IL86086A - Fusion protein with activities of interleukin-2 and gm-csf and pharmaceutical compositions containing said protein - Google Patents
Fusion protein with activities of interleukin-2 and gm-csf and pharmaceutical compositions containing said proteinInfo
- Publication number
- IL86086A IL86086A IL8608688A IL8608688A IL86086A IL 86086 A IL86086 A IL 86086A IL 8608688 A IL8608688 A IL 8608688A IL 8608688 A IL8608688 A IL 8608688A IL 86086 A IL86086 A IL 86086A
- Authority
- IL
- Israel
- Prior art keywords
- protein
- thr
- asp
- csf
- constituent
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 50
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 47
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 title claims description 28
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 8
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 title claims 4
- 108010002350 Interleukin-2 Proteins 0.000 title abstract description 28
- 102000000588 Interleukin-2 Human genes 0.000 title abstract 2
- 108020001507 fusion proteins Proteins 0.000 title description 7
- 102000037865 fusion proteins Human genes 0.000 title description 6
- 230000000694 effects Effects 0.000 title description 5
- 230000001588 bifunctional effect Effects 0.000 claims abstract description 18
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 6
- 201000011510 cancer Diseases 0.000 claims abstract description 5
- 150000001413 amino acids Chemical class 0.000 claims description 16
- 239000000470 constituent Substances 0.000 claims description 15
- 230000014509 gene expression Effects 0.000 claims description 11
- 210000004027 cell Anatomy 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 210000004899 c-terminal region Anatomy 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 claims 1
- 238000010276 construction Methods 0.000 claims 1
- 230000003211 malignant effect Effects 0.000 claims 1
- 230000004071 biological effect Effects 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000002281 colonystimulating effect Effects 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 32
- 102100020873 Interleukin-2 Human genes 0.000 description 26
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 23
- 239000013612 plasmid Substances 0.000 description 17
- 235000001014 amino acid Nutrition 0.000 description 10
- 238000003776 cleavage reaction Methods 0.000 description 7
- 230000007017 scission Effects 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 108010093581 aspartyl-proline Proteins 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 2
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000031018 biological processes and functions Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 210000003714 granulocyte Anatomy 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WXERCAHAIKMTKX-ZLUOBGJFSA-N Ala-Asp-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O WXERCAHAIKMTKX-ZLUOBGJFSA-N 0.000 description 1
- VPSHHQXIWLGVDD-ZLUOBGJFSA-N Asp-Asp-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O VPSHHQXIWLGVDD-ZLUOBGJFSA-N 0.000 description 1
- LKIYSIYBKYLKPU-BIIVOSGPSA-N Asp-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N)C(=O)O LKIYSIYBKYLKPU-BIIVOSGPSA-N 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 1
- 108010054278 Lac Repressors Proteins 0.000 description 1
- WIDZHJTYKYBLSR-DCAQKATOSA-N Leu-Glu-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WIDZHJTYKYBLSR-DCAQKATOSA-N 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 241000607715 Serratia marcescens Species 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- DSGIVWSDDRDJIO-ZXXMMSQZSA-N Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DSGIVWSDDRDJIO-ZXXMMSQZSA-N 0.000 description 1
- GRIUMVXCJDKVPI-IZPVPAKOSA-N Thr-Thr-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GRIUMVXCJDKVPI-IZPVPAKOSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 239000000824 cytostatic agent Substances 0.000 description 1
- 230000001085 cytostatic effect Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000002607 hemopoietic effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
Classifications
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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
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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
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- 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)
- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
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- General Engineering & Computer Science (AREA)
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- Biochemistry (AREA)
- Plant Pathology (AREA)
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- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (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.
[EP0288809A1]
Description
86086/2 FUSION PROTEIN WITH ACTIVITIES OF INTERLEUKIN-2 AND GM-CSF AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAID PROTEIN.
GM-CSF -■) 2-,ρρ,>Ί υ Ν n-mtnyon yi >m>N ii-i rv iniN D^ on rnnpn i^w3rri Abstract of the DiscLosure BifunctionaL proteins, obtainable by genetic manipulation, composed of an interleukin-2 and a granulocyte macrophage colony stimulating factor constituent 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.
InterLeuk in-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.
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 human treatment with and without administration of cytostatics. However, the stabilities of IL-2 and GM-CSF differ, which may result in problems on direct adm nistration 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 - Z or Met - Z - Y - X (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 human, IL-2, Y denotes a direct bond if the amino acid or amino acid sequence adjacent to the desired protein allows the desired protein to be cleaved off, or 2 86086/2 otherwise denotes a bridging member which is composed of one or more genetically encodable amino acids and which 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 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).
EP-Al-0 158 198 discloses a hybrid protein consisting of human IFN-^ and human IL-2, GM-CSF is not mentioned.
Schrader et al . (1986) disclose homologies between GM-CSF and IL-2. However, the homolgy is only 25% and Schrader et al. do not teach or suggest any hybrid protein between GM-CSF and IL-2.
The present invention however teaches for the first time that GM-CSF and IL-2 can be combined to result in a bifunctional protein, i.e. without loss of activity.
This was neither taught nor suggested by the above references. Moreover, because of the fact that the stabilities of IL-2 and GM-CSF differ, it was very surprising to obtain a bifunctional protein with greater stability than IL-2 or GM-CSF alone. A further advantage of the present .invention is that because of the greater stability a lower dosage for application is possible . - 2a - 86086/3 The present invention relates to a bifunctional protein composed of a biologically active interleukin-2 (IL-2) constituent and a bilogically active granulocyte macrophage colony stimulating factor (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.
Preferably the bridge corresponds to the formula (II) - Asp - (aa)x - Pro - (II) x denoting an integer from 1 to 18, and aa being a genetically encodable amino acid with the exception of Cys. The Bridging member (a.a)^ may denote the amino acid sequence -pro-Met-lle-Thr-Thr-Tyr-Ala-Asp-Asp- or -Pro-Met-lle-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr-lle-Asp-Asp- The invention further relates to a pharmaceutical composition comprising a protein as defined above, where appropriate combined with a pharmacologically suitable vehicle. Such compositions can be used for treatment of malignant neoplasms.
- - . 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 the 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 neopl asms .
The fusion protein used according to the invention is thus composed of two biologically active components, namely of an IL-2 constituen , 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 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 a'lso be used for the preparation of other novel bifunctional proteins.
The figure shows the construe tion of the plasmid pB30 which codes for a bifunctiona I protein according to the invention. - 3 - Modi ications 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 N-terminal 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.
The bridging member Y advantageously has the formula II - Asp - (aa)x - 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 the formula II for the IL-2 con-stituent 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 acid sequence -As p-Pro-Met-I I e-Thr-Thr-Tyr-A la-Asp-Asp-Pro or -Asp-Pro-Met-Ile-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr- I le-Asp-Asp-Pro-it again being preferable for the IL-2 constituent to be 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 in bacterial expression systems for the 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, B. subtilis, Salmonella typhimurium or Serratia marcescens, especially E. col .
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, P\_ or PR of phage λ, hsp, omp or a synthetic promoter, as described in, for example, German Of f enLegungsschr ft 3,430,683 and in EP-A 0,173,149. The tac promoter-operator sequence is advantageous and is now commercially available (for example '··'ρ Κ223-3 express ion vec tor , Pharmac ia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, page 63).
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 ct-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 - 5 - gtycosylat ion sites in the bifunctionaL protein have been destroyed by appropriate exchange of individual amino acid,s. 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 bifunctionaL proteins according to the invention corresponds to that of 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 p159/6 (EP-A2 0,163,249, Figure 5; (1) in the present figure) contains a synthetic gene coding for IL-2 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 Taql 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 EcoRI and Taql, and is isolated.
The plasmid pHG23 (3) which codes for GM-CSF is disclosed in EP-A2 0,183,350. The GM-CSF 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, the Pstl cleavage site at the 5' end and, on the other hand, a Pstl 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 SfaNI and Pstl. - 6 - The following oligonucleotide (5) is synthesized by the phosphite method: 128 ( 133) He lie Se r Thr Leu Asp Pro Met He CG ATC ATC TCT ACC CTG GAC CCG ATG ATC TAG TAG AGA TGG GAC CTG GGC TAC TAG (Taql ) (5) 1 2 Thr Thr Ty r Ala Asp Asp Pro (Ala) (Pro) ACC ACC TAT GCG GAC GAT CCG GC TGG TGG ATA CGC CTG CTA GGC CGT GGG (Sf aNI ) 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 oligonucleo-tide are located the nuc leotides which have been deleted from the cDNA by cutting with SfaNI.
The preparation of the expression pLasmid pEWIOOO (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 (Amann et 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 n this way. Insertion of the lac repressor (Farabaugh, Nature 274 (1978) 765 - 769) results in the plasmid p J F 118. 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 pEWIOOO (6) is obtained. Opening of this plasmid in the polylinker using the enzymes EcoRI and Pstl results in the linearized expression plasmid (7).
This linearized plasmid DNA (7) is now ligated with the DNA fragment (2) which codes for the IL-2 sequence, with - 7 - the synthetic oL i gonuc L eo t i de (5) and with the cDNA fragment (4). The result is the plasmid pB30 (8) which is transformed into the E. coli strain Mc1061. The plasmid DNA from individual clones is isolated and character zed by restriction analysis.
Example 2 If the following synthetic oligonucleotide 128 (133) He He Se r Thr Leu Asp Pro Met 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 (Taql ) 1 2 Leu Glu Glu Leu Thr He 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 (SfaNI) is used in place of oligonucleotide (5) in the example the result is the plasmid pB31.
Example 3 Competent cells of the E. coli strain W3110 are transformed 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 ug/ml ampicillin, and the growth is followed by measurement of the 00. At OD = 0.5 the culture is adjusted to a concentration of 2 mM in isopropyl-B-D-th iogalactopyra-noside (IPTG) and, after 150 - 180 minutes, the bacteria are spun down. These bacteria are treated in a buffer mixture (7M urea, 0.1% SDS, 0.1M 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 appro-pr iately .
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 dis- ( R ) rupted (French Press, Dynomill). The disrupted cells are spun down. The supernatant and sediment are analyzed by SDS polyacrylamide gel elec trophorese 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 5 M in guanidinium hydrochloride and 2 mM in d i t h i o t h re i tol (OTT). 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 s 100 ug/ml. It is now dialyzed against this tris buffer 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 bio-logical action both in the i n t e r I euk i n-2-dependent CTLL 2 cell proliferation assay and in the human bone marrow assay. Mixed colonies of granulocytes and macrophages are observed in these.
The bifunctional protein can be further purified by i n te r I euk i n-2-spec i f i c affinity chromatography. The protein is still active in both assays. In contrast, an E. coli extract of the untransf ormed strain W3110 which has been treated as described shows no activity. - 9 - 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 HPLC chromatography.
Claims (9)
1. , A bifunctional protein composed of a biologically actiye interle'ukin-2 (IL-2) constituent and a biologically actiye granulocyte macrophage colony stimulating factor (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,
2. · A protein as claimed in Claiml , wherein the bridge corresponds to the formula (II) - Asp - (aa)x - Pro - (II) x denoting an integer from 1 to 18, and aa being a genetically encodable amino acid with the exception of Cys .
3. A protein as claimed in Claim 2* wherein the bridging member (aa)x denotes the amino acid sequence -Pro-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Asp- or -Pro-Met-Ile-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr-Ile-Asp-Asp-.
4. A protein as claimed in Claim 2 or 3, wherein the IL-2 constituent is arranged N-terminal and the GM-CSF constituent is arranged C-terminal.
5. A process for the preparation of bifunctional proteins as claimed in any of Claims 1 to 4, which comprises construction, and expression in a host cell, of a gene coding for these proteins.
6. · A pharmaceutical composition comprising a protein as claimed in any of claims 1 to 4, where appropriate combined with a pharmacologically suitable vehicle.
7. The use of a protein as claimed in any of claims 1 to 4 for the preparation of a pharmaceutical composition,for the treatment of malignant 'neoplasms .substantially an hereinbefore described,. 86068/2 - 11 -
8. A pharmaceutical composition containing a bifunctional protein defined in claim 1 as active ingredient.
9. . A pharmaceutical composition for the treatment of malignant neoplasms containing as active ingredient a compound claimed in any of claims 1 to A 10 , Pharmaceutical compositions containing a bifunctional protein defined in claim 1, substantially as hereinbefore described.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19873712985 DE3712985A1 (en) | 1987-04-16 | 1987-04-16 | BIFUNCTIONAL PROTEINS |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| IL86086A0 IL86086A0 (en) | 1988-09-30 |
| IL86086A true IL86086A (en) | 1995-01-24 |
Family
ID=6325799
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL8608688A IL86086A (en) | 1987-04-16 | 1988-04-15 | Fusion protein with activities of interleukin-2 and gm-csf and pharmaceutical compositions containing said protein |
Country Status (20)
| Country | Link |
|---|---|
| EP (1) | EP0288809B1 (en) |
| JP (1) | JP2667193B2 (en) |
| 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) |
Families Citing this family (27)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5662896A (en) * | 1988-03-21 | 1997-09-02 | Chiron Viagene, Inc. | Compositions and methods for cancer immunotherapy |
| US6406697B1 (en) | 1989-02-23 | 2002-06-18 | Genentech, Inc. | Hybrid immunoglobulins |
| US5116964A (en) * | 1989-02-23 | 1992-05-26 | Genentech, Inc. | Hybrid immunoglobulins |
| US5225538A (en) * | 1989-02-23 | 1993-07-06 | Genentech, Inc. | Lymphocyte homing receptor/immunoglobulin fusion proteins |
| WO1990012877A1 (en) * | 1989-04-19 | 1990-11-01 | Cetus Corporation | Multifunctional m-csf proteins and genes encoding therefor |
| US5108910A (en) * | 1989-08-22 | 1992-04-28 | Immunex Corporation | DNA sequences encoding fusion proteins comprising GM-CSF and IL-3 |
| DK0489116T3 (en) * | 1989-08-22 | 1994-05-02 | Immunex Corp | Fusion proteins comprising GM-CSF and IL-3 |
| US5073627A (en) * | 1989-08-22 | 1991-12-17 | Immunex Corporation | Fusion proteins comprising GM-CSF and IL-3 |
| US5376367A (en) * | 1991-11-22 | 1994-12-27 | Immunex Corporation | Fusion proteins comprising MGF and IL-3 |
| US5723125A (en) * | 1995-12-28 | 1998-03-03 | Tanox Biosystems, Inc. | Hybrid with interferon-alpha and an immunoglobulin Fc linked through a non-immunogenic peptide |
| ES2221717T3 (en) | 1997-12-08 | 2005-01-01 | Emd Lexigen Research Center Corp. | USEFUL HETERODIMERAS FUSION PROTEINS FOR DIRECTED IMMUNOTHERAPY AND GENERAL IMMUNOSTIMULATION. |
| US7067110B1 (en) | 1999-07-21 | 2006-06-27 | Emd Lexigen Research Center Corp. | Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens |
| SK782002A3 (en) | 1999-07-21 | 2003-08-05 | Lexigen Pharm Corp | FC fusion proteins for enhancing the immunogenicity of protein and peptide antigens |
| US6617135B1 (en) | 1999-08-09 | 2003-09-09 | Emd Lexigen Research Center Corp. | Multiple cytokine protein complexes |
| CA2391080A1 (en) | 1999-11-12 | 2001-05-25 | Merck Patent Gesellschaft Mit Beschraenkter Haftung | Erythropoietin forms with improved properties |
| JP4553290B2 (en) * | 1999-11-30 | 2010-09-29 | 塩野義製薬株式会社 | Chemokine SLC-IL2 fusion protein and its gene |
| DK1252192T3 (en) | 2000-02-11 | 2006-11-20 | Merck Patent Gmbh | Enhancement of the serum half-life of antibody-based fusion proteins |
| RU2272644C2 (en) | 2000-06-29 | 2006-03-27 | Мерк Патент Гмбх | Enhancement of immune response wherein fused protein antibody-cytokine is mediator by combined treatment with agents increasing immunocytokine absorption |
| KR100900176B1 (en) | 2001-03-07 | 2009-06-02 | 메르크 파텐트 게엠베하 | Expression Techniques for Proteins Containing Hybrid Isotype Antibody Substructures |
| US6992174B2 (en) | 2001-03-30 | 2006-01-31 | Emd Lexigen Research Center Corp. | Reducing the immunogenicity of fusion proteins |
| CA2446087C (en) | 2001-05-03 | 2013-06-18 | Stephen D. Gillies | Recombinant tumor specific antibody and use thereof |
| EP2305312B1 (en) | 2001-10-10 | 2015-03-04 | ratiopharm GmbH | Remodelling and glycoconjugation of follicle-stimulating hormone (FSH) |
| BR0214650A (en) | 2001-12-04 | 2005-05-03 | Merck Patent Gmbh | Modulated selectivity immunocytokines |
| JP4494977B2 (en) | 2002-12-17 | 2010-06-30 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング | Humanized antibody (H14.18) of mouse 14.18 antibody that binds to GD2 and its IL-2 fusion protein |
| DE602004031341D1 (en) | 2003-07-21 | 2011-03-24 | Transgene Sa | MULTIFUNCTIONAL CYTOKINE |
| ATE500267T1 (en) * | 2003-07-21 | 2011-03-15 | Transgene Sa | MULTIFUNCTIONAL CYTOKINE |
| WO2010001414A1 (en) * | 2008-07-03 | 2010-01-07 | Lupin Limited | Expression of heterologous proteins in bacterial system using a gm-csf fusion tag |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1985000817A1 (en) * | 1983-08-10 | 1985-02-28 | Amgen | Microbial expression of interleukin ii |
| GB8327880D0 (en) * | 1983-10-18 | 1983-11-16 | Ajinomoto Kk | Saccharomyces cerevisiae |
| EP0158198A1 (en) * | 1984-03-29 | 1985-10-16 | Takeda Chemical Industries, Ltd. | DNA and use thereof |
| WO1985004673A1 (en) * | 1984-04-10 | 1985-10-24 | Takeda Chemical Industries, Ltd. | Novel dna and its use |
| DE3419995A1 (en) * | 1984-05-29 | 1985-12-05 | Hoechst Ag, 6230 Frankfurt | GENE TECHNOLOGICAL METHOD FOR PRODUCING HUMAN INTERLEUKIN-2 AND MEANS FOR CARRYING OUT THIS METHOD |
| AU588819B2 (en) * | 1984-10-29 | 1989-09-28 | Immunex Corporation | Cloning of human granulocyte-macrophage colony stimulating factor gene |
| JPS61128889A (en) * | 1984-11-27 | 1986-06-16 | Green Cross Corp:The | Recombinant dna and transformant by same |
| JPH0646957B2 (en) * | 1985-03-11 | 1994-06-22 | 武田薬品工業株式会社 | Method for producing interleukin-2 |
| WO1987002060A1 (en) * | 1985-10-03 | 1987-04-09 | Biogen N.V. | Human granulocyte-macrophage colony stimulating factor-like polypeptides and processes for producing them in high yields in microbial cells |
| DE3541856A1 (en) * | 1985-11-27 | 1987-06-04 | Hoechst Ag | EUKARYOTIC FUSION PROTEINS, THEIR PRODUCTION AND USE, AND MEANS FOR CARRYING OUT THE PROCESS |
| DE3545568A1 (en) * | 1985-12-21 | 1987-07-16 | Hoechst Ag | GM-CSF-PROTEIN, ITS DERIVATIVES, PRODUCTION OF SUCH PROTEINS AND THEIR USE |
-
1987
- 1987-04-16 DE DE19873712985 patent/DE3712985A1/en not_active Withdrawn
-
1988
- 1988-04-09 DE DE8888105693T patent/DE3873397D1/en not_active Expired - Fee Related
- 1988-04-09 EP EP88105693A patent/EP0288809B1/en not_active Expired - Lifetime
- 1988-04-09 AT AT88105693T patent/ATE79135T1/en not_active IP Right Cessation
- 1988-04-09 ES ES198888105693T patent/ES2033981T3/en not_active Expired - Lifetime
- 1988-04-14 NZ NZ224247A patent/NZ224247A/en unknown
- 1988-04-14 FI FI881743A patent/FI98830C/en not_active IP Right Cessation
- 1988-04-14 PT PT87237A patent/PT87237B/en active IP Right Grant
- 1988-04-15 HU HU881966A patent/HU204303B/en not_active IP Right Cessation
- 1988-04-15 DK DK209188A patent/DK170741B1/en not_active IP Right Cessation
- 1988-04-15 IL IL8608688A patent/IL86086A/en not_active IP Right Cessation
- 1988-04-15 JP JP63093321A patent/JP2667193B2/en not_active Expired - Fee Related
- 1988-04-15 AU AU14661/88A patent/AU613022B2/en not_active Ceased
- 1988-04-15 CA CA000564322A patent/CA1322157C/en not_active Expired - Fee Related
- 1988-04-15 IE IE114688A patent/IE61574B1/en not_active IP Right Cessation
- 1988-04-15 NO NO881658A patent/NO176922C/en not_active IP Right Cessation
- 1988-04-15 ZA ZA882659A patent/ZA882659B/en unknown
- 1988-04-15 AR AR88310585A patent/AR242991A1/en active
- 1988-04-16 KR KR1019880004345A patent/KR970000187B1/en not_active Expired - Fee Related
-
1989
- 1989-04-14 PH PH36799A patent/PH25327A/en unknown
-
1992
- 1992-11-04 GR GR920402185T patent/GR3006141T3/el unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DE3873397D1 (en) | 1992-09-10 |
| AU1466188A (en) | 1988-10-20 |
| KR970000187B1 (en) | 1997-01-06 |
| HUT47319A (en) | 1989-02-28 |
| IL86086A0 (en) | 1988-09-30 |
| NO176922C (en) | 1995-06-21 |
| NO881658D0 (en) | 1988-04-15 |
| FI98830B (en) | 1997-05-15 |
| NO176922B (en) | 1995-03-13 |
| AU613022B2 (en) | 1991-07-25 |
| FI881743A0 (en) | 1988-04-14 |
| DK209188A (en) | 1988-10-17 |
| GR3006141T3 (en) | 1993-06-21 |
| AR242991A1 (en) | 1993-06-30 |
| PT87237A (en) | 1988-05-01 |
| CA1322157C (en) | 1993-09-14 |
| KR880012760A (en) | 1988-11-29 |
| JPS63301898A (en) | 1988-12-08 |
| ATE79135T1 (en) | 1992-08-15 |
| FI881743L (en) | 1988-10-17 |
| NO881658L (en) | 1988-10-17 |
| PT87237B (en) | 1992-07-31 |
| NZ224247A (en) | 1990-04-26 |
| DK209188D0 (en) | 1988-04-15 |
| DE3712985A1 (en) | 1988-11-03 |
| IE61574B1 (en) | 1994-11-16 |
| EP0288809B1 (en) | 1992-08-05 |
| ZA882659B (en) | 1988-10-14 |
| PH25327A (en) | 1991-04-30 |
| HU204303B (en) | 1991-12-30 |
| FI98830C (en) | 1997-08-25 |
| EP0288809A1 (en) | 1988-11-02 |
| DK170741B1 (en) | 1996-01-08 |
| JP2667193B2 (en) | 1997-10-27 |
| IE881146L (en) | 1988-10-16 |
| ES2033981T3 (en) | 1993-04-01 |
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