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WO2016013725A1 - Composition pharmaceutique contenant, comme principe actif, une protéine mutante du facteur stimulant les colonies de granulocytes ou la protéine de fusion de transferrine correspondante - Google Patents

Composition pharmaceutique contenant, comme principe actif, une protéine mutante du facteur stimulant les colonies de granulocytes ou la protéine de fusion de transferrine correspondante Download PDF

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Publication number
WO2016013725A1
WO2016013725A1 PCT/KR2014/011269 KR2014011269W WO2016013725A1 WO 2016013725 A1 WO2016013725 A1 WO 2016013725A1 KR 2014011269 W KR2014011269 W KR 2014011269W WO 2016013725 A1 WO2016013725 A1 WO 2016013725A1
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WIPO (PCT)
Prior art keywords
protein
csf
expression vector
granulocyte colony
transferrin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2014/011269
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English (en)
Korean (ko)
Inventor
윤정혁
장병하
진봉석
배지영
김순남
박경수
박영규
김한조
서영주
정우성
강경태
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
EQUISNZAROO Co Ltd
Original Assignee
EQUISNZAROO Co Ltd
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Publication date
Priority claimed from KR1020140137414A external-priority patent/KR101623906B1/ko
Application filed by EQUISNZAROO Co Ltd filed Critical EQUISNZAROO Co Ltd
Priority to EP14898071.7A priority Critical patent/EP3173422B1/fr
Priority to ES14898071T priority patent/ES2735899T3/es
Priority to US15/327,838 priority patent/US10479822B2/en
Publication of WO2016013725A1 publication Critical patent/WO2016013725A1/fr
Anticipated expiration legal-status Critical
Priority to US16/575,612 priority patent/US11421009B2/en
Ceased legal-status Critical Current

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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • composition comprising granulocyte colony stimulator mutant protein or transferrin fusion protein thereof as an active ingredient
  • the present invention relates to a fusion protein in which transferrin is peptide-bound to the ends of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator (G-CSF) variant proteins.
  • G-CSF granulocyte colony stimulator protein
  • G-CSF granulocyte colony stimulator
  • G-CSF Granulocyte colony stimulating factor
  • G-CSF stimulates neutrophil granulopoiesis, and has been used as an anticancer adjuvant to prevent infectious complications caused by neutropenia. Reducing the incidence of side effects of pyrogenic neutropenia caused by cancer chemotherapy and radiation therapy, thereby reducing the death caused by cancer-chemotherapeutic treatment, has been shown to have clinical benefits that have great benefits for cancer patients.
  • G-CSF increases the number of hematopoietic progenitor cells and thus It is reported to mitigate.
  • G-CSF causes bone marrow stem cells to migrate to the ischemic heart, through blood vessels through myocardial regeneration. It is known to promote differentiation into cells and cardiomyocytes.
  • Recombinant human G-CSF has a pharmacological effect for a short period of time, requiring discomfort to be administered more than once a day during the treatment of ischemic disease or for the duration of leukopenia that occurs after cancer chemotherapy or radiotherapy. There is this.
  • a substance having a hepatic circulatory half-life (Hal fl i fe) will have an effect of reducing the number of administrations necessary for alleviating leukopenia and the like, and consequently preventing infectious complications.
  • PEG Polyethylene glycol
  • pegfilgrastim Pegf i lgrast itn
  • pegfilgrastim Pegf i lgrast itn
  • Cysteine the 17th amino acid of G-CSF
  • G-CSF variants in which cysteine 17 is substituted with serine have been reported to increase protein stability at neutral pH.
  • Transferrin in the third largest amount of plasma protein It exists and serves to transport iron ions in the blood to various tissues. It is shorter than albumin or immunoglobulin G (Immunoglobul in G, IgG) but has a relatively long half-life of 8 days. As a result, it is released outside the cell and circulates.
  • An object of the present invention is granulocyte colony stimulator protein (G-CSF) or granulocyte It is to provide a fusion protein in which the transferrin is peptide-bound to the terminal of the granulocyte colony stimulator (G-CSF) mutant protein substituted with the 116 th threonine ° 1 cysteine in the amino acid sequence of the colony stimulator protein.
  • the present invention relates to a granulocyte colony stimulator (G-CSF) mutant protein in which the 116th threonine is substituted with cysteine in the amino acid sequence of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator protein. Fusion protein peptide-bound to the terminal, and granulocyte colony stimulator (G-CSF) variant protein in which 116 th threonine of granulocyte colony stimulator protein is substituted with cysteine.
  • G-CSF granulocyte colony stimulator
  • the present invention is a granulocyte colony stimulator (G-CSF) mutant protein in which the 116th threonine is replaced with cysteine in the amino acid sequence of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator protein.
  • G-CSF granulocyte colony stimulator
  • Neutrophils comprising a fusion protein having a peptide-linked transferrin at the terminal or a granulocyte colony stimulator (G-CSF) mutant protein substituted with cysteine at 116 th threonine of granulocyte colony stimulator protein
  • pharmaceutical compositions for the prevention or treatment of diminished, ischemic disease, and anticancer adjuvant.
  • the present invention is a granulocyte colony stimulator (G-CSF) mutant protein in which the 116th threonine is replaced with cysteine in the amino acid sequence of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator protein.
  • G-CSF granulocyte colony stimulator
  • G-CSF granulocyte colony stimulator
  • the present invention is a granulocyte colony stimulator (G-CSF) mutation in which the 116th threonine is substituted with cysteine in the amino acid sequence of the over-globule colony stimulator protein (G-CSF) or granulocyte colony stimulator protein.
  • G-CSF granulocyte colony stimulator
  • the transferrin peptide coupled to ' end of the fusion protein or granulocyte 116th threonine (threonine) a granulocyte colony stimulating substituted with cysteine (cysteine) of the colony stimulating factor protein factor (G-CSF) mutations encoding a protein, wherein said protein
  • An expression vector comprising a gene to be provided, and a method for producing a transformant, wherein the expression vector is introduced into a host cell.
  • the present invention is a granulocyte colony stimulator (G-CSF) mutant protein in which the 116th threonine is replaced with cysteine in the amino acid sequence of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator protein.
  • G-CSF granulocyte colony stimulator
  • a fusion protein having a peptide-linked transferrin at the end of it, or a granulocyte colony stimulator (G-CSF) mutant protein in which the 116 th threonine of the granulocyte colony stimulator protein is substituted with cysteine, the vector or the transformation Provided is a method for preventing or treating neutropenia or ischemic disease comprising administering a sieve to an individual or an individual suffering from neutropenia or ischemic disease.
  • the present invention is a granulocyte colony stimulator (G-CSF) mutant protein in which the 116th threonine is replaced with cysteine in the amino acid sequence of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator protein.
  • Fusion protein peptide-linked transferrin at the end of Or neutrophils comprising administering to the subject a granulocyte colony stimulating factor (G-CSF) mutant protein, said vector or said transformant, wherein 116 th threonine of the granulocyte colony stimulating protein is substituted with cysteine
  • G-CSF granulocyte colony stimulating factor
  • the present invention is the 116th threonine (Threonine) in the amino acid sequence of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator protein for the treatment of neutropenia, anticancer adjuvant, ischemic disease Fusion-peptide-fused fusion protein at the end of the substituted granulocyte colony stimulator (G-CSF) mutant protein, or granulocyte colony stimulator wherein 116 th threonine of granulocyte colony stimulator protein is replaced with cysteine ( G-CSF) variant protein, said vector or said transformant are provided.
  • G-CSF granulocyte colony stimulator protein
  • G-CSF granulocyte colony stimulator protein
  • G-CSF granulocyte colony stimulator protein for the treatment of neutropenia, anticancer adjuvant, ischemic disease Fusion-peptide-fused fusion protein at the end of the substituted granulocyte colony stimulator (G-CSF)
  • the granulocyte colony stimulating factor (G-CSF) mutant protein or transferrin fusion protein thereof of the present invention has a markedly increased activity and blood stability and an increased half-life compared to conventional human G-CSF. Since the purification efficiency is higher than PEGylated G-CSF, it can be usefully used for preventing or treating ischemic disease or neutropenia.
  • 1 is a diagram showing a cleavage map of a pcDNA3.1 (+) / preTi vector.
  • Fig. 2 is a diagram showing a cleavage map of the pcDNA3.1 (+) / preTf (B) vector.
  • Fig. 3 shows a cleavage map of the pcDNA3.1 (+) / Tf (B) vector.
  • Fig. 4 shows a cleavage map of the pcDNA3.1 (+) / G 'CSF (T116C) -Tf vector.
  • FIG. 5 Expi293F cell line transformed with G-CSF (T116C) -Tf plasmid It is a figure which shows the expression amount of G-CSF (T116G) -Tf.
  • FIG. 6 is a diagram showing G_CSF (T116C) -Tf protein isolated by DEAE Af f-gel blue chromatography.
  • FIG. 7 is a diagram showing the preparation and concentration separation of the holo (preenzyme) form of G-CSF (T116C) -Tf to which iron (Fe3 +) is bound.
  • FIG. 8 is a diagram showing the transferoline receptor binding capacity of the holo form of G-CSF (T116C) -Ti.
  • FIG. 9 is a diagram showing cell proliferation activity of HL-60 cells treated with G-CSF, G-CSF-Tf, and G-CSF (T116C) -Tf.
  • FIG. 10 is a diagram showing a comparison of degradation resistance by protease of G-CSF and G-CSF (T116C) proteins.
  • FIG. 11 shows a comparison of plasma half-life of G-CSF-Tf and G-CSF (T116C) -Tf proteins in vivo.
  • FIG. 12 shows a comparison of the physiological activity of G-CSF, G-CSF (T116C) -Tf proteins in the rats of neutropenia.
  • the invention relates to the amino acid sequence of granulocyte colony stimulator protein.
  • G-CSF Mutant Protein Granulocyte Colony Stimulator Protein
  • G-CSF Granulocyte Colony Stimulator Protein
  • G-CSF Mutant Protein Granulocyte Colony Stimulator Protein
  • G-CSF Mutant Protein Granulocyte Colony Stimulator Protein
  • G-CSF Mutant Protein Granulocyte Colony Stimulator Protein with 116th Threonine Substituted with Other Amino Acids Fusion protein peptide-linked to the terminal of and provides a use thereof.
  • G-CSF or transferrin used in the present invention may be derived from animals, plants, microorganisms, preferably human-derived G-CSF or transferrin, but is derived from heterologous cells having the same activity as human-derived G-CSF or transferrin. Protein Can be.
  • the protein may additionally have modifications such as phosphorylation, acetylation, methylation, glycosylation, etc., and may be combined with other proteins, but may be regarded as the same as the protein before modification unless it is changed enough to lose the function of the protein.
  • the other amino acid substituted for the 116 th threonine may be a hydrophobic amino acid, and specifically, may be a cysteine capable of disulfide bond with the 17 th cysteine of the G-CSF sequence, but is not limited thereto.
  • the terminal to which the transferrin is linked may be either an amino terminal (5 'terminal N-terminus) or a carboxy terminal (3' terminal, C-terminus).
  • the granulocyte colony stimulator (G-CSF) protein is a protein represented by the sequence of ⁇ SEQ ID NO: 1>, or has the same activity as the protein or a gene encoding the granulocyte colony stimulator protein (G-CSF) on a chromosome As long as the position is the same, one or several amino acids of the protein may be added, deleted, or substituted.
  • the granulocyte colony stimulating factor (G-CSF) protein has at least 80% homology, more specifically at least 90% homology, most specifically 95%, 96%, 97%, 98% , But not limited to, sequences having at least 99% or 99.5% homology.
  • the transferrin is a protein represented by the sequence of ⁇ SEQ ID NO: 2>, but at least a gene having the same activity as the protein or the same gene position encoding the transferrin on a chromosome, one or several amino acids of the protein is added, It may be composed of a nucleotide sequence that is deleted, substituted.
  • the transferrin protein has at least 80% homology to the amino acid sequence of ⁇ SEQ ID NO: 2, more specifically at least 90% homology, most specifically at least 95%, 96%, 97%, 98%, 99% or 99.5% It is composed of, but not limited to, sequences having homology.
  • the granulocyte colony stimulating factor (G-CSF) mutant protein is a protein represented by the sequence of ⁇ SEQ ID NO: 3>, or having the same activity as the protein or having the same gene position encoding the transferrin on the chromosome ', One or several amino acids of may be composed of a base sequence that is added, deleted, substituted.
  • the granulocyte colony stimulating factor (G-CSF) mutant protein has more than 80% homology to the amino acid sequence of ⁇ SEQ ID NO 3>, more specifically, more than 90% homology, most specifically 95%, 96%, 97%, 98 It consists of, but is not limited to, sequences having homology of at least%, 99% or 99.5%.
  • the fusion protein peptide-linked fusion protein at the end of the granulocyte colony stimulator protein is at least 80% homology to the amino acid sequence of ⁇ SEQ ID NO: 4, more specifically at least 90% homology, most specifically 95%, 96%, It consists of sequences having at least 97%, 98%, 99% or 99.5% homology but is not limited to these.
  • the fusion protein in which the transferrin peptide is coupled to the terminal of the granulocyte colony stimulator mutant protein in which the 116 th threonine is replaced with another amino acid is 80 in the amino acid sequence of ⁇ SEQ ID NO: 5>.
  • More than% homology more specifically more than 90% homology, Most specifically, but not limited to, a sequence composed of at least 95%, 96%, 97%, 98%, 99% or 99.5% homology.
  • the nucleotide sequence of the restriction enzyme recognition site in the gene may be substituted with the same amino acid as the amino acid encoded by the nucleotide sequence, or a nucleotide sequence encoding another amino acid within a range that does not change the activity with the protein.
  • the protein may be a protein obtained by replacing thymine of BamfR restriction enzyme recognition site sequence (GGATCC) in a protein gene with cytosine, but is not limited thereto.
  • restriction enzymes include EcoRi, BamHl., Hind. , kpnl, Not I, Pst I,
  • the present invention is the terminal of the granulocyte colony stimulator (G-CSF) mutant protein in which the 116th threonine is substituted with another amino acid in the amino acid sequence of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator protein Neutropenia of the neutropenia comprising peptide-bound fusion protein or granulocyte colony stimulator (G-CSF) mutant protein in which 116 th threonine of granulocyte colony stimulator protein is substituted with another amino acid as an active ingredient.
  • G-CSF granulocyte colony stimulator
  • neutropenia refers to an abnormally reduced number of neutrophils, mild neutrophils per ⁇ ⁇ of blood, mild symptoms of less than 1500, moderate symptoms of less than 1000, and severe neutropenia of less than 500. Are classified. Leukopenia (neucopeni a) can be seen as a broad neutropenia.
  • Ischemic disease refers to cell damage caused by bleeding, embolism (embol) and infarct ion to stop blood flow to tissues, trauma, transplant rejection, stroke, cerebral infarction, ischemic Renal disease, ischemic lung disease, ischemic disease caused by infection, ischemic disease of limb, ischemic cardiomyopathy, myocardial infarction, heart failure, and the like.
  • the other amino acid substituted with the 116 th threonine may be a hydrophobic amino acid, and specifically, may be cysteine capable of disulfide bond, but is not limited thereto.
  • the granulocyte colony stimulator (G-CSF) protein is a protein represented by the sequence of ⁇ SEQ ID NO: 1>, or has the same activity as the protein or a gene encoding the granulocyte colony stimulator protein (G-CSF) on a chromosome As long as the positions are the same, one or several amino acids of the protein may be added, deleted or substituted.
  • the granulocyte colony stimulating factor (G-CSF) protein has at least 80% homology to the amino acid sequence of ⁇ SEQ ID NO: 1>, more specifically, at least 90% homology, most Specifically . Consists of, but is not limited to, sequences having at least 95%, 96%, 97%, 98%, 99% or 99.5% homology.
  • the transferrin is a protein represented by the sequence of ⁇ SEQ ID NO: 2>, or having the same activity as the protein or having the same gene position encoding the transferrin on a chromosome, one or a few amino acids of the protein are added, deleted, It may be composed of a base sequence to be substituted.
  • the transferrin protein has a homology of 8 subtypes to amino acid sequence of ⁇ SEQ ID NO: 2, more specifically 90% or more homology, most specifically 95%, 96% ,. It consists of, but is not limited to, sequences having at least 97%, 98%, 99% or 99.5% homology.
  • the granulocyte colony stimulating factor (G-CSF) mutant protein is a protein represented by the sequence of ⁇ SEQ ID NO: 3>, or having the same activity as that of the protein or the gene position encoding the transferrin on the chromosome, One or several amino acids may be composed of base sequences that are added, deleted, or substituted.
  • the granulocyte colony stimulating factor (G-CSF) mutant protein has more than 80% homology to the amino acid sequence of ⁇ SEQ ID NO 3>, more specifically, more than 90% homology, most specifically 95%, 96%, 97%, 98 %, 99% or 99.53 ⁇ 4> or more of the sequence having a homology of, but not limited to.
  • the fusion protein in which the transferrin peptide is bound to the terminal of the granulocyte colony stimulator protein is a protein represented by the sequence of ⁇ SEQ ID NO: 4>, or has the same activity as the protein or encodes the granulocyte colony stimulator protein or transferrin on a chromosome. As long as the gene position is the same, One or several amino acids of the protein may be composed of base sequences that are added, deleted, or substituted.
  • the fusion protein in which the transferrin peptide is coupled to the terminal of the granulocyte colony stimulator protein has at least 8 homology to the amino acid sequence of ⁇ SEQ ID NO: 4>, more specifically, at least 90% homology, most specifically 95%, 96%, 97 It consists of, but is not limited to, sequences having homology of at least%, 98%, 99% or 99.5%.
  • the fusion protein in which the transferrin peptide is coupled to the terminal of the granulocyte colony ⁇ stimulator mutant protein in which the 116 th threonine is replaced with another amino acid is represented by the sequence of ⁇ SEQ ID NO: 5>.
  • the granulocyte colony stimulator protein black is a nucleotide sequence in which one or several amino acids of the protein are added, deleted, or substituted at the same gene position encoding the transferrin. Can be configured.
  • the fusion protein in which the transferrin peptide is coupled to the terminal of the granulocyte colony stimulator mutant protein in which the 116 th threonine is replaced with another amino acid is 80 in the amino acid sequence of ⁇ SEQ ID NO: 5>.
  • G-CSF Granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor mutant protein
  • transferrin wherein 116 th threonine is substituted with another amino acid in amino acid sequence of granulocyte colony stimulating factor protein
  • the recognition site may be inserted.
  • B a! It may be a protein in which thymine of a restriction enzyme recognition site sequence (GGATCC) is replaced with cytosine, but is not limited thereto.
  • restriction enzymes examples include EcoR ⁇ , BamHl, Hincm, kpnl, Notl, Pstl, Smal, Xhol, Fokl, Alw26l, Bbvl, Bsrl, Earl, Hphl, Mbol, SfaNl, Tthlll I, Nael, Nhel, NgoMN, Eco57 ⁇ , Bcgl, Bpl, Bsp241, Bael, Cjel, EcoPl, Hin, StyLTl and the like, and any restriction enzymes used in the art can be used without limitation depending on the gene, expression vector, or genetic engineering environment to be used.
  • 'Expression vector' of the present invention is a means for introducing a nucleic acid sequence encoding a protein of interest into a host cell, and includes various forms such as polamide, cosmid, BAC, viral nucleic acid, and the like.
  • the vector generally includes a selective marker such as an antibiotic resistance gene which can confirm that the target gene has been successfully introduced into the host cell, and a promoter, an operator, an initiation codon, a stop codon, a polya to induce the expression of the target gene.
  • the denylation sequence, enhancer, Kozak sequence, shine-dalgano sequence and the like can be included in various ways.
  • Replicating expression vectors include the origin of replication.
  • the host cell may be any one selected from the group consisting of E.
  • compositions of the present invention may be in various oral or parenteral formulations. have. When formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like form at least one excipient such as starch, calcium carbonate, sucrose, lactose in one or more compounds. Or gelatin and the like.
  • Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.
  • various excipients for example, wetting agents, sweeteners, fragrances, and preservatives, may be included.
  • Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous and suspending solvents propylene glycol, polyethylene glycol, vegetable oils such as eurib oil, injectable esters such as ethyl oleate, and the like can be used.
  • base of the suppository wi tepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, according to the desired method, and may be administered externally or intraperitoneally, rectally, intravenously, intramuscularly, subcutaneously, intrathoracically or cerebrovascularly by injection. It is desirable to choose.
  • the dosage of the pharmaceutical composition of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion and severity of the disease of the patient, the scope of the present invention in any way It is not intended to be limiting.
  • Individual dosages specifically contain amounts in which the effective drug is administered at one time.
  • Single dose is 1 to 100 milligrams, preferably 3 to 50 milligrams, more preferably 6 to 50 milligrams based on the amount of protein 30 mg, can be administered once a day or divided into several times, it may be divided into several sites.
  • the pharmaceutical compositions of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological reaction modifiers.
  • the present invention is a granulocyte colony stimulator (G-CSF) mutant protein in which the 116th threonine is replaced with cysteine in the amino acid sequence of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator protein.
  • G-CSF granulocyte colony stimulator
  • G-CSF granulocyte colony stimulator
  • the protein can be obtained from chemically synthesized, naturally occurring cells, or purified from modified host cells using genetic recombination technology. There may be a modification and may be in a state in which it is combined with another protein, but this may be regarded as the same as the protein before the modification, unless the protein is changed enough to lose its function.
  • Obtaining the protein comprises protein precipitant, centrifugation, ultrasonic crushing, ultrafiltration, dialysis, gel filtration, adsorption chromatography; This can be done using purification techniques known in the art such as exchange chromatography, affinity chromatography and the like.
  • the restriction enzyme recognition sequence in the transferrin gene is removed, a restriction enzyme site sequence is inserted at the 5 'end, and a plasmid including a gene having a restriction enzyme site and a stop codon inserted at the 3' end.
  • the plasmid in which the tunosperin gene and the G—CSF gene were connected was prepared by inserting a gene from which the restriction enzyme recognition sequence was removed into the plasmid.
  • the restriction enzyme recognition sequence in the gene is removed, and the restriction enzyme site and A Kozak sequence was inserted and a plasmid containing the gene with the restriction enzyme site inserted at the 3 'end was prepared. Thereafter, the transferrin gene was fused with the plasmid to prepare a plasmid linked with the transferan gene and the G-CSF variant gene.
  • the present invention is granulocyte colony stimulating factor protein (G-CSF) or granulocyte colony stimulating factor protein ""
  • the threonine (Threonine) of the 116th in the amino acid sequence is replaced by a cysteine (Cysteine) granulocyte colony stimulating factor (G-CSF ) Fusion protein peptide-linked to the terminal of the mutant protein, or granulocyte colony stimulator (G-CSF) mutant protein wherein 116 th threonine of the granulocyte colony stimulator protein is replaced with cysteine.
  • An expression vector comprising a gene encoding the gene, and a transformant in which the expression vector is introduced into a host cell; or an neutropenia or ischemia
  • a method for preventing or treating neutropenia or ischemic disease comprising administering to a subject having a disease.
  • the granulocyte colony stimulating factor (G-CSF) mutant protein or transferrin fusion protein thereof of the present invention has a significantly increased specific activity and blood stability compared to conventional human G-CSF, and has a conventional half-life. Since the purification effect is higher than the increased PEGylated G-CSF, it can be usefully used for preventing or treating ischemic disease or neutropenia.
  • the present invention is a granulocyte colony stimulator (G-CSF) mutation in which the 116th threonine is substituted with cysteine in the amino acid sequence of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator protein A fusion protein having a peptide-linked transferrin at the end of the protein, or a granulocyte colony stimulator (G-CSF) mutant protein in which the 116 th threonine of the granulocyte colony stimulator protein is substituted with cysteine, the vector or the trait A method for neutrophil reduction comprising administering a convert to a subject.
  • G-CSF granulocyte colony stimulator
  • the granulocyte colony stimulating factor (G-CSF) mutant protein or transferrin fusion protein thereof of the present invention has a significantly increased specific activity and blood stability compared to conventional human G-CSF, and has a conventional half-life. Since the purification efficiency is higher than the increased PEGylated G-CSF, the protein can be usefully used to reduce neutrophils.
  • 116 th threonine in the amino acid sequence of granulocyte colony stimulator protein (G-CSF) or granulocyte colony stimulator protein for the treatment of neutropenia, anticancer adjuvant, ischemic disease is cysteine.
  • G-CSF Granulocyte colony stimulating factor
  • a fusion protein having a peptide-linked transferrin at the end of the protein, or a granulocyte colony stimulator (G-CSF) mutant protein in which 116 th threonine of the granulocyte colony stimulator protein is replaced with cysteine, the vector or the above Provides the use of the transformant.
  • the granulocyte colony stimulating factor (G-CSF) mutant protein or transferrin fusion protein thereof of the present invention has a markedly increased specific activity and hypertension stability compared to conventional human G-CSF, and has a conventional half-life.
  • HepG2 * a human liver cancer cell, was used to obtain total mRNA.
  • HepG2 cells were passaged at 37 ° C and 5% carbon dioxide incubator using DMEM (HyClone) containing 10% fetal bovine serum (WelGNE) and 1% penicillin-straptomycin (Gibco). After passage, the culture medium of HepG2 cells was removed, washed with phosphate buffer (HyClone), and the cell pellets were collected and centrifuged to remove the medium, and then stored at -70 ° C. Cells stored at ⁇ 70 ° C.
  • RNA samples were taken out and thawed and then subjected to the standard conditions given in the guidelines using RnaUs ToTal Tissue Premium RNA Preps (LeGene) to obtain total mRNA.
  • Total mRNA obtained was synthesized using ol igo (dT) by the Premium Express 1 st Strand cDNA synthesi s system (Legene). Polymerase chain reaction (PCR) was performed using DNA polymerase (Phusion) as a template of the prepared cDNA.
  • PCR Polymerase chain reaction
  • the primers used for reaction were custom-made by Cosmojintech, and the Nhe ⁇ restriction enzyme site, Kozak sequence was placed at the 5 'end of the transferrin gene, and the Xho I restriction enzyme site, Stop codon (TM) at the 3' end. ) To be positioned.
  • Sense primer Nhe ⁇ restriction enzyme site, Kozak sequence was placed at the 5 'end of the transferrin gene, and the Xho I restriction enzyme site, Stop codon (TM) at the 3' end.
  • the amplified transferrin gene was electrophoresed on a 1% agarose gel to confirm its size, and the DNA was purified using a MEGAquick-sp in TM Total Fragment DNA purification kit (iNtRoN) by cutting the agarose gel of the desired portion.
  • the transferrin gene and pcDNA3.1 (+) plasmid (Invitrogen) obtained through purification were treated with Nhel (Enzynomics) and Xhol (Enzynomics) restriction enzymes.
  • E. coli DH10B was transformed and E. coli colonies were selected by applying to agar plate containing ampicillin antibiotic (Agar) and incubated for 16 hours at 37 ° C. • PCR polymerase chain reaction (PCR) was used to confirm the successful insertion of the transferrin gene into the pcDNA3.1 (+) plasmid. E. coli colonies were used as a template by diluting in water and using a sense primer (5'-CATGCTAGCTCCACCATGAGGCTCGCCGTGGGAGCC-3 ', SEQ ID NO: 9) and an antisense primer (5, -AGACTCGAGTTMGGTCTACGGAAAGTGCAG-3', SEQ ID NO: 10) used for transferrin gene amplification.
  • a sense primer 5'-CATGCTAGCTCCACCATGAGGCTCGCCGTGGGAGCC-3 ', SEQ ID NO: 9
  • an antisense primer 5, -AGACTCGAGTTMGGTCTACGGAAAGTGCAG-3', SEQ
  • the reaction was repeated for 30 seconds at 98 ° C., the circulation program was repeated 30 times at 10 ° C. for 10 seconds, 30 seconds at 53 ° C., 1 minute at 72 ° C., and finally for 10 minutes at 72 ° C.
  • electrophoresis was performed on 1% agarose gel to confirm the gene, and the confirmed E. coli colonies were inoculated in LB liquid medium (1% tryptone, 0.5% yeast extract, l% NaCl) for 16 hours. The cells were cultured in a 37 ° C shaker. The cultured E.
  • the nucleic acid was replaced with thymine (T) to cytosine (C) to remove the Ban restriction enzyme site present in the transferrin gene, but the amino acid sequence was maintained with aspartic ac, BawHl restriction Enzyme site, GASC primers (5′-CTATGGGTCAAMGAGGACCCACAGACTrrCTATT— 3 ′, SEQ ID NO: 11) and anticent primer (5′-AATAGAAAGTCTGTGGGTCCTCTTTTGACCCATAG-3 ′, SEQ ID NO: 12) to replace nucleic acids with GGATCCC. Used by Cosmojin Tech.
  • the prepared pcDNA3.1 (+) / preTf plasmid was used as a template and the Muta-direct Site Directed Mutagenesis kit (iNtRON) was used as the instructions provided by the manufacturer. After completion of the polymerase chain reaction, the gene was confirmed by electrophoresis on 1% agarose gel. The identified E. coli colonies were inoculated in LB liquid medium containing an empicillin antibiotic and incubated in a 37 ° C shaker for 16 hours. The cultured E.
  • Transferrin is a secreted protein that contains a signal peptide that helps the protein synthesized at the N-terminus to the cell membrane, and the amino acid sequence is MRLAVGALLVCAVLGLCLA (SEQ ID NO: 13).
  • a plasmid was prepared in which the signal peptide present in the transferrin was removed and the restriction enzyme 3 ⁇ 4 //? M was placed at the 5' end.
  • Xho I and Stop codon (TM) were located.
  • Polymerase chain reaction was carried out using DNA polymerase as pcDNA3.K +) / preTf (B). .
  • the primers used for reaction were custom made by Cosmojin Tech.
  • a sense primer (5'-CTCGGATCCGTCCCTGATAAAACTGTGAGATG-3 ', SEQ ID NO: 14) and an antisense primer (5' -AGACTCGAGTTMGGTCTACGGAAAGTGCAG— 3 ', SEQ ID NO: 10) were used as 10 ⁇ ) 1 with template and distilled water in DNA polymerase and buffer solution. was added to bring the total volume to 50 ⁇ . The reaction was repeated for 30 seconds at 98 ° C using a PCR apparatus, and the circulation program was repeated 30 times for 1 minute at 98 ° C 10 seconds, at 53 ° C for 30 seconds, and at 72t; and finally for 10 minutes at 72 ° C. .
  • the amplified transferrin gene was electrophoresed on a 1% agarose gel to confirm its size, and the agarose gel was cut and purified using a MEGAquick-s in TM Total Fragment DNA purification kit iNtRoN.
  • Purified transferrin gene and pcDNA3.1 (+) plasmid were treated. 10 units of restriction enzyme and complete solution 2 (10 mM Tris-HCL H 7.9, 50 mM NaCl, 10 mM MgCl 2> 1 mM DTT, 100 yg / ml BSA) was added and reacted at 37 ° C for 2-3 hours.
  • E. coli colonies were used as a template by dilution in water and were used as a template for sense amplification (5'-CTCGGATCCGTCCCTGATAAAACTGTGAGATG-3 ', SEQ ID NO: 14) and antisense primer (5' -AGACTCGAGTTAAGGTCTACGGAAAGTGCAG-3 ', SEQ ID NO: 10) the use was and banung 30 seconds at 98 ° C, cycling program was banung 98 ° C 10 seconds, 30 seconds at 53 ° C, repeated for 1 minute 30 cycles at 72 ° C and finally added at 72 ° C 10 minutes.
  • reaction plasmid 45 ng of the reaction plasmid was amplified by PCR method with 10 pmol sense primer (5'-CATGCTAGCTCCACCA TGGCTGGACCTGCCACCCAG-3 ', SEQ ID NO: 15) and antisense primer (5' -CATGGATCCGGGCTGGGCAAGGTGGCG-3 ', SEQ ID NO: 16), 5 G-CSF gene fragment was prepared with Nhe restriction enzyme site and Kozak sequence at the end and Ba / nH restriction site at the 3 'end.
  • sense primer 5'-CATGCTAGCTCCACCA TGGCTGGACCTGCCACCCAG-3 ', SEQ ID NO: 15
  • antisense primer 5' -CATGGATCCGGGCTGGGCAAGGTGGCG-3 ', SEQ ID NO: 16
  • Sense primer (5'-GCCGACTTTGCCACCTGCATCTGGCAGCAGAT-3 ', SEQ ID NO: 19) and antisense primer (5 '-ATCTGCTGCCAGATGCAGGTGGCAAAGTCGGC-3', SEQ ID NO: 20), respectively, 10 ⁇ and mixed with pET21a (+) / G-CSF, and through a chain polymerization reaction in the same manner as in Example ⁇ 1-1> Regioselective mutations were performed to construct the G-CSF (T116C) gene.
  • the plasmid thus made is referred to herein as pET21a (+) / G-CSF (T116C).
  • pET21a (+) / G-CSF was placed on DNA agarose gel (0.5X TAE, 1% agarose) to check the size, and only the portion of the gel was cut out and purified by a gel extraction kit (CosmoGentech).
  • E. coli DH10B was transformed with purified pET21a (+) / G-CSF (T116C), and empicillin resistant colonies were selected. Selected colonies were inoculated in LB liquid medium (50 mg of tryptone per 5 mL, 50 mg of sodium chloride, 25 mg of yeast extract) incubated for 15 hours using an empicillin (100 ug / mL) and pET21a (plasmid extraction kit). After extracting +) / G-CSF (T116C), it was confirmed that the nucleotide sequence was successfully replaced by the nucleotide sequence method.
  • the sense primer (5'-GCCGACTTTGCCACCTGCATCTGGCAGCAGAT-3 ', SEQ ID NO: 19) and the antisense primer
  • each of the primers take each 10 pn l of the G-CSF gene inserted into pcDNA6 by the PCR method and the above Example Regioselective mutations were carried out in the same manner as in ⁇ 1-1> to obtain a G-CSF (T116C) gene.
  • the plasmid thus made is referred to herein as pcDNA6 / G-CSF (T116C).
  • pcDNA6 / G-CSF (T116C) was placed on a DNA agarose gel (0.5X TAE, 1% agarose) to check the size, and only the gel of the portion was cut out and purified by a gel extraction kit (CosmoGentech).
  • E. coli DH10B was transformed with purified PCDNA6 / G—CSF 116C) and empicillin resistant colonies were selected. Selected colonies were inoculated in LB liquid medium (50 mg of tryptone per 5 mL, 50 mg of sodium chloride, 25 mg of yeast extract) containing 15 ml of empicillin (100 / g / ml), and incubated for 15 hours, using pcDNA6 as a plasmid extraction kit.
  • reaction plasmid 45 ng was amplified by PCR using a 10 pm sense primer (5'-CATGCTAGCTCCACCATGGCTGGACCTGCCACCCAG-3 ', SEQ ID NO: 15) and an antisense primer (5' -CATGGATCCGGGCTGGGCAAGGTGGCG-3 ', SEQ ID NO: 16).
  • a G-CSF (T116C) DNA fragment was prepared having an A3 ⁇ 4el restriction enzyme site and a Kozak sequence at the end and a BamHY restriction site at the 3 'end. Prepared G-CSF T116C) DNA fragment 1 and pcDNA3.
  • pET21a (+) / G-CSF (T116C) fulllasmid 1 was inoculated into the expression strain Rosetta2 (DE3) 20 ⁇ and transformed by reaction at 42 ° C for 90 seconds.
  • the transformed expression strains were plated in LB medium poolate containing 50 ug / mL empicillin and incubated at 37 ° C. for 15 hours to obtain colonies. Colonies were inoculated into 100 mL LB medium containing 50 ug / mL empicillin and 25 ug / mL chloramphenicol and incubated at 37 ° C for 15 hours.
  • the proteins used in the present invention were expressed by secreting proteins out of cells using Expi293F TM cells (Gibco), a human embryonic kidney cancer cell line modified for suspension culture. After thawing the Expi293F TM cells contained in the Ex i293F TM Expression System Kit (Gibco), a sterile 125 mL flask (Thermo) containing 30 mL suspension-free media (Serum-free media for animal Cel lture, Gibco) ) was inoculated at 37 ° C, 5 carbon dioxide sections were incubated at a stirring speed of 125 rpm in an incubator.
  • G-CSF-Tf and G-CSF (T116C) -Tf plasmid DNA 22.5 were prepared and diluted with Opt i -MEM (Gibco) to prepare 45 ⁇ L of Exp i Feet amine TM 293 Reagent. Diluted with Opt i -MEM® medium and left for 5 minutes at room temperature. After mixing the plasmid and the Exp i Feet amine TM 293 Reagent mixture well, the mixture was left for 20 minutes in the chamber and evenly added to the prepared cells.
  • the cells were dissolved in crushing buffer (50mMTris-Cl ( ⁇ . ⁇ ), 2 mM EDTA, 1 mM PMSF), and the cells were crushed using an ultrasonic crusher (breaking time 5 minutes, ultrasonic 30 seconds, air 40 seconds). ). The supernatant was removed by centrifugation at 10,000 rpm for 30 minutes at 4 ° C., and then dissolved in a washed supernatant (50mMTris-Cl, pH8.0, 2mMEDTA, 0.3% Triton), and the precipitate was crushed using an ultrasonic grinder (crushing time 5 Minutes, ultrasonic 30 seconds, atmospheric 40 seconds).
  • crushing buffer 50mMTris-Cl ( ⁇ . ⁇ ), 2 mM EDTA, 1 mM PMSF
  • the supernatant was removed by centrifugation at 10,000 rpm for 30 minutes at 4 ° C., and then dissolved in a washed supernatant (50mMTris-Cl, pH8.0, 2mMEDTA, 0.
  • Purified G-CSF (T116C) was quantified using silver staining. After obtaining a standard quantitative curve using G-CSF standard grasine (less than 30 ng / SDS-PAGE well), the protein was quantified by comparing the unknown amount of each sample with the standard quantitative curve.
  • G-CSF-Tf black or black G-CSF (T116C) -Tf contained in potassium phosphate buffer was charged to a DEAE Affi-gel Blue column at a flow rate of 0.5 mL / min.
  • 72 mM potassium phosphate was passed in a step gradient, so that the proteins adsorbed on the resin were separated from the other proteins included in the culture (FIG. 6).
  • Quantification of purified protein was carried out by Western blot using transferrin antibodies.
  • Transferrin binds to the transferrin receptor expressed on the cell surface, enters the endocytosis into the cell, releases iron and moves back to the cell surface in combination with the receptor and is secreted into the blood.
  • the half-life is increased through in vivo recycling metabolism, which avoids the decomposition of intracellular lysosomes while supplying iron to cells.
  • the fusion of a target protein with a short half-life with transferrin allows the transfer of the transfer protein in vivo to be metabolized by following the in vivo recycling metabolism of transferrin.
  • a protein fused with G-CSF to transferrin is a transferrin receptor. It is necessary to maintain the binding capacity to effectively prevent the degradation metabolic process in vivo, so the extracellular surface transferrin receptor of the proteins of the present invention.
  • the human liver cancer cell line HepG2 having a high specific expression of TfR cell membrane was tested.
  • HepG2 cells were inoculated in 96-well poolates at 5xl0 5 cells per well and incubated for 24 hours.
  • HepG2 attached to the plate was washed with phosphate buffer, and then serum-free DMEM medium (HyClone) containing 1 mg / ml of bovine serum albumin (BSA) was added thereto and left for 30 minutes at 37 ° C and 5% carbon dioxide thermostat. By removing the endogenous transferrin existing in the cells.
  • serum-free DMEM medium HyClone
  • BSA bovine serum albumin
  • test substance and fluorescently labeled human transferrin Alexa Fluor ® 647 were added to serum-free DMEM medium containing 1 mg / mL BSA to a concentration of 133 nM and 5 / mL, respectively. After mixing, the mixture was treated with HepG2 cells prepared by the above technique and left for 30 minutes in a 37 ° C, 5% carbon dioxide incubator. Protein samples remaining in the medium were washed with cold phosphate buffer and the amount of fluorescence-transferrin bound to transferrin receptor. was measured using a fluorimeter. Fluorescence was measured by applying a light source at 650 nm and measuring the intensity of fluorescence emitted at 668 nm.
  • the transfero receptor binding capacity of the holo form of G-CSF (T116C) -Tf prepared in Example ⁇ 3-4> is shown in ⁇ Figure 8>.
  • the experimental results showed that the transferrin receptor binding capacity of the G-CSF-Tf and G-CSF (T116C) -Tf fusion proteins was similar to that of the native human hol-transferr in (SIGMA). Indicated.
  • SIGMA native human hol-transferr in
  • Human bone marrow-derived HL-60 cells used in the present invention were used for the experiments obtained from the Korea Institute of Bioscience and Biotechnology (KCTC).
  • the cell line was 371, 5% in RPMI-1640 medium containing 10% fetal calf serum. Incubated under carbon dioxide conditions and replaced with fresh medium every two to three days. Cells are not adhesion dependent. Reagents related to cell culture were purchased from HyClone. ⁇ 5-2> Accumulation of Cell Proliferation Activity of G-CSF Mutant Proteins
  • DMSO dimethyl sulfoxide, culture grade, SIGMA
  • G-CSF Lung-enzymatic human G-CSF (GenScript), native G-CSF (GenScript), test substance G-CSF-Tf, G-CSF (T116C) -Tf ⁇ G-CSF (T116C) proteins used as controls
  • G-CSF G-CSF, PEG.
  • G-CSF, G-CSF (T116C) ⁇ G-CSF-Tf, G-CSF (T116C) -Tf cell proliferation activity is shown in Figure 9.
  • G-CSF T116C Cell proliferation activity was inferior to that of native G-CSF and showed similar cell activity to PEGylated G-CSF.
  • the G-CSF (T116C) -Tf fusion protein, in which the G-CSF 116C) mutant protein is fused to transferrin has significantly increased cell proliferation activity compared to G-CSF0116C), and has much higher cell proliferation activity than PEGylated G-CSF. Increased.
  • G-CSF (T116C) -Tf has a higher cellular activity than commercially available PEGylated G-CSF and is expected to have pharmacological significance when used in the treatment of G—CSF.
  • the amount of G-CSF remaining in the serum at a predetermined time after the reaction of the protein with human serum (Serum) is determined. Measured.
  • G-CSF G-CSF protein content was determined by regression analysis.
  • Rabbit polyclonal anti-G-CSF antibody (K0MA BIOTECH) coated at a concentration of about 2 pg / ml High protein binding microtiter plates (Corning) were prepared.
  • Blocking buffer is prepared by using 1) IX phosphate buffer solution containing Casein sodium salt, and the standard solution and the sample solution are mixed with 10 times diluted buffering buffer.
  • Protein samples and reagents were dispensed at 100 y 1 per well, and each step was treated three times with 300 ⁇ l wash solution (1 ⁇ PBS; 0.05% Tween-20) per well to wash unreacted samples.
  • Protein samples and detection antibodies (rabbit polyclonal anti-G-CSF antibody, K0MA BIOTECH) were sequentially treated at room temperature for about 2 hours and then Streptavidin-horseradi sh peroxidase (Pierce) was reacted at 37 ° C. for about 30 minutes.
  • Substrates ⁇ (3,3 ', 5, 5'-tetramethylbenzidine), Pierce
  • G-CSF, G-CSF T116C time remaining residual G-CSF amount (3 ⁇ 4) is shown in Figure 10.
  • the G-CSF (T116C) protein was 15 hours after the native human G-CSF, and the amount of remaining G-CSF0116C) was about 30% higher than that of the native human G-CSF.
  • the formation of disulfide bonds between Cysll6 and Cysl7 helps to increase the protein's resistance to blood proteases.
  • Example 7 In Vivo Half-Life Measurement of G-CSF Mutant Proteins
  • the G-CSF variant of the present invention and its transferrin fusion protein were administered in vivo, the amount of G-CSF present in serum after administration of the protein in mice was measured to determine the biohalf life of the protein.
  • the "in vivo half-life” described above refers to the "half-life of protein in plasma", ie, the time zone in which G ⁇ CSF protein circulates in the plasma, remaining about 50% of its initial concentration. Pharmacokinetic calculations were performed using PKsoiver v2.0.
  • mice were purchased from 6 weeks old ICR, SPF, male mice from Samtaco. Individual body weights were measured at the end of the quarantine and purifying periods after a minimum of 6 days of woo-woong period. On the basis of the body weight, healthy animals without weight gain and general symptoms were selected and the average weight of each group was Randomly placed (35-45 g per animal) randomly composed 6 animals for each test group.
  • the dose of each individual was converted (5 mL / kg), followed by transferrin fusion protein of 20 G-CSF variant or 100 g G-CSF variant per weight (kg). Taken with a disposable syringe (lmL, 26G needle) and slowly injected into the Intravenous Bolus. Dosing date was defined as Day 1 of testing. Blood was collected from the animals by orbital collection at 0.25, 0.5, 1, 3, 6, 12, 24, 48 and 72 hours after injection. After collection, blood flowed for 1-2 minutes at 10, 000-13,000 rpm.
  • Plasma is separated by centrifugation, and the separated plasma is contained in about 1 ⁇ 50 u L / tube each in one tube marked with test classification, animal number, and blood collection time, and stored in ultra-cold storage (about -70 ° C) until the day of analysis. Stored.
  • the amount of active G-CSF in plasma was quantified by ELISA after thawing the sample on ice.
  • the quantification of G-CSF in plasma by ELISA was performed by purchasing Human G-CSF ELISA Kit, pink-ONE ( Koma biotech.), And the experimental method was performed according to the manual of the corresponding reagent.
  • a calibration curve of absorbance according to the protein concentration of the standard solution was prepared, and the content of G-CSF mutant proteins in the sample was determined by regression analysis.
  • Noncompartmental pharmacokinetic analysis from the mean concentration-time profile of each test compound is performed by applying IV Bolus non-compartment analys is input to the PKsolver v2.0 program.
  • Pharmacokinetic parameters assess terminal half-life (t 1/2 ).
  • the half-life of proteins in plasma of proteins is shown below.
  • the G-CSF (T116C) mutant protein showed a blood half-life similar to that of native human G-CSF. This is a result showing that the filtration rate in the kidney is similar because it has a similar protein radius to G-CSF.
  • G-CSF-Tf protein showed improved blood half-life compared to G-CSF.
  • the G-CSF (T116C) -Tf fusion protein in which the G-CSF (T116C) protein was fused to transferrin, had a longer half-life than the G-CSF-Tf protein and showed a half-life similar to that of the PEGylated G-CSF. This is a result of fusion of G-CSF (T116C) mutant protein with increased resistance to blood protease to transferrin, resulting in the reduction of renal filtration rate and improvement of blood stability, thereby maximizing blood half-life in vivo. 11).
  • Example 8 Pharmacodynamics of Leukopenia Rats of G "CSF Mutant Proteins
  • hematology analysis was performed using a leukopenia rat disease model. All processes such as breeding, administration and hematological analysis were commissioned by Cuvest Co., Ltd. Hematological analysis items were WBC, NEU, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, Differential leucocyte count, etc.
  • test substance was converted into individual doses (2 mL / kg) based on the weight measured immediately before administration on the day of administration, and then slowly injected into the Intravenous Bolus using a disposable syringe (l mL, 26G needle). do. Dosing date was defined as Day 1 of testing.
  • G-CSF (T116C) -Tf-treated group was treated with G-CSF and Peptide at 6 and 12 hours. Neutrophils higher than 3 ⁇ 4 G-CSF group To represent the daegap Confirmed.
  • G-CSF the absolute value of neutrophils was slightly higher than that of the control group at 24 hours, but there was no significant difference, whereas pegylated G-CSF and G-CSF (T116C) -Tf were treated in Cyclophosphamide-induced leukopenia rat disease model. The effect of increasing white blood cell (Whi te blood cel l, WBC) and neutrophil levels up to 24 hours was observed.
  • the G-CSF (T116C) -Tf fusion protein was circulated in vivo by transferrinol-mediated activation and showed a longer in vivo retention time than G-CSF and similar to PEGylated G-CSF.
  • the pharmacokinetic parameters (Cmax and AUCO-t) values were measured higher than PEGylated G-CSF, as shown in FIG. 9, to proliferate more neutrophi lic precursor cel l in the same time than PEGylated G-CSF. Judging from the result. ( Figure 12)
  • the substance derived from the present invention has the biological efficacy of G-CSF , which is clinically used, it is used for the treatment of ischemic diseases or stimulates neutrophi granules (neutrophi l granulopoiesis) for cancer chemotherapy and radiation therapy. It can be used as an anticancer adjuvant to prevent infectious complications caused by neutropenia.

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Abstract

La présente invention concerne une protéine de fusion dans laquelle la transferrine est liée par un peptide à une extrémité d'une protéine du facteur stimulant les colonies de granulocytes (G-CSF) ou d'une protéine G-CSF mutante dans laquelle la 116ème thréonine est substituée par une cystéine dans la séquence d'acides aminés de la protéine G-CSF. Plus particulièrement, la protéine G-CSF mutante de la présente invention ou une protéine de fusion de transferrine correspondante présente une activité spécifique et une stabilité dans le sang remarquablement augmentées par rapport à une protéine G-CSF humaine classique et présente une efficacité de purification élevée par rapport à la protéine G-CSF pégylée classique, présentant une demi-vie augmentée, et peut ainsi être utile pour prévenir ou traiter les maladies ischémiques ou une neutropénie.
PCT/KR2014/011269 2014-07-23 2014-11-21 Composition pharmaceutique contenant, comme principe actif, une protéine mutante du facteur stimulant les colonies de granulocytes ou la protéine de fusion de transferrine correspondante Ceased WO2016013725A1 (fr)

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KR20040084884A (ko) * 2001-07-11 2004-10-06 맥시겐 홀딩스 엘티디 지-씨에스에프 접합체
US8188032B2 (en) * 2003-10-10 2012-05-29 National Institutes Of Health (Nih) G-CSF transferrin fusion proteins
KR20120047338A (ko) * 2010-10-18 2012-05-14 대한민국(농촌진흥청장) 인간 과립구 콜로니 자극 인자(ｇ-ｃｓｆ)의 변이체를 포함하는 허혈성 질환 치료용 약학 조성물

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