WO2006135176A1 - Human granulocyte-colony stimulating factor isoforms - Google Patents
Human granulocyte-colony stimulating factor isoforms Download PDFInfo
- Publication number
- WO2006135176A1 WO2006135176A1 PCT/KR2006/002238 KR2006002238W WO2006135176A1 WO 2006135176 A1 WO2006135176 A1 WO 2006135176A1 KR 2006002238 W KR2006002238 W KR 2006002238W WO 2006135176 A1 WO2006135176 A1 WO 2006135176A1
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- WIPO (PCT)
- Prior art keywords
- colony stimulating
- stimulating factor
- granulocyte colony
- human granulocyte
- amino acid
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Classifications
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/193—Colony stimulating factors [CSF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Definitions
- the present invention relates to a novel human granulocyte colony stimulating factor isoform, by which in vivo lifetime of human granulocyte colony stimulating factor (rhG-CSF) is prolonged. More particularly, the present invention relates to a human granulocyte colony stimulating factor isoform, obtained from the covalent bonding of polyethylene glycol as a non-protein region to a human granulocyte colony stimulating factor isoform having cysteine added to the N-terminal or the C-terminal thereof.
- rhG-CSF human granulocyte colony stimulating factor
- Human granulocyte colony stimulating factor has the main biological function of stimulating specific leucocytes, known as neutrophilic granulocytes or neutrophils, to accomplish growth and development thereof in vivo (Welte et al., PNAS, 82, 1526-1530, 1985: Souza et al Science, 232, 61-65, 1986).
- neutrophilic granulocytes function to protect a biological species against infection with microorganisms, when they are discharged to blood flow.
- Human granulocyte colony stimulating factor is a protein capable of forming a complex with a receptor thereof in a ratio of 2:2 via the dimerization of the receptor (Horan et al., Biochemistry, 35, 4886-96, 1996).
- Aritomi et al. have shown the X-ray structure of the BN-BC domain complex of human granulocyte colony stimulating factor with a receptor thereof (Nature, 401, 713-717, 1999). It is reported by Aritomi et al. that amino acids of human granulocyte colony stimulating factor, which exist in contact region or adjacent region thereof when the receptor is bound to human granulocyte colony stimulating factor, include G4, P5, A6, S7, S8, L9, PlO, QI l, S12, L15, K16, E19, Q20, L108, D109, Dl 12, Tl 15, Tl 16, Ql 19, E122, E123 and L124.
- protein removal in the kidney depends on the physical properties of a protein or a polymer thereof, such as size (molecular diameter), symmetry, shape/ rigidity or charge, or the like.
- Protein removal caused by the receptor present on the surface of leucocytes is executed by the introduction of a receptor-bound polypeptide into cells, and by lysosomal degradation of protein in the presence of proteases present in endosomes.
- Degradation of protein by bonding of a receptor to human granulocyte colony stimulating factor are described in detail by Saker and Roufenburger (MoI. Pharmacol., 2003, 63, 147-158) and Saker (Nature biotech. 2002, 20, 908-913).
- adhesion of a polymer to a protein can interrupt proteases effectively and thus can prevent functions of non-specific proteases.
- polyethylene glycol is one of the polymers widely used to prepare therapeutic protein products.
- Surface modification of a protein molecule caused by bonding of a protein drug to a synthetic polymer can increase the solubility of the drug to water or organic solvents. Accordingly, it is possible to increase the biocompatibility of the drug, to reduce the immunoreactivity, to improve the in vivo stability, and to retard clearance in the intestinal system, kidney, spleen or liver. Clearance of small protein molecules is made by filtering in the intestinal system or kidney. Hence, when a high- molecular weight PEG is bound to such small protein molecules, it is possible to prevent such clearance (Knauf, M. J. et al, J. Hiol. Chem.
- PEG is attached to the surface of protein by forming a covalent bond with at least one free lysine residue.
- a site which is present on the protein surface and is directly related to the activity of the protein, is bound to PEG, the activity of the protein decreases.
- various kinds of PEG-bound protein conjugates are present in the form of a mixture, so that separation of a desired conjugate in a pure state becomes complicated and difficult.
- EP 0401384 discloses a material and a method for producing polyethylene glycol-added G-CSF.
- EP0335423 discloses a modified polypeptide having an activity of human granulocyte colony stimulating factor.
- the polyethylene glycol molecule cannot be bound to a predictable specific residue but bound to any reactive group present in the protein, such as a lysine residue, or the N-terminal in a nonspecific manner, resulting in the formation of a non-homogeneous product.
- USP5,766,897 and WO00/42175 disclose a method of bonding human growth hormone with PEG, wherein PEG is allowed to react selectively with cysteine by using PEG-maleimide so as to prevent PEG from reacting with the active region of human growth hormone.
- 2OkDa polyethylene glycol bound to the N-terminal has been developed and commercialized (Neulasta).
- the polyethylene glycol-polymerized human granulocyte colony stimulating factor has an increased half life, and thus can reduce administration frequency.
- human granulocyte colony stimulating factor examples include E. coli-derived filgrastim (trade name: Gran and Neupogen), lenograstim (trade name: Neutroginand Granocyte) produced in animal cells, such as Chinese Hamster Ovary (CHO) cells, and E. coli-derived nartograstim (trade name: Neu-up), in which mutation occurs at the N-terminal regions in five amino acid sequences in order to increase the efficacy of human granulocyte colony stimulating factor protein. Disclosure of Invention
- human granulocyte colony stimulating factor isoforms according to the prior art still have problems in that such amino acid sequence-modified human granulocyte colony stimulating factor isoforms have a low activity or insufficient half life in vivo, or non-homogeneous products are generated upon the bonding of PEG to human granulocyte colony stimulating factor.
- the present invention has been made in view of the above-mentioned problems. It is an object of the present invention to provide a human granulocyte colony stimulating factor isoform having an excellent activity and a significantly increased half life by bonding polyethylene glycol to amino acid sequence-modified human granulocyte colony stimulating factor, wherein the amino acid sequence is modified by substitution with or addition of cysteine in a specific site in order to facilitate bonding polyethylene glycol to human granulocyte colony stimulating factor, and then the modified site is partially bound to polyethylene glycol.
- human granulocyte colony stimulating factor isoforms have been generally produced by modifying the sequence in the polypeptide and performing pegylation at the position of a suitable amino acid in a specific site so as to increase the in vivo activity or lifetime.
- the inventors of the present invention have found that a novel isoform of human granulocyte colony stimulating factor, which shows an increased in vivo activity or lifetime compared to the isoforms according to the prior art, can be obtained by adding amino acids to both terminals of the polypeptide of human granulocyte colony stimulating factor and performing pegylation at the same addition sites. The present invention is based on this finding.
- a human granulocyte colony stimulating factor isoform which has at least one amino acid added before Thr of the N-terminal of human granulocyte colony stimulating factor represented by Sequence No. 1 or after Pro of the C-terminal thereof, wherein at least one of the amino acids added to human granulocyte colony stimulating factor is cysteine and at least one cysteine is bound to polyethylene glycol.
- the cysteine may be added between the Met and the threonine of the N-terminal.
- the above isoform is also included in the scope of the present invention.
- 1-12 amino acids may be added to each terminal of the sequence.
- one cysteine is added. More preferably, the cysteine is 175C, added to the C-terminal.
- polyethylene glycol is covalently bonded to the cysteine added to human granulocyte colony stimulating factor.
- polyethylene glycol has a molecular weight of 20 kDa ⁇ 40 kDa. More preferably, polyethylene glycol is branched.
- At least one amino acid in human granulocyte colony stimulating factor is substituted with another amino acid before polyethylene glycol is bound to human granulocyte colony stimulating factor.
- a pharmaceutical composition comprising the aforementioned human granulocyte colony stimulating factor isoform and a pharmaceutically acceptable carrier.
- an oligodeoxynucleotide used as a primer for modifying the amino acid sequence of human granulocyte colony stimulating factor into the aforementioned human granulocyte colony stimulating factor isoform.
- the primer is the oligodeoxynucleotide represented by any one of Sequence Nos. 2-7.
- the human granulocyte colony stimulating factor isoform according to the present invention shows a significantly increased in vivo activity or lifetime, and thus can reduce administration frequency.
- FIG. 1 illustrates the genetic sequence and protein sequence of human granulocyte colony stimulating factor
- FIG. 2 illustrates the amino acid sequence of a human granulocyte colony stimulating factor isoform, amino acid-modified at the N-terminal site
- FIG. 3 illustrates the amino acid sequence of a human granulocyte colony stimulating factor isoform, amino acid-modified at the C-terminal site
- FIG. 34 illustrates the amino acid sequence of a human granulocyte colony stimulating factor isoform, amino acid-modified at the C-terminal site
- FIG. 4 illustrates the amino acid sequence of a human granulocyte colony stimulating factor isoform, amino acid-modified at the middle part of the polypeptide;
- FIG. 5 illustrates the structure of a plasmid vector expressing a human granulocyte colony stimulating factor isoform;
- FIG. 6 is a graph showing the in vivo lifetime of human granulocyte colony stimulating factor isoforms in rats. Best Mode for Carrying Out the Invention
- FIG. 1 illustrates the genetic sequence and protein sequence of human granulocyte colony stimulating factor.
- Amino acid sequence No. 1 is the same as the amino acid sequence represented in FIG. 1 with the exception of Met in - 1 position.
- FIG. 2 illustrates the amino acid sequence of a human granulocyte colony stimulating factor isoform, amino acid-modified at the N-terminal site.
- AA represents amino acids
- n is the number of amino acids.
- N is an integer ranging from 1 to 12.
- the amino acids include at least one cysteine.
- FIG. 3 illustrates the amino acid sequence of a human granulocyte colony stimulating factor isoform, amino acid-modified at the C-terminal site.
- AA represents amino acids
- n is the number of amino acids.
- N is an integer ranging from 1 to 12.
- the amino acids include at least one cysteine.
- FIG. 4 illustrates the amino acid sequence of a human granulocyte colony stimulating factor isoform, amino acid-modified at the part comprising a sugar chain.
- 133-threonine is substituted with cysteine, and polyethylene glycol is covalently bonded with the same cysteine.
- This is a known isoform according to the prior art (see Korean Laid-Open Patent No. 2002-0079778).
- FIG. 5 illustrates the structure of a plasmid vector expressing a human granulocyte colony stimulating factor isoform.
- PR of a bacteriophage lamda is used.
- the plasmid vector includes cl 857 genes capable of inducing such expression in the plasmid vector.
- FIG. 6 is a graph showing the in vivo lifetime of human granulocyte colony stimulating factor isoforms in rats.
- PBS carrier is used as a control
- test samples include: T133C20kDa(Br) (G-CSF isoform obtained by substituting 133-Thr with cysteine and covalently bonding branched PEG with 20 kDa to the same cysteine according to Korean Laid-Open Patent No.
- N+20kDa(Br) G-CSF isoform obtained by adding cysteine between the threonine and methionine of the N-terminal (-1C) of G-CSF and covalently bonding branched PEG with 20 kDa to the same cysteine
- 175C+20kDa(Br) G-CSF isoform obtained by adding cysteine after 174-Pro of the C-terminal of G-CSF and covalently bonding branched PEG with 20 kDa to the same cysteine
- 175C+40kDa(Br) G-CSF isoform obtained by adding cysteine after 174-Pro of the C-terminal of G-CSF and covalently bonding branched PEG with 40 kDa to the same cysteine).
- Each isoform is diluted with PBS, and
- the inventors of the present invention have studied the protein structure of human granulocyte colony stimulating factor and the effect of the amino acids forming human granulocyte colony stimulating factor upon the activity thereof. Reference has been made to the structure of human granulocyte colony stimulating factor, information about the effect of the amino acids forming human granulocyte colony stimulating factor upon the activity thereof, and to the results of GOR4 protein secondary structure prediction used to determine the effect of the modification of amino acids upon the protein structure (GOR program in Secondary Structure Prediction, http:us.expasy.org).
- human granulocyte colony stimulating factor isoforms include analogs, variants, mutants, conjugates, or the like, which maintain the indigenous activity of human granulocyte colony stimulating factor, even though they experience modification of one amino acid residue into another amino acid residue in the original amino acid sequence of human granulocyte colony stimulating factor.
- amino acid position amino acid position
- amino acid position amino acid position
- cysteine amino acid position
- the primer used to induce the modification of an amino acid at a specific site is represented by the expression (single letter for an amino acid) (amino acid position) (single letter for another amino acid) 1 or 2, wherein suffix 1 represents the primer for a single strand template progressing in the direction of 5' ⁇ 3' in a double strand template, while suffix 2 represents the primer for a single strand template progressing in the direction of 3' ⁇ 5'.
- the human granulocyte colony stimulating factor genes obtained by the above process may be modified in the position at least one codon.
- modification refers to a change in the amino acid sequence of human granulocyte colony stimulating factor caused by substitution or addition of at least one codon in the genes encoding human granulocyte colony stimulating factor. More particularly, such modification includes: addition of cysteine before threonine, the first amino acid in the amino acid sequence of human granulocyte colony stimulating factor; addition of cysteine after proline of the C-terminal of the same amino acid sequence; sequence modification in the middle of the polypeptide sequence in addition to the modification at the terminal sites; or the like.
- T133C means substitution of threonine, the 133 r amino acid of natural human granulocyte colony stimulating factor, with cysteine.
- a synthetic oligonucleotide including a codon that encodes modification of a target amino acid in human granulocyte colony stimulating factor is constructed.
- an oligonucleotide with a length corresponding to about 27-36 nucleotides is used.
- the oligonucleotide preferably has 12-15 nucleotides complementary to the template at both sides of the nucleotide encoding modification.
- Such oligonucleotides may be sufficiently hybridized with the template DNA.
- a synthetic oligonucleotide used for the amino acid modification according to the present invention is shown in the following Table 1.
- the oligonucleotide may be prepared by a method known to those skilled in the art.
- DNA of human granulocyte colony stimulating factor isoform is constructed.
- PCR is performed by using human granulocyte colony stimulating factor DNA (Table 1) as a template and a synthetic oligonucleotide encoding modification as a primer. If the double- strand template is separated in the heating step of PCR, a complementary primer is hybridized with each single strand template.
- DNA polymerase allows the nucleotides complementary to the template to be linked continuously thereto from -OH groups of the primer encoding modification along the direction of 5' ⁇ 3'.
- the second strand includes the primer encoding modification, so that the resultant genes encode the target modification.
- the second strand functions as a template DNA during repeated replication steps, so that the genes encoding modification can be amplified continuously.
- T133C refers to substitution of threonine, the 133 r amino acid of natural human granulocyte colony stimulating factor, with cysteine.
- PCR is performed by using natural granulocyte colony stimulating factor DNA (FIG. 1) as a template and by introducing each of the primer pairs formed of N-term with T133C2 and T 133Cl with C-term. By doing so, two DNA fragments, having a modified codon corresponding to cysteine that substitutes for threonine, the 133 r amino acid, can be obtained.
- PCR is further carried out by introducing the two DNA fragments and using primer pairs of N-term and C-term.
- threonine is substituted with cysteine at the position of the 133 r amino acid.
- amino acid addition is made at the N-terminal or the C-terminal.
- Construction of human granulocyte colony stimulating factor having at least one modified amino acid can be performed at the N- terminal and the C-terminal.
- modification includes the sequence of Met- (AA)n-Thr- at the N-terminal (see FIG. 2) and the sequence of -Gin-Pro- (AA)n at the C-terminal (see FIG. 3).
- AA means any amino acid and particular examples thereof include an amino acid that has a low molecular weight and shows no specific function, such as glycine, serine or alanine.
- At least one AA includes cysteine.
- Suffix n means the number of amino acids and ranges from 1 to 12.
- At least about 12 amino acids are required for the in vivo recognition of the modified human granulocyte colony stimulating factor, to which amino acids are added, as a heterogeneous protein, followed by the production of an antibody. This is why the maximum number of amino acids is limited to 12.
- the N-terminal has the amino acid sequence of Met- (AA)n-Thr- and includes the DNA base sequence of ATG-(NNN)n-ACT-, while the C- terminal has the amino acid sequence of -Gln-Pro-(AA)n- and includes the DNA base sequence of -CAG-CCG- (NNN)n-TAA.
- NNN represents the DNA base sequence that encodes the corresponding amino acid.
- human granulocyte colony stimulating factor genes having at least one modified amino acid are constructed by using the primer including the DNA base sequence obtained as described above and human granulocyte colony stimulating factor genes as shown in FIG. 1.
- the amino acid sequence at the N-terminal may include the sequence of Met- (Gly-Cys-)Thr-.
- the primer used herein may be designed to have the sequence of GTAATAAATA-ATG- (GGT-TGT-) ACT-.
- modified human granulocyte colony stimulating factor genes having the sequence of Met-(Gly-Cys-)Thr- may be constructed from the above primer, by using the template of human granulocyte colony stimulating factor genes as shown in FIG. 1 and the C-term primer as shown in Table 1.
- the human granulocyte colony stimulating factor genes obtained as described above are further subjected to PCR by using N-term and C-term primers.
- - 1C/T133C includes addition of cysteine to the N-terminal and substitution of the threonine, the 133 r amino acid of the polypeptide of human granulocyte colony stimulating factor, with cysteine.
- PCR is performed by using preformed DNA of -1C human granulocyte colony stimulating factor isoform as a template and by introducing primer pairs of N-term with T122C2 and T133C1 with C- term. By doing so, two DNA fragments, having a modified codon corresponding to cysteine that substitutes for threonine, the 133 r amino acid, can be obtained. Then, PCR is further carried out by introducing the two DNA fragments and using primer pairs of N-term and C-term.
- Modification of amino acid in the middle of the polypeptide of the amino acid- added isoform including at least one cysteine at the N-terminal or the C-terminal can be performed at a site known to those skilled in the art.
- methods of substituting at least one amino acid in the middle of the polypeptide of human granulocyte colony stimulating factor with another amino acid are disclosed in Korean Patent Publication No. 0248111 (N-terminal polyethylene glycol-bound G-CSF or analogues thereof, and preparation thereof), Korean Laid-Open Patent Nos. 2002-0007297 (G-CSF conjugates), 2002-0079778 (G-CSF conjugates) and 2004-0084884(G-CSF conjugates), US Patent Nos.
- the DNA sequence that encodes the human granulocyte colony stimulating factor isoforms according to the present invention may be synthesized by the conventional method known to those in the art, for example, by using an automatic DNA synthesizer (available from Biosearch, Applied Biosystem , etc.).
- a recombinant expression vector including the DNA sequence encoding the human granulocyte colony stimulating factor isoform and a host cell transformed or transfected with the above expression vector.
- polypeptide may be used interchangeably with “protein” or “physiologically active protein”.
- the term “isoform” means a physiologically active protein, which has the main function of the original physiologically active protein but is modified with a genetic engineering means or others.
- modification refers to substitution, addition and loss in an amino acid sequence, and includes substitution with cysteine or addition of cysteine for carrying out bonding with polyethylene glycol. Further, such modification also includes a covalent bonding formed between cysteine and polyethylene glycol via a chemical reaction.
- the term "in vivo lifetime” is defined as follows: a physiologically active protein loses its function when it is administered to humans or animals, because the total amount and activity of the physiologically active protein decrease due to the protease-mediated removal and the removal in the kidney. Therefore, the term “in vivo lifetime” means the retention time of a protein during which the protein can perform its function in vivo, and is represented by the in vivo half life of the physiologically active protein.
- the term "vector” refers to a DNA molecule as a carrier capable of delivering foreign genes stably into a host cell. To be a useful vector, it is necessary for a vector to allow replication, to have a means, through which it can be introduced into a host cell, and to include a means for detecting itself. Additionally, the term “recombinant expression vector” refers to a cyclic DNA molecule formed by a vector linked operatively to foreign genes so that the foreign genes can be expressed in a host cell.
- a DNA vector having human granulocyte colony stimulating factor inserted thereto may be produced.
- the corresponding genes should be linked operatively to a transcription and translation expression regulatory sequence that functions in a selected host cell, as generally known in the art.
- the expression regulatory sequence and the corresponding genes are included in a single expression vector that further includes a bacterial selection marker and a replication origin.
- a suitable vector including not only the genes encoding human granulocyte colony stimulating factor isoforms but also the above elements (e.g. regulatory sequence) can be constructed by using basic recombinant DNA technique.
- individually separated DNA fragments should be linked to each other.
- each DNA fragment is cleaved first by using restriction enzymes, and then the DNA fragments are linked to each other considering a specific order and orientation.
- DNA may be cleaved by using a specific restriction enzyme in a suitable buffer.
- a specific restriction enzyme in general, about 0.2- ID of a plasmid or DNA fragment is used in about 2OD of a buffer solution along with about 1-2 units of the corresponding restriction enzyme.
- Suitable buffer, DNA concentration, reaction time and reaction temperature will be determined by the producer of the restriction enzyme. Generally, the reaction is carried out at 37 0 C for about 1-2 hours. However, some enzymes require a higher temperature. After the reaction, enzymes and other impurities are removed by extracting the digestion solution with a mixture of phenol and chloroform. DNA may be precipitated with ethanol to recover it from aqueous layer. At this time, terminals of DNA fragments to be linked to each other should be compatible with each other in order to link DNA fragments so that a functional vector can be formed.
- DNA electrophoresis may be performed through an agarose or poly- acrylamide matrix. Selection of the matrix depends on the size of DNA to be separated. After the electrophoresis, DNA is extracted from the matrix via electroelution, or extracted directly from the matrix. When a low-melting agarose matrix is used, agarose should be melted before the extraction of DNA.
- DNA fragments to be linked to each other should be added to a solution in an equimolar amount.
- the solution contains ATP, ligase buffer, and about 10 units ligase such as T4 ligase per 0.5D of DNA.
- the vector should be cleaved by a suitable restriction enzyme first to form a linear vector.
- a suitable restriction enzyme may be used after it is treated with alkali phosphatase or bovine intestinal hydrolase. Such treatment with hydrolase prevents the vector from self-ligation.
- the recombinant expression vector constructed as described above is used to perform transformation or transfection of a host cell.
- Polynucleotides can be introduced into a host cell according to a known method disclosed in laboratory manuals such as [Davis et al., Basic Methods in Molecular biology (1986)] and [Sambrook, J., et al. (1989) "Molecular Cloning” A laboratory Manual 2° edition]. Preferred methods of introducing polynucleotides into a host cell include calcium chloride transformation, electroporation, or the like.
- host cells are incubated in a nutrition medium suitable for the production of polypeptides by using a known technique.
- hose cells may be incubated in a laboratory or industrial fermenting instrument via small-scale or large-scale fermentation/shaking flask incubation with a suitable medium under the conditions permitting expression and/or secretion of polypeptides.
- Incubation is performed in a suitable nutrition medium containing a carbon source, a nitrogen source and inorganic salts by using a known technique.
- the medium is generally known to those skilled in the art and is commercially available. Otherwise, a medium prepared directly in a laboratory may be used. If polypeptides are secreted directly to the nutrition medium, the polypeptides can be separated directly from the medium. If polypeptides are not secreted to the nutrition medium, they can be separated from the cell lysate.
- polypeptides can be separated from the medium by a conventional method including centrifugal separation, filtration, extraction, spray drying, evaporation or precipitation.
- purification of polypeptides can be performed by various methods known to those skilled in the art, including chromatography (e.g. ion exchange chromatography, affinity chromatography, hydrophobic chromatography or size exclusion chromatography), electrophoresis, fractional solubility separation (e.g. ammonium sulfate precipitation), SDS-PAGE or extraction.
- the human granulocyte colony stimulating factor isoform purified as described above is subjected to pegylation in the manner as described hereinafter.
- Suitable "non-protein segments" used for the pegylation according to the present invention include polyethylene glycol, and branched polyethylene glycol is preferred.
- Other examples of such non-protein segments include, but are not limited to: at least one material selected from the group consisting of water soluble polymers such as polypropylene glycol (PPG), poly oxye thy lene (POE), polytrimethylene glycol, polylacatic acid and derivatives thereof, polyacrylic acid and derivatives thereof, poly(amino acid), poly(vinyl alcohol), polyurethane, polyphosphazenes, poly (L- lysine), poly alky lene oxide (PAO), polysaccharide, or the like, and nonimmune polymers such as dextran, polyvinyl pyrrolidone, polyvinyl alcohol (PVA), polyacryl amide, or the like.
- PPG polypropylene glycol
- POE poly oxye thy lene
- PEO polytrimethylene glycol
- the biocompatible polymer used in the preparation of the polymer derivatives according to the present invention preferably has a molecular weight of about 2,000-100,000, more preferably 20,000-40,000.
- the biocompatible polymer should be activated.
- the biocompatible polymer may be bonded with a reactive functional group.
- reactive polymer group means a group or a moiety capable of activating a biocompatible polymer so that it can be bonded with a biologically active material.
- one of the terminal groups of the biocompatible polymer is converted into a reactive functional group.
- This process is referred to as "activation".
- one of the hydroxyl terminal groups may be converted into a reactive group, such as carbonate.
- activated poly(alkylene oxide) can be obtained.
- the reactive group that may be used to activate a biocompatible non-protein segment polymer according to the present invention is selected from the group consisting of maleimide, acetamide, pentenoic amide, butenoic amide, isocyanate, isothiocyanate, cyanuric acid chloride, 1,4-benzoquinone, disulfide, or the like.
- Bonding PEG to a protein via a thiol group of the protein can be performed by a method generally known to those skilled in the art.
- PEG-maleimide is used to allow thiol groups to be bound to activated double bonds via Michael reaction [Ishii et al., Biophys J. 1986, 50:75-80].
- PEG- iodoacetamide reagent may be used. The latter method is advantageous in that a stable PEG-bound cysteine derivative, i.e. carboxymethyl cysteine, can be obtained by a strong acidic hydrolysis, wherein the derivative can be identified and determined by standard amino acid analysis [Gard FRN. Carboxymethylation, Method Enzymol 1972; B25: 424-49].
- the pegylation method in the following examples were performed by using mPEG20,000-maleimide, branched mPEG20,000-maleimide and branched mPEG40,000 available from NOF in a molar ratio of 20: 1 to human granulocyte colony stimulating factor.
- the pegylation reaction is performed in 0.1 M phosphate buffer (pH 7-8) at room temperature for 2 hours with stirring.
- compositions must be non-toxic to the subjects and be compatible with other constituents of the pharmaceutical preparation under the corresponding administration dose and concentration.
- pharmaceutical preparation should not contain any materials known to be harmful to polypeptides, such as oxidants or other materials.
- Suitable carriers include: buffers such as phosphoric acid, citric acid and other organic acids; antioxidants including ascorbic acid; low-molecular weight polypeptides; proteins such as blood plasma albumin, gelatin and immunoglobulin; hy- drophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamin, arginine, or lysine; monosaccharides, disaccharides or other carbohydrates including glucose, mannose or dextrin; chelate factors such as EDTA; metal ions such as zinc, cobalt or copper; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or non-ionic surfactants such as Tween, Pluronic or polyethylene glycol (PEG).
- buffers such as phosphoric acid, citric acid and other organic acids
- antioxidants including ascorbic acid
- low-molecular weight polypeptides proteins such as blood plasma albumin, gelatin
- a sugar chain-bound human granulocyte colony stimulating factor isoform should be sterilized before it is administered for the purpose of treatment. Such sterilization may be accomplished easily by way of filtering through a sterilized filtration membrane.
- a therapeutic sugar chain-bound human granulocyte colony stimulating factor isoform composition is stored in a container equipped with a sterilized access port, for example, an intravenous injection bag or vial having a septum through which a subcutaneous injection needle can pass.
- a sterilized access port for example, an intravenous injection bag or vial having a septum through which a subcutaneous injection needle can pass.
- the human granulocyte colony stimulating factor isoform according to the present invention may be administered directly to animals by suitable routes including parenteral administration routes, locally or systematically.
- a particular administration route will be determined by the history of a patient, including side effects recognized or expected upon the administration of human granulocyte colony stimulating factor isoform.
- Particular parenteral administration routes include subcutaneous, intramuscular, intravenous, intraarterial and intraperitoneal routes. Most preferably, administration is carried out by continuous infusion (e.g. mini-pump such as an osmotic pump) or injection, for example, intravenous injection or subcutaneous injection.
- a preferred administration route for human granulocyte colony stimulating factor isoform is subcutaneous administration.
- the human granulocyte colony stimulating factor isoform according to the present invention may be administered to a patient in a therapeutically effective amount.
- the term "therapeutically effective amount” refers to the amount sufficient to obtain a desired treating effect by using a given administration mode under a given condition.
- the therapeutic composition of human granulocyte colony stimulating factor should be formulated and administered conformed to the preferred medical practice, under the consideration of the particular condition to be treated, clinical conditions of an individual patient (particularly, side effects upon the single administration of human granulocyte colony stimulating factor), delivery sites of the composition of the human granulocyte colony stimulating factor isoform, administration modes, administration schedules and other factors generally known to those skilled in the art.
- the therapeutically effective amount of the human granulocyte colony stimulating factor isoform is determined considering the above factors. In general, daily dose of the human granulocyte colony stimulating factor isoform according to the present invention ranges from about ID/kg to 100D/kg.
- human granulocyte colony stimulating factor genes were synthesized as a template.
- the genes synthesized in this example have base sequences as shown in the following Table 1, and DNA sequence and amino acid sequence of human granulocyte colony stimulating factor, used as a template, are shown in FIG. 1.
- N-term ⁇ 175C correspond to Sequence Nos. 2-7, respectively.
- PCR was performed to construct -1C isoform by using DNA of natural human granulocyte colony stimulating factor as a template and primer pairs formed of -1C primer and C-term primer.
- the amplified DNA fragment was further subjected to PCR by using N-term primer and C-term primer so that restriction enzyme cleavage sites (Xba I and BamH I) could be introduced, and then was introduced into an expression vector.
- restriction enzyme cleavage sites Xba I and BamH I
- amino acid sequence of the N-terminal was methionine- cysteine-threonine-proline-leucine-glycine.
- T133C was constructed in order to provide a control as a known isoform or to realize amino acid modification in the middle of the polypeptide of human granulocyte colony stimulating factor.
- T133C having cysteine substituting for threoninine, the 133 amino acid of natural human granulocyte colony stimulating factor, was obtained by carrying out PCR using DNA of natural human granulocyte colony stimulating factor as a template and primer pairs formed of N-term primer with T133C2 and T 133Cl with C-term primer.
- N-term primer primer pairs formed of N-term primer with T133C2 and T 133Cl with C-term primer.
- the two DNA fragments were further subjected to PCR by using primer pairs formed of N-term primer and C-term primer to obtain modified genes of T133C, a human granulocyte colony stimulating factor isoform having cysteine substituting for threonine in the position of the 133 r amino acid.
- Introduction into an expression vector was performed in the same manner as described in the above 'Construction of -1C Human Granulocyte Colony Stimulating Factor Isoform'.
- -1C/T133C was constructed in order to realize amino acid addition to the N- terminal and amino acid modification in the middle of the polypeptide of human granulocyte colony stimulating factor.
- threoninine the 133 r amino acid of the preformed -1C human granulocyte colony stimulating factor isoform, was substituted with cysteine.
- PCR was performed by using DNA of the preformed -1C human granulocyte colony stimulating factor isoform as a template and primer pairs formed of N-term primer with T133C2 and T 133Cl with C-term primer.
- PCR was performed by using DNA of natural human granulocyte colony stimulating factor as a template and primer pairs formed of N-term primer and 175C primer.
- the amplified DNA fragment was further subjected to PCR by using N-term primer and C-term primer so that restriction enzyme cleavage sites (Xba I and BamH I) could be introduced, and then was introduced into an expression vector.
- restriction enzyme cleavage sites Xba I and BamH I
- Introduction into the expression vector was performed in the same manner as described in the above 'Construction of -1C Human Granulocyte Colony Stimulating Factor Isoform'.
- the amino acid sequence of the C-terminal was - alanine-glutamine-proline-cysteine.
- the two DNA fragments were further subjected to PCR by using primer pairs formed of N-term primer and C-term primer to obtain modified genes of 175C/T133C, a human granulocyte colony stimulating factor isoform having cysteine substituting for threonine in the position of the 133 r amino acid.
- Introduction into an expression vector was performed in the same manner as described in the above 'Construction of -1C Human Granulocyte Colony Stimulating Factor Isoform'.
- Transformation of plasmid vectors containing genes of human granulocyte colony stimulating factor isoforms into E. coli was performed to construct strains producing human granulocyte colony stimulating factor proteins.
- the seed cells culture was inoculated into a 500ml of Luria Broth (LB) medium in a 3,000ml triangular flask and was incubated at 3O 0 C.
- LB Luria Broth
- the incubator was heated to 42 0 C and the cells were further incubated for 4 hours to induce the expression of human granulocyte colony stimulating factor isoforms.
- the culture solution was subjected to centrifugal separation to collect the cells, and the cells were washed with distilled water.
- the washed cells were lysed in a microfluidizer (available from Microfluidics Co.) under 12,000 psi four times, and inclusion bodies were recovered via centrifugal separation, followed by washing three times with distilled water.
- the inclusion bodies were thoroughly suspended and dissolved in 10ml of 8M urea, 5OmM glycine (pH 11.0) at room temperature for at least 5 hours, and then the suspension was subjected to centrifugal separation at 10,000rpm for 30 minutes. The supernatant was collected to obtain a solution of human granulocyte colony stimulating factor isoform protein.
- glycine and distilled water were added to control the glycine concentration and the urea concentration to 5OmM and 2M, respectively.
- the resultant protein solution was titrated to pH 9.0 and was subjected to protein refolding overnight at 4 0 C.
- the solution was adjusted to pH 4.0, diluted with distilled water by three times or more, and was loaded to a column packed with 10ml of CM Sepharose FF (available from Amersham Biosciences), which have been previously equilibrated by using 2mM HCl in an amount of at least three times of the column volume.
- CM Sepharose FF available from Amersham Biosciences
- the column was equilibrated by using 2mM HCl in an amount of at least three times of the column volume, and washed by using 5OmM acetate sodium chloride buffer (pH 5.4) in an amount of at least three times of the column volume.
- human granulocyte colony stimulating factor isoforms were eluted by using 5OmM acetate sodium chloride buffer (pH 5.4) containing 35mM sodium chloride in an amount of at least twelve times of the column volume and were divided into 5ml fractions. The fractions were collected after the HPLC analysis.
- Sepharose FF Sepharose FF
- a Centricon Plus-80 filter unit with a molecular weight of 10,000Da at 3000rpm for 25 minutes to obtain protein concentrate having a final protein concentration of about 5mg/ml.
- Example 2 was introduced into 0.1M phosphate buffer (pH 7.0-8.0) and 200mg of branched mPEG (20,000)-maleimide (available from NOF) was added thereto. Reaction was performed at room temperature for 2 hours with stirring. The reaction mixture was quenched by reducing pH to 3.0 with IN HCl solution.
- Example 2 was introduced into 0.1M phosphate buffer (pH 7.0-8.0) and 400mg of branched mPEG (40,000)-maleimide (available from NOF) was added thereto. Reaction was performed at room temperature for 2 hours with stirring. The reaction mixture was quenched by reducing pH to 3.0 with IN HCl solution.
- each mPEG-G-CSF polymer was titrated to pH 3.0, diluted with distilled water by at least 10 times, and loaded to a column packed with 10ml of CM Sepharose FF (Amersham Bioscience), which have been previously equilibrated by using 2mM HCl in an amount of at least three times of the column volume.
- the column was equilibrated by using 2mM HCl in an amount of at least three times of the column volume, and washed by using 50 mM acetate sodium chloride buffer (pH 5.0) in an amount of three times of the column volume.
- mPEG-G-CSF polymers were eluted by using 50 mM acetate sodium chloride buffer (pH 5.4) containing 40 mM sodium chloride in an amount at least twelve times of the column volume.
- mPEG-G-CSF polymer was eluted by using 50 mM acetate sodium chloride buffer (pH 5.4) containing 20 mM sodium chloride in an amount at least twelve times of the column volume. The eluted solution was fractionated into a 5 ml fraction and each fraction was collection after the HPLC analysis.
- mPEG-G-CSF polymers purified by CM Sepharose FF, were concentrated by using a Centricon Plus- 80 filter unit with a molecular weight of 10,000Da at 3000rpm for 25 minutes to obtain protein concentrate having a final protein concentration of about 5mg/ml.
- the mPEG-G-CSF polymers prepared and purified according to Examples 4 and 5 were evaluated for the activity and in vivo lifetime by using 8-week aged rats (SD rats, male, five rats per test sample).
- the test samples include: PBS carrier as a carrier; G- CSF T133C20kDa(Br) prepared and purified according to Examples 1 to 4 (G-CSF isoform obtained by substituting 133-Thr with cysteine and covalently bonding branched PEG with 20 kDa to the same cysteine according to Korean Laid-Open Patent No.
- N+20kDa(Br) G-CSF isoform obtained by adding cysteine between the threonine and methionine of the N-terminal (-1C) of G-CSF and covalently bonding branched PEG with 20 kDa to the same cysteine
- 175C+20kDa(Br) G-CSF isoform obtained by adding cysteine after 174-Pro of the C-terminal of G-CSF and covalently bonding branched PEG with 20 kDa to the same cysteine
- 175C+40kDa(Br) G-CSF isoform obtained by adding cysteine after 174-Pro of the C-terminal of G-CSF and covalently bonding branched
- each blood sample was introduced into an EDTA anticoagulant tube and subjected to centrifugal separation to separate blood plasma.
- G-CSF concentration in the plasma was measured by using a human G-CSF ELISA kit (Quantokine, R&D Systems). Each sample was diluted serially to the linear zone allowing detection of concentration in the kit. Measurement was performed two times per sample. The results are shown in the following Table 3.
- the polyethylene gly col-bound human granulocyte colony stimulating factor isoforms having cysteine added to the N-terminal or C- terminal according to the present invention shows an increased in vivo activity and in vivo lifetime.
- N+20kDA(Br) and 175C+20kDa(Br) show a significant increase in activity from the point of 24 hours after the administration, when compared to the control.
- 175C+40kDa(Br) shows an increase in activity from the point of 24 hours after the administration and a similar aspect of neutrophil production, when compared to the control.
- 175C+40kDa (Br) can maintain its activity until the time of 72 hours after the administration.
- 175C+40kDa(Br) provides a significantly improved lifetime
- 175C+20kDa(Br) and N+20kDa(Br) provide a significantly improved activity.
- the human granulocyte colony stimulating factor isoforms according to the present invention provide a significantly increased in vivo lifetime and can be used for treating or preventing various human granulocyte colony stimulating factor-related diseases.
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EP (1) | EP1896081A4 (en) |
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KR (1) | KR100694994B1 (en) |
CN (1) | CN101198357A (en) |
BR (1) | BRPI0613332A2 (en) |
WO (1) | WO2006135176A1 (en) |
Cited By (6)
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WO2009103199A1 (en) * | 2008-02-18 | 2009-08-27 | 江苏恒瑞医药股份有限公司 | A g-csf conjugate modified by water-soluble polymer |
CN102164955A (en) * | 2008-10-02 | 2011-08-24 | 奥克塔法马生物制药股份有限公司 | Improved processing of recombinant human G-CSF |
WO2013068602A2 (en) | 2012-03-19 | 2013-05-16 | Richter Gedeon Nyrt. | Method for the production of polypeptides |
US9458207B2 (en) | 2012-03-19 | 2016-10-04 | Richter Gedeon Nyrt. | Methods for refolding G-CSF from inclusion bodies |
WO2019130344A1 (en) * | 2017-12-27 | 2019-07-04 | Council Of Scientific And Industrial Research | A polypeptide exhibiting granulocyte-colony stimulating factor activity |
EP3705492A4 (en) * | 2017-10-30 | 2021-08-18 | Hankook Korus Pharmaceutical Co. Ltd. | Method for preparing, in high yield, conjugate in which colony stimulating factor and polyol are conjugated |
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KR100886783B1 (en) * | 2006-06-12 | 2009-03-04 | 성균관대학교산학협력단 | N-terminal modified PEG-TRAIL, method for preparing and uses thereof |
TWI751154B (en) * | 2016-04-18 | 2022-01-01 | 大陸商江蘇恆瑞醫藥股份有限公司 | A preparation method of recombinant human granulocyte colony stimulating factor |
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CN111574597B (en) * | 2020-05-07 | 2023-03-31 | 中国科学院微生物研究所 | anti-HIV polypeptide modified by high molecular weight PEG (polyethylene glycol), preparation method and application thereof |
CN112121009B (en) * | 2020-09-24 | 2022-12-02 | 科兴生物制药股份有限公司 | New preparation of polyethylene glycol modified recombinant human granulocyte stimulating factor |
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WO2009103199A1 (en) * | 2008-02-18 | 2009-08-27 | 江苏恒瑞医药股份有限公司 | A g-csf conjugate modified by water-soluble polymer |
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US9458207B2 (en) | 2012-03-19 | 2016-10-04 | Richter Gedeon Nyrt. | Methods for refolding G-CSF from inclusion bodies |
EP3517621A1 (en) | 2012-03-19 | 2019-07-31 | Richter Gedeon Nyrt. | Method for the production of polypeptides |
EP3705492A4 (en) * | 2017-10-30 | 2021-08-18 | Hankook Korus Pharmaceutical Co. Ltd. | Method for preparing, in high yield, conjugate in which colony stimulating factor and polyol are conjugated |
WO2019130344A1 (en) * | 2017-12-27 | 2019-07-04 | Council Of Scientific And Industrial Research | A polypeptide exhibiting granulocyte-colony stimulating factor activity |
US11732016B2 (en) | 2017-12-27 | 2023-08-22 | Council Of Scientific & Industrial Research | Polypeptide exhibiting granulocyte-colony stimulating factor activity |
Also Published As
Publication number | Publication date |
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JP2008543304A (en) | 2008-12-04 |
EP1896081A1 (en) | 2008-03-12 |
BRPI0613332A2 (en) | 2011-01-04 |
KR100694994B1 (en) | 2007-03-14 |
CN101198357A (en) | 2008-06-11 |
US20100143288A1 (en) | 2010-06-10 |
KR20060129889A (en) | 2006-12-18 |
US8088899B2 (en) | 2012-01-03 |
EP1896081A4 (en) | 2011-05-25 |
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