesized entirely correspondingly, their nucleotide sequence being derived from DNA sequence II. 2. Enzymatic linkage of the single-stranded oligonucleotides to give gene fragments IL 2-1 to IL 2-IV.

For the phosphorylation of the oligonucleotides at the 5' end, 1 nmol of each of oligonucleotides Ia and lb was treated with 5 nmol of adenosine triphosphate and four units of T4 polynucleotide kinase in 20 pi of 50 mM tris HCl buffer (pH 7.6), 10 mM magnesium chloride and 10 mM dithiothreitol (DTT) at 37 °C for 30 minutes (C.C. Richardson, Progress in Nucl. Acids Res. 2 (1972) 825). The enzyme is inactivated by heating at 95’C for 5 minutes. Then oligonucleotides Ia and lb are hybridized together by heating them in aqueous solution at 95C for 2 minutes and then cooling slowly to 5°C.

Phosphorylation and pairwise hybridization are carried out analogously for oligonucleotides lc and Id, and Ie and If. Phosphorylation and pairwise hydridization are carried out on oligonucleotides Ila and lib etc. through JL V Hi and IIj for subfragment IL 2-II, on oligomers Ilia and Illb etc. through IHk and III1 for subfragment IL 2-III and on oligomers IVa and IVb etc. through IVi and IVj for subfragment IL 2-IV.

The three oligonucleotide pairs for the gene fragment IL 2-1, the five oligonucleotide pairs for the gene fragments IL 2-II and IL 2-IV, and the six oglionucleotide pairs for the gene fragment IL 2-III, which are thus obtained, are ligated in each case as follows: The double-stranded nucleotides are combined and ligated in each case over the course of 16 hours in 40 μΐ of 50 mM tris HCl buffer, 20 mM magnesium chloride and 10 mM DTT using 100 units of T4 DNA ligase at 15*C.

Gene fragments IL 2-1 to IL 2-IV are purified by gel electrophoresis on a 10% polyacrylamide gel (without addition of urea, 20 x 40 cm, 1 mm thick), the marker substance used being 0X 174 DNA (supplied by BRL) cut with Hinf I, or pBR 322 cut with Hae III , 3. Preparation of hybrid plasmids which contain gene fragments IL 2-1 to IL 2-IV a) Incorporation of gene fragment IL 2-1 in pUC 12 Plasmid pUC 12 corresponds to plasmid pUC 8 (Vieira et al., Gene 19 (1982) 259-268; Messing et al., ibid. 269-276), but contains a somewhat larger polylinker with the additional restriction sites for the enzymes Xbal and Sacl (Norrander et al., Gene 26 (1983) 101-106). The plasmid is incubated with the enzymes EcoRI and Pstl. By this means, a polynucleotide about 40 base pairs in size is cut out of the plasmid. The removal of this fragment from the opened plasmid is carried out either by chromatography on *SEPHADEX G50 or by electrophoresis on 1.5% agarose in a known manner (Maniatis). It is also possible to dispense with removal of the fragment since plasmids * Trade Mark il into which IL 2-1 has been inserted can readily be recognized by plating out (as is detailed below): The fragment IL 2-1 is inserted enzymatically into the opened plasmid as follows, the plasmid p 145/3 (Figure 1) being formed: The DNA is ligated in a mixture of 50 mM tris (pH 7.6), 5mM ATP, 5 mM dithiothreitol (DTT), 5 mM MgCl2 and about 100 units of T4 DNA ligase at 12’C for 16 hours. The plasmid is then transformed into E. coli K 12 (JM 103) made competent with 70 mM CaCl2.

Bacteria which contain the plasmid p 145/3 can be detected as white colonies on agar plates containing 50 ^g/ml ampicillin, 1 mM isopropyl thiogalactoside (IPTG) and 2% 5-bromo-4-chloro-3-indolyl 0-D-galactoside (X-Gal). Any unchanged or incompletely cut plasmid pUC 12 gives rise to blue bacterial colonies. About five of the white bacterial clones are cultivated, and the plasmids they contain are isolated in a known manner (Maniatis).

The size of the insert is checked by incubation with the restriction enzymes Pstl and EcoRi followed by electrophoresis on 10% polyacrylamide gels. This is followed by sequencing of the insert by the method of Maxam and Gilbert (Methods Enzymol. 65 (1980) 499) or Sanger and Coulson (J. Mol. Biol. 94 (1975) 441). b) Incorporation of the gene fragment IL 2-II in pUC 12 In analogy to a), the plasmid pUC 12 is reacted with the restriction enzymes Pst I and Xba I and, where appropriate after removal of the oligonucleotide which has been produced, the fragment IL 2-II is incorporated enzymaticlly. The plasmid p 147/1 (Figure 2) is produced. c) Incorporation of the gene fragment IL 2-III in pUC 12 -Λ.

In analogy to a), the plasmid pUC 12 is reacted with the restriction enzymes Xba I and Sac 1, and the fragment IL 2-III is incorporated enzymatically. The plasmid 138/25 (Figure 3) is produced. d) Incorporation of the gene fragment IL 2-IV in pUC 12 In analogy to a), the plasmid pUC 12 is cut with the restriction enzymes Sac I and Sal I, and the fragment IL 2-IV is incorporated enzymatically. The plasmid p 143/1 (Figure 4) is produced. 4. Linkage of the gene fragments After propagation of the plasmids p 145/3, p 147/1, p 138/25 and p 143/1 and confirmation of the sequence, the subfragments IL 2-1 to IL 2-IV are again cut out with the appropriate restriction enzymes and separated by electrophoresis on polyacrylamide. After immersion of the gels in an aqueous solution of ethidium bromide, the bands are identified under UV light, cut out and the DNA is eluted in a known manner (Maniatis).

The subfragments IL 2-1 to IL 2-IV are linked enzymatically as described under 3 a) and are incorporated into plasmid pUC 12 which has been opened by reaction with the restriction enzymes Eco RI and Sal I. The hybrid plasmid p 159/6 (Figure 5) is obtained, and its sequence is again confirmed by analysis.

. Construction of hybrid plasmids for the expression of DNA sequence I a) Incorporation in pKK 177.3 The expression plasmid pKK 177.3 (plasmid ptac 11, Amman et al., Gene 25 (1983) 167, in which a sequence, which contains a Sal I restriction site, has been incorporated synthetically into the Eco RI recognition site) is opened using the restriction enzymes Eco RI and Sal I. DNA sequence I is cut out of the plasmid p 159/6 (Figure 5) using the restriction enzymes Eco RI and Sal I and is applied to polyacrylamide or 2% low-melting agarose, is separated from the plasmid DNA, and the insert is recovered (Maniatis). A hybrid plasmid, in which an expression or regulation region is located upstream of the insert, is produced by ligation of the plasmid pKK 177.3, which has been cut open, with DNA sequence I. After addition of a suitable inducer, such as IPTG, a mRNA is formed, and this leads to expression of the polypeptide corresponding to DNA sequence I. b) Incorporation in p trp Hl The expression plasmid p trp Hl (Amann et al., Gene 25 (1983) 167-178) contains the control elements of the Trp operon (promoter and operator) followed by a Hind III restriction site (Fig. 6).

After isolation of DNA sequence I from p 159/6, the protruding ends are degraded using mung bean nuclease in accordance with the manufacturer's data (PL Biochemicals). p trp Hl is opened with Hind III, and the protruding ends are likewise removed with mung bean nuclease. DNA sequence I is then incorporated bluntended into the opened plasmid using T 4 DNA ligase (Fig. 6).

The starting point for protein synthesis is established by the triplet ATG for methionine at position 0. Expression is induced by the absence of tryptophan and/or the presence of indolylacetic acid. 6. Preparation of modifications a) Curtailments at the C-terminal end of the protein.

To prepare curtailed protein molecules having the biological activity of IL-2, the plasmid p 159/6 was reacted with the restriction enzyme Sal I and then incubated in a manner known per se with exonuclease III and S 1 nuclease. After reaction with Eco RI, part sequences of IL-2 were obtained, and these carry at one end the overlapping sequences for Eco Rl and at the other end are blunt ended. This curtailed DNA sequence can be incorporated into the same expression vectors after addition of a chemically synthesized DNA which is blunt-ended at one end, and has a stop codon here as the first codon, but carries at the other end the protrusion of a Sal I sequence. b) Curtailments at the N-terminal end of the protein.

To construct a hybrid plasmid which contains the DNA sequence for the expression of de-[Ala1]-IL-2, DNA sequence I, including the polylinker part of the plasmid, is cut out of the plasmid p 159/6 by methods known per se, using the restriction enzymes Eco RI and Hind III, is separated by electrophoresis from remaining plasmid and is then cut with the restriction enzyme Aha II. The isolated Aha Il-Hind III fragment is made blunt-ended as described in Example 5 b) and then cut with Sal I. Using the following adaptor ' AA TTC ATG 3' 3' G TAC 5' and after ligation with the plasmid pKK 177.3 which has been opened with Eco RI and Sal I, a new hybrid plasmid which contains the gene for the production of de-fAla1]IL-2 is obtained. 7. Transformation of the hybrid plasmids Competent E. coli cells are transformed with 0.1 to 1 μ$ of the hybrid plasmids which contain sequence I or derivatives thereof, and are plated onto agar plates containing ampiciliin. Subsequently, it is possible to identify clones which contain the correctly integrated IL-2 gene sequence, or derivatives thereof, in the appropriate plasmids by rapid DNA work-up (Maniatis loc. cit.). 8. Expression of the polypeptides exhibiting IL-2 activity Following transformation of the hybrid plasmids with DNA sequence I or derivatives thereof in E. coli, there is expression of a polypeptide which, apart from the IL-2 amino acid sequence or modified sequences, carries an additional methionyl group on the amino end. 9. Working up and purification The bacterial strains which have been cultivated to the desired optical density are incubated with a suitable inducer, for example IPTG, for an adequate time, for example 2-4 hours. The cells are then killed using 0.1% cresol and 0.1 mM benzylsulfonyl fluoride. After the induction, a protein band with a molecular weight of about 15,000 daltons is found on a SDS-PAA gel. Following centrifugation or filtration, the cell aggregate is taken up in a buffer solution (50 mM tris, 50 mM EDTA, pH 7.5) and disrupted mechanically, for example using a French press or *DYNO mill (supplied by Willy Bachofer, Basel), whereupon the insoluble constituents are removed by centrifugation.

After disruption of the cells, part of the IL-2 protein remains in the residue and can be solubilized using 8M urea, 6 M guanidinium hydrochloride, buffer solutions containing ionic or non-ionic detergents and similar solvents. The protein containing IL-2 activity is purified from these solutions by customary processes. Ion exchanger, adsorption or gel filtration columns, or affinity chromatography on antibody columns, are suitable. The enrichment and purity of the product are * Trade Mark checked by sodium dodecyl sulfate/acrylamide gel or HPLC analysis.

T cells which are strictly dependent on the presence of IL-2 in the medium are used for the biological characterization of the IL-2 protein. The rate of cell division of cell lines of this type is, within certain limits, proportional to the IL-2 concentration in the medium, which can thus be determined from the uptake of 3H-thymidine from the medium.

Claims (15)

1. DNA sequence I with the nucleotides 9 to 407 (amino acids 1 to 133) shown in Table 1, coding for a protein with the biological activity of human interleukin-2 (IL-2).

2. DNA sequence I with the nucleotides 6 to 407 (amino acids 0 to 133), shown in Table 1, followed by one or two stop codon(s), coding for a protein with the biological activity of human interleukin-2 (IL-2).

3. DNA sequences Ila (IL 2-1), lib (IL 2-II) , He (IL 2-III) and lid (IL 2-IV) shown in Table 2.

4. DNA oligonucleotides Ia to IVj shown in Table 2.

5. Hybrid plasmids which contain between an Eco RI and a Pst I cleavage site the DNA sequence Ila (IL 2-1) or between a Pst I and an Xba I cleavage site the DNA sequence lib (IL 2-II) or between an Xba I and a Sac I cleavage site the DNA sequence lie (IL 2III) or between a Sac I cleavage site and a Sal I cleavage site the DNA sequence lid (IL 2-IV) shown in Table 2.

6. Hybrid plasmids which contain between an Eco RI and a Sal-I cleavage site the DNA sequence I shown in Table 1.

7. Hybrid plasmids which contain the DNA sequence I (nucleotides 6-413) shown in Table 1.

8. Host cells, in particular E. coli, which contain hybrid plasmids as claimed in claim 5 to 7.

9. A genetic engineering process for the preparation of IL-2, which comprises employing a synthetic gene which contains the DNA sequence I (nucleotides 9 to 407 corresponding to amino acids 1 to 133) shown in Table 1.

10. The use of the DNA sequences shown in Tables 1 and 5 2 for preparing modified DNA sequences.

11. . A DNA sequence according to any one of claims 1 to 3, substantially as hereinbefore described and exemplified.

12. A DNA oligonucleotide according to claim 4, sub10 stantially as hereinbefore described and exemplified

13. A hybrid plasmid according to any one of claims 5 to 7, substantially as hereinbefore described and exemplified.

14. A process according to claim 9, substantially as 15. Hereinbefore described and exemplified.

15. IL-2 whenever prepared by a process claimed in a preceding claim.