IE61574B1 - Bifunctional proteins - Google Patents

Bifunctional proteins

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Publication number
IE61574B1
IE61574B1 IE114688A IE114688A IE61574B1 IE 61574 B1 IE61574 B1 IE 61574B1 IE 114688 A IE114688 A IE 114688A IE 114688 A IE114688 A IE 114688A IE 61574 B1 IE61574 B1 IE 61574B1
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IE
Ireland
Prior art keywords
protein
constituent
bifunctional
thr
medicament
Prior art date
Application number
IE114688A
Other versions
IE881146L (en
Inventor
Paul Habermann
Original Assignee
Hoechst Ag
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Publication date
Application filed by Hoechst Ag filed Critical Hoechst Ag
Publication of IE881146L publication Critical patent/IE881146L/en
Publication of IE61574B1 publication Critical patent/IE61574B1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

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.

Description

Interleukin-2, called 11,-2 hereinafter, acts as T-cell growth factor. 11,-2 potentiates the activity of killer cells such as NS (natural killer) cells, cytotoxic Tcells and LAS (lymphokine-activated killer) cells.
By contrast, granulocyte macrophage colony stimulating factor, called GM-CSF 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. without administration of cytostatics. However, the stabilities of IL-2 and GM-CSF differ, which may result in problems on direct administration of the two components and thus in a decrease in the therapeutic success.
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 - 2 or Met - 2 - ¥ - X (la) (lb) have already been proposed for the preparation, by genetic manipulation, of optionally modified GM-CSF in which £ essentially denotes the amino aeid sequence ox approximately the first 100 amino acids of, preferably, human IL-2, ϊ denotes a direct linkage if the amino acid or amino acid sequence adjacent to the desired protein allows the desired protein to b® cleaved off, or otherwise denotes & bridging member which is composed of one or more genetically encodable amino acids and which allows th© cleavage off, and S is e 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 th® end of the DNA 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 (EP-A) 0,228,018 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 of th® human body and to medicaments which contain fusion 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» The fusion protein used according to the invention is thus composed of two biologically active components, namely of an IL-2 constituent, which can be modified in a manner known per se, on the one hand, and of a Oi-CSF constituent,, which can likewise be modified, on the other hand and, where appropriate, of a bridging member corresponding to the definition. Z in the formulae given above. The arrangement of the two components preferably corresponds to the formula la. The principle according to th® invention can also be used for th® preparation of other novel bifunctional proteins.
The figure shows the construction of the plasmid p330 which codes for a bifunctional protein according to the invention.
Modifications of the IL-2 molecule have been disclosed, 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.
Furthermore , the not prior-published EP-A 0,219,839 proposes an IL-2 derivative in which the first seven Nterminal amino acids are deleted.
Modifications of the GM-CSF molecule have been proposed 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.
Th® bridging member Y advantageously has the formula II - 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.
It is advantageous in th® 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 th® right-hand end.
Particularly preferred embodiments of Y have the amino acid sequence -Asp-Pro-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Asp-Pro- or -Asp-Pro-Het-1 le-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-ThrIle-Asp-Asp-Proit again being preferable for th© IL-2 constituent to be arranged at the left-hand end and the GM-CSP constituent to b® arranged at the right-hand end.
The bifunctional proteins according to the invention can be expressed In a manner known peruse. It is possible in bacterial expression systems for th® route of direct 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 Serratla marcescens, especially Ξ. coli.
The DNA sequence which codes for the desired protein is Incorporated In a known manner Into a vector which ensures satisfactory expression in the chosen expression system.
It is expedient to choose for this purpose the promoter and operator from the group trp, lac, tac, PL or Pa of phage A, hsp, omp or a synthetic promoter, as described in, for example, German Of fenlegungsschrift 3,430,683 and in EP-A 0,173,149. Th® 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 S3).
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 codon in order to prevent any base-pairing at the level of the mRNA. Such modifications, such as deletions or additions 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 e-factor system, which 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 acids. This results in many possible combinations which may also influence the biological action.
The expression of IL-2 in yeast is disclosed in EP-A 0,142,268, and that of GM-CSF in EP-A 0,188,350.
The administration of the bi functional proteins according to the invention corresponds to that ox the two components. 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 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 IL2 between an EcoRI and a Sail cleavage site. The DNA sequence for this gene is represented in the said EP-A2 as DNA sequence I. A TaqI cleavage site is located in the region of triplets 127 and 128. The IL-2 partsequence (2) icS cut out of this plasmid by cutting with EcoRI and TaqI, and is Isolated.
The plasmid pHG23 (3) which codes for GM-CSF is disclosed in EP-A2 0,183,350. Th© GM-CSP cDNA Is represented In Figure 2 in this EP-A2. The plasmid pHG23 Is obtained when the cDNA sequence is incorporated in the Pstl cleavage site of pBR322, use being made of, on the one hand, th® Pstl cleavage site at the 5' end and, on the other hand, a Pstl site introduced at the 3* end fey 'SC tailing. Th® DMA sequence (4) which contains most of the GM-CSF gen® is isolated from this plasmid by cutting with Sf&MI and Pstl.
The following oligonucleotide (5) is synthesised by the phosphite methods 128 Leu (133) Asp Pro Met Ils lie Ile Ser Thr CG ATC ATC TCT ACC CTG GAC CCG ATG ATC TAG TAG AGA TGG GAC CTG GGC TAC TAG (TaqI) 1 2 Thr Thr Tyr Ala Asp Asp Pro (Ala) (Pro) ACC ACC TAT GCG GAC GAT CCG (SC TGG TGG ATA CGC CTG CTA GGC CGT GGG (SfaNI) The oligonucleotide (5) extends at the 5" end the DNA 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 th© cDNA by cutting with SfaNI.
The preparation of the expression plasmid pSWIOOO (6) is proposed in the (not prior-published) EP-A 0,227,938 (Figure 1). This plasmid Is a derivative of the plasmid ptac 11 (Jtean ©t al.,» Gene 25 (1983) 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 pKK 177.3 is obtained in this way. Insertion of the lac repressor (Farabaugh, Nature 274 (1978) 765 - 769) results in the plasmid pJP118. The latter Is opened at the unique restriction cleavage site for Aval,, and is shortened by about 1000 bp in a known manner by exonuclease treatment and is ligated. The plasmid pSW'1000 (6) is obtained. Opening of this plasmid in the polylinker using the enzymes BcoRI and Pstl results in the linearised expression plasmid (7).
This linearized plasmid DNA (7) 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 p330 (8) which is trans formed into the E. coli strain He 1061. Th® plasmid DNA from individual clones is isolated and characterised by restriction analysis.
Example 2 If th© following synthetic oligonucleotide 128 (133) Thr ACC TGG Thr ACC TGG Tyr TAT ATA Ils CG ATC TAG lie ATC TAG Ser TCT AGA Thr ACC TGG Leu CTG GAC Asp GAC CTG Pro CCG GGC Het ATG TAC lie ATC TAG 10 (TaqI) 1 2 Leu Glu Glu Leu Thr lie Asp Asp Pro (Ala) (Pro) CTA GAA 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 Sxample 1, the result is the plasmid pB3l.
Example 3 Competent cells of the E. coli strain W3110 are trans20 formed with the plasmid pS30 or pB31. An overnight culture of the strain is diluted in the ratio of about 1:100 with L3 medium (J. H. Miller, Experiments in Molec.
Gen., Cold Spring Harbor Lab., 1972), which contains 50 ptg/ml ampicillin, and the growth is followed by measurement of the OD. Ax GD = 0.5 the culture is adjusted to a. concentration of 2 mM in isopropyl-0-Dthiogalactopyranoside (IPTG) and, after 150 - 180 minutes, the bacteria are soun down. These bacteria are treated In a buffer mixture (7H urea, 0.1% SDS, Θ.1Μ r 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.
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.
Example 4 E. coli W3110 cells which contain the plasmid pB30 or pB31 are, after induction, spun down, resuspended in sodium phosphate buffer (pH 7) and again spun down. The bacteria are taken up in the same buffer and then disrupted (French Press, 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 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 concentration of S M in guanidinium hydrochloride and 2 mM in dithiothreitol (DTT). The mixture is stirred under nitrogen for about 30 minutes and then diluted with 50 mM tris buffer (pH 8.5) so that the protein concentration is 100 pg/ml. It is now dialyzed against this tris buffer and, after two changes of the buffer, dialysed against water. Th® protein treated in this way is sterile filtered and its biological activity is checked. It shows full biological action both In the interleukin-2dependent CTLL 2 cell proliferation assay and in the hnman bone marrow assay. Mixed colonies of granulocytes and macrophages are observed in these.
The bifunctional protein can be further purified by inter leukin-2-specific affinity chromatography. The protein is still active in both assays. In contrast, an Ξ. coli extract of the untransformed strain s311Q 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 ox the protein and its purification. Suitable purification processes - which are known per se - ion. exchange, adsorption,, gel filtration and preparative HPLC chromatography.

Claims (12)

1. Patent Claixns
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) s - Pro - (ZX) x denoting an integer from 1 to 18, and && being a genetically encodable amino acid with the exception of Cys.
4. A protein as claimed in claim 3, wherein the bridging member (aa) s denotes the amino acid sequence -Pro-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Aspor -Pro-Met -1 le-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr-Ile-Asp-Asp5. A protein es claimed in one or more of claims 2 to 4, wherein th® IL-2 constituent is arranged N-terminal and the GM-CSP 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. Λ 7. A medicament composed of a protein as claimed in any * of claims 1 to S, where appropriate combined with a
5. Pharmacologically suitable vehicle.
6. 8. The use of a protein as claimed in aay of claims 1 to 5 for the preparation of a medicament for the treatment of malignant neoplasms .
7. 9. A bifunctional protein composed of a
8. 10 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„ 15 substantially as hereinbefore described.
9. 11. A process according to Claim 6 for the preparation of bifunctional proteins? substantially as hereinbefore described and exemplified.
10. 12. A bifunctional protein whenever prepared by a 20 process claimed in Claim ό or 11.
11. 13. A medicament according to Claim 7? substantially as hereinbefore described.
12. 14. Use according to Claim 8? substantially as t hereinbefore described.
IE114688A 1987-04-16 1988-04-15 Bifunctional proteins IE61574B1 (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
DE19873712985 DE3712985A1 (en) 1987-04-16 1987-04-16 BIFUNCTIONAL PROTEINS

Publications (2)

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IE881146L IE881146L (en) 1988-10-16
IE61574B1 true IE61574B1 (en) 1994-11-16

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KR (1) KR970000187B1 (en)
AR (1) AR242991A1 (en)
AT (1) ATE79135T1 (en)
AU (1) AU613022B2 (en)
CA (1) CA1322157C (en)
DE (2) DE3712985A1 (en)
DK (1) DK170741B1 (en)
ES (1) ES2033981T3 (en)
FI (1) FI98830C (en)
GR (1) GR3006141T3 (en)
HU (1) HU204303B (en)
IE (1) IE61574B1 (en)
IL (1) IL86086A (en)
NO (1) NO176922C (en)
NZ (1) NZ224247A (en)
PH (1) PH25327A (en)
PT (1) PT87237B (en)
ZA (1) ZA882659B (en)

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HU204303B (en) 1991-12-30
PT87237A (en) 1988-05-01
FI98830B (en) 1997-05-15
JP2667193B2 (en) 1997-10-27
NO881658L (en) 1988-10-17
IL86086A (en) 1995-01-24
NO881658D0 (en) 1988-04-15
FI98830C (en) 1997-08-25
ES2033981T3 (en) 1993-04-01
GR3006141T3 (en) 1993-06-21
CA1322157C (en) 1993-09-14
IL86086A0 (en) 1988-09-30
JPS63301898A (en) 1988-12-08
IE881146L (en) 1988-10-16
NZ224247A (en) 1990-04-26
AR242991A1 (en) 1993-06-30
NO176922B (en) 1995-03-13
DE3712985A1 (en) 1988-11-03
DK170741B1 (en) 1996-01-08
NO176922C (en) 1995-06-21
DE3873397D1 (en) 1992-09-10
ZA882659B (en) 1988-10-14
HUT47319A (en) 1989-02-28
PT87237B (en) 1992-07-31
EP0288809A1 (en) 1988-11-02
EP0288809B1 (en) 1992-08-05
PH25327A (en) 1991-04-30
FI881743A (en) 1988-10-17
FI881743A0 (en) 1988-04-14
AU1466188A (en) 1988-10-20
KR970000187B1 (en) 1997-01-06
DK209188A (en) 1988-10-17
KR880012760A (en) 1988-11-29
ATE79135T1 (en) 1992-08-15
AU613022B2 (en) 1991-07-25
DK209188D0 (en) 1988-04-15

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