NO176922B - Process for the preparation of bifunctional proteins - Google Patents

Description

The present invention relates to a method for the production of "bifunctional proteins.

More specifically, the invention relates to the production of such proteins which consist of an active interleukin-2 and granulocyte-macrophage-colony-stimulating-factor (GM-CSF) part, the two biologically active protein parts being linked together via a bridge of 1 to approx. 20 genetically codeable amino acids.

Interleukin-2, hereinafter referred to as IL-2, acts as a T-cell growth factor. IL-2 enhances the activity of "Killer" cells such as NK (Natural Killer) cells, cytotoxic T cells and LAK (Lymphokine Activated Killer) cells.

Granulocyte-macrophage "Colony Stimulating" factor, hereinafter referred to as GM-CSF, on the other hand stimulates granulocyte and macrophage formation from hematopoietic precursor cells. The combination of the two biological activities is interesting for tumor treatment with and without cytostatic administration. However, IL-2 and GM-CSF are differently stable, which can lead to problems with the direct administration of the two components and thus to a reduction in the therapeutic result.

According to the invention, the problem of the different stability can be solved by linking these two proteins together to form a bifunctional protein.

Fusion proteins of the general formula Ia or Ib have already been proposed

for the genetic engineering production of possibly modified GM-CSF, in which X essentially means the amino acid sequence of the first approx. 100 amino acids and preferably human IL-2, Y means

a direct bond, if the amino acid or amino acid series located adjacent to the desired protein enables cleavage of the desired protein, or else a bridge link of one or more genetically coded amino acids that enables the cleavage and Z is a sequence of genetically coded amino acids, which is the desired GM-CSF protein. In this way, the DNA sequence coding for IL-2 can be utilized to a greater or lesser extent and thereby produce biologically active, possibly modified, IL-2 as a "by-product" (EP-A 0 228 018 resp. SA-86 /9557).

The present invention aims to improve the known technique and thereby relates to a method of the kind mentioned at the outset, and this method is characterized by combining a DNA sequence that codes for IL-2, a DNA sequence that codes for GM- CSF and a DNA sequence encoding the bridge, in an expression vector, transform a host cell with this and express and isolate the corresponding bifunctional protein.

The fusion proteins produced according to the invention thus consist of two biologically active components, namely on the one hand an IL-2 part which can be modified in a manner known per se, on the other hand, a GM-CSF part which likewise can be modified and possibly a bridge link corresponding to the definition Y in the above stated formulas. Preferably, the arrangement of the two components corresponds to formula (Ia). However, the principle according to the invention can also serve to produce other new bifunctional proteins.

The figure shows the construction of the plasmid pB30, which codes for a bifunctional protein produced according to the invention.

Modifications of the IL-2 molecule are known, but here only reference should be made to EP-A 0 091 539, 0109 748, 0 118 617, 0 136 489 and 0 163 249.

Furthermore, in the not previously published EP-A 0 219 839, an IL-2 derivative was proposed in which the N-terminal first seven amino acids have been deleted.

Modifications of the GM-CSF molecule were proposed in EP-A 0 228 018.

Further modifications of the two active molecular parts can be carried out in a manner known per se, as only the specific mutagenesis should be mentioned here as an example.

The bridge link Y advantageously has formula II,

where x means an integer up to 20, and AS means a desirable genetically codeable amino acid with the exception of cysteine.

Preferably, in formula (II) the IL-2 part is arranged at the left end and consequently the GM-CSF part at the right end.

Particularly preferred forms of Y have the amino acid sequence -Asp-Pro-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Asp-Pro- or -Asp-Pro-Met-Ile-Thr-Thr-Tyr-Leu-Glu- Glu-Leu-Thr-Ile-Asp-Asp-Pro-

the IL-2 part being likewise preferably arranged at the left end, and the GM-CSF part at the right end.

The expression of the bifunctional proteins produced according to the invention can take place in a manner known per se. In bacterial expression systems, direct expression can be used. All known host-vector systems with bacteria of the type Streptomyces, B.subtilis, Salmonella tuphymurium or Serratia marcenscens, especially E.coli as hosts, are suitable for this.

The DNA sequence which codes for the desired protein is incorporated in a manner known per se into a vector which, in the chosen expression system, ensures good expression.

In this way, the promoter and the operator are appropriately selected from the group trp. lac, tac, P1 or Pg of the phage \, hsp, omp or a synthetic promoter, as they are for example described in DE-OS 34 30 683 resp. EP-A 0 173 149. Advantageous is the tac-promoter-operator sequence which is meanwhile commercially available (e.g. expression vector pKK223-3, Pharmacia, "Molecular Biology, Chemicals and Equipment for molecular Biology", 1984, p . 63).

When expressing the protein according to the invention, it may prove appropriate to change certain triplets of the first amino acids after the ATG start codon to prevent possible base pairing at the level of mRNA. Such changes as deletions or additions of certain amino acids are common to those skilled in the art, and are likewise covered by the invention.

For expression in yeast - preferably S. cerevisiae - a secretion system is suitably used - for example the heterologous expression via the α-factor system which has been described several times.

For the expression of the bifunctional molecule in yeast, it is advantageous when in the bifunctional protein the basic peptide sequences and glycosylation sites are destroyed by corresponding exchange of individual amino acids. This creates many combination possibilities that can also affect the biological effect.

The expression of IL-2 in yeast is known from EP-A 0 142 568, of GM-CSF from EP-A 0 188 350.

The administration of the bifunctional proteins produced according to the invention corresponds to this of the two components. Due to the greater stability, however, it is possible in many cases with a smaller dosage that stays in the lower range of the hitherto proposed dosages.

The invention will be explained in more detail with the help of some examples. Percentages and ratios are calculated by weight if nothing else is stated.

Example 1.

The plasmid p159/6 (EP-A2 0 163 249, fig. 5; (1) in the present figure contains, between an EcoRI and a SalI cut site, a synthetic IL-2 coding gene. The DNA sequence for this gene is reproduced in said EP-A2 as "DNA sequence I". In the area of triplets 127 and 128, there is a Taql cut site. From this plasmid, the IL-2 subsequence (2) is excised using EcoRI and Taql and isolates it.

From EP-A2 0 183 350 the plasmid pEG23 (3) is known, which codes for GM-CSF. The GM-CSF cDNA is shown in fig. 2 of this EP-A2. Plasmid pHG23 is obtained when the cDNA sequence is incorporated into the Pstl cut site of pBR322, using on the one hand the Pstl cut site at the 5' end, and on the other hand a Pstl site at the 3' end. From this plasmid, the DNA sequence (4) containing the largest part of the CM-CSF gene is isolated by cutting with SfaNI and Pstl.

Following the phosphite method, the following oligonucleotide (5) is synthesized:

The oligonucleotide (5) supplements the DNA sequence of IL-2 at the 5' end, whereby however Asp. stands instead of Thr in position 133. At the 3' end of this oligonucleotide are found the nucleotides that have been lost by cutting with SfaNI from the cDNA.

The preparation of the expression plasmid pEW1000 (6) is proposed in (not previously published) EP-A 0 227 938 (Fig. 1). This plasmid is a derivative of the plasmid ptac 11 (Amann et al., "Gene" 25 (1983) 167-178), where a synthetic sequence containing a SalI cut site was built into the recognition site for EcoRI. The expression plasmid pKK 177.3 is thus obtained. By inserting the lac repressor (Farabaugh, "Nature" 274 (1978) 765 - 769), the plasmid pJF 118 is obtained. This is opened at the singular restriction site for Aval and shortened in a known manner with exonuclease treatment around approx. 100o bp and ligated. The plasmid pEWlOOO (6) is obtained. By opening this plasmid in polylinker with the enzymes EcoRI and PstI, the linearized expression plasmid (7) is obtained.

This linearized plasmid DNA (7) is now ligated with the DNA fragment (2) that codes for the IL-2 sequence, with synthetic oligonucleotide (5) and with the cDNA fragment (4). The plasmid pB30 (8) is obtained, which is transformed in the E.coli strain Mcl061. Plasmid DNA of individual clones is isolated and characterized by restriction analysis.

Example 2.

In example 1, instead of the oligonucleotide (5), the following synthetic oligonucleotide is used

then you get the plasmid pB31.

Example 3.

Competent cells of E.coli strain W3110 are transformed with plasmid pB30 or pB31. An overnight culture of the strain is diluted with LB medium (J.H. Miller, "Esperiments in Molec-Gen.", Cold Spring Harbor Lab., 1972), which contains 50 pg/ml ampicillin in a ratio of approx. 1:100, and the growth is followed by OD measurement. At OD = 0.5, the culture is adjusted to a concentration of 2mM isopropyl-p<->D-thiogalactopyranoside (IPTG) and the bacteria are centrifuged off after 150-180 minutes. The bacteria are treated approx. 5 min in a buffer mixture (7M, urea, 0.1% SDS, 0, IM sodium phosphate, pH 7.0), and samples are brought onto an SDS gel electrophoresis plate. The expression of the bifunctional protein is thus confirmed.

The specified conditions apply to shaking cultures for larger fermentations, where the OD value, nutrient medium and varied IPTG concentrations are suitably changed accordingly.

Example 4.

After induction, E.coli W3110 cells containing the plasmid pB30 or pB31 are filtered out, these are resuspended in sodium phosphate buffer pH (7), and centrifuged again. The bacteria are taken up in the same buffer, and then digested (French Press, "Dyno-Miihle"). The cell digest is centrifuged and the supernatant and sediment are analyzed using SDS-polyacrylamide electrophoresis as described in example 3. After staining the protein band, it turns out that the bifunctional protein is found in the sediment of the digest. The sediment is washed several times with chaotropic buffers and finally with water, as the desired protein is further enriched. In the aqueous protein suspension, the protein concentration is then determined. The suspension is now adjusted to a concentration of 5M guanidinium hydrochloride and 2 mM dithiothreitol (DTT). The mixture is stirred for approx. 30 minutes under nitrogen, and then diluted with 50 mM tris buffer (pH 8.5) so that the protein concentration is 100 µg/ml. You then dialyze against this tris buffer, and after changing the buffer twice, you dialyze against water. The thus treated protein is sterile filtered and examined for its biological activity. It shows full biological effect both in the interleukin-2-dependent CTLL-2 cell proliferation test and also in the human bone marrow test. Mixed colonies of granulocytes and macrophages are thereby observed.

The bifunctional protein can be further purified by interleukin-2-specific affinity chromatography. The protein is also then active in both samples. An E.coli extract of the non-transformed strain W3110 which was treated as discussed, however, shows no activity.

For the technical preparation of the product, other conditions are appropriate, for example for holding protein and its purification. As purification methods known per se, ion exchange and adsorption, gel filtration and preparative HPLC chromatography can be used.

Claims (4)

1. Process for producing bifunctional proteins consisting of an active interleukin-2 and granulocyte-macrophage-colony-stimulating-factor (GM-CSF) part, the two biologically active protein parts being linked together via a bridge of 1 to approx. 20 genetically codeable amino acids, characterized in that a DNA sequence that codes for IL-2, a DNA sequence that codes for GM-CSF and a DNA sequence that codes for the bridge are joined in an expression vector, transforms a host cell with this and experiments and isolates the corresponding bifunctional protein. 2. Method according to claim 1, characterized in that the bridge has the formula where x means an integer from 1-18, and As means a genetically codeable amino acid with the exception of Cys. 3. Method according to claim 2, characterized in that the bridge link (As)x stands for the amino acid sequence or 4. Method according to any one of claims 2 to 4, characterized in that the IL-2 part is arranged N-terminally and the GM-CSF part is arranged C-terminally.