IE881146L

IE881146L - Bifunctional proteins

Info

Publication number: IE881146L
Application number: IE881146A
Authority: IE
Inventors: Paul Habermann
Original assignee: Timothy Gerard Vaughan
Priority date: 1987-04-16
Filing date: 1988-04-15
Publication date: 1988-10-16
Also published as: GR3006141T3; ATE79135T1; AU1466188A; NZ224247A; HU204303B; NO881658D0; DE3712985A1; KR880012760A; ZA882659B; DK209188D0; AU613022B2; AR242991A1; FI98830C; IL86086A; FI881743A0; NO176922C; HUT47319A; KR970000187B1; FI881743L; DE3873397D1

Abstract

Bifunctional proteins obtainable by genetic engineering from an interleukin-2 and a granulocyte-macrophage "colony stimulating" factor fraction have the biological activity of both components, but are distinguished by increased stability. These proteins are thus medicaments which are suitable for the treatment of malignant neoplasms. [EP0288809A1]

Description

615 7 4 - i - Interleukin-2, called IL-2 hereinafter, acts as T-cell growth factor. IL-2 potentiates the activity of killer cells such as NK (natural killer) cells, cytotoxic T-cells and LAK (lymphokine-activated killer) cells. 5 By contrast,? granulocyte macrophage colony stimulating factor, called GM-C3F hereinafter, stimulates the formation of granulocytes and macrophages from hemopoietic precursor cells. Combination of the two biological activities is of interest for tumor treatment with and. 10 without administration of cytostatics - However, the stabilities of IL~2 and GM-CSF differ, whicli may result in problems on direct administration of the two components and thus in a decrease in the therapeutic success. 15 The problem of the difference in stability can be solved according to the invention by linking these two proteins to a bifunctional protein.

Fusion proteins of the general formula Met - X - Y - Z or Met - 2 - Y - X 20 (la) (lb) have already been proposed for the preparation, by genetic manipulation, of optionally modified GM-CSF in which X essentially denotes the amino acid sequence of approximately the first 100 amino acids of, preferably,. 25 human IL-2, Y denotes a direct linkage if the amino acid or amino acid sequence adjacent to the desired protein allows the desired protein to be cleaved off, or otherwise denotes a bridging member which is composed of on® or .more genetically encodable amino acids and which 30 allows the cleavage off, and Z is a sequence which is composed of genetically encodable amino acids and which represents the desired GM-CSF protein. It is also possible during this to make use - more or less - up to the - 2 - end of the DMA sequence coding for IL~2, and thus generate biologically active IL-2 - modified where appropriate - as a "by-product.** (not prior-published European Patent Application with the publication number (SP-A) 0,228,018 5 and South African Patent 86/9557).

In contrast to the earlier proposal, the invention relates not to the use of the proteins as intermediate but to the use in methods for the therapeutic treatment o£ the human body and to medicaments which contain fusion 10 proteins of this type or which are composed of fusion proteins of this type. A further aspect of the invention relates to the use of these fusion proteins for the preparation of a medicament for the treatment of malignant neoplasms. 15 The fusion protein used according to the invention is thus composed of two biologically active components, namely of an IL-2 constituents which can be modified in a manner known per se, on the one hand, and of a GM-CSF constituent, which can likewise be modified,,, on the other 20 hand and., where appropriate,, of a bridging member corresponding to the definition Y in the formulae given above. The arrangement of the two components preferably corresponds to the formula la. The principle according to the invention can also be used for the preparation of 25 other novel bifunctional proteins.

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

Modifications of the IL-2 molecule have been disclosed, 30 reference being made here only to EP-A 0,091,539, 0,109,748, 0,118,617, 0,,136,489 and 0,163,249 by way of example - - 3 - Furthermore the not prior-published EP-A 0t, 219 # 839 proposes an IL-2 derivative in which the first seven N-tennin&l amino acids are deleted.

Modifications of the GH-CSF raolecule have been proposed 5 in EP-A 0,228/018.

Further alterations to the two active constituents of the molecule can be carried out in a manner known per se, mention being made here only of specific mutagenesis by way of example. 10 The bridging member Y advantageously has the formula XI - Asp - (aa)_ - Pro - (II) in which x denotes an integer up to about 20, and aa denotes any desired genetically encodable amino acid with the exception of cysteine. 15 It is advantageous in the formula II for the IL-2 constituent to be arranged at the left-hand end, and consequently the GM-CSF constituent to be arranged at the right-hand end.

Particularly preferred embodiments of Y have the amino 20 acid sequence -Asp-Pro-Met-Ile-Thr-Thr~Tyr-Ala-Asp~Asp-Pro- or -Aap-Pro-Met-1 le-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr ~ lie-Asp-Asp-Pro-it again being preferable tor the IL-2 constituent to be 25 arranged at the left-hand end and the GM-CSF constituent to be arranged at the right-hand end.

The bifunctional proteins according to the invention can be expressed in a manner known per' se. It is possible isa bacterial expression systems for the route of direct 30 expression to be followed. Suitable for this purpose are all known host-vector systems with hosts such as bacteria of the species Streptomyces, 3. subtilis, Salmonella - 4 - typhimurium or Serratia marcescens, especially E. coli.

The DMA sequence which codes for the desired protein is incorporated in a know, manner Into a vector which ensures satisfactory expression in the chosen expression 5 system.

It is expedient to choose for this purpose the promoter and operator from the group trp, lac, tac,, P^ or Pa of phage A, hsp, omp or a synthetic promoter, as described in, for example, German Offenlegungsschrift 3,430,683 and 10 in EP-A 0,173,149. The tac promoter-operator sequence is advantageous and is now commercially available (for example pKK223-3 expression vector. Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, page 63). 15 On expression of the protein according to the invention, it may prove expedient to modify individual triplets for the first few amino acids after the ATG start codosi in order to prevent any base-pairing at the level of the mJRMA. Such modifications, such as deletions or additions 20 of individual amino acids, are familiar to the expert, and the invention also relates to them.

For expression in yeasts - preferably S. cerevisiae - it is expedient to use a secretion system, for example heterologous expression via the c-factor system, which 25 has been described several times.

It is advantageous for the expression of the bifunctional molecule in yeast if dibasic peptide sequences and glycosylation sites in the bifunctional protein have been destroyed by appropriate exchange of individual amino 30 acids. This results in many possible combinations which may also influence the biological action. - 5 - The expression of IL-2 in yeast is disclosed in SP-A 0,142,268, and that of GM-CSF in EP-A Q#188,350.

The administration of the bifunctional proteins according to the invention corresponds to that of the two cosa-5 ponents. However, because of the greater stability a lower dosage is possible in many cases, the dosage being In the lower part of the range of those hitherto proposed.

The invention is illustrated In detail in the examples 10 which follow. Unless indicated otherwise, percentage data and ratios relate to weight.

Example 1 The plasmid pl59/6 (EP-A2 0,163,249, Figure 5; (1) In the present figure) contains a synthetic gene coding for IL~ 15 2 between an EcoHl and a Sail cleavage site. The DNA sequence for this gene is represented in the said 2P-A2 as "DNA sequence I". A TaqI cleavage site is located in the region of triplets 127 and 128. The IL-2 part-sequence (2) is cut out of this plasmid by cutting with 20 EcoRI and TaqI, and is isolated.

The plasmid pHG23 (3) which codes for GM-CSF is disclosed in SP-A2 0,, 183350, The GM-CSP cDNA Is represented in Figure 2 in this EP-A2. The plasmid pKG23 Is obtained when the cDNA sequence Is Incorporated In the PstI 25 cleavage site of pBR322, use being made of, on the one hand, the PstI cleavage site at the 5' end and, on the other hand, a PstI site introduced at the 3* end by GC tailing. The DNA sequence (4) which contains most of the GM-CSF gene is isolated from this plasmid by cutting with 30 SfaNI and PstI.

The following oligonucleotide (5) is synthesized by the phosphite methods — 6 — 5 10 15 20 25 30 ■n ! 128 (133) 11® lie Ser Thr Leu Asp Pro Met lie ATC ATC TCT ACC CTG GAC CCG ATG ATC THUS TAG AGA TGG GAC CTG GGC TAC TAG 1 2 Thr Thr Tyr Ala Asp Asp Pro (Ala) (Pro) ACC ACC TAT GCG GAC GAT CCG CSC TGG TGG ATA CGC CTG CTA GGC CGT GGG CG (TaqI) (5) (SfaNI) The oligonucleotide (5) extends at the 5" and the DMA sequence of IL-2, there being, however, Asp in place of Thr in position 133. At the 3' end of this oligonucleotide ere located the nucleotides which have been deleted from the cDNA by cutting with SfaNI.

The preparation of the expression plasmid pEWlOOO (6) is proposed in the (not prior-published) EP-A 0,227,938 (Figure 1). This plasmid is a derivative of the plasmid pt&c II (Amann st al„,» Gene 25 (1083) 167 - 178), in which a synthetic sequence which contains a Sail cleavage site has been incorporated in the recognition site for EcoRI- The expression plasmid pXK 177.3 is obtained in this way. Insertion, of the lac repressor ^Farobaugh, Nature 274 (1978) 765 - 769) results in the plasmid pJF118. The latter is opened at the unique restriction cleavage site for Aval, and is shortened by abcmt 1000 bp in a known manner by exonuclease treatment and is ligated. The plasmid pEWlOOO (6) is obtained. Opening of this plasmid in the polylinker using the enzymes EcoHI and PstI results in the linearized expression plasmid (7).

This linearized plasmid DNA (/) is now ligated with the DNA fragment (2) which codes for the IL-2 sequence, with the synthetic oligonucleotide (5) and with the cDNA fragment (4). The result is the plasmid pB30 (8) which is ~ 7 ~ transformed into the E. coll strain MclOGl. The plasmid DMA from individual clones is isolated and characterised by restriction (analysis- c Example 2 5 If the following synthetic oligonucleotide * 128 (133) lis He Sar Thr Lau Asp Fro Net lie Thr Thr Tyr CG ATC ATC TCT ACC CTG GAC CCG ATG ATC ACC ACC TAT TAG TAG AGA TGG GAC CTG GGC TAC TAG TGG TGG ATA (TaqI) 1 2 Leu Glu Glu Leu Thr lie Asp Asp Pro (Ala) (Pro) CT k GM GAG CTC ACG ATC GAC GAT CCG GC GAT CTT CTC GAG TGC TAG CTG CTA GGC CGT GGG 15 (SfaNI) is used in place of oligonucleotide (5) in Example 1, the result is the plasmid pB3l.

Example 3 Competent cells of the B. coli strain W3110 .are trans-20 formed with the plasmid pB30 or pB31. An overnight culture of the strain is diluted in the ratio of about 1:100 with LB medium (J. H. Miller, Experiments in Molec. Gen., Cold Spring Harbor Lab., 1972), which contains 50 p,q/wl ampicillin, and the growth is followed by 25 measurement of the OD. At OD = 0.5 the culture is adjusted to a concentration ox 2 mK in isopropyl-/9-D-thiogalactopyranoside (IPTG) and, after 150 - 180 minutes, the bacteria are spun down. These bacteria are treated in a buffer mixture (7H urea, 0.1% SDS, 0.1H r- 30 sodium phosphate» pH 7.0) for about 5 minutes, and samples are applied to an SDS polyacrylamide gel electrophoresis plate. This confirms the expression of the bifunctional protein. - 8 - The stated conditions apply to shake cultures; for larger fermentations it is expedient to modify the OD values and nutrient media and vary the IPTG concentrations appropriately- 5 Example 4 E. coli W3110 cells which contain the plasmid pB30 or p331 are, after induction, spun down, resuspended in sodium phosphate buffer (jpH 7) and again spun down. The bacteria are taken up in the same buffer and then dis-10 rupted (French Press f {a)Dynomill). The disrupted cells are spun down. The supernatant and sediment are analyzed by SDS polyacrylamide gel electrophoresis as described in Example 3. Staining of the protein bands reveals that the bifunctional protein is located in the sediment from the 15 disruption. The sediment is washed several times with chaotropic buffers and finally with water, resulting in further enrichment of the desired protein. The protein concentration is then determined in the aqueous protein suspension. The suspension is now adjusted to a con-20 centration of 5 M in guanidinium hydrochloride and 2 inK in dithiothreitol (DTT). The mixture is stirred under nitrogen for about 30 minutes and then diluted with 50 wM tris buffer (pi 8.5) so that the protein concentration is 100 - It is now dialyzed against this tris buffer 25 and,, after two changes of the buffer, dialyzed against water. The protein treated in this way is sterile filtered and its biological activity is checked. It shows full biological action both in the interleukin-2-dependent CTLL 2 cell proliferation assay and in the 30 human bone marrow assay. Mixed colonies of granulocytes and macrophages are observed in these.

The bifunctional protein can be further purified by interleukin-2-spec if ic affinity chromatography- The protein is still active in both assays. In contrast, an 35 E. coli extract of the mvtrans formed strain W3110 which has been treated as described shows no activity.

Other conditions are expedient for the industrial preparation of the product, for example for the folding of the protein and its purification- Suitable purification processes - which are known per se - are ion exchange, adsorption,, gel filtration and preparative EPLC chromatography.

Claims

- 10 - Patent Claims

1. A bifunctional protein composed of a biologically active interleukin-2 (IL-2) constituent and granulocyte macrophage colony stimulating factor (GM-CSF) constituent, for use as medicament -

2. - A bifunctional protein having a biologically active IL-2 constituent and GM-CSF constituent.- wherein the two biologically active protein constituents are linked via a bridge composed of 1 to about 20 genetically encodable amino acids.

3. A protein as claimed in claim 2, wherein the bridge corresponds to the formula (II) - Asp - (ea)j - Pro ~ (II) x denoting an integer from 1 to 18 f and && being a genetically encodable aiaino acid with the exception of Cys.

4. A protein as claimed in claim 3, wherein the bridging member (aa)K denotes the amino acid sequence

5. -Pro-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Asp- or

6. -Pro-Met-Ile~Thr-Thr-Tyr~Leu~Glu-Glu-Leu-Thr-Ile-Asp-Asp-. 5 - A protein as claimed in one or more of claims 2 to 4, wherein the IL-2 constituent is arranged N-terminal and the GM-CSF constituent is arranged C-terminal. 6- A process for the preparation of bifunctional proteins as claimed in any of claims 1 to 5, which comprises construction,? and expression in a host cell, of a gene coding for these proteins.

7. A medicament composed of a protein as claimed in any of claims 1 to 5, where appropriate combined with a pharmacologically suitable vehicle.,

8. Th© use of a protein as claimed in any of claims 1 to 5 for the preparation of a medicament for the treatment of malignant neoplasms.

9. A bifunctional protein composed of a biologically active interleukin-2 (IL-2) constituent and a granulocyte macrophage colony stimulating factor (GM-CSF) constituent, for use as a medicament, substantially as hereinbefore described.

10. A bifunctional protein according to Claim 2, substantially as hereinbefore described.

11. h process according to Claim 6 for the preparation of bifunctional proteins, substantially as hereinbefore described and exemplified-

12. h bifunctional protein whenever prepared by a process claimed in Claim 6 or 11.

13. A medicament according to Claim 7, substantially as hereinbefore described.

14. use according to Claim 8, substantially as hereinbefore described. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.