CA2041439A1 - Muteins of the granulocyte colony stimulating factor (g-csf) - Google Patents
Muteins of the granulocyte colony stimulating factor (g-csf)Info
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- CA2041439A1 CA2041439A1 CA002041439A CA2041439A CA2041439A1 CA 2041439 A1 CA2041439 A1 CA 2041439A1 CA 002041439 A CA002041439 A CA 002041439A CA 2041439 A CA2041439 A CA 2041439A CA 2041439 A1 CA2041439 A1 CA 2041439A1
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- csf
- amino acids
- mature
- mutein
- amino acid
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
- B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
- B23K26/00—Working by laser beam, e.g. welding, cutting or boring
- B23K26/351—Working by laser beam, e.g. welding, cutting or boring for trimming or tuning of electrical components
-
- 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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Abstract
A b s t r a c t A granulocyte stimulating factor (G-CSF) or a G-CSF
variant differs from natural G-CSF in that one or several amino acids of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile at position 50 to 56 of the mature G-CSF with 174 amino acids or at position 53 to 59 of the mature G-CSF with 177 amino acids or/and at least one of the 4 His residues at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids are mutagenized. It is suitable for immunotherapy.
variant differs from natural G-CSF in that one or several amino acids of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile at position 50 to 56 of the mature G-CSF with 174 amino acids or at position 53 to 59 of the mature G-CSF with 177 amino acids or/and at least one of the 4 His residues at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids are mutagenized. It is suitable for immunotherapy.
Description
2 ~
~ e s c r i p t i o n The invention concerns muteins of the granulocyte stimulatinq factor G-CSF in the sequence 5~1 52 53 54 55 56 Leu-Gly-His-Ser-Leu-Gly-Ile at position 50 to 56 of the mature G-CSF with 174 amino acids or at position 53 to 59 of the mature G-CSF with 177 amino acids or/and at least one of the 4 His residues at positions 43, 79, 156 and 170 of the mature G-CSF with 174 amino acids or at positions 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids.
Lymphokines are involved in the maturation of blood cells. They stimulate the maturation of bone marrow stem cells to fully differentiated cells. G CSF is synthesized by activated monocytes, macrophages as well as by a series of other cell lines.
G-CSF was purified to homogeneity from cell culture supernatants of the human bladder carcinoma cell line 5637 (Welte et al., Proc. Natl. Acad. Sci 82 (1985), 1526). The sequence of the cDNA coding for native G-CS~' is known from Sunza et al., Science 232 (1986), 61. As a consequence of alternative splicing in the second intron two naturally occurring forms of G-CSF exist with 204 or 207 amino acids of which the first 30 represent a signal peptide (Lymphokines, IRL Press, Oxford, Washington D.C., Editors D. ~ale and C. Rickwood). The mature protein has a molecular weight of ca.l9.6 kD and has 5 cysteine residues which can form intermolecular or intramolecular disulphide bridges. Binding studies have shown that G-CSF binds to neutrophilic granulocytes.
None or only slight binding is observed with erythroid, lymphoid eosinophilic cell lines as well as with macrophages. The G-CSF receptor consists of a single peptide chain with a molecular weight of 150 kD (Nicola, Immunol. Today 8 (1987), 134). The number of receptors per cell generally increases with the maturation of the cells~and can amount to several hundred per cell. It is assumed that lymphokine receptors consist of an e~tracellular domain, which binds the ligands, a hydrophobic transmembrane re~ion and an intracellular domain. Binding of lymphokines to their receptor can cause the synthesis of cyclic nucleotides, hydrolysis of phosphatidylinositol-~,5-biphosphate as well as the activation of protein kinase C and an increase in the intracellular calcium level. There is a great interest in how these processes effect the metabolism of the cell but at present they are hardly understood. A further result of the binding of a ligand to its receptor can be the migration of the receptor-ligand complex into the inside of the cell by a receptor-dependent endocytosis.
This type of internalization apparently also occurs with lymphokines (e.g. G-CSF), however, the consequences for the metabolism of the cell are not yet understood.
Since G-CSF is able to substantially increase the population of neutrophilic granulocytes within a short period, considerable therapeutic fields of application arise for G-CSF. Thus, G-CSF could be used e.g. after chemotherapy in cancer, in which the cells of the immune system are destroyed. In addition G-CSF could be used in bone marrow transplantations, in severe burn wounds, in opportunistic infections caused by immune deficiency and in leukemia. For the different types of therapy it would be desirable to develop more active and also less active forrns of G-CSF. The object of the present invention is therefore to develop G-CSF molecules with a wide -`-` 20~1~39 spectrum of activity by the specific introduction of point mutations. In this~process the changes in activity should be achieved by chanqes in the Dino acid sequence which are as small as possible.
The object according to the present invention is achieved by a granulocyte stimulating factor (G-CSF) or a G-CSF variant, in which one or~several~amino acids of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile at position 50 to 56 of the mature G-CSF with~174 amino acids or at position 53 to 59 of the mature G-CSF with 177 amino acids or/and at least one of the 4 His residues at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids are mutagenized.
Surprisingly the introduction of new amino acids yields G-CSF muteins which have a broad spectrum of activity.
The determination of the activity can for example be carried out according to ~iochem. J. 253 (1988) 213-218;
Exp. Hematol. 17 (1989) }16-119; Proc. Natl. Acad. Sci.
USA 83 (1986) 5010.
The term G-CSF or G-CSF variant according to the present invention includes all naturally occurring variants of G-CSF with or without a leader sequence as well as G-CSF
proteins derived therefrom which are modified by recombinant DNA technology, in particular fusion proteins which contain further polypeptide sequences apart from the G-CSF moiety. In this sense a G-CSF
mutein is particularly preferred with a N-terminal Met residue at position -1 which is suitable for expression in prokaryotic cells. Also preferred is a recombinant, methionine-free G-CSF variant which can be produced according to PCT/EP 91/00 192. The term "mutagenized"
~, .
-`~` 2~4143g means that the respective amino acid is deleted or preferably substituted by another amino acid.
In this sense G-CSF muteins are preferred in which one of the 7 amino acids of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile is substituted by another amino acid.
However, more than one, in particular two amino acids, can also be replaced.
A G-CSF mutein is particularly preferred in which the Ser residue at position 53 of the mature G-CSF with 174 amino acids or at position 56 of the mature G-CSF with 177 amino acids is replaced by one of the other 19 amino acids, in particular by Thr.
Furthermore, it is preferred that the Leu residue at position 54 of the mature G-CSF with 174 amino acids or at position 57 of the mature G-CSF with 177 amino acids is substituted by one of the 19 other amino acids, in particular by Thr. By this means one obtains G-CSF
muteins with a broad variation of G-CSF activity.
In addition G-CSF muteins are preferred in which one of the 4 His residues at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids is substituted by another amino acid, in particular Gln.
The invention also provides a recombinant DNA which codes for a G-CSF mutein according to the present invention. The invention also provides a recombinant vector which contains at least one copy of a recombinant DNA according to the present invention. In this connection a recombinant vector is preferred which is suitable for gene expression in prokaryotic cells.
vectors of this type are known to one skilled in the art.
In addition the invention provides a celI which is transformed with a recombinant DNA according to the present invention or/and a recombinant vector according to the present invention. This cell is preferably a prokaryotic cell, particularly preferably an E. coli cell.
The invention also provides a process for the production of a recombinant DNA according to the present invention in which a DNA sequence which codes for G-CSF or a G-CSF
variant is site-specifically mutagenized. The usual molecular-biological methods for site-specific mutagenesis are known to one skilled in the art. The mutagenesis is preferably carried out by using synthetic oligonucleotides as mutagenesis primers on singIe-stranded DNA as the template. Common methods are for example described in Amersham No. 1523 "Oligonucleotide-; directed in vitro mutagenesis system"; Methods in Enzymology (Academic Press, Inc. Vol. 154, Part E, 367-382 (1987); Analytical Biochemistry 179 (1989) 309-311.
In addition the invention provides a process for producing a G-CSF mutein according to the present invention in which a cell is transformed with a recombinant DNA according to the present invention or/and a recombinant vector according to the present invention, the transformed cell is cultured in a suitable medium and the protein is isolated from the cells or the medium. The methods usually used in molecular biology for the isolation of recombinant proteins from eukaryotic or prokaryotic cells are known to one skilled in the art and do not need to be elucidated in detail.
Finally the invention also provides a pharmaceutical preparation based on a G-CSF mutein according to the present invention as the active substance, if desired, together with the usual pharmaceutical carrier, filling and auxiliary substances. Such a pharmaceutical preparation is particularly suitable for the therapeutic fields of application mentioned above and ~ven for further therapeutic proceedures in which the formation of neutrophilic granulocytes is to be stimulated.
The following examples are intended to elucidate the invention without however limiting its scope.
E x a m p l e Production of the vector m~l-G-CSF-B~
The 554 bp long EcoRI/BamHI fragment from the vector pKK
177-3 G-CSF-Bg (DSM 5867) containing the Shine Dalgarno sequence, ATG codon and coding sequence for the G-CSF
gene is cloned via a blunt-end ligation into the NcoI
cleavage site of the vector pPZ 07-mgl lac (W088/09373, Figure lO). The ATG start codon of the lac Z gene, which is located in the protruding single strand after NcoI
digestion, is digested beforehand by incubation with mung bean nuclease (Pharmacia). The resulting vector is denoted mgl-G-CSF-Bg.
-~` 2 0 ~ 3 E x a m p 1 e 2 Mutagenesis of the amino acid Leu ~X) in the sequence Gly-His-Ser-Leu-Gly-Ile ,~ :
The mutagenesis is carried out on the M13 template according to known techniques (Amersham No. 1523 "Oligonucleotide-directed in vitro mutagenesis system").
A 251 bp long G-CSF cDNA fragment is isolated via the cleavage site BstXI/AatII. The protruding single-strands are digested off by mung bean nuclease (Pharmacia) and the fragment is cloned into the vector M13mpl9 which was cleaved with EcoRI/SmaI (EcoRI protruding single strand was filled in for blunt-end cloning). After preparing single-stranded DNA, the oligonucleotide is hybridized to the single-stranded DNA and an elongation in the 5'-3' direction beyond the oligonucleotide is carried out using Klenow polymerase, ligase and the four nucleotide triphosphates (GTP, CTP, TTP, ATP). The DNA which is now double-stranded is transformed in E. coli cells which carry a F' episome so that infection by filamentous M13 phages is possible (e.g. JM101, obtainable from Stratagene, LaJolla, California). Individual plaques are picked out and the mutagenized M13 phages contained therein are used for the preparation of single-stranded DNA. A DNA sequencing is carried out according to known techniques (e.g. dideoxy method according to Sanger) and the exact substitution to form the desired mutation is checked in this way. After preparing double-stranded DNA
the mutated AvaI fragment of G-CSF is isolated and cloned in the expression vector mgl-G-CSF-Bg (cleaved with AvaI).
. .
2 ~
In order to reconstitute the complete G-CSF gene the DNA
is subsequently cleaved with HindIII, the protruding ends are filled in with Klenow polymerase and afterwards partially digested with AvaI so that the 5' AvaI site in the G-CSF gene (at ca 130 bp) is not cleaved. This DNA
is ligated with the approximately 240 bp G-CSF fragment AvaI/BamHI (BamHI site is filled in with Klenow polymerase) from the starting vector mgl-G-CSF-Bg.
After transformation in E. coli JM83, the expression of G-CSF is carried out in the manner described in W~88/09373.
The cDNA used has a sequence which codes for a G-CSF
with 175 amino acids (without a signal sequence, but with a Met residue at position -1) so that the preferred mutation is located at Leu at position 54 of the G-CSF
amino acid sequence (in this the N-terminal Met residue is not counted).
The sequence of the cDNA encoding G-CSF which codes for the amino acids 50 to 56 (with reference to the G-CSF
with 174 amino acids) reads:
(X) Leu-Gly-His-Ser-Leu-Gly-Ile 5'-CTC GGA CAC TCT CTG GGC ATC-3' The corresponding complementary opposite strand to be mutagenized reads:
5l-GAT GCC CAG AGA GTG TCC GAG-3' 20~1~}~3 g The following 19 oligonucleotides correspond:ing to the opposite strand are used for site-directed mutagenesis:
Wild-type: 5'-3 GAT GCC CAG AGA GTG TCC~GAG 3' Met l. S' GAT GCC CAT AGA GTG TCC GAG 3' :
Phe 2. 5' GAT GCC GAA AGA GTG TCC GAG 3' Gln 3. 5' GAT GCC CTG AGA GTG TCC GAG 3' ^: Glu 4. 5' GAT GCC CTC AGA GTG TCC GAG :3' Asp ~ 5. 5' GAT GCC GTC AGA GTG TCC GAG 3' ,~ : Cys 6. 5' GAT GCC GCA AGA GTG TCC GAG 3' , ~ , ~; Ala 7. 5' GAT GCC GGC AGA GTG TCC GAG 3' ~ .~
Gly 8. 5' GAT GCC AGG AGA GTG TCC GAG 3' His 9. 5' GAT GCC GTG AGA GTG TCC GAG 3' Ile 10. 5' GAT GCC GAT AGA GTG TCC GAG 3' ~, .
Lys 11. 5' GAT GCC CTT AGA GTG TCC GAG 3' Tyr 12. 5I GAT GCC ATA AGA GTG TCC GAG 3' Asn 13. 5' GAT GCC GTT AGA GTG TCC GAG 3' Pro 14. 5' GAT GCC GGG AGA GTG TCC GAG 3' Arg 15. 5' GAT GCC GCG AGA GTG TCC GAG 3' Ser 16. 5' GAT GCC GGA AGA GTG TCC GAG 3' Thr 17. 5' GAT GCC GGT AGA GTG TCC GAG 3' Val 18. 5' GAT GCC GAC AGA GTG TCC GAG 3' Trp 19. 5' GAT GCC CCA AGA GTG TCC GAG 3' E x a m p l e 3 Productlon_of a G-CSF with modified activity A G-CSF which is more enzymatically active compared to the wild type can be produced by substituting serine at position 53 by a threonine at position 53 of a G-CSF
with 174 amino acids (serine in the sequence Gly-His--` 20~14~9 Ser-Leu-Gly). The following double-stranded oligonucleotide was used for the mutagenesis:
His Thr Leu Gly Ile S' CCC GAG GAG CTG GTG CTG CTC GGA CAC ACC CTG GGC ATC CCC TGG GCT CCC CTG AGC 3' 3' C CTC GAC CAC GAC GAG CCT GTG TGG GAC CCG TAG GGG ACC CGA GGG GAC 5' For the cloning, the G-CSF cDNA fragment (ca 300 bp, EcoRI/EcoRV) from the vector pKK 177-3 G-CSF-Bg (DSM
5867) was ligated into the EcoRI/SmaI cleavage site of the vector pUC19 (Yannish-Perron et al., (1985), Gene 33, 103).
This DNA is cleaved with AvaI/SacI and directly ligated with the primer pair described above according to the usual techniques. The mutated BstIXlSacI fragment can now be isolated from this construct and cloned into the vector pXK 177-3 G-CSF-Bg (DSM 5867) (cleaved with BstXI/SacI). The final construction of the expression clone is carried out in analogy to Example l. The determination of activity is carried out as described in Example 5.
E x a m p l e 4 Alteration of ,t~,he enzvmatic properties of G-CSF_by mutation of amin,o acids which are not located in the active_centre.
In analogy to known serine esterases it is assumed that the serine of the active centre interacts with histidine for the development of enzymatic activity. Four histidines are present in the sequence of G-CSF and namely at positions 43, 79, 156 and 170 (numbered from ~-` 2 0 ~ 9 the 174 aa sequence without a signal peptide~. The histidine residue at position 52 (or at position 55 in the 177 amino acid form) is left out of consideration in this mutagenesis. In this process His (CCA, CTA) is substituted by Gln (CAG). The sequence on the opposite strand corresponding to the codon coding for Gln is CTG.
:`
A G-CSF fragment is subcloned in M13mpl9 as described in Example 1.
The following~oligonucleotides corresponding to the opposite strand are used for the mutagenesis:
l. 5' GCT CCT GGG CTG GCA CAG C 3' histidine 43 to glutamine 43 2. 5' GAA AAG GCC GCT CTG GAG TTG GCT C 3' histidine 79 to glutamine 79 ;: : :
3. 5' GCT CTG CAG CTG GCC TAG CAA CC 3' histidine 156 to glutamine 156 4. 5' GGG CTG CGC AAG CTG GCG TAG AAC G 3 histidine 170 to glutamine 170 f, ~ ;
The analytical procedure and the recloning in an expression vector is carried out in analogy to Example 1.
:
~ 20~3~
E x a m p 1 e 5 Determination of the G-~SF act;ivity The ac~tivity of G-CSF is tested with the murine leukaemia line NSF60 which is completely dependent on G-CSF as described in Biochem. J. 253 (1988) ~213-218, Exp.
Hematol. 17 (1989) 116-119, Proc. Natl. Acad. Sci. USA
83 (1986) 5010. In order that the factor-dependency of the cells is preserved, the medium (RPMI medium, Boehringer Mannheim GmbH, Order No. 2099445 with 10 %
foetal calf serum) for the maintenance culture permanently contains 1000 U/ml G-CSF.
The proliferation of the NSF60 cells stimulated by G-CSF
is measured directly in this test by the incorporation of 3H thymidine. The test is carried out as follows:
NSF60 cells which are in the exponential growth phase (cell density is maximally lx105 cells/ml) are transferred to microtitre plates (lx104 celIs/well) and cuItured with a decreasing G-CSF concentration. The maximum dose of G-CSF in well 1 corresponds to the concentration in the maintenance culture (1000 U/ml, specific activity lx108 U/mg protein). The dilution is carried out in steps of ten.
After about 24 hours incubation 3H thymidine (0.1 ~Ci/well) is added. Afterwards the cells are incubated for a further 16 hours.
In order to evaluate the test the cells in the microtitre plates are frozen in order to lyse them. The cell lysate is aspirated on a glass fibre filter, 20~3~
rinsed, dried and measured in a scintillation counter.
The incorporation of 3H thymidine is proportional to the G-CSF-induced proliferation of the NSF60 cells.
E x a m p 1 e 6 , Alt ration i~__he activity of G-CSF by_amino acid substitution in the active centre A G-CSF modified in amino acid position 54 can be produced by substitution of preferably one leucine at position 54 by a threonine at position 54 (Leu in the sequence Gly-His-Ser-Leu-Gly) in correspondence with the procedure described in Example 3 using a suitable double-stranded oligonucleotide which contains a nucleic acid triplet (e.g. ACC) coding for the amino acid Thr at the appropriate position. In this connection position 54 of the 174 amino acid form of G-CSF corresponds to position 57 of the 177 amino acid form.
The activity of a mutant having 174 amino acids with Thr at position 54 is reduced in the NSF60 cell test (see Example 5) in comparison to the wild-type G-CSF with 174 amino acids. Moreover, the activity of this G-CSF mutant is reduced in comparison to a G-CSF mutant with an amino acid substitution of a serine by a threonine at position 53 (described in Example 3).
~ e s c r i p t i o n The invention concerns muteins of the granulocyte stimulatinq factor G-CSF in the sequence 5~1 52 53 54 55 56 Leu-Gly-His-Ser-Leu-Gly-Ile at position 50 to 56 of the mature G-CSF with 174 amino acids or at position 53 to 59 of the mature G-CSF with 177 amino acids or/and at least one of the 4 His residues at positions 43, 79, 156 and 170 of the mature G-CSF with 174 amino acids or at positions 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids.
Lymphokines are involved in the maturation of blood cells. They stimulate the maturation of bone marrow stem cells to fully differentiated cells. G CSF is synthesized by activated monocytes, macrophages as well as by a series of other cell lines.
G-CSF was purified to homogeneity from cell culture supernatants of the human bladder carcinoma cell line 5637 (Welte et al., Proc. Natl. Acad. Sci 82 (1985), 1526). The sequence of the cDNA coding for native G-CS~' is known from Sunza et al., Science 232 (1986), 61. As a consequence of alternative splicing in the second intron two naturally occurring forms of G-CSF exist with 204 or 207 amino acids of which the first 30 represent a signal peptide (Lymphokines, IRL Press, Oxford, Washington D.C., Editors D. ~ale and C. Rickwood). The mature protein has a molecular weight of ca.l9.6 kD and has 5 cysteine residues which can form intermolecular or intramolecular disulphide bridges. Binding studies have shown that G-CSF binds to neutrophilic granulocytes.
None or only slight binding is observed with erythroid, lymphoid eosinophilic cell lines as well as with macrophages. The G-CSF receptor consists of a single peptide chain with a molecular weight of 150 kD (Nicola, Immunol. Today 8 (1987), 134). The number of receptors per cell generally increases with the maturation of the cells~and can amount to several hundred per cell. It is assumed that lymphokine receptors consist of an e~tracellular domain, which binds the ligands, a hydrophobic transmembrane re~ion and an intracellular domain. Binding of lymphokines to their receptor can cause the synthesis of cyclic nucleotides, hydrolysis of phosphatidylinositol-~,5-biphosphate as well as the activation of protein kinase C and an increase in the intracellular calcium level. There is a great interest in how these processes effect the metabolism of the cell but at present they are hardly understood. A further result of the binding of a ligand to its receptor can be the migration of the receptor-ligand complex into the inside of the cell by a receptor-dependent endocytosis.
This type of internalization apparently also occurs with lymphokines (e.g. G-CSF), however, the consequences for the metabolism of the cell are not yet understood.
Since G-CSF is able to substantially increase the population of neutrophilic granulocytes within a short period, considerable therapeutic fields of application arise for G-CSF. Thus, G-CSF could be used e.g. after chemotherapy in cancer, in which the cells of the immune system are destroyed. In addition G-CSF could be used in bone marrow transplantations, in severe burn wounds, in opportunistic infections caused by immune deficiency and in leukemia. For the different types of therapy it would be desirable to develop more active and also less active forrns of G-CSF. The object of the present invention is therefore to develop G-CSF molecules with a wide -`-` 20~1~39 spectrum of activity by the specific introduction of point mutations. In this~process the changes in activity should be achieved by chanqes in the Dino acid sequence which are as small as possible.
The object according to the present invention is achieved by a granulocyte stimulating factor (G-CSF) or a G-CSF variant, in which one or~several~amino acids of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile at position 50 to 56 of the mature G-CSF with~174 amino acids or at position 53 to 59 of the mature G-CSF with 177 amino acids or/and at least one of the 4 His residues at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids are mutagenized.
Surprisingly the introduction of new amino acids yields G-CSF muteins which have a broad spectrum of activity.
The determination of the activity can for example be carried out according to ~iochem. J. 253 (1988) 213-218;
Exp. Hematol. 17 (1989) }16-119; Proc. Natl. Acad. Sci.
USA 83 (1986) 5010.
The term G-CSF or G-CSF variant according to the present invention includes all naturally occurring variants of G-CSF with or without a leader sequence as well as G-CSF
proteins derived therefrom which are modified by recombinant DNA technology, in particular fusion proteins which contain further polypeptide sequences apart from the G-CSF moiety. In this sense a G-CSF
mutein is particularly preferred with a N-terminal Met residue at position -1 which is suitable for expression in prokaryotic cells. Also preferred is a recombinant, methionine-free G-CSF variant which can be produced according to PCT/EP 91/00 192. The term "mutagenized"
~, .
-`~` 2~4143g means that the respective amino acid is deleted or preferably substituted by another amino acid.
In this sense G-CSF muteins are preferred in which one of the 7 amino acids of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile is substituted by another amino acid.
However, more than one, in particular two amino acids, can also be replaced.
A G-CSF mutein is particularly preferred in which the Ser residue at position 53 of the mature G-CSF with 174 amino acids or at position 56 of the mature G-CSF with 177 amino acids is replaced by one of the other 19 amino acids, in particular by Thr.
Furthermore, it is preferred that the Leu residue at position 54 of the mature G-CSF with 174 amino acids or at position 57 of the mature G-CSF with 177 amino acids is substituted by one of the 19 other amino acids, in particular by Thr. By this means one obtains G-CSF
muteins with a broad variation of G-CSF activity.
In addition G-CSF muteins are preferred in which one of the 4 His residues at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids is substituted by another amino acid, in particular Gln.
The invention also provides a recombinant DNA which codes for a G-CSF mutein according to the present invention. The invention also provides a recombinant vector which contains at least one copy of a recombinant DNA according to the present invention. In this connection a recombinant vector is preferred which is suitable for gene expression in prokaryotic cells.
vectors of this type are known to one skilled in the art.
In addition the invention provides a celI which is transformed with a recombinant DNA according to the present invention or/and a recombinant vector according to the present invention. This cell is preferably a prokaryotic cell, particularly preferably an E. coli cell.
The invention also provides a process for the production of a recombinant DNA according to the present invention in which a DNA sequence which codes for G-CSF or a G-CSF
variant is site-specifically mutagenized. The usual molecular-biological methods for site-specific mutagenesis are known to one skilled in the art. The mutagenesis is preferably carried out by using synthetic oligonucleotides as mutagenesis primers on singIe-stranded DNA as the template. Common methods are for example described in Amersham No. 1523 "Oligonucleotide-; directed in vitro mutagenesis system"; Methods in Enzymology (Academic Press, Inc. Vol. 154, Part E, 367-382 (1987); Analytical Biochemistry 179 (1989) 309-311.
In addition the invention provides a process for producing a G-CSF mutein according to the present invention in which a cell is transformed with a recombinant DNA according to the present invention or/and a recombinant vector according to the present invention, the transformed cell is cultured in a suitable medium and the protein is isolated from the cells or the medium. The methods usually used in molecular biology for the isolation of recombinant proteins from eukaryotic or prokaryotic cells are known to one skilled in the art and do not need to be elucidated in detail.
Finally the invention also provides a pharmaceutical preparation based on a G-CSF mutein according to the present invention as the active substance, if desired, together with the usual pharmaceutical carrier, filling and auxiliary substances. Such a pharmaceutical preparation is particularly suitable for the therapeutic fields of application mentioned above and ~ven for further therapeutic proceedures in which the formation of neutrophilic granulocytes is to be stimulated.
The following examples are intended to elucidate the invention without however limiting its scope.
E x a m p l e Production of the vector m~l-G-CSF-B~
The 554 bp long EcoRI/BamHI fragment from the vector pKK
177-3 G-CSF-Bg (DSM 5867) containing the Shine Dalgarno sequence, ATG codon and coding sequence for the G-CSF
gene is cloned via a blunt-end ligation into the NcoI
cleavage site of the vector pPZ 07-mgl lac (W088/09373, Figure lO). The ATG start codon of the lac Z gene, which is located in the protruding single strand after NcoI
digestion, is digested beforehand by incubation with mung bean nuclease (Pharmacia). The resulting vector is denoted mgl-G-CSF-Bg.
-~` 2 0 ~ 3 E x a m p 1 e 2 Mutagenesis of the amino acid Leu ~X) in the sequence Gly-His-Ser-Leu-Gly-Ile ,~ :
The mutagenesis is carried out on the M13 template according to known techniques (Amersham No. 1523 "Oligonucleotide-directed in vitro mutagenesis system").
A 251 bp long G-CSF cDNA fragment is isolated via the cleavage site BstXI/AatII. The protruding single-strands are digested off by mung bean nuclease (Pharmacia) and the fragment is cloned into the vector M13mpl9 which was cleaved with EcoRI/SmaI (EcoRI protruding single strand was filled in for blunt-end cloning). After preparing single-stranded DNA, the oligonucleotide is hybridized to the single-stranded DNA and an elongation in the 5'-3' direction beyond the oligonucleotide is carried out using Klenow polymerase, ligase and the four nucleotide triphosphates (GTP, CTP, TTP, ATP). The DNA which is now double-stranded is transformed in E. coli cells which carry a F' episome so that infection by filamentous M13 phages is possible (e.g. JM101, obtainable from Stratagene, LaJolla, California). Individual plaques are picked out and the mutagenized M13 phages contained therein are used for the preparation of single-stranded DNA. A DNA sequencing is carried out according to known techniques (e.g. dideoxy method according to Sanger) and the exact substitution to form the desired mutation is checked in this way. After preparing double-stranded DNA
the mutated AvaI fragment of G-CSF is isolated and cloned in the expression vector mgl-G-CSF-Bg (cleaved with AvaI).
. .
2 ~
In order to reconstitute the complete G-CSF gene the DNA
is subsequently cleaved with HindIII, the protruding ends are filled in with Klenow polymerase and afterwards partially digested with AvaI so that the 5' AvaI site in the G-CSF gene (at ca 130 bp) is not cleaved. This DNA
is ligated with the approximately 240 bp G-CSF fragment AvaI/BamHI (BamHI site is filled in with Klenow polymerase) from the starting vector mgl-G-CSF-Bg.
After transformation in E. coli JM83, the expression of G-CSF is carried out in the manner described in W~88/09373.
The cDNA used has a sequence which codes for a G-CSF
with 175 amino acids (without a signal sequence, but with a Met residue at position -1) so that the preferred mutation is located at Leu at position 54 of the G-CSF
amino acid sequence (in this the N-terminal Met residue is not counted).
The sequence of the cDNA encoding G-CSF which codes for the amino acids 50 to 56 (with reference to the G-CSF
with 174 amino acids) reads:
(X) Leu-Gly-His-Ser-Leu-Gly-Ile 5'-CTC GGA CAC TCT CTG GGC ATC-3' The corresponding complementary opposite strand to be mutagenized reads:
5l-GAT GCC CAG AGA GTG TCC GAG-3' 20~1~}~3 g The following 19 oligonucleotides correspond:ing to the opposite strand are used for site-directed mutagenesis:
Wild-type: 5'-3 GAT GCC CAG AGA GTG TCC~GAG 3' Met l. S' GAT GCC CAT AGA GTG TCC GAG 3' :
Phe 2. 5' GAT GCC GAA AGA GTG TCC GAG 3' Gln 3. 5' GAT GCC CTG AGA GTG TCC GAG 3' ^: Glu 4. 5' GAT GCC CTC AGA GTG TCC GAG :3' Asp ~ 5. 5' GAT GCC GTC AGA GTG TCC GAG 3' ,~ : Cys 6. 5' GAT GCC GCA AGA GTG TCC GAG 3' , ~ , ~; Ala 7. 5' GAT GCC GGC AGA GTG TCC GAG 3' ~ .~
Gly 8. 5' GAT GCC AGG AGA GTG TCC GAG 3' His 9. 5' GAT GCC GTG AGA GTG TCC GAG 3' Ile 10. 5' GAT GCC GAT AGA GTG TCC GAG 3' ~, .
Lys 11. 5' GAT GCC CTT AGA GTG TCC GAG 3' Tyr 12. 5I GAT GCC ATA AGA GTG TCC GAG 3' Asn 13. 5' GAT GCC GTT AGA GTG TCC GAG 3' Pro 14. 5' GAT GCC GGG AGA GTG TCC GAG 3' Arg 15. 5' GAT GCC GCG AGA GTG TCC GAG 3' Ser 16. 5' GAT GCC GGA AGA GTG TCC GAG 3' Thr 17. 5' GAT GCC GGT AGA GTG TCC GAG 3' Val 18. 5' GAT GCC GAC AGA GTG TCC GAG 3' Trp 19. 5' GAT GCC CCA AGA GTG TCC GAG 3' E x a m p l e 3 Productlon_of a G-CSF with modified activity A G-CSF which is more enzymatically active compared to the wild type can be produced by substituting serine at position 53 by a threonine at position 53 of a G-CSF
with 174 amino acids (serine in the sequence Gly-His--` 20~14~9 Ser-Leu-Gly). The following double-stranded oligonucleotide was used for the mutagenesis:
His Thr Leu Gly Ile S' CCC GAG GAG CTG GTG CTG CTC GGA CAC ACC CTG GGC ATC CCC TGG GCT CCC CTG AGC 3' 3' C CTC GAC CAC GAC GAG CCT GTG TGG GAC CCG TAG GGG ACC CGA GGG GAC 5' For the cloning, the G-CSF cDNA fragment (ca 300 bp, EcoRI/EcoRV) from the vector pKK 177-3 G-CSF-Bg (DSM
5867) was ligated into the EcoRI/SmaI cleavage site of the vector pUC19 (Yannish-Perron et al., (1985), Gene 33, 103).
This DNA is cleaved with AvaI/SacI and directly ligated with the primer pair described above according to the usual techniques. The mutated BstIXlSacI fragment can now be isolated from this construct and cloned into the vector pXK 177-3 G-CSF-Bg (DSM 5867) (cleaved with BstXI/SacI). The final construction of the expression clone is carried out in analogy to Example l. The determination of activity is carried out as described in Example 5.
E x a m p l e 4 Alteration of ,t~,he enzvmatic properties of G-CSF_by mutation of amin,o acids which are not located in the active_centre.
In analogy to known serine esterases it is assumed that the serine of the active centre interacts with histidine for the development of enzymatic activity. Four histidines are present in the sequence of G-CSF and namely at positions 43, 79, 156 and 170 (numbered from ~-` 2 0 ~ 9 the 174 aa sequence without a signal peptide~. The histidine residue at position 52 (or at position 55 in the 177 amino acid form) is left out of consideration in this mutagenesis. In this process His (CCA, CTA) is substituted by Gln (CAG). The sequence on the opposite strand corresponding to the codon coding for Gln is CTG.
:`
A G-CSF fragment is subcloned in M13mpl9 as described in Example 1.
The following~oligonucleotides corresponding to the opposite strand are used for the mutagenesis:
l. 5' GCT CCT GGG CTG GCA CAG C 3' histidine 43 to glutamine 43 2. 5' GAA AAG GCC GCT CTG GAG TTG GCT C 3' histidine 79 to glutamine 79 ;: : :
3. 5' GCT CTG CAG CTG GCC TAG CAA CC 3' histidine 156 to glutamine 156 4. 5' GGG CTG CGC AAG CTG GCG TAG AAC G 3 histidine 170 to glutamine 170 f, ~ ;
The analytical procedure and the recloning in an expression vector is carried out in analogy to Example 1.
:
~ 20~3~
E x a m p 1 e 5 Determination of the G-~SF act;ivity The ac~tivity of G-CSF is tested with the murine leukaemia line NSF60 which is completely dependent on G-CSF as described in Biochem. J. 253 (1988) ~213-218, Exp.
Hematol. 17 (1989) 116-119, Proc. Natl. Acad. Sci. USA
83 (1986) 5010. In order that the factor-dependency of the cells is preserved, the medium (RPMI medium, Boehringer Mannheim GmbH, Order No. 2099445 with 10 %
foetal calf serum) for the maintenance culture permanently contains 1000 U/ml G-CSF.
The proliferation of the NSF60 cells stimulated by G-CSF
is measured directly in this test by the incorporation of 3H thymidine. The test is carried out as follows:
NSF60 cells which are in the exponential growth phase (cell density is maximally lx105 cells/ml) are transferred to microtitre plates (lx104 celIs/well) and cuItured with a decreasing G-CSF concentration. The maximum dose of G-CSF in well 1 corresponds to the concentration in the maintenance culture (1000 U/ml, specific activity lx108 U/mg protein). The dilution is carried out in steps of ten.
After about 24 hours incubation 3H thymidine (0.1 ~Ci/well) is added. Afterwards the cells are incubated for a further 16 hours.
In order to evaluate the test the cells in the microtitre plates are frozen in order to lyse them. The cell lysate is aspirated on a glass fibre filter, 20~3~
rinsed, dried and measured in a scintillation counter.
The incorporation of 3H thymidine is proportional to the G-CSF-induced proliferation of the NSF60 cells.
E x a m p 1 e 6 , Alt ration i~__he activity of G-CSF by_amino acid substitution in the active centre A G-CSF modified in amino acid position 54 can be produced by substitution of preferably one leucine at position 54 by a threonine at position 54 (Leu in the sequence Gly-His-Ser-Leu-Gly) in correspondence with the procedure described in Example 3 using a suitable double-stranded oligonucleotide which contains a nucleic acid triplet (e.g. ACC) coding for the amino acid Thr at the appropriate position. In this connection position 54 of the 174 amino acid form of G-CSF corresponds to position 57 of the 177 amino acid form.
The activity of a mutant having 174 amino acids with Thr at position 54 is reduced in the NSF60 cell test (see Example 5) in comparison to the wild-type G-CSF with 174 amino acids. Moreover, the activity of this G-CSF mutant is reduced in comparison to a G-CSF mutant with an amino acid substitution of a serine by a threonine at position 53 (described in Example 3).
Claims (19)
1. Granulocyte stimulating factor (G-CSF) or G-CSF
variant, w h e r e i n one or several amino acids of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile at position 50 to 56 of the mature G-CSF with 174 amino acids or at position 53 to 59 of the mature G-CSF with 177 amino acids or/and at least one of the 4 His residues at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids are mutagenized.
variant, w h e r e i n one or several amino acids of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile at position 50 to 56 of the mature G-CSF with 174 amino acids or at position 53 to 59 of the mature G-CSF with 177 amino acids or/and at least one of the 4 His residues at position 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids are mutagenized.
2. G-CSF mutein as claimed is claim 1, w h e r e i n it contains a N-terminal Met residue at position -1.
3. G-CSF mutein as claimed in claim 1 or 2, w h e r e i n one amino acid of the sequence Leu-Gly-His-Ser-Leu-Gly-Ile is substituted by another amino acid.
4. G-CSF mutein as claimed in one of the claims 1 to 3, w h e r e i n the Ser residue at position 53 of the mature G-CSF with 174 amino acids or at position 56 of the mature G-CSF with 177 amino acids is substituted by another amino acid.
5. G-CSF mutein as claimed in claim 4, w h e r e i n the other amino acid is Thr.
6. G-CSF mutein as claimed in one of the claims 1 to 3, w h e r e i n the Leu residue at position 54 of the mature G-CSF with 174 amino acids or at position 57 of the mature G-CSF with 177 amino acids is substituted by another amino acid.
7. G-CSF mutein as claimed in claim 6, w h e r e i n the other amino acid is Thr.
8. G-CSF mutein as claimed in claim 1 or 2, w h e r e i n one of the 4 His residues at position 43, 79, 156 or 170 of the mature G-CSF
with 174 amino acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids is substituted by another amino acid.
with 174 amino acids or at position 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids is substituted by another amino acid.
9. G-CSF mutein as claimed in claim 8, w h e r e i n the other amino acid is Gln.
10. Recombinant DNA, w h e r e i n it codes for a G-CSF mutein as claimed in one of the claims 1 to 9.
11. Recombinant vector, w h e r e i n it contains at least one copy of a recombinant DNA as claimed in claim 10.
12. Recombinant vector as claimed in claim 11, w h e r e i n it is suitable for gene expression in prokaryotic cells.
13. Cell, w h e r e i n it is transformed with a recombinant DNA as claimed in claim 10 or/and a recombinant vector as claimed in claim 11 or 12.
14. Cell as claimed in claim 13, w h e r e i n it is a prokaryotic cell.
15. Process for the production of recombinant DNA as claimed in claim 10, w h e r e i n a DNA
sequence which codes for G-CSF or for a G-CSF
variant is site-specifically mutagenized.
sequence which codes for G-CSF or for a G-CSF
variant is site-specifically mutagenized.
16. Process as claimed in claim 15, w h e r e i n synthetic oligonucleotides are used as mutagenesis primers.
17. Process for the production of a protein with G-CSF
activity as claimed in one of the claims 1 to 9, w h e r e i n a cell is transformed with a recombinant DNA as claimed in claim 10 or/and a recombinant vector as claimed in claim 11 or 12 the transformed cell is cultured in a suitable medium and the protein is isolated from the cells or from the medium.
activity as claimed in one of the claims 1 to 9, w h e r e i n a cell is transformed with a recombinant DNA as claimed in claim 10 or/and a recombinant vector as claimed in claim 11 or 12 the transformed cell is cultured in a suitable medium and the protein is isolated from the cells or from the medium.
18. Pharmaceutical preparation, characterized by one or several G-CSF muteins as claimed in one of the claims 1 to 9 as the active substance, if desired, together with the usual pharmaceutical carrier, filling and auxiliary substances.
19. Use of a G-CSF mutein as claimed in one of the claims 1 to 9 for immunotherapy.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4014750.9 | 1990-05-08 | ||
| DE4014750A DE4014750A1 (en) | 1990-05-08 | 1990-05-08 | MUTEINE OF THE GRANULOCYTE-STIMULATING FACTOR (G-CSF) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2041439A1 true CA2041439A1 (en) | 1991-11-09 |
Family
ID=6405959
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002041439A Abandoned CA2041439A1 (en) | 1990-05-08 | 1991-04-29 | Muteins of the granulocyte colony stimulating factor (g-csf) |
Country Status (17)
| Country | Link |
|---|---|
| EP (1) | EP0456200A1 (en) |
| JP (1) | JPH04225998A (en) |
| KR (1) | KR940000757B1 (en) |
| CN (1) | CN1057862A (en) |
| AU (1) | AU631312B2 (en) |
| CA (1) | CA2041439A1 (en) |
| CS (1) | CS133391A3 (en) |
| DE (1) | DE4014750A1 (en) |
| FI (1) | FI912203A (en) |
| HU (1) | HUT58801A (en) |
| IE (1) | IE911359A1 (en) |
| IL (1) | IL98070A0 (en) |
| NO (1) | NO911786L (en) |
| NZ (1) | NZ237975A (en) |
| PL (1) | PL290166A1 (en) |
| PT (1) | PT97611A (en) |
| ZA (1) | ZA913430B (en) |
Families Citing this family (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5718893A (en) * | 1984-04-15 | 1998-02-17 | Foster; Preston F. | Use of G-CSF to reduce acute rejection |
| GB9107846D0 (en) * | 1990-04-30 | 1991-05-29 | Ici Plc | Polypeptides |
| US5581476A (en) * | 1993-01-28 | 1996-12-03 | Amgen Inc. | Computer-based methods and articles of manufacture for preparing G-CSF analogs |
| US5536495A (en) * | 1994-04-15 | 1996-07-16 | Foster; Preston F. | Use of G-CSF to reduce acute rejection |
| US20030053982A1 (en) | 1994-09-26 | 2003-03-20 | Kinstler Olaf B. | N-terminally chemically modified protein compositions and methods |
| US5824784A (en) | 1994-10-12 | 1998-10-20 | Amgen Inc. | N-terminally chemically modified protein compositions and methods |
| KR100440460B1 (en) * | 1998-07-08 | 2004-10-08 | 주식회사유한양행 | Gene, recombinant vector and transformant of hG-CSF and method of producing hG-CSF using the same |
| US6245740B1 (en) | 1998-12-23 | 2001-06-12 | Amgen Inc. | Polyol:oil suspensions for the sustained release of proteins |
| DE19860801A1 (en) * | 1998-12-30 | 2000-07-06 | Rhein Biotech Proz & Prod Gmbh | Recombinant growth factor with the biological activity of a G-CSF (Granulocyte Colony Stimulating Factor) |
| AU9096001A (en) * | 2000-09-08 | 2002-03-22 | Amgen Inc | G-csf analog compositions and methods |
| ES2309167T3 (en) | 2001-02-19 | 2008-12-16 | Merck Patent Gmbh | METHOD FOR IDENTIFYING T-CELL EPITOTS AND METHOD FOR PREPARING MOLECULES WITH REDUCED IMMUNOGENICITY. |
| DE60236522D1 (en) | 2001-07-11 | 2010-07-08 | Maxygen Inc | G-CSF CONJUGATES |
| US7785601B2 (en) | 2002-12-31 | 2010-08-31 | Sygnis Bioscience Gmbh & Co. Kg | Methods of treating neurological conditions with hematopoietic growth factors |
| US7695723B2 (en) | 2002-12-31 | 2010-04-13 | Sygnis Bioscience Gmbh & Co. Kg | Methods of treating neurological conditions with hematopoietic growth factors |
| US7220407B2 (en) | 2003-10-27 | 2007-05-22 | Amgen Inc. | G-CSF therapy as an adjunct to reperfusion therapy in the treatment of acute myocardial infarction |
| DE102004041639A1 (en) | 2004-08-27 | 2006-03-02 | Bioceuticals Arzneimittel Ag | Method for obtaining biologically active human G-CSF from inclusion bodies |
| AU2005306894B2 (en) | 2004-11-05 | 2011-11-24 | Northwestern University | Use of SCF and G-CSF in the treatment of cerebral ischemia and neurological disorders |
| RU2007149238A (en) | 2005-06-01 | 2009-07-20 | Максиджен Холдингз Лтд. (Ky) | PEGylATED GKSF POLYPEPTIDES AND METHODS FOR PRODUCING THEM |
| DE102005033250A1 (en) | 2005-07-15 | 2007-01-18 | Bioceuticals Arzneimittel Ag | Process for purifying G-CSF |
| DE102006009437A1 (en) | 2006-03-01 | 2007-09-13 | Bioceuticals Arzneimittel Ag | G-CSF liquid formulation |
| WO2009023566A2 (en) | 2007-08-09 | 2009-02-19 | Genzyme Corporation | Method of treating autoimmune disease with mesenchymal stem cells |
| ES2524454T3 (en) | 2007-08-27 | 2014-12-09 | Ratiopharm Gmbh | G-CSF liquid formulation |
| DE102007040932A1 (en) | 2007-08-27 | 2009-03-05 | Biogenerix Ag | Liquid granulocyte colony-stimulating factor formulation, e.g. for treating cancer, neutropenia, HIV infections or neurological disorders, comprises a sugar alcohol, surfactant and acetate buffer |
| DE202008017456U1 (en) | 2007-08-27 | 2009-08-27 | Biogenerix Ag | Liquid formulation of G-CSF conjugates |
| US8758761B2 (en) | 2007-09-30 | 2014-06-24 | University Of Florida Research Foundation, Inc. | Combination therapies for treating type 1 diabetes |
| WO2011109556A2 (en) | 2010-03-04 | 2011-09-09 | Pfenex Inc. | Method for producing soluble recombinant interferon protein without denaturing |
| WO2011123570A2 (en) | 2010-04-01 | 2011-10-06 | Pfenex Inc. | Methods for g-csf production in a pseudomonas host cell |
| EP2399572A1 (en) | 2010-06-22 | 2011-12-28 | Sandoz AG | Long-term storage of non-glycosylated recombinant human G-CSF |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06102021B2 (en) * | 1985-12-03 | 1994-12-14 | 中外製薬株式会社 | Novel polypeptide |
| JPS63500636A (en) * | 1985-08-23 | 1988-03-10 | 麒麟麦酒株式会社 | DNA encoding multipotent granulocyte colony stimulating factor |
| WO1988001297A1 (en) * | 1986-08-11 | 1988-02-25 | Cetus Corporation | Expression of g-csf and muteins thereof |
| HU215241B (en) * | 1986-10-24 | 1998-11-30 | Cetus Corp. | Process for producing new forms of colony stimulating factor-1 and expression cassette, vector and recombinant host cells used in the process |
| NO176799C (en) * | 1986-12-23 | 1995-05-31 | Kyowa Hakko Kogyo Kk | DNA encoding a polypeptide, recombinant plasmid that encodes and can express a polypeptide and method for producing this polypeptide |
-
1990
- 1990-05-08 DE DE4014750A patent/DE4014750A1/en not_active Withdrawn
-
1991
- 1991-04-23 IE IE135991A patent/IE911359A1/en unknown
- 1991-04-29 NZ NZ237975A patent/NZ237975A/en unknown
- 1991-04-29 CA CA002041439A patent/CA2041439A1/en not_active Abandoned
- 1991-05-02 JP JP3100666A patent/JPH04225998A/en active Pending
- 1991-05-06 IL IL98070A patent/IL98070A0/en unknown
- 1991-05-06 AU AU76380/91A patent/AU631312B2/en not_active Ceased
- 1991-05-07 FI FI912203A patent/FI912203A/en not_active Application Discontinuation
- 1991-05-07 NO NO91911786A patent/NO911786L/en unknown
- 1991-05-07 CS CS911333A patent/CS133391A3/en unknown
- 1991-05-07 HU HU911529A patent/HUT58801A/en unknown
- 1991-05-07 EP EP91107429A patent/EP0456200A1/en not_active Withdrawn
- 1991-05-07 ZA ZA913430A patent/ZA913430B/en unknown
- 1991-05-08 CN CN91103929A patent/CN1057862A/en active Pending
- 1991-05-08 KR KR1019910007419A patent/KR940000757B1/en not_active IP Right Cessation
- 1991-05-08 PT PT97611A patent/PT97611A/en not_active Application Discontinuation
- 1991-05-08 PL PL29016691A patent/PL290166A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04225998A (en) | 1992-08-14 |
| IL98070A0 (en) | 1992-06-21 |
| CN1057862A (en) | 1992-01-15 |
| KR910020033A (en) | 1991-12-19 |
| AU7638091A (en) | 1991-11-14 |
| NO911786L (en) | 1991-11-11 |
| FI912203A0 (en) | 1991-05-07 |
| ZA913430B (en) | 1992-02-26 |
| PL290166A1 (en) | 1992-01-13 |
| HUT58801A (en) | 1992-03-30 |
| NZ237975A (en) | 1992-09-25 |
| KR940000757B1 (en) | 1994-01-29 |
| EP0456200A1 (en) | 1991-11-13 |
| PT97611A (en) | 1992-03-31 |
| AU631312B2 (en) | 1992-11-19 |
| FI912203A (en) | 1991-11-09 |
| NO911786D0 (en) | 1991-05-07 |
| IE911359A1 (en) | 1991-11-20 |
| DE4014750A1 (en) | 1991-11-14 |
| HU911529D0 (en) | 1991-11-28 |
| CS133391A3 (en) | 1992-01-15 |
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